FIELD AND BACKGROUND OF THE INVENTION

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 α-D- glucopyranosyl- (l<→2)-β-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.

Overconsumption of 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). When a large amount of foods that contain a high percentage of sucrose is consumed, beneficial nutrients can be displaced from the diet, which can contribute to an increased risk for chronic disease. It has been suggested that 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 melϋtus. Sucrose can contribute to development of metabolic syndromes. In an experiment with 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. Another study found that rats fed 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 -β-2-6 linked D- fructose units or type inulin - β-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. In addition, 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 (FOS) 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).

In vitro production of bacterial levansucrase has been studied extensively in recent years. Several bacterial species are able to produce the enzyme. These include Acetobacter diazotrophicus SRT4-sinonim Gluconacetobacter diazotrophicus SRT4 (5), Bacillus circulans (4), Bacillus natto (6), and Bacillus subtilis NRC 33a (7).

The ratio between polymerization needed for levan production and oligosaccharide synthesis needed for FOS production has been found to be species dependent.

Thus, several scientific reports describe the enzymatic transfer of fructosyl residues of sucrose to produce various FOS. Levansucrase of Gluconacetobacter diazotrophicus and Zymomonas mobilis have been shown to synthesize mainly short

FOS (kestose and nystose ) from 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 (10). Levansucrase from Acetobacter diazotrophicus generates high yield of oligofructoside during sucrose transformation (7,11). In contrast, levansucvrase of B subtilis catalyses the formation of high- molecular-mass levan without accumulation of FOS (5, 12, 13). Other Related Art:

WO2002/050311

WO2005/051102 SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a method of in-situ producing fructan in food, feed, dietary supplement or cosmetic supplement, the method 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. According to an aspect of some embodiments of the present invention there is provided 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. According to some embodiments of the invention, the method further comprising incubating the glycosyltransferase with sucrose so as to generate levan prior to the contacting.

According to some embodiments of the invention, 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.

According to some embodiments of the invention, the conditions comprise presence of a metal ion.

According to some embodiments of the invention, the metal ion is selected from the group consisting of Ca ²⁺ and Fe ³⁺ .

According to some embodiments of the invention, the glycosyltranferase comprises levansucrase (E.C. 2.4.1.10).

According to some embodiments of the invention, 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.

According to some embodiments of the invention, the levansucrase is a streptococcus salivarius levansucrase. According to some embodiments of the invention, the method is effected with the proviso that the food is not soybeans.

According to some embodiments of the invention, 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.

According to some embodiments of the invention, the food, feed, dietary supplement or cosmetic supplement does not comprise exogenously added levan.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings: 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; and

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.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION The present invention, in some embodiments thereof, relates to methods of in- situ production of fructan in foods.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

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. At the same time 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. Thus, according to an aspect of the present invention there is provided a method of in-situ producing fructan in food, feed, dietary supplement or cosmetic supplement. The method 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.

As used herein the term "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.

As used herein the term "fructan" refers to a fructose polymer of at least 15 units linked by β(2>l) and/or β(2>6) bonds. Exemplary types of fructans include levan, inulin and graminan.

According to an exemplary embodiment of the present invention the fructan is levan.

As used herein the term "levan" refers to a D-fructan which is characterized by β(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.

As used herein the term "fructo-oligosaccharide (FOS)" 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 (hereinafter "the product") 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).

According to some embodiments of the present invention, substantially all the sucrose in the product is converted to fructan. Thus, 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.

As used herein the phrase "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. According to exemplary embodiments, the corresponding product is devoid of exogenously added fructan.

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 + α-D-glucosyl-(l→2)-[(2→6)-β-D-fructosyl] _π = glucose + α-D-glucosyl-

(l→2)-[(2→6)-β-D-fructosyl] _π+1

Other names: sucrose 6-fructosyltransferase; β-2,6-fructosyltransferase; β-2,6- fructan:D-glucose 1-fructosyltransferase; sucrose:2,6-β-D-fructan 6-β-D- fructosyltransf erase; sucrose:(2-→6)-β-D-fructan 6-β-D-fructosyltransferase

Systematic name: sucrose:α-D-glucosyl-(l— »2)-(2→6)-β-D-fructan 6-β-D- fructosyltransferase

According to other embodiments of the present invention, the glycosyltransferase is inulosucrase (E.C. 2.4.1.9) that catalyzes the chemical reaction:

sucrose + (2,l-beta-D-fructosyl)n glucose + (2,l-beta-D-fructosyl)n ⁺ l According to some embodiments of the present invention, 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.

Examples of 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.

