Iron-containing green plant material concentrate

Abstract

A method for preparing an iron-containing green plant material concentrate is disclosed. The method comprises the step of suspending a green plant in an aqueous liquid to form a green plant material suspension. The green plant material suspensions are then blended, treated by physical means, and optionally dried. This iron-containing green plant material concentrate is also disclosed. In addition, food products, beverages, food supplements, cosmetic compositions or pharmaceutical compositions comprising such iron-containing green plant material concentrates, and methods for fortifying a food product or beverage with iron are also disclosed.

Description

TECHNICAL FIELD

[0001] The present invention relates generally to the field of iron-containing ingredients derived from plants. For example, the present invention relates to a process for preparing an iron-containing green plant material concentrate and to an iron-containing green plant material concentrate thereof. The present invention also relates to a food product, a beverage, a food supplement, a cosmetic composition or a pharmaceutical composition comprising such an iron-containing green plant material concentrate. The present invention also relates to a process for fortifying a food product or a beverage with iron, which process involves the use of such an iron-containing green plant material concentrate. BACKGROUND

[0002] According to the WHO, iron deficiency is the most common nutritional deficiency worldwide. Nearly 30% of the world’s population suffers from anemia, half of which is due to iron deficiency (Gupta et al., 2020). However, iron fortification is not an easy task, which requires considering the selection of the right source and mixing procedures to avoid the occurrence of sensory issues such as metallic taste and discoloration.

[0003] Commercially available solutions mainly correspond to inorganic iron salts. These inorganic solutions present many drawbacks. First, they are not natural as they are obtained by chemical reactions / extracted from mines. In addition, the consumer acceptance of iron salts is lower compared to the real food ingredients delivering the same micronutrient. Finally, inorganic iron salts are usually poorly absorbed as they can form low solubility complexes in the weakly basic environment of the upper small intestine.

[0004] An alternative solution to inorganic iron salts are organic iron compounds from animal sources, such as hemoglobin, myoglobin or cytochrome forms. These organic iron compounds from animal sources are better absorbed by the human body than their inorganic counterparts. However, they are not suitable for a vegetarian / vegan diet given their source.

[0005] Another alternative solution to inorganic iron salts are organic iron compounds from plant sources. In plants, iron is present as iron-protoporphyrin in cytochrome b or as plant ferritin. However, organic iron compounds from plant sources still present some drawbacks. First, plants contain anti-nutritional factors such as oxalic acid and phytic acid, which can chelate iron. Iron chelation induced by these anti-nutritional factors is the cause of the low bioavailability when the iron from plant sources is ingested by the human body. In addition, organic iron compounds from plant sources can generate sensory defects such as unpleasant off-flavors when used in food products, food supplements or beverages. For example, the iron solution on the market corresponds to a curry leaf extract. However, its use in food products, food supplements or beverages is limited due to the strong metallic off-flavor of the curry leaf extract.

[0006] It is therefore desirable to provide an ingredient that is a substantial source of iron that can be used to fortify food products and beverages or to prepare food supplements, cosmetic or pharmaceutical compositions that deliver iron. It is desirable that the ingredient is natural, suitable for a vegan / veggie diet, has a limited content of anti-nutritional factors that reduce or prevent iron absorption, has satisfactory bioaccessibility properties, and has good organoleptic properties, in particular a limited metallic off-taste.

[0007] No reference made herein to any prior art document is to be construed as an admission that such prior art is widely known or forms part of the common general knowledge in the art. SUMMARY

[0008] It is an object of the present application to improve the state of the art, in particular to provide methods, iron-containing green plant material concentrates, food products, beverages, food supplements, cosmetic compositions, pharmaceutical compositions, and methods for fortifying food products or beverages with iron that overcome the problems of the state of the art and address the needs described above, or at least provide a useful alternative.

[0009] The inventors have surprisingly found that the objects of the present application can be achieved by the subject matter of the independent claims. The dependent claims further develop the idea of the present application.

[0010] A first aspect of the present application therefore proposes a method for preparing an iron-containing green plant material concentrate, comprising the steps of:

[0011] a) suspending a green plant material in an aqueous liquid to form a green plant material suspension,

[0012] b) blending the green plant material suspension to obtain a green plant material slurry,

[0013] c) applying a physical means to the green plant material slurry to separate and obtain an iron-containing green plant material concentrate,

[0014] d) optionally, drying the iron-containing green plant material concentrate.

[0015] A second aspect of the present application proposes an iron-containing green plant material concentrate obtainable or obtained by the method according to the first aspect of the present application.

[0016] A third aspect of the present application proposes an iron-containing green plant material concentrate comprising at least 500 ppm of iron based on the dry weight of the iron-containing green plant material concentrate.

[0017] A fourth aspect of the present application proposes a food product or beverage comprising an iron-containing green plant material concentrate according to the second or third aspect of the present application.

[0018] A fifth aspect of the present application proposes a food supplement comprising the iron-containing green plant material concentrate according to the second or third aspect of the present application.

[0019] A sixth aspect of the present application proposes a cosmetic or pharmaceutical composition comprising the iron-containing green plant material concentrate according to the second or third aspect of the present application.

[0020] A seventh aspect of the present application proposes a method for fortifying a food product or beverage with iron, comprising preparing a food product or beverage, and adding to the food product or beverage the iron-containing green plant material concentrate according to the second or third aspect of the present application.

[0021] It has been found that the method of the present application allows to efficiently concentrate iron from plant material, while allowing to efficiently reduce the ratio of undesired compounds compared to iron, including insoluble plant material and anti-nutritional factors that reduce or prevent iron absorption. The concentrate obtained has a high amount of iron, while having a limited amount of anti-nutritional factors compared to iron, in particular anti-nutritional factors that reduce or prevent iron absorption in the body. The iron of the concentrate is from the plant material. It is thus from a natural source, and is suitable for a vegetarian / vegan diet. In addition, the concentrate has good organoleptic properties, and does not produce organoleptic defects or produces very limited organoleptic defects, in particular limited metallic off-taste, when used in a beverage, food supplement or food product. In addition, the concentrate has satisfactory iron bioavailability properties.

[0022] These and other aspects, features and advantages of the present application will become apparent to those of ordinary skill in the art from a review of the detailed description of the application's embodiments, taken in conjunction with the drawings. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Iron content (mg / kg) based on dry weight (DW) is shown for dry peppermint A, dry nettle, dry thyme A and B, and their corresponding iron-containing green plant material concentrate obtained from them according to the method of Example 1. In addition, the iron content of two commercial peppermint extracts, one commercial nettle extract and one commercial thyme extract is reported. Values represent the mean between triplicate independent samples, and error bars represent the standard deviation. Figure 1 By "dry peppermint", "dry nettle", "dry thyme A", "dry thyme B" in Table 1 is understood the whole of the leaves and stems of the corresponding dry plant, ground into a powder before being further processed in the concentration method of Example 1. For Figure 1 By "dry peppermint" in Table 1, the corresponding dry plant is dry peppermint A.

[0024] Figure 2Molar ratios (M / M) between the molar concentrations of iron and oxalate in dry peppermint A, dry nettle, iron-containing peppermint concentrate obtained from the dry peppermint A according to the method of Example 1 and iron-containing nettle concentrate obtained from the dry nettle according to the method of Example 1 are shown. Values represent the mean between two replicates. Figure 2 The terms "dry peppermint A" and "dry nettle" in the above table are to be understood as the whole of the respective leaves and stems of dry peppermint plant A and dry nettle plant ground into powder before being further processed in the concentration method of Example 1.

[0025] Figure 3 Molar ratios (M / M) between the molar concentrations of iron and phytic acid in dry peppermint A and B, nettle and thyme A and iron-containing concentrates obtained from the dry peppermint A and B, nettle and thyme, respectively, according to the method of Example 1 are shown. Values represent the mean between two replicates of independent samples and error bars represent the standard deviation. Figure 3 The terms "dry peppermint A", "dry peppermint B", "dry nettle", "dry thyme" in the above table are to be understood as the whole of the leaves and stems of the corresponding dry plants ground into powder before being further processed in the concentration method of Example 1. For Figure 3 The corresponding dry plant for "dry thyme" in the above table is dry thyme A.

[0026] Figure 4 Iron bioaccessibility of iron-containing peppermint concentrate prepared from dry peppermint B with water (according to Example 1) and iron-containing peppermint concentrate prepared from dry peppermint B with water in the presence of citric acid, hydrochloric acid, malic acid or ascorbic acid (according to Example 2) compared to ferric pyrophosphate is shown. Values represent the mean between two replicates of independent samples and error bars represent the standard deviation.

[0027] Figure 5 Iron bioaccessibility of iron-containing nettle concentrate prepared with water (according to Example 1) and iron-containing nettle concentrate prepared with water in the presence of citric acid and hydrochloric acid, respectively (according to Example 2). Values represent the mean between two replicates of independent samples and error bars represent the standard deviation.

[0028] Figure 6 Absolute amounts of bioaccessible iron contained in dry peppermint B and in iron-containing peppermint concentrate prepared from dry peppermint B with water according to the concentration method of Example 1 or with water in the presence of citric acid, hydrochloric acid, malic acid and ascorbic acid, respectively, according to the concentration method of Example 2 are shown. The absolute amount of bioaccessible iron was calculated by multiplying the iron content in the sample by the bioaccessibility value. Values represent the mean between two replicates of independent samples and error bars represent the standard deviation. Figure 6"dry peppermint" in the context of the present application is understood to mean the entirety of the leaves and stems of the dry peppermint plant B that are ground into a powder prior to being further processed in the concentration method of Example 1 or Example 2.

[0029] Figure 7 The absolute amount of bioaccessible iron contained in dry nettle, iron-containing nettle concentrate prepared from dry nettle with water (according to Example 1) and iron-containing nettle concentrate prepared from dry nettle with water in the presence of citric acid and hydrochloric acid, respectively (according to Example 2) is shown. The absolute amount of bioaccessible iron was calculated by multiplying the iron content in the sample by the bioaccessibility value. Values represent the mean between duplicate independent samples and error bars represent the standard deviation. Figure 7 "dry nettle" in the context of the present application is understood to mean the entirety of the leaves and stems of the dry nettle plant that are ground into a powder prior to being further processed in the concentration method of Example 1 or Example 2.

[0030] Figure 8 The iron bioaccessibility of iron-containing thyme concentrate prepared from dry thyme B with water (according to Example 1) and iron-containing thyme concentrate prepared from dry thyme B with water in the presence of citric acid and hydrochloric acid, respectively (according to Example 2) is shown. Values represent the mean between duplicate independent samples and error bars represent the standard deviation.

[0031] Figure 9 The absolute amount of bioaccessible iron contained in dry thyme B, iron-containing thyme concentrate prepared from dry thyme B with water (according to Example 1) and iron-containing thyme concentrate prepared from dry thyme B with water in the presence of citric acid and hydrochloric acid, respectively (according to Example 2) is shown. The absolute amount of bioaccessible iron was calculated by multiplying the iron content in the sample by the bioaccessibility value. Values represent the mean between duplicate independent samples and error bars represent the standard deviation. Figure 9 "dry thyme B" in the context of the present application is understood to mean the entirety of the leaves and stems of the dry thyme plant B that are ground into a powder prior to being further processed in the concentration method of Example 1 or Example 2.

