WO2019185939A1

WO2019185939A1 - Use of twin-chromanols as antioxidants in oil

Info

Publication number: WO2019185939A1
Application number: PCT/EP2019/058118
Authority: WO
Inventors: Laure CLASADONTE; André DUESTERLOH; Weerasinghe INDRASENA; Thomas Netscher; René Tobias STEMMLER
Original assignee: Dsm Ip Assets B.V.
Priority date: 2018-03-29
Filing date: 2019-03-29
Publication date: 2019-10-03

Abstract

The present invention is directed towards the use of at least one compound of formula (I) wherein R¹, R² and R³ are independently from each other H or linear C_1-6-alkyl or branched C_3-8-alkyl, as antioxidant in PUFA-containing edible oils such as marine oil, microbial oil, fungal oil, algal oil and PUFA-containing plant oil for human consumption. The present invention is further directed towards these PUFA- containing edible oils comprising at least one compound of formula (I). The present invention is further directed to a method of preserving the shelf life of PUFAs and/or their esters in an edible oil comprising the step of adding at least one compound of formula (I) to said edible oil, as well as to a method of limiting the amount of oxidation of PUFAs and/or their esters in an edible oil which is exposed to air, comprising adding at least one compound of formula (I) to said edible oil, preferably in an amount of said compound of formula (I) ranging from 10 to 500 ppm, preferably ranging from 30 to 300 ppm, more preferably ranging from 100 to 250 ppm, based on the total amount of said edible oil.

Description

USE OF TWIN-CHROMANOLS AS ANTIOXIDANTS IN OIL

The present invention is directed towards the use of at least one compound of formula (I) as antioxidant in oil,

wherein the oil contains polyunsaturated fatty acids and/or their esters, and wherein the oil is for human consumption, and

wherein R¹, R² and R³ are independently from each other H or linear Ci-₆- alkyl or branched C_3-8-alkyl.

Oils containing polyunsaturated fatty acids and/or their esters are gaining more and more attention, because of their beneficial health effects in humans.

Since these oils are only of limited stability, because they are oxidized very easily, there is a need to provide efficient antioxidants for their stabilization.

Detailed description of the invention

This need is fulfilled by the present invention, which is directed to the use of at least one compound of formula (I) as antioxidant in oil,

wherein R¹, R² and R³ are independently from each other H or linear Ci-₆- alkyl or branched C_3-8-alkyl;

and with the preferences for the substituents R¹ to R³ as given below.

Compounds of formula (I)

Preferred are compounds of formula (I), wherein R¹, R² and R³ are independently from each other H or linear Ci-₄-alkyl or branched C3-₄-alkyl.

More preferred are compounds of formula (I), wherein R¹ is H or methyl or ethyl or n-propyl or iso-propyl or tert- butyl; and/or R² and R³ are

independently from each other H or methyl or ethyl.

Even more preferred are compounds of formula (I), wherein R¹ is methyl or tert- butyl and/or R² and R³ are independently from each other H or methyl. Especially preferred compounds of the present invention include the following compounds:

Compound of formula (I) with R¹ = R² = R³ = methyl;

Compound of formula (I) with R¹ = tert- butyl and R² = R³ = H,

Compound of formula (I) with R¹ = methyl and R² = R³ = H,

Compound of formula (I) with R¹ = R³ = methyl and R² = H,

Compound of formula (I) with R¹ = R² = R³ = H. Most preferred is the following compound of formula (1 ) (3,9-di-tert-butyl- 12A7-6,12-methanodibenzo[d,g][1 ,3]dioxocine-2,10-diol):

Use as antioxidants

The compounds of the present invention are efficient as antioxidants in PUFA-containing oils for human consumption.

“PUFA(s)” means polyunsaturated fatty acid(s) such as docosahexaenoic acid (“DHA”) and/or eicosapentaenoic acid (“EPA”) and/or docosapentaenoic acid (“DPA”) and/or oleic acid and/or stearidonic acid and/or linoleic acid and/or alpha-linolenic acid (“ALA”) and/or gamma-linolenic acid and/or arachidonic acid (“ARA”) and/or the esters of all of them, whereby the term “esters” encompasses monoglycerides, diglycerides and triglycerides as well as Ci-₆-alkyl esters such as especially the methyl esters and the ethyl esters, whereby the triglycerides are often dominant.

