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WO2010061383A1 - Procédé permettant d’enrichir in-situ des aliments avec des fructanes - Google Patents

Procédé permettant d’enrichir in-situ des aliments avec des fructanes Download PDF

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Publication number
WO2010061383A1
WO2010061383A1 PCT/IL2009/001104 IL2009001104W WO2010061383A1 WO 2010061383 A1 WO2010061383 A1 WO 2010061383A1 IL 2009001104 W IL2009001104 W IL 2009001104W WO 2010061383 A1 WO2010061383 A1 WO 2010061383A1
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Prior art keywords
levansucrase
sucrose
food
fructan
levan
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Ceased
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PCT/IL2009/001104
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English (en)
Inventor
Oded Vashitz
Ester Gorelik
Roni Aharon Bornstein
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BIODALIA MICROBIOLOGICAL TECHNOLOGIES Ltd
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BIODALIA MICROBIOLOGICAL TECHNOLOGIES Ltd
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Publication of WO2010061383A1 publication Critical patent/WO2010061383A1/fr
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Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
    • C12Y104/0101—Glycine dehydrogenase (1.4.1.10)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/195—Proteins from microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin

Definitions

  • the present invention in some embodiments thereof, relates to methods of in- situ enrichment of foods with fructan.
  • Sucrose (common name: table sugar, also called saccharose) is a disaccharide (glucose + fructose) with the molecular formula C 12 H 22 O 11 . Its systematic name is ⁇ -D- glucopyranosyl- (l ⁇ 2)- ⁇ -D-fructofuranoside. It is best known for its role in human nutrition and is formed by plants but not by other organisms such as animals. Sucrose is an easily assimilated macronutrient that provides a quick source of energy to the body, provoking a rapid rise in blood glucose upon ingestion. However, pure sucrose is not normally part of a human diet balanced for good nutrition, although it may be included sparingly to make certain foods more palatable.
  • sucrose has been linked with some adverse health effects.
  • the most common is dental caries or tooth decay, in which oral bacteria convert sugars (including sucrose) from food into acids that attack tooth enamel.
  • Sucrose as a pure carbohydrate, has an energy content of 3.94 kilocalories per gram (or 17 kilojoules per gram).
  • beneficial nutrients can be displaced from the diet, which can contribute to an increased risk for chronic disease.
  • sucrose-containing drinks may be linked to the development of obesity and insulin resistance. The rapidity with which sucrose raises blood glucose can cause problems for predisposed population suffering from defects in glucose metabolism, such as hypoglycemia or diabetes mel ⁇ tus.
  • Sucrose can contribute to development of metabolic syndromes.
  • rats that were fed a diet one-third of which was sucrose the sucrose first elevated blood levels of triglycerides, which induced visceral fat and ultimately resulted in insulin resistance.
  • sucrose-rich diets developed high triglycerides, hyperglycemia, and insulin resistance.
  • Levansucrase catalyses transfructosylation from sucrose to a variety of acceptors including water (sucrose hydrolysis), glucose (exchange reaction), levan (polymerase reaction) and sucrose (oligofructoside synthesis).
  • This enzyme converts sucrose into fructan (type levan - ⁇ -2-6 linked D- fructose units or type inulin - ⁇ -2-1 linked D- fructose units) and fructoolligosaccharides-FOS (1).
  • the two catalytic products i.e., levan and FOS have different nutritional value.
  • Levan has some potential medical/ pharmaceutical application such as anti cancer and cholesterol lowering properties.
  • levan has a number of effects on the immunologic system, including tumor suppression and enhancement of leukocyte antitumor activity (2).
  • Levans are widely used in the food industry as any of a sweetener, filler/ bulking agent , substitue for gum arabic and other applications as an emulsifier, formulator, stabilizer, thickener, surface-finishing agent, encapsulating agent, carrier for flavor and fragrances, cosmetics (3) and the like.
  • Fructooligosaccharides are natural low calorie sweeteners, which have been shown to improve the health of humans and animals by selectively stimulating the growth of beneficial bacteria, such as bifidobacteria (4).
  • the ratio between polymerization needed for levan production and oligosaccharide synthesis needed for FOS production has been found to be species dependent.
  • FOS kestose and nystose
  • sucrose 8-9
  • Reacting Lactobacillus levansucrase with 0.4 M sucrose as fructosildonor and acceptor yielded 1-kestose, nystose and FOS with a DP of 5
  • Levansucrase from Acetobacter diazotrophicus generates high yield of oligofructoside during sucrose transformation (7,11).
  • levansucvrase of B subtilis catalyses the formation of high- molecular-mass levan without accumulation of FOS (5, 12, 13).
  • a method of in-situ producing fructan in food, feed, dietary supplement or cosmetic supplement comprising contacting the food, feed, dietary supplement or cosmetic supplement with a glycosyltransferase under conditions which favor fructan production over fructo-oligosaccharide (FOS) production, thereby in-situ producing the fructan.
