WO2011057340A1 - Procédé pour l'enrichissement en agent bioactif de plante - Google Patents

Procédé pour l'enrichissement en agent bioactif de plante Download PDF

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Publication number
WO2011057340A1
WO2011057340A1 PCT/AU2010/001510 AU2010001510W WO2011057340A1 WO 2011057340 A1 WO2011057340 A1 WO 2011057340A1 AU 2010001510 W AU2010001510 W AU 2010001510W WO 2011057340 A1 WO2011057340 A1 WO 2011057340A1
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WIPO (PCT)
Prior art keywords
plant
stage
edible product
edible
product
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PCT/AU2010/001510
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English (en)
Inventor
Louise Bennett
Gerald Muench
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Commonwealth Scientific And Industrial Research Organisation
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Priority claimed from AU2009905525A external-priority patent/AU2009905525A0/en
Application filed by Commonwealth Scientific And Industrial Research Organisation filed Critical Commonwealth Scientific And Industrial Research Organisation
Publication of WO2011057340A1 publication Critical patent/WO2011057340A1/fr

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Classifications

    • 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
    • A23L33/105—Plant extracts, 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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • 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
    • A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
    • 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
    • A23L5/10—General methods of cooking foods, e.g. by roasting or frying
    • A23L5/15—General methods of cooking foods, e.g. by roasting or frying using wave energy, irradiation, electrical means or magnetic fields, e.g. oven cooking or roasting using radiant dry heat
    • 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
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/27—Removal of unwanted matter, e.g. deodorisation or detoxification by chemical treatment, by adsorption or by absorption
    • A23L5/273—Removal of unwanted matter, e.g. deodorisation or detoxification by chemical treatment, by adsorption or by absorption using adsorption or absorption agents, resins, synthetic polymers, or ion exchangers
    • 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
    • A23L5/30—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation
    • A23L5/32—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation using phonon wave energy, e.g. sound or ultrasonic waves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to a process for making a water-dispersible emulsion from plant and/or fungal material to produce edible products.
  • the invention also relates to use of the edible products in food and drink products and for the treatment and prevention of disease.
  • Plant materials are known to contain a number of classes of compounds which exert bioactivity in various animals. Historically, these compounds have been considered to be somewhat non-nutritive, however, recent scientific evidence now suggests these compounds may play an important role in the maintenance of health.
  • Bioactive compounds extracted from plant materials are widely used in the food industry as functional ingredients. Many bioactive ingredients are well perceived by consumers for their beneficial properties, but their application in food products is either difficult or provides poor bioavailability.
  • the present inventors have developed a process for producing plant and/or fungal edible products without the need for an organic solvent to extract out the edible and biological active component in the first extraction stage.
  • the process of the invention enables the extraction of water-soluble and water-insoluble plant and/or fungal metabolites from dietary or other types of plants and/or fungus that have biological activity.
  • the present invention provides a process for obtaining a biologically active edible product, the method comprising: a) obtaining an aqueous medium comprising plant and/or fungal material, b) thermally treating the aqueous medium comprising plant and/or fungal material, and
  • the method further comprises emulsifying the biologically active edible product to produce a water-dispersible emulsion.
  • the water-dispersible emulsion is a soluble emulsion.
  • organic solvent may be present in the aqueous medium, for example ethanol may be present for anti-microbial stability if desired, the process of the invention advantageously does not require an organic solvent in order to produce a water-dispersible emulsion.
  • producing a water-dispersible emulsion does not require an organic solvent.
  • producing a water-dispersible emulsion is performed in the absence of an organic solvent.
  • the plant and/or fungal material is in particulate form.
  • the particulate plant and/or fungal material may comprise particles about 100 ⁇ to about 2 mm in diameter.
  • the aqueous medium comprising plant and/or fungal material is thermally treated at about 80°C to about 100°C.
  • the aqueous medium comprising plant and/or fungal material is thermally treated at about 100°C.
  • the aqueous medium comprising plant and/or fungal material is thermally treated for about 5 minutes to about 1 hour.
  • the aqueous medium comprising plant and/or fungal material is thermally treated for about 10 minutes.
  • the process of the invention comprises emulsifying the thermally treated aqueous medium comprising plant and/or fungal material to produce a water- dispersible emulsion. Any suitable means known in the art may be used to form the water-dispersible emulsion.
  • producing a water-dispersible emulsion comprises mechanical emulsification.
  • producing a water-dispersible emulsion comprises treating the aqueous medium comprising plant and/or fungal material with one or more emulsifying agents.
  • the one or more emulsifying agents is selected from a lipid hydrolysing agent, a carbohydrate hydrolysing agent, and an inorganic emulsifying agent.
  • the lipid hydrolysing agent and/or the carbohydrate hydrolysing agent is an enzyme.
  • the enzyme may be, for example, lipase and/or amylase.
  • the enzyme is amylase and the process further comprises adding starch to the aqueous medium comprising plant and/or fungal material.
  • the process comprises treating the aqueous medium comprising plant and/or fungal material with tetra sodium pyrophosphate.
  • producing a water-dispersible emulsion comprises treating the aqueous medium comprising plant and/or fungal material with a lipid hydrolysing agent, a carbohydrate hydrolysing agent, and an inorganic emulsifying agent.
  • the lipid hydrolysing agent is lipase
  • the carbohydrate hydrolysing agent is amylase
  • the inorganic emulsifying agent is tetra sodium pyrophosphate.
  • the process may further comprise adding starch to the aqueous medium comprising plant and/or fungal material.
  • the aqueous medium comprising plant and/or fungal material is treated with one or more emulsifying agents for about 1 hour to about 24 hours, for about 10 to 20 hours, or about 15 to 20 hours.
  • the aqueous medium comprising plant and/or fungal material is treated with one or more emulsifying agents at about 30°C to about 50°C, preferably at about 45°C.
  • producing the water-dispersible emulsion is performed at about pH 5 to about pH 6.
  • the enzymes are inactivated prior to mechanical emulsification.
  • the process further comprises filtering the edible product.
  • filtering is performed with an about 100 ⁇ to an about 200 ⁇ sieve.
  • the process may further comprise centrifuging the edible product at about 10,000 x g to about 20,000 x g to remove insoluble material.
  • the process further comprises homogenising the edible product.
  • the aqueous medium comprising plant and/or fungal material further comprises about 0.5% to about 2% glucose.
  • the process of the invention may further comprise the use of an edible adsorbent to separate the edible product into binding and non-binding products, thereby fractionating biologically active components. Fractionating the biologically active components results in an increase in biological activity in either the binding or non- binding edible product.
  • the process further comprises:
  • the edible product is contacted with the edible adsorbent for about 5 minutes to about 1 hour. In one particular embodiment, the edible product is contacted with the edible adsorbent for about 15 minutes.
  • step ii) comprises centrifuging the edible product and edible adsorbent.
  • the edible adsorbent is in a column.
  • the edible adsorbent is chitosan.
  • the edible product comprises the edible adsorbent.
  • the process further comprises testing the edible product for biological activity.
  • the present invention further provides an edible product produced by the process of the invention.
  • the plant and/or fungal material is selected from a vegetable, grain, fruit, nut, tea, coffee, cocoa, a herb and/or spice
  • the plant and/or fungal material is selected from grapes, pomegranate, mushrooms, cinnamon, ginger, tea, citrus, rosemary, green basil, Thai basil, clove, rhubarb, chives, bay leaf, potato, Illawara plum, quandong, Kakadu plum, Cedar Bay cherry, riberry, lettuce, tarragon, onion, oregano, zucchini, spent grain, and/or red potato.
  • the edible product may have biological activity such as for example antiinflammatory or pro-inflammatory activity, cardiovascular regulatory or modulatory activity, neurological activity and/or metabolic activity.
  • the edible product has biological activity selected from angiotensin inhibitory activity, angiotensin 1 receptor inhibitory activity, cyclooxyegenase 2 inhibitory activity, lipoxygenase inhibitory activity, antiproliferative activity, anti-inflammatory activity, metabolic activity and fibril assembly inhibitory activity.
  • the edible product of the invention has more than one biological activity.