According to an exemplary embodiment the levansucrase is from Bacillus subtilis. Examples of levansucrase derived from B. subtilis include, but are not limited to, CAA26513, AAN75494, CAB08015, AAA22725, NP391325, CAB15450,

CAL69697, CAI39224, CAI39222. According to an exemplary embodiment the levansucrase is from bacterial strain of DSMZ 347.

According to another exemplary embodiment 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.

As mentioned, 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.

The 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.

In accordance with exemplary embodiments, especially when the product has poor sucrose content (e.g., below 5 %), 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.

Table 1 -Sucrose content in fruits (gr Sucrose 1100 gr of food/ beverage)

Apples, raw, unpeeled

Apple juice

100%, canned, 1.7 unsweetened

Applesauce, canned, no info unsweetened

Apricots, raw 5.2

Apricots, dried 6.4

Avocados, raw 0.1

Bananas, raw 6.5

Bilberries, raw 0.6

Blackberries, _Λ , l.o raw

Blueberries, raw 0.5

Boysenberry, _{Q lξ)} raw

Cherries, raw 0.2

Cranberries, raw 0.5

Currents, black, „ _< . raw Currants, dried 0

Currants, red, „ _ raw

Currants, white, „ „ raw

Damsons, raw 1.0 Dates, dried, no „ sugar

Feijoa, raw 5.19

Figs, dried, no „ sugar

Figs, raw 0

Gooseberries, _{1 1} ripe, raw Grapefruit, raw 2.0

Grapes, black, _ raw

Grapes, white, _ raw

Greengages, raw 3.4

Guava, raw 0.48

Kiwi, raw 1.7

Lemon, raw 0.5

Lemon, juice, _ _ fresh

Lemon, rind, _ _ raw

Loganberries, _ _ raw

Lychees, raw 0.7

Mandarin, raw 5.95

Mango, raw 9.7

Medlars, raw 1.9

Melon, ₁ - Cantaloupe

Melon, _{4 g} Honeydew

Melon, Rock 1.5

Mulberries, raw 0

Nashi, raw 0.60

Nectarine, raw 5.1

Olives, no sugar 0

Orange, juice . _q 100%, fresh

Orange, raw 4.1

Passion fruit, ^ _„ raw

Pawpaw, raw 0

Peach, dried 36.3

Peach, raw 5.3

Pears, raw 3.67

Persimmon, raw 0.5

Pineapple, raw 6.7

Plum, raw 6.4

Pomegranate, 0 juice !00%, fresh

Prunes, dried 0

Quinces, raw 0.3

Raisins, dried T

Raspberries, raw 1.4

Rhubarb, stems, „ ₁ raw

Strawberries, . .. _ raw

Sultanas 0.1

Tamarillo, raw 1.65

Tangelo, raw 5.1

Tangerine, raw 5.0

Watermelon, _ raw

Table 2- content in baverages (gr Sucrose 1100 gr of food/beverage

Carrot juice,

J canned

Cocoa, powder, T 1 unsweetened

Cocoa, powder, 70 3 sweetened

Gatorade, liquid 4.03

Juice, apple 100% 2.1

Juice, grape 100% 0

Juice, grapefruit, unsweetened 0.3

100%

Juice, lemon 100% π U. S o? unsweetened

Juice, lime 100% π A

- unsweetened

Juice, orange

100% 3.0 unsweetened

Juice, pineapple,

100% 4.0 unsweetened

Juice, tomato

100% 0.9 unsweetened

Soft drink, cola, 0 diet

Soft drink, soda _ water

Tea, herbal 0

Water, bottled 0

Table 3- Sucrose content in Dairy (gr Sucrose 1100 gr offood/beverage

Butter, salted 0

Butter, ₀ unsalted Cheese, blue „ vein

Cheese, „ camembert Cheese, „ cheddar

Cheese, colby 0

Cheese, „ cottage Cheese, cottage, low 0 fat Cheese, cream 0

Cheese, cream, 0.5 reduced fat

Cheese, blue 0 Cheese, edam 0

Cheese, „ egmont

Cheese, feta 0 Cheese, gouda 0

Cheese, „ gruyere

Cheese, „ mozzarella

Cheese, „ parmesan

Cheese, ricotta 0 Cheese, swiss 0 Cream, sour 0

Cream, sour, T 1 reduced fat

Cream 0

Cream,

0 whipping

Ice Cream, 1 1Z O.11 softserve

Ice Cream, Not sucrose free Available

Ice Cream, vanilla, 13.1 economy

Ice Cream, vanilla, 13.6 premium

Ice Cream, vanilla, 14 standard

Milk, n U evaporated

Milk, 2% T

Milk, skim 0

Milk, whole 0

Milk, goat, π U whole

Milk, human, π U mature

Milk, human,

0 transitional

Milk, powder, instant, non0 fat

Quark 0

Yogurt, fruit flavor, sucrose 12.2 sweetened

Yogurt, π unsweetened Table 4- Sucrose content in Grains (gr Sucrose 1100 gr of food/beverage

