Low sugar food products with high fiber content

Abstract

The present invention provides processes for producing food products, particularly juice beverages, with reduced sugar content enriched with dietary fibers and the food products produced. The processed food products are low in calories while preserving the palatable test of the starting material, and contain beneficial amount of dietary fibers. The processed food product further contains sorbitol and gluconic acid.

Claims

1. A process for preparing a low sugar, high fiber food product comprising the step of contacting a starting food product containing sugar or a composition comprising same with at least one type of dead microbial cells, wherein the dead microbial cells are active in reducing the sugar content of the starting food product and in converting said sugar mono- or disaccharides to at least one oligosaccharide and/or polysaccharide, thereby obtaining a processed food product with reduced sugar content and elevated content of at least one oligosaccharide and/or polysaccharide compared to the starting food product.

2. The process of claim 1, wherein the dead microbial cells are immobilized in or on a matrix.

3. The process of claim 1, wherein the microbial cells are selected from the group consisting of yeast cells, bacterial cells, fungi cells and any combination thereof.

4. The process of claim 3, wherein the microbial cells are of yeast selected from the group consisting of Xanthophyllomyces dendrorhous, Kluyveromyces lactis, Ogataea polymorpha, Metschnikowia fructicola, Saccharomyces cerevisiae, yeast isolated from olives and any combination thereof.

5. The process of claim 3, wherein the bacterial cells are selected from the group consisting of Zymomonas mobilis, Bacillus licheniformis, Paenibacillus polymyxa, Acetobacter xylinum, Sarcina ventriculi, Gluconobacter xylinus, Pseudomonas sp. #142, Microbacterium laevaniformans, Bacillus subtilis, Bacillus macerans, Streptococcus Salivarius, Leuconostoc mesenteroides, Aerobacter levanicum, and any combination thereof.

6. The process of claim 3, wherein the species of the fungal cells is selected from the group consisting of Aspergillus japonicus, Aspergillus niger, Aspergillus foetidus, Aspergillus oryza Aureobasidium pullulans, Sclerotinia sclerotiorum and Scopulariopsis brevicaulis.

7. The process of claim 1, wherein the starting food product is selected from the group consisting of natural juice and a ready-to-drink product containing sugar.

8. The process of claim 2, wherein the immobilized dead microbial cells are packed in a bed or a column.

9. The process of claim 1, wherein the processed food product contains reduced amount of a sugar selected from the group consisting of glucose, fructose, sucrose and any combination thereof compared to the sugar content in the starting food product.

10. The process of claim 1, wherein the processed food product contains elevated amount of at least one oligosaccharide, at least one polysaccharide, at least one sugar alcohol, gluconic acid and any combination thereof compared to the amount in the starting food product.

11. The process of claim 1, said process is consisting of the step of contacting a starting food product containing sugar or a composition comprising same with at least one type of dead microbial cells.

12. The process of claim 1, said microbial cells consist of dead cells.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the glucose and fructose levels in processed apple juice after passing through a column containing dead Zymomonas mobilis cells compared to the levels in the starting, non-treated juice.

(2) FIG. 2 demonstrates the decrease in sucrose concentration as a function of time. A decrease of 29% after 18.5 hours using the yeast Aureobasidium pullulans is demonstrated.

(3) FIG. 3 demonstrates reduction in sugar content and production of sorbitol and gluconic acid in apple juice after incubation with dead, immobilized Zymomonas mobilis cells.

(4) FIG. 3A: sucrose content, FIG. 3B: sorbitol. FIG. 3C: gluconic acid. Control—starting apple juice. 2 h—content after two hours of incubation. 22 h—content after 22 hours of incubation.

(5) FIG. 4 shows the changes is the sugar content of apple juice during incubation with different microorganisms. FIG. 4A: Kluyveromyces lactis. FIG. 4B: Zymomonas mobilis. FIG. 4C: Gluconobacter xylinus.

(6) FIG. 5 shows the changes in BRIX of apple juice during incubation with different microorganisms.

(7) FIG. 6 shows the BRIX reduction by repeated contact with Bacillus licheniformis.

(8) FIG. 7 shows the effect of different growth conditions on the changes in BRIX of apple juice incubated with Aspergillus japonicus. FIG. 7A: effect of aeration and growth temperature. FIG. 7B: Effect of juice pH FIG. 7C: effect of addition of yeast extract. FIG. 7D: Effect of addition of ammonium nitrate. FIG. 7E: Effect of peptone or sucrose addition.