  • the edible product may have multiple biological activity such as combinations of, for example, anti-inflammatory and cardiovascular regulatory activity, inflammatory and neurological activity, neurological and metabolic activity, inflammatory and cardiovascular regulatory activity and neurological activity, inflammatory and neurological activity and metabolic, neurological and cardiovascular regulatory and metabolic activity, or any other combinations of two or more of these activities.
  • the edible product of the invention has more than one biological activity selected from angiotensin inhibitory activity, angiotensin 1 receptor inhibitory activity, cyclooxyegenase 2 inhibitory activity, lipoxygenase inhibitory activity, antiproliferative activity, anti-inflammatory activity, metabolic activity and fibril assembly inhibitory activity.
  • the edible product has angiotensin inhibitory activity and/or angiotensin 1 receptor inhibitory activity and the plant and/or fungal material the edible product is produced from is selected from grapes, pomegranate, mushrooms, cinnamon, ginger, tea, citrus, rosemary and basil.
  • the edible product has cyclooxyegenase 2 inhibitory activity and the plant and/or fungal material the edible product is produced from is selected from clove, citrus, tea and rhubarb.
  • the edible product has lipoxygenase inhibitory activity and the plant and/or fungal material the edible product is produced from is selected from chives, tea, rosemary, pomegranate, clove, cinnamon and bay leaf.
  • the edible product has antiproliferative activity and the plant and/or fungal material the edible product is produced from is selected from chives, bay leaf, tea, grape, rosemary and ginger.
  • the edible product has anti-inflammatory or proinflammatory activity and the plant and/or fungal material the edible product is produced from is selected from mushroom, rhubarb, clove, cinnamon, bay leaf, grape, tea, potato, red potato, Illawara plum, quandong, Kakadu plum, Cedar Bay cherry, riberry, onion, oregano, zucchini and spent grain.
  • the edible product has neurological activity through demonstration of anti-fibril assembly inhibitory activity and the plant and/or fungal material the edible product is produced from is selected from lettuce, rosemary, tarragon, cinnamon, clove, citrus, pomegranate, tea, grape, Illawara plum, quandong, Kakadu plum, Cedar Bay cherry, and riberry.
  • the present invention further provides a composition comprising the edible product of the invention.
  • the present invention further provides use of the edible product of the invention, or the composition of the invention in the manufacture of a food or drink product.
  • the food or drink product is a nutritional supplement.
  • the present invention further provides a food or drink product comprising the edible product of the invention, or the composition of the invention.
  • the present invention further provides use of the edible product of the invention, the composition of the invention, or the food or drink product of the invention in the manufacture of a medicament for the treatment or prevention of disease in a subject.
  • the present invention further provides the edible product of the invention, the composition of the invention or the food or drink product of the invention for use in the treatment or prevention of disease in a subject.
  • the disease is cardiovascular disease, cancer, inflammatory or autoimmune disease, a neurological disorder, and/or metabolic syndrome.
  • the cardiovascular disease is hypertension, atherosclerosis, and/or arteriosclerosis.
  • the cancer is selected from carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • the inflammatory or autoimmune disease is selected from asthma, rheumatoid arthritis, inflammatory bowel disease, psoriasis, and multiple sclerosis.
  • the neurological disorder is selected from Alzheimer's disease, Parkinson's disease, dementia, Huntington's disease, Shy-Drager syndrome, progressive supranuclear palsy, Lewy body disease and amyotrophic lateral sclerosis.
  • the present invention further provides a method for the treatment or prevention of disease in a subject, the method comprising administering to the subject the edible product of the invention, the composition of the invention, or the food or drink product of the invention.
  • the present invention further provides a method for identifying a biologically active edible product, the method comprising:
  • FIGURE 1 Optimisation of Stage 2 processing aid concentration, showing dependence of processing aid concentration on extracted solids for walnut, prepared according to Stage 1 process method, and pure limonene. All processing aids are present in each system at a concentration of 0.01%, unless the concentration is specified on the axis label and the 'water' control shows extraction of solutes from Stage 1 processing and in the absence of any processing aids in Stage 2 (but under simulated processing conditions).
  • the asterisks indicate the 'standard' conditions chosen for each additive in the optimised form of the Stage 2 'F0' process, for sample at 1% total solids.
  • FIGURE 2 Comparison of effects of different combinations of processing aids on the extraction of plant solids for (a) walnut and (b) limonene, in comparison with 10% ethanol.
  • FIGURE 3 Flow chart demonstrating processing steps from plant and fungal extracts through to preparing a crude extract at Stage 1 which can be further processed to stage 2, F0 product and finally to stage 3, F1+F2 product.
  • FIGURE 4 Comparison of the bioactivity of Stage 1 products made in the presence of reducing sugar or absence of reducing sugar.
  • A results from ATI receptor assay
  • B results from ACE assay
  • C results from Inhibition of 5-LOX assay
  • FIGURE 5 Transmission Electron Microscopy (TEM) images of amyloid beta peptide 1-42 ( ⁇ 1-42 at 25 ⁇ ) incubated for 200 mins at 37°C, in the absence (a) or presence of selected anti-fibril plant products at 0.5 mg/mL: (b) P101 1, pomegranate, (c) PI 043, black tea, (d) P0587, clove and (e) PI 071, grape seed extract.
  • TEM Transmission Electron Microscopy
  • FIGURE 6 Dose-dependent effects of processed forms of chives (PP0311); bay leaves (PP0589) and black tea (PP1043) on HT-29, SW-480, Caco-2 and HCT-116 cell lines on (A) proliferation and (B) apoptosis.
  • FIGURE 8 Comparison of Unprocessed (fresh, dried); ' Stage and 'Stage 2' forms of selected plant products showing percentage change in IFN- ⁇ compared with sample-free control for soluble extracts tested at 200 ⁇ g/ml in Whole Blood assay. Each data point in either assay represents the average of four donors ⁇ 1 standard deviation. Significant differences between data pairs are designated * (PO.05).
  • FIGURE 9 Stage 3 processing with chitosan of a selection of leads as shown, after Stage 2 (F0, plant milk) processing, showing effects of adsorption to chitosan on bioactivity of supernatants with respect to (a) 5-LOX, (b) COX2, (c) HT29 anti- proliferation and (d) apoptosis biological activities.
  • FIGURE 10 HPLC analysis of sample products.
  • the HPLC chromatograms compare 2 different methods of extraction demonstrating efficacy of Stage 2 'F0' processing, showing profiles for extraction of plant products generated from Stage 1, subsequently processed by either (A) 10% ethanol or (B) the standard Stage 2 'F0' method.
  • the term "subject" relates to an animal.
  • the subject is a mammal such as a human, or a companion animal such as a dog or cat, or a livestock animal such as horse, cow, pig or sheep.
  • the subject may be avian, for example, poultry such as a chicken, turkey or duck.
  • the subject is a human.
  • administering as used herein is to be construed broadly and includes administering an edible product as described herein to a subject as well as providing an edible product as described herein to a cell.
  • treating include administering a therapeutically effective amount of an edible product as described herein sufficient to reduce or delay the onset or progression of specified disease, or to reduce or eliminate at least one symptom of the disease.
  • preventing include administering a therapeutically effective amount of an edible product sufficient to stop or hinder the development of at least one symptom of the specified condition.
  • Plant or fungal material suitable for use in the process of the invention includes all plant and fungal material such as fruit, leaves, stems, roots, tubers, seeds, juice and pulp, as well as food processing co-products or waste material, for example spent grain from brewing. While any part of a plant of fungus may be used in the process of the invention, in some embodiments only parts of the plant and/or fungus that are commonly consumed are used and plant or fungal parts that are not commonly consumed, for example stems or skin, may be discarded.
