Phenolics Extraction And Use Patent Application (2025)

U.S. patent application number 13/264453 was filed with the patent office on 2012-09-20 for phenolics extraction and use. This patent application is currently assigned to OCEAN SPRAY CRANBERRIES, INC.. Invention is credited to Lowell Vernon Dravenstadt, Margarita Gomez, James Harvey Johnson, Paula Lent, Harold L. Mantius, Christopher McNamara, Stephen Joseph Nojeim, Lawrence E. Rose.

PHENOLICS EXTRACTION AND USE

Abstract

Methods for concentrating phenolics in a solution andcompositions related to the concentrated phenolics are provided Inparticular, methods and compositions are described relating toconcentrated phenolics obtained from cranberry feedstock extractscombined with fumaric acid as beverage additives Additionally,methods for obtaining phenolics from feedstocks using resinabsorption and elution are described.

Related U.S. Patent Documents

Claims

1. A method of extracting phenolics comprising: (i) obtaining aliquid feedstock comprising cranberry juice obtained by countercurrent extraction; (ii) contacting the feedstock with a resin thatbinds phenolics, and that does not substantially bind to sugar andorganic acids, for a time and under conditions sufficient forphenolics in the feedstock to bind to the resin, wherein the resinhas a surface area of greater than or equal to about 300 m.sup.2/g;(iii) contacting the resin with a wash solution, wherein the washsolution does not substantially reduce the amount of phenolicsbound to the resin; (iv) contacting the resin with an elutionsolution comprising a solvent, wherein the elution solutionsubstantially decreases the amount of phenolics bound to the resin;and (v) collecting the elution solution.

2. The method of claim 1, wherein the elution solution comprisesphenolics at a first concentration and further comprising: (vi)removing solvent from the elution solution; or (vii) concentratingthe solution, thereby providing a liquid extract comprisingphenolics at a second concentration, wherein the secondconcentration is greater than the first concentration, and whereinthe solution comprises at least anthocyanins and proanthocyanidins(PACs) and one or more of the following: a ratio of anthocyanins toPACs of about 1:5; a PACs oligomeric profile that is substantiallythe same as the PACs oligomeric profile in cranberries; a ratio ofPACs to total phenolics that is substantially the same as the ratioof PACs to total phenolics in cranberries; a ratio of PACs toanthocyanins that is not the same as the ratio of PACs toanthocyanins in cranberries; phenolics with an average molecularweight of less than 14,000 Daltons; less than about 5% organicacids; and/or less than about 5% sugars.

3. The method of claim 1, further comprising drying the solution,to thereby provide a dry extract.

4. The method of claim 1, wherein the resin is an aliphatic esterresin and/or has moisture holding capacity of about 61% to about69%.

5-7. (canceled)

8. The method of claim 1, wherein the resin has a surface area ofgreater than or equal to about 700 m.sup.2/g.

9. The method of claim 1, wherein the resin is a macroreticulararomatic polymer.

10. (canceled)

11. The method of claim 1, wherein the wash solution compriseswater or mixture of water and ethanol at a concentration of about5% by volume.

12. The method of claim 1, wherein the elution solution comprisesabout 95% ethanol by volume or about 90% acetone by volume.

13. The method of claim 1, further comprising adding the elutionsolution, liquid extract, or dry extract to a liquid suitable foringestion.

14-15. (canceled)

16. A composition comprising the elution solution obtained usingthe method of claim 1.

17. A composition comprising the liquid extract obtained using themethod of claim 2.

18. A composition comprising the dry extract obtained using themethod of claim 3.

19. An extract comprising at least anthocyanins andproanthocyanidins (PACs) and at least one of the following: a ratioof anthocyanins to PACs of about 1:5; a PACs oligomeric profilethat is substantially the same as the PACs oligomeric profile incranberry juice or cranberries; a ratio of PACs to total phenolicsthat is substantially the same as the ratio of PACs to totalphenolics in cranberry juice or cranberries; a ratio of PACs toanthocyanins that is not the same as the ratio of PACs toanthocyanins in cranberry juice or cranberries; phenolics with anaverage molecular weight of less than 14,000 Daltons; less thanabout 5% organic acids; and/or less than about 5% sugars.

20. The extract of claim 19, wherein the extract is a liquidextract.

21. The extract of claim 19, wherein the extract is a dry,dehydrated, or powdered extract.

22. (canceled)

23. A composition comprising isolated fumaric acid and isolatedphenolics, wherein the isolated phenolics comprisesproanthocyanidins (PACs) and wherein the ratio of fumaric acid toPACs is between about 4000:1 to about 100:1.

24. The composition of claim 23, wherein the ratio is about135:1.

25. The composition of claim 23, wherein the ratio is about238:1.

26. (canceled)

27. A composition comprising isolated fumaric acid and isolatedphenolics, wherein the isolated phenolics comprisesproanthocyanidins (PACs) and wherein the ratio of fumaric acid toPACs is between about 10:1-50:1.

28. The composition of claim 27, wherein the ratio is about14:1.

29-62. (canceled)

Description

CLAIM OF PRIORITY

[0001] This application claims priority to U.S. ProvisionalApplication Ser. No. 61/170,090, filed on Apr. 16, 2009, thecontent of which is hereby incorporated by reference in itsentirety.

TECHNICAL FIELD

[0002] The present disclosure provides, inter alia, compositionsand processes related to the concentration of phenolics.

BACKGROUND

[0003] Many phenolics, e.g., plant derived phenolics (e.g.,phenolics in fruits and vegetables), can be useful in food stuffsand/or as health products (e.g., dietary supplements), due to theirwell documented association with human health. Certain foods, forexample, fruits such as cranberries, provide a rich source ofphenolics. However, current methods for the recovery of plantderived phenolics are inefficient or yield undesirable mixtures ofphenolics. Improved methods for the selective recovery and/orconcentration of phenolics are desired. Such methods may allownovel opportunities in the field of health product development.

SUMMARY

[0004] Compositions and processes are provided for extracting,obtaining and/or concentrating (e.g., enriching) phenolics.

[0005] In sonic aspects, the present disclosure provides methods ofextracting phenolics. These methods can include steps of obtaininga liquid feedstock. Useful liquid feedstocks can include anyphenolics containing feedstock. In some embodiments, the liquidfeedstock includes a juice obtained from one or more phenolicscontaining fruits, vegetables, legumes and the like. For example,liquid feedstock can include juice obtained from cranberries (e.g.,cranberry juice). Cranberry juice can be obtained by any methodthat allows juice to be obtained from cranberries. In someinstances, cranberry juice can be obtained by crushing cranberriesand purifying the resulting cranberry juice. Methods suitable foruse in such application are known in the art and include, but arenot limited to, e.g., counter current extraction. Liquid feedstockcan then be contacted with a material that retains, captures, orbinds phenolics, and that does not substantially retain, capture,or bind sugars and organic acids. The duration of time andconditions under which the feedstock is contacted with such amaterial (e.g., a resin) can be modified such that at least aportion of the phenolics present in the liquid feedstock areretained, captured, or bound, without retaining, capturing, orbinding at least sugars and organic acids. In some aspects, theliquid feedstock is contacted with a resin. Suitable resinsinclude, but are not limited to, for example, resins with one ormore of the following physical properties: a surface area ofgreater than or equal to about 300 m.sup.2/g (e.g., greater than380 m.sup.2/g or equal to about 700 m2/g), aliphatic ester resins,a moisture holding capacity of about 61% to about 69%, a porosityof greater than about 0.5 ml/ml. In some aspects, the resin caninclude Amberlite.TM. XAD-7HP resin. In some aspects the resin caninclude Amberlite.TM. FPX-66. The material contacted with theliquid feedstock (e.g., the resin) can then be washed using asolution that does not substantially reduce the amount of phenolicsretained, captured, or bound therein. Useful wash solutions caninclude a solvent diluted in water to a concentration that does notsubstantially reduce the amount of phenolics retained, captured, orbound to the material (e.g., the resin). Exemplary solventsinclude, for example, ethanol at a concentration of about 5% byvolume. The portion of phenolics retained, captured, or bound tothe material (e.g., the resin) can then be obtained using asolution comprising a solvent, wherein the elution solutionsubstantially decreases the amount of phenolics bound to the resin.Suitable elution solutions can include, but are not limited to, forexample, about 95% ethanol by volume or about 90% acetone byvolume. In some embodiments, the resulting phenolics can beobtained. Additional steps to remove any solvent present in thephenolics solution can optionally be performed, and/or the solutioncan be concentrated.

[0006] The phenolics containing solution resulting from the abovedescribed process is referred to herein as an extract. Suchextracts can include phenolics at a second concentration, whereinthe second concentration is greater than the first concentrationand wherein the first concentration is the concentration of thephenolics present in the liquid feedstock. Further, the extract caninclude at least anthocyanins and proanthocyanidins (PACs) and oneor more of the following: a ratio of anthocyanins to PACs of about1:5; a PACs oligomeric profile that is substantially the same asthe PACs oligomeric profile in cranberries; a ratio of PACs tototal phenolics that is substantially the same as the ratio of PACsto total phenolics in cranberries; a ratio of PACs to anthocyaninsthat is not the same as the ratio of PACs to anthocyanins incranberries; phenolics with an average molecular weight of lessthan 14,000 Daltons; less than about 5% organic acids; and/or lessthan about 5% sugars. Such extracts, if liquid, can be dried tothereby provide a dry extract.

[0007] In some aspects, the present disclosure provides methods ofmaking a beverage suitable for ingestion by a subject (e.g., ahuman or non-human subject). Such methods can include obtainingphenolics using the process described above, and adding theresulting extract to a liquid suitable for ingestion by a humanand/or non-human animal. Exemplary beverages can include, but arenot limited to beverages that contain fruit juice or juices.

[0008] In some aspects, the present disclosure provides phenolicscontaining extracts. Such extracts can include at leastanthocyanins and proanthocyanidins (PACs) and at least one of thefollowing characteristics or properties: a ratio of anthocyanins toPACs of about 1:5; a PACs oligomeric profile that is substantiallythe same as the PACs oligomeric profile in cranberry juice orcranberries; a ratio of PACs to total phenolics that issubstantially the same as the ratio of PACs to total phenolics incranberry juice or cranberries; a ratio of PACs to anthocyaninsthat is not the same as the ratio of PACs to anthocyanins incranberry juice or cranberries; phenolics with an average molecularweight of less than 14,000 Daltons; less than about 5% organicacids; and/or less than about 5% sugars. These extracts can beliquid, dry, or partially dry (e.g., dehydrated, lyophilized, orpowdered), or gel extract.

[0009] In some aspects, the present disclosure providescompositions that include at least anthocyanins andproanthocyanidins (PACs) and at least one of the followingcharacteristics or properties: a ratio of anthocyanins to PACs ofabout 1:5; a PACs oligomeric profile that is substantially the sameas the PACs oligomeric profile in cranberry juice or cranberries; aratio of PACs to total phenolics that is substantially the same asthe ratio of PACs to total phenolics in cranberry juice orcranberries; a ratio of PACs to anthocyanins that is not the sameas the ratio of PACs to anthocyanins in cranberry juice orcranberries; phenolics with an average molecular weight of lessthan 14,000 Daltons; less than about 5% organic acids; and/or lessthan about 5% sugars. These extracts can be liquid, dry, orpartially dry (e.g., dehydrated, lyophilized, or powdered), or gelextract.