  • a food or feed comprising levansucrase, wherein a levan content in the food or feed is higher than in a corresponding food or feed that has not been contacted with the levansucrase and wherein the corresponding food or feed is devoid of an exogenously added levan.
  • the method further comprising incubating the glycosyltransferase with sucrose so as to generate levan prior to the contacting.
  • the conditions comprise a sucrose concentration of 1-80 %. According to some embodiments of the invention, the conditions comprise a pH at a range of 2-9.
  • the conditions comprise presence of a metal ion.
  • the metal ion is selected from the group consisting of Ca 2+ and Fe 3+ .
  • the glycosyltranferase comprises levansucrase (E.C. 2.4.1.10).
  • the conditions comprise a concentration of the levansucrase of 50-5000 u/1. According to some embodiments of the invention, the conditions comprise an incubation time of 1-96 hours. According to some embodiments of the invention, the levansucrase is a B. subtilis levansucrase.
  • the levansucrase is a streptococcus salivarius levansucrase.
  • the method is effected with the proviso that the food is not soybeans.
  • the levansucrase is purified. According to some embodiments of the invention, the levansucrase is comprised in bacterial cells. According to some embodiments of the invention, the food, feed, dietary supplement or cosmetic supplement does not comprise exogenously added sucrose.
  • the food, feed, dietary supplement or cosmetic supplement does not comprise exogenously added levan.
  • FIG. 1 is a line graph showing the effect of different pH conditions on levansucrase activity
  • FIG. 2 is a line graph showing the effect of temperature on levansucrase activity
  • FIG. 3 is a line graph showing the effect of different pH conditions on fructan production as determined by the viscosity of the reaction mixture
  • FIG. 4 is a line graph showing the effect of sucrose concentration on viscosity at different temperatures.
  • FIG. 5 is a line graph showing the effect of enzyme concentration at different temperatures on viscosity;
  • FIG. 6 is a line graph showing the effect of Ca 2+ on fructan production as determined by the viscosity of the reaction mixture
  • FIG. 7 is a bar graph showing the effect of Fe 3+ on fructan production as determined by the viscosity of the reaction mixture.
  • FIG. 8 is a line graph showing hydrolysis of sucrose (squares) and release of glucose (circles) by levansucrase.
  • FIG. 9 is a line graph showing HPLC analysis of saccharides profile of 30 % sucrose solution treated with 750 u/1 in 20 0 C. Note, 81 % hydrolysis of sucrose in 24 hrs. The high peak at the left is glucose and moving to the right is fructose, sucrose and FOS in increasing order. At the minimum is the transfer from FOS to Levan. Further right each peak describes one fructosyl unit added to the polysaccharide. The concentration of each Levan chain is described by the amplitude of each peak. The HPLC was Dionex with an amperometric sensor FIGs. 10A-B show sucrose conversion and fructan production in in-situ treated strawberry mash. 1 - control concentrate; 2 - concentrate; 3 - control mash; 4 - treated mash; 5 - fructose.
  • the present invention in some embodiments thereof, relates to methods of in- situ production of fructan in foods.
  • the food industry is in constant search for solutions for reducing sucrose content and caloric value in fruit products, used in processed food like fruit juices, fruit yogurts etc.
  • fructans are added to food products as dietary fibers for enhancement of the food health qualities.
  • the existing methods for sugar reduction lead to loss of nutritional ingredients and minerals.
  • the external addition of dietary fibers is expensive. While reducing embodiments of the present invention to practice, the present inventors have identified reaction conditions which favor in-situ production of fructan over FOS in foods. In-situ production of fructan is a cost-effective process which results in foods having low caloric value and glycemic index, increased dietary fiber content and viscosity.
  • a method of in-situ producing fructan in food, feed, dietary supplement or cosmetic supplement comprises contacting the food, feed, dietary supplement or cosmetic supplement with a glycosyltransferase such as levansucrase (E.C. 2.4.1.10) under conditions which favor fructan production over fructo-oligosaccharide (FOS) production, thereby in-situ producing the fructan.
  • a glycosyltransferase such as levansucrase (E.C. 2.4.1.10)
  • in-situ refers to a process in which the enzyme is directly contacted with the food, feed, dietary or cosmetic supplement to reduce sucrose content and increase fructan production in the product.
  • fructan refers to a fructose polymer of at least 15 units linked by ⁇ (2>l) and/or ⁇ (2>6) bonds.
  • exemplary types of fructans include levan, inulin and graminan.
  • the fructan is levan.
  • levan refers to a D-fructan which is characterized by ⁇ (2>6) binding of fructose molecules, reaching up to n (e.g., tens, hundreds, thousands and hundreds of thousand) fructose units per a carbohydrate chain.