[0032] Figure 10 The total phenolic content (mg gallic acid equivalent / g DW) in dry peppermint B and dry nettle and in the iron-containing concentrate obtained from said dry peppermint B and dry nettle, respectively, according to the method of Example 1 is shown. Values represent the mean between duplicate independent samples and error bars represent the standard deviation. Figure 10 "dry peppermint", "dry nettle" in the context of the present application is understood to mean the entirety of the leaves and stems of the corresponding dry plant that are ground into a powder prior to being further processed in the concentration method of Example 1. For Figure 3 For "dry peppermint" in the context of the present application, the corresponding dry plant is dry peppermint B. DETAILED DESCRIPTION

[0033] As used herein, the words “comprise,” “comprises,” and “comprising,” or the like, are to be interpreted inclusively rather than exclusively. Likewise, the terms “include,” “including,” and “e.g.” are to be interpreted inclusively rather than exclusively. However, the compositions / products disclosed herein can not include any element not specifically disclosed. Thus, the disclosure of embodiments using the term “comprise” includes disclosure of embodiments “consisting essentially of and embodiments “consisting of the recited components.

[0034] All numerical ranges are to be interpreted as being inclusive of every integer within the given range. Additionally, the numerical ranges should be interpreted as providing support for a claim to any number or subset of numbers within the given range. For example, a disclosure of 1 to 10 should be interpreted as supporting a range 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0035] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “green plant material” or “the green plant material” includes one green plant material, but also includes two or more green plant materials.

[0036] All percentages in the present specification are, where applicable, by weight, unless otherwise indicated.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0038] As used herein, the term “and / or,” used in the context of “X and / or Y,” should be interpreted as “X,” or “Y,” or “X and Y.” Similarly, “at least one of X or Y” should be interpreted as “X,” or “Y,” or “both X and Y.” For example, “dry leaves and / or fresh leaves” means “dry leaves” or “fresh leaves” or “both dry leaves and fresh leaves.”

[0039] As used herein, the terms “example” and “such as,” particularly when followed by a listing of terms, are merely exemplary and illustrative and should not be deemed exhaustive or comprehensive. However, the disclosure of embodiments using the terms “example,” “such as,” and “for example” includes disclosure of embodiments in which the terms are exclusive and / or comprehensive.

[0040] As used herein, “associated” means occurring at the same time, preferably meaning caused by the same underlying disease or condition, and most preferably meaning that one of the identified diseases or conditions is caused by the other identified condition or disease.

[0041] As used herein, the term "treatment" refers to administration of a composition disclosed herein to a subject having a disorder to alleviate, reduce, or improve at least one symptom associated with the disorder and / or to slow, decrease, or block progression of the disorder. The term "treatment" includes both prophylactic or preventative treatment (preventing and / or delaying the development or onset of a pathology disorder or condition of interest) and curative, therapeutic or disease-modifying treatment, including therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of an already diagnosed pathology disorder or condition; and treatment of patients at risk of contracting a disease or suspected to have contracted a disease, as well as patients who are ill or have been diagnosed with a disease or medical condition. The term "treatment" does not necessarily imply that a subject is treated until total recovery. The term "treatment" also refers to health maintenance and / or promotion in individuals who are not ill but who can be susceptible to developing an unhealthy condition. The term "treatment" is also intended to include strengthening or otherwise enhancing one or more primary prophylactic or therapeutic measures. Treatment can be performed by a patient, caregiver, physician, nurse, or another health care professional, by way of non-limiting example.

[0042] Both human and veterinary treatments are within the scope of the present disclosure.

[0043] As used herein, the term "prevention" means administration of a composition disclosed herein to a subject who shows no symptoms of a disorder to reduce or prevent development of at least one symptom associated with the disorder. Further, "prevention" includes reducing the risk, incidence, and / or severity of a disorder or condition.

[0044] As used herein, an "effective amount" is an amount that treats or prevents a deficiency, treats or prevents a disease or medical condition in an individual, or more generally, an amount that reduces symptoms, manages progression of the disease, or provides a nutritional, physiological, or medical benefit to the individual.

[0045] As used herein, the term "animal" includes, but is not limited to, mammals, which include, but are not limited to, rodents; aquatic mammals; domestic animals such as dogs, cats, and other pets; farm animals such as sheep, pigs, cows, and horses; and humans. Where "animal," "mammal," or a plural thereof is used, these terms also apply to any animal that is capable of the physiological benefit sought to be achieved by the subject context of the paragraph in which it appears, e.g., an animal that benefits from iron fortification. While the term "individual" or "subject" is often used herein to refer to humans, the present disclosure is not limited to this. Thus, the terms "individual" or "subject" refer to any animal, mammal, or human that can benefit from the methods and compositions disclosed herein.

[0046] As used herein, the term "pet" means any animal that can benefit from or enjoy the composition provided by the present disclosure. For example, the pet can be an avian animal, a bovine animal, a canine animal, an equine animal, a feline animal, a caprine animal, a lupine animal, a murine animal, an ovine animal, or a porcine animal, but the pet can also be any suitable animal. The term "companion animal" means a dog or a cat.

[0047] As used herein, the term "green plant material" refers to plant material comprising chlorophyll and chloroplasts. These plant materials are typically green due to the presence of chlorophyll as a photosynthetic pigment. Green plant material can also be referred to as chlorophyll-containing plant material.

[0048] As used herein, the term "added organic solvent" refers to an organic solvent that is exogenous to the green plant material and is added in addition to the green plant material for the preparation of the iron-containing green plant material concentrate. The term "added organic solvent" does not include organic solvents that are inherently present in the green plant material of the iron-containing green plant material concentrate.

[0049] As used herein, the term "vegan" refers to an edible composition that is completely free of animal products or animal-derived products.

[0050] As used herein, the term "vegetarian" refers to an edible composition that is free of meat, including fish.

[0051] As used herein, the term "bioavailability" refers to the fraction of the total amount of a substance that is potentially available for absorption. For example, this refers to the fraction of the substance that is potentially available for absorption in the body of a subject, in particular in the gastrointestinal tract of the subject.

[0052] As used herein, the term "GAE" refers to gallic acid equivalent. This term is used when the content of a component is quantified against a calibration curve for gallic acid. The gallic acid equivalent is the amount of each component that is equivalent to one molecule of gallic acid. In other words, 1 mg GAE / g of the quantified component is equivalent to 1 mg / g of the quantified component.

[0053] As used herein, the terms "blending" and "mixing" are used interchangeably.

[0054] In a first aspect, the present invention relates to a method for preparing an iron-containing green plant material concentrate.

[0055] The method comprises a step a) of suspending green plant material in an aqueous liquid to form a green plant material suspension.

[0056] In one embodiment, the ratio (w / v) of plant green plant material to aqueous liquid in the green plant material suspension is from 1 :3 to 1 :20, preferably from 1 :5 to 1 :20, more preferably from 1 :10 to 1 :20, most preferably from 1 :12 to 1 :18.

[0057] The green plant material comprises plant cells. Likewise, the green plant material suspension comprises plant cells, including intact plant cells. The plant cells of the green plant material suspension are from the green plant material.

[0058] The green plant material suspension is the resulting product of step a). The green plant material slurry is the resulting product of step b). The green plant material suspension and the green plant material slurry are different. In particular, in the green plant material slurry, the plant cells are disrupted and the intracellular material of the plant cells is released, whereas in the green plant material suspension, the plant cells are not disrupted such that the intracellular material of the plant cells is not released.

[0059] The aqueous liquid and thus the green plant material suspension is free of added organic solvents. For example, the aqueous liquid and thus the green plant material suspension is free of any added organic solvent selected from the list consisting of acetone, acetonitrile, benzene, 1 -butanol, 2-butanol, 3-butanone, tert-butanol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, diethylene glycol, diethyl ether, diethylene glycol dimethyl ether, 1,2-dimethoxyethane (glycol dimethyl ether, DME), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4 dioxane, 1,2-dichloroethane, ethanol, ethyl acetate, ethylene glycol, glycerol, heptane, hexamethylphosphoramide (HMPA), hexane, methanol, dichloromethane, N-methyl-2-pyrrolidone (NMP), nitromethane, naphthalene, pentane, 1 -propanol, 2-propanol, pyridine, toluene, triethylamine, tris(hydroxymethyl)methylaminomethane, tris(hydroxymethyl)aminomethane, tetrahydrofuran, o-xylene, m-xylene, p-xylene, and combinations thereof.

[0060] In one preferred embodiment, the aqueous liquid comprises at least 80 wt.% water, more preferably 90 wt.% water, even more preferably at least 95 wt.% water. Most preferably, the aqueous liquid is water.

[0061] In one embodiment, a permeabilizing agent can be further added to the green plant material suspension prior to step b). The permeabilizing agent can be selected from the list consisting of glucose, glycerol, sucrose, sorbitol, sodium chloride, potassium chloride or combinations thereof. Preferably, the permeabilizing agent is sucrose. The amount of permeabilizing agent to be added to the green plant material suspension can be readily determined by the person of ordinary skill in the art depending on the type of permeabilizing agent and the osmolality of the suspension. The permeabilizing agent can serve to adjust the osmotic pressure. Without wishing to be bound by theory, this can help to keep some plant structures that store iron intact and avoid their lysis under osmotic pressure. Without wishing to be bound by theory, this can allow to further improve iron stability.

[0062] In a preferred embodiment, an acid can be further added to the green plant material suspension prior to step b).

[0063] In one embodiment, an acid is added to the green plant material suspension prior to step b) until a pH of 2 to 5.5, preferably 2.5 to 4.5, most preferably 3 to 4 is reached.

[0064] The acid can be selected from the group consisting of hydrochloric acid, citric acid, malic acid, ascorbic acid, acetic acid, lactic acid, propionic acid, fumaric acid, tartaric acid, phosphoric acid, adipic acid, succinic acid, gluconic acid or mixtures thereof. Preferably, the acid is selected from the group consisting of ascorbic acid, malic acid, citric acid, hydrochloric acid or mixtures thereof. In a more preferred embodiment, the acid is selected from the group consisting of malic acid, citric acid, hydrochloric acid or mixtures thereof. In an even more preferred embodiment, the acid is selected from the group consisting of citric acid, hydrochloric acid or mixtures thereof. In a most preferred embodiment, the acid is hydrochloric acid. In another most preferred embodiment, the acid is citric acid. The acid can be provided as a pure acid solution, as a diluted acid solution or as an acid containing food ingredient. Examples of acid containing food ingredients include citrus juices such as lemon juice, lime juice, orange juice, tangerine juice and the like.

[0065] The green plant material suspension can comprise 0.01 to 5 wt.% of the acid.

[0066] When the acid is citric acid, the green plant material suspension can comprise 0.01 to 3 wt.% of citric acid, preferably 0.5 to 2.3 wt.% of citric acid.

[0067] When the acid is hydrochloric acid, the green plant material suspension can comprise 0.01 to 0.5 wt.% of hydrochloric acid, preferably 0.02 to 0.54 wt.% of hydrochloric acid.

[0068] When the acid is malic acid, the green plant material suspension can comprise 0.01% to 3% by weight of malic acid, preferably 0.5% to 2.5% by weight of malic acid.

[0069] The use of acid has a dual effect. In particular, the acid lowers the pH and chelates iron. The lowering of the pH and the iron chelation help to improve the iron solubility and thus the iron bioavailability in the iron-containing green plant material concentrate.

[0070] Advantageously, the green plant material is from a herbaceous plant or duckweed. In some embodiments, the green plant material can be a combination of green plant material from a herbaceous plant or duckweed combined with algae and / or cyanobacteria. Non-limiting examples of algae include Chlorella vulgaris. Non-limiting examples of cyanobacteria include Arthrospira platensis.

[0071] Duckweed is a floating aquatic green plant, which can also be referred to as water lettuce. In a preferred embodiment, the duckweed is from one of the following genera: Spirodela, Lemna, Wolffia, Landoltia or Wolffia. The use of duckweed allows the amount of iron to be maximized. Other advantages of using duckweed are that it has a high growth rate, it can tolerate extreme environments, and it can be cultivated in pots of non-tilled land, thereby avoiding the use of tilled land.