DHA, EPA, ALA and stearidonic acid are omega-3 fatty acids, whereas linoleic acid, gamma-linolenic acid and ARA are omega-6 fatty acids.

The term “DPA” encompasses two isomers, the omega-3 fatty acid clupanodonic acid (7Z,10Z,13Z,16Z,19Z-docosapentaenoic acid) and the omega-6 fatty acid osbond acid (4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid). In accordance with the invention, the polyunsaturated fatty acid (PUFA) is preferably DHA and/or EPA and/or DPA and/or any ester thereof, more preferably the polyunsaturated fatty acid (PUFA) is preferably DHA and/or EPA and/or any ester thereof.

Examples of PUFA-containing oils for human consumption are

- marine oil, such as preferably fish oil,

- microbial biomass containing polyunsaturated fatty acids and/or their esters (“microbial oil”), preferably containing high amounts of docosahexaenoic acid (“DHA”) and/or eicosapentaenoic acid (“EPA”) and/or docosapentaenoic acid (“DPA”) and/or their esters, and

- oil containing high amounts of PUFAs and/or their esters, preferably containing high amounts of docosahexaenoic acid (“DHA”) and/or eicosapentaenoic acid (“EPA”) and/or docosapentaenoic acid (“DPA”) and/or their esters, extracted from microbial biomass, such as fungae (“fungal oil”) or algae (“algal oil”), and

- plant oil with relatively high amounts of PUFAs and/or their esters, (“PUFA-containing plant oil”), such as e.g. canola seed oil, linseed/flaxseed oil, hempseed oil, pumpkin seed oil, evening primrose oil, borage seed oil, blackcurrent seed oil, sallow thorn/sea buckthorn oil, chia seed oil, argan oil and walnut oil.

Marine oils, microbial oils and algal oils are especially preferred.

Further objects of the present invention

Thus, in addition, the present invention is

(1 ) directed to the use of the compounds of formula (I) with the preferences as given above as antioxidants in marine oils, microbial oils, oils containing high amounts of PUFAs and/or their esters extracted from microbial biomass and plant oils with relatively high amounts of PUFAs and/or their esters for human consumption; as well as (2) directed to these PUFA-containing oils for human consumption comprising such compounds of formula (I) with the preferences as given above.

Furthermore, the present invention is directed to

(3) an edible oil comprising a compound of formula (I) and PUFAs and/or their esters;

(4) a method of preserving the shelf life of PUFAs and/or their esters in an edible oil comprising the step of adding at least one compound of formula (I) to said edible oil, preferably in an amount of said compound of formula (I) ranging from 10 to 500 ppm, preferably ranging from 30 to 300 ppm, more preferably ranging from 100 to 250 ppm, based on the total amount of the edible oil;

(5) a method of limiting the amount of oxidation of PUFAs and/or their esters in an edible oil which is exposed to air, comprising adding at least one compound of formula (I) to said edible oil, preferably in an amount of said compound of formula (I) ranging from 10 to 500 ppm, preferably ranging from 30 to 300 ppm, more preferably ranging from 100 to 250 ppm, based on the total amount of the edible oil.

For all these objects (1 ) to (5) of the present invention the preferences with respect to the compound of formula (I) and the PUFA-containing oil for human consumption, i.e. the PUFA-containing edible oil, as given above and below apply.

Further antioxidants

The compounds of formula (I) can be used in combination with one or more other antioxidants as described below.

In an embodiment of the present invention the PUFA-containing oils of the present invention comprising a compound of formula (I) additionally comprise ascorbyl palmitate and/or carnosic acid. Instead of ascorbyl palmitate other esters of ascorbic acid such as the esters of ascorbic acid with linear C12-20 alkanols, preferably the esters of ascorbic acid with linear C_14-18 alkanols, may also be used, so that further embodiments of the present invention are directed to PUFA-containing oils of the present invention comprising a compound of formula (I) that additionally comprise esters of ascorbic acid with linear C12-20 alkanols, preferably esters of ascorbic acid with linear C14-18 alkanols, more preferably ascorbyl palmitate.

The PUFA-containing oils of the present invention comprising a compound of formula (I) may also comprise additionally alpha-tocopherol and/or gamma- tocopherol, whereby either an ester of ascorbic acid with a linear C_12-20 alkanol with the preferences as given above may additionally be present.

