WO2019185941A1

WO2019185941A1 - Novel use of substituted chroman-6-ols with extended lipophilic side chains

Info

Publication number: WO2019185941A1
Application number: PCT/EP2019/058120
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 substituted chroman-6-ols with extended lipophilic side chains of formula (I) wherein one of the two substituents R¹ and R² is C_12-21-alkyl and the other of the two substituents R¹and R² is either hydrogen or C_1-5-alkyl or (CH₂)_n-OH with n being an integer from 1 to 5, and wherein A is CH(R³), and wherein R³, R⁴ and R⁶ are independently from each other H or C_1-4-alkyl, and wherein R⁵ is H or OH or C_1-4-alkyl or C_1-4-alkoxy; with the proviso that at least two of R⁴, R⁵ and R⁶ are not methyl, when R³ is H, and one of the substituents R¹ and R² is methyl and the other of the two substituents R¹ and R² is C_12-21-alkyl, as antioxidants 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 such substituted chroman-6-ols with extended lipophilic side chains 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 the edible oil.

Description

Novel use of substituted chroman-6-ols with extended lipophilic side chains

The present invention is directed to the use of a 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 one of the two substituents R¹ and R² is C_12-21 -alkyl and the other of the two substituents R¹ and R² is either hydrogen or Ci-₅-alkyl or (CH₂)_n-OH with n being an integer from 1 to 5, and

wherein A is CH(R³), and

wherein R³, R⁴ and R⁶ are independently from each other H or Ci-₄-alkyl, and wherein R⁵ is H or OH or Ci-₄-alkyl or Ci-₄-alkoxy; with the proviso that at least two of R⁴, R⁵ and R⁶ are not methyl, when R³ is H and one of the substituents R¹ and R² is methyl and the other of the two substituents R¹ and R² is C12-21 -alkyl.

With said proviso compounds of formula (I) with at least two of R⁴, R⁵ and R⁶ being methyl, R³ being H and one of the two substituents R¹ and R² being methyl and the other of the two substituents R¹ and R² being C_12-21 -alkyl are not encompassed by formula (I). Thus, if one of the two substituents R¹ and R² is methyl and the other of the two substituents R¹ and R² is 4,8,12- trimethyltridecyl, the compound is selected from alpha-tocopherol, beta- tocopherol and gamma-tocopherol, and thus, not encompassed by formula

(I ) ·

Compounds of formula (I) with at least two of R⁴, R⁵ and R⁶ being methyl, A being CH(R³), R³ being H and one of the two substituents R¹ and R² being methyl and the other of the two substituents R¹ and R² being C12-21 -alkyl may, however, still be present as additional antioxidants in the oils for human consumption containing polyunsaturated fatty acids and/or their esters of the present invention. Thus, if at least two of R⁴, R⁵ and R⁶ are methyl, R³ is H, and one of the two substituents R¹ and R² is methyl and the other of the two substituents R¹ and R² is 4,8,12-trimethyltridecyl, the compound is selected from alpha-tocopherol, beta-tocopherol and gamma- tocopherol, and these compounds may still be present as additional

antioxidants in the oils for human consumption containing polyunsaturated fatty acids and/or their esters of the present invention.

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 a 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 one of the two substituents R¹ and R² is C_12-21 -alkyl and the other of the two substituents R¹ and R² is either hydrogen or Ci-₅-alkyl or (CH₂)_n-OH with n being an integer from 1 to 5, and

wherein A is CH(R³), and

wherein R³, R⁴ and R⁶ are independently from each other H or Ci-₄-alkyl, and wherein R⁵ is H or OH or Ci-₄-alkyl or Ci-₄-alkoxy, with the proviso that at least two of R⁴, R⁵ and R⁶ are not methyl, when R³ is H, and one of the substituents R¹ and R² is methyl and the other of the two substituents R¹ and R² is C_12-21 -alkyl;

and with the preferences for the substituents R¹ to R⁶ as given below.

Compounds of formula (I)

“alkyl” and“alkoxy” in the context of the present invention encompass linear alkyl and branched alkyl, and linear alkoxy and branched alkoxy, respectively.