  • the plant or fungus is one that is typically consumed by animals or humans in either a raw or cooked state. More preferably, the plant or fungus is selected from Herbs and spices such as, for example: bay leaves, chives, cinnamon, ginger, garlic, basil, cloves, rosemary, oregano, tarragon, sage, thyme, parsley, coriander or from teas, coffee beans, cocoa beans; vegetables such as, for example: rhubarb, potato, onions, zucchini, cucumber, bok choi, broccoli, choi sum, lettuce, leeks, cabbage, choko, carrot, squash, turnip, parsnip, suede, beans, beetroot, pumpkin; fruits such as, for example: native and other plums, quandongs, cheeries, peaches, nectarines, Troong, coconut, berries (for example, blueberries, raspberries, blackberries, strawberries, riberry, boysenberries, cloudberries,
  • Herbs and spices such as, for example: bay leaves, ch
  • the plant or fungal material is dispersed in an aqueous medium.
  • all that may be required for example, is to chop, blend, grind or process in a food processor the plant or fungal material to obtain the plant or fungal material dispersed in an aqueous medium.
  • the plant or fungal material may be chopped, blended, ground or processed in a suitable aqueous medium to break up solids and to achieve a dispersion of plant or fungal material at an approximate ratio of about 1 :2 of solids to liquids.
  • the skilled person would understand that the ratio of solids to liquids may be adjusted to higher or lower ratios and still be suitable for use in the process of the invention.
  • aqueous medium refers to any liquid containing water and preferably which contains a majority of water.
  • the aqueous medium may contain components other than water such as for example salts, buffers, and anti-microbial compounds such as, for example, ethanol and/or ascorbic acid.
  • suitable aqueous media include, but are not limited to, water and buffers such as phosphate buffer, carbonate buffer, acetate buffer, borate buffer, citrate buffer and Tris buffer.
  • the aqueous medium will be substantially free of organic solvent.
  • the aqueous medium may contain no more than about 5 wt. % organic solvent, desirably no more than about 1 wt.
  • the aqueous medium will be free of organic solvent.
  • the aqueous medium may also comprise a reducing sugar such as glucose, fructose, galactose lactose, or maltose.
  • a reducing sugar such as glucose, fructose, galactose lactose, or maltose.
  • glucose is added to the aqueous medium to a final concentration of about 0.5% to about 2% glucose, but more preferably about 1% glucose.
  • the present inventors have found that thermally treating the aqueous medium comprising plant and/or fungal material allows for the selection of heat stable and/or heat modified products having biological activity.
  • the aqueous medium comprising plant and/or fungal material is thermally treated at about 80°C to about 100°C, preferably about 90°C to about 100°C, or more preferably at about the boiling point of the aqueous medium.
  • the temperature at which a given aqueous medium comprising plant and/or fungal material will boil is dependent on the composition of the aqueous medium and will be affected by the concentration of, for example, salts and other solutes in the aqueous medium comprising plant and/or fungal material.
  • the period of time for which the aqueous medium comprising plant and/or fungal material is thermally treated may be about 5 minutes or, alternatively, about 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes or about 1 hour.
  • the aqueous medium comprising plant and/or fungal material is thermally treated for about 10 minutes.
  • the aqueous medium comprising plant and/or fungal material may be thermally treated using any known suitable method for heating liquids or solids for example, using a stove, oven or microwave oven, boiler or steamer.
  • the aqueous medium comprising plant and/or fungal material is sonicated to provide a greater release of bioactive material.
  • Sonication works by providing localised high pressure/low pressure cycles with concomitant high temperatures. Sonication in the food industry is traditionally used to sterilise food ingredients as it breaks down cell walls. More recently it has been of use in extracting proteins, enzymes and organic compounds contained within plants and seeds that have been initially been solvent treated. Sonication can be achieved by any suitable means known in the art using commercially available ultrasound devices.
  • the aqueous medium comprising plant and/or fungal material may be sonicated with an ultrasonic device (such as manufactured by Hielscher USA, Inc.) under the following conditions:
  • ultrasonic device and probe will depend on factors such as the volume and nature of the material to be sonicated.
  • the material may be sonicated in batch, or alternatively in flow using single or clusters of ultrasonic devices.
  • ultrasonication of the aqueous medium comprising plant and/or fungal material may result in the formation of a water dispersible or soluble emulsion. For example, if the ultrasonication is performed for sufficient time and/or at a sufficient level of power, a soluble emulsion may be formed.
  • the thermally treated and sonicated aqueous medium comprising plant and/or fungal material is subject to a further step of emulsification.
  • the aqueous medium comprising plant and/or fungal material may be emulsified with at least one emulsifying agent including, by way of non-limiting example, a lipid hydrolysing agent, a carbohydrate hydrolysing agent and/or an inorganic emulsifying agent.
  • the term "emulsifying agent” refers to any agent that directly or indirectly promotes the formation of a stable water-dispersible or soluble emulsion and includes an agent, for example an enzyme, that is able to convert a substrate into a substance with emulsifying activity.
  • an agent for example an enzyme
  • This provides a process that may be applied to any plant and/or fungal material to achieve solubilisation of both hydrophilic and hydrophobic solutes, for example, lipids and hydrophobic metabolites without the use of an organic solvent.
  • the use a lipid hydrolysing agent and/or a carbohydrate hydrolysing agent is designed to release chemical species from the plant and/or fungal material that could act as endogenous emulsifiers.
  • starch for example, wheat starch may be added.
  • the at least one emulsifying agent is an enzyme, for example a lipid hydrolysing enzyme or a carbohydrate hydrolysing enzyme.
  • the enzyme is a lipase and/or an amylase.
  • Suitable lipases can be obtained from plant, fungal, bacterial, and animal sources.
  • One suitable lipase is wheat germ lipase.
  • Suitable fungal sources of lipases include Laetiporus sulphureus, Ganoderma spp, Mucor spp, Rhizopus spp, and Penicillium spp.
  • lipases from fungi such as Candida spp and Aspergillis spp.
  • Suitable bacteria include Pseudomonas spp, Rhizobium spp or Chromobacterium spp.
  • Suitable animal sources include pigs, goats, sheep, and bovine sources.
  • Lipases are also commercially available such as those obtainable from Valley Research Technologies, South Bend, Ind., which markets Validase 8000. Mixtures of individual lipases can be used. Lipases useful in the invention are preferably those described by enzyme No. E.C. 3.1.1.3 or CAS No. 9001.62-1.
  • the enzyme is an amylase.
  • Amylases are enzymes that hydrolyse starch, glycogen and related polysaccharides and oligosaccharides.
  • Amylases include oc-amylase (EC 3.2.1.1, also known as 1,4-a-D-glucan glucanohydrolase and glycogenase), ⁇ -amylase (EC 3.2.1.2; also known as 1,4-a-D-glucan maltohydrolase; glycogenase and saccharogen amylase) and ⁇ -amylase (EC 3.2.1.3, also known as Glucan 1,4-a-glucosidase; amyloglucosidase; Exo-l,4-a-glucosidase; glucoamylase; lysosomal a-glucosidase and 1,4-a-D-glucan glucohydrolase).
  • Commercial sources of amylase include Novozymes, Genecor and Enzyme Solutions.
  • carbohydrate hydrolysing enzymes could be used in the process of the invention.
  • carbohydrate hydrolysing enzymes include, but are not limited to, glucosidases, glucanases, isoamylases, cellulases, pectinases, arabinases, inunlinases, arabinases, fructosidases, chitinases, xylanases, mannanases and pullulanases.
  • emulsifying agents as known in the art may be used to emulsify the thermally treated aqueous medium comprising plant and/or fungal material.
  • known emulsifying agents include inorganic salts such as tetra sodium pyrophosphate, monosodium phosphate, disodium phosphate, dipotassium phosphate, trisodium phosphate, sodium metaphosphate, sodium acid pyrophosphate, sodium aluminum phosphate, sodium citrate, potassium citrate, calcium citrate, sodium tartrate, and sodium potassium tartrate.
  • emulsifying agents include phospholipids and fatty acids such as stearic acid, palmitic acid, oleic acid, linoleic acid, glycerol monostearate, glycerol triacetate, lecithin, monoglyceride and triglyceride, as well as esters of fatty acids (e.g. C16-22, especially C 18 fatty acids) and polyhydric alcohols (especially Ce alcohols) or polyoxyethylated derivatives thereof, in particular the span and tween non-ionic surfactants, especially polysorbate 80 (i.e.