[0072] In a preferred embodiment, the green plant material is from a herbaceous plant of the Lamiaceae family and / or a herbaceous plant of the Apiaceae family and / or a herbaceous plant of the Urticaceae family. Examples of herbaceous plants from the Lamiaceae family include peppermint, thyme, lemon balm, basil, sage, oregano, rosemary, rosemary, elecampane, savory, or a combination thereof. Examples of herbaceous plants from the Apiaceae family include parsley, coriander, dill, or a combination thereof. Examples of herbaceous plants from the Urticaceae family include nettle. In a more preferred embodiment, the green plant material is from a herbaceous plant, wherein the herbaceous plant is selected from the group consisting of parsley, coriander, peppermint, thyme, lemon balm, nettle, sage, oregano, rosemary, basil, dill, elecampane, savory, or a mixture thereof. Preferably, the herbaceous plant is selected from the group consisting of sage, oregano, parsley, coriander, peppermint, thyme, lemon balm, nettle, or a mixture thereof. More preferably, the herbaceous plant is selected from the group consisting of sage, oregano, parsley, coriander, peppermint, thyme, lemon balm, nettle, or a mixture thereof. Even more preferably, the herbaceous plant is selected from the group consisting of peppermint, thyme, lemon balm, nettle, sage, oregano, or a mixture thereof. Most preferably, the herbaceous plant is selected from the group consisting of peppermint, thyme, lemon balm, nettle, or a mixture thereof. The use of herbaceous plants, in particular of this list of selected herbaceous plants, allows the amount of iron in the iron-containing green plant material concentrate to be maximized, while imparting a pleasant flavor.

[0073] In an embodiment, the peppermint can be spearmint, peppermint, or a mixture thereof.

[0074] Parsley typically comprises 20 ppm - 600 ppm iron. Coriander typically comprises 20 ppm - 300 ppm iron. Peppermint typically comprises 200 ppm - 1000 ppm iron. Thyme typically comprises 200 ppm - 3000 ppm. Lemon balm typically comprises 300 ppm - 600 ppm iron. Nettle typically comprises 100 ppm - 1000 ppm iron. Sage typically comprises 200 ppm - 1300 ppm iron. Oregano typically comprises 200 ppm - 1000 ppm iron. Rosemary typically comprises 20 ppm - 500 ppm iron. Basil typically comprises 20 ppm - 900 ppm iron. Dill typically comprises 20 ppm - 500 ppm iron. Elecampane typically comprises 20 ppm - 400 ppm iron. Savory typically comprises 50 ppm - 400 ppm iron.

[0075] In some embodiments, parsley refers to any plant from the genus Petroselinum, preferably any edible plant from the genus Petroselinum. Preferably, parsley refers to Petroselinum crispum.

[0076] In some embodiments, coriander refers to any plant from the genus Coriandrum, preferably any edible plant from the genus Coriandrum. Preferably, coriander refers to Coriandrum sativum.

[0077] In some embodiments, mint refers to any plant from the genus Mentha, preferably any edible plant from the genus Mentha. More preferably, mint refers to a plant selected from the list consisting of Mentha spicata, Mentha x piperita, or a combination thereof.

[0078] In some embodiments, thyme refers to any plant from the genus Thymus, preferably any edible plant from the genus Thymus. Preferably, thyme refers to Thymus vulgaris.

[0079] In some embodiments, lemon balm refers to any plant from the genus Melissa, preferably any edible plant from the genus Melissa. Preferably, lemon balm refers to Melissa officinalis.

[0080] In some embodiments, nettle refers to any plant from the genus Urtica, preferably any edible plant from the genus Urtica. Preferably, nettle refers to Urtica dioica.

[0081] In some embodiments, sage refers to any plant from the genus Salvia, preferably any edible plant from the genus Salvia. Preferably, sage refers to a plant selected from the list consisting of Salvia officinalis, Salvia elegans, or a combination thereof.

[0082] In some embodiments, oregano refers to any plant from the genus Origanum, preferably any edible plant from the genus Origanum. Preferably, oregano refers to a plant selected from the list consisting of Origanum vulgare, Origanum majorana, or a combination thereof. More preferably, oregano refers to Origanum vulgare.

[0083] In some embodiments, rosemary refers to any plant from the genus rosemary, preferably any edible plant from the genus rosemary. Preferably, rosemary refers to rosemary (Rosmarinus officinalis).

[0084] In some embodiments, basil refers to any plant from the genus basil, preferably any edible plant from the genus basil. Preferably, basil refers to Ocimum baoricum.

[0085] In some embodiments, dill refers to any plant from the genus dill, preferably any edible plant from the genus dill. Preferably, dill refers to Anethum graveolens.

[0086] In some embodiments, chervil refers to any plant from the genus chervil, preferably any edible plant from the genus chervil. Preferably, chervil refers to Anthriscus cerefolium.

[0087] In some embodiments, savory refers to any plant from the genus savory, preferably any edible plant from the genus savory. Preferably, savory refers to a plant selected from the list consisting of Satejea hortensis, Satureja montana, or a combination thereof.

[0088] In some embodiments, the green plant material is not from the spice pimento, in particular the green plant material is not from Murraya koenigii and / or Bergera koenigii. Green plant material from the spice pimento is detrimental to the present application. For example, they can cause the iron-containing green plant material concentrate to have unpleasant organoleptic properties, including a metallic off-note.

[0089] Saffron spice is not a green plant material. In some embodiments, the green plant material is not from saffron, in particular not from Crocus sativus.

[0090] In some embodiments, the green plant material is different from Tagetes erecta L.

[0091] In some implementations, the green plant material is different tarragon (Artemisia dracunculus), cultivated chicory (Cichoria endivia), and lettuce (Lactuca sativa).

[0092] In some embodiments, the green plant material is not derived from berries. Examples of berries include goji berries, blueberries, cranberries, white currants, red currants, black currants, mulberries, blackberries, currants, raspberries, sea buckthorn, strawberries, strawberry berries, grapes, or combinations thereof. In some embodiments, the green plant material is not derived from blueberries, particularly American blueberries (Vaccinium sect. cyanococcus).

[0093] The green plant material can comprise any part of a green plant, such as leaves, stems, flowers, buds, and roots. In one embodiment, the green plant material comprises leaves. In another embodiment, the green plant material comprises both leaves and stems. Preferably, the green plant material comprises a large amount of leaves. The green plant material comprises at least 80% by weight of leaves, more preferably at least 90% by weight of leaves, even more preferably at least 95% by weight of leaves, and even more preferably at least 98% by weight of leaves. The remaining portion of the green plant material can be any part of the green plant disclosed herein other than leaves. In one embodiment, the remaining portion of the green plant material consists only of stems.

[0094] In a preferred embodiment, the green plant material consists only of leaves.

[0095] Leaves are preferred because they typically contain a high proportion of iron found in green plants and are edible. Therefore, leaves are a good edible starting material for concentrating large amounts of iron from green plants.

[0096] In one implementation, the green plant material is dried green plants and / or fresh green plant material. For example, the green plant material is dried leaves and / or fresh leaves. Advantageously, the green plant material is dried green plant material. Dried plant material is easier to process on an industrial scale because it has a longer shelf life than fresh plant material.

[0097] In one embodiment, when the green plant material comprises or is a dried green plant material, the dried green plant can be ground into a powder prior to step a). The dried green plant material can be ground via dry milling. Dry milling can be obtained using any machine that provides shearing or contains cutting devices. For example, dry milling can be performed by a hammer mill, a stone mill, a roller mill, a ball mill, a jet mill, a colloid mill, a stirred media mill, a bead mill, a pin mill, a roll mill, a roll refiner, a flash mill, a cryogenic mill, a rod mill, a vibration mill, a cutter mill, a disc mill, a perforated disc mill, a micro cutter mill, or an extrusion device.

[0098] The method further comprises a step b) of blending the green plant material suspension to obtain a green plant material slurry. For example, blending can be performed by any kind of shearing or mixing device. Examples of mixing devices are: a mixer, a kitchen mixer, a drum blender, a paddle mixer, a whisk, a flow impeller, a planetary mixer, a multi-shaft mixer, a Scanima mixer, or a Stephan mixer. In one embodiment, blending can be performed in step b) for at least 8 seconds, preferably for 8 seconds to 5 minutes, more preferably for 1 minute to 3 minutes. In one embodiment, blending can be performed in step b) at a temperature of 4°C to 80°C, preferably at a temperature of 4°C to 25°C, more preferably at a temperature of 10°C to 25°C. The preferred temperature range of 4°C to 25°C is advantageous as it limits the oxidation / chemical degradation of plant organelles that can occur at high temperatures (e.g. 60°C to 100°C). For example, blending can be performed at room temperature. This step allows the disruption of plant cells and the release of their intracellular content, including iron. This helps to improve the iron bioavailability of the final concentrate when ingested by a human.

[0099] The method further comprises a step c) of applying a physical means to the green plant material slurry to separate and obtain an iron-containing green plant material concentrate.

[0100] In one embodiment, step c) is performed by filtration and / or centrifugation and / or decantation and / or heat treatment.

[0101] Filtration of step c) can be performed with the same conditions or features as filtration step cl) provided hereafter in the section “Step cl) Filtration”.

[0102] Heat treatment of step c) can be performed with the same conditions or features as heat treatment step c2) provided hereafter in the section “Step c2) Heat treatment”.

[0103] Decantation or centrifugation of step c) can be performed with the same conditions or features as decantation or centrifugation step c3) provided hereafter in the section “Step c3) Decantation or centrifugation”.

[0104] In one embodiment, the step c) of applying physical means is performed by the following steps:

[0105] c1 ) filtering the green plant material slurry to obtain a permeate,

[0106] c2) optionally, heat treating the permeate,

[0107] c3) centrifuging or decanting the permeate to obtain an iron-containing green plant material concentrate.

[0108] Step c1 ) filtration

[0109] As mentioned above, in one embodiment, the method can comprise the step c1 ) of filtering the green plant material slurry of step b) to obtain a permeate.

[0110] After the filtration of step c1 ), a retentate and a permeate are obtained. The material that passes through the filter is called "permeate"; the material that does not pass through the filter and is recirculated is called "retentate". The retentate is removed after step c1 ) and the permeate is recovered after step c1 ) and further processed.

[0111] In a preferred embodiment, the filtration of step c1 ) is performed with a filter having a mesh size comprised between 25 pm and 1000 pm, preferably between 25 pm and 500 pm, more preferably between 100 pm and 200 pm. This mesh size helps to separate, concentrate the compound of interest, such as iron, and thus increase its purity, while discarding / reducing the undesirable compounds, such as insoluble plant compounds. The mesh size also reduces the particle size of the iron-containing green plant material concentrate to a level such that the concentrate is less prone to sedimentation, especially when used in a beverage or liquid / semi-liquid food product or liquid / semi-liquid food supplement or liquid / semi-liquid cosmetic composition or liquid / semi-liquid pharmaceutical composition.

[0112] The filtration of step c1 ) can be performed in one or several steps. In one embodiment, the filtration of step c1 ) can be performed in 1 to 10 steps, preferably in 1 to 5 steps. When the filtration of step c1 ) is performed in several steps, i.e. in 2 to 10 steps, preferably in 2 to 5 steps, the size of the mesh of the filter is decreased in each successive filtration step. In other words, the size of the mesh of the filter used in a predetermined filtration step (e.g. the first filtration step) is higher than the size of the mesh of the filter used in the successive and downstream filtration step (e.g. the second filtration step), and so on.

[0113] In a more preferred embodiment, the filtration of step cl) is performed in two steps, in particular the green plant material slurry is first filtered with a filter having a mesh of 400 to 500 microns, preferably 500 microns, and then filtered with a filter having a mesh of 50 to 200 microns, preferably 180 microns.