In a preferred embodiment of the present invention in compound of formula (I) one of the two substituents R¹ and R² is C_12-21 -alkyl, preferably one of the two substituents R¹ and R² is linear or branched Cis-₂o-alkyl, more preferably one of the two substituents R¹ and R² is linear or branched Ci₆-i₈-alkyl, most preferably one of the two substituents R¹ and R² is 4,8,12-trimethyltridecyl, and the other of the two substituents R¹ and R² is either hydrogen or C_1-5- alkyl, and R³, R⁴ and R⁶ are independently from each other H or Ci-₄-alkyl, and

R⁵ is H or OH or Ci-₄-alkyl or Ci-₄-alkoxy, with the proviso that at least two of R⁴, R⁵ and R⁶ are not methyl, when R³ is H, and one of the substituents R¹ and R² is methyl and the other of the two substituents R¹ and R² is C_12-21 - alkyl.

More preferably in compound of formula (I) one of the two substituents R¹ and R² is C_12-21 -alkyl, preferably one of the two substituents R¹ and R² is linear or branched Cis-₂o-alkyl, more preferably one of the two substituents R¹ and R² is linear or branched Ci6-i8-alkyl, most preferably one of the two substituents R¹ and R² is 4,8,1 2-trimethyltridecyl, and

the other of the two substituents R¹ and R² is either hydrogen or methyl or ethyl, and

R³ and R⁴ are independently from each other H or methyl or ethyl, and R⁶ is H or Ci-₄-alkyl, preferably R⁶ is H or methyl or ethyl, and

R⁵ is H or OH or methyl or ethyl or methoxy or ethoxy, with the proviso that at least two of R⁴, R⁵ and R⁶ are not methyl, when R³ is H, and one of the substituents R¹ and R² is methyl and the other of the two substituents R¹ and R² is C_12-21 -alkyl.

Even more preferably in compound of formula (I)

one of the two substituents R¹ and R² is C_12-21 -alkyl, preferably one of the two substituents R¹ and R² is linear or branched Cis-₂o-alkyl, more preferably one of the two substituents R¹ and R² is linear or branched Ci₆-i8-alkyl, most preferably one of the two substituents R¹ and R² is 4,8,1 2-trimethyltridecyl, and the other of the two substituents R¹ and R² is either hydrogen or methyl, and

R³ is H, and

R⁴ is H or methyl, and

R⁶ is H or Ci-₄-alkyl, preferably R⁶ is H or methyl or ethyl, and

R⁵ is H or OH or methyl or methoxy; with the proviso that at least two of R⁴, R⁵ and R⁶ are not methyl, when R³ is H, and one of the substituents R¹ and R² is methyl and the other of the two substituents R¹ and R² is C_12-21 -alkyl. Furthermore, preferably in compound of formula (I)

one of the two substituents R¹ and R² is C12-21 -alkyl, preferably one of the two substituents R¹ and R² is linear or branched Cis-2o-alkyl, more preferably one of the two substituents R¹ and R² is linear or branched Ci₆-i₈-alkyl, most preferably one of the two substituents R¹ and R² is 4,8, 12-trimethyltridecyl, and the other of the two substituents R¹ and R² is methyl, and

R³ is H, and R⁴ and R⁵ are independently from each other H or methyl, R⁶ is

H or Ci-₄-alkyl, preferably R⁶ is H or methyl or ethyl, and with the proviso that at least one of R⁴, R⁵ and R⁶ is H.

Furthermore, more preferably in compound of formula (I)

one of the two substituents R¹ and R² is C12-21 -alkyl, preferably one of the two substituents R¹ and R² is linear or branched Cis-₂o-alkyl, more preferably one of the two substituents R¹ and R² is linear or branched Ci₆-i₈-alkyl, most preferably one of the two substituents R¹ and R² is 4,8, 12-trimethyltridecyl, and the other of the two substituents R¹ and R² is methyl, and

R³ and R⁴ are H, and R⁵ is H or methyl, and R⁶ is H or Ci-₄-alkyl, preferably R⁶ is H or methyl or ethyl.

Most preferably in compound of formula (I) one of the two substituents R¹ and R² is C12-21 -alkyl, preferably one of the two substituents R¹ and R² is linear or branched Ci5-₂₀-alkyl, m

ore preferably one of the two substituents R¹ and R² is linear or branched Ci₆-i₈-alkyl, most preferably one of the two substituents R¹ and R² is 4,8, 12- trimethyltridecyl, and

the other of the two substituents R¹ and R² is methyl, and

R³ _, R⁴ and R⁵ are H, and R⁶ is H or Ci-₄-alkyl, preferably R⁶ is H or methyl, more preferably R⁶ is methyl.