DETAILED DESCRIPTION OF THE INVENTION

(9) The present invention provides a process for reducing the sugar content of food products containing sugar, particularly of natural juice beverages, and at the same time elevating the content of dietary fibers in the food product without negatively affecting other characteristics of the food product, particularly without negatively affecting the smell and the taste, only reducing its sweetness. The unique process of the invention is advantageous over hitherto known processes at least in that it is simple to operate and employs intact microbial cells, particularly dead cells that can be immobilized while preserving enzymatic capabilities, without the need to use costly and/or genetically engineered isolated enzymes. The resulted processed food product is low in calories while providing the consumer with a satiety feeling and preserves the aroma and taste of the starting, particularly natural, food product. The processed food product does not contain any significant amount of the microbial cells, nor of any enzymes. When the food product is juice, the juice obtained by the process of the present invention is not carbonated and is essentially devoid of alcohol.

(10) The present invention now discloses contacting a food product containing sugar with a particular type or types of microbial cells, particularly dead microbial cells preserving the capability of corresponding live microbial cells of sugar reduction and conversion of mono- and disaccharides to oligo- and/or polysaccharides. In particular aspects, the present invention discloses combinations of cells (dead, alive or a combination thereof) of yeast and bacteria or fungi species that provides for this dual activity.

Definitions

(11) The singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

(12) Unless the context clearly requires otherwise, throughout the specification, the words “comprise”, “comprising” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

(13) The word “about”, as used in the specification, should generally be understood to refer to both numbers in a range of numerals, and refers to the numeral ±5%. Moreover, all numerical ranges herein should be understood to include each whole integer within the range.

(14) As used herein, the term “monosaccharide” refers to a simple sugar, such as glucose or fructose that does not hydrolyze to yield other sugars.

(15) As used herein, the term “disaccharide” refers to any compound that comprises two covalently linked monosaccharide units. The term encompasses but is not limited to such compounds as sucrose, lactose and maltose. The term “sucrose” means a disaccharide comprised of 1 mole of D-glucose and 1 mole of D-fructose wherein the C-1 carbon atom of the glucose and the C-2 carbon atom of the fructose participate in the glycoside linkage.

(16) As used herein, the term “oligosaccharide” refers to a compound having 2 to 10 monosaccharide units joined by glycosidic linkages. The term “fructo-oligosaccharides” (FOS) means short chain oligosaccharides comprised of D-fructose and D-glucose units.

(17) For example some FOSs comprise of one molecule of D-glucose in the terminal position and from 2 to 6 D-fructose units. The linkage between fructose residues in FOSs are a β-(2-1) glycosidic links.

(18) The term “polysaccharide” as used herein refers to polymeric carbohydrate molecules composed of long chains of monosaccharide units bound together by glycosidic linkages and on hydrolysis give the constituent monosaccharides or oligosaccharides. They range in structure from linear to highly branch structure. According to certain embodiments, the polysaccharide of the invention is fructan. Fructans are built up of fructose residues, normally with a sucrose unit (i.e. a glucose-fructose disaccharide) at what would otherwise be the reducing terminus. The linkage position of the fructose residues determines the type of the fructan. Linkage normally occurs at one of the two primary hydroxyls (OH-1 or OH-6), and there are two basic types of simple fructan:

(19) 1-linked: in inulin, the fructosyl residues are linked by β-2,1-linkages.

(20) 6-linked: in levan (or phlein), the fructosyl residues are linked by β-2,6-linkages.

(21) A third type of fructans, the graminan-type, contains both β-2,1-linkages and β-2,6-linkages.

(22) The terms “dietary fibers” refers to the indigestible portion of food derived from plants. Dietary fibers include (1) soluble fiber, which dissolves in water, is readily fermented in the colon into gases and physiologically active byproducts, and can be prebiotic and viscous; it delays gastric emptying which in turn can cause an extended feeling of fullness; and (2) insoluble fiber, which does not dissolve in water, is metabolically inert and provides bulking, or it can be prebiotic and metabolically ferment in the large intestine; bulking fibers absorb water as they move through the digestive system, easing defecation. According to certain exemplary embodiments, the term “dietary fibers” is used herein to refer to soluble fibers.

(23) As used herein, the terms “alive cells”, “living cells” and “live cells” with reference to microbial cells are used herein interchangeably and refer to cells that proliferate when grown on compatible medium and are found to be viable when examined in a viability test (e.g. propidium iodide staining).