Barley, pearl, boiled Bulgur, ₀ boiled

Flour, „ arrowroot

Flour, cornflower

Flour, rice 0.5 Flour, rye 0

Flour, „ semolina

Flour, soy 11 Flour, _{0 3} wheat, white Flour, wheat, 0.95 wholemeal Macaroni, „ ₁ boiled

Noodles, „ ₁ egg, boiled Noodles, instant, beef 0 flavor

Noodles, „ rice, boiled

Oat, bran 3.0

Oatmeal T

Oats, rolled T Rice, white, _, boiled

Rye, whole . _ grain, flakes

Semolina, „ , cooked

Soy, yogurt, _{χ 22} unsweetened

Spaghetti, _{Q 1} boiled

Tapioca, _τ uncooked

Tofu 0.3 Wheat, whole grain, 0.86 raw

Wheat bran 3.2

Wheat germ 14.5

Thus, 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 . Examples of syrups include but are not limited to, maple syrup, strawberry syrup, blueberry syrup and boysenberry syrup. Examples of 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

(liquid or powder), yogurt, ketchup and mayonnaise. 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.

Additionally, as known in the art 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.

While under some conditions a low dose time may be required (e.g., 8 units * hours/gram sucrose) under other conditions a greater dose time may be required to provide the same degree of levan production. Thus, in order to completely convert 100 % sucrose to levan, an exemplary levansucrase concentration is 50-10,000 u/1, 100-10,000 u/1, 200-2000 u/1, 50-5000, u/1,

750-2000 u/1; a pH range of 2-9, 4-7, 5-6; time of reaction 1-96, 4-48, 12-30 hours; and a temperature range of 10-60, 20-55, 30-40 ⁰ C. 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.

The enzymatic reaction may be actively terminated such as by heat, pasteurization or change in pH conditions. A product (e.g., food) generated according to the present teachings comprises 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.

Thus, in some embodiments 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.

It is expected that during the life of a patent maturing from this application many relevant levansucrase will be developed and the scope of the term is intended to include all such new technologies a priori As used herein the term "about" refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". This term encompasses the terms "consisting of and "consisting essentially of.

The phrase "consisting essentially of means that the 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.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in 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. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "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.

As used herein, 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.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", VoIs. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley- Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. L, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference. EXAMPLE 1

General Materials and Methods

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. 2355537200, chem.pure), (NHU) ₂ HPO ₄ - 4.2 (Chen samuel chemicals ltd- cat. 503-13- 90-50), Yeast extract- 5 (Merck- cat.1.03753.05000), KCL- 0.47 (Frutarom-cat. 2355528800), MgCl ₂ - 0.42 (Acros organics- cat. 413415000), MnCl ₂ *H ₂ O- 0.62 (Chen samuel chemicals ltd- cat. 012-1311229994), FeCl ₃ - 0.05 (Merck- cat. 803945), ZnCl ₂ - 0.02 (Chen samuel chemicals ltd- cat. 0010-14424-9994), Antifoam - PPG-P-2000 - 0.5ml (Fluka- cat. 342033390. The pH was kept at 6.8 by the controlled addition of NaOH. The medium containing the above components was sterilized for 60 min at 121 ⁰ C and inoculated with the strain B. subtilis. The bioreactor inoculum (200ml) was grown in shake flask using rich media at 37 ⁰ C for 24h. Fermentation was conducted aerobically with stirring rate of 200-300 rpm at temperature 37 ⁰ C and initial pH of 6.8 for 41 hours. The aeration rate was maintained at 1 wm. Following fermentation the biomass was recovered from the fermentation broth by centrifugation (6000 x g, 15 min 4 ⁰ C). Levansucrase assays -Levansucrase activity was 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.

Preparation of Levansucrase concentrate - The fermentation broth was centrifugated at 10,000 x g, 4 ⁰ C for 30 min. The supernatant was used as a starting enzyme solution (crude enzyme solution) for the preparation of levansucrase concentrate. The crude enzyme solution was concentrated by (NILi) ₂ SO ₄ precipitation in the range of 50-60 % saturation at 4 °C and in the presence of acetone . Acetone 60% v/v was added slowly to the cold supernatant. Following 48 hours, the mixtures were centrifuged at 10,000 x g for 15 min, 4°C. The precipitates was resuspended in acetate buffer pH 5.2. A precipitate of 50 % saturation (NHU) ₂ SO ₄ was the most active, therefore, it was used for further studies of enzymatic synthesis of levan.