Especially preferred are the following compounds of formulae (1 ) and (2), whereby all possible diastereomers and enantiomers are included, meaning that also all possible isomers having any configuration at the chiral centers (marked with asterisks ^*) are included,

as well as the enantiomer of formula (1A), (2/?,4’/?,8’/?)-delta-tocopherol ((/?)-2,8-dimethyl-2-((4/?,8/?)-4,8, 12-trimethyltridecyl)chroman-6-ol),

Most preferred is the compound of formula (1A). 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.

Instead of ascorbyl palmitate other esters of ascorbic acid such as the esters of ascorbic acid with linear C_12-20 alkanols, preferably the esters of ascorbic acid with linear Ci₄-is 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 C_12-20 alkanols, preferably esters of ascorbic acid with linear C_14-18 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.

1 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, Raphidophytes, 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 Clodosporium, genus Mucor, genus Fusarium, genus Aspergillus, genus Rhodotorula, genus Entomophthora, genus Echinosporongium, 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, multirudimentole, 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

R,R,R- delta-tocopherol (compound of formula (1A)) is commercially available, e.g. from Sigma-Aldrich, Product no. T2028, (+)-d-tocopherol, >90%.

Example 1 : Synthesis of the compound of formula (2) (2-methyl-2- (4.8.12-trimethyltridecyl)chroman-6-ol = toco I) (see Fig. 2)

A 200 ml. 4-necked flask equipped with magnetic stirrer, oil bath, thermometer and argon supply was charged with 1 ,4-hydroquinone (12.0 g, 109 mmol, 99.8%, 4.0 mol equiv.), isophytol (8.06 g, 25.8 mmol, 95.1%, 1 .0 mol equiv.), ethylene carbonate (50 ml.) and heptane (50 ml.) forming a 2- phase system. Then, para-toluenesulfonic acid monohydrate (0.10 g, 0.52 mmol, 2 mol%) was added and the mixture was heated to reflux. After 80 min, the reaction mixture was cooled to 50 °C and the phases were separated. The lower ethylene carbonate phase was extracted with heptane (25 mL). The combined organic phases were extracted with water (25 ml_), dried over sodium sulfate and concentrated in vacuo (40°C/50-20 mbar). The residue was purified by column chromatography, eluent gradient heptane/EtOAc 95:5 to 85:15 (w/w). The combined pure fractions were concentrated in vacuo (40°C/200-10 mbar) and dried under high vacuum at 40°C, furnishing tocol as light beige oil (6.81 g, 96% purity by quant. NMR, 16.8 mmol, 65% yield).

GC-HRMS: 99.2 area%. Calcd. for C₂₆H₄₄O₂ (M⁺) 388.3341 , found 388.3344. Ή NMR (300 MHz, CHLOROFORM-d) d 0.82-0.91 (m, 12 H), 1.00-1 .45 (m, 18 H), superimposed by 1 .26 (s, 3 H), 1 .46-1 .67 (m, 3 H), 1 .68-1 .88 (m, 2 H), 2.71 (t, J = 6.8 Hz, 2 H), 4.26 (br s, 1 H, OH), 6.52-6.63 (m, 2 H), 6.66 (d, J = 8.5 Hz, 1 H) ppm.

¹³C NMR (75 MHz, CHLOROFORM-d) d 19.58, 19.62, 19.63, 19.66, 19.73, 21.07, 22.26, 22.61 , 22.70, 24.03, 24.42, 24.78, 26.39, 27.93, 30.82, 30.85,

32.65, 32.67, 32.73, 35.39, 37.25, 37.36, 37.41 , 37.46, 37.55, 39.34, 39.76, 39.80, 76.10, 1 14.62, 115.57, 1 17.74, 121 .97, 147.59, 148.39 ppm.

Comparison example: reaction carried out in one solvent only

“eq.” = mol equivalent(s); h = hours; p-TsOH = para-toluenesulfonic acid. Example 2: Antioxidant activities of compound of formula (2) in fish oil and algal oil

Compound of formula (2) was tested in fish oil and algal oil. The blank oil, i.e. oil without any antioxidant, and oil containing“MNT” 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” consists of mixed natural tocopherols and is commercially available

e.g. as“Tocomix 70 IP” from AOM (Buenos Aires, Argentina). Tocomix 70 IP

comprises d-alpha-tocopherol, d-beta-tocopherols, d-gamma-tocopherols

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

Compound of formula (2) 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 possible synergistic effect with

ascorbyl palmitate (“AP”) and carnosic acid (“CA”) was also determined

using the OSI data. 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 (2) 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 -40 psi. All samples were run in duplicate. The

Protection Factors (PF) for compound of formula (2) in oil were calculated

in percentage as: 100% x (OSI of the sample with antioxidant - OSI of the sample without antioxidant)