[0114] Performing the filtration step in several steps, in particular two steps, reduces the tendency of the filter to clog.

[0115] In an embodiment, the sequence of steps a), b) and cl) is repeated at least twice, preferably 2 to 5 times, before step c2), and starting from the second sequence of steps a), b) and cl), the green plant material of step a) is replaced by the retentate obtained in step cl) of the previous sequence of steps a), b) and cl). For the sake of clarity, starting from the second sequence of steps a), b) and cl), the retentate of the previous sequence of steps a), b) and cl) is suspended in the aqueous liquid of step a) of the successive sequence of steps a), b) and cl) instead of the green plant material. Thus, starting from the second sequence of steps a), b) and cl), the suspension of steps a) and b) is not a green plant material suspension, but a retentate suspension, and the slurry of steps b) and cl) is not a green plant material slurry, but a retentate slurry. In addition, starting from the second sequence of steps a), b) and cl), the permeate is still obtained in step cl). Furthermore, starting from the second sequence of steps a), b) and cl), the retentate is still obtained in step cl) as well. The obtained retentate can be further processed in successive sequences of steps a), b) and cl) and the like.

[0116] Step c2) heat treatment

[0117] As mentioned above, in an embodiment, the method can comprise a step c2) of optionally heat treating the permeate obtained in step cl). This heat treatment step allows to extend the shelf life of the final iron-containing green plant material concentrate. In an embodiment, this step c2) is not optional.

[0118] In an embodiment, the heat treatment of step c2) is performed at a temperature of at least 60°C for at least 2 seconds. Preferably, the heat treatment of step c2) is performed at a temperature of 60°C to 125°C for 2 seconds to 30 minutes. More preferably, the heat treatment of step c2) is performed at a temperature of 70°C to 85°C for 1 minute to 3 minutes.

[0119] Step c3) decanting or centrifugation

[0120] As mentioned above, in one embodiment, the method can comprise a step c3) centrifuging or decanting the permeate to obtain the iron-containing green plant material concentrate. Preferably, step c3) is a step of centrifuging the permeate. In one embodiment, step c3) centrifuging is performed at 500 g to 10000 g, preferably 1000 g to 5000 g, more preferably 1000 g to 3000 g. In one embodiment, step c3) centrifuging is performed for 2 minutes to 30 minutes, preferably 2 minutes to 20 minutes, more preferably 5 minutes to 15 minutes.

[0121] After centrifugation or decanting, a supernatant and a precipitate are obtained. The precipitate corresponds to the material forming a deposit at the bottom of the centrifugation / decanting vessel, which is usually solid or semi-solid, while the supernatant corresponds to the material floating or located above the precipitate, which is usually liquid. The supernatant is discarded. The precipitate is recovered. The precipitate obtained after step c3) corresponds to the iron-containing green plant material concentrate.

[0122] In one embodiment, step c3) centrifuging or decanting is performed once. In other words, the precipitate obtained in step c3) is not further centrifuged or decanted.

[0123] The method further comprises a step d) optionally drying the iron-containing green plant material concentrate to obtain the iron-containing green plant material concentrate in the form of a powder. After step d), the iron-containing green plant material concentrate is not in the form of a semi-solid, but in the form of a powder. For example, the drying step can be performed by spray drying, roller drying, air drying or freeze drying. In one embodiment, step d) drying is not optional.

[0124] As an alternative to drying the iron-containing green plant material concentrate into a powder, the water activity of the iron-containing green plant material concentrate can be reduced to improve its microbiological stability over time. Thus, in an alternative embodiment, the method can comprise a step d') reducing the water activity of the iron-containing green plant material concentrate after step c) applying physical means or step c3) centrifugation or decantation. After step d') reducing the water activity, the iron-containing green plant material concentrate has a water activity below 0.85, preferably between 0.5 and 0.85. After this step d') reducing the water activity, the iron-containing green plant material concentrate is not in powder form. Indeed, the iron-containing green plant material concentrate obtained after step d') is in the same form as the iron-containing green plant material concentrate obtained after step c) or step c3), i.e. in semi-solid form. However, the iron-containing green plant material concentrate obtained after step d') has a water activity lower than the water activity of the iron-containing green plant material concentrate just obtained after step c) or step c3). This step d') reducing the water activity can be performed by evaporation of the iron-containing green plant material concentrate, by drying the iron-containing green plant material concentrate or by adding a humectant to the iron-containing green plant material concentrate. Preferably, the humectant is sucrose. The person of ordinary skill in the art can easily determine the amount of humectant to be added to the iron-containing green plant material concentrate depending on the type of humectant and the targeted water activity. The drying step can be performed by freeze-drying, spray-drying, air-drying or roller-drying. The evaporation step can be performed with an evaporator.

[0125] In some embodiments, step d') reducing the water activity of the iron-containing green plant material concentrate and step d) drying the iron-containing green plant material concentrate can be performed sequentially. In this embodiment, step d') reducing the water activity of the iron-containing green plant material concentrate is performed before step d) drying the iron-containing green plant material concentrate.

[0126] In some embodiments, in particular when step d) drying is applied, the method does not comprise evaporation or drying before step d) drying.

[0127] In some embodiments, in particular when step d) drying is applied, the method does not comprise any evaporation or drying step.

[0128] In some embodiments, when step d) drying is not applied, the method does not comprise any evaporation or drying step.

[0129] In some embodiments, step c) is not performed by evaporation or does not comprise any use of an evaporation machine, such as a rotary evaporator. Likewise, steps cl, c2, c3 and c4 are not performed by evaporation or do not comprise any evaporation, any use of an evaporation machine, such as a rotary evaporator.

[0130] It has been observed that, compared to a process where drying or evaporation is applied directly on the permeate as a concentration method without additional physical separation, in particular without a centrifugation step, such as the process in RO132538A0, the concentration of iron in ppm in the final concentrate is significantly increased in the process of the present application.

[0131] In some embodiments, the process can comprise a step d") heat treatment of the concentrate of the iron-containing green plant material after step c) or step c3). The step d") heat treatment can be before or after step d'). The step d") heat treatment can be before or after step d). The step d") heat treatment can be performed at a temperature of at least 60°C for at least 2 seconds. Preferably, the step d") heat treatment is performed at a temperature of 60°C to 125°C for 2 seconds to 30 minutes.

[0132] The process allows efficient concentration of iron while allowing efficient reduction of the ratio of undesired compounds compared to iron, including insoluble plant material and anti-nutritional factors that reduce or prevent iron absorption in the body.

[0133] The concentrate obtained has a large amount of iron while having a limited amount of anti-nutritional factors compared to iron, in particular anti-nutritional factors that reduce or prevent iron absorption in the body. The iron of the concentrate comes from plant material. It is thus from a natural source and is suitable for a vegetarian / vegan diet. In addition, the concentrate has good organoleptic properties and does not produce organoleptic defects or produces very limited organoleptic defects, in particular limited metallic off-notes, when used in a beverage, a food supplement, a food product or a pharmaceutical composition. In addition, the iron of the concentrate has satisfactory bioaccessibility properties.

[0134] The process is essentially natural. It does not involve the use of added organic solvents, but still allows efficient concentration of iron and efficient reduction of the ratio of undesired compounds compared to iron, including anti-nutritional factors that reduce or prevent iron absorption in the body.

[0135] In a preferred embodiment, the method does not involve the use of any added organic solvent. For example, the method does not involve the use of any added organic solvent selected from the list consisting of acetone, acetonitrile, benzene, 1 -butanol, 2-butanol, 3-butanone, tert-butanol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, diethylene glycol, diethyl ether, diethylene glycol dimethyl ether, 1,2-dimethoxyethane (glycol dimethyl ether, DME), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4 dioxane, 1,2-dichloroethane, ethanol, ethyl acetate, ethylene glycol, glycerol, heptane, hexamethylphosphoramide (HMPA), hexane, methanol, dichloromethane, N-methyl-2-pyrrolidone (NMP), nitromethane, naphthalene, pentane, 1 -propanol, 2-propanol, pyridine, toluene, triethylamine, tris(hydroxymethyl)methylaminomethane, tris(hydroxymethyl)aminomethane, tetrahydrofuran, o-xylene, m-xylene, p-xylene, and combinations thereof.

[0136] Thus, in a preferred embodiment, the iron-containing green plant material concentrate is free of added organic solvent. For example, the iron-containing green plant material concentrate is free of any of the above listed added organic solvents.

[0137] The method of the present application allows to efficiently concentrate the iron of the green plant material. Advantageously, the concentration of iron in the iron-containing green plant material concentrate obtained in step c) or c3) is at least 2 times higher, preferably 2 to 10 times higher, in weight percent, than the concentration of iron in the green plant material of step a). For example, the concentration of iron in the concentrate and in the green plant material can be measured according to the method provided in the examples.

[0138] The method of the present application allows to efficiently reduce the ratio of undesired anti-nutritional factors compared to iron, in particular anti-nutritional factors that can reduce or prevent the absorption of iron in the body.

[0139] In particular, the molar ratio (M / M) of iron to oxalic acid is significantly increased by the method of the present application. Increasing the molar ratio of iron to oxalic acid is advantageous to reduce the impact of oxalic acid on iron. Indeed, oxalic acid can reduce or prevent the absorption of iron in the body. Advantageously, the molar ratio of iron to oxalic acid in the iron-containing green plant material concentrate obtained in step c) or c3) is at least 2 times higher, preferably at least 2 to 10 times higher, more preferably 3 to 10 times higher, than the molar ratio in the green plant material of step a). The molar ratio of iron to oxalic acid of the iron-containing green plant material concentrate is expressed by the dry weight of the iron-containing green plant material concentrate. The molar ratio of iron to oxalic acid of the green plant material is expressed by the dry weight of the green plant material. For example, the molar ratio of iron to oxalic acid in the concentrate and in the green plant material can be measured according to the method provided in the examples.

[0140] In some embodiments, the molar ratio of iron to phytic acid (M / M) is significantly increased by the method of the application. Increasing the molar ratio of iron to phytic acid is advantageous to reduce the effect of phytic acid on iron. Indeed, phytic acid can reduce or prevent the absorption of iron in the body. Advantageously, the molar ratio of iron to phytic acid in the iron-containing green plant material concentrate obtained in step c) or c3) is at least 2 times higher, preferably at least 2 to 10 times higher, more preferably 3 to 10 times higher than the molar ratio of iron to phytic acid in the green plant material of step a). The molar ratio of iron to phytic acid of the iron-containing green plant material concentrate is expressed by the dry weight of the iron-containing green plant material concentrate. The molar ratio of iron to phytic acid of the green plant material is expressed by the dry weight of the green plant material. The molar ratio of iron to phytic acid can be measured according to the method provided in the examples.

[0141] In addition, the amount of total phenolic compounds is significantly reduced by the method of the application. In particular, reducing phenolic compounds is advantageous because phenolic compounds can prevent the absorption of iron. Advantageously, the total phenolic compound concentration in the iron-containing green plant material concentrate obtained in step c) or c3) is at most 2 times, preferably at most 5 times, lower than the total phenolic compound concentration in the green plant material of step a) in weight percent. In particular, the total phenolic compound concentration in the iron-containing green plant material concentrate obtained in step c) or c3) is 2 to 15 times, preferably at most 5 to 15 times, lower than the total phenolic compound concentration in the green plant material of step a) in weight percent.

[0142] In some embodiments, the method does not comprise any step of adding an enzyme. For example, the method does not comprise any step of adding a protein-degrading enzyme, a carbohydrate-degrading enzyme, a fiber-degrading enzyme, an oxalate-degrading enzyme, a phytic acid-degrading enzyme, and / or a phenolic compound-degrading enzyme.