The PUFA-containing oils themselves are described in more detail below.

PUFA-containing oils

In the context of the present invention the term“PUFA-containing oil” encompasses

- marine oil, such as especially fish oil,

- microbial biomass containing polyunsaturated fatty acids (“PUFAs”), especially docosahexaenoic acid (“DHA”) and/or eicosapentaenoic acid (“EPA”) and/or docosapentaenoic acid (“DPA”) and/or their esters (“microbial oil”);

- oil containing high amounts of PUFAs, especially containing high amounts of DHA and/or EPA and/or DPA and/or their esters extracted from microbial biomass as e.g., fungi (“fungal oil”) or algae (“algal oil”);

- Plant oil with high amounts of PUFAs and/or their esters (“PUFA- containing plant oil”), such as e.g. canola seed oil, linseed/flaxseed oil, hempseed oil, pumpkin seed oil, evening primrose oil, borage seed oil, blackcurrent seed oil, sallow thorn/sea buckthorn oil, chia seed oil, argan oil and walnut oil. The term “DHA” does not only encompass the acid but also derivatives thereof such as monoglycerides, diglycerides and triglycerides as well as Ci- ₆-alkyl esters such as the methyl and ethyl esters. The same applies for “EPA” and“DPA” and all the other PUFAs.

Fish oil and algal oil are commonly used for human consumption. Instead of fish oil and algal oil also other PUFA-containing oils may be used for human consumption, i.e.:

- microbial biomass containing PUFAs (“microbial oil”)

- oil containing high amounts of PUFAs extracted from microbial

biomass, such as especially fungal oil, and

- plant oil with high amounts of PUFAs.

The above-mentioned PUFA-containing oils may not only be used as alternative of fish oil and algal oil, but also in addition.

Details of these PUFA-containing oils for human consumption are given below.

Marine oil

Examples of suitable marine oils include, but are not limited to, Atlantic fish oil, Pacific fish oil, or Mediterranean fish oil, or any mixture or combination thereof.

In more specific examples, a suitable fish oil can be, but is not limited to, pollack oil, bonito oil, pilchard oil, tilapia oil, tuna oil, sea bass oil, halibut oil, spearfish oil, barracuda oil, cod oil, menhaden oil, sardine oil, anchovy oil, capelin oil, herring oil, mackerel oil, salmonid oil, tuna oil, and shark oil, including any mixture or combination thereof. Other marine oils suitable for use herein include, but are not limited to, squid oil, cuttle fish oil, octopus oil, krill oil, seal oil, whale oil, and the like, including any mixture or combination thereof. For stabilizing marine oil an amount of at least one compound of formula (I) ranging from 10 to 500 ppm, preferably ranging from 30 to 300 ppm, more preferably ranging from 100 to 250 ppm, based on the total amount of the marine oil, is usually sufficient. The same applies for the other PUFA- containing oils such as microbial oil, algal oil, fungal oil and PUFA- containing plant oil.

A commercially available example of marine oil is the fish oil “MEG-3” (Bleached 30S TG Fish oil) from DSM Nutritional Products, LLC (US) whose specification and composition is shown in Tables 1 and 2 below:

Table 1

The peroxide value is defined as the amount of peroxide oxygen per 1 kilogram of oil. Traditionally this is expressed in units of milliequivalents or meq/kg.

Winterization is part of the processing of fish oil, and it is performed to remove solid fat in the oil. The“cold test” is performed to check if any solid fat is present and precipitated in the oil when cooled to 0°C within a specific period of time. In this fish oil (Product Code: FG30TG), any such precipitation is checked for 3 hours at 0°C.

Table 2

“TG” = triglyceride;

“A%” = “area

= area percentage by GC based on 24 peak analysis (meaning the 24 highest peaks have been analyzed)

Oil containing high amounts of PUFAs. especially containing high amounts of DHA and/or EPA and/or DPA and/or their esters, extracted from microbial biomass as e.g., fungi (“fungal oil”) or algae (“algal oil”) Algal oil

“Algal oil” is an oil containing high amounts of DHA and/or EPA and/or DPA and/or their esters extracted from algae as microbial source/biomass.

