WO2019155216A1 - Anthocyanins and uses thereof - Google Patents

Anthocyanins and uses thereof Download PDF

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Publication number
WO2019155216A1
WO2019155216A1 PCT/GB2019/050334 GB2019050334W WO2019155216A1 WO 2019155216 A1 WO2019155216 A1 WO 2019155216A1 GB 2019050334 W GB2019050334 W GB 2019050334W WO 2019155216 A1 WO2019155216 A1 WO 2019155216A1
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anthocyanin
liquid crystalline
ant
hydrogel
lycopene
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PCT/GB2019/050334
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French (fr)
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Ivan Petyaev
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Ip Science Limited
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/02—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation containing fruit or vegetable juices
    • A23L2/04—Extraction of juices
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/007—Other edible oils or fats, e.g. shortenings, cooking oils characterised by ingredients other than fatty acid triglycerides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/02—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation containing fruit or vegetable juices
    • A23L2/08—Concentrating or drying of juices
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/03—Organic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/10—Foods or foodstuffs containing additives; Preparation or treatment thereof containing emulsifiers
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins
    • A23L33/155—Vitamins A or D
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/40—Colouring or decolouring of foods
    • A23L5/42—Addition of dyes or pigments, e.g. in combination with optical brighteners
    • A23L5/43—Addition of dyes or pigments, e.g. in combination with optical brighteners using naturally occurring organic dyes or pigments, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/40—Colouring or decolouring of foods
    • A23L5/42—Addition of dyes or pigments, e.g. in combination with optical brighteners
    • A23L5/43—Addition of dyes or pigments, e.g. in combination with optical brighteners using naturally occurring organic dyes or pigments, their artificial duplicates or their derivatives
    • A23L5/44—Addition of dyes or pigments, e.g. in combination with optical brighteners using naturally occurring organic dyes or pigments, their artificial duplicates or their derivatives using carotenoids or xanthophylls
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/20—Ingredients acting on or related to the structure
    • A23V2200/222—Emulsifier
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/20—Ingredients acting on or related to the structure
    • A23V2200/238—Solubility improving agent
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/20—Ingredients acting on or related to the structure
    • A23V2200/244—Viscosity modification agent
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/21—Plant extracts
    • A23V2250/2104—Anthocyanins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/21—Plant extracts
    • A23V2250/211—Carotene, carotenoids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/21—Plant extracts
    • A23V2250/213—Lycopene

Definitions

  • the invention is aimed at addressing some of these shortcomings.
  • ANT anthocyanin-rich plant extracts or other anthocyanin-rich products
  • ANT anthocyanin-rich products
  • amphiphilic and hydrophobic molecules These types of complexes allow ANT to be used as emulsifiers or surfactants.
  • the inventor has shown that ANT can change the physical properties of hydrophobic and amphiphile bioactive molecules; examples are carotenoids and frans-resveratrol.
  • ANT facilitates blending and emulsifying of hydrophobic molecules and oily or fat-based products to incorporate them into aqueous solution, suspension or dispersions. This is achieved by using a matrix with ANT.
  • This protective property of ANT can be utilised in the preservation of other bioactive agents when they are used as beverage or food matrixes with an acidic environment. This includes for example some soft drinks or juices, as well as products of bacterial and / or fungal fermentation like yogurt, chocolate, cheese, etc.
  • ANT can be utilised to protect bioactive molecules or agents in orally ingestible products during their passage through an acidic stomach environment. As a result of this protection, more bioactives can pass through the stomach to reach the sites of their absorption in unmodified form. This results in an improved absorption rate.
  • the invention relates to ANT liquid crystalline and / or hydrogel, ANT-LCH, which can be used as a platform itself or in combination with a carotenoid, to emulsify or disrupt hydrophobic molecules or hydrophobic clusters of amphiphilic molecules, crystals, and other non-water soluble structures or products, to create stable solutions, suspensions, colloids or dispersions in aqueous media.
  • ANT can also be used to disrupt lipid folding of different fats or oils, or fat-, or oil-rich products in order to reduce their viscosity and spreadability, reduce their melting, boiling and freezing points. For example, reduction of cooking time (without an increase in temperature) can help preserve temperature sensitive vitamins and other nutrients in cooked food. It can also positively affect the taste of food.
  • disruption of the lipid folding by ANT can increase the size of the lipid droplets or globules and therefore reduce the rate of their digestion in the gastrointestinal tract. These properties could be used to create improved food or beverage products, which helps to control lipid metabolism and weight management.
  • Fig. 1 Lutein & meso-zeaxanthin - orange, turquoise - blend of blueberry anthocyanins and lutein & meso-zeaxanthin’ spectrum with long-wave shift in absorption of pick 9- 6. pH 2.5 - fumaric acid / H 2 0.
  • Fig. 14 trans- Resveratrol - blue, dark-red - malvidine, purple - blend of pelargonidin and frans-resveratrol’ spectrum with long-wave shift in absorption of pick 5- 6. H 7.4 - ethanol / H 2 0.
  • Fig. 16 trans- Resveratrol - blue, black - delphinidin, red - blend of delphinidin and frans-resveratrol’ spectrum with long-wave shift in absorption of pick 3- 3. pH 2.5 - fumaric acid / H20.
  • FIG. 18 A-B. Carotenoid emulsifying properties of ANT of red wine. A) ANT with Red; B) ANT with white. Fig. 19 A-G Carotenoid emulsifying effect of cranberry ANT-LCH
  • F-G carotenoids and cranberry ANT-LCH dispersions in H 2 0; F) ANT-LCG + lycopene; G) ANT-LCG + astaxanthin in H 2 0.
  • Figure 20 A) Optical microscopy of Blueberry ANT-LCH micelles with Astaxanthin and B) the solubility of a blend Astaxanthin & Blueberry ANT-LCH in H 2 0.
  • Figure 21 A-E demonstrates a similar facilitating dispersion effect of oil formulations of carotenoids, lycopene and astaxanthin, by cherry ANT-LCH.
  • Figure 22 A-M. Dairy Butter emulsifying properties of carotenoids.
  • FIG 23 A-D. Effect of ANT-LCH and lutein on dairy butter fat droplets.
  • Figure 24 A-B A) Effect of anthocyanins and lutein on the viscosity and the meting time, the vertical axis, of dairy butter.
  • Figure 25 A-B.
  • FIG. 26 A-F. Cocoa Butter emulsifying properties of complexes of aronia ANT-LCH with carotenoids.
  • a - C ANT-LCH with astaxanthin in H 2 0.
  • D - F ANT-LCH with lycopene in H 2 0;
  • Figure 27 A-B A) Effect of anthocyanins and lutein on the viscosity and the meting time, the vertical axis, of cocoa butter.
  • Figure 28 A-L Disruptive effect of carotenoids on lipid folding of olive oil.
  • a - C astaxanthin in H 2 0
  • Figure 29 A-B A) control olive oil; B) Aronia ANT-LCH promotes fusing and enlargement of lipid droplets of olive oil.
  • Figure 30 A-B).
  • B) Aronia ANT-LCH promotes fusing and enlargement of lipid droplets of hazelnut oil.
  • Figure 31 A-B).
  • B) Aronia ANT-LCH promotes fusing and enlargement of lipid droplets of DHA.
  • FIG 32 A-K. DHA 40% in sunflower oil - emulsifying properties of aronia ANT-LCH and its complexes with carotenoids.
  • A-D DHA + ANT-LCH & carotenoids; A) Astaxanthin W; B) Astaxanthin 3.8; C) Lycopene W; D) Lycopene 3.8.
  • F - G interaction of carotenoids with DHA; F) DHA + lycopene; G) DHA + astaxanthin; H-K) interaction of carotenoids with DHA in aqueous solution pH 3.8; H) DHA + lycopene at pH 3.8; I) DHA + lycopene in water; J) DHA + astaxanthin at pH 3.8; K) DHA + astaxanthin in water.
  • Figure 33 ANT-LCH - astaxanthin particles survived in the acid environment of the dark chocolate matrix.
  • Figure 35 Comparison of epicatechin metabolites pharmacokinetics after ingestion of two aronia extracts in the form of the ANT-LCG and spray-dried powder.
  • the invention relates to a liquid crystalline hydrogel comprising an anthocyanin. This can have an appearance and behaviour of a gel or a paste, semi-solid and spreadable mass.
  • Hydrogels are materials that are composed of a network of polymer chains that are hydrophilic and can hold a certain levels of bound water. In one embodiment, the hydrogel does not comprise free water. In one embodiment, the hydrogel comprises bound water. In one embodiment, the amount of bound water is from 1 % to 90% w/w, for example 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% w/w.
  • Anthocyanins are multi-glycosylated anthocyanidins.
  • Anthocyanins are pigments, which naturally appear red, purple, or blue. Frequently, the color of anthocyanins is dependent on pH.
  • Anthocyanins are naturally found in flowers, where they provide bright-red and -purple colours.
  • Anthocyanins are also found in vegetables and fruits.
  • Anthocyanins are useful as dyes or colouring agents, and furthermore, anthocyanins are known to have antioxidant properties.
  • anthocyanin as used herein is intended to include both glycosylated anthocyanins (anthocyanosides) as well as the aglycon of the anthocyanoside (anthocyanidin). It also includes synthetic anthocyanins or those isolated from natural sources, i.e. purified anthocyanins. In one embodiment, the anthocyanin is a plant extract or comprised in a plant extract.
  • extract is intended to mean anthocyanin materials obtained from plant sources, such as leaves, twigs, bark, roots, stem, seeds, flowers, berries, fruit, for example, by routine isolation methods from suitable plants sources noted, but not limited to, those described herein. There are various methods for the extraction of anthocyanins known to those of skill in the art.
  • anthocyanin-containing plants include, but are not limited to, fruits, vegetables, flowers and other plants selected from the group consisting of acai, Acer macrophyllum, Acer platanoides, acerola, Ajuga reptans, apple, apricot, aronia, Artict bramble, avocado, banana, baobab, barberry, barley, Begonia semperfiorens, Beilis perennis, Bletilla striata, bilberry, black beans, black soybeans, black, blue and purple potatoes, blackberry, blueberry, bog whortleberry, boysenberry, buckwheat, cacao, Camellia sinensis, canarygrass, Caucasian blueberry, Chimonanthus praecox, celery, Cerasus avium, cherry, cherry Morello, cherry laurel, chicory, chive, chokeberry, Coffee beans, Coffee cherries, Cornelian cherry, cornflower, Laceaster, cowberry, cranberry, crow
  • anthocyanin extracts of particular interest include bilberry extract, blackcurrant extract, cranberry extract, black soybean extract, cowberry extract, blueberry extract and mixtures of two or more thereof.
  • the extract is concentrated by various methods to provide a solution enriched in anthocyanins.
  • ultrafiltration can be used to remove unwanted components by molecular weight cut offs.
  • the retentate from the filtration can be stored as a liquid or, for example, can then be further concentrated into a powder by spray drying, freeze drying, flash drying, fluidized bed drying, ring drying, tray drying, vacuum drying, radio frequency drying or microwave drying.
