Iron-containing green plant material concentrate

Abstract

A method for preparing an iron-containing green plant material concentrate is disclosed. This method includes the step of suspending green plants in an aqueous liquid to form a green plant material suspension. The green plant material suspension is then blended, treated by physical means, and optionally dried. Such an iron-containing green plant material concentrate is also disclosed. Furthermore, a food product, beverage, nutritional supplement, cosmetic composition or pharmaceutical composition containing such an iron-containing green plant material concentrate, and a method for fortifying a food product or beverage with iron are also disclosed.

Description

[Technical Field]

[0001] This invention generally relates to the field of iron-containing raw materials derived from plants. For example, this invention relates to a method for preparing an iron-containing green plant material concentrate, and to the iron-containing green plant material concentrate itself. This invention also relates to food products, beverages, nutritional supplements, cosmetic compositions, or pharmaceutical compositions containing such an iron-containing green plant material concentrate. This invention also relates to a method for fortifying a food product or beverage with iron, including the use of such an iron-containing green plant material concentrate. [Background technology]

[0002] According to the WHO, iron deficiency is the most common nutritional deficiency in the world. Nearly 30% of the world's population suffers from anemia, and half of these cases are due to iron deficiency (Gupta et al., 2020). However, iron fortification is not straightforward, and careful consideration is needed in selecting appropriate sources and mixing procedures to avoid sensory issues such as metallic taste and discoloration.

[0003] Commercially available solutions primarily consist of inorganic iron salts. These inorganic solutions have several drawbacks. Firstly, they are not natural, as they are achieved through chemical reactions / extracted from minerals. Furthermore, iron salts are less acceptable to consumers compared to real food ingredients that deliver the same micronutrients. Finally, inorganic iron salts are often poorly absorbed because they tend to form complexes with low solubility in the mildly alkaline environment of the upper intestines.

[0004] Alternative solutions to inorganic iron salts include organic iron compounds derived from animal sources, such as hemoglobin, myoglobin, or cytochrome. These organic iron compounds derived from animal sources are absorbed more readily by the human body than their corresponding inorganic counterparts. However, due to their origin, they are not suitable for vegetarian / vegan diets.

[0005] Another alternative solution to inorganic iron salts is organic iron compounds derived from plant sources. In plants, iron exists as iron protoporphyrin in cytochrome b or as phytoferritin. However, organic iron compounds derived from plant sources still have several drawbacks. Firstly, plants contain antitrophic factors that can chelate iron, such as oxalic acid and phytic acid. Iron chelation induced by these antitrophic factors reduces the bioavailability of plant-derived iron when ingested by humans. Furthermore, organic iron compounds derived from plant sources may impart sensory defects, such as an unpleasant off-flavor, when used in food products, dietary supplements, or beverages. For example, commercially available iron solutions are equivalent to curry leaf extract. However, the use of this curry leaf extract in food products, dietary supplements, or beverages is limited due to its strong metallic off-flavor.

[0006] Therefore, it is desirable to provide a raw material that is a substantial source of iron and can be used to fortify food products and beverages, or to prepare iron-delivering nutritional supplements, cosmetic compositions, or pharmaceutical compositions. This raw material should be natural, suitable for vegetarian / vegan diets, have a limited content of anti-nutrient factors that reduce or inhibit iron absorption, possess sufficient bioaccessibility, and have good sensory characteristics, particularly limited metallic off-flavors.

[0007] No reference to prior art documents in this specification should be considered to imply that such prior art is well known or that it forms part of a common general understanding in the art. [Overview of the project]

[0008] The present invention aims to improve the current state of the art, and in particular to overcome the problems of the prior art and address the above needs by providing a process, an iron-containing green plant material concentrate, a food product, a beverage, a nutritional supplement, a cosmetic composition, a pharmaceutical composition, and a method for fortifying food products or beverages with iron, or at least a useful alternative.

[0009] The inventors have surprisingly found that the object of the present invention can be achieved by the subject matter of the independent claim. The dependent claims further develop the idea of ​​the present invention. Therefore, a first aspect of the present invention is a method for preparing an iron-containing green plant material concentrate, a) A step of suspending green plant material in an aqueous liquid to form a green plant material suspension, b) A step of blending green plant material suspensions to obtain a green plant material slurry, c) A step of applying physical means to a green plant material slurry to separate and obtain an iron-containing green plant material concentrate, d) Optionally, a step of drying the iron-containing green plant material concentrate, We propose a method that includes this.

[0010] A second aspect of the present invention proposes an iron-containing green plant material concentrate that can or can be obtained by the method according to the first aspect of the present invention.

[0011] A third aspect of the present invention proposes an iron-containing green plant material concentrate containing at least 500 ppm of iron by dry weight.

[0012] A fourth aspect of the present invention proposes a food product or beverage comprising an iron-containing green plant material concentrate according to the second or third aspect of the present invention.

[0013] A fifth aspect of the present invention proposes a nutritional supplement comprising an iron-containing green plant material concentrate according to the second or third aspect of the present invention.

[0014] A sixth aspect of the present invention proposes a cosmetic composition or pharmaceutical composition comprising an iron-containing green plant material concentrate according to the second or third aspect of the present invention.

[0015] A seventh aspect of the present invention proposes a method for fortifying a food product or beverage with iron, comprising the steps of preparing a food product or beverage and adding an iron-containing green plant material concentrate according to a second or third aspect of the present invention to the food product or beverage.

[0016] The present invention has been found to enable the effective concentration of iron from plant materials while effectively reducing the ratio of undesirable compounds to iron, such as insoluble plant materials and anti-trophic factors that reduce or inhibit iron absorption. The resulting concentrate contains a substantial amount of iron and a limited amount of anti-trophic factors compared to iron, particularly anti-trophic factors that reduce or inhibit iron absorption in the body. The iron in the concentrate is derived from plant materials. Therefore, the concentrate is from natural raw materials and is suitable for vegetarian / vegan diets. Furthermore, when used in beverages, nutritional supplements, or food products, the concentrate has good sensory properties and does not impart any sensory defects, or imparts only very limited sensory defects, particularly limited metallic off-flavors. Moreover, the concentrate has sufficient iron bioaccessibility properties.

[0017] These and other aspects, features and advantages of the present invention will become further apparent to those skilled in the art from the detailed description of embodiments of the invention in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0018] [Figure 1]The iron content (mg / kg) based on dry weight (DW) of dried peppermint A, dried nettle, dried thyme A and B, and the corresponding iron-containing green plant material concentrates obtained from them according to the method of Example 1 is shown. Furthermore, the iron content of two commercially available peppermint extracts, one commercially available nettle extract, and one commercially available thyme extract is reported. The values ​​represent the mean between three independent samples, and the error bars represent the standard deviation. In Figure 1, “dried peppermint,” “dried nettle,” “dried thyme A,” and “dried thyme B” are understood to be a combination of the leaves and stems of the corresponding dried plants that have been ground into powder before further processing in the concentration process of Example 1. For “dried peppermint” in Figure 1, the corresponding dried plant is dried peppermint A. [Figure 2] Figure 2 shows the molar ratio (M / M) of iron to oxalic acid in dried peppermint A, dried nettle, iron-containing peppermint concentrate obtained from dried peppermint A according to the method of Example 1, and iron-containing nettle concentrate obtained from dried nettle according to the method of Example 1. The values ​​represent the average between two consecutive measurements. In Figure 2, "dried peppermint" and "dried nettle" are understood to be a combination of the leaves and stems of dried peppermint plant A and dried nettle plant, respectively, which were ground into a powder before further processing in the concentration process of Example 1. [Figure 3] Figure 3 shows the molar ratio (M / M) of iron to phytic acid in the iron-containing concentrates obtained from dried peppermint A and B, nettle, and thyme A, respectively, according to the method of Example 1. The values ​​represent the average between two independent samples, and the error bars represent the standard deviation. In Figure 3, "dried peppermint A," "dried peppermint B," "dried nettle," and "dried thyme" are understood to be a combination of the leaves and stems of the corresponding dried plants that were ground into powder before further processing in the concentration process of Example 1. For "dried thyme" in Figure 3, the corresponding dried plant is dried thyme A. [Figure 4]The bioaccessibility of iron in an iron-containing peppermint concentrate prepared from dried peppermint B using water (according to the method of Example 1), and in an iron-containing peppermint concentrate prepared from dried peppermint B using water in the presence of citric acid, hydrochloric acid, malic acid, or ascorbic acid (according to the method of Example 2), respectively, is shown compared to iron pyrophosphate. Values represent the mean between duplicate independent samples, and error bars represent the standard deviation. [Figure 5] The bioaccessibility of iron in an iron-containing nettle concentrate prepared using water (according to Example 1), and in an iron-containing nettle concentrate prepared using 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. [Figure 6] The absolute amount of bioaccessible iron contained in dried peppermint B and in iron-containing peppermint concentrates prepared from dried peppermint B using water according to the concentration method of Example 1 or using water in the presence of citric acid, hydrochloric acid, malic acid, and ascorbic acid according to the concentration method of Example 2, respectively, 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. In Figure 6, "dried peppermint" is understood to be the combined leaves and stems of the dried peppermint plant B that were ground into powder before further processing during the concentration process of Example 1 or Example 2. [Figure 7] The absolute amount of bioaccessible iron contained in dried nettle, in an iron-containing nettle concentrate prepared using water (according to Example 1), and in an iron-containing nettle concentrate prepared using 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. In Figure 7, "dried nettle" is understood to be the combined leaves and stems of the dried nettle plant that were ground into powder before further processing during the concentration process of Example 1 or Example 2. [Figure 8] Bioaccessibility of iron in the iron-containing thyme concentrate prepared from drying time B with water (according to the method of Example 1), and the iron-containing thyme concentrates prepared from drying time B with water in the presence of citric acid and hydrochloric acid (according to the method of Example 2), respectively, are shown. Values represent the average between duplicate independent samples, and error bars represent the standard deviation. [Figure 9] Absolute amounts of bioaccessible iron contained in drying time B, the iron-containing thyme concentrate prepared from drying time B with water (according to the method of Example 1), and the iron-containing thyme concentrates prepared from drying time B with water in the presence of citric acid and hydrochloric acid (according to the method of Example 2), respectively, 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 average between duplicate independent samples, and error bars represent the standard deviation. In Figure 9, "drying time" is understood to be the combination of the leaves and stems of dried thyme plant B ground into powder before further processing during the concentration process of Example 1 or Example 2. [Figure 10] Total phenolic contents (mg gallic acid equivalent / g DW) in dried peppermint B and dried nettle, and the iron-containing concentrates obtained from the above dried peppermint B and dried nettle according to the method of Example 1, respectively, are shown. Values represent the average between duplicate independent samples, and error bars represent the standard deviation. In Figure 10, "dried peppermint" and "dried nettle" are understood to be the combination of the corresponding dried plant leaves and stems ground into powder before further processing during the concentration process of Example 1. For "dried peppermint" in Figure 3, the corresponding dried plant is dried peppermint B. **BEST MODE FOR CARRYING OUT THE INVENTION**

[0019] When used herein, terms such as “comprise” and “comprising” should be interpreted not as exclusive or exhaustive, but as encompassing, i.e., “including, but not limited to.” Similarly, all terms “include,” “including,” and “or” should be interpreted as encompassing, unless such interpretation is clearly prevented by the context. However, compositions / products disclosed herein may not include elements not specifically disclosed herein. Therefore, disclosures of embodiments using the term “comprising” include disclosures of embodiments that “essentially include / essentially consist of” the specified components, and embodiments that “consist of” the specified components.