[0010] In some aspects, the present disclosure providescompositions that include fumaric acid (e.g., isolated or purifiedfumaric acid) and phenolics (e.g., isolated, purified, or enrichedphenolics (e.g., PACs)). In some embodiments, the ratio of fumaricacid to phenolics (e.g., proanthocyanidins (PACs) within suchcompositions is between about 4000:1 to about 100:1. For example,the ratio can be about 135:1, or about 238:1. In some embodiments,these compositions can be present in a beverage. Such beverages canhave a pH of between about pH 2.0 to about pH 3.49. In someembodiments, the pH of such beverages can be equal to or greaterthan pH 3.49. For example, the pH can be about pH 2.0 to about pH4.1 (e.g., pH 3.7 or pH 4.1). In some embodiments, the beverage canbe a beverage that includes apple juice or the beverage can beapple juice. In some aspects, compositions comprising isolatedfumaric acid and isolated phenolics can include a ratio of fumaricacid to phenolics (e.g., PACs) of between about, e.g., 10:1-50:1,or about 14:1. Such compositions can also be presented in abeverage. In some instances, such beverages can have a pH ofgreater than or equal to about pH 3.5, and/or the beverage cancontain orange juice. In some embodiments, the phenolics present inthese compositions can include, e.g., at least anthocyanins andPACs, and one of the following of the following: a ratio ofanthocyanins to PACs of about 1:5; a PACs oligomeric profile thatis substantially the same as the PACs oligomeric profile incranberry juice or cranberries; a ratio of PACs to total phenolicsthat is substantially the same as the ratio of PACs to totalphenolics in cranberry juice or cranberries; a ratio of PACs toanthocyanins that is not the same as the ratio of PACs toanthocyanins in cranberry juice or cranberries; phenolics with anaverage molecular weight of less than 14,000 Daltons; less thanabout 5% organic acids; and/or less than about 5% sugars.Alternatively, or in addition, the phenolics can be obtained usingthe methods disclosed herein. In some embodiments, compositionsthat include fumaric acid (e.g., isolated or purified fumaric acid)and phenolics (e.g., isolated, purified, or enriched phenolics(e.g., PACs)) can be powdered or liquid. In some embodiments,compositions that include fumaric acid (e.g., isolated or purifiedfumaric acid) and phenolics (e.g., isolated, purified, or enrichedphenolics (e.g., PACs)) can be in a container. In some embodiments,the composition comprises isolated fumaric acid and isolatedphenolics, e.g. within a suitable container.

[0011] In some aspects, the disclosure provides beverages (e.g.,beverages suitable for ingestion by a human or non-human animal(e.g., fruit juice beverages) that includes phenolics containing atleast PACs, and fumaric acid. In such aspects the concentration ofPACs can be between about 4.2.times.10.sup.-4 mg/mL and8.29.times.10.sup.-3 mg/mL and the concentration of fumaric acidcan be between about 0.01% (weight/volume (w/v)) and 0.15% (w/v).In some embodiments the concentration of PACs can be about4.2.times.10.sup.-3 mg/mL and the concentration of fumaric acid canbe about 0.1% (w/v). In some instances, the pH of beveragescontaining such compositions can be between about pH 2.0 to aboutpH 3.49, or the beverage can be apple juice or a beveragecomprising apple juice. In some embodiments, the phenolics in suchbeverages can include at least anthocyanins and proanthocyanidins(PACs) and at least one of the following: a ratio of anthocyaninsto PACs of about 1:5; a PACs oligomeric profile that issubstantially the same as the PACs oligomeric profile in cranberryjuice or cranberries; a ratio of PACs to total phenolics that issubstantially the same as the ratio of PACs to total phenolics incranberry juice or cranberries; a ratio of PACs to anthocyaninsthat is not the same as the ratio of PACs to anthocyanins incranberry juice or cranberries; phenolics with an average molecularweight of less than 14,000 Daltons; less than about 5% organicacids; and/or less than about 5% sugars, or the phenolics can beobtained using the methods disclosed herein.

[0012] In some aspects, such beverages can include a concentrationof PACs of between about 4.2.times.10.sup.-4 mg/mL and 0.1 mg/mLand a concentration of fumaric acid of between about 0.01%(weight/volume (w/v)) and 0.15% (w/v). For example, in someinstances the concentration of PACs can be about 5.times.10.sup.-2mg/mL and the concentration of fumaric acid can be about 0.07%(w/v). In some instances, the pH of beverages containing suchcompositions can be greater than or equal to about pH 3.5 and/orthe juice can be a beverage comprising orange juice.

[0013] In some aspects, the present disclosure provides methods ofmaking compositions for reducing bacterial contamination (e.g.,Alicyclobacillus (ACB) contamination) of a beverage. Such methodcan include obtaining isolated fumaric acid and isolated phenolicscomprising PACs; and combining the isolated phenolics and fumaricacid to yield a ratio of fumaric acid to PACs of between about4000:1 to about 100:1 when the composition is added to thebeverage. In some embodiments, the ratio can be about 135:1 orabout 238:1.

[0014] In some aspects, the present disclosure provides methods ofmaking compositions for reducing bacterial contamination (e.g.,Alicyclobacillus (ACB) contamination) of a beverage. Such methodscan include obtaining isolated fumaric acid and isolated phenolicscomprising PACs; and combining the isolated phenolics and fumaricacid to yield a ratio of fumaric acid to PACs of between about 10:1to about 50:1. In some embodiments, the ratio can be about14:1.

[0015] In some aspects, the present disclosure encompasses methodsof making a beverages that include obtaining a beverage; and addingto the beverage phenolics comprising PACs and fumaric acid, whereinthe final concentration of exogenously added PACs is between about4.2.times.10.sup.-4 mg/mL and 8.29.times.10.sup.-3 mg/mL and theconcentration of fumaric acid is between about 0.01% (weight/volume(w/v)) and 0.15% (w/v). In some embodiments, the concentration ofPACs can be about 4.2.times.10.sup.-3 mg/mL and the concentrationof fumaric acid is about 0.1% (w/v), and/or the pH of the beveragecan be between about pH 2.0 to about pH 3.49. In some embodiments,the beverage can be apple juice or a beverage comprising applejuice. In such embodiments, the concentration of exogenously addedPACs can be about 4.2.times.10.sup.3 mg/mL and the concentration offumaric acid can be 0.1% (w/v). In some embodiments, the phenolicsincluded in such beverages can include at least anthocyanins andproanthocyanidins (PACs) and at least one of the following: a ratioof anthocyanins to PACs of about 1:5; a PACs oligomeric profilethat is substantially the same as the PACs oligomeric profile incranberry juice or cranberries; a ratio of PACs to total phenolicsthat is substantially the same as the ratio of PACs to totalphenolics in cranberry juice or cranberries; a ratio of PACs toanthocyanins that is not the same as the ratio of PACs toanthocyanins in cranberry juice or cranberries; phenolics with anaverage molecular weight of less than 14,000 Daltons; less thanabout 5% organic acids; and/or less than about 5% sugars. In someembodiments, these methods can include adding to the beveragephenolics comprising PACs and fumaric acid, wherein the finalconcentration in the beverage of exogenously added PACs is betweenabout 4.2.times.10.sup.-4 mg/mL and 0.1 mg/mL and the concentrationof fumaric acid is between about 0.01% (weight/volume (w/v)) and0.15% (w/v). In some embodiments, the concentration of PACs can beabout 5.times.10.sup.-2 mg/mL and the concentration of fumaric acidcan be about 0.07% (w/v), and/or the beverage can have a pH ofgreater than or equal to about pH 3.5, and/or the beverage can beorange juice or a beverage comprising orange juice. In someembodiments, the beverage is orange juice and the concentration ofexogenously added PACs is about 5.times.10.sup.-2 mg/mL and theconcentration of fumaric acid is 0.07% (w/v).

[0016] Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Methodsand materials are described herein for use in the presentinvention; other suitable methods and materials known in the artcan also be used. The materials, methods, and examples areillustrative only and not intended to be limiting. Allpublications, patent applications, patents, sequences, databaseentries, and other references mentioned herein are herebyincorporated by reference in their entirety. In case of conflict,the present specification, including definitions, will control.

[0017] Other features and advantages of the invention will beapparent from the following description and figures, and from theclaims.

DESCRIPTION OF DRAWINGS

[0018] FIG. 1 is a flow diagram providing one embodiment of theprocess disclosed herein. A through K correspond to the Sample IDsshown in Table 7.

[0019] FIG. 2 is a line graph showing the growth ofAlicyclobacillus species in the presence of concentrations ofextract and fumaric acid.

[0020] FIGS. 3A-3B are bar graphs showing the log number ofAlicyclobacillus CFU in apple juice treated with extract (CE, mgPAC/8 oz) and/or fumaric acid (%). Mean+sem, n=3. (A) Significantdifferences are indicated by different letters. (B) Log reductionin the number of Alicyclobacillus CFU.

[0021] FIGS. 4A-4B are bar graphs showing the log number ofAlicyclobacillus CFU in orange juice treated with extract (CE, mgPAC/8 oz) and/or fumaric acid (%). Mean+sem, n=3. (A) Significantdifferences are indicated by different letters. (B) Log reductionin the number of Alicyclobacillus CFU.

DETAILED DESCRIPTION

[0022] The present disclosure is based, at least in part, on thefinding that phenolics can be extracted, obtained, and/orconcentrated from a feedstock containing phenolics using theprocess disclosed herein. Furthermore, the present disclosureprovides that these extracted phenolics can be used in thedevelopment of health products and food preservatives. Accordingly,the present disclosure provides, inter alia, processes that can beused to extract, obtain, and/or concentrate (e.g., enrich)phenolics (e.g., naturally occurring phenolics, e.g., plantphenolics) from feedstocks containing phenolics, and compositions(e.g., extracts) containing phenolics obtained using theseprocesses (e.g., phenolics enriched extracts). Suchcompositions--which are termed herein as "phenolics," "enrichedextracts" or simply "extracts"--can include, for example, at leastanthocyanins and proanthocyanidins (PACs).

[0023] Processes

[0024] Referring to FIG. 1, a flow diagram is provided illustratingone exemplary embodiment of a process for extracting phenolics froma phenolics containing feedstock (e.g., cranberries or a solutionobtained from cranberries). The process can use a feedstock thatcontains phenolics. This feedstock can be a solid or liquid. Solidfeedstocks can be liquefied or solubilized and optionally filteredto generate a liquid feedstock prior to use in the disclosedprocess.

[0025] The process can begin with a phenolics containing liquidfeedstock (e.g., cranberry juice obtained by countercurrentextraction (CCE), as described in U.S. Pat. Nos. 5,320,861 and5,419,251). Liquid feedstocks can contain known amounts of solidsper volume (e.g., about 5 pounds of solids per gallon or about 50Brix). If required (e.g., to decrease or increase the concentrationof the solids in the liquid), the liquid feedstock can be diluted(e.g., using water or reverse osmosis (RO) water) in "mix tank" 10to yield a lower concentration of solids per volume (e.g., about2.3 pounds of solids per gallon or about 25 Brix), or concentratedto yield a higher concentration of solids per volume (e.g., about8.6 pounds of solids per gallon or about 75 Brix). Theconcentration of solids in the liquid feedstock can be modified toyield a viscosity, e.g., for allowing optimal passage or flow ofthe material through columns 20 and 30 (e.g., the concentration ofsolids can be about 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or about 8.6 pounds of solids pergallon). The liquid feedstock can be held in mix tank 10 prior tobeing fed to columns 20 and 30.

[0026] Prior to being contacted with the liquid feedstock, resin incolumns 20 and 30 can be contacted with a volume of liquid (e.g.,water or Reverse Osmosis (RO) water) sufficient to remove or dilutemedia (e.g., ethanol) used to store the resin. The liquid feedstockcan then be fed from mix tank 10 to resin columns 20 and 30,wherein the resin is contacted by the liquid feedstock for a timeand under conditions sufficient for phenolics (e.g., a portion ofphenolics) present in the feedstock to bind to the resin (e.g. arecaptured or retained by the resin). Liquid exiting columns 20 and30 is referred to as feedstock flow through. An initial volume offeedstock flow through can be discarded as waste or redirected forfurther processing. The remaining volume of feedstock flow throughcan be collected as reduced-phenolics containing permeate.

[0027] A volume of wash solution can then be fed from wash tank 50to resin columns 20 and 30 to remove residual feedstock. All washsolution flow through can be collected as permeate.

[0028] A volume of elution solution containing a solvent cansubsequently be fed from elution tank 60 to resin columns 20 and 30to remove bound phenolics (e.g., a substantial portion of boundphenolics) from the resin. The entire volume, or a portion thereof,of elution solution flow through can be collected in holding vessel70.