  • Tween® 80 ethoxylated/propoxylated block polymers, for example, poloxamers, alkylpolyglycosides, and polyacrylic acid polymers, for example, Carbopol and Pemulen type emulsifiers.
  • mechanical emulsification may be used in place of, or in addition to the use of emulsifying agents in the process of the invention.
  • any suitable form of mechanical emulsion know in the art could be used in the process of the invention, including by way of non-limiting example highspeed stirring emulsification machines such as TK homomixer (manufactured by Primix Corporation), Filmics (manufactured by Primix Corporation), Polytron (manufactured by KTNEMATICA AG), Hiscotron (manufactured by microtec nition), Cleamix W-Motion (manufactured by M Technique Corporation), high-pressure emulsification machines such as microfluidizer (manufactured by Mizuho Industrial Co., Ltd.), Ultimizer system (manufactured by Sugino Machine Limited), nanomizer (manufactured by Yoshida Kikai Co., Ltd.), Manton-Gaulin homogenizer, colloid mill
  • the process of the invention may further comprise the use of an edible adsorbent to chromatographically separate the edible product into adsorbent binding and non-binding (supernatant) edible products.
  • an edible adsorbent to chromatographically separate the edible product into adsorbent binding and non-binding (supernatant) edible products.
  • biologically active components may be fractionated and increased in either the binding or non-binding products.
  • Any suitable food-compatible insoluble material may be used as an adsorbent.
  • suitable adsorbents include chitosan, cellulose, insoluble carboxymethylcellulose, and chitin.
  • the edible adsorbent is chitosan.
  • the edible product is contacted with the edible adsorbent for a time sufficient for at least some compounds in the edible product to bind to the edible adsorbent.
  • the edible product may be contacted with the edible adsorbent for about 1 minute to about 2 hours, or about 5 minutes to about 1 hour, or about 5 minutes to about 30 minutes, or more preferably for about 15 minutes.
  • the edible product may be fractionated by chromatography in any suitable manner known in the art, including in batch or in a column.
  • the adsorbent and edible product may be gently agitated to allow compounds in the edible product to bind to the adsorbent. After agitation has ceased, and solids have been allowed to settle, the non- binding product may be removed as supernatant.
  • the adsorbent binding edible product may be released from the adsorbent by washing, or alternatively the adsorbent binding edible product may comprise the adsorbent.
  • the edible adsorbent is placed in a column, and the edible product applied to the column for time sufficient for at least some compounds in the edible product to bind to the edible adsorbent.
  • the non-binding edible product may then be collected as column flow through (supernatant).
  • the adsorbent binding edible product may be washed from the column (eluate) or alternatively the adsorbent binding edible product may be collected together with the edible adsorbent.
  • the edible products of the invention may exhibit biological activity and be useful for the modulation of biological pathways associated with disease.
  • the edible product of the invention is preferably a biologically active edible product.
  • a biologically active edible product, or a an edible product having “biological activity” may be one which is capable of regulating or modulating for example, the cardiovascular system such as by modulating blood pressure, the immunohemopoietic system, or which may affect the viability, growth and differentiation of a variety of normal or neoplastic cells in the body.
  • the biologically active edible product may be one which is capable of affecting immune regulation or which is capable of enhancing or inducing resistance to infection of cells and tissues, or which modulates energy metabolism as relevant to diabetes and metabolic disease.
  • assays There are many assays known to those skilled in the art for determining whether an edible product of the invention has a particular biological activity. Such assays include, by way of non-limiting example, assays for determining whether an edible product of the invention has cardiovascular regulatory or modulatory activity, by measuring, for example, angiotensin converting enzyme (ACE) inhibition activity or determining whether an edible product of the invention has angiotensin II receptor inhibition activity.
  • ACE angiotensin converting enzyme
  • ACE inhibition activity may be determined, for example, by monitoring the decrease in absorbance at 340 nm as a result of ACE-mediated hydrolysis of the substrate 3-(2-furylacryloyl)-L-phenylalanylglyclglycine (FAPGG) (Sigma-Aldrich, Sydney, Australia) to 3-(2-furylacryloylphenylalanine (FAP) and glyclglycine (GG) (Harjanne, 1984) in the presence of an edible product of the invention.
  • FAP 2-furylacryloyl)-L-phenylalanylglyclglycine
  • GG glyclglycine
  • Anti-inflammatory activity may be determined, for example, by generating a Thl response in human peripheral blood mononuclear cells culture by the addition of IL-7 and IL-12.
  • the stimulation of naive T-cells to mature into Thl cells causes the production of Thl associated cytokines including IFN- ⁇ (Gutcher and Becher, 2007).
  • a decrease in the level of IFN- ⁇ in a sample comprising an edible product of the invention relative to a control culture is indicative of anti-inflammatory biological activity.
  • anti-inflammatory activity may be determined by testing for cyclooxygenase-2 (COX-2) inhibitory activity or for 5 -lipoxygenase inhibitory activity. Assays for testing for COX-2 inhibitory activity and 5-lipoxygenase inhibitory activity are known in the art.
  • Anticancer activity can be assessed by measuring antiproliferative effects on certain cancer cell lines.
  • One example of a suitable assay is testing for inhibition of HT29 human adenocarcinoma cell proliferation and apoptosis as described herein.
  • Other cell lines suitable for use in antiproliferation assays include Caco-2, SW480 and HCT1 16 human colon cancer cell lines.
  • an assay for determining fibril inhibition activity of an edible product fraction utilises reduced and carboxymethylated ⁇ -casein (RCM-K-CN) which forms amyloid fibrils under physiological conditions.
  • RCM-K-CN carboxymethylated ⁇ -casein
  • Other assays for determining fibril inhibition activity include assays to test for inhibition of amyloid fibril formation of ⁇ 1 -42 and assays to test for inhibition of beta secretase.
  • an edible product obtained by the process of the invention is included in a food or drink product.
  • Food or drink products according to the invention can be prepared by the skilled person using known techniques. Non-limiting examples of such food or drink products are baked goods, dairy type foods and drinks, snacks, etc.
  • the amount of the edible product of the invention to be used in a food or drink product can be variable as the edible products themselves are nutrients.
  • the food or drink product is a nutritional supplement.
  • a "nutritional supplement” is an orally ingestible product consumed to improve overall nutrition, health, well-being, or performance of a subject in an activity and/or an orally ingestible product which provides additional perceived nutritional or biological benefit to a subject.
  • the nutritional supplement may be provided in a concentrated form, thus allowing for the addition of the nutritional supplement to a food or drink product to allow for the consumption of a desired quantity of an edible product of the invention in a reasonable serving size.
  • the food or drink product of the present invention can include additional ingredients including an orally ingestible diluent or carrier.
  • an orally ingestible diluent or carrier are known in the food sciences. These include, but are not limited to, manufactured cereals, fruit or vegetable products, beverages or beverage concentrates, ground meat products or vegetable analogues thereof, and any inert diluent, carrier, or excipient known in the pharmaceutical art.
  • the edible products of the current invention constitute from about 0.0001 to about 20% by weight of the food or drink product.
  • the bioactive edible product could be incorporated into processed foods including but not limited to: breads, pastas, biscuits, sauces, soups, bars, dairy products (for example, milk, yoghurts, cheese, ice cream), cakes, ready-to- eat prepared meals, and breakfast cereals.
  • the nutritional supplement of the current invention can include additional ingredients.
  • more than one of the edible products of the current invention can be included in the same nutritional supplement formulation.
  • Other additional ingredients include any ingestible product.
  • Preferred additional ingredients include, but are not limited to, other active food supplement ingredients such as micro- nutrients: vitamins and minerals or macro-nutrients such as polyunsaturated fatty acids or fibre.
  • the food additive may also include acceptable dispersing and suspending agents, and water.
  • Other conventional nutritional supplements can also be included if desired.
  • the nutritional supplement can take many forms including, but not limited to, powders, tablets, capsules, solutions, concentrates, syrups, suspensions, or dispersions.
  • the present invention provides compositions comprising an edible product of the invention and a suitable carrier or excipient.