[0020] All numerical ranges should be understood to include all integers, whole numbers, or fractions within that range. Furthermore, these numerical ranges should be interpreted as supporting claims that cover any number or subset of a number within that range. For example, a disclosure of 1 to 10 should be interpreted as corresponding to ranges such as 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, and 3.5 to 9.9.

[0021] As used herein, the singular “one” (“a,” “an,” and “the”) includes multiple references unless otherwise indicated. Therefore, for example, a reference to “a green plant material” or “the green plant material” includes one green plant material, but also includes two or more green plant materials.

[0022] Unless otherwise stated, all percentages herein refer to weight percentages, where applicable.

[0023] Unless otherwise defined, all technical and scientific terms have the same meaning as those commonly understood by those skilled in the art in the field to which this invention pertains, and should be given the same meaning.

[0024] Where used herein, the term "and / or" 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, "dried leaves and / or fresh leaves" means "dried leaves" or "fresh leaves" or "both dried leaves and fresh leaves".

[0025] When used herein, the terms “example” and “such as” are merely illustrative and descriptive, and should not be considered exclusive or inclusive, especially when followed by a list of terms. However, disclosures of embodiments using the terms “example,” “such as,” and “for example” include disclosures of embodiments in which these terms are exclusive and / or inclusive.

[0026] As used herein, “related” means occurring simultaneously, preferably caused by the same underlying disease or condition, most preferably caused by one of the specified diseases or conditions being caused by the other specified condition or disease.

[0027] As used herein, the terms “treatment” and “treatment” mean administering a composition disclosed herein to an object having a condition for the purpose of reducing, reducing or improving at least one symptom associated with that condition, and / or delaying, reducing or preventing the progression of that condition. The terms “treatment” and “treatment” include both deterrent or preventive treatment (treatment to prevent and / or delay the onset or progression of a targeted pathological condition or disorder) and curative, therapeutic, or disease-modifying treatment, such as therapeutic means for the cure, delay, reduction of symptoms, and / or cessation of progression of a diagnosed pathological condition or disorder, and treatment of patients at risk of or suspected of having the condition, and patients in poor health, or patients diagnosed with a disease or medical condition. The terms “treatment” and “treatment” do not necessarily mean treating until the object is fully recovered. The terms “treatment / therapy” and “to treat / therapy” also refer to maintaining and / or promoting the health of an individual who is not suffering from a disease but is susceptible to unhealthy conditions. The terms “treatment / therapy” and “to treat / therapy” also aim to include the synergistic effect, or otherwise enhancement, of one or more primary preventive or therapeutic measures. In non-limiting examples, treatment / therapy may be performed by a patient, caregiver, physician, nurse, or other healthcare professional.

[0028] The treatment of both humans and animals is within the scope of this disclosure.

[0029] As used herein, the terms “prevent” and “prevention” mean administering a composition disclosed herein to a subject who is not exhibiting any symptoms of the condition in order to suppress or prevent the onset of at least one symptom associated with the condition. Furthermore, “prevention” includes reducing the risk, incidence, and / or severity of the condition or disorder.

[0030] As used herein, “effective amount” means an amount that, in an individual, treats or prevents a deficiency, treats or prevents a disease or medical condition, or, more generally, reduces symptoms, controls the progression of a disease, or provides nutritional, physiological or medical benefit to the individual.

[0031] As used herein, the term “animal” includes, but is not limited to, mammals, including rodents; aquatic mammals; domestic animals, e.g., dogs, cats, and other pets; livestock, e.g., sheep, pigs, cattle, and horses; and humans. Where “animal,” “mammal,” or their plural forms are used, these terms also apply to any animal that can obtain the effects indicated or intended to be indicated in that context, e.g., an animal that benefits from iron fortification. The terms “individual” or “subject” are often used herein to refer to humans, but this disclosure is not limited in that sense. Thus, the terms “individual” or “subject” refer to any animal, mammal, or human that can benefit from the methods and compositions disclosed herein.

[0032] As used herein, the term “pet” means any animal that can benefit from or enjoy the compositions provided herein. For example, a pet may be a bird, a bovine, a canid, a equine, a feline, a goat, a wolves, a mouse, a sheep, or a pig, but a pet can be any suitable animal. The term “companion animal” means a dog or a cat.

[0033] As used herein, the term “green plant material” refers to plant material containing chlorophyll and chloroplasts. These plant materials are generally green due to the presence of chlorophyll, a photosynthetic pigment. Green plant material may also be referred to as chlorophyll-containing plant material.

[0034] As used herein, the term “added organic solvent” refers to an organic solvent that is exogenous to the green plant material and is added to the green plant material in addition to the green plant material for the preparation of an iron-containing green plant material concentrate. The term “added organic solvent” excludes organic solvents that are originally present in the green plant material of the iron-containing green plant material concentrate.

[0035] As used herein, the term "vegan" refers to an edible composition that contains no animal products or animal-derived products whatsoever.

[0036] As used herein, the term "vegetarian" refers to an edible composition that does not contain meat, such as fish.

[0037] As used herein, the term “bioaccessibility” refers to the proportion of the total amount of a substance that may be available for absorption. For example, it refers to the fraction of the substance in an object that may be available for absorption, particularly in the gastrointestinal tract of the object.

[0038] As used herein, the term "GAE" refers to gallic acid equivalent. This term is used when the content of a component is quantified relative to a gallic acid calibration curve. Gallic acid equivalent means that each quantified component is considered to be equivalent to one molecule of gallic acid. In other words, 1 mg GAE / g of a quantified component is equivalent to 1 mg / g of that quantified component.

[0039] As used herein, the terms “blend” and “mixture” may be used interchangeably.

[0040] In a first aspect, the present invention relates to a method for preparing an iron-containing green plant material concentrate.

[0041] This method includes step a) suspending green plant material in an aqueous liquid to form a green plant material suspension.

[0042] In one embodiment, the ratio (w / v) of plant green material to aqueous liquid in the green plant material suspension is 1:3 to 1:20, preferably 1:5 to 1:20, more preferably 1:10 to 1:20, and most preferably 1:12 to 1:18.

[0043] Green plant material contains plant cells. Similarly, green plant material suspension contains plant cells, which are intact plant cells. The plant cells in green plant material suspension are derived from green plant material.

[0044] The green plant material suspension is a product resulting from step a). The green plant material slurry is a product resulting from step b). The green plant material suspension and the green plant material slurry are different. Specifically, in the green plant material slurry, plant cells are destroyed and intracellular substances of the plant cells are released, whereas in the green plant material suspension, plant cells are not destroyed, and as a result, intracellular substances of the plant cells are not released.

[0045] The aqueous liquid, and therefore the suspension of green plant material, does not contain any added organic solvents. For example, an aqueous liquid, and therefore a suspension of green plant material, does not contain added organic solvents selected from the list consisting of acetone, acetonitrile, benzene, 1-butanol, 2-butanol, 3-butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, diethylene glycol, diethyl ether, diethylene glycol dimethyl ether, 1,2-dimethoxyethane (Glym, DME), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, 1,2-dichloroethane, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, hexamethylphosphoramide (HMPA), hexane, methanol, methylene chloride, N-methyl-2-pyrrolidinone (NMP), nitromethane, naphthalene, pentane, 1-propanol, 2-propanol, pyridine, toluene, triethylamine, tricine, tris, tetrahydrofuran, o-xylene, m-xylene, p-xylene, and combinations thereof.

[0046] In a preferred embodiment, the aqueous liquid contains at least 80% by weight of water, more preferably 90% by weight of water, and even more preferably at least 95% by weight of water. Most preferably, the aqueous liquid is water.

[0047] In one embodiment, an osmotic agent may be further added to the green plant material suspension before step b). The osmotic agent may be selected from a list consisting of glucose, glycerol, sucrose, sorbitol, sodium chloride, potassium chloride, or a combination thereof. Preferably, the osmotic agent is sucrose. The amount of osmotic agent added to the green plant material suspension can be easily determined by those skilled in the art, depending on the type of osmotic agent and the osmotic pressure of the suspension. The osmotic agent may be used to adjust the osmotic pressure. Although not bound by theory, the osmotic agent may contribute to preserving certain plant structures that store iron and avoiding their dissolution due to osmosis. Although not bound by theory, this may make it possible to further improve the stability of iron.

[0048] In a preferred embodiment, an acid may be further added to the green plant material suspension before step b).

[0049] In one embodiment, the acid is added to the green plant material suspension before step b) until the pH reaches 2 to 5.5, preferably 2.5 to 4.5, most preferably 3 to 4.

[0050] The acid may 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 the most preferred embodiment, the acid is hydrochloric acid. In another most preferred embodiment, the acid is citric acid. The acid may 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, and tangerine juice.

[0051] The green plant material suspension may contain 0.01% to 5% by weight of acid.

[0052] When the acid is citric acid, the green plant material suspension may contain 0.01 to 3% by weight of citric acid, preferably 0.5 to 2.3% by weight of citric acid.

[0053] If the acid is hydrochloric acid, the suspension of green plant material may contain 0.01% to 0.5% by weight of hydrochloric acid, preferably 0.02% to 0.54% by weight of hydrochloric acid.

[0054] When the acid is malic acid, the suspension of green plant material may contain 0.01% to 3% by weight of malic acid, preferably 0.5% to 2.5% by weight of malic acid.

[0055] The use of acid has a dual effect. In particular, the acid lowers the pH and chelates iron. Lowering the pH and chelating iron contribute to improving the solubility of iron, and therefore to improving the bioaccessibility of iron in iron-containing green plant material concentrates.

[0056] Advantageously, the green plant material is derived from herbs or duckweed. In some embodiments, the green plant material may be a combination of green plant material derived from herbs or duckweed, combined with algae and / or cyanobacteria. Non-limiting examples of algae include Chlorella vulgaris. Non-limiting examples of cyanobacteria include Spirulina platensis.