  • fructo-oligosaccharide refers to short chain (i.e., less than 10 fructose residues) oligosaccharides comprised of D-fructose and D-glucose units.
  • the linkage between fructose residues in FOS is beta-(2-l) glycosidic link.
  • Methods of analyzing the carbohydrate profile of the treated food, feed, dietary supplement or cosmetic supplement are well known in the art, e.g., HPLC. Other methods include chromatography and NMR. In the absence of a hydrolytic reaction, the formation of each fructosyl molecule yields a glucose molecule which may be measured by a wide variety of methods. Furthermore, since fructan is a bulking agent, formation of same can be measured as a function of product viscosity.
  • the viscosity is measured as the time for flow of defined volume of liquid through a restriction (see Examples section which follows).
  • substantially all the sucrose in the product is converted to fructan.
  • the sucrose content in the treated product is about 30 % lower, about 35 % about lower, about 40 % lower, about 45 % lower, about 50 % lower, about 55 % lower, about 60 % lower, about 65 % lower, about 70 % lower, about 75 % lower, about 80 % lower, about 85 % lower, about 90 % lower, about 95 % lower, or more than a corresponding product that has not been contacted with levansucrase according to the present teachings.
  • corresponding product refers to a product (e.g., food, feed, dietary or cosmetic supplement) that has not been contacted with the enzyme according to the present teachings, but has been otherwise treated the same as the subject product.
  • the corresponding product is devoid of exogenously added fructan.
  • levansucrase As used herein the term “levansucrase” (E.C. 2.4.1.10) refers to an enzyme of the glycosyltransferase family, which catalyzes the following reaction:
  • sucrose + ⁇ -D-glucosyl-(l ⁇ 2)-[(2 ⁇ 6)- ⁇ -D-fructosyl] ⁇ glucose + ⁇ -D-glucosyl-
  • sucrose 6-fructosyltransferase ⁇ -2,6-fructosyltransferase
  • ⁇ -2,6- fructan:D-glucose 1-fructosyltransferase sucrose:2,6- ⁇ -D-fructan 6- ⁇ -D- fructosyltransf erase
  • the glycosyltransferase is inulosucrase (E.C. 2.4.1.9) that catalyzes the chemical reaction:
  • the enzyme is naturally occurring. According to other embodiments the enzyme is recombinantly produced using methods well known in the art. According to some embodiments the glycosyltransferase is purified i.e., isolated from the host organism. According to some embodiments, the glycosyltransferase is provided in the living host organism (e.g., bacterial cell) which may be inactivated (e.g., irradiated) or in a proliferative state. In this case when the food product is Natto the enzyme is preferably not provided in B.subtilis Natto spp.
  • Levansucrase used in accordance with the present teachings may be derived from bacterial sources, fungal sources and plants.
  • enzymes which can be used in accordance with the present teachings include but are not limited to (SwissProt Accession Numbers) P21130, P05655,Q46654,Q43998, 052408, O68609,O54435,P11701,Q55242, Q60114.
  • the levansucrase is from Bacillus subtilis.
  • levansucrase derived from B. subtilis include, but are not limited to, CAA26513, AAN75494, CAB08015, AAA22725, NP391325, CAB15450,
  • the levansucrase is from bacterial strain of DSMZ 347.
  • the levansucrase is from Streptococcus salivarius e.g., GenBank Accession Numbers: Q55242, AAA71925.
  • the enzyme may be used in a soluble form or the enzyme may be immobilized to a solid phase for economic use.
  • the enzyme is contacted with the product.
  • the product may be supplemented with sucrose or used "as is” such that the processed sucrose is endogenous to- i.e., naturally occurring in, the product.
  • the food or feed in accordance with some embodiments of the present invention typically refers to a solid, semi-solid or liquid food or beverage.
  • sucrose content in some foods is provided below. To achieve optimal fructan synthesis the sucrose content is above 5 %, as will be further discussed hereinbelow.
  • the glycosyltransferase is incubated with sucrose so as to generate fructan prior to contacting with the product Thereafter the whole mixture (which comprises the enzyme) is contacted with the product so as to convert residual (i.e., endogenous) sucrose in the product to fructan.
  • sucrose content e.g., below 5 %
  • the food product may be a beverage or a sweetener such as a syrup.
  • Preferred beverages include but are not limited to, orange juice, apple juice, grapefruit juice, grape juice , pineapple juice , cranberry juice , lemon juice , prune juice , and line juice .
  • syrups include but are not limited to, maple syrup, strawberry syrup, blueberry syrup and boysenberry syrup.
  • food products which can are subject to the present teachings.
  • Other foods which may be treated in accordance with the present teachings include but are not limited to infant formula, cereals, milk
  • Feed products which may be treated in accordance with the present teachings may be in any suitable form; for example in dried form, semi-moist form and wet form, produced using conventional methods.
