阅读说明：本技术 包含栎草亭和没食子酸的抗氧化剂组合物 (Antioxidant composition comprising quercitrin and gallic acid ) 是由 J·埃斯特韦斯·麦地那 M·帕莱拉·贝瑟 S·卡内·弗鲁克托索 于 2020-02-07 设计创作，主要内容包括：本发明提供一种以65:1至1.2:1的重量比包含栎草亭和没食子酸的组合,其中没食子酸是以酸形式或以其盐或酯的形式。本发明还提供包含本发明组合的食品、动物饲料、药用或美容组合物。本文还提供本发明的组合在食品、动物饲料、药用、兽医用或美容组合物中作为抗氧化剂的用途。本发明的组合对于防止包含脂质的组合物的氧化非常有用。(The present invention provides a combination comprising quercitrin and gallic acid in a weight ratio of 65:1 to 1.2:1, wherein the gallic acid is in the acid form or in the form of a salt or ester thereof. The invention also provides a food, animal feed, pharmaceutical or cosmetic composition comprising the combination of the invention. Also provided herein is the use of a combination of the invention as an antioxidant in a food, animal feed, pharmaceutical, veterinary or cosmetic composition. The combination of the invention is very useful for preventing oxidation of a lipid containing composition.)

1. A combination comprising quercitrin and gallic acid in a weight ratio of 65:1 to 1.2:1, wherein the gallic acid is in the acid form, or in the form of a salt or ester thereof.

2. The combination of claim 1, wherein the weight ratio between quercus and gallic acid is from 20:1 to 1.5: 1.

3. The combination of any one of claims 1-2, wherein the weight ratio between quercus and gallic acid is from 10:1 to 2: 1.

4. The combination of any one of claims 1-3, wherein the weight ratio between quercus and gallic acid is from 8.6:1 to 2.85: 1.

5. The combination according to any one of claims 1-4, wherein the gallic acid is in the acid form.

6. The combination of any one of claims 1-5, wherein the quercitrin is provided in the form of a plant extract.

7. The combination according to claim 6, wherein the plant extract is a marigold (Tagetes erecta) extract.

8. The combination according to claim 7, wherein the marigold extract is a pomace extract.

9. The combination according to any one of claims 6-8, wherein the extract is an alcoholic extract.

10. A food, animal feed, pharmaceutical, veterinary or cosmetic composition comprising a combination according to any one of claims 1-9.

11. The food, animal feed, pharmaceutical, veterinary or cosmetic composition of claim 10, wherein the composition comprises a lipid.

12. A food, animal feed, pharmaceutical, veterinary or cosmetic composition according to any one of claims 10-11, wherein the amount of the combination is from 0.001% w/w to 1% w/w.

13. Use of a combination according to any one of claims 1-9 as antioxidant in food, animal feed, pharmaceutical, veterinary or cosmetic compositions.

14. A method of making a combination according to any one of claims 1-9, comprising the steps of:

-providing a quercus pavilion;

-addition of gallic acid;

-optionally, adding a suitable liquid carrier; and

-mixing and homogenization.

Technical Field

The present invention belongs to the field of antioxidant. In particular, it relates to antioxidants for preserving food, feed, pharmaceutical or cosmetic compositions. The antioxidants of the present invention are particularly useful for preserving lipid-containing compositions.

Background

Industrial processed foods, pharmaceutical or cosmetic products must be capable of being stored for a considerable period of time in order to reach the consumer in an unaltered state. Furthermore, when a consumer purchases a product, it is expected that the product will further be stored for at least several weeks, and sometimes even months. Most products, especially food products, if untreated, can spoil within a few days due to physicochemical changes and fungal or bacterial growth.

Among the physicochemical changes affecting these kinds of products, oxidation is highly relevant. It affects especially fats, leading to lipid oxidation. In fact, lipid oxidation is a major cause of spoilage in stored products, leading to flavor deterioration, color change and bad odor. Polyunsaturated fatty acids (PUFAs) having labile double bonds are oxidized, and the decomposition of the PUFAs produces secondary oxidized compounds such as hexanal, pentanal, heptanal, and octanal. These volatile aldehydes primarily promote the development of oxidative rancidity known as a cooked-over flavour (WOF). Not only does secondary oxidation compounds cause a deterioration in the quality of the food product, but the ingested oxidation products can also be a risk to human and animal health.