[0057] Duckweed is a floating aquatic green plant, also known as water lentil. In preferred embodiments, duckweed belongs to one of the following genera: Spirodela, Landoltia, Lemna, Wolffia, or Wolffiella. The use of duckweed allows for the maximization of iron content. Other advantages of using duckweed include its high growth rate, its ability to withstand extreme environments, and its ability to be cultivated in uncultivated ponds, thereby avoiding the use of cultivated land.

[0058] In a preferred embodiment, the green plant material is derived from herbs of the Lamiaceae family and / or herbs of the Apiaceae family and / or herbs of the Urticaceae family. Examples of Lamiaceae herbs include mint, thyme, lemon balm, basil, sage, oregano, rosemary, chervil, savory, or combinations thereof. Examples of Apiaceae herbs include parsley, coriander, dill, or combinations thereof. An example of Urticaceae herb is nettle. In a more preferred embodiment, the green plant material is derived from herbs, which are selected from the group consisting of parsley, coriander, mint, thyme, lemon balm, nettle, sage, oregano, rosemary, basil, dill, chervil, savory, or mixtures thereof. Preferably, the herbs are selected from the group consisting of sage, oregano, parsley, coriander, mint, thyme, lemon balm, nettle, or mixtures thereof. More preferably, the herbs are selected from the group consisting of sage, oregano, parsley, coriander, mint, thyme, lemon balm, nettle, or mixtures thereof. Even more preferably, the herbs are selected from the group consisting of mint, thyme, lemon balm, nettle, sage, oregano, or mixtures thereof. Most preferably, the herbs are selected from the group consisting of mint, thyme, lemon balm, nettle, or mixtures thereof. The use of herbs, in particular the use of the above-listed list of herbs, makes it possible to maximize the amount of iron in the iron-containing green plant material concentrate while simultaneously imparting a pleasant flavor.

[0059] In one embodiment, the mint may be spearmint, peppermint, or a mixture thereof.

[0060] Parsley generally contains 20-600 ppm of iron. Coriander generally contains 20-300 ppm of iron. Mint generally contains 200-1000 ppm of iron. Thyme generally contains 200-3000 ppm of iron. Lemon balm generally contains 300-600 ppm of iron. Nettle generally contains 100-1000 ppm of iron. Sage generally contains 200-1300 ppm of iron. Oregano generally contains 200-1000 ppm of iron. Rosemary generally contains 20-500 ppm of iron. Basil generally contains 20-900 ppm of iron. Dill generally contains 20-500 ppm of iron. Chervil generally contains 20-400 ppm of iron. Savory generally contains 50-400 ppm of iron.

[0061] In some embodiments, parsley refers to any plant of the genus Petroselinum, preferably any edible plant of the genus Petroselinum. Preferably, parsley refers to parsley (Petroselinum crispum).

[0062] In some embodiments, coriander refers to any plant of the genus Coriandrum, preferably any edible plant of the genus Coriandrum. Preferably, coriander refers to coriander (Coriandrum sativum).

[0063] As used herein, the term “mint” refers to any plant of the genus Mentha, preferably any edible plant of the genus Mentha. More preferably, mint refers to a plant selected from the list consisting of Mentha spicata, Mentha × piperita, or a combination thereof.

[0064] In some embodiments, thyme refers to any plant of the genus Thymus, preferably any edible plant of the genus Thymus. Preferably, thyme refers to Thymus vulgaris.

[0065] In some embodiments, lemon balm refers to any plant of the genus Melissa, preferably any edible plant of the genus Melissa. Preferably, lemon balm refers to lemon balm (Melissa officinalis).

[0066] In some embodiments, nettle refers to any plant of the genus Urtica, preferably any edible plant of the genus Urtica. Preferably, nettle refers to Urtica dioica.

[0067] In some embodiments, sage refers to any plant of the genus Salvia, preferably any edible plant of the genus Salvia. Preferably, sage refers to a plant selected from a list consisting of common sage (Salvia officinalis), pineapple sage (Salvia elegans), or a combination thereof.

[0068] In some embodiments, oregano refers to any plant of the genus Origanum, preferably any edible plant of the genus Origanum. Preferably, oregano refers to a plant selected from the list consisting of oregano (Origanum vulgare), marjoram (Origanum majorana), or a combination thereof. More preferably, oregano refers to oregano (Origanum vulgare).

[0069] In some embodiments, rosemary refers to any plant of the genus Rosmarinus, preferably any edible plant of the genus Rosmarinus. Preferably, rosemary refers to rosemary (Rosmarinus officinalis).

[0070] In some embodiments, basil refers to any plant of the genus Ocimum, preferably any edible plant of the genus Ocimum. Preferably, basil refers to basil (Ocimum basilicum).

[0071] In some embodiments, dill refers to any plant of the genus Anethum, preferably any edible plant of the genus Anethum. Preferably, dill refers to Anethum graveolens.

[0072] In some embodiments, chervil refers to any plant of the genus Anthriscus, preferably any edible plant of the genus Anthriscus. Preferably, chervil refers to chervil (Anthriscus cerefolium).

[0073] In some embodiments, savory refers to any plant of the genus Satureja, preferably any edible plant of the genus Satureja. Preferably, savory refers to a plant selected from a list consisting of summer savory (Satureja hortensis), winter savory (Satureja montana), or a combination thereof.

[0074] In some embodiments, the green plant material is not derived from curry tree, and in particular, the green plant material is not derived from Murraya koenigii and / or Bergera koenigii. Green plant material derived from curry tree is not advantageous for the present invention. For example, green plant material derived from curry tree may result in an iron-containing green plant material concentrate having unpleasant sensory properties, including a metallic off-flavor.

[0075] Saffron spice is not a green plant material. In some embodiments, the green plant material is not derived from saffron, and in particular not from saffron (Crocus sativus).

[0076] In some embodiments, the green plant material is different from Tagetes erecta L. In some embodiments, the green plant material is different from tarragon (Artemisia dracunculus), endive (Cichoria endivia), and lettuce (Lactuca sativa).

[0077] In some embodiments, the green plant material is not derived from berries. Examples of berries include wolfberries, blueberries, cranberries, white currants, red currants, blackcurrants, mulberries, blackberries, gooseberries, raspberries, sea buckthorn, strawberries, arbutus berries, grapes, or combinations thereof. In some embodiments, the green plant material is not derived from blueberries, in particular from the Vaccinium sect. cyanococcus. The green plant material may include any part of a green plant, such as leaves, stems, flowers, buds, roots, etc. In one embodiment, the green plant material includes leaves. In one embodiment, the green plant material includes leaves and stems. Preferably, the green plant material includes a substantial amount of leaves. The green plant material includes 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 remainder of the green plant material may be any part of a green plant other than the leaves disclosed herein. In one embodiment, the remainder of the green plant material consists only of stems.

[0078] In the most preferred embodiment, the green plant material consists only of leaves.

[0079] Leaves are generally edible and are preferable because they contain a high proportion of iron compared to other green plants. Therefore, leaves are a good edible raw material for concentrating a considerable amount of iron from green plants.

[0080] In one embodiment, the green plant material is dried green plant material 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 has a longer shelf life than fresh plant material, making it more convenient for handling on an industrial scale.

[0081] In one embodiment, if the green plant material includes or is dried green plant material, the dried green plant material may be pulverized into a powder before step a). The dried green plant material may be pulverized by dry grinding. Dry grinding can be achieved using any machine that provides shearing or includes a cutting device. For example, dry grinding can be performed using a hammer mill, stone mill, roller mill, ball mill, jet mill, colloid mill, agitated medium mill, bead mill, pin mill, roller grinder, roller refiner, impact mill, freeze grinding, rod mill, vibrating mill, cutting mill, disc mill, perforated disc mill, microcut mill, or extruder. The method further includes step b) of blending the green plant material suspension to obtain a green plant material slurry. For example, the blending step may be carried out by any type of shearing or mixing device. Examples of mixing devices include mixers, kitchen mixers, tumbler blenders, paddle mixers, agitators, flow impellers, planetary mixers, multi-screw mixers, Scanima mixers, or Stephan mixers. In one embodiment, the blending may be carried out in step b) for at least 8 seconds, preferably 8 seconds to 5 minutes, more preferably 1 minute to 3 minutes. In one embodiment, the blending may be carried out in step b) at a temperature of 4 to 80°C, preferably 4 to 25°C, more preferably 10 to 25°C. The preferred temperature range of 4 to 25°C is advantageous because it limits the oxidation / chemical decomposition of plant organelles that can occur at higher temperatures, e.g., 60°C to 100°C. For example, the blending may be carried out at room temperature. This step disrupts plant cells and releases their intracellular materials, including iron. This contributes to improved bioaccessibility of iron in the final concentrate when ingested by humans.

[0082] The method further includes step c) applying physical means to a green plant material slurry to separate and obtain an iron-containing green plant material concentrate.

[0083] In one embodiment, step c) is carried out by filtration and / or centrifugation and / or decantation and / or heat treatment.

[0084] The filtration in step c) may be carried out under the same conditions or characteristics as the filtration step c1) provided in the section "Filtration step c1)" below.

[0085] The heat treatment in step c) may be carried out under the same conditions or characteristics as the heat treatment step c2) provided in the section "Heat Treatment Step c2)" below.

[0086] The decantation or centrifugation in step c) may be carried out under the same conditions or characteristics as the decantation or centrifugation step c3) provided in the section “Decantation or centrifugation step c3)” below. In one embodiment, step c) applying physical means is c1) A step of filtering a green plant material slurry to obtain a permeate, c2) Optionally, a step of heat-treating the permeate, c3) This is carried out by the step of centrifuging or decanting the permeate to obtain an iron-containing green plant material concentrate. Filtration process c1) As described above, in one embodiment, the method may include step c1) of filtering the green plant material slurry of step b) to obtain a permeate.

[0087] After filtration step c1), a retenate and a permeate are obtained. The substance that passes through the filter is called the "permeate," and the substance that does not pass through the filter and is recycled is called the "retenate." The retenate is removed after step c1), and the permeate is recovered after step c1 and further processed.

[0088] In a preferred embodiment, the filtration step c1) is carried out using a filter having a mesh of 25 μm to 1000 μm, preferably 25 μm to 500 μm, more preferably 100 μm to 200 μm. This mesh size contributes to improving purity by separating and concentrating the desired compound, such as iron, while discarding / reducing 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 where the concentrate tends to settle less, particularly when used in beverages or liquid / semi-liquid food products, or liquid / semi-liquid nutritional supplements, or liquid / semi-liquid cosmetic compositions, or liquid / semi-liquid pharmaceutical compositions.

[0089] The filtration step c1) can be carried out in one or several steps. In one embodiment, the filtration step c1) can be carried out in 1 to 10 steps, preferably 1 to 5 steps. When the filtration step c1) is carried out in several steps, i.e., 2 to 10 steps, preferably 2 to 5 steps, the mesh size of the filter decreases with each successive filtration step. In other words, the mesh size of the filter used in a given filtration step (e.g., the first filtration step) is larger than the mesh size of the filter used in a successive downstream filtration step (e.g., the second filtration step), etc.