[0029] The above steps represent a single cycle of the exemplaryprocess. This cycle can be repeated any number of times (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, or more, times), as required to obtain atarget volume of elution solution, in a single run. Repeat cyclescan be batch type, semi-continuous, or continuous. Prior toadditional cycles, the columns can be flushed. Alternatively, iffurther cycles are not required, resin in columns 20 and 30 can besubmerged in ethanol for storage.

[0030] When required, e.g., once a sufficient volume of elutionsolution flow through has been collected in holding tank 70, theelution solution flow through can be fed to evaporator feed tank 80and then cycled through to evaporator 90 and flash pot 100 (e.g.,once or multiple times or as required to reduce the solvent contentof the elution solution flow through to less than 90 parts permillion using, e.g., evaporator 130), where much of the solvent isrecovered by evaporation to yield a volume of solution containing areduced amount of solvent and water and concentrated phenolics. Thereduced solvent solution (i.e., the extract, i.e., the liquidextract containing, e.g., water and extracted phenolics) can be fedto tank 110 and/or tote 120.

[0031] In some instances, the liquid extract can optionally bedried, e.g., spray dried.

[0032] The above described steps constitute a single run of asingle process.

[0033] The foregoing is a description of one embodiment of theprocess. Those skilled in the art will be able to modify theprocess and will appreciate that any number of variations arepossible and within the present disclosure.

[0034] As noted above, the process disclosed herein can include oneor more cycles encompassed by a single run. Runs can be repeated asrequired.

[0035] Feedstock that can be used in the foregoing process caninclude any naturally occurring and/or synthetic materials (e.g.,solutions and liquids) containing phenolics (e.g., containinglevels (e.g., naturally occurring levels) of at least anthocyaninsand/or proanthocyanidins (e.g., type A proanthocyanidins)).Phenolics can include, for example, the art recognized class ofcompounds, which may also be known as phenols, and all compoundsknown in the art to be encompassed by this class of compounds. Thephenolics class encompasses a diverse range of naturally occurringand synthetic compounds. The simplest of the phenolics is phenol,which contains a single hydroxyl group directly bonded to anaromatic group. Phenolics also include the polyphenols, whichcontain more than one phenol unit per molecule. The most commonlyoccurring polyphenols are classified as flavonoids. All flavonoidscontain a nucleus consisting of two phenolic rings and anoxygenated heterocycle. Flavonoids are further categorized, basedupon their oxidation state. Exemplary classes of flavonoids includeflavonols, flavanols, catechins, flavanones, anthocyanidins, andisoflavonoids. Proanthocyanidins (PACs), or condensed tannins, area form of flavanol that are composed of polymer chains ofcatechins. (see e.g., Cheynier V., Am. J. Clin. Nutr, 81:223S-229S, 2005). PACs are reported to be formed through reactions ofanthocyanins with compounds containing a polarizable double bond.Accordingly, PACs differ in the nature of their constitutive units,sequence, the positions of interflavanic linkages, chain length,and the presence of substituents (e.g., galloyl or glucosylgroups). PACs, including higher-molecular weight PACs, aregenerally soluble in aqueous media or hydroalcoholic media. PACprotein affinity and astringency correlates (e.g., increases) withthe degree of PAC polymerization and galloylation. Specifically,higher-molecular weight PACs are more astringent than oligomericPACs (Vidal et al., J. Sci. Food Agric., 83:564-573, 2003).Furthermore, PACs with a low degree of polymerization, e.g., adegree of polymerization of 2 to 4 (dimer to tetramer), i.e.,oligomeric PACs, are highly bioactive. Exemplary lists of compoundsencompassed by the class are publicly available and can be found,for example, on the World Wide Web (see, for example, World WideWeb address en.wikipedia.org/wiki/Category:Phenols (accessed onNov. 27, 2009, and last modified on 23 Nov. 2009 at 14:07), in textbooks, and in published periodicals. The term phenolics includesthose compounds encompassed by the phenols, polyphenols, flavonols,flavanols, catechins, flavanones, anthocyanidins, andisoflavonoids, and proanthocyanidins (PACs) chemical classes.

[0036] Phenolics containing feedstocks can include, but are notlimited to, for example, fruits from plants of the genus Vaccinium,cranberries (e.g., juice, seeds, skin, pulp, and leaves), thejuice, seeds, and skins of grapes, apples, fruit of locusts,cowberry fruit, bilberry, blueberry (and juice obtained therefrom),lingonberry, huckleberry, black current, chokeberry, blackchokeberry, and pine bark, peanuts (e.g., peanut skins), ginkgo(e.g., ginkgo leaves), cola nuts, Rathania (e.g., Rathania roots),cinnamon, cocoa, black tea, and green tea. In some instances,feedstock can include, but is not limited to, (1) wholecranberries, (2) cranberry skins, (2) cranberry seeds, (3)cranberry pulp, (4) cranberry leaves, (5) whole cranberry plants,(6) and any combination of (1)-(6). In addition, materials otherthan the disclosed can be used in the processes disclosed herein ifthe material contains phenolics. Methods for identifying andquantifying phenolics in materials or feedstocks are known in theart and include, but are not limited to, for example, directspectroscopy at 280 nm; indirect spectroscopy using, e.g., artrecognized and commercially available reagents and assays, e.g.,Vanillan assay, Folin-Denis assay, Folin-Ciocalteu assay, PrussianBlue assay, Bate-Smith assay, and Porter assay; and liquidchromatography, e.g., using ultraviolet, fluorescence, massspectroscopy, and nuclear magnetic resonance (NMR). Phenolicsdetection techniques are also disclosed in the literature (see,e.g., Fereidoon and Naczk, Food Phenolics, Technomic Publishing Co.Inc, 1995).

[0037] Feedstock can be solid or liquid and fresh or frozen. Solidfeedstocks can be liquefied or solubilized, e.g., put intosolution, prior to commencing the process. Frozen feedstocks can bethawed, e.g., prior to use. Feedstock can also be used with orwithout modification. Exemplary useful modifications can includeselection, refinement, and/or mechanical processing. For example,the materials can be cleaned to remove debris (e.g., material thatdoes not contain PACs), e.g., debris, and/or sorted to selectmaterial of a defined size. When the material is fruit (e.g.,cranberry), the material can be cut into slices, and/or skinned toexpose the inner flesh of the fruit (e.g., the cranberry pulp) andto increase the surface area of the material. In some cases, skinsand pulp can then be used together or can be separated and usedseparately.

[0038] Feedstock can also include juice (e.g., cranberry juice)produced by traditional pressing, enzymatic digestion, and/or bycountercurrent extraction (CCE) (CCE is described in U.S. Pat. Nos.5,320,861 and 5,419,251, which are hereby incorporated by referencein their entirety)), or juice as described in or as obtained usingthe methods described in U.S. Pat. Nos. 6,733,813; 6,977,092; and7,022,368, each of which is hereby incorporated by reference in itsentirety.

[0039] As used herein, juice refers, e.g., to the liquid expressedor extracted from one or more of the fruits or vegetables disclosedin the paragraphs above (e.g., cranberries) or a puree of theedible portions of a fruit or vegetable that is used as abeverage.

[0040] In some embodiments, feedstock is not an ultrafiltrateand/or is not pretreated using ultrafiltration, e.g.,ultrafiltration, e.g., as disclosed in U.S. Publication No.20090035432.

[0041] The volume of liquid feedstock used in the process (e.g., ina single cycle of the process) can be varied as required. Forexample, the volume of feedstock in a single cycle of the processcan include from about 100 mL to about 10 gallons for small scaleruns, and about 100 gallons to about 1000 gallons, and up to, e.g.,20,000 gallons for large scale runs.

[0042] In some instances, the volume and/or concentration of liquidfeedstock used can be based upon the adsorbant capacity of theresin, which can be based on the volume of resin present in thecolumn), such that the volume and/or concentration of liquidfeedstock is optimized to not exceed the adsorbant capacity of theresin. For example, the volume of liquid feedstock can be, lessthan, equal to, or greater than the adsorbant capacity of theresin.

[0043] The volume of liquid feedstock can be increased or decreasedto provide a chosen concentration of solids. In some instances, thevolume of a first feedstock with a first concentration of solidscan be increased or decreased to provide a second feedstock withmore or less concentrated solids. In some instances, theconcentration of solids in a liquid feedstock can be selected toprovide a certain viscosity, e.g., such that the liquid feedstockallows certain flow rates. Exemplary concentrations of solids thatcan be present in a liquid feedstock include, but are not limitedto, about 1-10%, 11-20%, 21-30%, 31-40%, 41-50%, over 50%, or 25%(the concentration of solids in a liquid feedstock can also beshown in terms of percent or Brix). Methods for increasing thevolume of a liquid feedstock include, for example, adding a volumeof a suitable solution (e.g., water) to the liquid feedstock, e.g.,to increase the total volume of the liquid feedstock and therebyreduce the concentration of solids in the feedstock.

[0044] Methods for decreasing the volume of a liquid feedstockinclude, for example, reverse osmosis, or evaporation, or both,e.g., to decrease the total volume of the liquid feedstock andthereby increase the concentration of solids in the feedstock. Insome instances, the volume of liquid feedstock can be about 346gallons and the concentration of solids in the liquid feedstock canbe about 50%, e.g., per column, per cycle. If required, the volumeof the feedstock can be increased to provide a liquid feedstockcontaining about 25% solids prior to contacting the liquidfeedstock with the resin, e.g., by adding an equal volume (e.g.,about 346 gallons) of liquid (e.g., water) to the liquidfeedstock.

[0045] In some embodiments, preparation of a feedstock for use inthe processes disclosed herein does not include an extraction step,e.g., an acid or alkaline extraction step.

[0046] The size (e.g., volume or capacity or area (e.g., inm.sup.2) within a single column) of a column for use in the abovedisclosed process can be varied according to the scale of theprocess. For example, a small scale process (e.g., a laboratoryscale process) can use columns (e.g., one or more, e.g., 2, 3, 4,5, 10, or 20) with a capacity from about 100 mL to about 10 gallons(e.g., 1 liter), and a large scale process (e.g., an industrial orcommercial scale process) can use columns (e.g., one or morecolumns, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more than 20columns) with a capacity from about 10 gallons to about 1000gallons (e.g., about 141 gallons).

[0047] The volume of liquid (e.g., water) that can be used toremove or dilute media used to store resin in the resin columns caninclude any volume of liquid that is sufficient to completelyreplace the media or dilute the media by at least about 10% (e.g.,at least about 20%, 50%, 80%, 90%, 95%, or 99%).

[0048] Resin suitable for use in the processes disclosed hereininclude, for example, a resin (e.g., a synthetic resin) that canbind phenolics present in a phenolics containing feedstock. Suchresins can include, for example, resin with (1) a first bindingaffinity for, and that binds (e.g., that binds specifically)phenolics; and (2) a second binding affinity for organic acidsand/or sugars, wherein the second binding affinity is lower thanthe first binding affinity, e.g., such that the resin does not bind(e.g., does not substantially bind), organic acids and/or sugars ina feedstock.

[0049] In some instances, the resin can (i) bind to non-polar tomedium polarity phenolics, (ii) be styrene-based having one or morebromine substituents, (iii) be hydrophobic, be a nonionic aliphaticacrylic polymer, (iv) provide a large binding surface area, (v) bean organic resin, (vi) be an ion-exchange resin, (vii) be anaromatic resin, (viii) be a (meth)acrylic acid resin, (ix) be a(meth)acrylate resin, (x) be a acrylonitrile aliphatic resin,and/or any combination of (i)-(x).