  • Anthocyanin extracts can be further purified by one or more methods known in the art, such as chromatography, gel chromatography, high performance liquid chromatography, crystallization, affinity chromatography, partition chromatography and the like. Identification of the particular anthocyanin(s) can be accomplished by methods know to those skilled in the art and include ⁇ 1 >H NMR, chemical degradation, chromatography and spectroscopy, especially homo- and heteronuclear two-dimensional NMR techniques for the characterization of the isolated anthocyanin compounds.
  • purified or“isolated” is used in reference to the purification and/or isolation of one or more anthocyanins from an anthocyanin extract as described above. Using conventional methods known in the art, various components of the anthocyanin extract can be separated into purified materials. In one aspect of the invention, the anthocyanin(s) of the extract are substantially purified and isolated by techniques known in the art.
  • the purity of the purified compounds is generally at least about 90%, preferably at least about 95%, and most preferably at least about 99% and even more preferably at least about 99.9% (e.g. about 100%) by weight.
  • the anthocyanin extract contains one or more anthocyanins and/or anthocyanidins e.g. selected from the group consisting of peonidin, cyanidin, pelargonidin, delphinldin, petunidin, malvidin, apigenindin, auratinidin, capensinidin, europinidin, hirsutidin, 6- hydroxycyanidin, luteolinidin, 5-methylcyanidin, pulchellidin, rosinidin, tricetnidin, derivatives and mixtures thereof.
  • anthocyanins and/or anthocyanidins e.g. selected from the group consisting of peonidin, cyanidin, pelargonidin, delphinldin, petunidin, malvidin, apigenindin, auratinidin, capensinidin, europinidin, hirsutidin, 6- hydroxycyanidin, luteolinidin,
  • the anthocyanins and anthocyanidins are selected from the group consisting of cyanidin, peonidin, malvidin, petunidin, delphinidin, their glycoside derivatives, and mixtures thereof.
  • the extract contains at least one cyanidin-based anthocyanin.
  • anthocyanin as used herein is intended to refer not only to monomeric anthocyanins, but also refers to dimeric and polymeric (i.e. containing from 3 to 20 anthocyanidin monomer residues) forms of anthocyanins and to leucoanthocyanidins (also known as flavan-3,4-diols).
  • the anthocyanins can comprise substitutions (e.g. alkyl, alkoxy groups etc.) and in particular can be O-glycosylated, as described above.
  • the anthocyanin as used herein can be a single anthocyanin or comprise a mixture of anthocyanins.
  • the anthocyanin is selected from the group consisting of: aurantidin, malvidin, cyanidin, delphinidin, europindin, paeonidin, pelargonidin, peonidin and petunidin, and glycosides thereof.
  • a typical example is malvin (malvidin diglucoside) chloride, which is commercially available in a purified form.
  • the anthocyanin can be obtained by extracting anthocyanin containing plants such as aronia, grape, black carrot, red cabbage, aubergine, baobab, barberry, barley, bilberry, cranberry, cherry, blackberry, blackcurrant, blueberries, buckwheat, hemp, flaxseed, redcurrant, raspberry, and the like as described above.
  • the anthocyanin is selected from cyanidin, delphinidin, malvidine, peonidin, petunidin, europinidin, aurantinidin, rosinidin.
  • the hydrogel may further comprise a carotenoid compound.
  • Carotenoid compounds are tetraterpenoids which contain long polyene chains. Carotenoid compounds include xanthophylls such as lutein and zeaxanthin, and carotenes, such as beta-carotene, alpha-carotene, zeto-carotene, and lycopene compounds.
  • the carotenoid is a xanthophyll.
  • the xanthophyll is selected from the group consisting of a-cryptoxantin, b-cryptoxantin, adonirubin, adonixanthin, alloxanthin, amarouciaxanthin A, antheraxanthin, astaxanthin, auroxanthin, caloxanthin, cantaxanthin, capsanthin, capsanthin-5-6-epoxide, capsorubin, crocoxanthin, diadinoxanthin, diatoxanthin, echinenone, fucoxanthin, fucoxanthinol, iso-fucoxanthin, iso-fucoxanthinol, lutein, luteoxanthin, mutatoxanthin, neoxanthin, nostoxanthin, violaxanthin, zeaxanthin and combinations thereof.
  • the carotenoid is a carotene.
  • the carotene is selected from the group consisting of a-carotene, b-carotene, y-carotene, d-carotene, e-carotene, z-carotene, lycopene, neurosporene, phytoene, phytofluene and/or combinations thereof.
  • the carotenes and xantophylles described above refer to the all-trans forms thereof.
  • the xantophylles and carotenes for use in the present invention include derivatives containing one or more cis double bond.
  • the carotenoid compound is a lycopene compound.
  • Lycopene compounds may include lycopene, l-HO-3 ', 4'- didehydrolycopene, 3, 1 ' - (HO) 2-gamma-carotene, 1 , 1 ' - (HO) 2-3 , 4, 3', 4 ' - tetradehydrolycopene, 1 , 1 '- (HO) 2-3, 4-didehydrolycopene.
  • the carotenoid compound is a lycopene compound such as lycopene.
  • Lycopene is an open-chain unsaturated C40 carotenoid of structure I (Chemical Abstracts Service Registry Number 502-65-8, C 40 H 56 ).
  • Lycopene occurs naturally in plants such as tomatoes, guava rosehip, watermelon and pink grapefruit and any such sources of lycopene may be, for instance, employed.
  • Lycopene for use as described herein may comprise one or more different isomers.
  • lycopene may include cis- lycopene isomers, trans-lycopene isomers and mixtures of the cis- and trans-isomers.
  • Lycopene may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% , at least 80%, at least 90%, or at least 95% (Z)- isomers, (all-E) -isomers , or cis-isomers, such as 5-cis- or 9- cis- or 13-cis-isomers, which have improved bioavailability relative to trans isomers.
  • Trans isomers may isomerise into cis forms in vivo, or during storage and processing.
  • Carotenoid compounds such as lycopene, for use as describe herein may be natural i.e. obtained from a natural source, for example, extracted from a carotenoid-rich fruit, vegetable or other plant, such as a tomato or melon, or from fungi, algae or bacteria.
  • the carotenoid compound may be, or comprise, oleoresin, particularly tomato oleoresin.
  • a range of methods for extracting, concentrating and/or purifying carotenoids from plants are known in the art.
  • solvent extraction using ethanol, DMSO, ethyl acetate, hexane, acetone, soya or other vegetable oil, or non-vegetable oils may be employed.
  • Carotenoid compounds such as lycopene, for use as described herein may be synthetic i.e. produced by artificial means, for example, by chemical synthesis.
  • a range of methods for chemical synthesis of lycopene and other carotenoids are known in the art.
  • a three-stage chemical synthesis based on the standard Wittig olefination reaction scheme for carotenoid synthesis may be employed, in which an organic solution of Ci5 phosphonium methanesulfonate in dichloromethane (DCM) and an organic solution of CiO dialdehyde in toluene are produced, and the two organic solutions are gradually combined with sodium methoxide solution and undergo a condensation reaction to form crude lycopene.
  • DCM dichloromethane
  • CiO dialdehyde in toluene an organic solution of CiO dialdehyde in toluene
  • the crude lycopene may then be purified using routine techniques, for example by adding glacial acetic acid and deionized water to the mixture, stirring vigorously, allowing the aqueous and organic phases to separate, and extracting the organic phase containing DCM and crude lycopene with water. Methanol is added to the organic phase and the DCM removed via distillation under reduced pressure. The crude methanolic lycopene solution is then be heated and cooled to crystalline slurry that is filtered and washed with methanol. The lycopene crystals may then be recrystalized and dried under heated nitrogen. Synthetic carotenoids, such as lycopene, are also available from commercial suppliers (e.g. BASF Corp, NJ USA).
  • Synthetic carotenoid compounds such as lycopene, may comprise an increased proportion of cis isomers relative to natural carotenoid compounds.
  • synthetic lycopene may be up to 25% 5-cis, 1 % 9- cis, 1 % 13-cis, and 3% other cis isomers, whilst lycopene produced by tomatoes may be 3-5% 5-cis, 0-1 % 9-cis, 1 % 13-cis, and ⁇ 1 % other cis isomers. Since cis- lycopene has increased bioavailability relative to trans- lycopene, synthetic lycopene is preferred in some embodiments.
  • Derivatives of carotenoids as described above may be produced by chemical synthesis analogous to the synthesis described above or by chemical modification of natural carotenoids extracted from plant material.
  • the concentration of anthocyanin in the hydrogel may be 0.01 - 50 mg / g.
  • the concentration of carotenoids in the hydrogel may be 0.1 - 50 mg / g.
  • the ratio of anthocyani carotenoids in the hydrogel may be from 1 :0.002 to 1 :5,000.
  • the pH may be from 2.5 to 7 or greater than 7.
  • the invention relates to a method for making a liquid crystalline hydrogel, for example as shown in the examples herein.
  • the method may comprise
  • the anthocyanin may be comprised in plant material, i.e. a plant source of anthocyanin is incubated, or may be a purified or synthetic anthocyanin.
  • the invention in another aspect, relates to a composition, foodstuff, beverage, nutraceutical or pharmaceutical product comprising a liquid crystalline hydrogel as described above.
  • the foodstuff can be a functional or medical food or beverage, a dietary supplement, or a nutraceutical product.
  • said foodstuff is a diary product. In one embodiment, said food stuff is a liquid or solid fat. In one embodiment, said foodstuff is butter, margarine, ice cream oil shortening, lard, chocolate, peanut butter, fat based creams and spreads, milk, cream, ice cream, yogurt.
  • Food products can be in solid, frozen, semi-solid, gel, molten, semi-liquid or in liquid form.
  • the invention also relates to the use of an anthocyanin or an anthocyanin hydrogel as described above as a preservative.
  • an anthocyanin or an anthocyanin hydrogel as described above can be used as a viscosity modifier or emulsifying agent. This has advantages in producing better foodstuff that contains fat or oil, in particular in increasing the bioavailability and stability of bioactive compounds, in particular in increasing their stability in the acidic gastric environment.
  • the anthocyanin or an anthocyanin hydrogel thus allows control of viscosity in liquid and semi-liquid products.
  • the invention thus also relates to the use of an anthocyanin or an anthocyanin hydrogel as described above as an emulsifying agent or as a viscosity modifier.
  • the invention also relates to the use of an anthocyanin or an anthocyanin hydrogel as described as above to reduce viscosity and/or lower the melting point of a fat or oil.
  • the invention also relates to the use of an anthocyanin or an anthocyanin hydrogel as described above to increase the bioavailability of a bioactive compound and increasing the stability of the bioactive in an acidic environment.
  • the bioactive compound is selected from a carotenoid.
  • the invention also relates to a method for reducing viscosity and/or lowering the melting point of a fat or oil comprising blending an anthocyanin or an anthocyanin hydrogel as described above with the fat or oil.