  • the amount of glycosyltransferase used in the process according to the present teachings will vary depending on a number of parameters. These parameters include, but are not limited to, the product, the amount of fructan to be produced, the treatment time, the inclusion of an enzyme catalyst (e.g., metal ions such as Ca 2+ and Fe 3+ ), the reaction temperature. Optimization of some reaction conditions is provided in the Examples section which follows.
  • an enzyme catalyst e.g., metal ions such as Ca 2+ and Fe 3+
  • enzyme dose and reaction time are inversely proportional, and therefore it is useful to calculate the product of dose and reaction time as a measure of the degree of reaction.
  • an exemplary levansucrase concentration is 50-10,000 u/1, 100-10,000 u/1, 200-2000 u/1, 50-5000, u/1,
  • Fructan production may take place any time during the manufacturing process of the product and may be allowed to continue during storage prior to consumption.
  • a product e.g., food
  • glycosyltransferase e.g., the levansucrase (active or inactivated such as by denaturation), has a levan content that is higher than in a corresponding product that has not been contacted with the enzyme and is devoid of an exogenously added fructan.
  • the fructan content in the product is about 2%, about 5 %, about 10 %, about 15 %, about 20 %, about 25 %, about 30 %, about 35 %, about 40 %, about 45 %, about 50% or more.
  • composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • the term "treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
  • Strains, media and growth conditions - Strains, media and growth conditions - B. subtilis (culture collection DSMZ 347) was grown in shake flasks at 37 0 C in rich media having the following composition (g/1): Peptone from casein- 5 (Merck- cat.1.07213), Meat extract- 3 (Fluka- cat. 1342492), Yeast extract- 2 (Merck- cat. 1.03753.05000), pH- 7 (NaOH/HCl).
  • Bacterial growth was effected in 250 ml Erlenmeyer flask (50 ml media). The medium containing the above components was sterilized in a conventional manner for 30 min at 121 0 C and inoculated with the strain B. subtilis DSMZ 347. An aerobic fermentation was effected in a 20 liter NBS bioreactor (Bioflo IV) with a working volume of 10 liters.
  • Industrial fermentation medium for production of levansucrase comprised the following (g/1): Sucrose- 100 (original sugar), Citric acid - 11.7 (Chen samuel chemicals ltd- cat. 508-01-10000, Na 2 SO 4 - 4 (Frutarom- cat.
  • Levansucrase assays were measured as the glucose released by sucrose hydrolysis. Glucose concentration was determined by reflectometric method (reflectometer RQflex plus, Merck). Levansucrase enzyme unit is defined as the amount of enzyme releasing one micromole of glucose per min under the following conditions: 20 % sucrose in 0.1 mol sodium acetate buffer, pH 5.2 at 37 0 C. To evaluate the effect of pH on levansucrase activity, enzyme samples were incubated with sucrose at 37 0 C in 0.1 mol sodium citrate buffer covering the pH range 4.2-7.1.
  • the Effect of temperature on levansucrase activity was determined by incubating enzyme samples with sucrose at 37, 40, 50, 60 0 C. Samples were withdrawn at different intervals of time and the enzymatic reaction terminated by heat at 100 0 C for 10 minutes.
  • Levan synthesis - Levan synthesis was effected by incubating levansucrase with sucrose. Hydrolysis of sucrose and release of glucose were determined by as described above. The influence of pH, temperature, enzyme concentration, sucrose concentration, and metal-ions- Ca 2+ and Fe 3+ were measured by relative viscosity (time of flow of a constant volume of samples).
  • sucrose concentration - Reaction mixtures were prepared containing different concentration of sucrose (1 %to 35 %) in 0.1 mol buffer acetate, pH 5.2 and 1000 u/1 levansucrase, incubated at 20 0 C up to 24 hours.
  • reaction temperature The effect of reaction temperature on levansucrase activity is shown in Figure 2.
  • the effect of temperature on the enzyme activity was determined in the range of 35-60 0 C.
  • the optimal temperature was determined to be 48 0 C.
  • Levansucrase activity decreased above 50 0 C.
  • Each reaction mixture contained 1000 u/1 levansucrase, 25 % sucrose in 0.1 mol sodium citrate buffer, with pH ranging from 2.2 to 7.2. Temperature of incubation was 20 0 C. Production of levan was determined by relative viscosity. Maximal viscosity was observed at pH 5.2.
  • levansucrase 1000 u/1 was incubated in acetate buffer with various sucrose concentrations and as at various temperatures from 20 to 45 0 C. Production of levan was determined by viscosity. Effect of enzyme concentration - The synthesis of levan by the enzyme in the presence of various concentrations of enzyme was examined. Results are shown in Figure 5. Each reaction mixture was incubated at 20 and 30 0 C in the presence of O.lmol sodium acetate buffer ,pH 5.2.