(24) As used herein, the term “dead cells” with reference to microbial cells refers to cells that do not proliferate when grown on compatible medium and are found to be nonviable when examined in a viability test (e.g. propidium iodide staining), while keeping the capability of corresponding living cells to reduce the sugar content within a food product and to convert mono- or disaccharides to oligosaccharide and/or polysaccharide, at least to some extent.

(25) As used herein, the terms “free enzyme” or “free enzymes” refer to enzyme or enzymes not associated with microbial cells (dead or alive) or to isolated enzyme. According to certain embodiments, the term refers to isolated enzyme(s) deliberately added to the staring food product.

(26) As used herein, the terms “column volume/hour”, “bed volume/hour” and “matrix volume/hour” are used herein interchangeably and refer to 1 unit volume of the fluent passed through to the same unit volume of the matrix/bed/column in an hour.

(27) According to one aspects, the present invention provides a process for preparing a low sugar, high fiber food product comprising the step of contacting a starting food product containing sugar or a composition comprising same with at least one type of dead or alive microbial cells, wherein the microbial cells have a capability of reducing the sugar content of the food product and of converting mono- or disaccharides to oligosaccharide and/or polysaccharide, thereby obtaining a food product with reduced sugar content and elevated oligosaccharide and/or polysaccharide content.

(28) According to certain exemplary embodiments, the microbial cells are dead cells.

(29) Method for killing the microbial cells while preserving the capability of the dead microbial cells to convert mono- or disaccharide to oligo- and/or polysaccharide are known in the art. Methods commonly used in the art include incubating living cells in a solution comprising ethanol; bile salts or bile-salt like compounds (e.g. sodium cholate); synthetic detergents (e.g. Tween 20, SDS, Triton X100); aldehydes like glutaraldehyde; or formaldehyde. The solution concentration and the microbial density of the cells are determined according to the type of the solution and the microbial cells, as is known to a person skilled in the Art.

(30) According to certain exemplary embodiments, the dead cells are obtained by isolating living microbial cells from the growing media by centrifugation and incubating the pellet with ethanol 70% at a concentration of 1 gr of cells in 1 liter of the 70% ethanol for 1 hour. According to certain embodiments, at the end of the incubation, the cells are separated from the solution and immobilized. According to some exemplary embodiments, the immobilization matrix is alginate. The matrix containing the dead microbial cells may be further sterilized by circulation of 70% ethanol within the matrix.

(31) According to certain embodiments, the starting food product is a natural juice or a ready-to-drink product containing sugar.

(32) Any method as is known in the art for forcing the juice out of the source material and optionally pre-treating the obtained juice before it is subjected to the process of the present invention can be used with the teachings of the present invention.

(33) According to certain embodiments, the juice is forced out of the source material by squeezing or crushing. The terms “squeezing” and “crushing” as used herein, are intended to comprise any disintegrating procedure that provides from the edible parts of the source material a fluid, a paste, or a suspension of any density or coarseness. Said squeezing or crushing provides a natural juice from which the juice products of the invention are manufactured, wherein natural juice may have a consistency of liquid, suspended pulp, mash, slurry, or puree. According to certain exemplary embodiments, the source material is fruit.

(34) Any fruit or vegetable juice containing sugar can be processed according to the teachings of the present invention. Thus, the process is equally applicable to apple, cranberry, pear, peach, plum, apricot, nectarine, grape, cherry, currant, raspberry, gooseberry, blackberry, blueberry, strawberry, lemon, orange, grapefruit, potato, tomato, celery, rhubarb, carrot, beet, cucumber, pineapple, custard-apple, coconut, pomegranate, kiwi, mango, papaya, banana, watermelon and cantaloupe. Each possibility represents a separate embodiment of the present invention. According to some exemplary embodiments, the juice is of apple, pear, cranberry, orange, strawberry, grape or cherry. Each possibility represents a separate embodiment of the present invention.

(35) The process of the invention combines reduction of the sugar content of the natural juice with production of dietary fibers within the food product, particularly juice, in an efficient, cost effective way. Using microorganisms as producers of the enzymes required for such transformation of simple sugars to dietary fibers eliminates the need to isolate and purify the enzymes. Using dead microbial cells is even more advantageous as the dead cells, once produced, are not sensitive to environmental conditions, and furthermore, the enzyme activity is not subjected to rate-limiting conditions, for example substrate feedback inhibition. Unexpectedly, conversion of simple sugar to oligo- and/or polysaccharides according to the teachings of the invention occurred also at acidic pH. Hitherto, enzyme active in such conversion were shown to be active only around natural pH. According to certain exemplary embodiments, the dead microbial cells are immobilized within a matrix, typically beads. The microbial cell-containing matrix can directly be added to the natural juice or a composition comprising same such that contacting the natural juice with the microbial cells occurs during incubation. Alternatively, the microbial cell-containing matrix is packed to form a bed or a column and the natural juice contacts with the microbial cells by passing the natural juice through the bed or column.