[0143] In a second aspect, the application relates to an iron-containing green plant material concentrate obtainable or obtained by the method according to the first aspect of the application. The features of the iron-containing green plant material concentrate of the third aspect of the application also apply to the iron-containing green plant material concentrate of the second aspect of the application.

[0144] The concentrate has a high amount of iron and has a limited amount of undesirable anti-nutritional factors compared to iron, in particular anti-nutritional factors that can reduce or prevent the absorption of iron.

[0145] The iron of the concentrate is from the plant material. It is thus from a natural source and is suitable for a vegetarian / vegan diet.

[0146] In addition, the concentrate has good organoleptic properties and does not produce organoleptic defects or produces very limited organoleptic defects when used in a beverage, food supplement, food product or pharmaceutical composition, in particular limited metallic off-notes.

[0147] In addition, the iron of the concentrate has satisfactory bioaccessibility properties.

[0148] The concentrate is natural and can be used for the fortification of food supplements, food products and beverages with iron, including vegetarian / vegan food supplements, food products and beverages.

[0149] In a third aspect, an iron-containing green plant material concentrate comprising at least 500 ppm of iron based on dry weight of the iron-containing green plant material concentrate.

[0150] In one embodiment, the iron-containing green plant material concentrate comprises at least 1000 ppm of iron based on dry weight, preferably at least 1500 ppm of iron based on dry weight of the iron-containing green plant material concentrate. In one embodiment, the iron-containing green plant material concentrate comprises at most 15000 ppm of iron, preferably at most 4000 ppm based on dry weight of the iron-containing green plant material concentrate. In one embodiment, the iron-containing green plant material concentrate is derived from a green plant material. In particular, the iron-containing green plant material concentrate comprises a green plant material. The green plant material can be a green plant material as provided in the first aspect of the application. For example, the concentration of iron of the iron-containing green plant material concentrate can be measured according to the method provided in the examples.

[0151] In some embodiments, the iron-containing green plant material comprises less than 100 mg, preferably less than 50 mg GAE / g total phenolic compounds based on dry weight of the iron-containing green plant material concentrate.

[0152] In some embodiments, the iron-containing green plant material concentrate has an iron bioaccessibility of at least 3%, preferably at least 10%, more preferably at least 15%. In some further embodiments, the iron-containing green plant material concentrate has an iron bioaccessibility of at most 50%, preferably at most 35%. The iron bioaccessibility of the iron-containing green plant material concentrate can be measured as provided in the examples.

[0153] In one embodiment, the iron-containing green plant material concentrate has an absolute amount of bio-accessible iron of at least 35 ppm, preferably at least 100 ppm, more preferably at least 300 ppm, even more preferably at least 450 ppm. In some further embodiments, the iron-containing green plant material concentrate has an absolute amount of bio-accessible iron of at most 1500 ppm, preferably at most 1000 ppm, more preferably at most 800 ppm, even more preferably at most 500 ppm, even more preferably at most 200 ppm. For example, the absolute amount of bio-accessible iron of the iron-containing green plant material concentrate can be measured according to the method provided in the examples.

[0154] In some embodiments, the iron-containing green plant material concentrate has a molar ratio of iron to oxalate (M / M) of at least 0.3, preferably from 0.3 to 3, more preferably from 0.4 to 3, most preferably from 0.4 to 1.5. The molar ratio of iron to oxalate is expressed by dry weight of the iron-containing green plant material concentrate. For example, the molar ratio of iron to oxalate of the iron-containing green plant material concentrate can be measured according to the method provided in the examples.

[0155] In some embodiments, the iron-containing green plant material concentrate comprises less than 15000 ppm, preferably less than 12000 ppm, more preferably less than 10500 ppm of oxalate by dry weight of the iron-containing green plant material concentrate. For example, the concentration of oxalate in the iron-containing green plant material concentrate can be measured according to the method provided in the examples.

[0156] In some embodiments, the iron-containing green plant material concentrate has a molar ratio of iron to phytic acid (M / M) of at least 5, preferably at least 7, more preferably at least 7.5. In particular embodiments, the iron-containing green plant material concentrate has a molar ratio of iron to phytic acid (M / M) of from 5 to 80, preferably from 7 to 80, more preferably from 7.5 to 80, even more preferably from 7.5 to 60, most preferably from 7.5 to 50. The molar ratio of iron to phytic acid is expressed by dry weight of the iron-containing green plant material concentrate. For example, the concentration of iron can be measured according to the method provided in the examples. For example, the molar ratio of iron to phytic acid of the iron-containing green plant material concentrate can be measured according to the method provided in the examples.

[0157] In some embodiments, the iron-containing green plant material concentrate comprises less than 3000 ppm, preferably less than 2000 ppm, more preferably less than 1650 ppm of phytic acid by dry weight of the iron-containing green plant material concentrate. For example, the concentration of phytic acid of the iron-containing green plant material concentrate can be measured according to the method provided in the examples.

[0158] In one embodiment, the iron-containing green plant material concentrate is free of added organic solvent, in particular free of any added organic solvent listed in the first aspect of the application.

[0159] In one embodiment, the iron-containing green plant material concentrate has a pH of 3 to 8.

[0160] In one embodiment, the iron-containing green plant material concentrate comprises 0.01 wt% to 5% wt% of an acid. The acid can be as provided in the first aspect of the application. Preferably, the acid is selected from the group consisting of ascorbic acid, malic acid, citric acid, hydrochloric acid or mixtures thereof. In a more preferred embodiment, the acid is selected from the group consisting of malic acid, citric acid, hydrochloric acid or mixtures thereof. In an even more preferred embodiment, the acid is selected from the group consisting of citric acid, hydrochloric acid or mixtures thereof. In a most preferred embodiment, the acid is hydrochloric acid. In another most preferred embodiment, the acid is citric acid.

[0161] The acid can be provided as a pure acid solution, as a dilute acid solution or as an acid-containing food ingredient. Examples of acid-containing food ingredients include citrus juices such as lemon juice, lime juice, orange juice, tangerine juice and the like.

[0162] When the acid is citric acid, the iron-containing green plant material concentrate can comprise 0.01 wt% to 3 wt%, preferably 0.5 wt% to 2.3 wt% of citric acid.

[0163] When the acid is hydrochloric acid, the iron-containing green plant material concentrate can comprise 0.01 wt% to 0.5 wt% of hydrochloric acid, preferably 0.05 wt% to 0.5 wt% of hydrochloric acid.

[0164] When the acid is malic acid, the iron-containing green plant material concentrate can comprise 0.01 wt% to 3 wt% of malic acid, preferably 0.5 wt% to 2.5 wt% of malic acid.

[0165] In one embodiment, the iron-containing green plant material concentrate can comprise a permeating agent. The permeating agent can be as provided in the first aspect of the application.

[0166] In one embodiment, the iron-containing green plant material concentrate can comprise a humectant. The humectant can be as provided in the first aspect of the application.

[0167] In one embodiment, the iron-containing green plant material concentrate can comprise a total sucrose content of 1 wt% to 50 wt%, preferably 5 wt% to 20 wt%. The sucrose in the iron-containing green plant material concentrate can be used as a humectant and / or a permeating agent. The total sucrose content ranges provided herein apply regardless of whether the sucrose is used as a humectant and / or a permeating agent.

[0168] The features of the iron-containing green plant material concentrate of the third aspect of the present invention also apply to the iron-containing green plant material concentrate provided in the first and second aspects of the present invention and vice versa. The concentrate has a high amount of iron and has a limited amount of undesirable anti-nutritional factors compared to iron, in particular anti-nutritional factors that can reduce or prevent iron absorption.

[0169] The iron of the concentrate is derived from plant material. It is thus from a natural source and is suitable for a vegetarian / vegan diet.

[0170] In addition, the concentrate has good organoleptic properties and does not produce or produces very limited organoleptic defects, in particular very limited metallic off-notes, when used in a food supplement, a beverage, a food product or a pharmaceutical composition.

[0171] In addition, the iron of the concentrate has satisfactory bioaccessibility properties.

[0172] The concentrate is natural and can be used for iron fortification of food products, food supplements and beverages, including vegetarian / vegan food products, food supplements and beverages.

[0173] In a fourth aspect, the present invention relates to a food product or a beverage comprising the iron-containing green plant material concentrate according to the second or third aspect of the present invention. The iron-containing green plant material concentrate of the present invention can be used for iron fortification of food products and beverages. The iron-containing green plant material concentrate has good organoleptic properties and does not produce or produces very limited organoleptic defects, in particular metallic off-notes, when used in a beverage or a food product.

[0174] In one embodiment, the food product or beverage can have a pH of 2.5 to 8.

[0175] In one embodiment, the food product or beverage can comprise an acid content of 0.01 wt% to 0.2 wt%, preferably 0.02 wt% to 0.1 wt%. The acid can be an acid as provided in the first aspect of the present invention. Preferably, the acid is selected from the group consisting of ascorbic acid, malic acid, citric acid, hydrochloric acid or mixtures thereof. In a more preferred embodiment, the acid is selected from the group consisting of malic acid, citric acid, hydrochloric acid or mixtures thereof. In an even more preferred embodiment, the acid is selected from the group consisting of citric acid, hydrochloric acid or mixtures thereof. In a most preferred embodiment, the acid is hydrochloric acid. In another most preferred embodiment, the acid is citric acid.

[0176] The acid can be provided as a pure acid solution, a dilute acid solution or as an acid containing food ingredient. Examples of acid containing food ingredients include citrus juices such as lemon juice, lime juice, orange juice, tangerine juice and the like.

[0177] In one embodiment, the food product or beverage can have a total sucrose content of 0 wt% to 40 wt%, preferably 0 wt% to 10 wt%, more preferably 0.5 wt% to 4 wt%.

[0178] In one embodiment, the food product or beverage can have a total iron content of at least 2.1 mg per serving. The serving can vary depending on the beverage or food product. Servings of different beverages or food products are well known and can be readily determined by the person skilled in the art. For example, for a beverage, the serving can be 200 mL. For example, for a sauce, the serving can be 15 g. For example, for a fermented dairy product, the serving can be 125 g. For example, for a seasoning, the serving can be 30 g.

[0179] In one embodiment, the food product can be selected from the list consisting of: bouillon, fruit and / or vegetable puree, confectionery product, ice cream, sherbet, culinary cream, sauce, seasoning, cheese, fermented dairy product, dairy dessert, pet food product, dairy dessert, nutritional bar, cereal product, fermented cereal-based product, food supplement, nutritional composition, nutritionally complete formula, infant nutrition product, nutritional bar, enteral nutrition product, plant-based meat analogue, plant-based cheese substitute, or a mixture thereof. In one embodiment, the beverage can be selected from the list consisting of: milked juice, soft drink, water-based beverage, soup, milk beverage, plant-based milk substitute, coffee, tea, cocoa beverage, flavored water, soup, mineral water, malt beverage, creamer, fermented milk beverage, plant-based fermented milk beverage substitute, or a mixture thereof.

[0180] In one embodiment, the food product or beverage is vegetarian or vegan. In particular, the iron-containing green plant material concentrate is suitable for use in a vegan / vegetarian diet, and can be used to fortify vegan or vegetarian food products and beverages with iron.

[0181] In a fifth aspect, the present application relates to a food supplement comprising the iron-containing green plant material concentrate according to the second or third aspect of the present application. The iron-containing green plant material concentrate of the present application can be used to prepare a food supplement that allows for the delivery of a significant amount of iron, for example in humans, animals or pets. In one embodiment, the food supplement is provided in the form of a capsule, gelatin capsule, soft capsule, tablet, sugar-coated tablet, pill, paste or lozenge, chewing gum, drinkable solution or emulsion, syrup or gel.