Thus, there is a need to enhance oxidative stability to maintain the safety and quality of lipid containing products such as food, animal feed, pharmaceutical, or even cosmetic products for human consumption.

In recent years, naturally occurring antioxidant compounds have been preferred for use due to potential health benefits and safety. Some examples are tocopherols and phospholipids such as lecithin. Also, many natural plant extracts are under intense investigation due to their high antioxidant content, including flavonoids and other polyphenols.

However, the antioxidants used so far suffer from various drawbacks, for example, tocopherol-based antioxidants are expensive and their efficacy varies with preservatives.

Thus, there remains a need for safe, economical and effective natural compounds to prevent oxidation of industrial products such as food, feed, pharmaceutical and cosmetic compositions.

Disclosure of Invention

The present inventors have developed a novel combination of compounds for improving the oxidative stability of various types of compositions. The combination of the invention comprises a mixture of quercitrin (queretagetin) and gallic acid in a specific ratio. While the above compounds are known to have some antioxidant effect, their use in combination in the specific ratios herein has not been previously disclosed.

By searching for antioxidant combinations of natural compounds through extensive experimentation, the present inventors have surprisingly found that a combination of quercitrin and gallic acid in a specific concentration ratio exhibits an extraordinary synergistic antioxidant effect (see fig. 1). Notably, the effect was not observed when quercitrin was combined with other antioxidant compounds such as tocopherol, BHA or BHT (table 1), or when the combination of quercitrin and gallic acid was performed at different ratios (fig. 1).

As shown below, the present inventors found that the combination of quercus robusta and gallic acid can improve their antioxidant activity by up to 48%.

On the other hand, it is well known that some antioxidants do not perform well in lipid containing compositions. This strongly hinders their use in most food or cosmetic compositions. Unexpectedly, as shown in the examples below, the inventors have discovered a synergistic antioxidant effect of the combination of quercus robur and gallic acid in lipid containing compositions such as lard.

In view of the above, the antioxidant combinations provided herein constitute a significant advance in the field of product preservation, particularly in the preservation of fat-containing foods.

Thus, in a first aspect, the present invention provides a combination comprising quercitrin and gallic acid in a weight ratio of from 65:1 to 1.2:1, wherein the gallic acid is in the acid form, or in the form of a salt or ester thereof.

In a second aspect, the invention provides a food, animal feed, pharmaceutical, veterinary or cosmetic composition comprising a combination as defined above.

In a third aspect, the present invention provides the use of a combination as defined above as an antioxidant in a food product, animal feed, pharmaceutical, veterinary or cosmetic composition.

In a fourth aspect, the present invention provides a method of preparing a combination as defined above, the method comprising the steps of: providing a quercus pavilion; adding gallic acid; optionally, adding a suitable liquid carrier; and mixing and homogenizing.

Drawings

Figure 1 shows the antioxidant efficacy of marigold (Tagetes erecta) extract (quercitrin), gallic acid and combinations thereof at the indicated concentrations. The y-axis represents the Protection Factor (PF) (calculated as described in the examples below) and the x-axis represents the concentration of active (antioxidant) tested. The values obtained with gallic acid are represented by open squares, the TE extract by open circles, the TE extract and gallic acid in weight ratioA 15:1 mixture is represented by black triangles, a 5:1 by weight mixture by black circles, a 1.67:1 by weight mixture by open triangles, and a 1:1.8 by weight mixture by black triangles. N-4-8 replicates/antioxidant concentration tested. Two-way analysis of variance (Tukey test),^*comparative TE P<0.05，^#Contrast Gallic acid P<0.01。

Figure 2 shows the antioxidant efficacy of marigold extract, quercitrin, gallic acid and combinations thereof at the indicated concentrations. The y-axis represents the Protection Factor (PF) (calculated as described in the examples below) and the x-axis represents the concentration of active (antioxidant) tested. The values obtained with gallic acid are represented by open circles, quercitrin by open squares, TE extract by open triangles, the mixture of TE extract and gallic acid at a weight ratio of 15:1 by black triangles with pointed corners pointing downwards, the mixture of TE extract and gallic acid at a weight ratio of 5:1 by black triangles with pointed corners pointing upwards, and the mixture of quercitrin and gallic acid at a weight ratio of 9:1 by black circles. N-4-14 replicates/antioxidant concentration tested. Two-way analysis of variance (Tukey test), different letters in each assessed concentration represent significant differences (P < 0.05).