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).

Effect of 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.

Effect of enzyme concentration - Reaction mixtures containing different levansucrase concentration ranging from 100 to 2000 u/1 were used. Each reaction mixture contained 25 % sucrose in 0.1 mol buffer acetate, pH 5.2 was incubated at 20 ⁰ C for up to 24 hours. Effect of the temperature - Reaction mixtures containing 1000 u/1 levansucrase and 25 % sucrose in O.lmol buffer acetate, pH 5.2 were incubated at 20 ⁰ C, 30 ⁰ C, 40 ⁰ C and 50 ⁰ C for up to 24 hours.

Effect of pH - Reaction mixtures containing 1000 u/1 levansucrase and 25 % sucrose in O.lmol buffer acetate were incubated at 20 ⁰ C for up to 24 hours in 0.1 mol sodium citrate buffer covering the pH range 2.2, 3.2, 4.2, 5.2, 6.2 and 7.2.

Effect of metal ions- Ca ²⁺ and Fe ³⁺ - Reaction mixtures containing 1000 u/1 levansucrase and 25 % sucrose in 0.1 mol buffer acetate were incubated at 20 ⁰ C for up to 24 hours with different concentration of 0.ImM, 0.5 mM,l mM, 2 mM, 5 mM Ca ²⁺ and with a concentration of 0.4 mM Fe ³⁺ .

EXAMPLE 2 Activity of levansucrase

The influence of several factors (pH, temperature) on activity of levansucrase from B. subtilis was tested. Effect of pH on levansucrase activity is shown in Figure 1. A high level of levansucrase activity was observed in the pH ranges from 4.2 to 7.1 and reached maximum at pH 5.8. Of note, acid stability of levansucrase is very important for industrial application of the enzyme .

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. There was a significant difference in levansucrase activity between 50 ⁰ C (9000 u/1) and 60 ⁰ C (1000 u/1).

EXAMPLE 3

Effect of incubation conditions on levan production as determined by viscosity analysis

Effect of pH - The influence of pH, temperature, enzyme concentration , sucrose concentration and metal ion Ca ²⁺ ' Fe ³⁺ were studied using levansucrase concentrate, to define the best conditions for production of levan ( Figures 3-8).

The influence of pH on levan production and viscosity of reaction mixture is shown in Figure 3. 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.

Effect of temperature - Temperature is an important factor for polymer production, either increasing or decreasing the polymerization of product.

The influence of temperature on levan production is shown in Figure 4.

Specifically, 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.

Effect of metal ions - The effect of metal-ions- Ca ²⁺ and Fe ³⁺ on levan production is shown in Figures 6 and 7.

Specifically, 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. As shown, the addition of both metal ions resulted in enhancement of levan production and viscosity.

Optimum conditions for levan production were observed at a temperature of 2O ⁰ C, pH 5.2, levansucrase concentrate from 750 tolOOO u\l with addition 0.4 Mm Fe ³⁺ ion and 2 mM Ca ²⁺ to the reaction mixture.

High concentration of sucrose (>5%) and long reaction time (24h) are required for the synthesis of levan. Reaction optimization The hydrolysis of sucrose by levansucrase was investigated using the above-described optimized parameters.

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.

At those conditions levansucrase displays a relatively high sucrose hydrolytic activity. At the final stage of the reaction time (26 hours incubation) levansucrase converted about 92 % of sucrose into transfructosylation products. These results suggests that sucrose can be completely hydrolysed at the end of the reaction.

EXAMPLE 4 In-situ production offructan in strawberry mash

Transfructosylation in strawberry jam was effected using the levansucrase concentrate from B. subtilis 347,

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.

Table 5

Note formation of each fructosyl molecule yields a glucose molecule. Conversion of sucrose to fructan is assayed by calculating the ratio of sucrose hydrolysis to glucose formation. Low glucose concentration is related to high FOS concentration. From the data presented in Figure 1OA and Table 5 above, it is evident that the product is fructan. Figure 1OB shows sucrose conversion and fructan production in in-situ treated strawberry mash. 1 - control concentrate; 2 - enzyme treated concentrate; 3 — control mash; 4 — treated mash; 5 — fructose. Levan stays at the starting point, FOS and low saccharides move to the top.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

REFERENCES

(other references are cited throughout the application)

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