(36) As used herein, the terms “reducing the sugar content” or “reduced sugar content” refer to a concentration level of sugar, particularly glucose, fructose and sucrose in a food product, particularly natural juice that is less than the concentration level of sugar in a corresponding food product, which has not been contacted with the microbial cells according to the teachings of the invention.

(37) According to certain embodiment, the total sugar content in the processed food product obtained by the process of the present invention is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% or more compared to the total sugar content in the starting food product. According to some embodiments, the fructose content in the processed food product is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% or more compared to its content in the starting food product. According to some embodiments, the sucrose content in the processed food product is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% or more compared to its content in the starting food product. According to some embodiments, the glucose content in the processed food product is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% or more compared to its content in the starting food product.

(38) According to certain embodiments, the process of the present invention provides for a stable production of oligo- and/or polysaccharides, i.e. there is no or negligible reduction in the oligo- and/or polysaccharide content in the processed food product.

(39) According to certain exemplary embodiments, when the processed food product obtained by the process of the invention is juice, the juice comprises oligo- and/or polysaccharide at a concentration of from 1 mg to 15 gr per 100 ml juice (0.001% to 15% w/v).

(40) According to certain embodiments, the process comprises contacting the starting food product with a combination of dead or living cells of at least one yeast species and at least one bacteria or fungi species.

(41) According to certain embodiments, the starting food product is simultaneously contacted with the combination of cells of at least one yeast species and at least one bacteria or fungi species. According to other embodiments, the starting food product is contacted with the cells of the at least on yeast species and with cells of the at least one bacteria or fungi species sequentially. According to certain exemplary embodiments, the starting food product is first contacted with the at least one yeast species and thereafter with the at least one bacteria or fungi species.

(42) The resulting low calorie, high dietary fiber food product, particularly beverage, contains essentially the same nutritional benefit of the original food product, fruit or vegetable juice, but with significantly lower calories due to removal and conversion of the sugar to dietary fibers and sugar alcohol. Advantageously, the resulted processed food product may further contain gluconic acid, which is known in the food industry as an acidity regulator, thus may contribute to the stability of the resulted product. Furthermore, gluconic acid does not add any calories to the obtained food product (its calorie value is 0), and it may serves as a carrier for iron, calcium and other ions, based on its capability to form gluconate salt with such ions, which may be present in the food product. The gluconate salts provides for better bioavailability of these essential microelements.

(43) It is however to be understood that not all the sugar must be removed/converted to obtain the low calories, high dietary fiber processed food product of the present invention.

(44) According to certain embodiments, the food product of the present invention may have a calorie content of less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or less of the food product from which it is derived. At the same time, the reduced calorie food product may have a flavor profile and mouth feel which are comparable to the starting food product.

(45) In some food products, the reduced intrinsic sugar content may affect the taste of the product that consumers would otherwise expect. In some situations, to provide added sweetness, a natural or artificial sweetener can be added. The sweetener(s) may be selected according to the desired nutritional characteristics, taste profile, and other factors. In certain embodiments, the at sweetener is selected from the group consisting of, but not limited to, erythritol, tagatose, sorbitol, mannitol, xylitol, rhamnose, trehalose, aspartame, cyclamates, saccharin, sucralose, glycyrrhizin, malitol, lactose, Lo Han Guo (“LHG”), rebaudiosides, steviol glycosides, xylose, arabinose, isomalt, lactitol, maltitol, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

(46) When the food product of the present invention is juice beverages it can also be used as a flavoring agent or as a supplement of dietary fibers in beverages or foods. The sugar-reduced beverage can further be used as an ingredient for low calorie products (e.g., jellies, fillings, fruit preparations, candies, cakes, or the like).

(47) The low calories high dietary fiber beverages of the present invention can optionally be concentrated by 10%-90% or more. This concentrate results in a significant reduction in volume compared to standard juice concentrates due to the reduction of sugars. Processing cost of concentrating juices is reduced significantly due to the lack of sugars in the juice streams. Sugar reduced concentrates would result in significant less frozen shipping and frozen storage cost compared to standard concentrates due to the lower volume. Thermal, flavor, and nutritional degradation of the juice is also reduced since the concentration process requires less heat and time.