  • Figure 6 shows the influence of Ca 2+ on levan production and viscosity of reaction mixture.
  • Levansucrase 1000 u/1 was incubated in acetate buffer with 25 % sucrose, at a temperature of 20 0 C with different Ca 2+ concentrations of 0.1 mM, 0.5 mM, 1 mM, 2 mM and 5 mM.
  • Production of levan was determined by relative viscosity. Increasing the concentration of Ca 2+ from 0.1 mM to 2 mM, increased the viscosity, but further increase from 2 mM to 5 mM, resulted in reduction in the viscosity and production of levan.
  • Figure 7 shows the influence of Fe 3+ on levan production and viscosity of reaction mixture.
  • Levansucrase (lOOOu/1) was incubated in acetate buffer with 25 % sucrose , at a temperature of 20 0 C with 0.4 mM Fe 3+ and 2 mM Ca 2+ .
  • Production of levan was determined by viscosity.
  • the addition of both metal ions resulted in enhancement of levan production and viscosity.
  • the hydrolysis of sucrose and release of glucose by levansucrase in reaction mixture is shown in Figure 8.
  • the reaction mixture contained 1000 u/1 levansucrase, 25 % sucrose in 0.1 mol sodium acetate buffer with pH 5.2. Temperature of incubation was 20 0 C. Samples were taken at different time intervals during the incubation.
  • the reaction mixture consisted of 28 ml strawberry jam (1% sucrose) and 2 ml enzyme concentrate ( ⁇ 7000 u/1), precipitated by (NHU) 2 SO 4 , as described above.
  • Sucrose concentration was adjusted in the jam to 143 g/1 and pH was adjusted to 5.1.
  • the temperature of incubation was set to 37°C.

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Abstract

La présente invention concerne un procédé permettant de produire in-situ des fructanes dans des produits alimentaires, des aliments, des compléments alimentaires ou des compléments cosmétiques, ledit procédé comprenant la mise en contact desdits produits alimentaires, aliments, compléments alimentaires ou compléments cosmétiques avec une glycosyltransférase, dans des conditions favorisant la production de fructanes par rapport à la production de fructo-oligosaccharides (FOS), et permettant ainsi la production in-situ des fructanes.
PCT/IL2009/001104 2008-11-26 2009-11-24 Procédé permettant d’enrichir in-situ des aliments avec des fructanes Ceased WO2010061383A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110293782A1 (en) * 2007-02-19 2011-12-01 Gan Shmuel Foods Ltd. Fruit juice and puree with a lowered amount of available sugars
CN105907815A (zh) * 2016-05-25 2016-08-31 四川大学 一种高均一度大分子量Levan型果聚糖的制备方法
WO2019100541A1 (fr) * 2017-11-27 2019-05-31 陈程 Procédé de préparation d'une enzyme pour réguler des fonctions gastro-intestinales
US10806769B2 (en) 2016-03-31 2020-10-20 Gojo Industries, Inc. Antimicrobial peptide stimulating cleansing composition
US10874700B2 (en) 2016-03-31 2020-12-29 Gojo Industries, Inc. Sanitizer composition with probiotic/prebiotic active ingredient
EP3781121A4 (fr) * 2018-04-15 2022-01-19 Gan Shmuel Foods Ltd. Compositions comprenant du lévane et leur utilisation
US11564879B2 (en) 2016-11-23 2023-01-31 Gojo Industries, Inc. Sanitizer composition with probiotic/prebiotic active ingredient

Citations (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3791932A (en) 1971-02-10 1974-02-12 Akzona Inc Process for the demonstration and determination of reaction components having specific binding affinity for each other
US3839153A (en) 1970-12-28 1974-10-01 Akzona Inc Process for the detection and determination of specific binding proteins and their corresponding bindable substances
US3850752A (en) 1970-11-10 1974-11-26 Akzona Inc Process for the demonstration and determination of low molecular compounds and of proteins capable of binding these compounds specifically
US3850578A (en) 1973-03-12 1974-11-26 H Mcconnell Process for assaying for biologically active molecules
US3853987A (en) 1971-09-01 1974-12-10 W Dreyer Immunological reagent and radioimmuno assay
US3867517A (en) 1971-12-21 1975-02-18 Abbott Lab Direct radioimmunoassay for antigens and their antibodies
US3879262A (en) 1972-05-11 1975-04-22 Akzona Inc Detection and determination of haptens