  • the invention also relates to a method for emulsifying an amphiphilic or hydrophobic compound in aqueous solution comprising adding an anthocyanin or an anthocyanin hydrogel as described above to the amphiphilic or hydrophobic compound.
  • the invention also relates to a method for increasing resistance, bioavailability or stability of a bioactive compound, comprising providing the bioactive compound together with an anthocyanin.
  • the method protects the bioactive compound from oxidative modification / damage by acidic environment.
  • the compound may be part of foodstuff or beverage matrix, a nutraceutical or pharmaceutical product.
  • the invention also relates to a method for increasing bioavailability of an orally administered a bioactive compound incorporated in a food or beverage matrix, a nutraceutical or pharmaceutical product, in an acidic environment comprising providing the bioactive compound together with an anthocyanin.
  • a fat can be selected from products comprising fatty acids, monoglycerides or diglycerides or triglycerides or other glycerolipids, phosphatic acid or phosphatidylethanolamine or phosphatidylcholine or phosphatidylserine or phosphatidylinositol or other glycerophospholipids, ceramides or sphingolipids, sterols, waxes, fat-soluble vitamins, prenols, saccharolipids, polyketides, or their derivatives in pure, or blended, or co-synthesised, or co-produced, or co-existing with each other from the above list, or with other molecules or substances, forms.
  • An oil can be a vegetable, or nut, or seed or fish oil.
  • a vegetable oil can be, for example but not limited, from corn, rapeseed, sunflower, palm, palm kernel, soybean, olive, rice bran, grape seed, avocado, canola, cotton seed, linseed, sesame, acai, jambu, graviola, tucuma, carapa, passion fruit, pracaxi.
  • a nut oil can be, for example, but not limited, from coconut, hazelnut, walnut, brazil nut, almond, peanut.
  • anthocyanin can change the physical properties of hydrophobic and amphiphile bioactive molecules: examples for carotenoids and trans- resve ratro I .
  • results presented in figures 1 to 1 1 indicate that blending any of these anthocyanins with either of these carotenoids results in appearance of a long-wave shift, which indicate formation of a physical complexes akin electron transfer ones.
  • These complexes are not new chemical entities, but physical electron-donor couples between these two types of molecules. As a result of these interactions they will typically have new red-ox and physical properties, which are different from properties of individual molecules when they are in free forms. It was interesting to note that formation of these complexes was observed in a broad range of pH, from 2.5 to above 7.
  • Results presented in figures 12 to 17 indicate that blending any of the selected anthocyanins and blueberry extract with f-RSV results also in appearance of a long-wave shift in optical absorption spectra. This indicates formation of not new chemical entities but physical electron transfer complexes, which should have new properties, different from these molecules when they are in their free forms. These complexes of ANT with f-RSV, like their complexes with carotenoids were also stable in a broad range of pH, from 2.5 to above 7.
  • ANT matrix For functional food and beverages, and also for nutraceutical formulations we propose to use more convenient, more efficient, better standardised and alcohol free new form of an ANT matrix.
  • Blueberry extract ‘potato ricer’ or similar, centrifuge, glass dish, temperature adjustable water bath, fan, plastic spatula or blade.
  • a number of dishes can be used for a larger volume of liquid.
  • a relatively shallow depth of liquid gives a faster drying time.
  • Blueberry, or Cherry, extract is not dried to a solid mass, the viscous semi-liquid mass is easier to collect and use in formulations.
  • the viscous mass is collected using a semi-rigid plastic spatula or blade to remove it from the glass dish.
  • the Blueberry, or Cherry, concentrate could be covered and stored in the glass dish.
  • the method can be varied by putting more Blueberry, or Cherry, liquid into the dish and varying air flow over the dish. Speed of drying depends on the interaction between liquid depth, surface area and air-flow. Temperature of the water bath should not exceed 45°C in order to protect anthocyanin content.
  • the drying and concentrating process should be focused on removing it.
  • the acceptable level would be between 0.075% and 0.241 %.
  • the second objective of this drying process is to achieve almost full evaporation of free water, which should not exceed 0.1 % in the finished product.
  • the main objective is by removing free water allow bound water to create liquid crystalline gel status.
  • the finished LCH contained between 9.5 and 10.5% of bound water.
  • the percentage of the bound water can vary and be dependent on a number of factors:
  • Figures 19 B-E demonstrates that blending of astaxanthin into the hydrogel increase its dispersibility even in a minimally added aqueous solvent, whether it is water or acetate buffer with pH 3.8.
  • Saturated fatty acids, SFA, or products rich with them have more compact folding, which harder to disrupt and emulsify than products with unsaturated fatty acids, UFA.
  • FIG. 23A-D Evidence that anthocyanins can indeed emulsify lipids of the dairy butte and create micelles with its fat globules is presented in the Figure 23A-D. Comparison of these globules after the blending with ANT-LCH shows that anthocyanins can disrupt their structure, which resulted in an increase of their size. Pre blending of this hydrogel with carotenoids, either lycopene or lutein, led to further enlargement of the globules, which indicate deeper disruption of their lipid folding (Fig. 23A-C). A closer microscopy reveals that in the core of the ANT and fat globules micelles is a hydrophobic lipid mass, which is surrounded by amphiphilic pigment molecules of aronia, anthocyanins (Fig. 23D).
  • Figure 24 A-B demonstrates that ANT-LCH blended butter had significantly low melting point and lower viscosity.
  • the experiment was done as follows: pieces of 200 mg of control and the blended butter were placed on the surface of the laboratory slides and incubated at 37°C. Melting time fixed with laboratory timer (QUANTUM).
  • Fig. 24A was taken after 30 sec on incubation, which shows that while ANT butter was already melted the control sample remains in the solid phase.
  • the graph in Fig. 24B demonstrates that the melting time of was twice as fast for the former than for the latter.
  • carotenoids, lycopene or lutein can further disrupt lipid folding of dairy butter they do not additionally decrease its melting time.
  • Figure 27A-B demonstrate that ANT-LCH blended cocoa butter had a significantly low melting point and lower viscosity. The experiment was done similar to one above with the dairy butter. Although carotenoids, lycopene or lutein, can further disrupt lipid folding of cocoa butter they do not additionally decrease its melting time.
  • Fig. 27A was taken after 30 sec on incubation, which shows that while cocoa butter blended with aronia hydrogel was already melted the control sample remains in the solid phase.
  • the graph in Fig. 27B demonstrates that the melting time of was more than twice faster for the former than for the latter.
  • Olive oil is a product where the main lipid is the monounsaturated oleic fatty acid, MFA.
  • FIG. 30A-B Microscopic evidence that anthocyanins can disrupt MFA rich hazelnut oil is presented on Figure 30A-B.
  • Fig. 30A with control oil sample, there are no visible lipid droplets under the magnification used.
  • Fig. 30B where was oil with blended in ANT-LCH there are clearly visible enlarged lipid structures of fused droplets.
  • Docosahexaenoic acid which belongs to the group of Omega 3, is a polyunsaturated fatty acid, PUFA. Microscopic evidence that anthocyanins can disrupt DHA is presented on Figure 31A-B. In Fig. 31A, with control sample, there are no visible lipid droplets under the magnification used. However, in Fig. 31 B, where DHA was blended in ANT-LCH, there are clearly visible enlarged lipid structures of fused droplets.
  • Blending of lycopene and astaxanthin with DHA preparation demonstrates some level of affinity between those two types of products, which illustrated by penetration of these pigments into oil phase of Omega 3. (Fig. 32F-G).
  • Anthocyanins improve bioavailability / absorption of other bioactive molecules
  • trans-Res veratrol f-RSV
  • concentration of trans-Res veratrol, f-RSV in blood serum was measured by using competitive ELISA with the specific monoclonal antibodies [1 ]
  • Concentration of serum epicatechins was expressed as a combined concentration of two main metabolites of epicatechin metabolites, epicatechin sulphate and O- methylcatechin sulphate, which were measured by UPLC MS/MS analysis [2].
  • AUC area under the curve
  • epicatechins were ingested in products containing anthocyanins their absorption rate / pharmacokinetics was noticeably stronger than in both chocolates: for blueberry LCH the ratio was 1 :7.6 for cranberry, 1 :3, for aronia LCH 1 :2.4 and for cherry LCH 1 :1 .5.
  • epicatechin molecules were from 6 to 12 times better absorbed from when they were ingested in a form of ANT-LCH than in the dark chocolate.
  • Blueberry extract ‘potato ricer’ or similar, centrifuge, glass dish, temperature adjustable water bath, fan, plastic spatula or blade.
  • a number of dishes can be used for a larger volume of liquid.
  • a relatively shallow depth of liquid gives a faster drying time.
  • the water level outside the dish should be just above the level of the blueberry liquid inside.
  • the drying process is speeded up by blowing air over the dish using a fan (see photo on p.2)
  • blueberry extract hydrogel
  • the viscous semi-liquid mass is easier to collect and use in formulations.
  • the viscous mass is collected using a semi-rigid plastic spatula or blade to remove it from the glass dish.
  • the blueberry concentrate, hydrogel could be covered and stored in the glass dish.
  • the method can be varied by putting more blueberry liquid into the dish and varying air flow over the dish. Speed of drying depends on the interaction between liquid depth, surface area and airflow.
  • Temperature of the water bath should not exceed 45°C in order to protect anthocyanin content.

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Abstract

The invention relates to methods and uses of anthocyanins, for example in food products.

Description

Anthocyanins and uses thereof
Introduction
The nutraceutical market reached $180 billion in 2015. Hydrophobic oily ingredients and products represent a significant part of this market. For example, the Omega-3 market alone was worth $25 billion last year and is set to grow to £35 billion by 2020.
At the same time, the functional beverage market reached $90 billion in 2015. The only way to introduce oils or hydrophobic ingredients into drink products currently available is to use encapsulation technologies, most of which are based on using polysaccharides and starch in particular. Although this is useful to disperse oils in aqueous solutions and to incorporate them into a broad range of beverage products from milk to recovery sport or hospital drinks, this technique has one significant drawback - it reduced absorption rate of the encapsulated bioactives. This is the result of the polysaccharides interference with a number of essential digestive reactions in the gastrointestinal tract. They include slowing down the emulsification process of the oily substances, which is critical for their digestion. Products of polysaccharide digestion may also interfere with interactions between released ‘cargo’ molecules and the membrane of enterocytes, which are responsible for their absorption and further processing.
Even fortification of beverages with water-soluble bioactives has some limitations. This is particularly the case for many soft drinks, fruit juices and other beverages of which the pH is acidic as the acidic environment can be detrimental to many bioactives and result in their degradation. There are a number of foods that are products of bacterial and / or fungal fermentation, and as a result, their pH is acidic. Examples include diary products such as yogurt, cheese and chocolate.
In order to fortify these products with valuable bioactives, including vitamins and other food supplements, it is important to guarantee their “survival” in an acidic environment to increase their bioavailability and efficacy.
The invention is aimed at addressing some of these shortcomings.