[0050] Examples of commercially available resins that can be usedin the processes disclosed herein include, but are not limited to,SP207 Sepabeads.TM. (Mitsubishi Chemical), SP700 Sepabeads.TM.(Mitsubishi Chemical), Diaion HP20 (Mitsubishi Chemical), DiaionSP70 (Mitsubishi Chemical), Diaion SP825 (Mitsubishi Chemical),Diaion SP850 (Mitsubishi Chemical), Diaion HP2MG methacrylate(Mitsubishi Chemical), ADS-5 (Nankai University, Tianjin, China),ADS-17 (Nankai University, Tianjin, China), Amberlite.TM. XAD-4(manufactured by Organo Co. and distributed globally by Rohm &Hass), Amberlite.TM. XAD-16 (manufactured by Organo Co. anddistributed globally by Rohm & Hass), Amberlite.TM. XAD-1600(manufactured by Organo Co. and distributed globally by Rohm &Hass), Amberlite.TM. XAD-2 (manufactured by Organo Co. anddistributed globally by Rohm & Hass), Amberlite.TM. XAD-1180(manufactured by Organo Co. and distributed globally by Rohm &Hass), Amberlite.TM. XAD-2000 (manufactured by Organo Co. anddistributed globally by Rohm & Hass), Amberchrom.TM. CG300-C(Rohm & Hass), and any combination thereof. In someembodiments, the resin is not the commercially available C18resin.

[0051] In some embodiments, the resin is commercially availableAmberlite.TM. FPX66 (Rohm & Hass). FPX66 has the followingproperties: FPX66 consists of white beads that form a matrixconsisting of a macroreticular aromatic polymer. The moistureholding capacity of the resin is 60-68% and its specific gravity is1.015 to 1.025. The resin has a uniformity coefficient of less thanor equal to 2.0, a harmonic mean size of 0.600-0.750 mm, a finecontent of less than 0.300 mm, and a surface area of greater thanor equal to 700 m.sup.2/g. The porosity of the resin is 1.4cc/g.

[0052] In some embodiments, the resin is commercially availableAmberlite.TM. XAD-7HP resin (manufactured by Organo Co. anddistributed globally by Rohm & Hass). Information on XAD-7HPcan be found at the Rohm & Hass world wide web siteamberlyst.com/xad7hp_typical.htm. Specifically, XAD-7HP resin hasthe following properties: XAD-7HP is a macroreticular aliphaticcrosslinked polymer ester resin that consists of white translucentbeads that have a moisture holding capacity of 61-69%. The resinhas an high surface area (e.g., approximately or about 300-500m.sup.2/g (e.g., greater than 380 m.sup.2/g)), a specific gravityof 1.06 to 1.08, an average pore size of approximately 450Angstroms, a mean diameter of approximately 560 .mu.m, and both acontinuous polymer phase and a continuous pore phase. The harmonicmean size of the beads is 0.56-0.71 mm with a uniformitycoefficient of less than or equal to 2.0. The maximum operatingtemperature of the resin is 80-100.degree. C. (i.e.,175-210.degree. F.).

[0053] The amount of resin used can be varied and is dependent uponthe scale of the process and/or the volume or capacity of thecolumn.

[0054] The time and conditions sufficient for phenolics present inthe feedstock to bind to the resin include those times andconditions under which at least and/or about 1% or 10% (e.g., atleast and/or about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99% and 100%, or a range between any two of these values)of phenolics present in the liquid feedstock bind to the resin.Methods for assessing the percentage of phenolics present in thefeedstock that have bound to the resin can include, for example,steps of first assessing the level of phenolics in the liquidfeedstock, and then assessing the level of phenolics present in thefeedstock flow through and/or the non-phenolics containingpermeate, wherein any difference in the level of phenolics is anindication of the level of phenolics bound to the resin. Methodsfor detecting phenolics are known in the art and are exemplifiedabove.

[0055] The time and conditions sufficient for phenolics present inthe feedstock to bind to the resin can be controlled, e.g., byvarying the flow rate of the feedstock into the resin (e.g., thetime the feedstock is contacted with the resin), and/or thetemperature within the resin column. For example the flow rate ofthe feedstock into the resin column can include, but is not limitedto, about 1.0-6.0 gallons per minute (e.g., about 1.0, 1.5, 2.0,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5.0, 5.5, 6.0 gallons per minute), and thetemperature within the resin column can be selected to minimizemicrobial growth (e.g., 35.degree. F.-80.degree. F.).

[0056] As noted above, a volume of feedstock flow through can bediscarded as waste or redirected for further processing. Exemplaryvolumes that can be discarded include at least and/or about, 0.5%,1%, 5%, 10%, and about 20%.

[0057] Wash solutions useful in the processes disclosed herein caninclude, for example, water based solutions containing one or moresolvents that will not reduce (e.g., substantially reduce) theamount of phenolics bound to the resin. Exemplary wash solutionscan include water or water mixed with one or more solvents, forexample, water containing up to about 25% solvent (e.g., up toand/or about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, and/orbelow 0.5% solvent). Suitable solvents include, but are not limitedto, e.g., alcohol (e.g., methanol, ethanol, propanol), acetone,hexane, and/or mixtures thereof.

[0058] In some instances, the wash solution is a water basedsolution containing 5%.+-.1% total ethanol (e.g., 1 part ethanol in19 parts water). The volume of wash solution can be adapted to thevolume or capacity of the resin-containing column, whereinone-times the volume of resin in the column is referred to as onebed volume. For example, the volume of wash solution can include,e.g., less than one bed volume, about one bed volume, about two bedvolumes, or more than two bed volumes.

[0059] In some instances, the volume of wash solution can be about282 gallons or about 2 bed volumes and the wash solution caninclude about 5%.+-.1% total ethanol (e.g., 1 part ethanol in 19parts water).

[0060] In some instances, the volume of wash solution can be about282 gallons or about 2 bed volumes and the wash solution caninclude about 5%.+-.1% Standard Denatured Alcohol (SDA), e.g., SDA35A 190, in water (further information regarding SDA 35A 190 can befound at world wide web addresssasoltechdata.com/tds/sda35A.sub.--190.pdf).

[0061] A single cycle can include 1 or more wash steps (e.g., 1, 2,3, 4, 5, or more wash steps) per each cycle. Furthermore, the washsteps can be performed for a time and under conditions that allowoptimal removal of non-phenolics from the resin. For example theflow rate of the wash solution into the resin column can include,but is not limited to, 1.0-6.0 gallons per minute (e.g., about 1.0,1.5, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4,4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.5, 6.0 gallons per minute), and thetemperature within the resin column can be selected to minimizemicrobial growth (e.g., 35.degree. F.-80.degree. F.).

[0062] Elution solutions that can be used in the processesdisclosed herein can include water based solutions containing oneor more solvents at any concentration that will decrease (e.g.,substantially decrease) the association between resin-boundphenolics and resin, such that phenolics are released from theresin. Exemplary elution solutions can include solvent or a mixtureof solvent and water (e.g., wherein the concentration of thesolvent is about 100%, or less than 100%, e.g., about 99%, 98%,97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 60%, 55%). Suitablesolvents can include, but are not limited to, e.g., alcohol (e.g.,methanol, ethanol, propanol), acetone, and hexane. In someinstances, the elution solution is a water based solutioncontaining 95%.+-.1% total ethanol. The volume of elution solutioncan include, e.g., less than one bed volume, about one bed volume,about two bed volumes, or more than two bed volumes.

[0063] In some instances, the volume of elution solution can beabout 346 gallons or about 2.8 bed volumes and the elution solutioncan include 95% total ethanol in water.

[0064] In some instances, the volume of elution solution can beabout 346 gallons or about 2.8 bed volumes and the elution solutioncan include 95% Standard Denatured Alcohol (SDA), e.g., SDA 35A190, in water.

[0065] A single cycle can include 1 or more elution steps (e.g., 1,2, 3, 4, 5, or more elution steps) per each cycle. Furthermore, theelution steps can be performed for a time and under conditions thatallow optimal removal of phenolics from the resin. For example theflow rate of the wash solution into the resin column can include,but is not limited to, 1.0-6.0 gallons per minute (e.g., about 1.0,1.5, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4,4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.5, 6.0 gallons per minute), and thetemperature within the resin column can be selected to minimizemicrobial growth (e.g., 35.degree. F.-80.degree. F.).

[0066] In some embodiments, elution flow through is collectedwithout further processing. In other embodiments, elution flowthrough is further processed. For example, phenolics containingsolutions can be evaporated using at least one evaporation step(e.g., 1, 2, 3, 4, 5, or more evaporation steps), e.g., to reducethe amount of solvent present in the solution, resulting in asolution containing phenolics, water, and a reduced amount ofsolvent as compared to the elution solution. In some instances, theevaporation step can be repeated to further reduce the amount ofsolvent present in the solution. Evaporation methods can include,but are not limited to, e.g., batch evaporation methods andcontinuous evaporation methods, falling film methods, rising filmmethods, falling plus rising film methods (e.g., using plates andtubes), multiple effects methods, single effects methods, and vaporrecompression.

[0067] In some instances, the evaporation step can be repeated,e.g., until the amount of solvent in the solution is less thanabout 90 parts per million. Evaporation methods can include, forexample, the use of temperature (e.g., heat) and/or pressure (e.g.,vacuum) sufficient to reduce the amount of solvent in solution.Exemplary conditions can include a temperature of at least about70.degree. C. (e.g., about 71.degree. C., 72.degree. C., 73.degree.C., 74.degree. C., 75.degree. C., 76.degree. C., 77.degree. C.,78.degree. C., 79.degree. C., 80.degree. C., 81.degree. C.,82.degree. C., 83.degree. C., 84.degree. C., 85.degree. C.,86.degree. C., 87.degree. C., 88.degree. C., 89.degree. C.,90.degree. C. and above 90.degree. C. (or the Fahrenheitequivalent)) and pressure of at least about 50 mBar (e.g., 50 mBar,60 mBar, 70 mBar, 80 mBar, 90 mBar and above 90 mBar). In someembodiments, evaporation conditions can be about 124.degree. F. andabout 130 mBar. Other exemplary evaporation conditions includecombinations of temperature and vacuum shown in Table 1.

TABLE-US-00001 TABLE 1 Exemplary Evaporation Conditions Temperature(.degree. F.) Vacuum (mBar) 104 73.77 114 98.61 124 130.35 134170.46 144 220.67 154 282.93 164 359.48 174 452.8 184 565.72 194701.24 204 862.9 212 2605.4

[0068] In some instances, an evaporation step can be carried out inconjunction with a step to reduce the concentration of solids inthe liquid (e.g., a dilution step).

[0069] In some embodiments, evaporation can include (i) removingsolvent from the phenolics containing solutions and (ii)concentrating the phenolics containing solutions. In someinstances, (i) and (ii) are performed simultaneously using e.g., arotary evaporator (Rotovap). Alternatively, (i) can be performedusing, e.g., distillation columns and (ii) can be performed eithersimultaneously or subsequently. Other exemplary methods forremoving solvent include, but are not limited to, the use oftemperature and/or vacuum as described above using, e.g., a rotaryevaporator (Rotovap).

[0070] In some embodiments, evaporation can include (i) removingsolvent from the phenolics containing solution using distillationcolumns and (ii) concentrating the phenolics containing solutionsusing a rising film plate evaporator. Alternatively or in addition,evaporation can include the use of forced circulation evaporators,or Pfaudler kettles.

[0071] The solution resulting from the evaporation step is a liquid(e.g., water) containing phenolics (a liquid extract). This extractdoes not comprise fruit juice, e.g., fruit juice expressed orextracted from a feedstock disclosed above in the absence of theprocess disclosed herein; or fruit juice produced by traditionalpressing, enzymatic digestion, or by CCE; or juice as described inor as obtained using the methods described in U.S. Pat. Nos.6,733,813; 6,977,092; and 7,022,368. In some instances, the liquidextract can be obtained and optionally analyzed, e.g., to assessthe level of phenolics and/or to characterize the types ofphenolics present.

[0072] The liquid extract can be concentrated, e.g., to increasethe concentration of solids in the extract. Concentration methodsinclude, but are not limited to, e.g., one or more of, membraneconcentration, heat concentration, vacuum (reduced pressure)concentration, and freeze concentration. In some instances, thisliquid extract can be obtained and optionally analyzed, e.g., toassess the level of phenolics and/or to characterize the types ofphenolics present.