An example of algal oil is the commercially available“Algal oil containing EPA+DPA” from DSM Nutritional Products, LLC (US) whose composition is shown in the Table 3 below:

Table 3

A further example of a crude oil containing high amounts of DHA and/or EPA extracted from microbial sources as e.g., algae, is the oil extracted from Algae Schizochytrium Biomass, whose specification is given in the following

Table 4.

Table 4

Microbial biomass containing polyunsaturated fatty acids (“PUFAs”). especially docosahexaenoic acid and/or eicosapentaenoic acid and/or docosapentaenoic acid (“DPA”) and/or their esters

The biomass preferably comprises cells which produce PUFAs hetero- trophically. According to the invention, the cells are preferably selected from algae, fungi, particularly yeasts, bacteria, or protists. The cells are more preferably microbial algae or fungi.

Suitable cells of oil-producing yeasts are, in particular, strains of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

Oil produced by a microorganism or obtained from a microbial cell is referred to as“microbial oil”. Oil produced by algae and/or fungi is referred to as an algal and/or a fungal oil, respectively.

As used herein, a "microorganism" refers to organisms such as algae, bacteria, fungi, protist, yeast, and combinations thereof, e.g., unicellular organisms. A microorganism includes but is not limited to, golden algae (e.g., microorganisms of the kingdom Stramenopiles); green algae; diatoms; dinoflagellates (e.g., microorganisms of the order Dinophyceae including members of the genus Crypthecodinium such as, for example,

Crypthecodinium cohnii or C. cohnii); microalgae of the order

Thraustochytriales; yeast ( Ascomycetes or Basidiomycetes); and fungi of the genera Mucor, Mortierella, including but not limited to Mortierella alpina and Mortierella sect, schmuckeri, and Pythium, including but not limited to Pythium insidiosum.

In one embodiment, the microorganisms of the kingdom Stramenopiles may in particular be selected from the following groups of microorganisms:

Hamatores, Proteromonads, Opalines, Developayella, Diplophrys,

Labrinthulids, Thraustochytrids, Biosecids, Oomycetes,

Hypochytridiomycetes, Commotion, Reticulosphaera, Pelagomonas,

Pelagococcus, Ollicola, Aureococcus, Parmales, Diatoms, Xanthophytes, Phaeophytes (brown algae), Eustigmatophytes, Rophidophytes, Synurids, Axodines (including Rhizochromulinales, Pedinellales, Dictyochales), Chrysomeri dales, Sarcinochrysidales, Hydrurales, Hibberdiales, and

Chromulinales. In one embodiment, the microorganisms are from the genus Mortierella, genus Crypthecodinium, genus Thraustochytrium, and mixtures thereof. In a further embodiment, the microorganisms are from Crypthecodinium Cohnii. In a further embodiment, the microorganisms are from Mortierella alpina. In a still further embodiment, the microorganisms are from

Schizochytrium sp. In yet an even further embodiment, the microorganisms are selected from Crypthecodinium Cohnii, Mortierella alpina,

Schizochytrium sp., and mixtures thereof.

In a still further embodiment, the microorganisms include, but are not limited to, microorganisms belonging to the genus Mortierella, genus Conidiobolus, genus Pythium, genus Phytophthora, genus Penicillium, genus Cladosporium, genus Mucor, genus Fusarium, genus Aspergillus, genus Rhodotorula, genus Entomophthora, genus Echinosporangium, and genus Saprolegnia.

In an even further embodiment, the microorganisms are from microalgae of the order Thraustochytriales, which includes, but is not limited to, the genera Thraustochytrium (species include arudimentale, aureum, benthicola, globosum, kinnei, motivum, multirudimentale, pachydermum, proliferum, roseum, striatum); the genera Schizochytrium (species include aggregatum, limnaceum, mangrovei, minutum, octosporum); the genera Ulkenia (species include amoeboidea, kerguelensis, minuta, profunda, radiate, sailens, sarkariana, schizochytrops, visurgensis, yorkensis); the genera Aurantiacochytrium; the genera Oblongichytrium; the genera Sicyoidochytium; the genera Parientichytrium; the genera Botryochytrium; and combinations thereof. Species described within Ulkenia will be considered to be members of the genus Schizochytrium. In another embodiment, the microorganisms are from the order Thraustochytriales. In yet another embodiment, the microorganisms are from Thraustochytrium.