[0090] In a more preferred embodiment, the filtration step c1) is carried out in two steps, specifically, the green plant material slurry is first filtered through a filter having a mesh of 400-500 μm, preferably 500 μm, and then filtered through a filter having a mesh of 50-200 μm, preferably 180 μm.

[0091] Implementing multiple filtration processes, especially two, reduces the tendency for filters to clog.

[0092] In one embodiment, the sequence of steps a), b), and c1) is repeated at least twice, preferably 2 to 5 times, before step c2), and at the point of the second sequence of steps a), b), and c1), the green plant material of step a) is replaced by the retaining liquid obtained in step c1) of the preceding sequence of steps a), b), and c1). For clarification, in the second and subsequent sequences of steps a), b), and c1), the retaining liquid from the preceding sequence of steps a), b), and c1) is suspended in place of the green plant material in the aqueous solution of step a) of the subsequent sequence of steps a), b), and c1). Thus, in the second and subsequent sequences of steps a), b), and c1), the suspensions of steps a) and b) are retaining liquid suspensions, not green plant material suspensions, and the slurries of steps b) and c1) are retaining liquid slurry, not green plant material slurry. Furthermore, in the subsequent steps a), b), and c1), a permeate is still obtained in step c1). Furthermore, in the subsequent steps a), b), and c1), a retaining liquid is still obtained in step c1). The obtained retaining liquid can be further processed in a series of consecutive steps such as a), b), and c1). Heat treatment process c2) As described above, in one embodiment, the method may optionally include step c2) of heat-treating the permeate obtained in step c1). This heat-treating step allows for an extension of the shelf life of the final iron-containing green plant material concentrate. In one embodiment, step c2) is not optional.

[0093] In one embodiment, the heat treatment step c2) is carried out at a temperature of at least 60°C for at least 2 seconds. Preferably, the heat treatment step c2) is carried out at a temperature of 60 to 125°C for 2 seconds to 30 minutes. More preferably, the heat treatment step c2) is carried out at a temperature of 70 to 85°C for 1 minute to 3 minutes. decantation or centrifugation step c3) As described above, in one embodiment, the method may include step c3) of obtaining an iron-containing green plant material concentrate by centrifuging or decanting the permeate. Preferably, step c3) is a step of centrifuging the permeate. In one embodiment, the centrifugal separation step c3) is carried out with 500 to 10000 g, preferably 1000 g to 5000 g, more preferably 1000 g to 3000 g. In one embodiment, the centrifugal separation step c3) is carried out for 2 to 30 minutes, preferably 2 to 20 minutes, more preferably 5 to 15 minutes.

[0094] After centrifugation or decantation, a supernatant and a precipitate are obtained. The precipitate corresponds to a generally solid or semi-solid material that forms a deposit at the bottom of the centrifugation / decantation vessel, while the supernatant corresponds to a generally liquid material that floats or spreads over the precipitate. Discard the supernatant. Collect the precipitate. The precipitate obtained after step c3) corresponds to an iron-containing green plant material concentrate.

[0095] In one embodiment, the centrifugation or decantation step c3) is performed only once. In other words, the precipitate obtained in step c3) is not subjected to further centrifugation or decantation.

[0096] The method of the present invention further comprises step d) optionally drying an iron-containing green plant material concentrate to obtain an iron-containing green plant material concentrate in powder form. After step d), the iron-containing green plant material concentrate is in powder form rather than semi-solid form. For example, the drying step may be carried out by spray drying, roller drying, air drying, or freeze-drying. In one embodiment, drying step d) is not optional.

[0097] Instead of drying the iron-containing green plant material concentrate into a powder, the long-term microbiological stability of the iron-containing green plant material concentrate may be improved by reducing its water activity. Therefore, in an alternative embodiment, the method may include a step d') of reducing the water activity of the iron-containing green plant material concentrate after step c) of applying physical means or step c3) of centrifugation or decantation. After step d') of reducing water activity, the iron-containing green plant material concentrate has a water activity of less than 0.85, preferably 0.5 to 0.85. After step d') of this water activity reduction, the iron-containing green plant material concentrate is not in powder form. In fact, 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., a semi-solid form. However, the iron-containing green plant material concentrate obtained after step d') has a lower water activity than the iron-containing green plant material concentrate obtained immediately after step c) or step c3). This step d') to reduce the water activity can be carried out by evaporating the iron-containing green plant material concentrate, drying the iron-containing green plant material concentrate, or adding a water-retaining agent to the iron-containing green plant material concentrate. Preferably, the water-retaining agent is sucrose. The amount of water-retaining agent added to the iron-containing green plant material concentrate can be easily determined by those skilled in the art, depending on the type of water-retaining agent and the target water activity. Drying may be carried out by freeze-drying, spray-drying, air-drying, or roller-drying. The evaporation step may be carried out using an evaporator.

[0098] In some embodiments, the step d') of reducing the water activity of the iron-containing green plant material concentrate and the step d) of drying the iron-containing green plant material concentrate may be performed consecutively. In this embodiment, the step d') of reducing the water activity of the iron-containing green plant material concentrate is performed before the step d) of drying the iron-containing green plant material concentrate.

[0099] In some embodiments, particularly when step d) drying is applied, the method does not involve evaporation or drying before step d).

[0100] In some embodiments, particularly when step d) drying is applied, the method does not include any steps of evaporation or drying.

[0101] In some embodiments, if drying step d) is not applied, the method does not include any evaporation or drying steps.

[0102] In some embodiments, step c) is not carried out by evaporation, or does not involve any use of an evaporation apparatus, such as a rotary evaporator. Similarly, steps c1, c2, c3, and c4 are not carried out by evaporation, or do not involve any evaporation, or any use of an evaporation apparatus, such as a rotary evaporator.

[0103] The iron concentration in ppm in the final concentrate was observed to be significantly increased in the method of the present invention compared to methods in which drying or evaporation is applied directly to the permeate as a concentration method, without additional physical separation, particularly without a centrifugation step, such as the method described in Romanian Published Patent Publication No. 132538(A0).

[0104] In some embodiments, the method may include step d'') of heat-treating the iron-containing green plant material concentrate after step c) or step c3). This heat-treating step d'') may be performed before or after step d'). This heat-treating step d'') may be performed at a temperature of at least 60°C for at least 2 seconds. Preferably, the heat-treating step d'') is performed at a temperature of 60 to 125°C for 2 seconds to 30 minutes.

[0105] The method allows for the effective concentration of iron while effectively reducing the ratio of undesirable compounds, such as insoluble plant materials and anti-nutrient factors that reduce or inhibit iron absorption, to iron.

[0106] The resulting concentrate contains a substantial amount of iron and, relative to the iron, a limited amount of anti-nutrient factors, particularly those that reduce or inhibit iron absorption in the body. The iron in the concentrate is derived from plant materials. Therefore, the concentrate is derived from natural raw materials and is suitable for vegetarian / vegan diets. Furthermore, when used in beverages, nutritional supplements, food products, or pharmaceutical compositions, the concentrate has good sensory properties, does not impart any sensory defects, or imparts very limited sensory defects, particularly limited metallic off-flavors. Moreover, the iron in the concentrate has sufficient bioaccessibility properties.

[0107] This method is substantially natural. Although it does not involve the use of added organic solvents, it still allows for effective concentration of iron and an effective reduction in the ratio of undesirable compounds, such as anti-trophic factors that reduce or inhibit iron absorption in the body, to iron.

[0108] In preferred embodiments, the method does not involve the use of added organic solvents. For example, the method does not involve the use of added organic solvents selected from the list consisting of acetone, acetonitrile, benzene, 1-butanol, 2-butanol, 3-butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, diethylene glycol, diethyl ether, diethylene glycol dimethyl ether, 1,2-dimethoxyethane (Glym, DME), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, 1,2-dichloroethane, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, hexamethylphosphoramide (HMPA), hexane, methanol, methylene chloride, N-methyl-2-pyrrolidinone (NMP), nitromethane, naphthalene, pentane, 1-propanol, 2-propanol, pyridine, toluene, triethylamine, tricine, tris, tetrahydrofuran, o-xylene, m-xylene, p-xylene, and combinations thereof.

[0109] As a result, in preferred embodiments, the iron-containing green plant material concentrate does not contain added organic solvents. For example, the iron-containing green plant material concentrate does not contain the added organic solvents listed above.

[0110] The method of the present invention enables the effective concentration of iron in green plant materials. Advantageously, the weight percentage concentration of iron in the iron-containing green plant material concentrate obtained in step c) or c3) is at least twice as high, preferably 2 to 10 times higher, than in the iron-containing green plant material of step a). For example, the iron concentration in the concentrate and the green plant material can be measured according to the method provided in the examples.

[0111] The method of the present invention makes it possible to effectively reduce the ratio of undesirable anti-nutrient factors, particularly anti-nutrient factors that may reduce or inhibit the absorption of iron in the body, to iron.

[0112] In particular, the molar ratio of iron to oxalate (M / M) is significantly increased by the method of the present invention. Increasing the molar ratio of iron to oxalate is advantageous in order to reduce the effect of oxalate on iron. Indeed, oxalate can reduce or inhibit the absorption of iron in the body. Advantageously, the molar ratio of iron to oxalate in the iron-containing green plant material concentrate obtained in step c) or c3) is at least twice as high, preferably 2 to 10 times higher, and more preferably 3 to 10 times higher, than in the green plant material of step a). The molar ratio of iron in 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 in the green plant material is expressed by the dry weight of the green plant material. For example, the molar ratio of iron in the concentrate and the green plant material can be measured according to the method provided in the examples.

[0113] In some embodiments, the molar ratio of iron to phytic acid (M / M) is significantly increased by the method of the present invention. Increasing the molar ratio of iron to phytic acid is advantageous in order to reduce the effect of phytic acid on iron. Indeed, phytic acid can reduce or inhibit 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 twice as high, preferably 2 to 10 times higher, and more preferably 3 to 10 times higher, than in the green plant material of step a). The molar ratio of iron to phytic acid in 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 in 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.

[0114] Furthermore, the total amount of phenolic compounds is significantly reduced by the method of the present invention. In particular, since phenolic compounds can inhibit the reduction or absorption of iron, it is advantageous to reduce the amount of phenolic compounds. Advantageously, the weight percentage concentration of the total phenolic compounds in the iron-containing green plant material concentrate obtained in step c) or c3) is 1 / 2 or less, preferably 1 / 5 or less, of that in the green plant material of step a). In particular, the weight percentage concentration of the total phenolic compounds in the iron-containing green plant material concentrate obtained in step c) or c3) is 1 / 2 to 1 / 15, preferably 1 / 5 to 1 / 15, of that in the green plant material of step a).