Summary
This invention explores a new unexpected property of anthocyanins, anthocyanin-rich plant extracts or other anthocyanin-rich products (collectively termed “ANT”), which is based on their ability to create physical complexes with amphiphilic and hydrophobic molecules. These types of complexes allow ANT to be used as emulsifiers or surfactants. The inventor has shown that ANT can change the physical properties of hydrophobic and amphiphile bioactive molecules; examples are carotenoids and frans-resveratrol. The inventor has demonstrated that ANT facilitates blending and emulsifying of hydrophobic molecules and oily or fat-based products to incorporate them into aqueous solution, suspension or dispersions. This is achieved by using a matrix with ANT.
Moreover, the inventor has shown that molecules, which are incorporated into these complexes can be protected from the oxidising effects of acidic pH.
This protective property of ANT can be utilised in the preservation of other bioactive agents when they are used as beverage or food matrixes with an acidic environment. This includes for example some soft drinks or juices, as well as products of bacterial and / or fungal fermentation like yogurt, chocolate, cheese, etc.
Moreover, this property of ANT can be utilised to protect bioactive molecules or agents in orally ingestible products during their passage through an acidic stomach environment. As a result of this protection, more bioactives can pass through the stomach to reach the sites of their absorption in unmodified form. This results in an improved absorption rate.
We have also shown that these properties can be enhanced by creating complexes of ANT with carotenoid molecules or carotenoid-rich products.
The invention relates to ANT liquid crystalline and / or hydrogel, ANT-LCH, which can be used as a platform itself or in combination with a carotenoid, to emulsify or disrupt hydrophobic molecules or hydrophobic clusters of amphiphilic molecules, crystals, and other non-water soluble structures or products, to create stable solutions, suspensions, colloids or dispersions in aqueous media.
These properties of ANT can also be used to disrupt lipid folding of different fats or oils, or fat-, or oil-rich products in order to reduce their viscosity and spreadability, reduce their melting, boiling and freezing points. For example, reduction of cooking time (without an increase in temperature) can help preserve temperature sensitive vitamins and other nutrients in cooked food. It can also positively affect the taste of food.
Moreover, disruption of the lipid folding by ANT can increase the size of the lipid droplets or globules and therefore reduce the rate of their digestion in the gastrointestinal tract. These properties could be used to create improved food or beverage products, which helps to control lipid metabolism and weight management.
Moreover, we describe the use of ANT to increase bioavailability and stability of a bioactive as its breakdown in an acidic environment is reduced.
Figures The invention is further illustrated in the following non-limiting figures. Graphs are marked to identify compounds for ease of reference.
Fig.1 Delphinidin - purple, astaxanthin - green, yellow - blend of delphinidin and astaxanthin’ with long wave shift in absorption of pick 1 . pH 7.4 - ethanol / H20.
Fig 2. Malvidine - purple, astaxanthin - green, red - blend of malvidine and astaxanthin’ spectrum with long wave shift in absorption of pick 1 . pH 7.4 - ethanol / H20.
Fig. 3. Malvidine - blue, grey - astaxanthin, red - blend of malvidine and astaxanthin’ spectrum with long wave shift in absorption of pick 1 . pH 2.5 - fumaric acid / H20.
Fig. 4. Peonidin - turquoise, green - astaxanthin, red - blend of peonidin and astaxanthin’ spectrum with long-wave shift in absorption of pick 1 . pH 2.5 - fumaric acid / H20.
Fig. 5. Cyanidin - green, blue - astaxanthin, orange - blend of cyanidin and astaxanthin’ spectrum with long wave shift in absorption of pick 1 . pH 2.5 - fumaric acid / H20.
Fig. 6. Pelargonidin - brown, blue - astaxanthin, turquoise - blend of peonidin and astaxanthin’ spectrum with long-wave shift in absorption of pick 1 . pH 2.5 - fumaric acid / H20.
Fig. 7. Petunidin - brown, green astaxanthin, orange - blend of petunidin and astaxanthin’ spectrum with long-wave shift in absorption of pick 1 . pH 2.5 - fumaric acid / H20.
Fig. 8. Lutein & meso-zeaxanthin - orange, purple - blend of cyanidin and lutein & meso-zeaxanthin’ spectrum with long-wave shift in absorption of pick 9- 5. H 7.4 - ethanol / H20.
Fig. 9. Lutein & meso-zeaxanthin - orange, turquoise - blend of malvidine and lutein & meso-zeaxanthin’ spectrum with long-wave shift in absorption of pick 9- 6. pH 2.5 - fumaric acid / H20.
Fig. 10. Lutein & meso-zeaxanthin - orange, turquoise - blend of petunidin and lutein & meso-zeaxanthin’ spectrum with long-wave shift in absorption of pick 9- 5. pH 2.5 - fumaric acid / H20.
Fig. 1 1 . Lutein & meso-zeaxanthin - orange, turquoise - blend of blueberry anthocyanins and lutein & meso-zeaxanthin’ spectrum with long-wave shift in absorption of pick 9- 6. pH 2.5 - fumaric acid / H20.
Fig. 12. trans- Resveratrol - blue, pink - pelargonidin, orange - blend of pelargonidin and frans-resveratrol’ spectrum with long-wave shift in absorption of pick 4- 3. H 7.4 - ethanol / H20.
Fig. 13. trans- Resveratrol - blue, grey - cyanidin, red - blend of pelargonidin and frans-resveratrol’ spectrum with long-wave shift in absorption of pick 4- 3. H 7.4 - ethanol / H20.
Fig. 14. trans- Resveratrol - blue, dark-red - malvidine, purple - blend of pelargonidin and frans-resveratrol’ spectrum with long-wave shift in absorption of pick 5- 6. H 7.4 - ethanol / H20.
Fig. 15. trans- Resveratrol - blue, turquoise - pelargonidin, brown - blend of pelargonidin and frans- resveratrol’ spectrum with long-wave shift in absorption of pick 2-4- 2. pH 2.5 - fumaric acid / H20.
Fig. 16. trans- Resveratrol - blue, black - delphinidin, red - blend of delphinidin and frans-resveratrol’ spectrum with long-wave shift in absorption of pick 3- 3. pH 2.5 - fumaric acid / H20.
Fig. 17. Astaxanthin - blue, red - frans-resveratrol, green - blueberry ANT extract, black - blend of frans- resveratrol with blueberry ANT and astaxanthin’ spectrum with long-wave shift in absorption of pick 4- 2. pH 2.5 - fumaric acid / H20.
Figure 18 A-B. Carotenoid emulsifying properties of ANT of red wine. A) ANT with Red; B) ANT with white. Fig. 19 A-G Carotenoid emulsifying effect of cranberry ANT-LCH
A) blend of carotenoids with cranberry ANT-LCH without any solvents; B-E) blend of carotenoids and cranberry ANT-LCH with 1 ml of aqueous solvents; B - C) ANT-LCH + lycopene in aqueous solvents, B) H20 and C) pH 3.8; D - E) ANT-LCH + astaxanthin in aqueous solvents D) H20 and E) pH3.8.
F-G) carotenoids and cranberry ANT-LCH dispersions in H20; F) ANT-LCG + lycopene; G) ANT-LCG + astaxanthin in H20.
Figure 20. A) Optical microscopy of Blueberry ANT-LCH micelles with Astaxanthin and B) the solubility of a blend Astaxanthin & Blueberry ANT-LCH in H20.
Figure 21 A-E demonstrates a similar facilitating dispersion effect of oil formulations of carotenoids, lycopene and astaxanthin, by cherry ANT-LCH. A) carotenoids placed on top of cherry ANT-LCH without any solvents; B - E) blend of carotenoids and cherry ANT-LCH with 1 ml of aqueous solvents. B - C) ANT- LCH + lycopene in aqueous solvents B) H20; C) pH 3.8. D - E) ANT-LCH + astaxanthin in aqueous solvents; D) H20 and E) pH 3.8.
Figure 22 A-M. Dairy Butter emulsifying properties of carotenoids. A) butter in water; B - D) lycopene in H20; B) incorporation of dairy butter with lycopene in H20; C) blending of butter and lycopene in H20; D) butter and lycopene in solvent; E - G) lycopene in solvent of pH 3.8 E) incorporation of dairy butter with lycopene at pH 3.8; F) blending of butter and lycopene at pH 3.8; G) butter and lycopene in solvent at pH 3.8; H - J) astaxanthin in H20; H) incorporation of astaxanthin with dairy butter in H20; I) blending of butter with astaxanthin; J) butter and astaxanthin in solvent; K - M) astaxanthin in solvent at pH 3.8; K) incorporation of astaxanthin in dairy butter at pH 3.8; L) blending of butter and astaxanthin in solvent at pH 3.8; M) astaxanthin and butter in solvent at pH 3.8.
Figure 23 A-D. Effect of ANT-LCH and lutein on dairy butter fat droplets. A) control butter; B) anthocyanins increase size of dairy butter fat droplets (butter + ANT-LCH); C) lutein boosts this effect (butter + ANT- LCH+Lutein); D) structure of fat globules ANT micelles - hydrophobic lipid mass is in the center and anthocyanins pigmented outer layer.
Figure 24 A-B. A) Effect of anthocyanins and lutein on the viscosity and the meting time, the vertical axis, of dairy butter. B) In the bar chart from left to right: dairy butter, ANT+LCH+dairy butter, dairy butter+ANT- LCH+lutein.
Figure 25 A-B. A) Cocoa butter fat globules in control sample; B) Fusion of fat globules of cocoa butter caused by its blending with aronia ANT-LCH at x1000.
Figure 26 A-F. Cocoa Butter emulsifying properties of complexes of aronia ANT-LCH with carotenoids. A - C) ANT-LCH with astaxanthin in H20. A) incorporation of ANT-LCH with cocoa butter blends, + ANT-LCH with astaxanthin in H20; B) blending with a solvent; C) final result of blending cocoa butter blends with ANT-LCH with astaxanthin; D - F) ANT-LCH with lycopene in H20; D) incorporation of cocoa butter blends with ANT-LCH with lycopene in H20; E) blending with solvent; F) final result of blending cocoa butter blends with ANT-LCH with lycopene in H20.
Figure 27 A-B. A) Effect of anthocyanins and lutein on the viscosity and the meting time, the vertical axis, of cocoa butter. B) In the bar chart form left to right: cacao butter, cocoa butter+ ANT-LCH, cocoa butter+ ANT-LCH+lutein.
Figure 28 A-L. Disruptive effect of carotenoids on lipid folding of olive oil. A - C) astaxanthin in H20 A) incorporation of astaxanthin with olive oil blends in H20; B) blending of the astaxanthin and olive oil; C) addition of solvent to the blend of olive oil with astaxanthin in water; D - F) astaxanthin in a solvent with pH 3.8; D) incorporation of astaxanthin with olive oil blends with a pH of 3.8; E) blending of astaxanthin with olive oil at pH 3.8; F) addition of solvent to the olive oil blend and astaxanthin at pH 3.8; G - I) Lycopene in H20; G) incorporation of olive oil blends with lycopene in water; H) blending of olive oil with lycopene in water; I) addition of solvent to lycopene and olive oil in water; J - L) lycopene in solvent at pH 3.8; J) incorporation of lycopene with olive oil blends with a pH of 3.8; K) blending of lycopene with olive oil at pH 3.8; L) addition of solvent to the olive oil blend and lycopene at pH 3.8.