In still a further embodiment, the microorganisms are from Schizochytrium sp. In certain embodiments, the oil can comprise a marine oil. Examples of suitable marine oils are the ones as given above.

The biomass according to the invention preferably comprises cells, and preferably consists essentially of such cells, of the taxon

Labyrinthulomycetes ( Labyrinthulea , net slime fungi, slime nets), in particular, those from the family of Thraustochytriaceae . The family of the Thraustochytriaceae ( Throustochytrids ) includes the genera Althomia, Aplanochytrium, Aurantiochytrium, Botryochytrium, Elnia, Japonochytrium, Oblongichytrium, Parietichytrium, Schizochytrium, Sicyoidochytrium, Thraustochytrium, and Ulkenia. The biomass particularly preferably comprises cells from the genera Aurantiochytrium, Oblongichytrium, Schizochytrium, or Thraustochytrium, more preferably from the genus Schizochytrium.

In accordance with the invention, the polyunsaturated fatty acid (PUFA) is preferably DHA and/or EPA and/or their esters as defined above.

The cells present in the biomass are preferably distinguished by the fact that they contain at least 20 weight-%, preferably at least 30 weight-%, in particular at least 35 weight-%, of PUFAs, in each case based on cell dry matter.

In a very preferred embodiment of the current invention, cells, in particular a Schizochytrium strain, is employed which produces a significant amount of EPA and DHA, simultaneously, wherein DHA is preferably produced in an amount of at least 20 weight-%, preferably in an amount of at least 30 weight-%, in particular in an amount of 30 to 50 weight-%, and EPA is produced in an amount of at least 5 weight-%, preferably in an amount of at least 10 weight-%, in particular in an amount of 10 to 20 weight-% (in relation to the total amount of lipid as contained in the cells, respectively). Preferred species of microorganisms of the genus Schizochytrium, which produce EPA and DHA simultaneously in significant amounts, as mentioned before, are deposited under ATCC Accession No. PTA-10208, PTA-10209, PTA-10210, or PTA-10211 , PTA-10212, PTA-10213, PTA-10214, PTA-10215.

DHA and EPA producing Schizochytrium strains can be obtained by consecutive mutagenesis followed by suitable selection of mutant strains which demonstrate superior EPA and DHA production and a specific EPA:DHA ratio. Any chemical or nonchemical (e.g. ultraviolet (UV) radiation) agent capable of inducing genetic change to the yeast cell can be used as the mutagen. These agents can be used alone or in combination with one another, and the chemical agents can be used neat or with a solvent.

Methods for producing the biomass, in particular, a biomass which comprises cells containing lipids, in particular PUFAs, particularly of the order

Thraustochytriales, are described in detail in the prior art (see e.g. WO 91 / 07498, WO 94/08467, WO 97/37032, WO 97/36996, WO 01 /54510). As a rule, the production takes place by cells being cultured in a fermenter in the presence of a carbon source and a nitrogen source, along with a number of additional substances like minerals that allow growth of the

microorganisms and production of the PUFAs. In this context, biomass densities of more than 100 grams per litre and production rates of more than 0.5 gram of lipid per litre per hour may be attained. The process is preferably carried out in what is known as a fed-batch process, i.e. the carbon and nitrogen sources are fed in incrementally during the

fermentation. When the desired biomass has been obtained, lipid

production may be induced by various measures, for example by limiting the nitrogen source, the carbon source or the oxygen content or combinations of these.

In a preferred embodiment of the current invention, the cells are grown until they reach a biomass density of at least 80 or 100 g/l, more preferably at least 120 or 140 g/l, in particular at least 160 or 180 g/l (calculated as dry-matter content). Such processes are for example disclosed in US

7,732,170.

Preferably, the cells are fermented in a medium with low salinity, in particular, so as to avoid corrosion. This can be achieved by using chlorine- free sodium salts as the sodium source instead of sodium chloride, such as, for example, sodium sulphate, sodium carbonate, sodium hydrogen carbonate or soda ash. Preferably, chloride is used in the fermentation in amounts of less than 3 g/l, in particular, less than 500 mg/l, especially preferably less than 100 mg/l.