[0115] In some embodiments, the method does not involve the addition of enzymes. For example, the method does not involve any addition of proteases, carbohydrateases, fiberases, oxalases, phytases, and / or phenolic compound-degrading enzymes.

[0116] In a second embodiment, the present invention relates to an iron-containing green plant material concentrate obtainable or obtainable by a method according to the first embodiment of the present invention. The features of the iron-containing green plant material concentrate of the third embodiment of the present invention also apply to the iron-containing green plant material concentrate of the second embodiment of the present invention.

[0117] The concentrate contains a substantial amount of iron and, in comparison to the iron, a limited amount of undesirable antitrophic factors, particularly those that can reduce or inhibit iron absorption.

[0118] The iron in the concentrate is derived from plant materials. Therefore, the concentrate is made from natural ingredients and is suitable for vegetarian / vegan diets.

[0119] Furthermore, when used in beverages, nutritional supplements, food products, or pharmaceutical compositions, the concentrate possesses excellent sensory properties, does not impart any sensory defects, or imparts only very limited sensory defects, particularly limited metallic off-flavors.

[0120] Furthermore, the concentrated iron possesses sufficient bioaccessibility properties.

[0121] The concentrate is natural and can be used to fortify dietary supplements, food products, and beverages with iron, including vegetarian / vegan dietary supplements, food products, and beverages.

[0122] A third embodiment is an iron-containing green plant material concentrate containing at least 500 ppm of iron by dry weight.

[0123] In one embodiment, the iron-containing green plant material concentrate contains at least 1000 ppm of iron, preferably at least 1500 ppm of iron, on a dry weight basis. In one embodiment, the iron-containing green plant material concentrate contains up to 15000 ppm of iron, preferably up to 4000 ppm, on a dry weight basis. In one embodiment, the iron-containing green plant material concentrate is derived from green plant material. In particular, the iron-containing green plant material concentrate contains green plant material. The green plant material may be the green plant material provided in the first aspect of the present invention. For example, the iron concentration of the iron-containing green plant material concentrate can be measured according to the method provided in the examples.

[0124] In some embodiments, the iron-containing green plant material contains less than 100 mg GAE / g, preferably less than 50 mg GAE / g, of total phenolic compounds by dry weight of the iron-containing green plant material concentrate.

[0125] In some embodiments, the iron-containing green plant material concentrate has an iron bioaccessibility of at least 3%, preferably at least 10%, and more preferably at least 15%. In some further embodiments, the iron-containing green plant material concentrate has an iron bioaccessibility of up to 50%, preferably at least 35%. The iron bioaccessibility of the iron-containing green plant material concentrate can be measured as provided in the examples.

[0126] In one embodiment, the iron-containing green plant material concentrate has an absolute amount of bioaccessible iron of at least 35 ppm, preferably at least 100 ppm, more preferably at least 300 ppm, and even more preferably at least 450 ppm. In some further embodiments, the iron-containing green plant material concentrate has an absolute amount of bioaccessible iron of up to 1500 ppm, preferably at least 1000 ppm, more preferably at least 800 ppm, even more preferably at least 500 ppm, and even more preferably at least 200 ppm. For example, the absolute amount of bioaccessible iron in an iron-containing green plant material concentrate can be measured according to the method provided in the examples.

[0127] In one embodiment, the iron-containing green plant material concentrate has a molar ratio (M / M) of iron to oxalate of at least 0.3, preferably 0.3-3, more preferably 0.4-3, and even more preferably 0.4-1.5. The molar ratio of iron to oxalate is expressed by the dry weight of the iron-containing green plant material concentrate. For example, the molar ratio of iron to oxalate in the iron-containing green plant material concentrate can be measured according to the method provided in the examples.

[0128] In some embodiments, the iron-containing green plant material concentrate contains less than 15,000 ppm, preferably less than 12,000 ppm, and more preferably less than 10,500 ppm of oxalic acid by dry weight of the iron-containing green plant material concentrate. For example, the concentration of oxalic acid in the iron-containing green plant material concentrate can be measured according to the method provided in the examples.

[0129] In some embodiments, the iron-containing green plant material concentrate has a molar ratio (M / M) of iron to phytic acid of at least 5, preferably at least 7, and more preferably at least 7.5. In specific embodiments, the iron-containing green plant material concentrate has a molar ratio (M / M) of iron to phytic acid of 5 to 80, preferably 7 to 80, more preferably 7.5 to 80, even more preferably 7.5 to 60, and most preferably 7.5 to 50. The molar ratio of iron to phytic acid is expressed by the dry weight of the iron-containing green plant material concentrate. For example, the iron concentration may be measured according to the method provided in the examples. For example, the molar ratio of iron to phytic acid in the iron-containing green plant material concentrate may be measured according to the method provided in the examples.

[0130] In some embodiments, the iron-containing green plant material concentrate contains less than 3000 ppm, preferably less than 2000 ppm, and 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 in the iron-containing green plant material concentrate can be measured according to the method provided in the examples.

[0131] In one embodiment, the iron-containing green plant material concentrate does not contain any added organic solvents, and in particular does not contain any of the added organic solvents listed in the first aspect of the present invention.

[0132] In one embodiment, the iron-containing green plant material concentrate has a pH of 3 to 8.

[0133] In one embodiment, the iron-containing green plant material concentrate contains 0.01% to 5% by weight of acid. The acid may be an acid provided in a 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 the most preferred embodiment, the acid is hydrochloric acid. In another most preferred embodiment, the acid is citric acid.

[0134] The acid may be provided as a pure acid solution, 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, and tangerine juice.

[0135] When the acid is citric acid, the iron-containing green plant material concentrate may contain 0.01 to 3% by weight, preferably 0.5 to 2.3% by weight, of citric acid.

[0136] When the acid is hydrochloric acid, the iron-containing green plant material concentrate may contain 0.01% to 0.5% by weight of hydrochloric acid, preferably 0.05% to 0.5% by weight of hydrochloric acid.

[0137] When the acid is malic acid, the iron-containing green plant material concentrate may contain 0.01% to 3% by weight of malic acid, preferably 0.5% to 2.5% by weight of malic acid.

[0138] In one embodiment, the iron-containing green plant material concentrate may contain an osmotic regulator. The osmotic regulator may be an osmotic regulator provided in the first aspect of the present invention.

[0139] In one embodiment, the iron-containing green plant material concentrate may include a water-retaining agent. The water-retaining agent may be a water-retaining agent provided in the first aspect of the present invention.

[0140] In one embodiment, the iron-containing green plant material concentrate may have a total sucrose content of 1 to 50% by weight, preferably 5 to 20% by weight. The sucrose in the iron-containing green plant material concentrate may be used as a water-retaining agent and / or an osmotic pressure regulator. The total sucrose content range provided herein applies whether or not sucrose is used as a water-retaining agent and / or an osmotic pressure regulator.

[0141] The features of the iron-containing green plant material concentrate according to the third aspect of the present invention also apply to the iron-containing green plant material concentrates provided in the first and second aspects of the present invention, and vice versa. The concentrate contains a substantial amount of iron and a limited amount of undesirable anti-trophic factors, particularly anti-trophic factors that can reduce or inhibit iron absorption, relative to the iron.

[0142] The iron in the concentrate is derived from plant materials. Therefore, the concentrate is made from natural ingredients and is suitable for vegetarian / vegan diets.

[0143] Furthermore, when used in nutritional supplements, beverages, food products, or pharmaceutical compositions, the concentrate possesses excellent sensory properties, does not impart any sensory defects, or imparts only very limited sensory defects, and in particular, imparts very limited metallic off-flavors.

[0144] Furthermore, the concentrated iron possesses sufficient bioaccessibility properties.

[0145] The concentrate is natural and can be used to fortify food products, dietary supplements and beverages with iron, including vegetarian / vegan food products, dietary supplements and beverages.

[0146] In a fourth aspect, the present invention relates to a food product or beverage comprising an iron-containing green plant material concentrate according to a second or third aspect of the present invention. The iron-containing green plant material concentrate of the present invention can be used to fortify food products and beverages with iron. When used in beverages or food products, the iron-containing green plant material concentrate has good sensory properties, does not impart sensory defects, or imparts very limited sensory defects, and in particular imparts very limited metallic off-flavors.

[0147] In one embodiment, the food product or beverage may have a pH of 2.5 to 8.

[0148] In one embodiment, the food product or beverage may have an acid content of 0.01 to 0.2% by weight, preferably 0.02 to 0.1% by weight. The acid may be an acid provided in a 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 the most preferred embodiment, the acid is hydrochloric acid. In another most preferred embodiment, the acid is citric acid.

[0149] The acid may be provided as a pure acid solution, 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, and tangerine juice.

[0150] In one embodiment, the food product or beverage may have a total sucrose content of 0 to 40%, preferably 0 to 10% by weight, and more preferably 0.5 to 4% by weight.

[0151] In one embodiment, a food product or beverage may have a total iron content of at least 2.1 mg per serving. Servings may vary depending on the beverage or food product. Serving sizes for various beverages or food products are well known and can be easily determined by those skilled in the art. For example, a serving may be 200 mL for a beverage. For example, a serving may be 15 g for a sauce. For example, a serving may be 125 g for a fermented dairy product. For example, a serving may be 30 g for a dressing.

[0152] In one embodiment, food products may be selected from a list consisting of broths, fruit and / or vegetable purees, confectionery products, ice cream, sorbets, cooking creams, sauces, dressings, cheeses, fermented dairy products, dairy desserts, pet food products, dairy desserts, nutrition bars, cereal products, fermented cereal-based products, nutritional supplements, nutritional compositions, complete nutrition formulas, infant nutrition products, nutrition bars, enteral nutrition products, plant-based meat substitutes, plant-based cheese substitutes, or mixtures thereof. In one embodiment, beverages may be selected from a list consisting of smoothies, soft drinks, aqueous beverages, soups, dairy beverages, plant-based milk substitutes, coffee, tea, cocoa beverages, flavored waters, soups, mineral waters, malt beverages, creamers, fermented dairy beverages, plant-based fermented dairy beverage substitutes, or mixtures thereof.

[0153] In one embodiment, the food product or beverage is vegetarian or vegan. In particular, iron-containing green plant material concentrates are suitable for vegan / vegetarian diets and can be used to fortify vegan or vegetarian food products and beverages with iron.

[0154] In a fifth aspect, the present invention relates to a dietary supplement comprising an iron-containing green plant material concentrate according to a second or third aspect of the present invention. The iron-containing green plant material concentrate of the present invention may be used to prepare a dietary supplement that enables the delivery of a substantial amount of iron, for example, to humans, animals, or pets. In one embodiment, the dietary supplement is provided in the form of capsules, gelatin capsules, soft capsules, tablets, sugar-coated tablets, pills, pastes or lozenges, gums, drinking solutions or emulsions, syrups or gels.