  • the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
  • the edible products obtained by the methods of the invention are of use in the inhibition of biological pathways associated with disease including cardiovascular disease, inflammatory diseases, neurological disorders, cancer and metabolic syndrome.
  • the present invention provides methods of treating or preventing disease by administering to a subject an edible product of the invention or a composition of the invention.
  • an edible product of the invention may be used in the treatment or prevention of cardiovascular disease in a subject, such as for example, for lowering blood pressure in a subject.
  • Using the edible products of the invention to lower blood pressure in a subject may decrease the risk of, for example, heart failure, kidney failure, and the harmful effects of diabetes.
  • the edible product of the invention may be used for the treatment or prevention of cardiovascular diseases including, but not limited to hypertension, atherosclerosis and arteriosclerosis.
  • the edible products of the invention are useful in the treatment or prevention of cancer in a subject, including, but not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, bladder cancer, hepatoma, colorectal cancer, uterine cervical cancer, endometrial carcinoma, salivary gland carcinoma, mesothelioma, kidney cancer, vulval cancer, thyroid cancer, hepatic carcinoma, skin cancer, melanoma, brain cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma and peripheral neuroepithelioma.
  • carcinoma lymphoma
  • blastoma
  • the edible product of the invention may be used for the treatment or prevention of inflammatory and autoimmune disease.
  • Inflammatory disorders and autoimmune diseases which may be treated with peptide fractions of the invention include asthma, rheumatoid arthritis, inflammatory bowel disease, psoriasis and multiple sclerosis.
  • Autoimmune diseases arise from an overactive immune response of the body against substances and tissues normally present in the body.
  • autoimmune diseases include Addison's disease, alopecia areata, ankylosing spondylitis, antiphospho lipid antibody syndrome (APS), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid, coeliac disease, chagas disease , chronic obstructive pulmonary disease, Crohns disease (one of two types of idiopathic inflammatory bowel disease "IBD”), dermatomyositis, diabetes mellitus type 1, endometriosis, goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's disease, hidradenitis suppurativa, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Interstitial cystitis, lupus erythematosus, mixed connective tissue disease, morphea, multiple sclerosis (MS), myasth
  • the edible product of the present invention may also be used for the treatment or prevention of a neurological disorder, such as, but not limited to Alzheimer's disease, Parkinson's disease, dementia, Huntington's disease, Shy-Drager syndrome, progressive supranuclear palsy, Lewy body disease or amyotrophic lateral sclerosis.
  • a neurological disorder such as, but not limited to Alzheimer's disease, Parkinson's disease, dementia, Huntington's disease, Shy-Drager syndrome, progressive supranuclear palsy, Lewy body disease or amyotrophic lateral sclerosis.
  • the edible product of the present invention may also be used for the treatment or prevention of metabolic syndrome.
  • “Metabolic syndrome” also known as syndrome X or insulin resistance syndrome
  • a patient has metabolic syndrome if insulin resistance and/or glucose intolerance is present together with two or more of the following conditions: 1) reduced glucose tolerance or diabetes; 2) reduced insulin sensitivity (under hyperinsulinemic, euglycemic conditions corresponding to a glucose uptake below the lower quartile for the background population); 3) increased blood pressure (above 140/90 mmHg); 4) increased plasma triglyceride (above 1.7 mmol/1) and/or low HDL cholesterol ( ⁇ 0.9 mmol/1 for men; ⁇ 1.0 mmol/1 for women); 5) central adiposity (waist/hip ratio for men: >0.90 and for women >0.85) and/or Body Mass Index>30 kg/M 2 ); 6) micro albuminuria (urine albumin excretion
  • the edible product of the present invention may provide protective or prophylactic benefits from one or more of the above diseases or disorders.
  • the edible product of the present invention may provide protective or prophylactic benefits from one or more of the above diseases or disorders.
  • the edible product may provide protective or prophylactic benefits from one or more of the above diseases or disorders.
  • the onset of neurological disease may be delayed, which may be a result of both the anti-inflammatory and anti-fibril activity associated with the product of the invention.
  • Food plant and fungal samples were washed and prepared so only edible fractions remained. That is, all plant parts such as leaves, stems, stalks, roots, seeds, skin that are typically eaten were prepared. In some cases the skin, stems and seeds that are not normally consumed were discarded.
  • the raw edible plant and fungal material was processed in a food processor before dividing into two batches for further processing with either water or 1% glucose solution. Edible plant and fungal materials were dispersed in water (approximately 1 :2 ratio of solids to liquid), and blended to break up solids. Samples were sealed in a microwave rice cooker and boiled by microwave heating for 10 minutes.
  • the dried crude products were reconstituted at 2% total solids into purified water and stirred for 2 hours.
  • processing aids wheat-based cornflour (0.01% w/v, Home brand, Australia); Clarase G Plus (0.001% w/v, Enzyme Solutions, Australia); Fungal Lipase 8000 (0.01% w/v, Enzyme Solutions, Australia); tetra sodium pyrophosphate (0.01% w/v, BDH, England), and the pH adjusted to 5.5 using 1.0 M sodium hydroxide (Merck, Darmstadt, Germany) or 1.0 M hydrochloric acid (Chem Supply, Sydney, Australia).
  • the development of the Stage 2 processing method was intended to represent a generic, food-compatible processing method for application to any non-protein-rich food material.
  • concentrations of processing aids wheat starch (WS), Clarase (C), tetra-sodium pyrophosphate (TSP) and lipase (L) were selected as based on optimal extraction of total soluble solids for the model systems: walnut (subjected to Stage 1 process method) and limonene (pure standard).
  • WS wheat starch
  • C Clarase
  • TSP tetra-sodium pyrophosphate
  • L lipase
  • These substances provided model systems for (a) high content of insoluble carbohydrate and lipid (walnut) and (b) a pure standard hydrophobic metabolite, of the carotenoid chemical class (limonene).
  • the additives and enzyme treatments were intended to collectively provide 3 types of natural emulsifier, including fatty acid, non-ionic carbohydrate and inorganic (ie, TSP), to stabilise hydrophobic extracted species of a broad range of phytochemical classes.
  • natural emulsifier including fatty acid, non-ionic carbohydrate and inorganic (ie, TSP), to stabilise hydrophobic extracted species of a broad range of phytochemical classes.
  • bioactivity could be further enhanced or moderated by further purification.
  • Adsorption of certain undesirable inhibitors could be achieved by chromatography.
  • chitosan IMCD, China
  • IMCD methyl methacrylate
  • a suspension of F0 product at 10% dry solids w/w (100 g) was mixed and gently agitated with 10 g of washed chitosan for 15 minutes.
  • the chitosan non-binding solute sample was recovered by collecting the supernatant and storing at 4°C. The chitosan was then washed with 4 volumes of 10 mM sodium chloride (Ajax, Australia) by agitation for 15 minutes at room temperature. After centrifugation (J6HC model centrifuge, Beckman Coulter, California, USA, 4000 rpm for 5 minutes at 4°C), the optical density of the filtered 0.2 ⁇ syringe filter, Pall Filtration, New York, USA) supernatant was determined (Shimadzu, Japan) at 500 nm. Washing was repeated until no further elution of solutes absorbing at 500 nm.
  • the chitosan was similarly washed with purified water until there was no further evidence of elution of solutes, before combining all supernatants and freeze drying.
  • the dried product containing chitosan non-binding solutes was referred to as the Fl+2 product.
  • the dried chitosan together with chitosan-bound solutes was referred to as the F3 product. All products so prepared were edible, with the option of removal of the salt present in the Fl+2 fraction by nano-filtration as required. In some cases, the supernatants eluted without and with salt, Fl and F2, respectively were not mixed and dried and tested separately. These samples are designated Fl and F2 instead of Fl+2.
  • the assay for the determination of pig kidney ACE (EC 3.4.15.1) (Sigma- Aldrich, Sydney, Australia) employed a COBAS BIO autoanalyser (Roche Diagnostics Ltd, Rotnch, Switzerland) which monitored the decrease in absorbance at 340 nm as a result of ACE-mediated hydrolysis of the substrate 3-(2-furylacryloyl)-L- phenylalanylglyclglycine (FAPGG) (Sigma-Aldrich, Sydney, Australia) to 3-(2- furylacryloylphenylalanine (FAP) and glyclglycine (GG) (Harjanne A 1984).