OSI of the sample without antioxidant Storage stability

Two different concentrations of compound of formula (2) were used for the storage stability study. Compound of formula (2) and MNT were added, each individually, to 40 g of fish oil samples in 60 ml amber bottles at 0.5 mg/g and 2 mg/g levels, thoroughly mixed and stored at ambient temperature storage for 19 days. All sample bottles were stored open to air, away from light. Compound of formula (2) was soluble in oil. Peroxide values (PV) and p-anisidine values (p-AV) were determined at different times for 19 days. Results

OSI values of the fish oil samples containing compound of formula (2), in comparison to the same levels of MNT, are shown in Table 5 and 6.

Table 5: Oil Stability Indices (OSI) of FG30TG fish oil stabilized with compound of formula (2) (SD = standard deviation)

Table 6: Protection Factors (PF) of compound of formula (2) in fish oil (80°C)

Based on the Oil Stability Index data, the compound of formula (2) showed higher OSI values than MNT in fish oil. OSI values and Protection Factors (PF) of the algal oil samples containing compound of formula (2), in comparison to the same levels of MNT, are shown in Table 7 and 8. Table 7: Oxidative stability of crude algal oil stabilized with compound of formula (2) (SD = standard deviation)

Table 8: Protection Factors (PF) of compound of formula (2) in crude algal oil (80°C)

Based on the Oil Stability Index data, the compound of formula (2) showed also higher OSI values than MNT in algal oil.

Peroxide values of fish oil samples at low (0.5 mg/g) and high levels (2 mg/g) are shown in Tables 9 and 10 respectively.

Table 9: Peroxide values (PV, meq/kg) during storage at 25°C (0.5 mg/g level)

Table 10: Peroxide values (PV, meq/kg) during storage at 25°C (2 mg/g level)

With regard to primary oxidation products (hydroperoxides) generated in the fish oil samples containing compound of formula (2) or MNT, determined as peroxide values (PV), the samples did not show a considerable difference at both concentration levels used although the untreated oil produced higher peroxide values than those containing compound of formula (2) or MNT, most of the times.

The p-AV of the same samples at low (0.5 mg/g) and high levels (2 mg/g) are shown in Tables 1 1 and 12 respectively.

Table 1 1 : p-Anisidine value (p-AV) during storage at 25°C (0.5 mg/g level)

Table 12: p-Anisidine value (p-AV) during storage at 25°C (2 mg/g level)

Also, there was no considerable difference in the variation of p-anisidine values (p-AV) in all these samples except the sample which did not contain any antioxidants (“untreated sample”). The untreated sample had relatively higher p- AV values than all other samples showing that compound of formula (2) has antioxidant properties.

Claims

1 . Use of a compound of formula (I) as antioxidant in oil,

wherein A is CH(R³), and

wherein R³, R⁴ and R⁶ are independently from each other H or Ci_-4- alkyl, and

wherein R⁵ is H or OH or Ci-₄-alkyl or Ci-₄-alkoxy;

with the proviso that at least two of R⁴, R⁵ and R⁶ are not methyl, when R³ is H, and one of the substituents R¹ and R² is methyl and the other of the two substituents R¹ and R² is Ci_2-2i-alkyl.

2. The use according to claim 1 , whereby in compound of formula (I) one of the two substituents R¹ and R² is C_12-21 -alkyl and the other of the two substituents R¹ and R² is either hydrogen or Ci-₅-alkyl, and

wherein R³, R⁴ and R⁶ are independently from each other H or Ci-₄- alkyl, and

wherein R⁵ is H or OH or Ci-₄-alkyl or Ci-₄-alkoxy,

with the proviso that at least two of R⁴, R⁵ and R⁶ are not methyl, when

R³ is H, and one of the substituents R¹ and R² is methyl and the other of the two substituents R¹ and R² is C_12-21 -alkyl.

3. The use according to claim 1 or 2, whereby in compound of formula (I) one of the two substituents R¹ and R² is C_12-21 -alkyl and the other of the two substituents R¹ and R² is either hydrogen or methyl or ethyl, and wherein R³ and R⁴ are independently from each other H or methyl or ethyl, and R⁶ is H or Ci-₄-alkyl, preferably R⁶ is H or methyl or ethyl, wherein R⁵ is H or OH or methyl or ethyl or methoxy or ethoxy, with the proviso that at least two of R⁴, R⁵ and R⁶ are not methyl, when R³ is H, and one of the substituents R¹ and R² is methyl and the other of the two substituents R¹ and R² is C_12-21 -alkyl.