[0155] In one embodiment, the dietary supplement may be a dietary supplement for use in the prevention or treatment of an iron deficiency-related condition or disease in a subject. In one embodiment, the iron deficiency-related condition or disease may be selected from the list consisting of iron deficiency anemia, chronic heart failure, or pulmonary hypertension. In this embodiment, the dietary supplement contains an effective amount, preferably a therapeutic effective amount or a preventive effective amount, of iron-containing green plant material concentrate, particularly iron. Further details regarding the terms “therapeutic effective amount” or “preventive effective amount” are provided in a sixth embodiment of the present invention.

[0156] In one embodiment, the dietary supplement is vegetarian or vegan. In particular, iron-containing green plant material concentrates are suitable for vegan / vegetarian diets and can be used to fortify vegan or vegetarian dietary supplements with iron.

[0157] In a sixth aspect, the present invention relates to a cosmetic composition or pharmaceutical composition comprising an iron-containing green plant material concentrate according to a second or third aspect of the present invention. The iron-containing green plant material concentrate of the present invention may be used to prepare a cosmetic composition or pharmaceutical composition that enables the delivery of a substantial amount of iron, for example, to humans, animals, or pets.

[0158] The pharmaceutical compositions may be administered for prophylactic and / or therapeutic purposes. In therapeutic use, the compositions according to the present invention are administered to patients already suffering from a disease in an amount sufficient to cure or at least partially suppress the symptoms of the disease and its complications, as described below herein. The amount sufficient to achieve this is defined as the “therapeutic effective dose.” The effective dose varies depending on the severity of the disease and the patient’s weight and overall health. In prophylactic use, the compositions according to the present invention are administered to patients who are susceptible to or otherwise at risk of a particular disease. Such a dose is defined as the “preventive effective dose.” In this use as well, the exact dose varies depending on the patient’s health and weight.

[0159] In one embodiment, the pharmaceutical composition may be a pharmaceutical composition for use in the prevention or treatment of an iron deficiency-related condition or disease in a subject. In one embodiment, the iron deficiency-related condition or disease may be selected from the list consisting of iron deficiency anemia, chronic heart failure, or pulmonary hypertension. In this embodiment, the pharmaceutical composition contains an effective amount, preferably a therapeutically effective amount or a preventively effective amount, of an iron-containing green plant material concentrate, particularly iron.

[0160] The pharmaceutical compositions of the present invention are preferably administered with a pharmaceutically acceptable carrier, the properties of which are adapted to the target route, such as the oral route. Desired formulations can be prepared using various excipients, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate.

[0161] In preferred embodiments, the pharmaceutical composition may be in the form of tablets, capsules, pills, liquids, suspensions, syrups, dry oral supplements, wet oral supplements, ointments, aerosols, patches, creams, gels, sprays, or suppositories.

[0162] In preferred embodiments, the pharmaceutical composition is a pharmaceutical oral composition. In particular, the pharmaceutical oral composition may be a tablet, capsule, pill, liquid, suspension, syrup, dry oral supplement, or wet oral supplement.

[0163] Those skilled in the art will understand, based on their knowledge, that they should select appropriate components and galenos formulations to target the target body tissue / compartment, such as the skin, colon, blood, veins, arteries, lungs, heart, stomach, eyes, kidneys, or liver, taking into consideration the intended route of administration, such as oral administration.

[0164] The cosmetic composition may be in the form of tablets, capsules, pills, liquids, suspensions, syrups, dry oral supplements, wet oral supplements, ointments, patches, creams, gels, or sprays. Other cosmetic active ingredients may also be added. Excipients or colorants commonly used in cosmetics may also be added to the composition.

[0165] In preferred embodiments, the cosmetic composition is an oral cosmetic composition. In particular, the oral cosmetic composition may be a tablet, capsule, pill, liquid, suspension, syrup, dry oral supplement, or wet oral supplement.

[0166] It will be understood that the concept of the present invention can also be applied as an adjuvant therapy to supplement currently used drugs. Since the iron-containing green plant material concentrate of the present invention can be easily administered together with food materials, it can be applied to special clinical foods containing large amounts of the iron-containing green plant material concentrate. By reading this specification in conjunction with the appended claims, it will be apparent to those skilled in the art that various different alternative forms to the specific embodiments described herein will be envisioned.

[0167] In a seventh aspect, the present invention relates to a method for fortifying a food product or beverage with iron, comprising the steps of preparing a food product or beverage and adding an iron-containing green plant material concentrate according to a second or third aspect of the present invention to the food product or beverage.

[0168] In one embodiment, the food product may be a food product provided in a fourth aspect of the present invention. In one embodiment, the beverage may be a beverage provided in a fourth aspect of the present invention.

[0169] Iron-containing green plant material concentrates can be used to fortify food products and beverages with iron, including vegan or vegetarian food products / beverages. When used in beverages or food products, iron-containing green plant material concentrates have good sensory properties and do not impart metallic off-flavors, or impart very limited metallic off-flavors.

[0170] Those skilled in the art will understand that all features of the present invention disclosed herein can be freely combined. In particular, features described for the products of the present invention may be combined with methods or processes of the present invention, and vice versa. Furthermore, features described for different embodiments of the present invention may be combined.

[0171] Furthermore, where known equivalents exist for a particular feature, such equivalents are incorporated as specifically referred to herein. Further advantages and features of the present invention are evident from the drawings and non-limiting embodiments. [Examples]

[0172] Example 1 - Method of the present invention for concentrating iron from dried herbs without using acid Dried herbs, including leaves and stems, particularly dried nettle (European nettle), dried peppermint A or B (European mint), or dried thyme A or B (wild thyme) were ground into a powder. Dried thyme A was from France, and dried thyme B was from Morocco. Dried peppermint A was from France, and dried peppermint B was 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 500 μm mesh filter. The permeate was collected and subsequently filtered through a 180 μm mesh filter. The permeate was collected and heat-treated to a temperature of 71°C, held for 2 minutes. After cooling to 4°C, the permeate was centrifuged at 2500 g for 10 minutes. The precipitate was collected, and an iron-containing green plant material concentrate was formed. The concentrate can be optionally dried. Example 2 - Method of the present invention for concentrating iron from dried herbs in the presence of acid Dried herbs, including leaves and stems, particularly dried peppermint (Mentha piperita), dried nettle (Urtica dioica), or dried thyme (Thymus vulgaris), were ground into a powder. A suspension was prepared by mixing the powder (50 g) with water (700 mL) in a ratio of 1:15 (w:v). Pure citric acid (4.5 g, 15.8 g, or 6.1 g of anhydrous citric acid for peppermint, nettle, or thyme, respectively), ascorbic acid (16.8 g of ascorbic acid for peppermint), hydrochloric acid (3.6 mL, 15 mL, or 5.3 mL of 6 M HCl solution for peppermint, nettle, or thyme, respectively), or malic acid (4.7 g of malic acid for peppermint) was added to the suspension to adjust the pH to 3.5. After adding the acid, the suspension was allowed to stand for 2 minutes to ensure that the powder in the suspension was properly hydrated. Next, the suspension was blended for 2 minutes to obtain a slurry. The obtained slurry was filtered through a 500 μm mesh filter. The permeate was collected and subsequently filtered through a 180 μm mesh filter. The permeate was collected and the resulting permeate was heat-treated to a temperature of 71°C and held for 2 minutes. After cooling to 4°C, the permeate was centrifuged at 2500 g for 10 minutes. The precipitate was collected, and an iron-containing green plant material concentrate was formed. Optionally, the concentrate can be dried.

[0173] Example 3 - Beverage Formulation Add citric acid, malic acid, lime concentrate, or a mixture thereof to 500 mL of water to lower the pH to 3.5. Add sucrose to bring the concentration to 20-40 g / L. Add the iron-containing green plant material concentrate from Example 1 or 2 to bring the iron content to 2.1 mg. Adjust the final pH to pH 3.

[0174] Example 4 - Quantitative determination of iron Materials and methods The iron content of the raw materials (i.e., the dried herb powder obtained after grinding) and the iron-containing green plant material concentrate obtained in either Example 1 or 2 was measured by atomic emission spectroscopy using microwave plasma atomic emission spectroscopy (MP-AES) 4200 (Agilent, Switzerland). For MP-AES analysis, samples (approximately 100-400 mg) were mineralized in a double-stage process using an Xpress microwave bomb in a Microwave Digestion System (Mars 6, CEM, USA) with 4 mL of ultra-high purity 70% HNO3 (Sigma-Aldrich, St. Louis, MO, USA) and 1 mL of 30% H2O2 (Merck KGaA, Darmstadt, Germany). The mineral solution was then transferred to a 50 mL Falcon tube and adjusted to 20 mL with Milli-Q water. Iron content was measured using external calibration with multi-element standards at a wavelength of 371 nm. The accuracy of the analysis was confirmed by analyzing a standard reference material (SRM 3233, Typical Diet; NIST, MD, USA).

[0175] result Figure 1 shows the iron content (based on DW) in dried peppermint A, dried nettle, dried thyme A and B, and the corresponding iron-containing green plant material concentrates obtained according to the method of Example 1. Furthermore, the iron content of four commercially available peppermint extracts (two extracts, Martin Bauer 11000005 Peppermint Extract Powder, Martin Bauer 11000192 Peppermint Fine Powder), nettle (one extract, Martin Bauer 15100000 Nettle Leaf Extract Powder), and thyme (one extract, Martin Bauer 17100001 Thyme Extract Powder) is reported.

[0176] Dried peppermint, dried nettle, and dried thyme are understood to be a combination of dried plant leaves and stems that have been ground into a powder before further processing in a concentration method.

[0177] The concentration method of the present invention significantly increased the iron concentration from 298 ppm in dried peppermint A to 2973 ppm in iron-containing peppermint concentrate A (= iron-containing peppermint A concentrate). The concentration method of the present invention significantly increased the iron concentration from 288 ppm in dried nettle to 1355 ppm in iron-containing nettle concentrate. The concentration method of the present invention significantly increased the iron concentration from 180 ppm in dried thyme A to 1294 ppm in iron-containing thyme concentrate A (= iron-containing thyme A concentrate). In another batch of thyme (i.e., thyme B), the concentration method of the present invention significantly increased the iron concentration from 1903 ppm in dried thyme B to 6142 ppm in iron-containing thyme concentrate B (= iron-containing thyme B concentrate).

[0178] Commercially available water extracts of peppermint, nettle, and thyme all exhibit very low iron concentrations, specifically less than 165 ppm, demonstrating that the concentration method of the present invention has a clear advantage over standard water concentration methods in obtaining concentrates with high iron concentrations.