US3901654A (en) 1971-06-21 1975-08-26 Biological Developments Receptor assays of biologically active compounds employing biologically specific receptors
US3935074A (en) 1973-12-17 1976-01-27 Syva Company Antibody steric hindrance immunoassay with two antibodies
US3984533A (en) 1975-11-13 1976-10-05 General Electric Company Electrophoretic method of detecting antigen-antibody reaction
US3996345A (en) 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
US4034074A (en) 1974-09-19 1977-07-05 The Board Of Trustees Of Leland Stanford Junior University Universal reagent 2-site immunoradiometric assay using labelled anti (IgG)
US4098876A (en) 1976-10-26 1978-07-04 Corning Glass Works Reverse sandwich immunoassay
US4666828A (en) 1984-08-15 1987-05-19 The General Hospital Corporation Test for Huntington's disease
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4801531A (en) 1985-04-17 1989-01-31 Biotechnology Research Partners, Ltd. Apo AI/CIII genomic polymorphisms predictive of atherosclerosis
US4879219A (en) 1980-09-19 1989-11-07 General Hospital Corporation Immunoassay utilizing monoclonal high affinity IgM antibodies
US5011771A (en) 1984-04-12 1991-04-30 The General Hospital Corporation Multiepitopic immunometric assay
EP0457919A1 (fr) * 1989-12-11 1991-11-27 Kabushiki Kaisya Advance Aliment fonctionnel
US5192659A (en) 1989-08-25 1993-03-09 Genetype Ag Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes
US5272057A (en) 1988-10-14 1993-12-21 Georgetown University Method of detecting a predisposition to cancer by the use of restriction fragment length polymorphism of the gene for human poly (ADP-ribose) polymerase
US5281521A (en) 1992-07-20 1994-01-25 The Trustees Of The University Of Pennsylvania Modified avidin-biotin technique
US5334524A (en) 1992-05-18 1994-08-02 Solvay Enzymes, Inc. Process for producing levan sucrase using Bacillus licheniformis
WO2002050311A2 (fr) 2000-12-21 2002-06-27 Societe Des Produits Nestle S.A. Souche de lactobacille produisant une levane et son utilisation dans des produits alimentaires destines aux humains ou aux animaux domestiques
US20020127681A1 (en) * 2000-05-25 2002-09-12 Van Hijum Sacha Adrianus Fokke Taco Novel fructosyltransferases
WO2004013343A2 (fr) * 2002-08-06 2004-02-12 Danisco A/S Composition
WO2005051102A1 (fr) 2003-11-27 2005-06-09 Technische Universität München Procede d'enrichissement d'aliments par fermentation au moyen de fructo-oligosaccharides
WO2007061918A2 (fr) 2005-11-22 2007-05-31 Genencor International, Inc. Production de fructo-olicosaccharides et reduction de sucrose in situ
WO2008102336A2 (fr) * 2007-02-19 2008-08-28 Gan Shmuel Foods Ltd. Jus et compote de fruits à teneur réduite en sucres disponibles
EP1987726A1 (fr) * 2007-05-01 2008-11-05 Friesland Brands B.V. Produit alimentaire de goût agréable contenant un agent de neutralisation des composés indésirables

Patent Citations (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3850752A (en) 1970-11-10 1974-11-26 Akzona Inc Process for the demonstration and determination of low molecular compounds and of proteins capable of binding these compounds specifically
US3839153A (en) 1970-12-28 1974-10-01 Akzona Inc Process for the detection and determination of specific binding proteins and their corresponding bindable substances
US3791932A (en) 1971-02-10 1974-02-12 Akzona Inc Process for the demonstration and determination of reaction components having specific binding affinity for each other
US3901654A (en) 1971-06-21 1975-08-26 Biological Developments Receptor assays of biologically active compounds employing biologically specific receptors
US3853987A (en) 1971-09-01 1974-12-10 W Dreyer Immunological reagent and radioimmuno assay
US3867517A (en) 1971-12-21 1975-02-18 Abbott Lab Direct radioimmunoassay for antigens and their antibodies
US3879262A (en) 1972-05-11 1975-04-22 Akzona Inc Detection and determination of haptens
US3850578A (en) 1973-03-12 1974-11-26 H Mcconnell Process for assaying for biologically active molecules
US3935074A (en) 1973-12-17 1976-01-27 Syva Company Antibody steric hindrance immunoassay with two antibodies
US3996345A (en) 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