  • reagent buffer as 100 mM Tris-HCl pH 8.3, 300 mM NaCl, 10 ⁇ ZnCl 2 containing ACE at 0.032 U/ml was loaded from a buffer boat and 20 xL of test sample (10-50 mg/ml) or known ACE inhibitors such as captopril (1-100 nM) or the tripeptide valinealanineproline (VAP, 0.1-20 ⁇ ) (Auspep Pty Ltd, Melbourne, Australia) found in as 1 -casein (Yamamoto N 1997) from the sample carousel are transferred into the multiple cuvette and spun in 2 minutes to warm to 37°C.
  • test sample 10-50 mg/ml
  • known ACE inhibitors such as captopril (1-100 nM) or the tripeptide valinealanineproline (VAP, 0.1-20 ⁇ ) (Auspep Pty Ltd, Melbourne, Australia) found in as 1 -casein (Yamamoto N 1997) from the sample carousel are transferred into
  • the solution was filtered through two pieces of nylon mesh and washed through with 5 ml of isolation media and centrifuged at 3,000 x g for rat liver or 6,000 x g for pig heart in a J2-21 centrifuge (Beckman Instrument Co., Palo Alto, CA) for 30 min at 4°C.
  • the supernatant was decanted and retained and the pellet resuspended with a rubber policeman in isolation medium and recentrifuged as before.
  • the supernatants were combined and spun at 46,000 x g for 30 min.
  • the final pellet was suspended (in mM) 50 Tris, 10 MgCl 2 , 1 EGTA, pH 7.4 assay buffer with 5 stokes of a dounce glass/teflon homogeniser that contained protease inhibitors diluted into the buffer at 1 : 1000 (vol/vol).
  • the stock protease inhibitor concentrations were (mg/ml, solvent) phenylmethylsulfonyl fluoride 17.4, DMSA; aprotinin 1, H 2 0; iodoacetate 184, H 2 0; and pepstatin A 1.37, DMSO.
  • the final stock concentration of liver or heart membrane preparations was 1-2 mg/ml and stored in aliquots at -80°C.
  • Receptor radioligand binding activity was determined for rat liver membrane fraction (3,000-46,000 x g) with the All antagonist II (2200 Ci/mmol, 10 ⁇ in 200 ⁇ assay buffer diluted to 1 nM stock) (Perkin Elmer, MA, USA) which is specific for the ATi receptor at sub 3 ⁇ 4 concentration of 0.1 nM (Del Carmen Caro M et al, 1998; Leifert et al, 2009).
  • the assay final volume of 75 ⁇ in a 4 ml plastic tube contained (in mM) 90 Tris, 10 MgCl 2 , 1 EGTA, pH with 0.2% BSA with buffer or the antagonists losartan (10-1000 nM), saralasin (1-100 nM), PD12319 (10-10,000 nM, AT 2 antagonist) or agonist angiotensin II (1-100 nM) (all Sigma-Aldrich, Sydney, Australia) or the prepared library fractions or products.
  • Plant or fungal products were dissolved in 10% ethanol (v/v) in (mM) 90 Tris, 10 MgCl 2 , 1 EGTA adjusted to pH 7.4 with 2 N NaOH, spun at 8,000 x g (Sigma 113 Laborzentrifugen, Ostrode am Harz, Germany) and added at 0.01-10 mg/mL final in the assay.
  • the assay contents were pre-incubated with of 7 ⁇ of rat liver membrane preparation containing approximately 7 ⁇ g protein for at least 10 min.
  • the assay is commenced by the addition of 7.5 ⁇ 1 nM [ 125 I]-[Sar 1 -Ile 8 ]-Angiotensin II (0.1 nM final) and the binding reaction allowed to proceed for 50 min while shaking at 25°C (Del Carmen Caro, M. et al, 1998). Non specific binding was determined in the presence of unlabelled All or losartan and did not exceed 5%.
  • Linoleic acid, N-N-N'-N'- tetra-methyl-p-phenylenediamine (TMPD) and fine chemicals were obtained from Sigma (St. Louis, MO, USA).
  • Cyclooxygenase-2 prostaglandin H synthase, ovine
  • DuP697 and arachidonic acid were obtained from Cayman Chemicals (MI, USA).
  • Cyclooxygenase-2 (COX-2, ovine) enzyme catalyses the formation of prostaglandin ⁇ (PGG 2 ) from arachidonic acid (oxidation reaction), and its subsequent reduction to prostaglandin-H 2 (PGH 2 ).
  • COX-2 enzyme was mixed with an ice-cold solution of 4 ⁇ hematin, 0.1% (v/v) Tween-20 and 0.1% (w/v) gelatine in 100 mmol/L phosphate buffer pH 6.4.
  • the mixture was dispensed into a 96 well plate at 180 ⁇ per well for an equivalent of 50 U COX-2 enzyme per well (and an absorbance change of 0.025 per min in the blank reaction).
  • Four of test solutions, blank (10% ethanol) or the selective COX-2 inhibitor DuP697 were added to each well in duplicate and the plate mixed for 3 s and incubated at 30°C.
  • DuP697 was serially diluted for final concentrations of 24, 2.4 and 0.24 ⁇ .
  • After 10 min the reaction was started by the addition of 20 xL TMPD and arachidonic acid pre-mixed immediately prior to use in phosphate buffer.
  • the reaction mix contained a final concentration of 100 mmol/L TMPD and arachidonic acid.
  • the final inhibition of the enzyme activity during the reduction (peroxidation) reaction was determined by measurement of the colour produced by the co-substrate TMPD in a Spectramax 250 spectrophotometer at 610 nm for 20 min.
  • NDGA Nordihydroguaiaretic acid
  • linoleic acid and fine chemicals were obtained from Sigma (St. Louis, MO, USA).
  • 5 -lipoxygenase (5LOX, potato) was obtained from Cayman Chemicals (MI, USA). Plant and fungal extracts were mixed at 50 mg/mL in 10% ethanol for 2 h, centrifuged at 10,000 x g for 4 min and the supernatant retained for testing. Using a 96 well plate format 10 xL of blank, test solutions or NDGA inhibitor were mixed with 200 ⁇ L lipoxygenase solution (10 U of 5 -lipoxygenase in 100 mmol/L phosphate buffer, pH 7.0).
  • 5-lipoxygenase activity was also determined by measurement of oxygen consumption in a Hanstech Oxygraph.
  • Ten ⁇ , of the control, inhibitor, or test solution were added to 200 ⁇ , (50 U) lipoxygenase solution in 100 mmol/L phosphate buffer, pH 6.3 and gently mixed in a tube.
  • the mixture was transferred to a Hansatech oxygraph chamber maintained at 25°C. Recording of oxygen consumption was continuously monitored while stirring the mixture until a steady state was reached after which time 10 ⁇ L of linoleic acid substrate was added (for a final concentration of 200 ⁇ /L) to start the reaction.
  • the rate of oxygen consumption was recorded during the linear phase of the reaction (during the first 30 sec).
  • the decrease in oxygen consumption by the known inhibitor or the test solution was expressed as percentage inhibition.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS Fetal bovine serum
  • FBS antibiotic/antimycotic solution
  • CellTiter-Blue ® and Apo-ONE ® assay kits were purchased from Promega (WI, USA).
  • Human colon adenocarcinoma (HT29), cells were obtained from the American Tissue Culture Collection (ATTC; Rockville, MD, USA).
  • Monolayers of cells were cultured in T-75 flasks and maintained in DMEM (Sigma) supplemented with 10% (v/v) FBS, 1% antibiotic/antimycotic solution, 0.375% NaHC0 3 and 25 mM HEPES pH 7.4, in a humidified atmosphere of 5% CO 2 in air at 37°C.
  • DMEM fetal calf serum
  • 1% antibiotic/antimycotic solution 0.375% NaHC0 3 and 25 mM HEPES pH 7.4
  • cells were dispersed with Trypsin-EDTA (0.05% Trypsin and 0.02% EDTA) for maintenance and passaging or plated in black flat bottom 96-well plates for cell proliferation studies.