[0179] Example 5 - Quantification of anti-trophic factors Materials and methods: Oxalic acid was extracted from the sample using water under mechanical stirring. The oxalic acid was measured by mass spectrometry (SCIEX Triple Quad 5500 with Selexion) coupled to ion chromatography (Dionex ICS-5000 with Dionex Ion PAC AS16 REFIC Analytical column (250×2mm)).

[0180] The total phenol content was quantified as follows: The sample was suspended in methanol and shaken for 1 minute every 5 minutes for 30 minutes to extract phenol. The sample was then centrifuged (750 g, 10 minutes, 20°C), and the supernatant was collected. The pellet was re-extracted with methanol, centrifuged, and the supernatant was collected and pooled with the previous supernatant. The extract (1 mL) was mixed with 15 mL of water and 1 mL of forin-thiocaltophenol reagent, mixed well, and allowed to stand for 6 minutes. Sodium carbonate solution (3 mL, 20%) was added to each sample and mixed well. The samples were incubated at 30°C for 2 hours, and then the absorbance at 765 nm was read. The total phenol content was quantified against the gallic acid calibration curve. Since each phenol compound was equivalent to one molecule of gallic acid, the results are expressed as gallic acid equivalents.

[0181] Phytic acid was measured according to the "Phytic Acid (Phytate) / Total Phosphorus" Megazyme kit. This kit allows for the quantification of free and total phosphorus in a sample by colorimetric detection. Total phosphorus is defined as phosphorus derived from phytic acid and other sources, and is measured after the sample is first treated with phytase and then with alkaline phosphatase. Free phosphorus, on the other hand, is defined as phosphorus derived from non-phytic acid sources in the sample, and is measured from the kit without enzymatic treatment. Briefly, 1 g of the sample was mixed with 20 mL of HCl acid (0.66 M) and vigorously stirred 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). A neutralized sample extract (0.05 mL) was mixed with distilled water (0.60 mL), the provided buffer I (0.20 mL), and a phytase suspension for total phosphorus quantification (0.02 mL). A control sample was prepared by mixing a sample extract (0.05 mL) with distilled water (0.62 mL) and the provided buffer I (0.20 mL), and free phosphorus was quantified. Both samples were vortexed and incubated at 40°C for 10 minutes. Distilled water (0.02 mL) and the provided buffer 3 (0.2 mL) were added to the control, and buffer 3 (0.20 mL) and suspension 4 (ADP, 0.02 mL) were added to the total phosphorus sample. 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 measurement of phosphorus. The sample was mixed with 0.5 mL of colorimetric reagent. The colorimetric 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 sample with the colorimetric reagent, it was incubated at 40°C for 1 hour, and then the absorbance at 655 nm was read.

[0182] The phosphorus concentration was calculated using the measured absorbance, and this was used to calculate the PA concentration. The concentrations of free phosphorus and phosphorus in the total phosphorus reaction were calculated using equation (1).

number

[0183] Equation (2) is used to convert the calculated concentration of bound phosphorus to the PA concentration. It is assumed that the measured amount of bound phosphorus is only from PA.

Number

[0184]

Table 1

[0185] Figure 2 shows the molar ratio of iron to oxalic acid in dried peppermint A and dried nettle powder, as well as in iron-containing peppermint A and nettle concentrate obtained according to the method of Example 1. The concentration method of the present invention increased the ratio of iron to oxalic acid from 0.25 to 0.52, indicating a lower concentration of iron that can be chelated by oxalic acid in the iron-containing peppermint concentrate, and therefore a potentially greater amount of iron available for absorption. A further significant improvement in the ratio of iron to oxalic acid was observed from dried nettle (0.23) to iron-containing nettle concentrate (1.11).

[0186] Figure 10 shows the total phenol content (mg gallic acid equivalent / g DW) of dried peppermint B and dried nettle, as well as iron-containing peppermint B and nettle concentrates obtained according to the method of Example 1. The concentration method of the present invention resulted in a significant decrease in total phenol from 47.3 mg GAE / g DW of dried peppermint to 16.4 mg GAE / g DW of iron-containing peppermint concentrate, suggesting a possible reduction in iron absorption inhibitors (anti-trophic factors). A similar trend was observed for nettle; total phenol decreased from 8.38 mg GAE / g DW of dried nettle to 0.88 mg GAE / g DW of iron-containing nettle concentrate.

[0187] These results suggest that the concentration of iron that can be chelated by phenolic compounds in iron-containing concentrates is lower, and therefore may be more readily available for absorption.

[0188] Figure 3 shows the molar ratios of iron and phytic acid in dried peppermint A and B, nettle, thyme A powder, and iron-containing concentrates obtained therefrom according to the method of Example 1. The concentration method of the present invention increased the ratio of iron to phytic acid from 2.2 in dried peppermint A to 7.7 in the iron-containing peppermint A concentrate.

[0189] The concentration method of the present invention increased the ratio of iron to phytic acid from 6.6 in dried peppermint B to 44 in iron-containing peppermint B concentrate (= iron-containing peppermint concentrate B).

[0190] The concentration method of the present invention increased the ratio of iron to phytic acid from 1.3 in dried nettle to 8.9 in iron-containing nettle concentrate.

[0191] The concentration method of the present invention increased the ratio of iron to phytic acid from 2.3 in dried thyme to 23 in iron-containing thyme concentrate A.

[0192] These results indicate that the concentration of iron that can be chelated by phytic acid in iron-containing concentrates is lower, and therefore potentially more readily available for absorption.

[0193] Example 6 - In vitro digestion for quantifying iron bioaccessibility Materials and methods: Briefly, 1 g of iron-containing green plant material concentrate prepared according to the concentration methods of Examples 1 and 2 was mixed with 10 mL of KCl 5 mmol + NaCl 140 mmol pH 2. After adjusting the pH to 2, 0.5 mL of pepsin solution (prepared by dissolving 200 mg of pepsin in 10 mL of 0.1 M HCl) was added, and the sample was incubated at 37°C for 1 hour. After 1 hour, the pH was adjusted to 5.5 with 1 M NaHCO3. The volume of the sample was adjusted to 15 mL by adding 6.7 μL of KCl 5 mmol + NaCl 140 mmol. Pancreatin solution (2.5 mL, prepared by adding 87.5 mg of pancreatin and 525 mg of bile extract to 44 mL of 0.1 M NaHCO3) was added, and the sample was incubated at 37°C for 2 hours. An aliquot (2.5 g) of the complete digest was analyzed for iron content by MPAES. The remaining sample was centrifuged at 10,000 g for 30 minutes at 4°C, and 2.5 g of the supernatant was analyzed for iron content by MPAES. Iron bioaccessibility was defined as follows:

number

[0194] result: Figure 4 shows the iron bioaccessibility of iron-containing peppermint concentrates prepared from dried peppermint B using water according to the concentration method of Example 1, or prepared in the presence of an acid (i.e., citric acid, hydrochloric acid, malic acid, or ascorbic acid) according to the concentration method of Example 2. The iron bioaccessibility of each concentrate was compared with that of iron salts, particularly iron pyrophosphate. The use of citric acid during concentration in the present invention significantly increased the iron bioaccessibility (24%) compared to concentrates prepared using the concentration method of the present invention with water in the absence of acid (11%) or in the presence of another acid, such as ascorbic acid (10%). Hydrochloric acid had a positive effect on iron bioaccessibility, but to a lesser extent than citric acid, resulting in an iron bioaccessibility of 20%. Malic acid also had a positive effect on iron bioaccessibility, but to a lesser extent than citric acid, resulting in an iron bioaccessibility of 18%. Iron-containing peppermint concentrates prepared with citric acid, hydrochloric acid, or malic acid showed higher iron bioaccessibility than iron pyrophosphate, a commonly used iron fortifier.

[0195] Figure 5 shows the bioaccessibility of iron in iron-containing nettle concentrates prepared with water (according to Example 1) or with water in the presence of citric acid and hydrochloric acid (according to Example 2), respectively. Citric acid and hydrochloric acid had a positive effect on the bioaccessibility of iron in the iron-containing nettle concentrates.

[0196] Figure 6 shows the absolute amount of bioaccessible iron contained in dried peppermint material B and in iron-containing peppermint concentrates prepared from dried peppermint material B using water according to the concentration method of Example 1, or prepared using water in the presence of citric acid, hydrochloric acid, malic acid, and ascorbic acid, respectively, according to the concentration method of Example 2. The absolute amount of bioaccessible iron was calculated by multiplying the iron content in the sample by the bioaccessibility value. The iron-containing peppermint concentrate prepared with hydrochloric acid has a clear advantage because it contains a larger amount of bioaccessible iron compared to dried peppermint (601 ppm in the iron-containing peppermint concentrate compared to 288 ppm in dried peppermint).

[0197] Figure 7 shows the absolute amount of bioaccessible iron contained in dried nettle and in iron-containing nettle concentrates prepared with water according to the concentration method of Example 1, or with water in the presence of citric acid and hydrochloric acid, respectively, according to the method of Example 2. The absolute amount of bioaccessible iron was calculated by multiplying the iron content in the sample by the bioaccessibility value. Iron-containing nettle concentrates prepared with water, citric acid, or hydrochloric acid have a clear advantage over dried nettle, as they contain a larger amount of bioavailable iron (28 ppm in dried nettle, compared to 39 ppm, 114 ppm, and 147 ppm in the iron-containing nettle concentrates prepared with water, citric acid, and hydrochloric acid, respectively).

[0198] Figure 8 shows the bioaccessibility of iron in iron-containing thyme concentrates prepared from dried thyme B using water (according to Example 1), or using water in the presence of citric acid and hydrochloric acid, respectively (according to Example 2). Citric acid and hydrochloric acid had a positive effect on the bioaccessibility of iron in the iron-containing thyme concentrates.

[0199] Figure 9 shows the absolute amount of bioaccessible iron contained in dried thyme B and in iron-containing thyme concentrates prepared from dried thyme B using water according to the concentration method of Example 1, or prepared using water in the presence of citric acid and hydrochloric acid, respectively, according to the method of Example 2. The absolute amount of bioaccessible iron was calculated by multiplying the iron content in the sample by the bioaccessibility value. Iron-containing thyme concentrates prepared using either citric acid or hydrochloric acid have a clear advantage over dried thyme, as they contain a larger amount of bioaccessible iron (59 ppm in dried thyme B, compared to 190 ppm and 224 ppm in the iron-containing thyme concentrates prepared from dried thyme B using citric acid and hydrochloric acid, respectively).

[0200] Example 7 - Peppermint / Thyme Beverage Iron-containing peppermint concentrate and iron-containing thyme concentrate were prepared according to Example 1. The iron-containing concentrate blend was prepared by mixing 95% iron-containing peppermint concentrate A and 5% iron-containing thyme concentrate A.