(48) The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

(49) Materials and Methods

(50) Microorganism Growth Conditions

(51) The bacteria Zymomonas mobilis were grown for 48 hours (or up to a concentration of 10.sup.9 cells/ml) in a growth medium containing 2% yeast extract and 2% glucose under anaerobic conditions at 30° C.

(52) The yeast Aureobasidium pullulans were grown for 48 hours in a growth medium of 2.4% potato dextrose broth under aerobic conditions at 30° C.

(53) The yeast Kluyveromyces lactis, Ogataea polymorpha, Metschnikowia fructicola, Xanthophyllomyces dendrorhous or a yeast isolated from olives; the bacteria Gluconobacter xylinus, Pseudomonas sp. #142, Microbacterium laevaniformans, Paenibacillus polymyxa, or Bacillus licheniformis; or the fungus Aspergillus japonicus were grown for 48 hours each in its specific growth medium. X. dendrorhous was incubated at 20′C. K. lactis, Pseudomonas sp. #142, yeast isolated from olives, P. polymyxa, A. japonicus and G. xylinus were incubated at 30′C; O. polymorpha and M. fructicola were grown at room temperature and B. licheniformis at and M. laevaniformans at 37′C. All cultures were rotated at 200 RPM in a 500 ml flask while Z. mobilis was incubated anaerobically at 30′C.

(54) Obtaining Dead Microbial Cells

(55) Bacteria, yeast or fungi were grown as described above. Once the culture has reached a desired cell density, cells were centrifugation and immediately suspended in 70% ethanol for 1 h. The suspension was then centrifuged to form a cell pellet. A sample of the cells from the pellet was placed on a growth medium to check the ability of the cells to grow. If no growth was observed, the cells in the pellet were designated “dead microbial cells”. Optionally, viability examination using propidium iodide staining or other known viability staining procedure is performed before designating the cells as “dead cells”.

(56) Sugar Determination:

(57) Sugars (disaccharides, monosaccharides and sugar alcohols) were separated in an analytical HPLC system (Pump System 320, Kontron, Switzerland) fitted with a Sugar-Pak I column (6.5×300 mm, Waters, Milford, Mass.) using a refractive-index detector (LDC Analytical, Riviera Beach, Fla.). Water was used as the solvent at a flow rate of 0.5 ml/min.

(58) FOS/Levan Quantification

(59) FOS/levan were precipitated from treated and non-treated juice samples using 1.5 volumes of iso-propanol. The sediment was air dried and either further dried at 37° C. for 48 hours and weighed or resuspended in 0.1M HCl and hydrolyzed for 1 hour at 100° C. The resulting monosaccharides were quantified by HPLC.

(60) Acetate Analysis

(61) Quantitative analysis of acetate ion in the sample was performed using ion chromatograph equipped with ion-conductivity detector (Dionex ICS 3000). Anions were separated on hydroxide-selective anion-exchange analytical column (AS11, 250×4 mm, Dionex).

(62) Ethanol Analysis

(63) The qualitative and quantitative analyses of ethanol was made using an Agilent model 5973N MSD mass spectrometer (MS) with a 7683 auto-sampler and a model 6890 gas chromatograph (GC) equipped with a 30 m×0.25 μm i.d. HP-5 (cross-linked phenylmethylsiloxane) column with 0.25 μm i.d. film thickness (Agilent, Palo Alto, Calif.). The initial oven temperature was held at 40° C. for 6 min. The temperature was then increased at a rate of 2.5° C./min to 150° C. and finally at a rate of 90° C./min to 250° C. The injection port and ionizing source were kept at 250° C. and 280° C., respectively. The split ratio is 10:1 with 2 μL of sample injected. There was a solvent delay of 2 min, after which the mass spectrum was collected from m/z 35 to 300, generating 5.27 scans/s. Compound identification was made by comparison of the mass spectra and retention times with those of a corresponding reference standard (Aldrich Chemical Co., St. Louis, Mo.; Bedoukian Research, Inc., Danbury, Conn.). For the purpose of quantifying identified component, linear regression models were determined using standard dilution techniques with cyclohexanone as internal standard. Target ions were used in the identification and quantification of each component by the mass spectrometry system.