Detailed Description

Unless otherwise indicated, all terms as used herein are to be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms used in the present application are as described below and are intended to apply uniformly throughout the specification and claims, unless an otherwise expressly set out definition provides a broader definition.

The indefinite articles "a" and "an" as used herein are synonymous with "at least one" or "one or more". As used herein, unless otherwise indicated, definite articles such as the term also include plural forms of the term.

As mentioned above, the present invention provides a combination of quercus robusta and gallic acid in a weight ratio of 65:1 to 1.2:1, wherein when the gallic acid is in the acid form or in the salt or ester form thereof. When gallic acid is in its salt or ester form, the weight ratio correlates with the amount of gallic acid present.

The term "weight ratio" refers to the weight relationship of the indicated molecules or compounds. For example, a weight ratio (weight/weight) between quercitrin and gallic acid is 65:1, meaning 65 units of quercitrin and 1 unit of gallic acid. As used herein, "weight percent" or "% w/w" of a component refers to the amount of the individual component relative to the total weight of the composition or, if specifically mentioned, other components. In the present invention, the weight of the extract means the weight or mass of the dry extract.

Quercetin is also called 2- (3, 4-dihydroxyphenyl) -3,5,6, 7-tetrahydroxychromen-4-one, CAS number 90-18-6. Quercus has formula (I):

gallic acid is also known as 3,4, 5-trihydroxybenzoic acid, CAS number 149-91-7. The gallic acid has formula (II):

several gallates and esters, known as gallates, are known to also have antioxidant activity and may therefore be used in the combinations of the present invention. For example, alkyl esters of gallic acid such as methyl gallate, propyl gallate, octyl gallate and dodecyl (lauryl) gallate, or gallic acid salts such as sodium gallate, can be combined with quercitrin to obtain a synergistic antioxidant combination.

Quercus and gallic acid are commercially available and available from several chemical suppliers.

Thus, in one embodiment of a combination of the invention, optionally in combination with any of the embodiments provided above or below, the gallic acid ester is an alkyl ester, e.g. C₁-C₁₂An alkyl ester. In a more specific embodiment, the gallic acid ester is selected from the group consisting of methyl gallate, nonfat foodEthyl gallate, propyl gallate, octyl gallate, dodecyl gallate and mixtures thereof.

The gallate salt can be prepared by methods known in the art. For example, they may be prepared from the parent compound containing an acidic moiety by conventional chemical methods. Typically, such salts are prepared, for example, by reacting the free acid form of gallic acid with a stoichiometric amount of the appropriate base in water or an organic solvent or a mixture thereof. Likewise, the preparation of the gallic acid ester can be performed by methods known in the art. Generally, gallic acid esters are prepared, for example, by reacting the free acid form of gallic acid with a stoichiometric amount of the appropriate alcohol at elevated temperatures.

In another embodiment of the combination of the invention, optionally in combination with any of the embodiments provided above or below, the gallic acid is in the acid form