  • Plant extracts were mixed at 50 mg/mL in 10% ethanol for 2 h, centrifuged at 10,000 x g for 4 min and the supernatant retained for testing.
  • Cell proliferation was determined using the CellTiter-Blue ® assay which provides a homogenous, fluorometric method of estimating cell viability in multiwell plates.
  • Cells were plated at 15,000 cells/well in a black, flat bottomed 96-well plate in DMEM culture media described above for 24 h.
  • Plant and fungal test extracts at 50 mg/mL in 10% ethanol were prepared in the DMEM culture media containing 50 mM HEPES pH 7.4, and sterile filtered through 0.22 ⁇ filters.
  • the medium in the plate was replaced with 100 ⁇ of the culture medium containing the test compound, 10% ethanol blank or 10 mM of the inhibitor butyrate.
  • This assay was similar to the above HT29 cell assay with the following exceptions.
  • Caco-2, SW480 and HCT 1 16 human colon cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). Caco-2 cells were cultured in DMEM supplemented with FBS (20% (v/v), 1% 100 x sodium pyruvate and 1% 100 x NEAA. SW480 cells were cultured in RPMI-1640 media supplemented with 10% FBS and HCT1 16 cells were cultured in MCoy's 5 A media supplemented with 10% FBS. Anti-T Helper Cell Type I-Mediated Inflammation Activity
  • Thl response in a human Peripheral Blood Mononuclear Cell (PMBC) culture system was generated by the addition of interleukin 7 (IL7), for homeostatic proliferation and survival of naive T-cells (Rathmell et al., 2001); and interleukin 12 (IL12) to promote the stimulation of naive T-cells to mature into Thl cells.
  • IL7 interleukin 7
  • IL12 interleukin 12
  • the stimulation of naive T- cells to mature into Thl cells causes the production of Thl associated cytokines including IFN- ⁇ .
  • Anti-inflammatory effects of edible products were demonstrated by introducing the test substance into a culture containing PBMC, IL-12 and IL-7 and monitoring levels of IFN- ⁇ . A decrease in the levels of IFN- ⁇ relative to a control culture, where the edible product was not present, was taken as indicating an anti-inflammatory effect while an increase in the level of IFN- ⁇ relative to control was taken to indicate a proinflammatory response.
  • RCM-K-CN carboxymethylated ⁇ -casein was reduced and carboxymethylated (RCM- kCn) as previously described (Bennett et al, 2009).
  • Our model target protein, RCM-K- CN which forms amyloid fibrils under physiological conditions, was used in a primary high throughput microtitre plate screen for potential inhibitors of amyloid formation.
  • RCM-K-CN was incubated at 37 °C in 50 mM phosphate buffer, pH 7.4 at 0.5 mg/mL. Screened samples were prepared by weighing out appropriate quantities and diluted to double final concentration in the assay using 10% ethanol for plant samples.
  • ⁇ and ⁇ 5 represents the change in ThT fluorescence after 1000 mins for RCM-K-CN in the absence and presence of the tested sample respectively.
  • the samples were used at a number of concentrations and the percent inhibition data were used to calculate the IC50 for that tested samples (i.e. the concentration of solids able to inhibit the increase in ThT fluorescence by 50%) using PRISM software.
  • Beta-Amyloid (1-42) was purchased from Anaspec Company (San Jose, CA, USA) and then dissolved with 60 ⁇ _ 1.0% NH 4 OH. MilliQ water was added to make a 250 ⁇ stock solution, which was aliquoted and stored at - 80 °C until use.
  • Amyloid fibril formation by ⁇ 1-42 was monitored using an adapted in situ ThT binding assay similar to that described above for RCM-K-CN. 25 ⁇ ⁇ 1-42 in 50 mM phosphate buffer (pH 7.4) with 100 mM NaCl was incubated at 37 °C in black ⁇ 96-microwell plates (Greiner Bio-One, Stonehouse, UK) either in the presence or absence of tested samples. The percent protection afforded by each sample was calculated from the ThT fluorescence data at 200 mins by the same equation as used for the RCM-K-CN ThT assay (see above).
  • Dried plant samples were dispersed in 10% ethanol at approximately 50 mg/mL total solids by vortexing and extracted for 30 mins using gentle agitation, at room temperature.
  • Supernatants containing extracted solutes were diluted by 1/100 into sodium acetate buffer (0.1 M, pH 4.5).
  • a working sample aliquot (25 ⁇ ) was pre- incubated (15 mins at room temperature) with BACE1 enzyme (25 ⁇ L, 4 ⁇ g/mL, Calbiochem) before adding the fluorescent substrate (50 ⁇ ,, 25 ⁇ , Calbiochem) and commencing monitoring of the reaction.
  • the positive control for the assay was a peptide inhibitor of ⁇ -Secretase (APP ⁇ -Secretase Inhibitor, Calbiochem, 171601).
  • the enzyme inhibition assay was adapted to a high-throughput format using 96-well plate format and microplate fluorescence reader (Biotek FL600, Bio-Tek Instruments, VT, USA), monitoring for 2 hours at room temperature, at excitation and emission wavelength settings of 360 and 485 nm, respectively. Enzyme activity was detected as a linear increase in florescence over the 2 hour monitoring timeframe, and inhibition was measured as the percentage reduction in the fluorescence increase, compared to the control reaction. For dose response experiments, the concentration of extracts was the only variable altered, with all other conditions remaining constant.
  • RAW264.7 cells obtained from Ian Cassidy, University of Queensland, Australia
  • Ni l microglia obtained from the University of Tublingen, Germany
  • DMEM Dulbecco's Modified Eagle's Medium
  • FCS fetal calf serum
  • penicillin 200U/ml
  • streptomycin 200 ⁇ g/ml
  • Fungizone 2.6 ⁇ g/ml
  • Samples to be tested were dissolved in DMSO, 95% ethanol or ddH 2 0 to concentrations of lOOmg/ml. Dilutions were then made in media from these concentrates, so that the maximum solvent content did not exceed 0.05% of the final well volume. All stock solutions were stored at -20°C and dilutions in media were stored at 4°C for no longer than one week.
  • Nitric oxide production was monitored by measuring the concentration of nitrite in the media using the 'Griess reagent' (Fox et al., 1982).
  • Conditioned media (50 ⁇ ) from each well was transferred to a fresh 96-well plate and 25 ⁇ 1 of Reagent 1 (l%w/v sulfanilamide in ddH 2 0) and 25 ⁇ of Reagent 2 (0.1%w/v naphthyethylene-diamine in 5% HCl) were added and the absorbance at 540nm determined using a plate reader (Multiskan Ascent with Ascent software v2.4, Labsystems).
  • the concentration of TNF, following 24 hours of incubation of cells with LPS was determined by a Sandwich Enzyme -Linked Immunosorbent Assay (ELISA), according to the manufacturer's manual (Peprotech). Briefly, capture antibody was used at a concentration of ⁇ g/ml in PBS (1.9mM NaH 2 P0 4 , 8.1mM Na 2 HP0 4 , 154mM NaCl) (pH 7.4). Serial dilutions of TNF standard from 0 to 10000 pg/ml in diluent (0.05% Tween-20, 0.1% BSA in PBS) were used as internal standard. TNF was detected with a biotinylated second antibody and an Avidin peroxidase conjugate with ABTS as detection reagent. Colour was determined at 405nm in a 96 well plate reader.
  • ELISA Sandwich Enzyme -Linked Immunosorbent Assay
  • MTT 3-(4,5-dimethylthiazol-2-yl)2,5- diphenyl tetrazolium bromide
  • Stage 1 and Stage 2 edible products produced by the method of the invention were tested in assays related to anti-oxidant activity and sugar metabolism.
  • the Stage 1 and Stage 2 products were prepared as described above.