PUFA-containing plant oils: Plant oils with relatively high amounts of PUFAs, especially with high amounts of DHA and/or EPA such as e.g. , canola seed oil

The plant cells may, in particular, be selected from cells of the families Brassicaceae, Elaeagnaceae and Fabaceae. The cells of the family

Brassicaceae may be selected from the genus Brassica, in particular, from oilseed rape, turnip rape and Indian mustard; the cells of the family

Elaeagnaceae may be selected from the genus Elaeagnus, in particular, from the species Oleae europaea ; the cells of the family Fabaceae may be selected from the genus Glycine, in particular, from the species Glycine max.

Examples:

- Canola seed oil with a content of DHA of at least 9% by weight, of at least 12% by weight, of at least 15% by weight, or of at least 20% by weight, based on the total weight of the canola seed oil;

- Canola seed oil with a content of EPA of at least 9% by weight, of at least 12% by weight, of at least 15% by weight, or of at least 20% by weight, based on the total weight of the canola seed oil. Examples of PUFA-containing plant oils containing high amounts of other PUFAs than EPA and/or DHA and/or DPA and/or their esters are linseed/flaxseed oil, hempseed oil, pumpkin seed oil, evening primrose oil, borage seed oil, blackcurrent seed oil, sallow thorn/sea buckthorn oil, chia seed oil, argan oil and walnut oil.

The invention is now further illustrated in the following non-limiting examples.

Examples

Example 1 : Synthesis of compound of formula (1 )

3,9-Di-tert-butyl-12A7-6, 12-methanodibenzo[d,g][1 ,3]dioxocine-2, 10-diol was prepared following a procedure by N. Seto and M. Morigaki (N. Seto, M. Morigaki, Silver Halide Color Photographic Sensitive Material, 1992,

JPH04330440).

Example 2: Antioxidant activities of compound of formula (1 ) in fish oil and algal oil

The antioxidant activity of compound of formula (1 ) was evaluated in both fish and algal oil in comparison with mixed natural tocopherols (MNT). Fish oil did not contain any antioxidants (= blank oil) whereas algal oil contained about 1 .5 mg/g of MNT. Thus, the blank oil, i.e. oil without any antioxidant, (= fish oil) and oil containing“MNT” (= algal oil) have been used as benchmark. Any compound better in antioxidant activity than the blank oil indicates that it has antioxidant activity. The comparison with MNT gives an indication about the amount of the antioxidant effect, relative to the activity of MNT.

“MNT” are mixed natural tocopherols commercially available as e.g., “Tocomix 70 IP” from AOM (Buenos Aires, Argentina). Tocomix 70 IP comprises d-alpha-tocopherol, d-beta-tocopherol, d-gamma-tocopherol and d-delta-tocopherol, whereby the total amount of tocopherols is at least 70.0

weight-% and the amount of non-alpha tocopherols is at least 56.0 weight-%.

Materials and methods

The compound of formula (1 ) was used in both fish and algal oils to see its

antioxidant effect in these oils. Antioxidant effect was determined using

mainly the Oil Stability Index (OSI). A storage stability study was performed

to compare the variation of primary oxidation products, the hydroperoxides,

generated during oxidation, measured in terms of peroxide value (PV) and

the secondary oxidation products which were measured and determined as

anisidine reactive substances or p-anisidine value (p-AV) of oil samples

containing this compound.

Oxidative stability

Two concentration levels were used. Compound of formula (1 ) was added in

the concentrations of 0.5 mg/g (low level) and 2 mg/g (high level) to 5 g of

oil and used in the Oxidative Stability Instrument operated at 80°C with the

continuous air flow rate at 5.5 psi. All samples were run in duplicate. The

Protection Factors (PF) for compound of formula (1 ) in oil were calculated

in percentage as:

10CK-₃ x (OSI of the sample with antioxidant - OSI of tie sample without antioxidant

PF {%) ^~ -

OSI of the sample without antioxidant

Results

OSI values of the fish oil samples containing compound of formula (1 ), in

comparison to the same levels of MNT, are shown in Tables 5 and 6. The

compound of formula (1 ) provides slightly lower protection in fish oil than

MNT.

Table 5: Oxidative stability of FG30TG fish oil with compound of formula (1 )

(SD = standard deviation)

Table 6: Protection Factor of compound of formula (1 ) in fish oil

The antioxidant effect of the compound of formula (1 ) in algal oil is different from the effect in fish oil. As shown in Table 7 the compound of formula (1 ) showed higher OSI values than the crude algal oil indicating a considerable antioxidant activity of said compound in algal oil. The compound of formula (1 ) clearly showed much better Protection Factor than MNT at both concentration levels (Table 8).