In another embodiment of the combination of the invention, optionally in combination with any of the embodiments provided above or below, the weight ratio between quercitrin and gallic acid or salts or esters thereof is 65:1 to 1.2:1, 64:1 to 1.2:1, 63:1 to 1.2:1, 62:1 to 1.2:1, 61.2:1 to 1.2:1, 60:1 to 1.2:1, 59:1 to 1.2:1, 58:1 to 1.2:1, 57:1 to 1.2:1, 56:1 to 1.2:1, 55:1 to 1.2:1, 54:1 to 1.2:1, 53:1 to 1.2:1, 52:1 to 1.2:1, 51.2:1 to 1.2:1, 50:1 to 1.2:1, 49:1 to 1.2:1, 48:1 to 1.2:1, 1.2:1 to 1, 1.2:1, 1 to 1.2:1, 1: 1.2:1 to 1, 45 to 1.2:1, 1: 1.2:1, 1:1, 1:1, 1:1, 43:1, 1:1, 43: 1.2:1, 43:1, 1:1, 2:1, 1:1, 43:1, 2:1, 43:1, 2:1, 1.2:1, 1:1, 40:1 to 1.2:1, 39:1 to 1.2:1, 38:1 to 1.2:1, 37:1 to 1.2:1, 36:1 to 1.2:1, 35:1 to 1.2:1, 34:1 to 1.2:1, 33:1 to 1.2:1, 32:1 to 1.2:1, 31.2:1 to 1.2:1, 30:1 to 1.2:1, 29:1 to 1.2:1, 28:1 to 1.2:1, 27:1 to 1.2:1, 26:1 to 1.2:1, 25:1 to 1.2:1, 24:1 to 1.2:1, 23:1 to 1.2:1, 22:1 to 1.2:1, 21.2:1 to 1.2:1, 20:1 to 1.2:1, 19:1 to 1.2:1, 1:1 to 1.2:1, 1:1, 19: 1.2:1 to 1.2:1, 1: 1.2:1, 1:1 to 1:1, 1: 1.2:1, 1: 1.2:1, 1: 1.2:1, 1: 1.2:1, 1:1, 1:1, 1: 1.2: 1: 1.2:1, 1:1, 1:1, 1: 1.2:1, 7:1 to 1.2:1, 6:1 to 1.2:1, 5:1 to 1.2:1, 4:1 to 1.2:1, 3:1 to 1.2:1, 2.9:1 to 1.2:1, 2.8:1 to 1.2:1, 2.7:1 to 1.2:1, 2:1 to 1.2:1, 1.9:1 to 1.2:1, 1.8:1 to 1.2:1, 1.7:1 to 1.2:1, 1.6:1 to 1.2:1, 1.5:1 to 1.2:1, 1.4:1 to 1.2:1, or 1.3:1 to 1.2: 1.

In another embodiment of the combination of the invention, optionally in combination with any of the embodiments provided above or below, the weight ratio between quercitrin and gallic acid or salts or esters thereof is from 10:1 to 1.2:1, from 10:1 to 1.3:1, from 10:1 to 1.4:1, from 10:1 to 1.5:1, from 10:1 to 1.6:1, from 10:1 to 1.7:1, from 10:1 to 1.8:1, from 10:1 to 1.9:1, from 10:1 to 2:1, from 10:1 to 2.1:1, from 10:1 to 2.2:1, from 10:1 to 2.3:1, from 10:1 to 2.4:1, from 10:1 to 2.5:1, from 10:1 to 2.6:1, from 10:1 to 2.7:1, from 10:1 to 2.8:1, or from 10:1 to 2.9: 1.

In another embodiment of the combination of the invention, optionally in combination with any of the embodiments provided above or below, the combination consists of quercitrin and gallic acid in a weight ratio of 65:1 to 1.2: 1.

In another embodiment of the combination of the invention, optionally in combination with any of the embodiments provided above or below, the combination comprises quercitrin and gallic acid in a weight ratio of from 65:1 to 1.2:1, more particularly from 20:1 to 1.5:1, more particularly from 10:1 to 2:1, more particularly from 4:1 to 2:1, or even more particularly from 8.6:1 to 2.85: 1. More particularly, the combination comprises quercus and gallic acid in a weight ratio of 49:1 to 1.5: 1. More particularly, the combination comprises quercus and gallic acid in a weight ratio of 9:1 to 1.5: 1.

In another embodiment of the combination of the invention, optionally in combination with any of the embodiments provided above or below, the quercus-citrin is provided in the form of a plant extract.

As used herein, "plant extract" or "extract" refers to a concentrated preparation of plant parts obtained by isolating active ingredients such as antioxidants from plants by suitable means. Depending on the physicochemical characteristics of these active ingredients, suitable means for their isolation may be used, such as aqueous solutions, alcoholic solutions, organic solvents, microwave or supercritical fluid extraction. The extract contains one or more active ingredients and can be incorporated into the composition in a variety of forms, including pure or semi-pure components, solid or liquid extracts, or solid plant matter.