[0182] In one embodiment, the food supplement can be a food supplement for use in preventing or treating a condition or disease associated with iron deficiency in a subject. In one embodiment, the condition or disease associated with iron deficiency can be selected from the list consisting of: iron deficiency anemia, chronic heart failure or pulmonary arterial hypertension. In this embodiment, the food supplement comprises an effective amount, preferably a therapeutically or prophylactically effective amount, of the iron-containing green plant material concentrate, in particular iron. The sixth aspect of the present invention provides further details on the term "therapeutically effective amount" or "prophylactically effective amount".

[0183] In one embodiment, the food supplement is vegetarian or vegan. In particular, the iron-containing green plant material concentrate is suitable for a vegan / vegetarian diet and can be used for fortifying a vegan or vegetarian food supplement with iron.

[0184] In a sixth aspect, the present invention relates to a cosmetic or pharmaceutical composition comprising the iron-containing green plant material concentrate according to the second or third aspect of the present invention. The iron-containing green plant material concentrate of the present invention can be used for preparing a cosmetic or pharmaceutical composition allowing the delivery of a significant amount of iron, for example in humans, animals or pets.

[0185] The pharmaceutical composition can be administered for both prophylactic and / or therapeutic treatment. In therapeutic applications, compositions according to the present invention are administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as "therapeutically effective amount". Amounts effective for this purpose will depend on the severity of the disease and the weight and general state of the patient. In prophylactic applications, compositions according to the present invention are administered to a patient susceptible of, or otherwise at risk of, suffering from a particular disease. Such an amount is defined to be "prophylactically effective amount". The exact amount in this case will also depend on the state of health and weight of the patient.

[0186] In one embodiment, the pharmaceutical composition can be a pharmaceutical composition for use in preventing or treating a condition or disease associated with iron deficiency in a subject. In one embodiment, the condition or disease associated with iron deficiency can be selected from the list consisting of: iron deficiency anemia, chronic heart failure or pulmonary arterial hypertension. In this embodiment, the pharmaceutical composition comprises an effective amount, preferably a therapeutically or prophylactically effective amount, of the iron-containing green plant material concentrate, in particular iron.

[0187] The pharmaceutical composition of the present invention is preferably administered with a pharmaceutically acceptable carrier, the nature of which will depend on the intended route of administration, for example oral. Various excipients can be used to prepare the desired formulations, including for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate.

[0188] In a preferred embodiment, the pharmaceutical composition can be a tablet, a capsule, a pill, a solution, a suspension, a syrup, a dry oral supplement, a wet oral supplement, an ointment, an aerosol, a patch, a cream, a gel, a spray, a suppository.

[0189] In a preferred embodiment, the pharmaceutical composition is a pharmaceutical oral composition. In particular, the pharmaceutical oral composition can be a tablet, a capsule, a pill, a solution, a suspension, a syrup, a dry oral supplement, a wet oral supplement.

[0190] It will be appreciated that the skilled person will select appropriate components and galenical forms to target the active compound to the tissue / compartment of interest of the body, e.g. the skin, the colon, the blood, the veins, the arteries, the lungs, the heart, the stomach, the eyes, the kidneys or the liver, in view of the target route of administration, such as the oral route of administration.

[0191] The cosmetic composition can be a tablet, a capsule, a pill, a solution, a suspension, a syrup, a dry oral supplement, a wet oral supplement, an ointment, a patch, a cream, a gel, a spray. Other cosmetically active ingredients can also be added. Excipients or colorants commonly used in cosmetics can also be added to the composition.

[0192] In a preferred embodiment, the cosmetic composition is a cosmetic oral composition. In particular, the cosmetic oral composition can be a tablet, a capsule, a pill, a solution, a suspension, a syrup, a dry oral supplement, a wet oral supplement.

[0193] It will be appreciated that the concept of the present application can equally be used as an adjuvant therapy to assist the current use of pharmaceuticals. Since the iron-containing green plant material concentrate of the present application can easily be administered together with food material, it is possible to apply a special clinical food containing a large amount of the iron-containing green plant material concentrate. It will be apparent to those skilled in the art, upon reading the present specification and appended claims, that various different alternatives to the specific embodiments mentioned herein will be envisaged.

[0194] In a seventh aspect, the present application relates to a method for fortifying a food product or a beverage with iron, the method comprising preparing a food product or a beverage, and adding to the food product or beverage an iron-containing green plant material concentrate according to the second or third aspect of the present application.

[0195] In an embodiment, the food product can be a food product as provided in the fourth aspect of the present application. In an embodiment, the beverage can be a beverage as provided in the fourth aspect of the present application.

[0196] The iron-containing green plant material concentrate can be used to fortify food products and beverages, including vegan or vegetarian food products / beverages, with iron. The iron-containing green plant material concentrate has good organoleptic properties and does not, or very limited, produce a metallic off-taste when used in a beverage or food product.

[0197] The person skilled in the art will understand that they can freely combine all features of the application disclosed herein. In particular, features described for the product of the application can be combined with the method or process of the application, or vice versa. In addition, features described for different embodiments of the application can be combined.

[0198] Furthermore, for a specific feature if there are known equivalents, such equivalents are incorporated as if explicitly set forth in the specification. Additional advantages and features of the application will become apparent after review of the following drawings and non-limiting examples.

[0199] Example

[0200] Example 1 - Method of the invention to concentrate iron from dried herbal plants without acid

[0201] Dry herbaceous plants, in particular dry nettle (Urtica dioica) or dry peppermint A or B (Mentha piperita) or dry thyme A or B (Thymus vulgaris) comprising leaves and stems were ground into a powder. Dry thyme A originates from France, while dry thyme B originates from Morocco. Dry peppermint A originates from France, while dry peppermint B originates from Egypt. The powder was mixed with water in a ratio of 1 : 15 (w:v) and the powder was hydrated for 5 minutes to form a suspension. The suspension was then blended for 1 minute to obtain a slurry. The obtained slurry was filtered through a filter with a mesh size of 500 pm. The permeate was recovered and subsequently filtered through a filter with a mesh size of 180 pm. The permeate was recovered and the obtained permeate was heat treated to a temperature of 71 °C for 2 minutes. After cooling to 4 °C, the permeate was centrifuged at 2500 g for 10 minutes. The sediment was recovered and an iron-containing green plant material concentrate was formed. Optionally, the concentrate can be dried.

[0202] Example 2 - Method of the invention to concentrate iron from dried herbal plants in the presence of acid

[0203] Dry herbaceous plants, in particular dry peppermint (Mentha piperita), dry nettle (Urtica dioica) or dry thyme (Thymus vulgaris) comprising leaves and stems were ground to a powder. The powder (50 g) was mixed with water (700 mL) in a ratio of 1 : 15 (w:v) to prepare a suspension and pure citric acid (4.5 g, 15.8 g or 6.1 g anhydrous citric acid for peppermint, nettle or thyme, respectively) or ascorbic acid (16.8 g ascorbic acid for peppermint) or hydrochloric acid (3.6 mL, 15 mL or 5.3 mL 6 M HC1 solution for peppermint, nettle or thyme, respectively) or malic acid (4.7 g malic acid for peppermint) was added to the suspension to reach a pH of 3.5. After acid addition, the suspension was left for 2 minutes to ensure proper hydration of the powder in the suspension. The suspension was then blended for 2 minutes to obtain a slurry. The obtained slurry was filtered through a filter with a mesh size of 500 pm. The permeate was recovered and subsequently filtered through a filter with a mesh size of 180 pm. The permeate was recovered and the obtained permeate was heat treated to reach a temperature of 71 °C for 2 minutes. After cooling to 4 °C, the permeate was centrifuged at 2500 g for 10 minutes. The precipitate was recovered and a green plant material concentrate was formed. Optionally, the concentrate can be dried.

[0204] Example 3 - Formulation of a beverage

[0205] Citric acid or malic acid or lime concentrate or mixtures thereof were added to 500 ml water to reduce the pH to 3.5. Sucrose was added to reach a concentration between 20 g / L and 40 g / L. The green plant material concentrate of Example 1 or 2 was added to reach an iron content of 2.1 mg iron. The final pH was adjusted to pH 3.

[0206] Example 4 - Iron quantification

[0207] Materials and methods

[0208] The iron content of the raw material (i.e. the dry herb powder obtained after grinding) and the iron-containing green plant material concentrate obtained in Example 1 or 2 was determined by atomic emission spectrometry using a microwave plasma atomic emission spectrometer (MP-AES) 4200 (Agilent, Switzerland). For MP-AES analysis, samples (approximately 100 mg to 400 mg) were mineralized in duplicate using a Xpress microwave bomb in a microwave digestion system (Mars 6, CEM, USA) with 4 mL of 70% HNO3 ultra-pure mass (Sigma-Aldric, St. Louis, MO, USA), 1 mL of 30% H2O2 (Merck KGaA, Darmstadt, Germany). The mineralized solution was then transferred into a 50 mL Falcon tube and the volume was adjusted to 20 mL with Milli-Q water. The iron content was measured at a wavelength of 371 nm using an external calibration with multi-element standards. The accuracy of the analysis was checked by analyzing a standard reference material (SRM 3233, typical diet; NIST, MD, USA).

[0209] Results

[0210] Figure 1 The iron content (based on DW) of dry peppermint A, dry nettle, dry thyme A and B and their corresponding iron-containing green plant material concentrate obtained according to the method of Example 1 is shown. In addition, the iron content of four commercial extracts of peppermint (2 extracts, Martin Bauer 11000005 Peppermint powder extract, Martin Bauer 11000192 Peppermint extra fine powder), nettle (1 extract, Martin Bauer 15100000 Nettle leaf powder extract) and thyme (1 extract, Martin Bauer 17100001 Thyme powder extract) is reported.

[0211] Dry peppermint, dry nettle and dry thyme are understood to be the whole of the leaves and stems of the dry plants which are ground into a powder before being further processed in the concentration method.

[0212] The concentration process of the present application achieved a significant increase in iron concentration from 298 ppm in dry peppermint A to 2973 ppm in the iron-containing peppermint concentrate A (= iron-containing peppermint A concentrate). The concentration process of the present application achieved a significant increase in iron concentration from 288 ppm in dry nettle to 1355 ppm in the iron-containing nettle concentrate. The concentration process of the present application achieved a significant increase in iron concentration from 180 ppm in dry thyme A to 1294 ppm in the iron-containing thyme concentrate A. For another batch of thyme (i.e. thyme B), the concentration process of the present application achieved a significant increase in iron concentration from 1903 ppm in dry thyme B to 6142 ppm in the iron-containing thyme concentrate B (= iron-containing thyme B concentrate).

[0213] The commercial water extracts of peppermint, nettle and thyme all showed very low iron concentrations, specifically below 165 ppm, indicating a clear advantage of the concentration process of the present application over standard water concentration processes in obtaining a concentrate with a high iron concentration.

[0214] Example 5 - Quantification of anti-nutritional factors

[0215] Materials and methods

[0216] Oxalic acid was extracted from the samples with water under mechanical stirring. Oxalic acid was determined by ion chromatography (Dionex ICS-5000 with column Dionex Ion PAC AS16 REFIC Analytical (250 x 2 mm)) coupled with mass spectrometry (SCIEX Triple Quad 5500 with Selexion).