[0201] Of these, citric acid was added to 500 mL of water to lower the pH to 3.5. Sucrose was added to bring the concentration to 20-40 g / L. An iron-containing concentrate blend was added to bring the iron content to 2.1 mg. The final pH was adjusted to pH 3.

[0202] Example 8 - Sensory Data Materials and Methods: Ten different beverages with the same iron content (2.1 mg of iron per 200 mL of beverage) were prepared.

[0203] Beverage A: A beverage prepared according to Example 3, using an iron-containing peppermint concentrate prepared according to the method of Example 1.

[0204] Beverage B: A beverage prepared according to Example 3, using an iron-containing peppermint concentrate prepared according to the method of Example 2 using citric acid.

[0205] Beverage C: A beverage prepared according to Example 3 using an iron-containing peppermint concentrate prepared according to the method of Example 2 using hydrochloric acid.

[0206] Beverage D: A beverage prepared according to Example 3 using an iron-containing nettle concentrate prepared according to the method of Example 1.

[0207] Beverage E: A beverage prepared according to Example 3 using an iron-containing nettle concentrate prepared according to the method of Example 2 using citric acid.

[0208] Beverage F: A beverage prepared according to Example 3 using an iron-containing nettle concentrate prepared according to the method of Example 2 using hydrochloric acid.

[0209] Beverage G: A beverage prepared according to Example 3 using iron-containing thyme concentrate A prepared according to the method of Example 1.

[0210] Beverage H: A beverage prepared according to Example 7.

[0211] Beverage I: A beverage prepared according to Example 3, but using reference curry leaf extract (MoFerrin 21, Biogena) instead of iron-containing green plant material concentrate.

[0212] Beverage J: A beverage prepared according to Example 3, except that a reference iron salt, i.e., iron pyrophosphate, was used instead of an iron-containing green plant material concentrate.

[0213] Beverages A-G and I-J were prepared using citric acid (instead of malic acid or lime concentrate) as the acid, according to the method provided in Example 3.

[0214] Next, the sensory characteristics of various beverages were evaluated using a monadic method by individuals trained to assess metallic off-flavors perceived in the mouth. The intensity of the metallic off-flavor was evaluated using a score ranging from 0 (no metallic off-flavor perceived in the mouth) to 5 (strong metallic off-flavor perceived in the mouth).

[0215] result The beverages of Examples 3 and 7, i.e., beverages A to J, containing the iron-containing green plant material according to the present invention, have good sensory properties. In particular, no unpleasant metallic off-flavor was perceived. Conversely, beverage K, using reference curry leaf extract, and beverage L, using iron pyrophosphate, have a strong, unpleasant metallic off-flavor.

[0216] Therefore, the method of the present invention makes it possible to provide concentrates, as well as foods, nutritional supplements, and beverages, that contain a substantial amount of iron while having good sensory properties, and in particular, having limited metallic off-flavors.

[0217] Example 9 - Method of the present invention for concentrating iron from fresh herbs without using acid Iron-containing nettle, peppermint, and thyme concentrates were prepared in the same manner as in Example 1, except for the following adjustments.

[0218] Instead of dried nettle, dried peppermint, and dried thyme, fresh nettle, fresh peppermint, and fresh thyme, including both leaves and stems, were used.

[0219] Do not grind fresh nettles, fresh peppermint, and fresh thyme into a powder before mixing with water.

[0220] Mix fresh nettles, fresh peppermint, and fresh thyme with water in a ratio of 1:18 (w:v).

[0221] Example 10 - Method of the present invention for concentrating iron from fresh herbs using acid Iron-containing nettle, peppermint, and thyme concentrates were prepared in the same manner as in Example 2, except for the following adjustments: Instead of dried nettle, dried peppermint, and dried thyme, fresh nettle, fresh peppermint, and fresh thyme, including both leaves and stems, were used.

[0222] Do not grind fresh nettles, fresh peppermint, and fresh thyme into a powder before mixing with water.

[0223] Mix fresh nettles, fresh peppermint, and fresh thyme with water in a ratio of 1:18 (w:v).

[0224] Example 11 - Method of the present invention for concentrating iron from duckweed without using acid An iron-containing duckweed concentrate was prepared in the same manner as in Example 1, except for the following adjustments: Instead of dried herbs, fresh whole duckweed plants, especially fresh whole duckweed (Lemna minor), were used.

[0225] Do not crush the whole fresh duckweed into a powder before mixing it with water.

[0226] Mix the whole fresh duckweed with water in a ratio of 1:12 (w:v).

[0227] Example 12 - Method of the present invention for concentrating iron from duckweed using acid An iron-containing duckweed concentrate was prepared in the same manner as in Example 2, except for the following adjustments: Instead of dried herbs, fresh duckweed whole plants, especially fresh winter duckweed whole plants, were used.

[0228] Do not crush the whole fresh duckweed into a powder before mixing it with water.

[0229] Mix the whole fresh duckweed with water in a ratio of 1:12 (w:v).

[0230] Example 13 - Iron concentration method of the present invention from dried herbs using water in the presence of sucrose Iron-containing nettle, peppermint, and thyme concentrates were prepared in the same manner as in Example 1, except for the following adjustments: Sucrose was added to the suspension of dried herb powder and water at a concentration ranging from 1% to 20% by weight, preferably 5% to 12% by weight, before blending.

[0231] Example 14 - Comparison of the present invention's method for concentrating iron from dried herbs using acid with another iron concentration method reported in Romanian Published Patent Publication No. 132538(A0). Materials and methods: An iron-containing spearmint concentrate was prepared in the same manner as in Example 2, except for the following adjustment: Dried pulverized spearmint and water were mixed in a ratio of 1:10 (w:v), and 3% citric acid was added to obtain a pH of 3. The suspension was mixed at 40°C for 10 minutes. The resulting slurry was filtered through a 200 μm mesh filter. The permeate was collected and subsequently filtered through a 50 μm mesh filter. The permeate was collected and centrifuged at 2500 g for 10 minutes. The precipitate was collected to form an iron-containing spearmint concentrate. This was freeze-dried.

[0232] An alternative iron concentration method was carried out as reported in Romanian Published Patent Publication No. 132538(A0). Dried pulverized spearmint and water were mixed in a ratio of 1:10 (w:v), and 3% citric acid was added to obtain a pH of 3. The suspension was mixed at 40°C for 10 minutes. The resulting slurry was filtered through a 200 μm mesh filter. The permeate was collected and subsequently filtered through a 50 μm mesh filter. The permeate was collected and water was removed by freeze-drying to obtain freeze-dried spearmint permeate.

[0233] Iron was measured in the iron-containing spearmint concentrate and the lyophilized spearmint permeate as described in Example 4.

[0234] result: The crushed spearmint had an iron concentration of 335.0 ± 0.6 ppm. Based on the quantitative results of iron, the iron concentration method of the present invention yielded an iron-containing spearmint concentrate containing 1652.7 ± 1.5 ppm (based on dry weight). The centrifugation step is crucial for concentrating the iron-containing material, removing the iron-free material from the supernatant (measured iron content was 30.2 ± 0.1 ppm based on dry weight). In contrast, based on the quantitative results of iron, the iron concentration method of Romanian Published Patent Publication No. 132538(A0), which does not involve a centrifugation step and directly freeze-dries the permeate, yielded an iron content of 246.0 ± 0.5 ppm. These results clearly demonstrate the advantages of the invention presented herein for obtaining iron-concentrated plant material, specifically, iron-concentrated plant material having at least twice the iron content of the original green plant material.

[0235] Although the present invention has been described using examples, it should be understood that modifications and alterations can be made without departing from the scope of the invention as defined in the claims.

Claims

1. A method for preparing an iron-containing green plant material concentrate, a) A step of suspending green plant material in an aqueous liquid to form a green plant material suspension, b) A step of blending the green plant material suspension to obtain a green plant material slurry, c) A step of applying physical means to the green plant material slurry to separate and obtain an iron-containing green plant material concentrate, d) Optionally, a step of drying the iron-containing green plant material concentrate, Methods that include...

2. The method according to claim 1, wherein step c) is carried out by filtration and / or centrifugation and / or decantation and / or heat treatment.

3. Step c) in which physical means are applied c １ ) A step of filtering the green plant material slurry to obtain a permeate, c ２ )Optionally, a step of heat-treating the permeate, c ３ ) A step of obtaining an iron-containing green plant material concentrate by centrifuging or decanting the permeate, The method according to claim 1 or 2, as implemented by...

4. The method according to any one of claims 1 to 3, wherein the green plant material is derived from a herb or duckweed.

5. The method according to claim 4, wherein the herb is selected from the group consisting of parsley, coriander, mint, thyme, lemon balm, nettle, sage, oregano, rosemary, basil, dill, chervil, savory, or a mixture thereof.

6. The method according to any one of claims 1 to 5, wherein the green plant material includes leaves, preferably consisting only of leaves.

7. The method according to any one of claims 1 to 6, wherein the green plant material is a dried green plant material and / or a fresh green plant material.

8. The method according to any one of claims 1 to 7, wherein the green plant material is a dried green plant material, and the dried green plant material is pulverized into a powder before step a).

9. The method according to any one of claims 1 to 8, wherein the ratio (w / v) of plant green plant material to aqueous liquid in the green plant material suspension is 1:3 to 1:

20.

10. The method according to any one of claims 1 to 9, wherein an acid is further added to the green plant material suspension before step b).

11. The method according to claim 10, wherein the acid is selected from the list consisting of hydrochloric acid, citric acid, malic acid, ascorbic acid, or a mixture thereof.

12. The filtration step c) or c １ The method according to any one of claims 2 to 11, wherein the method is carried out using a filter having a mesh of 25 μm to 1000 μm.

13. The weight percentage concentration of iron is in process c) or c ３ The method according to any one of claims 1 to 12, wherein the iron-containing green plant material concentrate obtained in step a) is at least twice as high, preferably 2 to 10 times higher, than the iron in the green plant material of step a).

14. The molar ratio of iron to oxalic acid is, in step c) or c ３ The method according to any one of claims 1 to 13, wherein the iron-containing green plant material concentrate obtained in step a) is at least twice, preferably three to ten times, higher than that in the green plant material of step a).

15. An iron-containing green plant material concentrate that can be obtained or obtained by the method described in any one of claims 1 to 14.

16. An iron-containing green plant material concentrate containing at least 500 ppm of iron by dry weight.

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

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

19. A nutritional supplement comprising an iron-containing green plant material concentrate according to any one of claims 15 to 17.

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

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

22. A nutritional supplement according to claim 19, or a pharmaceutical composition according to claim 20, for use in the treatment or prevention of a condition or disease related to iron deficiency in a subject.

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