Figure 29 A-B). A) control olive oil; B) Aronia ANT-LCH promotes fusing and enlargement of lipid droplets of olive oil.
Figure 30 A-B). A) control hazelnut oil; B) Aronia ANT-LCH promotes fusing and enlargement of lipid droplets of hazelnut oil.
Figure 31 A-B). A) control DHA; B) Aronia ANT-LCH promotes fusing and enlargement of lipid droplets of DHA.
Figure 32 A-K. DHA 40% in sunflower oil - emulsifying properties of aronia ANT-LCH and its complexes with carotenoids. A-D) DHA + ANT-LCH & carotenoids; A) Astaxanthin W; B) Astaxanthin 3.8; C) Lycopene W; D) Lycopene 3.8. E) DHA + ANT-LCH at pH 3.8. F - G) interaction of carotenoids with DHA; F) DHA + lycopene; G) DHA + astaxanthin; H-K) interaction of carotenoids with DHA in aqueous solution pH 3.8; H) DHA + lycopene at pH 3.8; I) DHA + lycopene in water; J) DHA + astaxanthin at pH 3.8; K) DHA + astaxanthin in water.
Figure 33. ANT-LCH - astaxanthin particles survived in the acid environment of the dark chocolate matrix. Figure 34. A-B). A) ANT-LCH - lycopene particles survive in the milk chocolate, optical microscopy, ANT- LCH - lycopene particles extracted from chocolate x50; B) water solubility of lycopene particles extracted from the milk chocolate.
Figure 35. Comparison of epicatechin metabolites pharmacokinetics after ingestion of two aronia extracts in the form of the ANT-LCG and spray-dried powder.
Detailed description
The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
In one embodiment, the invention relates to a liquid crystalline hydrogel comprising an anthocyanin. This can have an appearance and behaviour of a gel or a paste, semi-solid and spreadable mass.
Hydrogels are materials that are composed of a network of polymer chains that are hydrophilic and can hold a certain levels of bound water. In one embodiment, the hydrogel does not comprise free water. In one embodiment, the hydrogel comprises bound water. In one embodiment, the amount of bound water is from 1 % to 90% w/w, for example 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% w/w.
Anthocyanins are multi-glycosylated anthocyanidins. Anthocyanins are pigments, which naturally appear red, purple, or blue. Frequently, the color of anthocyanins is dependent on pH. Anthocyanins are naturally found in flowers, where they provide bright-red and -purple colours. Anthocyanins are also found in vegetables and fruits. Anthocyanins are useful as dyes or colouring agents, and furthermore, anthocyanins are known to have antioxidant properties.
The term “anthocyanin” as used herein is intended to include both glycosylated anthocyanins (anthocyanosides) as well as the aglycon of the anthocyanoside (anthocyanidin). It also includes synthetic anthocyanins or those isolated from natural sources, i.e. purified anthocyanins. In one embodiment, the anthocyanin is a plant extract or comprised in a plant extract.
The term“extract” is intended to mean anthocyanin materials obtained from plant sources, such as leaves, twigs, bark, roots, stem, seeds, flowers, berries, fruit, for example, by routine isolation methods from suitable plants sources noted, but not limited to, those described herein. There are various methods for the extraction of anthocyanins known to those of skill in the art. Examples of suitable anthocyanin-containing plants include, but are not limited to, fruits, vegetables, flowers and other plants selected from the group consisting of acai, Acer macrophyllum, Acer platanoides, acerola, Ajuga reptans, apple, apricot, aronia, Artict bramble, avocado, banana, baobab, barberry, barley, Begonia semperfiorens, Beilis perennis, Bletilla striata, bilberry, black beans, black soybeans, black, blue and purple potatoes, blackberry, blueberry, bog whortleberry, boysenberry, buckwheat, cacao, Camellia sinensis, canarygrass, Caucasian blueberry, Chimonanthus praecox, celery, Cerasus avium, cherry, cherry Morello, cherry laurel, chicory, chive, chokeberry, Coffee beans, Coffee cherries, Cornelian cherry, cornflower, cotoneaster, cowberry, cranberry, crowberry, chrysanthemum, Cynomorium coccineum, Dahlia variabilis, danewort, deerberry, Dendrobium, dwarf dogwood, Echinacea purpea, eggplant, elderberry, fababean, Fatsia japonica, feijoa, fig, flaxseed, garlic, gerbera, ginseng, Globe artichoke, goji, gooseberry, grapes, guava, hawthorn, hemp, hibiscus or roselle, Hibiscus Sabdaiffa, highbush blueberry, hollyhock, honeysuckle, Ipomoea purpurea, Iris ensata, Java plum, Jerusalem artichoke, kokum, Laeliocattleya, lentil, loganberry, lupine, lychee, maize, mango, mangosteen, maqui, Matthiola incana, meconopsis, Metrosideros excelsa, millet, mountain ash berry, mulberry, myrtle berry, olive, onion, orange, ornamental cherry, passion fruit, pea, peach, peanut, pear, perilla, petunia, Phalaenopsis, Phalsa, Pharbitis, Pineapple, pistachio, plum, pomegranate, Phragmites australis, purple carrot, quince, rabbiteye blueberry, radish, red and black currant, red and black raspberry, red cabbage, rice, rhubarb, rosehip, rye, saffron, sarracenia, sea buckthorn, sheepberry, Sophronitis coccinea, sorghum, sparkleberry, strawberry, Fragada Vesca, sugarcane, sunflower, sweet cherry, sweet potato, tamarillo, tamarind, taro, tart cherry, Theobroma cacao, Tulip greigii, turnip, water lily, Weigela, wheat, wild rice, Verbena hybrida, yam and mixtures thereof.
Suitable and non-limiting examples of anthocyanin extracts of particular interest include bilberry extract, blackcurrant extract, cranberry extract, black soybean extract, cowberry extract, blueberry extract and mixtures of two or more thereof.
Typically, the extract is concentrated by various methods to provide a solution enriched in anthocyanins. For example, ultrafiltration can be used to remove unwanted components by molecular weight cut offs. The retentate from the filtration can be stored as a liquid or, for example, can then be further concentrated into a powder by spray drying, freeze drying, flash drying, fluidized bed drying, ring drying, tray drying, vacuum drying, radio frequency drying or microwave drying.
Anthocyanin extracts can be further purified by one or more methods known in the art, such as chromatography, gel chromatography, high performance liquid chromatography, crystallization, affinity chromatography, partition chromatography and the like. Identification of the particular anthocyanin(s) can be accomplished by methods know to those skilled in the art and include <1 >H NMR, chemical degradation, chromatography and spectroscopy, especially homo- and heteronuclear two-dimensional NMR techniques for the characterization of the isolated anthocyanin compounds.
The term “purified” or“isolated” is used in reference to the purification and/or isolation of one or more anthocyanins from an anthocyanin extract as described above. Using conventional methods known in the art, various components of the anthocyanin extract can be separated into purified materials. In one aspect of the invention, the anthocyanin(s) of the extract are substantially purified and isolated by techniques known in the art. The purity of the purified compounds is generally at least about 90%, preferably at least about 95%, and most preferably at least about 99% and even more preferably at least about 99.9% (e.g. about 100%) by weight.
In accordance with the present invention, the anthocyanin extract contains one or more anthocyanins and/or anthocyanidins e.g. selected from the group consisting of peonidin, cyanidin, pelargonidin, delphinldin, petunidin, malvidin, apigenindin, auratinidin, capensinidin, europinidin, hirsutidin, 6- hydroxycyanidin, luteolinidin, 5-methylcyanidin, pulchellidin, rosinidin, tricetnidin, derivatives and mixtures thereof. In one embodiment, the anthocyanins and anthocyanidins are selected from the group consisting of cyanidin, peonidin, malvidin, petunidin, delphinidin, their glycoside derivatives, and mixtures thereof. In yet another embodiment, the extract contains at least one cyanidin-based anthocyanin.
The term“anthocyanin” as used herein is intended to refer not only to monomeric anthocyanins, but also refers to dimeric and polymeric (i.e. containing from 3 to 20 anthocyanidin monomer residues) forms of anthocyanins and to leucoanthocyanidins (also known as flavan-3,4-diols). The anthocyanins can comprise substitutions (e.g. alkyl, alkoxy groups etc.) and in particular can be O-glycosylated, as described above.
The anthocyanin as used herein can be a single anthocyanin or comprise a mixture of anthocyanins. In particular, the anthocyanin is selected from the group consisting of: aurantidin, malvidin, cyanidin, delphinidin, europindin, paeonidin, pelargonidin, peonidin and petunidin, and glycosides thereof. A typical example is malvin (malvidin diglucoside) chloride, which is commercially available in a purified form. Alternatively, the anthocyanin can be obtained by extracting anthocyanin containing plants such as aronia, grape, black carrot, red cabbage, aubergine, baobab, barberry, barley, bilberry, cranberry, cherry, blackberry, blackcurrant, blueberries, buckwheat, hemp, flaxseed, redcurrant, raspberry, and the like as described above.
In one embodiment, the anthocyanin is selected from cyanidin, delphinidin, malvidine, peonidin, petunidin, europinidin, aurantinidin, rosinidin.
In one embodiment, the hydrogel may further comprise a carotenoid compound. Carotenoid compounds are tetraterpenoids which contain long polyene chains. Carotenoid compounds include xanthophylls such as lutein and zeaxanthin, and carotenes, such as beta-carotene, alpha-carotene, zeto-carotene, and lycopene compounds.
In a particular embodiment, the carotenoid is a xanthophyll. In one embodiment, the xanthophyll is selected from the group consisting of a-cryptoxantin, b-cryptoxantin, adonirubin, adonixanthin, alloxanthin, amarouciaxanthin A, antheraxanthin, astaxanthin, auroxanthin, caloxanthin, cantaxanthin, capsanthin, capsanthin-5-6-epoxide, capsorubin, crocoxanthin, diadinoxanthin, diatoxanthin, echinenone, fucoxanthin, fucoxanthinol, iso-fucoxanthin, iso-fucoxanthinol, lutein, luteoxanthin, mutatoxanthin, neoxanthin, nostoxanthin, violaxanthin, zeaxanthin and combinations thereof.
In one embodiment, the carotenoid is a carotene. In another embodiment, the carotene is selected from the group consisting of a-carotene, b-carotene, y-carotene, d-carotene, e-carotene, z-carotene, lycopene, neurosporene, phytoene, phytofluene and/or combinations thereof.
In one embodiment, the carotenes and xantophylles described above refer to the all-trans forms thereof. In other embodiment, the xantophylles and carotenes for use in the present invention include derivatives containing one or more cis double bond.
In one embodiment, the carotenoid compound is a lycopene compound. Lycopene compounds may include lycopene, l-HO-3 ', 4'- didehydrolycopene, 3, 1 ' - (HO) 2-gamma-carotene, 1 , 1 ' - (HO) 2-3 , 4, 3', 4 ' - tetradehydrolycopene, 1 , 1 '- (HO) 2-3, 4-didehydrolycopene.