[0217] Total phenolic content was quantified as follows. Phenolics were extracted by suspending the sample in methanol, shaking it every 5 minutes for 1 minute for 30 minutes. The sample was then centrifuged (750 g, 10 minutes, 20°C) and the supernatant was recovered. The pellet was re-extracted with methanol, centrifuged, and the supernatant was recovered and combined with the previous supernatant. The extract (1 mL) was mixed with 15 mL water and 1 mL Folin-Ciocalteu reagent, mixed well, and it was left to stand for 6 minutes. A sodium carbonate solution (3 mL, 20%) was added to each sample and mixed well. The samples were incubated for 2 hours at 30°C, and then the absorbance was read at 765 nm. Total phenolic content was quantified according to a gallic acid calibration curve, so the results are expressed as gallic acid equivalents, since each phenolic compound is equivalent to one molecule of gallic acid.

[0218] ​Phytic acid (phytate) was measured according to the Megazyme kit for "Phytic acid (phytate) / total phosphorus". This kit enables the quantification of free and total phosphorus in a sample by colorimetric detection. Total phosphorus is defined as the phosphorus originating from phytic acid as well as other sources and is measured after first treating the sample with phytase followed by treatment with alkaline phosphatase. Free phosphorus, on the other hand, is defined as the phosphorus originating from non-phytate sources within the sample and is measured without enzyme treatment from the kit. In short, 1 g of sample was mixed with 20 mL of HC1 acid (0.66 M) and stirred vigorously for 3 hours to form an extract. The extract (1 mL) was centrifuged at 13000 rpm for 10 minutes and 0.5 mL of the resulting supernatant was neutralized with 0.5 mL of NaOH solution (0.75 M). The neutralized sample extract (0.05 mL) was mixed with distilled water (0.60 mL), buffer I provided (0.20 mL) and a phytase suspension (0.02 mL) for the quantification of total phosphorus. A control sample was prepared by mixing the sample extract (0.05 mL) with distilled water (0.62 mL) and buffer I provided (0.20 mL) for the quantification of free phosphorus. Both samples were vortexed and incubated at 40 °C for 10 minutes. Distilled water (0.02 mL) and buffer 3 provided (0.2 mL) were added to the control, while buffer 3 (0.20 mL) and suspension 4 (ADP, 0.02 mL) were added to the sample for total phosphorus. The samples were vortexed and incubated at 40 °C for 15 minutes. The reaction was stopped by adding 0.30 mL of trichloroacetic acid (50% w / v). The samples were centrifuged at 13000 rpm for 10 minutes. The supernatant (1 mL) was used for the colorimetric determination of phosphorus. The samples were mixed with 0.5 mL of color reagent. The color reagent was prepared by mixing 1 part of ammonium molybdate solution (5% w / v) with 5 parts of ascorbic acid (10% w / v) / sulfuric acid (1 M) solution. After mixing the samples with the color reagent, they were incubated at 40 °C for 1 hour, after which the absorbance was read at 655 nm.

[0219] The measured absorbance was used to calculate the concentration of phosphorus, which in turn was used to calculate the concentration of PA. The concentration of phosphorus in the free phosphorus as well as total phosphorus reaction was calculated using equation (1).

[0220]

[0221] c 磷 = concentration of phosphorus in the sample [g / 100g]

[0222] average M = average of the phosphorus standards [pg / AA 标准物 ], where M = pg of phosphorus in the standard solution (i.e. 0.5-7.5) divided by AA 标准物 (i.e. A 标准物X -A 标准物0 )

[0223] vHCl = volume of original sample extract [mL]

[0224] F = dilution factor (in this study, F = 1)

[0225] AA 样品 = absorbance difference of sample (i.e. A 样品总 - A 样品游离 )

[0226] w = weight of original sample material [g]

[0227] v = volume of sample used in colorimetric assay step [mL] (= 1 mL)

[0228] 10000 = conversion from pg / g to g / 100g

[0229] The calculated bound phosphorus concentration is converted to PA concentration using equation (2). This assumes that the amount of bound phosphorus measured is solely from PA sources.

[0230]

[0231] c PA= Concentration of PA in sample [g / 100g]

[0232] c 磷 = concentration of bound phosphorus in sample [g / 100g]

[0233] 0.282 = mass fraction of phosphorus in PA

[0234] The molar ratio between iron and phytic acid is then calculated.

[0235] Results :

[0236] Table 1 shows the amount of oxalic acid and phytic acid on a dry weight basis for iron-containing peppermint concentrates A and B, iron-containing nettle concentrate, and iron-containing thyme concentrate A obtained according to the method of Example 1.

[0237]

[0238] Figure 2 The molar ratio between iron and oxalic acid molarity is shown for dry peppermint A and dry nettle powder, and iron-containing peppermint A and nettle concentrates obtained according to the method of Example 1. The concentration method of the present application achieves an increase in the ratio of iron to oxalic acid from 0.25 to 0.52, indicating a lower concentration of iron potentially sequestered by oxalic acid in the iron-containing peppermint concentrate, and thus potentially more iron available for absorption. An even greater improvement in the ratio of iron to oxalic acid is observed from dry nettle (0.23) to the iron-containing nettle concentrate (1.11).

[0239] Figure 10 The total phenolic content (mg gallic acid equivalents / g DW) in dry peppermint B and dry nettle and iron-containing peppermint B and nettle concentrates obtained according to the method of Example 1 is shown. The concentration method of the application achieved a significant reduction in total phenolics from 47.3 mg GAE / g DW in dry peppermint to 16.4 mg GAE / g DW in the iron-containing peppermint concentrate, which indicates a potential reduction in iron uptake inhibitors (anti-nutritional factors). A similar trend was observed for nettle; total phenolics were reduced from 8.38 mg GAE / g DW in dry nettle to 0.88 mg GAE / g DW in the iron-containing nettle concentrate.

[0240] These results indicate that the concentration of iron potentially sequestered by phenolic compounds in the iron-containing concentrates is lower, and therefore potentially more iron is available for uptake.

[0241] Figure 3 The molar ratio between iron and phytic acid molarity in dry peppermint A and B, nettle and thyme A powders and iron-containing concentrates obtained therefrom according to the method of Example 1 is shown. The concentration method of the application achieved an increase in the ratio of iron to phytic acid from 2.2 in dry peppermint A to 7.7 in the iron-containing peppermint A concentrate.

[0242] The concentration method of the application achieved an increase in the ratio of iron to phytic acid from 6.6 in dry peppermint B to 44 in the iron-containing peppermint B concentrate (= iron-containing peppermint concentrate B).

[0243] The concentration method of the application achieved an increase in the ratio of iron to phytic acid from 1.3 in dry nettle to 8.9 in the iron-containing nettle concentrate.

[0244] The concentration method of the application achieved an increase in the ratio of iron to phytic acid from 2.3 in dry thyme to 23 in the iron-containing thyme concentrate A.

[0245] These results indicate that the concentration of iron potentially sequestered by phytic acid in the iron-containing concentrates is lower, and therefore potentially more iron is available for uptake.

[0246] Example 6 - In vitro digestion to quantify iron bioavailability

[0247] Materials and methods :

[0248] In short, 1 g of the concentrated iron-containing green plant material concentrate prepared according to the concentration method of examples 1 and 2 was mixed with 10 mL of KCI 5 mmol + NaCI 140 mmol at pH 2. After the pH was adjusted to 2, 0.5 mL of a pepsin solution (prepared by dissolving 200 mg of pepsin in 10 mL of 0.1 M HC1) was added and the sample was incubated for 1 hour at 37°C. After 1 hour, the pH was adjusted to 5.5 with 1 M NaHC03. The volume of the sample was adjusted to 15 mL by adding 6.7 KCI 5 mmol + NaCI 140 mmol. A trypsin solution (2.5 mL, prepared by adding 87.5 mg of trypsin and 525 mg of bile extract to 44 mL of 0.1 M NaHC03) was added and the sample was incubated for 2 hours at 37°C. The iron content of a 2.5 g aliquot of the total digest was analyzed by MP AES. The remaining sample was centrifuged at 10000 g for 30 minutes at 4°C and the iron content of 2.5 g of the supernatant was analyzed by MP AES. Iron bioaccessibility was defined as:

[0249]

[0250] Iron bioaccessibility refers to the fraction of the total amount of iron that is theoretically available for absorption.

[0251] Results :

[0252] Figure 4 Iron bioaccessibility of iron-containing peppermint concentrates prepared from dry peppermint B with water according to the concentration method of example 1 or in the presence of an acid, i.e. citric acid or hydrochloric acid or malic acid or ascorbic acid, according to the concentration method of example 2 is shown. The iron bioaccessibility of the different concentrates is compared to the iron bioaccessibility of iron salts, in particular of ferric pyrophosphate. The use of citric acid during the concentration method of the application significantly increases the iron bioaccessibility (24%) compared to the concentrate prepared with water using the concentration method of the application in the absence of an acid (11%) or in the presence of another acid such as ascorbic acid (10%). Hydrochloric acid has a positive effect on the iron bioaccessibility, but to a lesser extent than citric acid, resulting in an iron bioaccessibility of 20%. Malic acid has a positive effect on the iron bioaccessibility, but to a lesser extent than citric acid, resulting in an iron bioaccessibility of 18%. The iron-containing peppermint concentrates prepared with citric acid, hydrochloric acid or malic acid show a higher iron bioaccessibility than ferric pyrophosphate, which is commonly used as an iron fortifier.

[0253] Figure 5 Iron bioaccessibility of iron-containing nettle concentrates prepared with water (according to example 1) or with water in the presence of citric acid and hydrochloric acid, respectively (according to example 2) is shown. Citric acid and hydrochloric acid have a positive effect on the iron bioaccessibility of the iron-containing nettle concentrate.

[0254] Figure 6 The absolute amount of bioaccessible iron contained in dry peppermint B and in iron-containing peppermint concentrates prepared from dry peppermint B with water according to the concentration method of Example 1 or with water in the presence of citric acid, hydrochloric acid, malic acid and ascorbic acid according to the concentration method of Example 2 is shown. The iron content in the samples is multiplied by the bioaccessibility value to calculate the absolute amount of bioaccessible iron. The iron-containing peppermint concentrate prepared with hydrochloric acid has a clear advantage over dry peppermint because it contains a higher amount of bioaccessible iron (601 ppm in the iron-containing peppermint concentrate compared to 288 ppm in dry peppermint).

[0255] Figure 7 The absolute amount of bioaccessible iron contained in dry nettle and in iron-containing nettle concentrates prepared from dry nettle with water according to the concentration method of Example 1 or with water in the presence of citric acid and hydrochloric acid according to the method of Example 2 is shown. The iron content in the samples is multiplied by the bioaccessibility value to calculate the absolute amount of bioaccessible iron. The iron-containing nettle concentrates prepared with water, citric acid or hydrochloric acid have a clear advantage over dry nettle because they contain a higher amount of bioaccessible iron (39 ppm, 114 ppm, 147 ppm in the iron-containing nettle concentrates prepared with water, citric acid, hydrochloric acid, respectively, compared to 28 ppm in dry nettle).

[0256] Figure 8 The iron bioaccessibility of iron-containing thyme concentrates prepared from dry thyme B with water according to Example 1 or with water in the presence of citric acid and hydrochloric acid according to Example 2 is shown. Citric acid and hydrochloric acid have a positive effect on the iron bioaccessibility of the iron-containing thyme concentrates.

[0257] Figure 9 The absolute amount of bioaccessible iron contained in dry thyme B and in iron-containing thyme concentrates prepared from dry thyme B with water according to the concentration method of Example 1 or with water in the presence of citric acid and hydrochloric acid according to the concentration method of Example 2 is shown. The iron content in the samples is multiplied by the bioaccessibility value to calculate the absolute amount of bioaccessible iron. The iron-containing thyme concentrates prepared with citric acid or hydrochloric acid have a clear advantage over dry thyme because they contain a higher amount of bioaccessible iron (190 ppm, 224 ppm in the iron-containing thyme concentrates prepared from dry thyme B with citric acid and hydrochloric acid, respectively, compared to 59 ppm in dry thyme B).