In a particular embodiment of the combination of the invention, optionally in combination with any of the embodiments provided above or below, the plant extract comprises quercitrin in an amount equal to or higher than 30% w/w of the mass of the dry extract, more particularly equal to or higher than 40% w/w of the mass of the dry extract, more particularly equal to or higher than 50% w/w of the mass of the dry extract, more particularly between 35 and 60% w/w of the mass of the dry extract.

In a particular embodiment of the combination of the invention, optionally in combination with any of the embodiments provided above or below, said plant extract is a marigold extract. In a more specific embodiment, the marigold extract is a flower residue extract (flower bagass extract). In a more particular embodiment, the marigold flower residue extract comprises quercitrin in an amount of 35 to 60% w/w on dry extract mass.

"marigold extract" is understood to mean a concentrated preparation of compounds obtained from marigold plants. In the present invention, it is preferable to use the flower residue of marigold for preparing the extract. The pomace is the residue remaining after extraction of the oleoresin from the flowers. The primary antioxidant compound of marigold pomace extract is quercet, making up more than 94% of its antioxidant (see examples below).

Tagetes erecta (also known as Mexican marigold (Mexican marigold), Aztec marigold (Aztec marigold), African marigold (African marigold), or marigold for short) is a herb belonging to the family Compositae. Such plants are commonly used in traditional medicine and are common ornamental plants in the world. Most of the research on marigold flowers has focused on the extraction of lutein oleoresin. Marigold oleoresin is used industrially as a source of natural carotenoids to produce pigments which are added, for example, to broiler or laying hen feed. Carotenoid-rich oleoresin was obtained from marigold dried flowers with hexane, producing a large amount of defatted flower residue or pomace. These residues are currently used as fiber sources for animals, as fertilizers, or simply discarded.

Thus, another advantage of the combination of the invention is that quercitrin can be obtained from abundant and cheap compounds that are normally discarded in the industry, for example from quercitrin-enriched marigold residue extracts.

In a particular embodiment of the combination of the invention, optionally in combination with any of the embodiments provided above or below, said plant extract is selected from the group consisting of aqueous extracts, alcoholic extracts and organic extracts. In a more specific embodiment, the plant extract is an alcoholic extract.

The term "aqueous extract" as used herein refers to a plant extract that is extracted using water as the sole solvent. The term "organic extract" refers to a plant extract that is extracted using an organic solvent other than alcohol. The term "alcoholic extract" refers to a plant extract that is extracted using a mixture of water and an alcohol, such as methanol or ethanol.

In another embodiment of the combination of the invention, optionally in combination with any of the embodiments provided above or below, said extract is a dry extract.

It will be apparent to those skilled in the art that if the quercitrin is provided in the form of a plant extract, the weight ratio of the extract to gallic acid should be adjusted according to the amount of quercitrin contained in the extract to obtain the ratios of the present invention. For example, as shown in the examples below, when a marigold extract containing 57% w/w quercetn is used, in order to obtain a 2.85:1 weight ratio of quercetn to gallic acid, a combination of the extract and gallic acid in a 5:1 weight ratio must be made.

Thus, in another embodiment of the combination of the invention, optionally in combination with any of the embodiments provided above or below, the combination comprises marigold extract and gallic acid in a weight ratio of 65:1 to 1.8:1, more particularly 18:1 to 3:1, more particularly 16:1 to 4:1, more particularly 15:1 to 5: 1.

Thus, in another embodiment of the combination of the invention, optionally in combination with any of the embodiments provided above or below, the combination comprises marigold extract and added gallic acid in a weight ratio of 90:1 to 1.8:1, more particularly 40:1 to 3:1, more particularly 20:1 to 4:1, more particularly 15:1 to 5: 1.

In another embodiment of the combination of the invention, optionally in combination with any of the embodiments provided above or below, the combination comprises the tagetes alcohol extract and added gallic acid in a weight ratio of 90:1 to 1.8:1, more particularly 40:1 to 3:1, more particularly 20:1 to 4:1, more particularly 15:1 to 5: 1.