US4034074A (en) 1974-09-19 1977-07-05 The Board Of Trustees Of Leland Stanford Junior University Universal reagent 2-site immunoradiometric assay using labelled anti (IgG)
US3984533A (en) 1975-11-13 1976-10-05 General Electric Company Electrophoretic method of detecting antigen-antibody reaction
US4098876A (en) 1976-10-26 1978-07-04 Corning Glass Works Reverse sandwich immunoassay
US4879219A (en) 1980-09-19 1989-11-07 General Hospital Corporation Immunoassay utilizing monoclonal high affinity IgM antibodies
US5011771A (en) 1984-04-12 1991-04-30 The General Hospital Corporation Multiepitopic immunometric assay
US4666828A (en) 1984-08-15 1987-05-19 The General Hospital Corporation Test for Huntington's disease
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683202B1 (fr) 1985-03-28 1990-11-27 Cetus Corp
US4801531A (en) 1985-04-17 1989-01-31 Biotechnology Research Partners, Ltd. Apo AI/CIII genomic polymorphisms predictive of atherosclerosis
US5272057A (en) 1988-10-14 1993-12-21 Georgetown University Method of detecting a predisposition to cancer by the use of restriction fragment length polymorphism of the gene for human poly (ADP-ribose) polymerase
US5192659A (en) 1989-08-25 1993-03-09 Genetype Ag Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes
EP0457919A1 (fr) * 1989-12-11 1991-11-27 Kabushiki Kaisya Advance Aliment fonctionnel
US5334524A (en) 1992-05-18 1994-08-02 Solvay Enzymes, Inc. Process for producing levan sucrase using Bacillus licheniformis
US5281521A (en) 1992-07-20 1994-01-25 The Trustees Of The University Of Pennsylvania Modified avidin-biotin technique
US20020127681A1 (en) * 2000-05-25 2002-09-12 Van Hijum Sacha Adrianus Fokke Taco Novel fructosyltransferases
WO2002050311A2 (fr) 2000-12-21 2002-06-27 Societe Des Produits Nestle S.A. Souche de lactobacille produisant une levane et son utilisation dans des produits alimentaires destines aux humains ou aux animaux domestiques
WO2004013343A2 (fr) * 2002-08-06 2004-02-12 Danisco A/S Composition
WO2005051102A1 (fr) 2003-11-27 2005-06-09 Technische Universität München Procede d'enrichissement d'aliments par fermentation au moyen de fructo-oligosaccharides
WO2007061918A2 (fr) 2005-11-22 2007-05-31 Genencor International, Inc. Production de fructo-olicosaccharides et reduction de sucrose in situ
WO2008102336A2 (fr) * 2007-02-19 2008-08-28 Gan Shmuel Foods Ltd. Jus et compote de fruits à teneur réduite en sucres disponibles
EP1987726A1 (fr) * 2007-05-01 2008-11-05 Friesland Brands B.V. Produit alimentaire de goût agréable contenant un agent de neutralisation des composés indésirables

Non-Patent Citations (36)

* Cited by examiner, † Cited by third party
Title
"Animal Cell Culture", 1986
"Basic and Clinical Immunology", 1994, APPLETON & LANGE
"Cell Biology: A Laboratory Handbook", vol. I-III, 1994
"Current Protocols in Immunology", vol. I-III, 1994
"Current Protocols in Molecular Biology", vol. I-III, 1994
"Genome Analysis: A Laboratory Manual Series", vol. 1-4, 1998, COLD SPRING HARBOR LABORATORY PRESS
"Immobilized Cells and Enzymes", 1986, IRL PRESS
"Methods in Enzymology", vol. 1, ACADEMIC PRESS, pages: 317
"Nucleic Acid Hybridization", 1985
"Oligonucleotide Synthesis", 1984
"PCR Protocols: A Guide To Methods And Applications", 1990, ACADEMIC PRESS
"Selected Methods in Cellular Immunology", 1980, W. H. FREEMAN AND CO.
"Transcription and Translation", 1984
AMMAR Y.; MATSUBARA T.; ITO K. ET AL.: "Some properties of levansucrase of Bacillus natto stabilized with periodate oxidized yeast glucomannan", ENZYME MICROB. TECHNOL., vol. 30, 2002, pages 875 - 882
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1989, JOHN WILEY AND SONS
FATTAH A.F.; MAHMOUD D.A.; ESAWY M.A.: "production of levansucrase from Bacillus subtilis NRC 33a and enzymic synthesis of levan and fructooligosaccharides", CURRENT MICROBIAL., vol. 51, 2005, pages 402 - 407, XP019365651
FLEITES C.M.: "Crytal structure of levansucrase from the Gram -negative Bacterium Gluconacetobacter diazotrophicus", BIOCHEM.J., vol. 390, 2005, pages 19 - 27
FRESHNEY: "Culture of Animal Cells - A Manual of Basic Technique", 1994, WILEY-LISS