(64) BRIX Determination

(65) BRIX (or Degrees Brix, symbol ° Bx) is the sugar content of an aqueous solution. One degree BRIX is 1 gram of sucrose in 100 grams of solutions. BRIX was determined using a ref-85 digital refractometer.

(66) Immobilizing Microbial Dead Cells within and/or onto Beads

(67) A matrix of immobilized beads and dead microbial cells was prepared as follows:

(68) A 1,000 ml composite solution was prepared as follows:

(69) Solution A: 10 g Na-alginate (SIGMA) in 500 ml of 100 mg/l Na.sub.2-EDTA solution.

(70) Solution B: 10 g Gelrite (SIGMA) in 500 ml of 100 mg/l Na.sub.2-EDTA solution.

(71) Solution A and Solution B were sterilized separately for 20 min at 120° C., and then mixed while still warm.

(72) The composite solution was then cooled to room temperature, and the dead microbial cell pellet prepared as described hereinabove was re-suspended in the solution. Cell concentration was at least 4% wet weight to volume (wet w/v). Typically, the cell concentration was 6% wet w/v.

(73) Cell Entrapment

(74) Before the entrapment procedure, the mixture of composite solution-cells was homogenized. Debris remained in the homogenate were filtered out from the homogenate.

(75) The mixed composite cells solution was then added drop-wise into 1% CaCl.sub.2 solution pH 6.2-6.8 containing 0.005% Chitosan (high molecular weight, FLUKA). The preferred average diameter of the resulting beads should be 2 mm or less to enlarge the ratio between the surface areas of the bead to its mass. After the beads were formed, the solution was stirred for 4 hours at 25° C.; the solution containing the microbial dead cell beads was then transferred to 4° C. for 24 hours. The beads were transferred to the column using a wide opened funnel; the lower valve was open to ensure the removal of access liquid and keeping all the beads in the column. The column was washed with 10 bed volumes at the flow of 1 bed volume/hour to remove any remnant of calcium chloride.

Example 1: Changes in Sugar Content of Apple Juice Contacted with Immobilized Dead Cells of Zymomonas mobilis

(76) Dead cells of Zymomonas mobilis were immobilized and packed in a column as described hereinabove. Commercially available apple juice was passed through the column at a rate of 0.2 bed volume (BV)/hr. Glucose and fructose levels were determined as described in the “Material and Methods” section hereinabove. FIG. 1 clearly shows a reduction in the content of both sugars in the treated juice.

Example 2: Changes in Sugar Content of Apple Juice Contacted with Immobilized Dead Cells of Aureobasidium pullulans Over Time

(77) Commercially available apple Juice was incubated with chitosan beads containing immobilized dead Aureobasidium pullulans yeast cells for 18.5 at 30° C. for 18.5 h. Samples were collected at 0, 7 and 18.5 hours. Sucrose concentration was measured by HPLC at the Agriculture Research Organization, Volcani center.

(78) FIG. 2 shows that the sugar content in the juice is continuously reduced. After 18.5 of incubation with the immobilized yeast Aureobasidium pullulans, the sugar content was reduced by 29%.

Example 3: Production of Polysaccharides in Apple Juice Contacted with Immobilized Dead Cells of Zymomonas mobilis

(79) FOS and/or levan production in apple juice contacted with immobilized dead Zymomonas mobilis cells under various temperatures was examined. The juice was passed through the column containing the immobilized dead bacteria at a rate of 0.2 BV/hr. 10 ml of the treated juice and of the source, untreated starting juice product were taken for analysis of FOS/levan as described in the “Material and Methods” section hereinabove. The results are presented in Table 1 hereinbelow.

(80) TABLE-US-00001 TABLE 1 production of levan/FOS in apple juice passed through immobilized dead cells of Z. mobilis under different temperatures Temperature (° C.) 10 30 45 55 Levan/FOS (mg/100 ml juice) 3 70 58 99

(81) As is apparent from Table 1, the highest levans/FOS content was obtained when the juice was contacted with the immobilized Z. mobilis dead cells at a temperature of 55° C.

(82) The possibility that higher levans/FOS levels can be obtained by re-incubating the juice obtained from a first run (at 1 BV/h) with the immobilized Z. mobilis dead cells for longer periods of time was examined. The juice was thus incubated with the immobilized Z. mobilis dead cells for 11 or 23 days. The results are presented in Table 2 herein below (day “0” denoting the juice after the first run).