In some embodiments, the carotenoid compound is a lycopene compound such as lycopene. Lycopene is an open-chain unsaturated C40 carotenoid of structure I (Chemical Abstracts Service Registry Number 502-65-8, C40H56).
Structure I
Figure imgf000010_0001
Lycopene occurs naturally in plants such as tomatoes, guava rosehip, watermelon and pink grapefruit and any such sources of lycopene may be, for instance, employed.
Lycopene for use as described herein may comprise one or more different isomers. For example, lycopene may include cis- lycopene isomers, trans-lycopene isomers and mixtures of the cis- and trans-isomers. Lycopene may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% , at least 80%, at least 90%, or at least 95% (Z)- isomers, (all-E) -isomers , or cis-isomers, such as 5-cis- or 9- cis- or 13-cis-isomers, which have improved bioavailability relative to trans isomers. Trans isomers may isomerise into cis forms in vivo, or during storage and processing.
Carotenoid compounds, such as lycopene, for use as describe herein may be natural i.e. obtained from a natural source, for example, extracted from a carotenoid-rich fruit, vegetable or other plant, such as a tomato or melon, or from fungi, algae or bacteria. In one instance, the carotenoid compound may be, or comprise, oleoresin, particularly tomato oleoresin.
A range of methods for extracting, concentrating and/or purifying carotenoids from plants are known in the art. For example, solvent extraction using ethanol, DMSO, ethyl acetate, hexane, acetone, soya or other vegetable oil, or non-vegetable oils may be employed.
Carotenoid compounds, such as lycopene, for use as described herein may be synthetic i.e. produced by artificial means, for example, by chemical synthesis. A range of methods for chemical synthesis of lycopene and other carotenoids are known in the art. For example, a three-stage chemical synthesis based on the standard Wittig olefination reaction scheme for carotenoid synthesis may be employed, in which an organic solution of Ci5 phosphonium methanesulfonate in dichloromethane (DCM) and an organic solution of CiO dialdehyde in toluene are produced, and the two organic solutions are gradually combined with sodium methoxide solution and undergo a condensation reaction to form crude lycopene. The crude lycopene may then be purified using routine techniques, for example by adding glacial acetic acid and deionized water to the mixture, stirring vigorously, allowing the aqueous and organic phases to separate, and extracting the organic phase containing DCM and crude lycopene with water. Methanol is added to the organic phase and the DCM removed via distillation under reduced pressure. The crude methanolic lycopene solution is then be heated and cooled to crystalline slurry that is filtered and washed with methanol. The lycopene crystals may then be recrystalized and dried under heated nitrogen. Synthetic carotenoids, such as lycopene, are also available from commercial suppliers (e.g. BASF Corp, NJ USA).
Synthetic carotenoid compounds, such as lycopene, may comprise an increased proportion of cis isomers relative to natural carotenoid compounds. For example, synthetic lycopene may be up to 25% 5-cis, 1 % 9- cis, 1 % 13-cis, and 3% other cis isomers, whilst lycopene produced by tomatoes may be 3-5% 5-cis, 0-1 % 9-cis, 1 % 13-cis, and <1 % other cis isomers. Since cis- lycopene has increased bioavailability relative to trans- lycopene, synthetic lycopene is preferred in some embodiments.
Derivatives of carotenoids as described above may be produced by chemical synthesis analogous to the synthesis described above or by chemical modification of natural carotenoids extracted from plant material.
In one embodiment, the concentration of anthocyanin in the hydrogel may be 0.01 - 50 mg / g.
In one embodiment, the concentration of carotenoids in the hydrogel may be 0.1 - 50 mg / g.
In one embodiment, the ratio of anthocyani carotenoids in the hydrogel may be from 1 :0.002 to 1 :5,000.
In one embodiment, the pH may be from 2.5 to 7 or greater than 7.
In one aspect, the invention relates to a method for making a liquid crystalline hydrogel, for example as shown in the examples herein.
For example, in one embodiment, the method may comprise
a) incubating an anthocyanin with water, ethanol methanol or another organic solution in different concentrations from 10% to 100%;
b) removing any free supernatant;
c) optionally adding further plant material, for example crushed berries, and mixing this with the supernatant;
e) centrifuging this blend, for example for about 2 minutes at about 3,000 rpm to remove any small particles and collect supernatant;
f) concentrating the liquid, for example by drying at about 45°C or at any temperature above up to about 100°C until a viscous gel-type mass is obtained.
The anthocyanin may be comprised in plant material, i.e. a plant source of anthocyanin is incubated, or may be a purified or synthetic anthocyanin.
In another aspect, the invention relates to a composition, foodstuff, beverage, nutraceutical or pharmaceutical product comprising a liquid crystalline hydrogel as described above. The foodstuff can be a functional or medical food or beverage, a dietary supplement, or a nutraceutical product.
In one embodiment, said foodstuff is a diary product. In one embodiment, said food stuff is a liquid or solid fat. In one embodiment, said foodstuff is butter, margarine, ice cream oil shortening, lard, chocolate, peanut butter, fat based creams and spreads, milk, cream, ice cream, yogurt.
Food products can be in solid, frozen, semi-solid, gel, molten, semi-liquid or in liquid form. The invention also relates to the use of an anthocyanin or an anthocyanin hydrogel as described above as a preservative.
The inventor has surprisingly found that an anthocyanin or an anthocyanin hydrogel as described above can be used as a viscosity modifier or emulsifying agent. This has advantages in producing better foodstuff that contains fat or oil, in particular in increasing the bioavailability and stability of bioactive compounds, in particular in increasing their stability in the acidic gastric environment. The anthocyanin or an anthocyanin hydrogel thus allows control of viscosity in liquid and semi-liquid products.
The invention thus also relates to the use of an anthocyanin or an anthocyanin hydrogel as described above as an emulsifying agent or as a viscosity modifier.
The invention also relates to the use of an anthocyanin or an anthocyanin hydrogel as described as above to reduce viscosity and/or lower the melting point of a fat or oil.
The invention also relates to the use of an anthocyanin or an anthocyanin hydrogel as described above to increase the bioavailability of a bioactive compound and increasing the stability of the bioactive in an acidic environment. In one embodiment, the bioactive compound is selected from a carotenoid.
The invention also relates to a method for reducing viscosity and/or lowering the melting point of a fat or oil comprising blending an anthocyanin or an anthocyanin hydrogel as described above with the fat or oil.
The invention also relates to a method for emulsifying an amphiphilic or hydrophobic compound in aqueous solution comprising adding an anthocyanin or an anthocyanin hydrogel as described above to the amphiphilic or hydrophobic compound.
The invention also relates to a method for increasing resistance, bioavailability or stability of a bioactive compound, comprising providing the bioactive compound together with an anthocyanin.
In one embodiment, the method protects the bioactive compound from oxidative modification / damage by acidic environment. The compound may be part of foodstuff or beverage matrix, a nutraceutical or pharmaceutical product.
The invention also relates to a method for increasing bioavailability of an orally administered a bioactive compound incorporated in a food or beverage matrix, a nutraceutical or pharmaceutical product, in an acidic environment comprising providing the bioactive compound together with an anthocyanin.
A fat can be selected from products comprising fatty acids, monoglycerides or diglycerides or triglycerides or other glycerolipids, phosphatic acid or phosphatidylethanolamine or phosphatidylcholine or phosphatidylserine or phosphatidylinositol or other glycerophospholipids, ceramides or sphingolipids, sterols, waxes, fat-soluble vitamins, prenols, saccharolipids, polyketides, or their derivatives in pure, or blended, or co-synthesised, or co-produced, or co-existing with each other from the above list, or with other molecules or substances, forms. An oil can be a vegetable, or nut, or seed or fish oil. A vegetable oil can be, for example but not limited, from corn, rapeseed, sunflower, palm, palm kernel, soybean, olive, rice bran, grape seed, avocado, canola, cotton seed, linseed, sesame, acai, jambu, graviola, tucuma, carapa, passion fruit, pracaxi. A nut oil can be, for example, but not limited, from coconut, hazelnut, walnut, brazil nut, almond, peanut.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.
All documents mentioned in this specification are incorporated herein by reference in their entirety.
"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.
The invention is further described in the non-limiting examples.
EXAMPLES
Evidence that anthocyanin (ANT) can change the physical properties of hydrophobic and amphiphile bioactive molecules: examples for carotenoids and trans- resve ratro I .
Anthocyanin - Carotenoid complexes
To study possible establishment of these complexes we selected some of the most common anthocyanins: Delphinidin, Malvidine, Peonidin, Cyanidin, Pelargonidin, Petunidin (Sigma, US). Moreover, we also study not only single molecule synthetic ANC, but also natural anthocyanidin-rich extract from blueberry. As for carotenoids, we studied astaxanthin (AstaReal, Sweden) and a blend of lutein with its isomer meso- zeaxanthin (Lycored, Switzerland) in a ratio 1 :1 . Results presented in figures 1 to 1 1 indicate that blending any of these anthocyanins with either of these carotenoids results in appearance of a long-wave shift, which indicate formation of a physical complexes akin electron transfer ones. These complexes are not new chemical entities, but physical electron-donor couples between these two types of molecules. As a result of these interactions they will typically have new red-ox and physical properties, which are different from properties of individual molecules when they are in free forms. It was interesting to note that formation of these complexes was observed in a broad range of pH, from 2.5 to above 7.
Anthocyanin - trans-Resveratrol complexes
In another experiment we used the same selection of the most common anthocyanins and 98% crystal form of trans- Resveratrol, f-RSV (Symrise, Germany).
Results presented in figures 12 to 17 indicate that blending any of the selected anthocyanins and blueberry extract with f-RSV results also in appearance of a long-wave shift in optical absorption spectra. This indicates formation of not new chemical entities but physical electron transfer complexes, which should have new properties, different from these molecules when they are in their free forms. These complexes of ANT with f-RSV, like their complexes with carotenoids were also stable in a broad range of pH, from 2.5 to above 7.
Anthocyanin emulsifying properties
Anthocyanin emulsification - examples on carotenoids
Physical changes hydrophobic molecules, caused by disruption of their folding by anthocyanins or ANT-rich products, can result in an increase of their emulsification. This can be illustrated, for example, by ability of red wine to disperse carotenoids to a higher degree than white wine of the same manufacturer and the same alcohol content, it was 12% in the experiment presented in figure 18 A-B. This experiment demonstrates that finer particles of astaxanthin, and their clusters, and even their partial solubility were observed when this carotenoid, in its oil form, was blended with red wine than with the white one.
Preparation of ANT liquid crystalline hydrogel
For functional food and beverages, and also for nutraceutical formulations we propose to use more convenient, more efficient, better standardised and alcohol free new form of an ANT matrix. To facilitate blending of hydrophobic molecules and oily or fat-based products it is important to create a matrix of ANT from rich plant extracts, which on the one hand would have no free water, and on another to be in a liquid, or near liquid albeit structured form. To have a remaining viscosity for this concentrated but not dried extract, it is important to maximise its blending in and disrupting clusters of hydrophobic molecules or lipid folding of oil / fat based products.