GHALY A.E.; ARAB F.; MAHMOUD N.D. ET AL.: "Production of levan by bacillus licheniformis for use as a soil sealant in earthen manure storage structures", AM. J. BIOCHEM. BIOTECHNOL., vol. 3, no. 2, 2007, pages 57 - 54
HERNANDEZ L.; ARRIETA J.; MENENDEZ C. ET AL.: "Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane", BIOCHEM. J., vol. 309, 1995, pages 113 - 118, XP000917365
HERNANDEZ L.; SOTOLONGO M.; ROSABAL Y. ET AL.: "Structural levansucrase gene (IsdA) constitutes a functional locus conserved in the species", GLUCONACETOBACTER DIAZOTROPHICUS, vol. 174, no. 1-2, 2000, pages 120 - 124
KIM M.G.; SEO J.W.; SONG K.B. ET AL.: "Levan and fructosyl derivatives formation by a recombinant levansucrase from Rahnella aquatilis", BIOTECH LETT, vol. 20, 1998, pages 333 - 336
KORAKLI M ET AL: "PRODUCTION OF MANNITOL BY LACTOBACILLUS SANFRANCISCENSIS", ADVANCED IN FOOD SCIENCES, TECHNISCHE UNIVERSITAET MUENCHEN, MUENCHEN, DE, vol. 22, no. 1/02, 1 January 2000 (2000-01-01), pages 1 - 04, XP001016281, ISSN: 1431-7737 *
LUKASZ K.O.; SLAVKO K.; MARK L.E.C. ET AL.: "The levansucrase and inulosucrase enzymes of Lactobacillus reuteri 121 catalyse processive and non-processive transglycosylation reactions", MICROBIOLOGY, vol. 152, 2006, pages 1187 - 1196
MARSHAK ET AL.: "Strategies for Protein Purification and Characterization - A Laboratory Course Manual", 1996, CSHL PRESS
MURO A.C.; RODRIGUEZ E.; ABATE C.M. ET AL.: "Levan production using mutant strains of Zymomonas mobilis in different culture conditions", BIOTECH LETT., vol. 22, 2000, pages 1639 - 1642
OSEGUERA P.; GUERECA L.; LOPEZ-MUNGUIA A. ET AL.: "Properties of levansucrase from Bacillus circulans", APPL. MICROBIOL. BIOTECHNOL., vol. 45, 1996, pages 465 - 471
PERBAL, B., A PRACTICAL GUIDE TO MOLECULAR CLONING, 1984
PERBAL: "A Practical Guide to Molecular Cloning", 1988, JOHN WILEY & SONS
ROBERT L.; JAYARAMA K., PROCESS FOR PRODUCING LEVAN SUCROSE USING BACILLUS LICHENIFORMIS, 1994
SAMBROOK ET AL., MOLECULAR CLONING: A LABORATORY MANUAL, 1989
SONG D.; JACQUES N.A.: "Purification and enzymic properties of the fructosyltransferase of Streptococcus salivarius ATCC 25975", BIOCHEM. J., vol. 341, 1999, pages 285 - 291
SZWENGIEL A.; CZARNECKA M.; ROSZYK H. ET AL.: "Levan production by bacillus subtilis DSM 347 strain", FOOD-SCIENCE AND TECHNOLOGY, vol. 7, no. 2, 2004
TIEKING ET AL.: "Exopolysaccharides from cereal-associated lactobacilli", TRENDS IN FOOD SCIENCE & TECHNOLOGY, vol. 16, no. 1-3, 2005, pages 79 - 84, XP002578280, ISSN: 0924-2244 *
TIEKING: "In situ production of exopolysaccharides during sourdough fermentation ny cereal and intestinal isolates of lactic acid bacteria", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 69, no. 2, February 2003 (2003-02-01), pages 945 - 952, XP002578281 *
WATSON ET AL.: "Recombinant DNA", SCIENTIFIC AMERICAN BOOKS

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US20110293782A1 (en) * 2007-02-19 2011-12-01 Gan Shmuel Foods Ltd. Fruit juice and puree with a lowered amount of available sugars
US9375027B2 (en) * 2007-02-19 2016-06-28 Gan Shmuel Foods Ltd. Fruit juice and puree with a lowered amount of available sugars
US10806769B2 (en) 2016-03-31 2020-10-20 Gojo Industries, Inc. Antimicrobial peptide stimulating cleansing composition
US10874700B2 (en) 2016-03-31 2020-12-29 Gojo Industries, Inc. Sanitizer composition with probiotic/prebiotic active ingredient
US11633451B2 (en) 2016-03-31 2023-04-25 Gojo Industries, Inc. Antimicrobial peptide stimulating cleansing composition
US11998575B2 (en) 2016-03-31 2024-06-04 Gojo Industries, Inc. Sanitizer composition with probiotic/prebiotic active ingredient
CN105907815A (zh) * 2016-05-25 2016-08-31 四川大学 一种高均一度大分子量Levan型果聚糖的制备方法
US11564879B2 (en) 2016-11-23 2023-01-31 Gojo Industries, Inc. Sanitizer composition with probiotic/prebiotic active ingredient
WO2019100541A1 (fr) * 2017-11-27 2019-05-31 陈程 Procédé de préparation d'une enzyme pour réguler des fonctions gastro-intestinales
EP3781121A4 (fr) * 2018-04-15 2022-01-19 Gan Shmuel Foods Ltd. Compositions comprenant du lévane et leur utilisation
US11964041B2 (en) 2018-04-15 2024-04-23 Gan Shmuel Foods Ltd. Compositions comprising levan and use thereof

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