(83) TABLE-US-00002 TABLE 2 production of levan and/or FOS in apple juice incubated with immobilized dead cells of Z. mobilis as a function incubation time Time at 4° C. (Days) 0 11 23 Levan/FOS (mg/100 ml juice) 99 67 36

(84) As can be taken from table 2, there is no advantage in further incubation of the juice obtained after the first run.

Example 4: Changes in Sugar Content of Apple Juice Contacted with Immobilized Dead Cells of Zymomonas mobilis

(85) Beads with immobilized dead cells of the bacterium Zymomonas mobilis were prepared as described hereinabove. Commercially available apple juice was incubated with the immobilized dead bacterial cells for 18.5 hours Samples were collected at 0, 7 and 18 hours. Sugar content was measured using GC-MS at the Interdepartmental unit at the Faculty of Agriculture, Food and Environment, the Hebrew University of Jerusalem.

(86) Sucrose content was significantly reduced as fast as after 2 hours of incubation. At the end of the experiment, the sucrose content was about 18% of its initial concentration (Table 3). Unexpectedly, under these conditions, sorbitol (FIG. 3, Table 3) and gluconic acid (FIG. 3) were produced. As is apparent from FIGS. 1 and 3, using the process of the present invention the concentration of all the main sugars of the apple juice (sucrose, glucose and fructose) was significantly reduced. As presented in Table 2 hereinabove, the sugars were converted to the fructooligosaccharide (FOS) and fructopolysaccharide (levan).

(87) TABLE-US-00003 TABLE 3 Sugar reduction and sorbitol accumulation in apple juice incubated with immobilized Zymomonas mobilis dead cells Sucrose Sorbitol mg/ml mg/ml Control 40.4 4.9 After 2 h of incubation 30.9 16.6 After 22 h of incubation 7.4 18.1

Example 5: Effect of Culture Conditions of Living Microbial Cells on Juice Sugar Content

(88) Microbial cells first incubated with diluted apple juice were collected, washed and then re-suspended in apple juice to examine their ability to reduce the sugar content.

(89) K. lactis, O. polymorpha, M. fructicola, Z. mobilis G. xylinus, Pseudomonas sp. #142, yeast isolated from olives, B. licheniformis P. polymyxa, X. dendrorhous or M. laevaniformans were grown for 48 hours in their specific growth medium, diluted 1:200 into 100 ml of apple juice adjusted to pH 7.0 by KOH (except X. dendrorhous, which was diluted into fresh apple juice) and grown for additional 48 hours. The cells were centrifuged and the pellet washed twice in distilled water (DW). The pellets were resuspended in 10 ml of apple juice to give a concentration of 1.8×10.sup.9 or 1.8×10.sup.10 CFU/ml for yeast and bacteria respectively.

(90) The cells were then incubated with rotation as described hereinabove. Samples were taken at designated times and tested for OD.sub.600, sugar content, and FOS content. BRIX value was also determined. Designated samples were analyzed for ethanol or acetic acid content.

(91) All microorganisms tested (except Pseudomonas sp. #142) could reduce sugar content but at different rates. Representative data are presented in FIG. 4. K. lactis reduced sucrose content from 2.8% to 0.065% in only about 0.5 hour; glucose content from 3% to 0.005% in 2.5 hours and fructose content from 7% to 0.33% in 3 hours. Z. mobilis reduced all sugar to nearly zero after 25.5 hours. G. xylinus could only reduce glucose content and only after 105.5 hours of incubation. Analysis for acetic acid production revealed that G. xylinus produces acetic acid under these experimental conditions. It was therefore concluded that G. xylinus is not suitable for use in the process of the present invention.

Example 6: Changes in BRIX of Apple Juice During Incubation with Different Live Microbial Cells

(92) As is shown in FIG. 5, the yeast isolated from olives reduced the BRIX of apple juice in the fastest manner, getting to BRIX of about 6 in about 2 hours of incubation. Reducing the apple juice BRIX to the same value (about 6) required 2.5 hours of incubation with M. fructicola, 3.5 hours with K. lactis, 5.5 hours with Z. mobilis and 19.5 hours of incubation with O. polymorpha. M. levaniformans and P. polymyxa were also found to have minor effect, reducing the BRIX to about 10 after 23 hours. B. licheniformis was found not to be effective in reducing the BRIX value which was maintained throughout the 24 hours of incubation.