Thus, in another embodiment of the combination of the invention, optionally in combination with any of the embodiments provided above or below, the combination comprises the alcohol extract of marigold flower residue and added gallic acid in a weight ratio of 90:1 to 1.8:1, more particularly 40:1 to 3:1, more particularly 20:1 to 4:1, more particularly 15:1 to 5: 1.

As mentioned above, the present invention provides in a second embodiment a food, animal feed, pharmaceutical, veterinary or cosmetic composition comprising a combination as defined above. The compositions of the present invention may additionally incorporate other active ingredients which enhance the beneficial effects of the combinations of the present invention.

As used herein, "food composition" refers to any solid, semi-solid, or liquid composition suitable for human consumption, as well as drinkable liquids. The term "animal feed composition" refers to any solid, semi-solid, or liquid composition suitable for consumption by a non-human animal, as well as drinkable liquids; thus, it also includes pet food. Both food and feed compositions are meant to also include the raw materials and materials used to make them, as well as dietary supplements. The term "dietary supplement" refers to a formulation intended to supplement the diet and provide nutrients such as vitamins, minerals, fiber, fatty acids or amino acids that may be deficient or may be ingested in insufficient amounts in the diet of a subject.

As used herein, "cosmetic composition" refers to a composition for improving the appearance or hygiene of a subject, comprising a cosmetically acceptable excipient or carrier. The term "cosmetically acceptable" refers to an excipient or carrier suitable for use in contact with human skin without undue toxicity, incompatibility, instability, allergic response, and the like.

The term "pharmaceutical composition" or "veterinary composition" refers to a composition for improving or maintaining the health of a subject or animal, respectively, and comprises a pharmaceutically acceptable excipient or carrier.

The composition comprising the combination of the invention may be in any form, e.g. solid or liquid.

The combination of the invention is particularly useful for preventing oxidation of a lipid-containing composition. Thus, in another embodiment, optionally in combination with any of the embodiments provided above or below, the food product, animal feed, pharmaceutical, veterinary or cosmetic composition comprises a lipid. More particularly, the composition comprises a mass of fat equal to or higher than 1% w/w, more particularly equal to or higher than 10% w/w.

As used herein, "lipid" refers to an organic molecule that is insoluble in water and soluble in a non-polar solvent, such as a fatty acid, steroid, or triglyceride (fat). The combination of the invention is particularly useful for preventing fat oxidation.

In another embodiment, optionally in combination with any of the embodiments provided above or below, the food product, animal feed, pharmaceutical, veterinary or cosmetic composition comprises a vitamin or carotenoid.

In another embodiment, optionally in combination with any of the embodiments provided above or below, the food, animal feed, pharmaceutical, veterinary or cosmetic composition comprises the combination of the invention in an amount of 0.001% w/w to 1% w/w, more particularly 0.005% w/w to 0.5% w/w, even more particularly 0.01% w/w to 0.1% w/w.

In another embodiment, optionally in combination with any of the embodiments provided above or below, the food, animal feed, pharmaceutical, veterinary or cosmetic composition comprises quercitrin in an amount from 20ppm to 5000 ppm. More particularly, 25ppm to 500ppm, more particularly, 50ppm to 200 ppm.

In another embodiment, optionally in combination with any of the embodiments provided above or below, the food, animal feed, pharmaceutical, veterinary or cosmetic composition comprises gallic acid in an amount from 20ppm to 5000 ppm. More particularly, 25ppm to 500ppm, more particularly, 50ppm to 200 ppm.

As mentioned before, the present invention provides in a third aspect the use of a combination as defined above as an antioxidant in a food product, animal feed, pharmaceutical, veterinary or cosmetic composition. The combination of the invention may be used to increase the oxidative stability of any composition comprising an oxidation sensitive molecule, such as a lipid. Thus, in a particular embodiment, the combination as defined above is used as an antioxidant in a food, animal feed, pharmaceutical, veterinary or cosmetic composition, wherein the composition comprises a lipid, more particularly it comprises an amount of lipid equal to or higher than 1% w/w, more particularly equal to or higher than 10% w/w.

As mentioned above, the present invention provides in a fourth aspect a method of preparing a combination as defined above, the method comprising the steps of:

-providing a quercus pavilion;

-adding gallic acid or salts or esters thereof;

-optionally, adding a suitable liquid carrier; and

-mixing and homogenization.