[0073] Liquid extracts can be dried to provide a dry extractcontaining phenolics. Methods for drying the liquid extracts caninclude, but are not limited to, for example, freeze drying, vacuumdrying, spray drying, drum drying, shelf drying, and drying bymicrowave.

[0074] If required, a liquid or dry extract can be analyzed, e.g.,to assess the level of phenolics present and/or to characterize thephenolics present (e.g., to determine the relative amounts ofphenolics (e.g., anthocyanins and PACs) present in the extract.

[0075] Extracts can also be optionally sterilized. Sterilizationcan be performed by a method commonly used by those skilled in theart, such as high-pressure sterilization, heat sterilization,filter sterilization, and microwave sterilization.

[0076] Extracts

[0077] The extracts (e.g., phenolics enriched extracts) obtainedusing the processes disclosed herein can be liquid, dry, semi dry,or powdered extracts (e.g., powdered, dehydrated, or lyophilizedextracts) containing at least anthocyanins and proanthocyanidins(PACs). Such extracts can be additionally characterized as havingor containing a total amount of anthocyanins of at least about 1%(weight to volume (w/v), weight to weight (w/w), or volume tovolume (v/v)), as assessed (e.g., quantified) using HPLC. Forexample, extracts can contain at least about or about 1%, e.g., atleast about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20% (w/v, w/w, or v/v), or at leastabout 21% (w/v, w/w, or v/v), or a range between any two of thesevalues, anthocyanins, as assessed by HPLC. Such extracts can alsocontain at least about 10% (w/v, w/w, or v/v) PACs, as assessed(e.g., quantified) using, e.g., HPLC. For example, extracts cancontain at least about or about 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60% (w/v, w/w, or v/v), more than 61%, 65%, 70%,75%, or at least about 80% (w/v, w/w, or v/v), or a range betweenany two of these values, PACs, as assessed by HPLC.

[0078] The levels of PACs in an extract can be assessed orquantified using DMAC (the DMAC method is disclosed in Cunninghamet al., Analysis and Standardization of Cranberry Products, QualityManagement of Nutraceuticals, ACS Symposium Series, 803ed.,American Chemical Society, Washington D.C., pages 151-166, 2002,which is hereby incorporated by reference). In such instances,extracts containing at least anthocyanins and proanthocyanidins(PACs) can contain about or at least about 40% (w/v, w/w, or v/v)PACs, as assessed (e.g., quantified) using, e.g., DMAC. Forexample, extracts can contain at least about or about 40%, 50%,55%, 60%, 70%, 80% (w/v, w/w, or v/v), more than 80% (w/v, w/w, orv/v), or a range between any two of these values PACs, as assessedby DMAC; and/or

[0079] a ratio of anthocyanins to PACs of about 1:5 (e.g., about1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1.4.5, 1:5, 1:5.5, 1:6, 1.6.5, 1:7,1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, or about 1:10), wherein anthocyaninsare assessed (e.g., quantified) using, e.g., HPLC, and PACS areassessed (e.g., quantified) using, e.g., HPLC or DMAC; and/or

[0080] a PACs oligomeric profile that substantially the same orsimilar, (e.g., substantially similar) to the PACs oligomericprofile present in CCE cranberry juice feedstock. Alternatively orin addition, the PACs oligomeric profile can include higher amountsof 2-mer and greater than 10-mers than other PACs oligomers.Alternatively or in addition, the PACs oligomeric profile caninclude ratios of PACs oligomers of about6(1mer):28(2mer):11(3mer):8(4mer):6(5mer):7(6mer):3(7mer):4(8mer):2(9mer)-:26(>10 mer); and/or

[0081] a ratio of PACs to total phenolics that is substantially thesame (e.g., roughly equal) to the ratio of PACs to total phenolicspresent in cranberries or the fruit from which the phenolics wereextracted, e.g., present in cranberries or counter currentextracted cranberry juice; and/or

[0082] a ratio of PACs to quercetin, quercgalac, quercitrin,myricetin, and/or quercaraban that is the same (e.g., substantiallythe same) or similar, (e.g., substantially similar) to the ratio ofPACs to quercetin, quercgalac, quercitrin, myricetin, and/orquercaraban present in CCE cranberry juice; and/or

[0083] a ratio of PACs to total anthocyanins that is not the sameas the ratio of PACs to total anthocyanins in cranberries or thefruit from which the phenolics were extracted, e.g., present incranberries or counter current extracted cranberry juice;and/or.

[0084] phenolics (e.g., polymeric phenolics) with a molecularweight (e.g., an average molecular weight) of less than 14,000Daltons; and/or

[0085] PACs (e.g., 10% or more of total PACs in the extract) withpolymer chain lengths of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greaterthan 10, or combinations thereof; and/or

[0086] a higher concentration of anthocyanin and PACs than ispresent in cranberries or the fruit from which the phenolics wereextracted, e.g., present in cranberries or counter currentextracted cranberry juice feedstock, e.g., a higher dry weightconcentration.

[0087] Extracts containing at least anthocyanins andproanthocyanidins (PACs) can be optionally further characterizedbased on the levels of organic acids (e.g. total organic acids) andsugars (e.g., total sugars) in the extract. For example, extractscan contain less than 5% (w/v, w/w, or v/v) organic acids (e.g.,about 5% or less than about 5%, 4%, 3%, 2%, 1% organic acids, lessthan 1% organic acids, no organic acids (e.g., the extract can befree (e.g., substantially free) of organic acids), or a rangebetween any two of these values), and/or less than 5% sugar (e.g.,about 5% or less than about 5%, 4%, 3%, 2%, 1% sugar, less than 1%sugar, no sugar (e.g., the extract can be free (e.g., substantiallyfree) of sugar), or a range between any two of these values).

[0088] The phenolics extracted using the process described hereincan be soluble in aqueous media.

[0089] An extract can be formulated as a composition for use in ananimal (e.g., a human and/or non-human animal), e.g., for ingestionor consumption by an animal (e.g., a human and/or non-humananimal). Such compositions can include excipients, e.g., toincrease the stability, solubility, shelf-life, taste, tostandardize the level of a particular compound in the composition,and/or bioabsorption of the extract. Examples of includableexcipients include but are not limited to, calcium carbonate,calcium phosphate, various sugars and types of starch, cellulosederivatives, gelatin, vegetable oils, polyethylene glycols,propylene glycol, and inhibitors of enzymes that degrade and/ormodify phenolics, such as inhibitors of polyphenoloxidases,peroxidases, glycosidases, decarboxylases, and esterases.Alternatively or in addition, the extracts can be combined withagents that protect them from oxidative reactions (e.g.,anti-oxidants). Different diafiltration media (e.g., acidifiedwater) can be employed to stabilize and/or adjust the color of theextract

[0090] Use of Extracts

[0091] In some embodiments, the extracts disclosed herein can beused in or as nutriceuticals and/or as food supplements. Forexample, the extracts can be formulated to as powders, pills,tablets, capsules or syrups for administration to an individual(e.g., a human or non-human) by any route, e.g., by ingestion.Alternatively or in addition, the extracts can be used tosupplement a food or beverage to enhance the health benefitsconferred by the food or beverage. For example, such an extractcould be applied to (e.g., coated onto or infused into) fruits,vegetable, legumes, and the like (e.g., dried cranberries) tocreate a food product with enhanced health benefits. Alternativelyor in addition, extracts can be used to supplement beverages, e.g.,juice beverages including, but not limited to, e.g., fruit juicesand fruit juice drinks (e.g., cranberry juice cocktails and juiceblends), tea (e.g., herbal and non-herbal tea), leaf tea, yogurt,milk, smoothies, chewing gum, dietary supplements, water, flavoredwaters, energy drinks, and milk (e.g., liquid and powderedmilk).

[0092] Compositions Comprising Phenolics and Fumaric Acid and UsesThereof

[0093] Species of the genus Alicyclobacillus (ACB) include, forexample, acidophilic, thermophilic, and spore forming bacteria suchas Alicyclobacillus acidoterrestris and Alicyclobacillusacidocaldarius. ACB contamination of juice beverages can causespoilage due to the production of guaiacol, an organic compoundthat imparts an unpleasant flavor and odor.

[0094] ACB contamination of juice beverages can be caused by thepresence of soil residue in the juice beverage. Accordingly,careful washing of fruit with uncontaminated water duringprocessing can reduce ACB contamination. Some fruits, however, aredifficult to wash thoroughly. Such methods are also inefficient andcan not be applied to previously processed and packaged juicebeverages (e.g., packaged juice). ACB contamination can also bepresent in raw or refined sugar added to juices. Once ACB ispresent in a production line it can be difficult to eliminatebecause ACB spores are heat resistant. Pasteurization cannot alwaysbe used to eliminate ACB because the high temperatures required toeliminate ACB spores can be detrimental to juice quality. Certaintypes of filtration and irradiation can also be used to eliminateACB, but such methods are not suitable for all products. Highconcentrations of phenolics can reduce ACB contamination inbeverages such as fruit juice. The application of such methods arelimited, however, because the level of phenolics required to causean undesirable change in the color of the beverage. Fumaric acidalone can also reduce ACB contamination in juice beverages, but notwithout undesirably altering the taste of the juice.

[0095] Provided herein are compositions comprising (e.g.,comprising, consisting essentially of, or consisting of) phenolics(e.g., concentrated, isolated, or purified phenolics (e.g., theextracts disclosed herein)) and fumaric acid. These compositionscan be added to juice beverages that are susceptible to microbial(e.g., bacterial contamination or ACB contamination), to reduce orprevent microbial (e.g., bacterial contamination or ACBcontamination) contamination therein.

[0096] Juice beverages susceptible to ACB contamination include,but are not limited to, for example, juice beverages contaminatedwith soil, juice beverages contaminated with raw or refined sugarcontaining ACB or ACB spores, and juice beverages containing ACBspores. Alternatively, the compositions can be added to juicebeverages to reduce or prevent ACB contamination in ACBcontaminated juices, e.g., juices containing viable ACB microbes.As ACB contamination reduces the shelf-life of juice beverages, thecompositions described herein can be used to increase theshelf-life of juice beverages, or as juice beverages preservatives.Furthermore, such results can be achieved without undesirablyaltering the taste or appearance of the juice beverage due to thesynergistic activity between the two components. As used herein,"synergy" or "synergistic activity" and the like refer to acombined effect of two components that is greater than theindividual effects of the same components alone or when addedtogether. For example, as used herein, synergy refers to a level ofreduction in ACB contamination of a juice beverage, a reduction inACB growth, or an increase in the death of ACB in the presence offumaric acid and phenolics that is not observed in the presence offumaric acid or phenolics alone.

[0097] In some embodiments, the compositions and methods disclosedherein prevent or reduce ACB growth, kill quiescent or dividing ACBcells, and/or eliminate ACB spores.

[0098] In some embodiments, compositions comprising ratios ofphenolics to fumaric acid can include ratios of phenolics (e.g.,PACs) to fumaric acid that are useful in higher acidity juicebeverages. Exemplary higher acidity juice beverages can include,but are not limited to, apple juice (e.g., about pH 2.9-3.3 orabout pH 2.9-4.1, e.g., pH 3.7), lemon juice (e.g., about pH 2.3),cranberry juice (e.g., about pH 2.3-2.5), tropical fruit blends,grapefruit juice (e.g., about pH 2.9-3.5), pineapple juice (e.g.,about pH 3.4), grape juice (e.g., about pH 2.8-3.3), and juiceblends containing two or more of these juices alone or incombination with low pH juice beverages. In some embodiments,higher acidity juice beverages have a pH of between about pH 2-pH3.49.

[0099] In some embodiments, compositions comprising ratios ofphenolics to fumaric acid can include ratios of phenolics (e.g.,PACs) to fumaric acid that are useful in lower pH (e.g., loweracidity) juice beverages. Exemplary low pH juice beverages caninclude, but are not limited to, for example, orange juice (e.g.,about pH 3.5-4.2, pH 3.9, or pH 4.6) and/or vegetable juice (e.g.,about pH 3.9-4.3). In some embodiments, lower pH juices have a pHof greater (i.e., more alkaline) than about pH 3.5.