An example of laboratory preparation of Blueberry, or Cherry, liquid crystalline hydrogel. Materials and Equipment Required:
Blueberry extract,‘potato ricer’ or similar, centrifuge, glass dish, temperature adjustable water bath, fan, plastic spatula or blade.
Method:
Remove free supernatant from Blueberry, or Cherry preparation, containing berries. Store the free supernatant in a suitable container. Crush the Blueberries, or Cherries, to collect any free liquid extract from the berries. A‘potato ricer’ or similar can be used for this.
Add this liquid to the free supernatant removed earlier and mix thoroughly.
Centrifuge the liquid mixture for 2 minutes at 3,000 rpm to remove any small particles and collect supernatant by pouring carefully into a suitable container.
Concentrate the liquid by drying 100ml in a glass dish of approx. 30 x 18cm. Weigh the dish before adding Blueberry, or Cherry, extract so final concentrated mass can be determined by subtraction.
A number of dishes can be used for a larger volume of liquid. A relatively shallow depth of liquid gives a faster drying time.
Place the dish in a water bath at 45°C. Ensure that there is adequate ventilation for any ethanol vapour. The water level outside the dish should be just above the level of the Blueberry, or Cherry, liquid inside. The drying process is speeded up by blowing air over the dish using a fan.
Using these conditions 100ml Blueberry, or Cherry, extract takes about 2 hours to be concentrated to a viscous mass.
Note that Blueberry, or Cherry, extract is not dried to a solid mass, the viscous semi-liquid mass is easier to collect and use in formulations. The viscous mass is collected using a semi-rigid plastic spatula or blade to remove it from the glass dish. Alternatively, the Blueberry, or Cherry, concentrate could be covered and stored in the glass dish.
The method can be varied by putting more Blueberry, or Cherry, liquid into the dish and varying air flow over the dish. Speed of drying depends on the interaction between liquid depth, surface area and air-flow. Temperature of the water bath should not exceed 45°C in order to protect anthocyanin content.
Since the original extract contained 50% ethanol the drying and concentrating process should be focused on removing it. The acceptable level would be between 0.075% and 0.241 %.
The second objective of this drying process is to achieve almost full evaporation of free water, which should not exceed 0.1 % in the finished product.
However, the main objective is by removing free water allow bound water to create liquid crystalline gel status. Under conditions described the finished LCH contained between 9.5 and 10.5% of bound water.
The percentage of the bound water can vary and be dependent on a number of factors:
- type of plant, or its part, or its cultivar, or the soil and a climate where it is grow, or the year and time of the harvest, etc.
- the method of extraction. All of these factors would affect spectrum and concentration of the anthocyanins and its glycosylated forms, presence of proteins, tannins, fibers and other polysaccharides, which all together would affect gelation process and properties of the final LCH products.
Formation of ANT-Carotenoid micelles and their aqueous dispersions
Evidence that anthocyanins in a form of liquid crystalline hydrogel of blueberry extract can create water dispersible complexes with lycopene and astaxanthin are presented on the figures 19A-G and 20A-B. Microscopy of the astaxanthin blend with the ANT-LCH, which does not contain any solvents (fig. 19A), is presented in Fig. 20 A.
Figures 19 B-E demonstrates that blending of astaxanthin into the hydrogel increase its dispersibility even in a minimally added aqueous solvent, whether it is water or acetate buffer with pH 3.8.
Addition of more of these solvents creates stable dispersions of these carotenoids, Fig. 1 9F-G.
ANT lipid folding disruption use with or without carotenoids:
increase of lipid droplets size,
increase lipids and incorporated into them other bioactive molecules to acidic oxidation / degradation,
reduce fat and oil viscosity,
increase of emulsification,
reduction of melting and freezing point.
Saturated Fatty Acids
Dairy Butter
Saturated fatty acids, SFA, or products rich with them, have more compact folding, which harder to disrupt and emulsify than products with unsaturated fatty acids, UFA.
Therefore, we have chosen dairy butter, which is rich with SFA, and tried to assess whether ANT-LCH would have any emulsifying effect on it. Figures 22B-22G demonstrates that indeed this hydrogel helps to breakdown a single piece of butter, and under acidic pH this effect more prominent.
An image of the control piece of butter in water is presented in Figure 22A.
The data presented in Fig. 22B - Fig. 22M shows that premixing of ANT-LCH with carotenoids, such as lycopene or astaxanthin, can significantly boost butter dispersibility even further both under acidic or neutral pH.
Evidence that anthocyanins can indeed emulsify lipids of the dairy butte and create micelles with its fat globules is presented in the Figure 23A-D. Comparison of these globules after the blending with ANT-LCH shows that anthocyanins can disrupt their structure, which resulted in an increase of their size. Pre blending of this hydrogel with carotenoids, either lycopene or lutein, led to further enlargement of the globules, which indicate deeper disruption of their lipid folding (Fig. 23A-C). A closer microscopy reveals that in the core of the ANT and fat globules micelles is a hydrophobic lipid mass, which is surrounded by amphiphilic pigment molecules of aronia, anthocyanins (Fig. 23D).
This figure also demonstrates that carotenoids indeed can disrupt lipids of the butter, which results in increasing their viscosity. However, this results in increase of the density of the lipid butter (droplets) and instead of floating this piece of becomes heavier than water. These results indicate that carotenoids themselves do not have sufficient ability to affect butter emulsification.
Figure 24 A-B demonstrates that ANT-LCH blended butter had significantly low melting point and lower viscosity. The experiment was done as follows: pieces of 200 mg of control and the blended butter were placed on the surface of the laboratory slides and incubated at 37°C. Melting time fixed with laboratory timer (QUANTUM).
Fig. 24A was taken after 30 sec on incubation, which shows that while ANT butter was already melted the control sample remains in the solid phase. The graph in Fig. 24B demonstrates that the melting time of was twice as fast for the former than for the latter. Although carotenoids, lycopene or lutein, can further disrupt lipid folding of dairy butter they do not additionally decrease its melting time.
Cocoa Butter
Another SFA product, which we tried to emulsify with anthocyanins, was cocoa butter. Under microscopy we observed that ANT-LCH could disrupt fat globules of cocoa butter, which resulted in their impregnation with amphiphilic anthocyanins and a fusion of these globules (fig. 25A-B).
However, a significantly stronger emulsifying effect was observed, like in the experiment with daily butter, when ANT-LCH was pre-blended with carotenoids, either astaxanthin or lycopene (fig. 26A-F).
Therefore, to achieve disruptive lipids effect on SFA rich products, which would result in their maximum emulsification, it is important to use a premix of both anthocyanins, preferably in liquid crystalline hydrogel forms, and carotenoids.
Figure 27A-B demonstrate that ANT-LCH blended cocoa butter had a significantly low melting point and lower viscosity. The experiment was done similar to one above with the dairy butter. Although carotenoids, lycopene or lutein, can further disrupt lipid folding of cocoa butter they do not additionally decrease its melting time. Fig. 27A was taken after 30 sec on incubation, which shows that while cocoa butter blended with aronia hydrogel was already melted the control sample remains in the solid phase. The graph in Fig. 27B demonstrates that the melting time of was more than twice faster for the former than for the latter.
Monounsaturated Fatty Acids For our study we selected two products rich with monounsaturated oleic fatty acid, MFA, olive and hazelnut oils. Both of then contain between 70 to 85% of oleic acid (C18:1 ).
Olive oil
Olive oil is a product where the main lipid is the monounsaturated oleic fatty acid, MFA.
Like in experiments described above anthocyanins, and in particular CNT-LCH, or carotenoids alone could disrupt lipid droplets of this oil. For example, it’s blending with astaxanthin or lycopene resulted in an increase in the density of this oil, and instead of floating on the surface of the aqueous solutions it precipitated to the bottom of the experimental glass vessel (Fig. 28A-L).
Microscopic evidence that anthocyanins can disrupt lipid folding of olive oil particles is presented on Figure 29A-B. In Fig.29A with control oil sample, there are no visible lipid droplets under the magnification used. However, in Fig.29B, where was oil with blended in ANT-LCH there are clearly visible fused lipid droplets.
Hazelnut oil
Microscopic evidence that anthocyanins can disrupt MFA rich hazelnut oil is presented on Figure 30A-B. In Fig. 30A, with control oil sample, there are no visible lipid droplets under the magnification used. However, in Fig. 30B, where was oil with blended in ANT-LCH there are clearly visible enlarged lipid structures of fused droplets.
Polyunsaturated Fatty Acids
Omega 3 Docosahexaenoic Acid
Docosahexaenoic acid, DHA, which belongs to the group of Omega 3, is a polyunsaturated fatty acid, PUFA. Microscopic evidence that anthocyanins can disrupt DHA is presented on Figure 31A-B. In Fig. 31A, with control sample, there are no visible lipid droplets under the magnification used. However, in Fig. 31 B, where DHA was blended in ANT-LCH, there are clearly visible enlarged lipid structures of fused droplets.
A group of experiments presented in the Figure 32A-K demonstrates that ANT-LCH alone perhaps provide some level of DHA emulsification, however significant lipid vesicles are still clearly floating on the surface of the liquid (Fig. 32E).
Blending of lycopene and astaxanthin with DHA preparation demonstrates some level of affinity between those two types of products, which illustrated by penetration of these pigments into oil phase of Omega 3. (Fig. 32F-G).
Neither of these carotenoids alone could disrupt the DHA matrix but their blending resulted, like in the case of butter above, in changes of the viscosity of the lipids and increase of their density. As a result of this there were no lipid vesicle observed and some of the oil material instead of floating on the surface precipitated on the bottom of glass containers used in this experiment (Fig. 32H-K). However, pre-blending of either lycopene or astaxanthin with ANT-LCH resulted in a product, which could significantly boost emulsification of DHA (Fig. 33). As a result of this emulsification there was no lipid vesicles observed on the surface of either aqueous acidic solution, acetic buffer pH 3.8, or in water itself.
Therefore, to maximize disruptive lipids effect on PUFA or MFA rich products, which would result in their maximum emulsification, it is important to use a premix of both anthocyanins, preferably in liquid crystalline hydrogel forms, and carotenoids.
Anthocyanins protect bioactive molecules in acidic environment Yogurt
Incubation of a Greek Yogurt blend with astaxanthin at +8°C for 7 days resulted in 93% loss in measurable concentration of this carotenoid in this acidic matrix. When the same amount of astaxanthin was pre-mixed with aronia ANT-LCH, before it was blended with the same yogurt, after one week of the incubation, at the same temperature, there were no changes observed in the carotenoid concentration (table 1 ).