[0258] Example 7 - Peppermint / thyme beverage

[0259] Iron-containing peppermint concentrate and iron-containing thyme concentrate were prepared according to example 1. An iron-containing concentrate blend was prepared by mixing 95% iron-containing peppermint concentrate A and 5% iron-containing thyme concentrate A.

[0260] Their citric acid was added to 500 ml water to reduce the pH to 3.5. Sucrose was added to reach a concentration between 20 g / L and 40 g / L. The iron-containing concentrate blend was added to reach an iron content of 2.1 mg iron. The final pH was adjusted to pH 3.

[0261] Example 8: Sensory data

[0262] Materials and methods Ten different beverages were prepared with the same iron content (2.1 mg iron per 200 mL beverage):

[0263] - Beverage A: a beverage prepared according to example 3 containing an iron- containing peppermint concentrate prepared according to the method of example 1.

[0264] - Beverage B: a beverage prepared according to example 3 containing an iron- containing peppermint concentrate prepared according to the method of example 2 with citric acid.

[0265] - Beverage C: a beverage prepared according to example 3 containing an iron- containing peppermint concentrate prepared according to the method of example 2 with hydrochloric acid.

[0266] - Beverage D: a beverage prepared according to example 3 containing an iron- containing nettle concentrate prepared according to the method of example 1.

[0267] - Beverage E: a beverage prepared according to example 3 containing an iron- containing nettle concentrate prepared according to the method of example 2 with citric acid.

[0268] - Beverage F: a beverage prepared according to example 3 containing an iron- containing nettle concentrate prepared according to the method of example 2 with hydrochloric acid.

[0269] - Beverage G: a beverage prepared according to example 3 containing an iron- containing thyme concentrate A prepared according to the method of example 1.

[0270] - Beverage H: a beverage prepared according to example 7.

[0271] - Beverage I: a beverage prepared according to example 3 but the iron- containing green plant material concentrate was replaced by a reference curry leaf extract (MoFerrin 21, Biogena).

[0272] - Beverage J: a beverage prepared according to example 3 but the iron- containing green plant material concentrate was replaced by a reference iron salt, i.e. ferric pyrophosphate.

[0273] Beverages A-G and I-J were prepared according to the method provided in example 3 with citric acid as acid (instead of malic acid or lime concentrate).

[0274] The sensory properties of the different beverages were then evaluated one by one by a person trained to assess the metallic off-taste perceived in the mouth. The intensity of the metallic off-taste was characterized by providing a score from 0 (no metallic off-taste perceived in the mouth) to 5 (high metallic off-taste perceived in the mouth).

[0275] Results

[0276] Beverages of example 3 and example 7 comprising the iron-containing green plant material according to the application, i.e. beverages A-J, have good sensory properties. In particular, no unpleasant metallic off-taste is perceived. In contrast, beverage K containing the reference curry leaf extract and beverage L containing ferric pyrophosphate have a strong and unpleasant metallic off-taste.

[0277] Thus, the method of the application allows to provide concentrates containing a significant amount of iron while having good sensory properties, in particular limited metallic off-taste, as well as such food, food supplements and beverages.

[0278] Example 9 - Method of the invention to concentrate iron from fresh herbal plants without acid

[0279] Iron-containing nettle, peppermint and thyme concentrates were prepared as in example 1 but with the following adaptations:

[0280] - dry nettle, dry peppermint and dry thyme were replaced by fresh nettle, fresh peppermint and fresh thyme containing leaves and stems, respectively.

[0281] - fresh nettle, fresh peppermint and fresh thyme were not ground into a powder before mixing with water.

[0282] - fresh nettle, fresh peppermint and fresh thyme were mixed with water at a ratio of 1 : 18 (w:v).

[0283] Example 10 - Method of the invention to concentrate iron from fresh herbal plants in the presence of acid

[0284] Iron-containing nettle, peppermint and thyme concentrates were prepared as in example 2 but with the following adaptations:

[0285] - dry nettle, dry peppermint and dry thyme were replaced by fresh nettle, fresh peppermint and fresh thyme containing leaves and stems, respectively.

[0286] - fresh nettle, fresh peppermint and fresh thyme were not ground into a powder before mixing with water.

[0287] - Fresh nettle, fresh peppermint and fresh thyme are mixed with water in a ratio of 1 : 18 (w:v).

[0288] Example 11 - Method of the invention to concentrate iron from duckweed without acid

[0289] An iron-containing duckweed concentrate is prepared according to the method of Example 1, but with the following adjustments:

[0290] - The dry herbs are replaced by fresh whole duckweed, in particular fresh whole Lemna minor, respectively.

[0291] - The fresh whole duckweed is not ground into a powder before being mixed with water.

[0292] - The fresh whole duckweed is mixed with water in a ratio of 1 : 12 (w:v).

[0293] Example 12 - Method of the invention to concentrate iron from duckweed in the presence of acid

[0294] An iron-containing duckweed concentrate is prepared according to the method of Example 2, but with the following adjustments:

[0295] - The dry herbs are replaced by fresh whole duckweed, in particular fresh whole Lemna minor, respectively.

[0296] - The fresh whole duckweed is not ground into a powder before being mixed with water.

[0297] - The fresh whole duckweed is mixed with water in a ratio of 1 : 12 (w:v).

[0298] Example 13 - Method of the invention to concentrate iron from dried herbal plants with water in the presence of sucrose

[0299] An iron-containing nettle, peppermint and thyme concentrate is prepared as in Example 1, but with the following adjustments:

[0300] - Sucrose is added to the suspension of dry herb powders and water before blending at a concentration ranging between 1% and 20% by weight, preferably 5 to 12% by weight.

[0301] Example 14 - Comparison of the method of the invention to concentrate iron from dried herbal plants in the presence of acid with another method of concentrating iron reported in RO132538A0 Materials and methods .

[0302] Results :

[0303] An iron-containing spearmint concentrate was prepared as in Example 2, but with the following adjustments: dry ground spearmint was mixed with water in a 1 : 10 (w:v) ratio and 3% citric acid was added, resulting in a pH of 3. The suspension was incubated at 40°C for 10 minutes. The obtained slurry was filtered through a filter with a mesh size of 200 pm. The permeate was recovered and subsequently filtered through a filter with a mesh size of 50 pm. The permeate was recovered and centrifuged at 2500 g for 10 minutes. The precipitate was recovered and formed into an iron-containing spearmint concentrate, which was freeze-dried.

[0304] An alternative iron concentration method was performed as reported in patent RO132538A0. Dry ground spearmint was mixed with water in a 1 : 10 (w:v) ratio and 3% citric acid was added, resulting in a pH of 3. The suspension was incubated at 40°C for 10 minutes. The obtained slurry was filtered through a filter with a mesh size of 200 pm. The permeate was recovered and subsequently filtered through a filter with a mesh size of 50 pm. The permeate was recovered and the water was removed by freeze-drying to obtain a freeze-dried spearmint permeate.

[0305] The iron in the iron-containing spearmint concentrate and in the freeze-dried spearmint permeate was measured as described in Example 4.

[0306] ​ :

[0307] The ground spearmint had an iron concentration of 335.0 ± 0.6 ppm. Based on the iron quantification results, the iron concentration method of the present invention resulted in an iron-containing spearmint concentrate containing 1652.7 ± 1.5 ppm (dry weight basis) of iron. The centrifugation step was essential for concentrating the iron-containing material, while removing the less iron-rich material in the supernatant (measured iron content of 30.2 ± 0.1 ppm ppm dry weight basis). Differently, based on the iron quantification results, the iron concentration method of RO132538A0 (where there was no centrifugation step, but the permeate was directly freeze-dried) resulted in an iron content of 246.0 ± 0.5 ppm. These results clearly show the advantage of the invention presented herein to obtain an iron-concentrated plant material, in particular where the iron content is at least 2 times higher than the original green plant material.

[0308] Although the present invention has been described by way of example, it is to be appreciated that modifications and alterations can occur to others upon reading the description. It is intended to cover in the appended claims all such modifications and alterations as fall within the scope of the invention.

Claims

1. Process for the preparation of an iron-containing green plant material concentrate, the process comprising the following steps: a) suspending a green plant material in an aqueous liquid to form a green plant material suspension, b) blending the green plant material suspension to obtain a green plant material slurry, c) applying a physical means to the green plant material slurry to separate and obtain an iron-containing green plant material concentrate, d) optionally, drying the iron-containing green plant material concentrate.

2. Process according to claim 1, wherein step c) is performed by filtration and / or centrifugation and / or decantation and / or heat treatment.

3. Process according to any one of the preceding claims, wherein step c) of applying a physical means is performed by the following steps: cl) filtering the green plant material slurry to obtain a permeate, c2) optionally, heat treating the permeate, c3) centrifuging or decanting the permeate to obtain an iron-containing green plant material concentrate.

4. Process according to any one of the preceding claims, wherein the green plant material is from a herbaceous plant or duckweed.

5. Process according to claim 4, wherein the herbaceous plant is selected from the group consisting of parsley, coriander, mint, thyme, lemon balm, nettle, sage, oregano, rosemary, basil, dill, eleutherococcus, savory or mixtures thereof.

6. Process according to any one of the preceding claims, wherein the green plant material comprises leaves, preferably consists only of leaves.

7. Process according to any one of the preceding claims, wherein the green plant material is a dried green plant material and / or a fresh green plant material.

8. Process according to any one of the preceding claims, wherein the green plant material is a dried green plant material, and wherein the dried green plant material is ground to a powder prior to step a).

9. Process according to any one of the preceding claims, wherein the ratio (w / v) of plant green plant material to aqueous liquid in the green plant material suspension is from 1 :3 to 1 :

20.

10. Process according to any one of the preceding claims, wherein further an acid is added to the green plant material suspension prior to step b).

11. Process according to claim 10, wherein the acid is selected from the list consisting of hydrochloric acid, citric acid, malic acid, ascorbic acid or mixtures thereof.

12. Process according to any one of claims 2 to 11, wherein the filtration step c) or cl) is performed with a filter having a mesh size of 25 pm to 1000 pm.

13. Process according to any one of the preceding claims, wherein the iron concentration in the iron-containing green plant material concentrate obtained in step c) or c3) is at least 2 times higher, preferably 2 to 10 times higher, in weight percent than the iron concentration in the green plant material of step a).

14. The method according to any one of the preceding claims, wherein the molar ratio of iron to oxalic acid in the iron-containing green plant material concentrate obtained in step c) or c3) is at least 2 times higher, preferably 3 to 10 times higher, than the molar ratio in the green plant material of step a).

15. Iron-containing green plant material concentrate obtainable or obtained by the method according to any one of claims 1 to 14.

16. Iron-containing green plant material concentrate comprising at least 500 ppm of iron based on the dry weight of the iron-containing green plant material concentrate.

17. The iron-containing green plant material concentrate according to claim 16 having a molar ratio of iron to oxalic acid of at least 0.3 and / or a molar ratio of iron to phytic acid of at least 5.

18. Food product or beverage comprising the iron-containing green plant material concentrate according to any one of claims 15 to 17.

19. Food supplement comprising the iron-containing green plant material concentrate according to any one of claims 15 to 17.

20. Cosmetic or pharmaceutical composition comprising the iron-containing green plant material concentrate according to any one of claims 15 to 17.

21. Method for fortifying a food product or beverage with iron comprising preparing a food product or beverage and adding to the food product or beverage the iron-containing green plant material concentrate according to any one of claims 15 to 17.

22. The food supplement according to claim 19 or the pharmaceutical composition according to claim 20 for use in the treatment or prevention of a condition or disease associated with iron deficiency in a subject.

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