[0100] Useful ratios of fumaric acid to phenolics (e.g., PACs) caninclude, but are not limited to, e.g., ratios of fumaric acid toPACs of between about 4000:1-100:1, 3571:1-121:1, about 135:1, orabout 238:1. In some instances, such ratios can be useful in higheracidity juice beverages.

[0101] Other useful ratios of fumaric acid to phenolics (e.g.,PACs) for use in high pH juice beverages can include, but are notlimited to, e.g., ratios of fumaric acid to PACs of between about10:1-50:1, or 14:1. In some instances, such ratios can be useful inlower pH juice beverages.

[0102] In some instances, phenolics and fumaric acid can be presentin a juice beverage at synergistic concentrations. With respect tofumaric acid, such concentrations can be defined using any artrecognized units (e.g., percent weight/volume (e.g., g/100 mL) orpercent volume/volume). Concentrations of phenolics can also bedefined using any art recognized term (e.g., percent weight/volume(e.g., g/100 mL) or percent volume/volume) and can be expressedeither as total phenolics or by specific phenolics (e.g.,proanthocyanidins (PACs)). In some embodiments, phenolics can beextracts obtained using the processes disclosed herein and theamount of PACs in the extract can be about 55%.

[0103] In some embodiments, concentrations of added phenolics andfumaric acid in a beverage can include, e.g., 4.2.times.10 mg/mLPACs (i.e., 0.1 mg PACs/8 oz)-8.291.times.10.sup.-3 mg/mL PACs(i.e., 1.99 mg PACs/8 oz), 2.1.times.10.sup.-3 mg/mLPACs-8.3.times.10.sup.-3 mg/mL PACs, 2.1.times.10.sup.-3 mg/mLPACs-6.3.times.10.sup.-3 mg/mL PACs, or about 4.2.times.10.sup.-3mg/mL PACs (i.e., 1 mg PACs/8 oz), and between about 0.01% (e.g.,0.001%-0.05%) to about 0.15% (e.g., about 0.10%-0.2%) fumaric acid,or about 0.1% fumaric acid by weight (e.g., w/w if the extract andfumaric acid are dry, v/w, or vice-versa, if one of the extract orliquid is dry).

[0104] In other embodiments, concentrations of added phenolics andfumaric acid in a beverage can be, e.g., 4.2.times.10.sup.-4 mg/mLPACs (i.e., 0.1 mg PACs/8 oz)-100 mg/mL PACs, 4.2.times.10.sup.-4mg/mL PACs-80 mg/mL PACs, 4.2.times.10.sup.-4 mg/ml, PACs-60 mg/mLPACs, 4.2.times.10.sup.-4 mg/mL PACs-40 mg/mL PACs,4.2.times.10.sup.-4 mg/mL PACs-20 mg/mL PACs, 4.2.times.10.sup.-4mg/mL PACs-10 mg/mL PACs, 4.2.times.10.sup.-4 mg/mL PACs-5 mg/mLPACs, 4.2.times.10.sup.-4 mg/ml PACs-1 mg/mL PACs,4.2.times.10.sup.4 mg/mL PACs-0.5 mg/mL PACs, 4.2.times.10.sup.-4mg/mL PACs-0.1 mg/mL PACs, 4.2.times.10.sup.-4 mg/mL PACs-0.05mg/mL PACs, 4.2.times.10.sup.-4 mg/mL PACs-0.04 mg/mL PACs,4.2.times.10.sup.-4 mg/mL PACs-0.03 mg/mL PACs, 4.2.times.10.sup.-4mg/mL PACs-0.02 mg/mL PACs, 4.2.times.10.sup.-4 mg/mL PACs-0.01mg/mL PACs, 4.2.times.10.sup.-4 mg/mL PACs-0.005 mg/mL PACs,4.2.times.10.sup.-4 mg/mL PACs-0.001 mg/mL PACs, or 0.05 mg/mL PACs(i.e., 12 mg PACs/8 oz), and between about 0.01% (e.g.,0.001%-0.05%) to about 0.15% (e.g., about 0.10%-0.2%) fumaric acid,or about 0.1% fumaric acid by weight (e.g., w/w if the extract andfumaric acid are dry, v/w, or vice-versa, if one of the extract orliquid is dry).

[0105] In some embodiments, concentrations of phenolics and fumaricacid can include, but are not limited to, for example, no more thanabout 0.04 mg/ml PACs, or no more than about 1 mg/ml PACs, or about0.04 mg/ml to about 0.17 mg/ml (e.g., 0.15 mg/ml-0.19 mg/ml) PACs,and about 0.01% (e.g., 0.001%-0.05%) to about 0.15% (e.g., about0.10%-0.2%) fumaric acid by weight.

[0106] In some embodiments, compositions comprising phenolics andfumaric acid (e.g., dry phenolics and fumaric acid) can be preparedin amounts that are sufficient to yield synergistic concentrationsof phenolics and fumaric acid when added to a volume of juicebeverage. Such compositions can be prepared according to any of theabove ratios alone or in amounts sufficient to provide asynergistic concentration of phenolics and fumaric acid when thecomposition is added to a defined volume of a juice beverage. Suchcompositions are within the present invention. Exemplary volumes ofjuice beverage to which such compositions can be prepared and/oradded include, but are not limited to, 0.1, 0.5, 1, 10, 20, 50,100, 200, 250, 300, 330, and 500 mLs, 1, 2, 2.5, 5, 10, 15, 20, 30,50, 100, 150, 200, 250, 500, 750, 1000, 10,000, 25,000, 50,000,100,000, 500,000, 1000,000 L, and above 1000,000 L (or theequivalent volumes in ounces and gallons), or a range between anytwo of the afore-listed integers.

[0107] Methods of Making Compositions Comprising Phenolics andFumaric Acid

[0108] Compositions comprising phenolics and fumaric acid caninclude concentrated, purified, or isolated phenolics that includeat least PACs, wherein the phenolics are concentrated and/orisolated from any phenolics containing feedstock, e.g., anyphenolics containing feedstock disclosed herein. Methods forconcentrating and/or isolating phenolics are known in the art andinclude, but are not limited to, for example, filtration. and thosemethods disclosed in, for example, U.S. Pat. Nos. 5,840,322,6,440,471, 6,210,681, 5,650,432, 5,646,178, 5,474.774, 5,525,341,6,720,353, and 6,608,102.

[0109] In some embodiments, phenolics are the extracts disclosedhere or are obtained using the processes disclosed herein.

[0110] In some embodiments, extracts for use in or as foodpreservatives contain about 90% PACs (e.g., type A PACs).

[0111] Fumaric acid, as included in the compositions disclosedherein, can include any commercially available fumaric acid and thesalts and esters thereof (e.g., fumarates). Fumaric acid is alsoreferred to in the art as trans-butenedioic acid, has the chemicalformula HO.sub.2CCH.dbd.CHCO.sub.2H, and has a molecular mass of116.07 g/mol. Compositions comprising phenolics and fumaric acidcan be prepared using any combination of liquid or dry phenolicsand fumaric acid. Similarly, compositions comprising phenolics andfumaric acid can themselves be liquid or dry (e.g., spray dried).In some embodiments, compositions comprising phenolics and fumaricacid are dry (e.g., spray dried). In some embodiments, compositionscomprising phenolics and fumaric acid can include isolatedphenolics and isolated acid. Such compositions can also consist ofor consist essentially of isolated phenolics and isolated fumaricacid. The compositions can be contained in any suitable container,vessel or vial suitable for storing or distributing the compositionand/or adding the composition to a juice beverage. For example,compositions can be disposed within a container in amountssufficient to yield synergistic concentrations of phenolics andfumaric acid when the composition is added to a liquid volume ofjuice beverage. Exemplary suitable containers include, but are notlimited plastic, glass, metal, and paper vessels suitable forsingle use or multiple use. In some embodiments, containers can bemarked or labeled to illustrate either the amount of thecomposition contained therein or that volume of juice beverage towhich the composition should be added, e.g., to yield a synergisticconcentration of phenolics and fumaric acid. For example, acontainer containing sufficient levels or amounts of phenolics andfumaric acid to provide synergistic concentrations of phenolics andfumaric acid in 1 L of juice beverage can be marked or labeled "1L."

EXAMPLES

[0112] The invention is further described in the followingexamples, which do not limit the scope of the invention describedin the claims.

Example 1

Extract Characterization (Small Scale Extraction)

[0113] Extract obtained using small scale (laboratory scale)processes was characterized to determine the types of phenolicspresent and the relative amounts of the different phenolics present(i.e., the relative amounts of one type of phenolic to another typeof phenolic). Non-phenolic material was also assessed.

[0114] Briefly, feedstock (i.e., counter current extraction (CCE)cranberry juice) was concentrated using reverse osmosis to increasethe Brix content from 1 Brix (the concentration obtained followingCCE) to 18 Brix and evaporation to further increase the Brixcontent from 18 Brix to 50 Brix. The 50 Brix CCE feedstock was thendiluted in water to 25 Brix. Diluted feedstock was contacted withAmberlite.TM. XAD-7HP resin. Flow through was collected asreduced-phenolics permeate. Resin was washed using 5% SDA ethanolwash solution. Flow through was collected as wash solution flowthrough. Bound phenolics were then eluted using 95% SDA ethanolelution solution. Elution solution flow through was collected andconcentrated using evaporation (heat and vacuum) to produce aliquid extract containing 25% solids. Extract was then spray driedusing a NIRO mobile minor spray drier Volumes used in the abovesmall scale process are shown in Table 2.

TABLE-US-00002 TABLE 2 Volumes Used in Single Cycle of Small ScaleProcess Small (Laboratory) Scale (mL unless shown) Column Volume 1L Volume of Resin in Column 943 Volume of feedstock (50 Brix) 2452Volume of Feedstock (25 Brix) 4904 Volume of feedstock notcollected 709 as feedstock flowthrough Volume of reduced- 4195phenolics feedstock flow through Volume of Wash Solution 2000Volume of Elution Solution 2452 Flow rate (all steps) 15.6mL/minute Re-equilibration volume 2678

[0115] Distinct extracts from multiple small scale single cycleruns were analyzed to determine the levels of phenolics, organicacids, and sugars. Data from each run was combined and meanscalculated. The results of these experiments are shown in Table3.

TABLE-US-00003 TABLE 3 Extract Characterization Av. (%) Min. (%)Max. (%) Std. Dev. % Solids 95.52 95.18 97.25 0.97 Quinic 0.07 0.030.12 0.05 Malic 0.17 0.11 0.25 0.07 Citric 0.26 0.11 0.38 0.11Total Organic Acids 0.50 0.39 0.76 0.17 PACs .sup.2 55.00 55.0055.00 0.00 PACs .sup.1 18.20 18.20 18.20 0.00 Phenolics (Folin)44.40 35.49 55.44 8.50 Anthocyanins .sup.1 6.86 4.94 9.78 2.24Dextrose 0.17 0.00 0.67 0.33 Fructose 0.00 0.00 0.00 0.00 Sucrose0.00 0.00 0.00 0.00 Total Sugars 0.17 0.00 0.67 0.33 Quercetin 0.540.17 1.22 0.59 Quercitrin 3.03 0.95 4.23 1.81 Hyperoside 2.24 0.934.71 2.14 Myricetin 0.24 0.09 0.52 0.24 Rutin 0.00 0.00 0.00 0.00Kaempferol 0.00 0.00 0.00 0.00 Isorhamnetin 0.00 0.00 0.00 0.00Isoquercitrin 0.00 0.00 0.00 0.00 Total Flavonols 6.05 5.17 7.060.95 Standardizing carrier 20.58 9.50 32.10 12.57 Flow Agent 0.800.80 0.80 0.00 Total Recovery 121.44 101.18 144.77 .sup.1 Assessedby HPLC .sup.2 Assessed by DMAC .sup.1 and 2 are normalizedvalues

[0116] As shown in Table 3, the primary component of extract isphenolics (see "Phenolics (Folin)" in Table 3) with PACs andanthocyanidins present at the highest levels. Levels of flavanolsquercetin, quercitrin, hyperoside, and myricetin were alsodetected.