Table 7: Oxidative stability of crude algal oil with compound of formula (1 ) (SD = standard deviation)

Table 8: Protection Factor of compound of formula (1 ) in crude algal oil

Also, the synergistic effect of the compound of formula (1 ) with ascorbyl palmitate (AP) and/or carnosic acid (CA) was determined using the OSI values (for the results see Table 9) and the Protection Factor (for the results see Table 10).

Table 9: Improvement of the effect of the compound of formula (1 ) in FG30TG fish oil using AP and/or CA (SD = standard deviation)

Table 10: Improvement of the Protection Factor of the compound of formula (1 ) in FG30TG fish oil using AP and/or CA (SD = standard deviation)

Storage stability A storage stability study for fish oil (XBUFG30TG) containing 2 different levels of compound of formula (1 ) (0.5 and 2 mg/g) was performed to compare the variation of primary and secondary oxidation products generated during storage at ambient temperature as a result of oxidation of the oil in the presence of these compounds.

Hereby, accurately weighed amounts of compound of formula (1 ) (0.5 mg/g and 2 mg/g) were added, each individually, to 60 g of fish oil samples in glass amber bottles, thoroughly mixed under nitrogen and stored at ambient temperature storage for 2 weeks. All sample bottles were left open to air, away from light. 0.5 mg/g of the compound of formula (1 ) were soluble in fish oil upon thorough mixing, whereas in an amount of 2 mg/g it was only partially soluble in fish oil. The primary oxidation products, hydroperoxides, were determined in terms of peroxide value (PV), and the secondary oxidation products were measured and determined as anisidine reactive substances or p-anisidine value (p-AV). Conjugated dienoic acid (CD) levels which are also indicative of the primary oxidation were also determined. Peroxide values (PV), p- anisidine values (p-AV) and conjugated dienes as percentage dienoic acid were determined at different times for 2 weeks.

Tables 1 1 , 12 and 13 show the PV (peroxide value), p-AV (p-anisidine value) and CD (Conjugated dienoic acid %) of the fish oil samples stabilized with the compound of formula (1 ) at low (0.5 mg/g) and high levels (2 mg/g).

Table 1 1 : Variation of PV [meq/kg] with compound of formula (1 ) in fish oil FG30TG

Table 12: Variation of p- AV (p-anisidine value) with compound of formula (1 ) in fish oil FG30TG

Table 13: Variation of CD (conjugated dienoic acid in %) with compound of formula (1 ) in fish oil FG30TG

Results and discussion All fish oil samples containing the compound of formula (1 ) had lower levels of PV than those of the samples which did not contain said compound or any other antioxidants indicating their antioxidant effect against the oxidation of PUFA in fish oil.

Although there was no considerable difference among the samples in the p- AV during storage, the samples containing high levels of MNT (2 mg/g) and the sample containing 0.5mg/g of compound 1 showed the highest level of p-AV after 15 days of storage (Table 12). All other samples containing the compound of formula (1 ) did not show clear difference in p-AV among them. There was no change in the values of conjugated dienes at all during the study period (Table 13).

Conclusion

The compound of formula (1 ) showed antioxidant activity in both fish and algal oil.

Claims

Use of at least one compound of formula (I) as antioxidant in oil,

whereby R¹, R² and R³ are independently from each other H or linear Ci-6-alkyl or branched C₃-8-alkyl.

2. The use according to claim 1 , whereby in compound of formula (I) R¹, R² and R³ are independently from each other H or linear Ci-₆-alkyl or branched C3-s-alkyl.

3. The use according to claim 1 and/or 2, whereby in compound of

formula (I) R¹, R² and R³ are independently from each other H or linear Ci-4-alkyl or branched C3-₄-alkyl.

4. The use according to claim 1 and/or 2, whereby in compound of

formula (I) R¹ is H or methyl or ethyl or n-propyl or /so-propyl or tert- butyl; and/or R² and R³ are independently from each other H or methyl or ethyl.

5. The use according to any one or more of claims 1 to 4, whereby in compound of formula (I) R¹ is methyl or tert- butyl and/or R² and R³ are independently from each other H or methyl.