In a particular embodiment of the method, optionally in combination with any of the embodiments provided above or below, the suitable liquid carrier is selected from an edible carrier and a pharmaceutically acceptable carrier. In a more particular embodiment, suitable liquid carriers are selected from the group consisting of alcohols (e.g., ethanol or isopropanol), organic acids, esters (e.g., citrate, mono-and diglycerides), lecithin, lipids (e.g., fats or oils), other liquid extracts, and water.

As used herein, "pharmaceutically acceptable carrier" refers to a carrier suitable for use in contact with the tissues or organs of human and non-human animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications commensurate with a reasonable benefit/risk ratio. By "edible carrier" is meant a carrier that can be ingested by animals, including humans, without significant deleterious health consequences.

Throughout the specification and claims, the word "comprise" and variations of the word are not intended to exclude other technical features, additives, components or steps. Furthermore, the word "comprising" covers the case of "consisting of … …". Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and figures are provided as illustrations and are not intended to limit the invention. Any reference signs placed between parentheses in the claims and associated with the drawings are intended for increasing the intelligibility of the claims and shall not be construed as limiting the scope of the claims. Moreover, the present invention encompasses all possible combinations of the specific and preferred embodiments described herein.

Examples

Preparation of extracts

Marigold dregs are obtained by oleoresin extraction of flowers (H.B. Sowbhagya et al, "Natural colors from Marigold-Chemistry and Technology" from Marigold Chemistry and Technology ", Food Reviews International, 2004, vol.20, p.33-50).

The flower residue was then subjected to alcoholic solvent extraction according to standard procedures. In short, the extraction can be carried out using water containing methanol and/or ethanol as solvent in a volume ratio of 1:3 to 3:1, over a period of time of 0.5 to 2 hours, at a temperature range of 30 to 50 ℃, and in a solid-to-liquid ratio of 1:30 to 1:50(w/v), depending on the nature of the starting material.

The solid-liquid intermediate was then filtered and the extract was spray dried to give a concentrated yellow to brown powder. The set of operating conditions for the concentration-filtration and spray-drying of the extract depends on the specific characteristics of each batch of extract, which is influenced by the quality of the original crop obtained. The ingredients added to the formulation to be spray dried also determine the operating conditions. The solid residue and viscosity between extraction batchesAnd (5) standardizing the degree. A two-fluid nozzle with a cap hole diameter of 0.5mm was used. The aqueous dispersion was mixed for about 30 minutes and then spray dried within the reference range of operating conditions: inlet temperature of drying air, 100-; volumetric flow rate of atomizing air: 300-800 l/h; feeding volume flow rate: 3-15 ml/min; feed concentration, 3-10% (w/w); volume flow rate of drying air: 25-50m³/h。

The resulting extract was quantified by external mode using liquid chromatography with arrayed photodiodes and a mass spectrometer detector with a triple quadrupole rod detector with MRM (UPLC-PDA-XevoTQS), "characterization of phenolic compounds from the processed fiber from the fruit juice industry" (chromatography of phenolic compounds in processed fibers), "Food chem., 2015, 172, 575 584 pages) and indicated to be flavonoid-rich (50-70%) with quercus content of 30% to 65%.

The distribution of the most abundant antioxidant substances in the two extract samples was as follows:

-quercus: sample-A: 573.68mg/g, sample-B: 567.29 mg/g.

-quercetin: sample-A: 26.12mg/g, sample-B2: 27.15 mg/g.

-gallic acid methyl ester: sample-A: 7.02mg/g, sample-B: 7.17 mg/g.

-gallic acid: sample-A: 1.18mg/g, sample-B: 1.20 mg/g.

Thus, quercitrin represents almost all of the antioxidant substances of the extract (94.24% of the active substances evaluated in these samples). Quercetin accounted for 4.39% of the phenols and flavonoids evaluated, while gallic acid, including methyl gallate and gallic acid, accounted for 1.37% (1.17% and 0.20%, respectively).

Rancimat method

Rancimat was measured using 892Professional Rancimat according to the manufacturer's instructions (Metrohm, switzerland) to evaluate the antioxidant efficacy of quercetn (provided in the form of the marigold residue extract described above) in combination with various antioxidants.