[0117] In contrast, extract is substantially free of sugars (see"Total Sugars" in Table 3) and organic acids. Moreover, totalorganic acid content was less than 1% and total sugar content wasless than 1%. These observations indicate that the processdisclosed herein can be used to obtain extract from a CCE cranberryjuice feedstock that contains high amounts of PACs andanothocyanins and that is substantially free of organic acids andsugars. Furthermore, the low standard deviation values shownconfirm that between batch variation is low, or that the levelsshown in Table 3 can be consistently obtained.

[0118] As noted above, PACs include molecules of various chainlengths. Experiments were performed to determine the chain lengthsof PACs present in Extract. Experiments were also performed todetermine the chain lengths of PACs present in multiple CCEfeedstock samples used to obtain extract to allow comparison ofPACs profiles in feedstock and extract. The results are shown inTable 4.

TABLE-US-00004 TABLE 4 PACs Oligomeric Content of Extract (gPACOligomers/100 g PACs) Sample 1mers 2mers 3mers 4mers 5mers 6mers7mers 8mers 9mers 10mers >10mers ID (%) (%) (%) (%) (%) (%) (%)(%) (%) (%) (%) total % CCE 1 6.87 25.31 10.35 6.10 4.27 5.11 1.461.59 2.39 0.00 36.55 100.00 CCE 2 5.11 30.86 11.52 9.13 6.67 8.123.40 3.23 1.81 0.00 20.14 100.00 CCE 3 7.02 29.39 9.98 7.14 5.136.11 3.25 3.88 2.81 0.00 25.28 100.00 CCE 4 6.27 26.22 10.08 7.895.82 7.06 4.01 4.03 2.70 0.00 25.92 100.00 CCE 5 5.52 28.95 11.388.40 6.19 7.53 3.99 4.15 2.75 0.00 21.15 100.00 Mean 6.2 28.1510.66 7.73 5.62 6.79 3.22 3.4 2.49 0 25.8 Extract 6.35 28.23 10.718.08 5.56 7.06 4.00 4.21 1.93 0.00 23.87 100.00

[0119] As shown in Table 4, the PACs profile in extract issubstantially similar to the PACs profiles detected in multiple CCEfeedstock samples. This observation suggests that the processesdisclosed herein can be used to obtains PACs at levels present in aCCE feedstock. More specifically, 2mer and >10mer PACs are mostprevalent in both extract and CCE feedstock. Levels of other PACsoligomers are also preserved in extract as compared to CCEfeedstock. As revealed by these experiments, the ratios of PACsoligomers in both extract and CCE feedstock is about6(1mer):28(2mer):11(3mer):8(4mer):6(5mer):7(6mer):3(7mer):4(8mer):2(9mer)-:26(>10 mer). Therefore, the processes disclosed herein can beused to obtain an abstract containing anthocyanins and PACs,wherein PACs oligomers are present at levels present in thefeedstock.

[0120] Experiments were next performed to assess the levels of PACsto other phenolics, including anthocyanins and total phenolics, inextract and feedstock. These experiments were performed to allowdetermination of whether the ratios of PACs to other phenolicspresent in feedstock are preserved in extract. The results of theseexperiments are shown in Tables 5 and 6.

TABLE-US-00005 TABLE 5 Ratios of PACs and Phenolics in CCEFeedstock and Extract Sample ID 1 2 3 4 Sample Type CCE Extract CCEExtract CCE Extract CCE Extract Total PACs (%)- dry weight 2.6 97.91.6 77.6 1.8 81.1 1.9 85.2 Total Phenolics (%)- dry weight 2.2 62.51.6 52.6 1.8 56.1 1.7 58.5 PACs:total Phenolics 1.3 1.6 1.2 1.5 1.11.4 1.3 1.4 PACs:total anothocyanin (TAcy) 5.7 14.0 4.9 11.2 5.313.2 6.7 16.3 Total sugars- dry weight 64.1 0.0 70.3 0.0 69.2 0.066.5 0.0 Total organic acids- dry weight 40.5 0.3 48.3 0.3 44.5 0.342.9 0.3 PACs (ppm)/Quercetin (ppm): 279.7 207.2 231.4 191.6 215.7194.1 43.5 51.1 PACs (ppm)/QuercGalac (ppm): 39.1 22.3 21.9 13.018.8 15.3 28.3 24.4 PACs (ppm)/Quercitrin (ppm): 152.8 99.2 92.375.3 99.6 93.5 96.9 97.5 PACs (ppm)/Myricetin (ppm): 187.6 170.3221.2 183.1 144.7 148.7 36.3 38.8 PACs (ppm)/QuercAraban (ppm):108.3 76.8 85.7 74.8 64.1 64.1 74.7 75.1 Total Solids (%) 55.4 95.758.4 96.0 54.7 97.1 56.3 97.9

TABLE-US-00006 TABLE 6 Average Ratios of PACs and Phenolics in CCEFeedstock and Extract Average ratios CCE Extract PACs:Phenolics 1.21.5 PACs:total anthocyanins (TAcy) 5.65 13.7 PACs:Quercetin 192.6161 PACs:Quercetin galactoside 27.0 18.8 (QuercGalac)PACs:Quercitrin 110.4 91.4 PACs:Myricetin 147.5 135.2PACs:Quercetin arabanoside 83.2 72.7 (QuercAraban)

[0121] As shown in Table 5, the levels of total PACs and totalphenolics are increased in extract as compared to CCE feedstock.This observation confirms that the processes disclosed herein canbe used to concentrate phenolics. Table 5 also presents dataconfirming that the levels of sugars and organic acids are reducedin extract as compared to CCE feedstock. This observation validatesthe data shown in Table 3, that extract contains reduced amounts ofsugars and organic acids, and confirms that the processes disclosedherein can be used to separate phenolic compounds from sugars andacids and obtain a phenolics extract.

[0122] As shown in Tables 5 and 6, the ratio of PACs to totalphenolics present in feedstock is preserved in extract. Similarly,the ratio of PACs to quercetin, quercgalac, quercitrin, myricetin,and quercaraban present in feedstock are also preserved in extract.In contrast, although anthocyanins are present in extract, theratio of PACs to anthocyanins present in feedstock is not preservedin extract (see PACs:TAcy).

[0123] Therefore, the data presented herein demonstrate that theprocesses disclosed herein can be used to obtain an extractcontaining anthocyanins and PACs, wherein PACs oligomers arepresent at levels present in the feedstock, and wherein the ratioof PACs to total phenolics and PACs to PACs to quercetin,quercgalac, quercitrin, myricetin, and quercaraban in extract arethe same as the ratios for the same phenolics in feedstock.

Example 2

Process Optimization

[0124] Certain steps or materials used in the process described inExample 1 were substituted in an attempt to further optimize theprocess. Briefly, the process described in Example 1 was repeatedwith the following modifications: (1) the wash step was performedusing water instead of using 5% ethanol; (2) the wash step andelution step were performed using acetone; (3) the Amberlite.TM.XAD-7HP resin was substituted for FPX-66 resin and the wash stepwas performed using water instead of using 5% ethanol; (5) theAmberlite.TM. XAD-7HP resin was substituted for FPX-66 resin; and(6) the Amberlite.TM. XAD-7HP resin was substituted for FPX-66resin and the wash step and elution step were performed usingacetone. The yield of phenolics extracted, extract purity, andextract stability were then assessed. The results are shown inTable 7.

TABLE-US-00007 TABLE 7 Process Optimization Modification # (seetext in Ex. 3) 1 2 3 4 5 6 Resin XAD- XAD- XAD- FPX-66 FPX-66FPX-66 7HP 7HP 7HP Wash Water 5% EtOH 5% Water 5% EtOH 5% acetoneacetone Elution 90% 90% 90% 90% 90% 90% EtOH EtOH acetone EtOH EtOHacetone Yield Data PAC Yield (% 92.59 97.57 101.44 86.32 88.4396.76 of feed-Av) Phenolic 77.98 77.78 79.35 67.45 80.37 84.55Yield (folin- Av) Purity Data PACs (% dwb- 51.49 64.40 71.02 41.6642.96 44.76 Av) Phenolics 48.68 51.34 51.88 36.55 38.83 41.32(Folin-Av) Phenolics 47.55 61.86 67.70 32.74 38.43 39.72(Folin-Bruns) Phenolics 93.97 155.63 108.32 81.56 88.12 87.45(HPLC-OS) Acys (ppm- 65056 83085 46977 58855 66269 50301 OS)Quercetin 4120 Not done Not done 3180 3300 3140 (ppm-OS) ORAC 81319701 10379 6756 7410 6633 (Bruns) Beverage Stability Bev Haze 2.871.52 1.93 2.44 2.14 2.68 Slope v. time Bev Color -3.27 -4.66 -3.35-3.50 -1.92 -2.65 Slope v. time

[0125] As shown in Table 6, both XAD-7HP and FPX-66 resin yieldedhigh levels of PACs and total phenolics. Overall, however, XAD-7HPprovided higher results than FPX-66, including haze stability. Incontrast, FPX-66 provided slightly better results for colorstability. Additionally, all wash and elution conditions testedprovided good results. Acetone was observed to be the best wash andelution solution for PAC and phenolics recovery and purity.

[0126] These results suggest that while the process described inExample 1 is effective for obtaining extract containinganthocyanins and PACs, wherein PACs oligomers are present at levelspresent in the feedstock, and wherein the ratio of PACs to totalphenolics and PACs to PACs to quercetin, quercgalac, quercitrin,myricetin, and quercaraban in extract are the same as the ratiosfor the same phenolics in feedstock; the process may be modifiedusing the changes shown here without compromising efficiency.

Example 3

Comparison of Small Scale and Large Scale Extractions

[0127] Experiments were performed to confirm that the processdescribed in Example 1 can be performed on a large scale (i.e., acommercial scale) and that extracts obtained using large scaleprocess represent those extracts described in Example 1.

[0128] The volumes of materials used in a single cycle of a largescale process are shown in Table 7.

TABLE-US-00008 TABLE 7 Volumes Used in Single Cycle of Small ScaleProcess Large (Commercial) Scale (gallons unless shown) ColumnVolume 141 Volume of Resin in Column 133 Volume of feedstock (50Brix) 346 Volume of Feedstock (25 Brix) 692 Volume of feedstock notcollected 100 as feedstock flowthrough Volume of reduced- 592phenolics feedstock flow through Volume of Wash Solution 282 Volumeof Elution Solution 346 Flow rate (all steps) 2.2 gallons/minuteRe-equilibration volume 378

[0129] Extracts obtained using a small scale process were thencompared to extract obtained using large scale process.

[0130] Briefly, a small scale process was performed using cranberryCCE as described in Example 1 using the volumes shown in Table 2and a large scale process was performed using the process describedin Example 1 with the volumes shown in Table 7.

[0131] A schematic representation of the above process is providedin FIG. 1. FIG. 1 includes points at which certain samples weretaken. The required characteristics of each of the samples shown inFIG. 1 are detailed in Table 9.

[0132] Extracts resulting from small scale and large scaleextractions were then analyzed and compared. The results of thesestudies are shown in Table 8.

TABLE-US-00009 TABLE 8 Comparison of Extracts Obtained Using LargeScale and Small Scale Extraction Sample ID Large Small Small SmallSmall Small Small Small Small Scale Scale 1 Scale 2 Scale 3 Scale 4Scale 5 Scale 6 Scale 7 Scale 8 Solids (%) 97.25 96.15 97.44 97.296.45 95.72 96.02 97.13 97.85 Total organic acids 0.85 0.42 0.800.61 0.62 0.26 0.31 0.27 0.31 (%) Total Sugars (%) 0.75 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 PACs (DMAC) 61.61 91.26 67.98 82.8980.44 97.86 77.60 81.13 85.17 Anthocyanins (HPLC) 10.96 6.59 7.127.94 7.18 6.71 6.66 5.99 5.12 Total Phenolics (Folin) 51.39 54.7947.58 51.98 57.54 58.73 55.02 5.99 5.12

[0133] As shown in Table 8, the results reported in Example 2 andTable 4, obtained using small scale processes were reproducible inmultiple small scale processes and an commercial scaleextraction.