(93) Samples for ethanol analysis were taken at time points when the samples reached a BRIX of about 9 and about 6. Ethanol content analysis revealed that K. lactis, Z. mobilis and M. fructicola produced ethanol under the experimental conditions tested as described in tables 4-8. O. polymorpha produced the least amount of ethanol when the samples reached a BRIX of about 9; M. fructicola produced the least amount of ethanol when the samples reached a BRIX of about 6. B. licheniformis did not produce any ethanol. It is to be noted that when the baker yeast S. cerevisiae was tested under the same conditions, the amounts of ethanol were similar to those obtained with Z. mobilis.

(94) TABLE-US-00004 TABLE 4 BRIX value and ethanol content in apple juice incubated with K. lactis BRIX Ethanol (%) 9.1 0.48 5.8 2.84

(95) TABLE-US-00005 TABLE 5 Ethanol content in apple juice incubated with Z. mobilis Time (Hours) Ethanol (%) 9.1 1.46 6.4 3.13

(96) TABLE-US-00006 TABLE 6 Ethanol content in apple juice incubated with M. fructicola Time (Hours) Ethanol (%) 8.5 0.16 5.9 1.71

(97) TABLE-US-00007 TABLE 7 Ethanol content in apple juice incubated with O. polymorpha Time (Hours) Ethanol (%) 9.2 0.07 5.7 2.15

(98) TABLE-US-00008 TABLE 8 Ethanol content in apple juice incubated with the yeast isolated from olives Time (Hours) Ethanol (%) 8.7 0.13 5.2 2.3

(99) Since B. licheniformis did not produce any ethanol it was examined whether incubating apple juice with two different batches of bacteria will help reducing the BRIX levels. It was found that soaking about 10.sup.10 CFU/ml of B. licheniformis in apple juice during 30 minutes reduced the BRIX to 10.1 while soaking an additional batch of bacteria in the same juice reduced the BRIX to 8.8 (FIG. 6). Advantageously, no ethanol or acetic acid was produced.

Example 7: Production of Polysaccharides

(100) FOS production by live Z. mobilis was examined as follows: bacteria were immersed in apple juice for 22 hours. The FOS were then precipitated from the treated juice or from a non-treated sample and hydrolyzed. Monosaccharide content was then analyzed and revealed a 1.6 fold increase in glucose content and 1.4 fold increase in fructose content in the juice treated with bacteria. This result indicates the formation of FOS by Z. mobilis in this experimental system.

(101) FOS content was further evaluated during the incubation time using Z. mobilis, P. polymyxa, or X. dendrorhous. Samples were taken at the designated times and FOS was precipitated and weighed. The results are presented in Table 9 below.

(102) TABLE-US-00009 TABLE 9 FOS content (mg/100 ml juice) Time (hours) Z. mobilis P. polymyxa X. dendrorhous 0 20 20 20 5.5 120 160 120 12 100 110 150 24 70 90

(103) As is apparent from Table 9, the highest FOS content was obtained at different incubation times for the different microorganisms. Incubation of 5.5 hours was found to be most effective for Z. mobilis and P. polymyxa while incubation time of 12 hours was the most effective for X. dendrorhous. M. laevaniformans formed negligible amounts of FOS under the experimental conditions.

(104) The possibility that higher FOS levels can be obtained by re-incubating apple juice treated once with FOS forming microorganisms for 5.5 hours with a second batch of the microorganisms for 5 hours was examined. After the second incubation time, the FOS content in the apple juice incubated with P. polymyxa was 70 mg/100 ml of juice; 110 mg/100 ml in apple juice incubated with Z. mobilis and 190 mg/100 ml in apple juice incubated with X. dendrorhous. Thus, only for X. dendrorhous a second incubation time was significant for further production of FOS.

Example 8: Examination of the Effect of Various Growth Conditions on the BRIX Reduction Activity of Live A. japonicus

(105) The growth experiment using A. japonicus was repeated using different growth conditions in order to examine their effect on BRIX reduction and FOS yield. The control conditions were as described hereinabove (Aerobic, 30° C.) but with greater aeration. The following conditions were examined: aeration and growth temperature (FIG. 7A); juice pH of 5.6 or juice pH titrated daily to pH=7 (FIG. 7B); addition of 0.1% or 1% yeast extract (FIG. 7C); addition of 0.1% or 1% ammonium nitrate (FIG. 7D); addition of 1% peptone or 1% sucrose (FIG. 7E). BRIX was checked at the indicated times.

(106) As is apparent from FIG. 7, growing the fungus in apple juice containing 1% Yeast extract, at 30° C. and under aerobic conditions resulted in the most significant decrease in the BRIX value. Under all designated growth conditions FOS production by A. Japonicus was negligible.

(107) The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.