The temperature of the heating block was fixed at 120 ℃. When the device reached the desired temperature, 60mL of deionized water was added to each cell and its conductivity was measured until stable (1.5-2 h).

Commercial lard without antioxidant was melted in a heater at 50 ℃. A homogenous lard stock solution with 1% antioxidant was prepared and diluted with lard to reach the desired concentration to be tested. The estimated concentrations of the antioxidant were 25, 75 and 150 ppm. The concentration of antioxidant in marigold extract used was 60%.

To identify synergistic relationships between antioxidants, different ratios of marigold extracts were tested in combination with gallic acid, BHT, BHA and tocopherol. The weight ratio used was 15:1 (90% active from extract-10% active from another antioxidant); 5:1 (75% active from extract-25% active from another antioxidant); 1.67:1 (50% active from TE extract-50% active from another antioxidant); and 1:1.8 (25% active from TE extract-75% active from another antioxidant)

Once the antioxidant-containing lard samples were prepared, 3g of sample was added per reaction vessel. The sample was exposed to a stream of air at 120 ℃. Volatile oxidation products (mainly formic acid) are transferred by the air stream into the measuring vessel and absorbed there in distilled water, in which the water conductivity is determined. The water conductivity, mainly caused by the increase of formic acid, was continuously recorded by software installed in a computer according to time, thereby obtaining an oxidation curve with an inflection point as an induction period. This induction period is the time required for the nature of the oxidation process to transition from a very slow oxidation state to a much more oxygen-consuming state, resulting in a rapid increase in the peroxide value. All tests included a negative control without any antioxidant throughout.

The antioxidant protection factor is determined by the ratio of the induction period with antioxidant to the induction period of the corresponding negative control (no antioxidant). The higher the antioxidant protection factor, the better the efficacy of the antioxidant evaluated. All statistical analyses were performed using a version 9.1.3 SAS software book (SAS institute).

Results of TE extract in combination with gallic acid

The results of the rancimat assay showed that the TE extract alone provided a higher protection factor than gallic acid, BHA or BHB, but lower than tocopherol (table 1 and figure 1).

TABLE 1

n 2 (number of repetitions per concentration)

The combination of TE extract with other antioxidants showed no improvement in oxidative protection when the extract was mixed with tocopherol, BHA or BHB. However, when TE extracts were combined with gallic acid, an unexpected synergistic antioxidant effect was observed (figure 1, black triangles and black circles), increasing their antioxidant capacity by 48% (N ═ 4-8 replicates/measured antioxidant concentrations, two-way anova (Tukey test),^*comparative TE P<0.05，^#Contrast Gallic acid P<0.01). This synergy disappeared when the amount of gallic acid was increased beyond the 1.67:1 (extract/gallic acid) weight ratio (figure 1, triangles and solid squares).

Thus, these results demonstrate that the combination comprising quercitrin and gallic acid provides a powerful synergistic antioxidant effect.

Results of isolated quercus robusta in combination with gallic acid

The antioxidant efficacy of pure quercitrin (> 95% purity) in combination with gallic acid (> 99% purity) was also assessed by the Rancimat method, as described above.

As shown in fig. 2, the addition of gallic acid to quercus robusta in a 9:1 weight ratio provided a synergistic effect (black circles) that exceeded the antioxidant capacity of quercus robusta alone (open squares), gallic acid alone (open circles) and TE extract alone (open triangles).

Thus, these results demonstrate that a combination containing only quercitrin and gallic acid provides a potent synergistic antioxidant effect.

Furthermore, the antioxidant capacity of the combination of quercitrin and gallic acid was comparable to that provided by the combination of TE extract and gallic acid (black triangles), demonstrating that the synergistic antioxidant effect observed by the combination of TE extract and gallic acid is indeed provided by the combination of quercitrin present in the TE extract and added gallic acid.

Citations

Sowbhagya et al, "Natural colorants from Marigold Chemistry and Technology", Food Reviews International, 2004, Vol.20, pp.33-50.

Antoni Delpino-Rius et al, "characterization of phenolic compounds in processed fibers from the juice industry" (Food chem., 2015, Vol.172, page 575-.