[0134] These results suggest that extract containing anthocyaninsand PACs, wherein PACs oligomers are present at levels present inthe feedstock, and wherein the ratio of PACs to total phenolics andPACs to PACs to quercetin, quercgalac, quercitrin, myricetin, andquercaraban in extract are substantially the same as the ratios forthe same phenolics in feedstock can be obtained using large scaleprocesses.

TABLE-US-00010 TABLE 9 Sample Characteristics of Extraction ProcessSample ID Description Analysis Specification A CCE 1 Haze Totalplate <1000/g count (TPC) Yeast <100/g Mould <100/g BReverse Osmosis TPC <1000/g water Yeast <100/g mould<100/g C CCE 2 % Solids Read and record (RR)(%) .degree.Brix RR(%) Specific gravity RR (g/mL) D Liquid feedstock % Solids 25 .+-.1% for feeding to PACs .gtoreq.1.5% dwb resin column Phenolics RR(% dwb) % titratable RR (%) acidity Haze RR (NTU) Anthocyanins RR(ppm) TPC <1000/g Yeast <100/g Mould <100/g E Washsolution % Etoh 5 .+-. 1% (v/v) F Phenolics free .degree.Brix.gtoreq.2 permeate G Elution solution % water .ltoreq.20&% byKarl Fisher H Feedstock flow % solids RR (%) through PACs RR (%dwb) TPC <1000/g Yeast <100/g Mould <100/g I Evaporated %Solids 25 .+-. 2% extract 1 % Etoh RR (%) J Evaporated % Solids 25.+-. 2% extract 2 % Etoh RR (%) K Liquid extract % Solids 25 .+-.2% Etoh <90 ppm PACs .gtoreq.56% dwb Phenolics R (% dwb)Anthocyanins RR (% dwb) Appearance Deep red/purple liquid AromaCranberry aroma TPC <1000/g Yeast <100/g Mould <100/g

Example 4

Use of Extracts in Food Preservation

[0135] Experiments were performed to determine whether thecombination of extract obtained using the processes disclosedherein and fumaric acid can be used to reduce spoilage.

[0136] Briefly, various juices were inoculated with approximately100 spores of a mixture of the ACB strains listed in Table 10.Spores were then harvested from PDA (Potato Dextrose Agar) platesand heat inactivated.

TABLE-US-00011 TABLE 10 ACB Strains Used ID Source (origin) 230Hassia apple juice 231 NFPA (National Food Processors Association)233 Hassia apple juice 245 Craving-less sugar Tropical 247 Peachjuice 250 Pink grapefruit 100% juice

[0137] Extract suitable for use in the methods disclosed in thisExample can be obtained using any convenient technique and from anysuitable feedstock. In this case, extract was obtained fromcranberries, which are a particularly rich source of PACs, usingthe processes disclosed herein. Briefly, cranberry juice at 25 Brixwas loaded on a Rohm & Haas XAD-7HP resin column. The columnwas washed with a solution containing 5% ethanol/95% water to elutesugars, acids and other unwanted components. Extract was eluted bywashing the column using a solution containing 90% ethanol and 10%water. Eluate was concentrated by evaporation to 25% solids, thenspray dried into a powder. The PAC level in the powder ranged from55 to 85% by weight.

[0138] PAC concentration was assessed for the experiments in thisExample as follows. Briefly, a sample to be analyzed was applied toa Sephadex LH-20 column. The column was then washed with distilledwater and then 25% ethanol/75% water. These washes elute sugars,organic acids, anthocyanins and monomeric phenolic compounds. ThePACs were then eluted by washing the column with 70% acetone in 30%water. The eluate is allowed to react with a solution of 0.1%dimethylaminocinnamaldehyde (DMAC) in 30% hydrochlonic acid in 70%methanol. DMAC can act as an electrophile condensing with aromaticrings in an acidic media and is highly specific for flavanols. Whena prepared DMAC reagent is added to a solution containing PACs analdehyde condensation reaction occurs with the terminal monomer ofa polymeric proanthocyanidins at the eight carbon position of theflavanoid A-ring. The resulting colored adducts have a maximumabsorption of 640 nm. A standard curve of absorbance at 640 nm wasdeveloped using solutions of known contents of purified cranberryPACs.

[0139] Sample juices were supplemented with either cranberry juice,extract, fumaric acid, or both, in the presence and absence ofabout 100 ACB spores/ml, as shown in Table 11. Sample juices werethen stored at 43.degree. C. Aliquots of juice were removedperiodically and tested for ACB by subculturing onto PDA plates.Sensory testing was also performed by a panel of 2-4 people trainedto detect the presence of guaiacol. The tests were performed atseveral intervals during a 1 month period. Negative controls(non-inoculated juice) were included in the tests and tested at thesame time as the inoculated product.

TABLE-US-00012 TABLE 11 Food Preservation Results % Extract Fumaric% Mg acid Cranberry TA Micro Sample juice PAC/8 oz (wt/wt) juice pHBrix (%) (Growth) Sensory CranPomBlue 0 0 7.33 3.56 12.52 0.45 + +CranPomBlue 40 0 7.33 3.5 10.54 0.48 - - Tropical 0 0.14 0 3.4313.35 0.46 + + Citrus Tropical 40 0.14 0 3.4 13.4 0.46 - - CitrusTropical 30 0.14 0 3.41 13.13 0.46 - - Citrus Tropical 20 0.14 03.41 13.18 0.46 - - Citrus Tropical 10 0.14 0 3.42 13.21 0.46 - -Citrus Extract water 40 0 0 2.67 2.74 0.17 - - beverage Tropicalwith 5 0.14 + Ruby Red Tropical with 7.5 0.14 - Ruby Red Tropicalwith 9.5 0.14 - Ruby Red Tropical with 10 0.14 - Ruby Red Tropicalwith 10.5 0.14 - Ruby Red Microbiological results [+] = microbialgrowth; [-] no growth detected. Sensory results [+] = off-flavors,typical of the presence of guaiacol; [-] no off-flavors.

[0140] As shown in Table 11, the combination of extract and fumaricacid to juices prevented ACB growth and guaiacol production.Similar results were observed with apple juice and orangejuice.

[0141] These observations support that the combination of extractand fumaric acid results in a synergistic effect at someconcentrations. Specifically, the reduction in contaminationpromoted by the combination of extract and fumaric acid is greaterthan the additive effect of the components. This synergism wasfurther evaluated in the following example.

Example 5

Minimum Inhibitory Concentration of Compositions Comprising Extractand Fumaric Acid

[0142] The ability of the compositions described in Example 4(i.e., compositions comprising extract and fumaric acid) to inhibitACB growth were evaluated over a range of different concentrationsto establish the minimum inhibitory concentration (MIC) of thecomposition.

[0143] Extract was obtained using the processes described hereinand contained 55% PACs as determined using DMAC. Fumaric acid wasobtained from a commercial vendor (Tate and Lyle, IL, lot numberFT7C2301B4).

[0144] Apple juice was inoculated with spores from a mixture of ACBstrains (eight strains of A. acidoterrestris and one ACB strainobtained from apple juice). These spores were suspended inphosphate buffered saline (PBS) and heat shocked at 76.degree. C.for 10 minutes. Spore concentration was determined using ahemocytometer under phase contrast microscopy. Juices wereinoculated with 1000 spores/mL. Inoculated samples were incubatedfor 48 hours and were then cultured on acidified potato dextroseagar (PDA+TA) at 43.degree. C. for 72 hours. Colony forming unitswere then counted. The ratios of extract to fumaric acid used inthese experiments are shown in Table 12.

TABLE-US-00013 TABLE 12 Ratio of Extract to Fumaric Acid Extract(mg PAC/8 oz) Fumaric acid (%) 0 1.0 2.0 3.0 4.0 0 0/0 1/0 2/0 3/04/0 0.05 0/0.05 1/0.05 2/0.05 3/0.05 4/0.05 0.10 0/0.10 1/01.02/0.10 3/0.10 4/0.10 0.15 0/0.15 1/01.5 2/0.15 3/0.15 4/0.15 0.200/0.20 1/0.20 2/0.20 3/0.20 4/0.20

[0145] Data was plotted in the format of a isobologram. Such graphsare useful in assessing synergy. Specifically, two compounds thatresult in an additive effect yield a straight line. Deviation tothe left of this line indicates that the combination of twocompounds is synergistic, while deviation to the right of the lineindicates that the combination of the two compounds is antagonistic(Vigil et. al., Methods for activity assay and evaluation ofresults, Antimicrobials in Food, 3.sup.rd Edition, CRC Press,Edited by Davidson, Sofos, and Branen, 2005).

[0146] As shown in FIG. 2, compositions comprising extract andfumaric acid inhibited ACB growth at a range of concentrations.Specifically, ACB growth was inhibited at concentrations of 0.1%fumaric acid and 0.01 mg PAC/8 oz. Furthermore, FIG. 2 shows aclear synergistic effect at concentrations of between 0.15%-0.1%fumaric acid and 0.01-1.99 mg PAC/8 oz. As noted above, the resultsshown in FIG. 2 represent experiments performed using applejuice.

Example 6

Log Reduction Experiments

[0147] Inhibition of ACB growth by compositions comprising extractand fumaric acid was confnined in 100% apple juice (OceanSpray--see FIG. 3) and 100% orange juice (Ocean Spray--see FIG. 4).Compositions tested for each juice type are shown in Table 13.

TABLE-US-00014 TABLE 13 Ratio of Extract to Fumaric Acid Extract(mg PAC/8 oz) Apple juice Orange juice Fumaric acid (%) 0.0 2.5 0.012.0 0.0 0.0/0.0 2.5/0.0 0.0/0.0 12.0/0.0 0.07 -- -- 0.0/0.0712/0.07 0.14 0.0/0.14 2.5/0.14 -- --

[0148] Apple juice was inoculated with nine strains of ACB (eightstrains of A. acidoterrestris and one ACB strain obtained fromapple juice) and orange juice was inoculated with four strains ofA. acidoterrestris. Spores were heat shocked and enumerated asdescribed in Example 5. Juice samples were then inoculated with 500spores and incubated at 43.degree. C. for 48 hours (apple) or 72hours (orange) before being plated on PDA+TA. Plates were incubatedat 43.degree. C. and CFU for 72 hours. CFU were quantified asdescribed in Example 5. CFU counts were log transformed andanalyzed by to analysis of variance (ANOVA) followed by Fisher'sLSD multiple comparison test. Log reductions were calculated forfumaric acid and extract alone and in combination. Results areshown in FIGS. 3 and 4.

[0149] As shown in FIGS. 3 and 4, the magnitude of ACB inhibitionby extract and fumaric acid was quantified in apple juice andorange juice. Both extract and fumaric acid significantly inhibitedACB growth in apple juice (FIG. 3A). The combination of extract andfumaric acid, however resulted in a synergistic reduction in ACBgrowth (ANOVA: p<0.001). There was a six log reduction in ACBgrowth in apple juice with the combined application of extract andfumaric acid (FIG. 3B). These data support that the combination ofextract and fumaric acid synergistically reduced the growth of ACBin apple juice, resulting in a >6 log reduction in the number ofCFU.

[0150] As shown in FIG. 4, in orange juice, ACB was inhibited byfumaric acid alone. Furthermore, the combination of extract andfumaric acid synergistically inhibited ACB in orange juice (ANOVA:p<0.001). There was no effect of extract alone (FIG. 4A). Thecombination of extract and fumaric acid resulted in >2 logreduction in ACB growth (FIG. 4B). These data support that thecombination of extract and fumaric acid synergistically reduced thegrowth of ACB in orange juice, resulting in a >2 log reductionin the number of CFU.

[0151] Accordingly, the data presented herein support that thecombination of extract and fumaric acid can used effectively toreduce spoilage of fruit juices by ACB.

Other Embodiments

[0152] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit thescope of the invention, which is defined by the scope of theappended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

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