  • Antioxidant activity of edible products was measure by ORAC assay (Huang et al, 2002). Capacity for inhibition of digestion of starch by a-Amylase was determined by colorimetric reaction for detection of residual starch by iodine, at 630 nm (Hall et al, 1970). Capacity for inhibition of digestion of trioleoylglycerol (4-MUO) by pancreatic lipase was determined by fluorescence detection of the product at 320/450 nm excitation and emission wavelengths (Jacks and Kircher, 1967). Capacity for inhibition of digestion of maltose by Maltase was determined by detection of the reaction product glucose (Giorgi et al, 1992). Capacity for inhibition of digestion of sucrose by Sucrase was determined by detection of the reaction product glucose (Giorgi et al., 1992).
  • Stage 1 processing was carried out in the presence of glucose for a select subset of plants and fungi.
  • Figure 4 illustrates a summary set of data (from the cardiovascular or inflammatory assays).
  • COX-2 inhibitory activity of a product made from Brewers Spent Grain was discovered.
  • the bioactivity was found to be dependent on several aspects of the processing method including: (a) presence of glucose (See Table 11); (b) use of ultrasonics; and (c) use of membrane filtration to recover low molecular mass fraction and increase specific activity of product.
  • the effects of variation in conditions of ultrasonication on COX-2 inhibitory activity of Stage 1 products of Brewers Spent grain is provided in Figure 7.
  • a selection of plant products were further studied to exemplify the effects of processing on either pro- or anti-inflammatory bioactivity.
  • Processed (Stage 1 and Stage 2 products) forms of clove and bay leaf exhibited anti-inflammatory activity while cinnamon and rhubarb were selected as examples of plant products for which Stage 2 processing significantly concentrated pro-inflammatory as opposed to antiinflammatory bioactivity.
  • Plant extracts were mixed at 10 mg/mL in water for 2 h, centrifuged at 10,000 x g for 4 min and the supernatant retained for chitosan treatment.
  • 1 mL supernatant was mixed with 100 mg chitosan for 1 h and centrifuged as above.
  • a second chitosan treatment was applied for 1 h with 0 to 50 mg chitosan mixed with 250 mL supernatant and centrifuged.
  • the second chitosan-treated supernatant was used to assess the residual ability of the solution to inhibit lipoxygenase and cyclooxygenase activity at 323 ⁇ !7 ⁇ and 200 ⁇ !7 ⁇ respectively.
  • Cell proliferation and apoptosis of HT-29 human adenocarcinoma cells were assessed using the above chitosan-treated solution at 500 ⁇ . final concentration in the assays.
  • Figure 9 illustrates the results of the various assay testing.
  • the adsorption of 5- LOX inhibitory species onto chitosan was gradual over the chitosan titration range with 20-30% of bioactivity being lost from the supernatant and adsorbing onto chitosan, for black tea, green tea and pomegranate skin, from an initial sample loading of 320 ⁇ g of total soluble solids ,
  • Table 1 Summary of Angiotensin Converting Enzyme inhibition bioactivity of food plant products.
  • the reference positive control for the assay was Captopril giving 73.1% inhibition at 29 nM.
  • Table 2 Summary of Angiotensin 1 receptor inhibition bioactivity of food plant products.
  • the reference positive control for the assay was Losartan giving 60.3% inhibition at 100 nM.
  • Table 3 Summary of Cycloxygenase 2 enzyme inhibition bioactivity of food plant products.
  • the reference positive control for the assay was DuP697 with IC50 of 20 ⁇ .
  • Table 4 Summary of 5-Lipoxygenase enzyme inhibition bioactivity of food plant products.
  • the reference positive control for the assay was NDGA with IC50 of 10 [
  • Table 5 Summary of inhibition of HT29 cellular proliferation and apoptosis, of food plant products.
  • the reference positive control for the assay was butyrate at 10 mM which inhibited HT29 cell proliferation by 50 to 80% at 10 mM and with apoptosis reading of 3900 relative fluorescence units.
  • Table 7 Summary of inhibition of RCM-kCn fibril assembly bioactivity of food plant products.
  • Table 9 Summary of inhibition of human beta secretase bioactivity by food plant products.
  • the positive control used in the assay was APP ⁇ -Secretase Inhibitor with an IC50 of 242 nM.
  • Table 11 Summary of IC50 of COX2 inhibitory activity in the Stage 1 product of Brewers Spent grain (processed with glucose), showing that bioactivity was enriched in the molecular size range of ⁇ 4.3 kDa.
  • Table 13 Summary of Plant library products with highest a-Amylase inhibitory activity.

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Abstract

La présente invention concerne un procédé à étages pour préparer une émission dispersible dans l'eau à partir de matériau végétal et/ou fongique pour préparer des produits comestibles bioactifs. Ce procédé est basé sur une combinaison de traitements thermiques et physiques. L'invention concerne en outre l'utilisation des produits comestibles dans des produits alimentaires et des boissons et pour le traitement et la prévention de maladie.
PCT/AU2010/001510 2009-11-11 2010-11-11 Procédé pour l'enrichissement en agent bioactif de plante WO2011057340A1 (fr)

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WO2014175929A2 (fr) * 2013-04-25 2014-10-30 Jeffrey Alan Mckinney Compositions et procédés pour l'amélioration de l'insuffisance rénale chronique et des symptômes associés chez des animaux domestiques
DE102014018564A1 (de) * 2014-12-15 2016-06-16 Heinz Saborowski Pflanzlichter Wirkstoff, Hemopatisches Erzeugnis Schmerzmittel, Heilmittel aus der Frucht der Bio Zuchinipflanze. Der Wirkstoff ist einsetzbar gegen Rheumatische Schmerzen wie Rücken, Schulter, Gicht Gelenke, Artritis, schmerzende Beine
WO2020014745A1 (fr) * 2018-07-18 2020-01-23 Mycelium Biotech Assets Pty Ltd Extrait bioactif
WO2021009450A1 (fr) 2019-07-15 2021-01-21 Genialis Procede d'amelioration de la biodisponibilite de composes actifs hydrophiles dans une solution aqueuse

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EP1702675A1 (fr) * 2003-12-18 2006-09-20 Gat Formulation Procede de multi-microencapsulage en continu pour l'amelioration de la stabilite et stockage d'ingredients biologiquement actifs
WO2007090614A1 (fr) * 2006-02-06 2007-08-16 Dsm Ip Assetts B.V. Compositions d'ingredients actifs
WO2009092754A1 (fr) * 2008-01-22 2009-07-30 Hamlet Protein A/S Composition comprenant des protéines et des graisses dispersées
WO2010126839A2 (fr) * 2009-05-01 2010-11-04 Parma Laboratories Inc. Aliments pour animaux, aliments et biocarburants traités et leurs procédés de fabrication et d'utilisation

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014175929A2 (fr) * 2013-04-25 2014-10-30 Jeffrey Alan Mckinney Compositions et procédés pour l'amélioration de l'insuffisance rénale chronique et des symptômes associés chez des animaux domestiques
WO2014175929A3 (fr) * 2013-04-25 2015-01-22 Jeffrey Alan Mckinney Compositions et procédés pour l'amélioration de l'insuffisance rénale chronique et des symptômes associés chez des animaux domestiques
DE102014018564A1 (de) * 2014-12-15 2016-06-16 Heinz Saborowski Pflanzlichter Wirkstoff, Hemopatisches Erzeugnis Schmerzmittel, Heilmittel aus der Frucht der Bio Zuchinipflanze. Der Wirkstoff ist einsetzbar gegen Rheumatische Schmerzen wie Rücken, Schulter, Gicht Gelenke, Artritis, schmerzende Beine
WO2020014745A1 (fr) * 2018-07-18 2020-01-23 Mycelium Biotech Assets Pty Ltd Extrait bioactif
US11857587B2 (en) 2018-07-18 2024-01-02 Mycelium Biotech Assets Pty Ltd Bioactive extract
WO2021009450A1 (fr) 2019-07-15 2021-01-21 Genialis Procede d'amelioration de la biodisponibilite de composes actifs hydrophiles dans une solution aqueuse
FR3098732A1 (fr) * 2019-07-15 2021-01-22 Genialis Procede d’amelioration de la biodisponibilite de composes hydrophiles dans une solution aqueuse

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