Table 1 . Effect pre-blending of astaxanthin with aronia ANT-LCH on its stability in Greek Yogurt, pH 4.5, storage at +8°C
Figure imgf000019_0001
Chocolate
The protective effect of anthocyanins was observed when astaxanthin was embedded into dark chocolate. Without this protection, after 3 months of storage under room temperature, concentration of this carotenoid in the dark chocolate reduced more than 91 %. Pre-mixing astaxanthin with ANC-LCH resulted in a significant improvement in its stability; the reduction of the concentration, for the same period and under the same conditions, was about 4% only (table 2). Table 2. Effect pre-blending of astaxanthin with blueberry ANT-LCH on its stability in dark chocolate, pH 3.5, storage at +20 °C
Figure imgf000020_0001
A similar protective effect of ANT-LCH in the dark chocolate was observed for another carotenoid, lycopene. In addition, this hydrogel could protect both of these carotenoids in milk chocolate matrix too (data are not presented).
To verify that it is actually formation of hydrogel micelles / particles of ANT-LCH with carotenoids have the protective effect on the latter in chocolate, we made an attempt to extract and identify these particle out of this food matrix.
For this purpose we used methyl chloride to dissolved hydrophobic mass of the chocolate. As results of this we succeeded to obtain these particles from both dark and milk chocolate.
It was interesting to observe that astaxanthin containing particles could be well preserved under atmospheric conditions (Fig. 33). On the other hand ANT-LCH - Lycopene particles were highly hygroscopic and easily absorbed atmospheric moisture (Fig. 34A-B).
Both particles were easily dispersed and even partially solved in water.
Anthocyanins improve bioavailability / absorption of other bioactive molecules
Crossover clinical study
This was a multi-arm crossover study on eight clinically healthy volunteers, 4 men and 4 women, with age between 35 and 66 years old. All products were ingested with a half glass of warm water in the morning after 12 hours of fasting, and no any other food was taken for the duration of the each arm of the study.
Concentration of trans- Resveratrol, f-RSV, in blood serum was measured by using competitive ELISA with the specific monoclonal antibodies [1 ] Concentration of serum epicatechins was expressed as a combined concentration of two main metabolites of epicatechin metabolites, epicatechin sulphate and O- methylcatechin sulphate, which were measured by UPLC MS/MS analysis [2]. trans-Res veratrol
Results of a multi-arm crossover study on pharmacokinetics of orally ingested different products containing frans- resveratrol, f-RSV, are presented in the table 3.
They show that intake of those products where anthocyanins were present, particular in their hydrogel form, were resulted in superior level of absorption of f-RSV.
For example, area under the curve, AUC, for the first 4 hours after ingestion of ANT-LCFI of such berries as cranberry, blueberry, aronia and cherry, which contained only about 30 pg per dose was comparable with ingestion of 100 mg of f-RSV but in a purified crystal form.
To assess effect of ANT on the absorption of f-RSV of red wine, we designed a following experiment. Volunteers were asked to consume white wine together with a capsule containing the same amount of resveratrol, which was present in the glass of the red wine taken in the previous experiment.
It was interested to notice that the AUC of f-RSV after drinking of red wine was more than 10-20 fold than the same amount of resveratrol ingested with the same amount of the white wine.
Table 3. Comparison of pharmacokinetics of trans- Resveratrol delivered in in different products in crossover clinical study.
Figure imgf000021_0001
* 350ml of Chardonnay Burgundy, 350ml of Pinot Noir Burgundy.
Catechins
Comparison in the crossover clinical study of the absorption of catechin, and in particular epicatechins, which are present in cocoa, berries, some other fruits and plants demonstrated that presence of anthocyanins could significantly boost pharmacokinetics of these group of molecules (table 4).
For example, ingestion of 12,660 pg epicatechins in a form dark chocolate resulted in AUC for the first 4 hours to be 696 ng/ml of serum. If we assume AUC as a measure reflecting amount of the epicatechins absorbed, the ratio with their ingested quantity would be 1 :1 8 for the dark chocolate. In other words, under assumption we made, that only one molecule of epicatechin absorbed out of eighteen molecules ingested in the form of this chocolate.
For the milk chocolate this ratio was even higher, 1 :28, which would mean that the bioavailability of epicatechins ingested in this type of chocolate was significantly lower than when these molecules were consumed in the dark chocolate.
When epicatechins were ingested in products containing anthocyanins their absorption rate / pharmacokinetics was noticeably stronger than in both chocolates: for blueberry LCH the ratio was 1 :7.6 for cranberry, 1 :3, for aronia LCH 1 :2.4 and for cherry LCH 1 :1 .5. In another words epicatechin molecules were from 6 to 12 times better absorbed from when they were ingested in a form of ANT-LCH than in the dark chocolate.
Table 4. Comparison of pharmacokinetics of epicatechins delivered in in different products in crossover clinical study.
Figure imgf000022_0001
In the crossover clinical study it was demonstrated that consumption of the same amount aronia extract but in a form of LCH resulted in a significantly stronger absorption of epicatechins than from the aronia extract in a from the dried powder (Fig. 35).
Preparation of Blueberry Hydrogel
Materials and Equipment Required:
Blueberry extract,‘potato ricer’ or similar, centrifuge, glass dish, temperature adjustable water bath, fan, plastic spatula or blade.
Method:
1 . Divide blueberries in equal portions.
Incubate them at +4°C overnight with water, or ethanol in different concentrations: 10%, 20%, 50%, 100%.
2. Remove free supernatant from blueberry preparation containing berries.
Store the free supernatant in a suitable container.
3. Crush the blueberries to collect any free liquid extract from the berries. A‘potato ricer’ or similar can be used for this.
4. Add this liquid to the free supernatant removed earlier and mix thoroughly.
5. Centrifuge the liquid mixture for 2 minutes at 3,000 rpm to remove any small particles and collect supernatant by pouring carefully into a suitable container.
6. Concentrate the liquid by drying 100ml in a glass dish of approx. 30 x 18cm. Weigh the dish before adding blueberry extract so final concentrated mass can be determined by subtraction.
A number of dishes can be used for a larger volume of liquid. A relatively shallow depth of liquid gives a faster drying time.
Place the dish in a water bath at 45°C. Ensure that there is adequate ventilation for any ethanol vapour.
The water level outside the dish should be just above the level of the blueberry liquid inside.
The drying process is speeded up by blowing air over the dish using a fan (see photo on p.2)
Using these conditions 100ml blueberry extract takes about 2 hours to be concentrated to a viscous gel- type mass.
Note that blueberry extract, hydrogel, is not dried to a solid mass, the viscous semi-liquid mass is easier to collect and use in formulations.
The viscous mass is collected using a semi-rigid plastic spatula or blade to remove it from the glass dish. Alternatively, the blueberry concentrate, hydrogel, could be covered and stored in the glass dish.
The method can be varied by putting more blueberry liquid into the dish and varying air flow over the dish. Speed of drying depends on the interaction between liquid depth, surface area and airflow.
Temperature of the water bath should not exceed 45°C in order to protect anthocyanin content.
References
1 . Ivan M. Petyaev, Valeriy V. Tsibezov, Sergey N. Osipov, Nigel H. Kyle,
Daria V. Vorobjeva, and Yuriy K. Bashmakov - Generation of Monoclonal Antibody Against trans- Resveratrol. Hybridoma (2012), v.31 . No6. 2. Ivan M. Petyaev, Dmitry Pristenskiy, Tatyana Bandaletova, Natalia E. Chalyk, Victor Klochkov, Nigel H. Kyle - Lycosome Formulation of Dark Chocolate Increases Absorption Cocoa Catechins and Augments Their Anti-Inflammatory and Antioxidant Properties. American Journal of Food Science and Nutrition (2016), 3(3): 37-44.

Claims

Claims
1 . A liquid crystalline hydrogel comprising an anthocyanin.
2. The liquid crystalline hydrogel of claim 1 wherein the anthocyanin is a natural or synthetic anthocyanin.
3. The liquid crystalline hydrogel of claim 1 or 2 further comprising a bioactive, stabilising, filling or co purified natural or synthetic molecule.
4. The liquid crystalline hydrogel of claim 2 or 3 wherein the anthocyanin is comprised in a plant extract.
5. The liquid crystalline hydrogel of any of claims 1 to 4 wherein the anthocyanin is selected from cyanidin, delphinidin, malvidine, peonidin, petunidin, europinidin, aurantinidin or rosinidin.
6. The liquid crystalline hydrogel of a preceding claim further comprising a carotenoid compound.
7. The liquid crystalline hydrogel of any of claims 1 to 6 wherein the carotenoid compound is selected from a lycopene, lutein, zeaxanthin, meso-zeaxanthin, astaxanthin, b-carotene, other carotenes, cryptoxanthins, flavoxanthin, neoxanthin or tetraterpenoids.
8. The liquid crystalline hydrogel of a preceding claim wherein the hydrogel does not comprise free water.
9. The liquid crystalline hydrogel of a preceding claim wherein the amount of bound water is from 1 % to 90% w/w.
10. A composition, foodstuff, beverage, nutraceutical of pharmaceutical product comprising a liquid crystalline hydrogel of any preceding claim.
1 1 . A composition or foodstuff of claim 10 further comprising a carotenoid.
12. Use of an anthocyanin or a liquid crystalline hydrogel according to any of claims 1 to 9 as a preservative.
13. Use of an anthocyanin as an emulsifying agent.
14. Use of an anthocyanin or a liquid crystalline hydrogel according to any of claims 1 to 9 to reduce viscosity and/or lowering the melting point of a fat or oil.
15. Use of an anthocyanin or a liquid crystalline hydrogel according to any of claims 1 to 9 to increase the bioavailability of a bioactive compound.
16. The use of any of claims 12 to 15 wherein the anthocyanin is a natural or synthetic anthocyanin.
17. The use of claim 16 wherein the anthocyanin is a plant extract.
18. The use of claim 16 or 17 wherein the anthocyanin is selected from cyanidin, delphinidin, malvidine, peonidin, petunidin, europinidin, aurantinidin, rosinidin, aurantidin or pelargonidin.
19. The use of any of claims 15 to 18 wherein the bioactive compound is selected from a carotenoid.
20. A method for reducing viscosity and/or lowering the melting and / or freezing point of a fat or oil comprising blending an anthocyanin with the fat or oil.
21 . A method for emulsifying an amphiphilic or hydrophobic compound in aqueous solution comprising adding an anthocyanin to the amphiphilic or hydrophobic compound.
22. A method for increasing resistance or stability of a bioactive compound comprising of the bioactive compound together with an anthocyanin from any claims 1 to 9.
23. A method for increasing bioavailability of an orally administered bioactive compound incorporated in a food or beverage matrix, a nutraceutical or pharmaceutical product comprising providing the bioactive compound together with an anthocyanin.
24. A method of any of claims 20 to 23 wherein the anthocyanin is in the form of a liquid crystalline hydrogel according to any of claims 1 to 9.
25. A method for making a liquid crystalline hydrogel according to claim 1 comprising
a) incubating a plant source of anthocyanins with water, ethanol methanol or another organic solution in different concentrations from 10% to 100%;
b) removing any free supernatant;
c) optionally adding further plant material, for example crushed berries, and mixing this with the supernatant collected earlier;
e) centrifuging this blend;
f) concentrating the liquid.
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