6. The use according to any one or more of claims 1 to 5, whereby the compound of formula (I) is a compound of formula (I) with R¹ = R² = R³

= methyl or a compound of formula (I) with R¹ = tert- butyl and R² = R³ = H or a compound of formula (I) with R¹ = methyl and R² = R³ = H or a compound of formula (I) with R¹ = R³ = methyl and R² = H, or a compound of formula (I) with R¹ = R² = R³ = H or any mixture thereof.

7. The use according to any one or more of claims 1 to 5, whereby the compound of formula (I) is the compound of formula (1 ) (3,9-di-tert- butyl-12A7-6, 12-methanodibenzo[d,g][1 ,3]dioxocine-2,10-diol):

8. Oil containing polyunsaturated fatty acids (PUFAs) and/or their esters for human consumption comprising at least one compound of formula

(I),

wherein R¹, R² and R³ are independently from each other H or linear Ci-₆-alkyl or branched C_3-8-alkyl.

9. The oil containing polyunsaturated fatty acids (PUFAs) and/or their esters for human consumption according to claim 8, whereby the PUFA- containing oil is marine oil or microbial oil or fungal oil or algal oil or PUFA-containing plant oil, preferably whereby the PUFA-containing oil is marine oil or algal oil, more preferably whereby the PUFA-containing oil is algal oil.

10. The oil containing polyunsaturated fatty acids (PUFAs) and/or their esters for human consumption according to claim 8 and/or claim 9 additionally comprising esters of ascorbic acid with linear C_12-20 alkanols, preferably esters of ascorbic acid with linear C_14-18 alkanols, more preferably ascorbyl palmitate.

11 . The oil containing polyunsaturated fatty acids (PUFAs) and/or their esters for human consumption according to any one or more of claims 8 to 10 additionally comprising alpha-tocopherol and/or gamma- tocopherol.

12. Marine oil for human consumption comprising at least one compound of formula (I),

13. The marine oil according to claim 12 additionally comprising esters of ascorbic acid with linear C12-20 alkanols, preferably esters of ascorbic acid with linear C14-18 alkanols, more preferably ascorbyl palmitate.

14. The marine oil according to claim 12 and/or claim 13 additionally

comprising alpha-tocopherol and/or gamma-tocopherol.

15. Microbial oil for human consumption comprising at least one compound of formula (I),

16. The microbial oil according to claim 15 additionally comprising esters of ascorbic acid with linear C12-20 alkanols, preferably esters of ascorbic acid with linear C14-18 alkanols, more preferably ascorbyl palmitate.

17. The microbial oil according to claim 15 and/or claim 16 additionally comprising alpha-tocopherol and/or gamma-tocopherol.

18. Algal oil for human consumption comprising at least one compound of formula (I),

wherein R¹, R² and R³ are independently from each other H or linear Ci-₆-alkyl or branched C3-s-alkyl.

19. The algal oil according to claim 18 additionally comprising esters of ascorbic acid with linear C12-20 alkanols, preferably esters of ascorbic acid with linear C14-18 alkanols, more preferably ascorbyl palmitate.

20. The algal oil according to claim 18 and/or claim 19 additionally

comprising alpha-tocopherol and/or gamma-tocopherol.

21 . An edible oil comprising a compound of formula (I) and PUFAs and/or their esters,

22. The edible oil according to claim 21 , whereby the edible oil is marine oil or microbial oil or fungal oil or algal oil or PUFA-containing plant oil, preferably whereby the edible oil is marine oil or algal oil, more preferably whereby the edible oil is algal oil.

23. A method of preserving the shelf life of PUFAs and/or their esters in an edible oil comprising the step of adding at least one compound of formula (I) to said edible oil,

24. The method according to claim 23, whereby the compound of formula

(I) is added to said edible oil in an amount ranging from 10 to 500 ppm, preferably ranging from 30 to 300 ppm, more preferably ranging from 100 to 250 ppm, based on the total amount of the edible oil.

25. A method of limiting the amount of oxidation of PUFAs and/or their esters in an edible oil which is exposed to air, comprising adding at least one compound of formula (I) to said edible oil,

26. The method according to claim 25, whereby the compound of formula (I) is added to said edible oil in an amount ranging from 10 to 500 ppm, preferably ranging from 30 to 300 ppm, more preferably ranging from 100 to 250 ppm, based on the total amount of the edible oil.