4. The use according to any one or more of claims 1 to 3, whereby in

compound of formula (I) one of the two substituents R¹ and R² is C_12-21 - alkyl and the other of the two substituents R¹ and R² is either hydrogen or methyl, and wherein

R³ is H, and wherein

R⁴ is H or methyl, and wherein

R⁶ is H or Ci-₄-alkyl, preferably R⁶ is H or methyl or ethyl, and wherein R⁵ is H or OH or methyl or methoxy;

with the proviso that at least two of R⁴, R⁵ and R⁶ are not methyl, when R³ is H and one of the substituents R¹ and R² is methyl and the other of the two substituents R¹ and R² is C_12-21 -alkyl.

5. The use according to any one or more of claims 1 to 4, whereby in

compound of formula (I) one of the two substituents R¹ and R² is C_12-21 - alkyl and the other of the two substituents R¹ and R² is methyl, and wherein R³ is H, and R⁴ and R⁵ are independently from each other H or methyl, R⁶ is H or Ci-₄-alkyl, preferably R⁶ is H or methyl or ethyl, with the proviso that at least one of R⁴, R⁵ and R⁶ is H.

6. The use according to any one or more of claims 1 to 5, whereby in

compound of formula (I) one of the two substituents R¹ and R² is C_12-21 - alkyl and the other of the two substituents R¹ and R² is methyl, and wherein R³ and R⁴ are H, and R⁵ is H or methyl, and R⁶ is H or Ci-₄-alkyl, preferably R⁶ is H or methyl or ethyl.

7. The use according to any one or more of claims 1 to 6, whereby in

compound of formula (I) one of the two substituents R¹ and R² is C12-21 - alkyl and the other of the two substituents R¹ and R² is methyl, and wherein R³ _, R⁴ and R⁵ are H, and R⁶ is H or Ci-₄-alkyl, preferably R⁶ is H or methyl, more preferably R⁶ is methyl.

8. The use according to claim 1 , whereby the compound of formula (I) is compound of formula (1 ) or compound of formula (2) or any mixture thereof.

9. Oil containing polyunsaturated fatty acids (PUFAs) and/or their esters for human consumption comprising a compound of formula (I)

wherein one of the two substituents R¹ and R² is C12-21 -alkyl and the other of the two substituents R¹ and R² is either hydrogen or Ci-5-alkyl or (CH₂)_n-OH with n being an integer from 1 to 5, and

wherein R³, R⁴ and R⁶ are independently from each other H or Ci-₄-alkyl, and wherein R⁵ is H or OH or Ci-₄-alkyl or Ci-₄-alkoxy, with the proviso that at least two of R⁴, R⁵ and R⁶ are not methyl, when R³ is H, and one of the substituents R¹ and R² is methyl and the other of the two substituents R¹ and R² is C12-21 -alkyl.

10. The oil containing polyunsaturated fatty acids (PUFAs) and/or their esters for human consumption according to claim 9, 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.

11 . The oil containing polyunsaturated fatty acids (PUFAs) and/or their esters for human consumption according to claim 9 and/or claim 10 additionally comprising esters of ascorbic acid with linear C12-20 alkanols, preferably esters of ascorbic acid with linear Ci_4-is alkanols, more preferably ascorbyl palmitate.

12. The oil containing polyunsaturated fatty acids (PUFAs) and/or their esters for human consumption according to any one or more of claims 9 to 1 1 additionally comprising alpha-tocopherol and/or gamma- tocopherol.

13. Marine oil for human consumption comprising a compound of formula

(I)

wherein A is CH(R³), and

wherein R³, R⁴ and R⁶ are independently from each other H or Ci-₄-alkyl, and wherein R⁵ is H or OH or Ci-₄-alkyl or Ci-₄-alkoxy; with the proviso that at least two of R⁴, R⁵ and R⁶ are not methyl, when R³ is H, and one of the substituents R¹ and R² is methyl and the other of the two substituents R¹ and R² is C12-21 -alkyl.

14. The marine oil according to claim 13 additionally comprising esters of ascorbic acid with linear C12-20 alkanols, preferably esters of ascorbic acid with linear Ci₄-ie alkanols, more preferably ascorbyl palmitate.

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

comprising alpha-tocopherol and/or gamma-tocopherol.

16. Microbial oil for human consumption comprising a compound of formula

(I)

wherein A is CH(R³), and

17. The microbial oil according to claim 16 additionally comprising esters of ascorbic acid with linear C12-20 alkanols, preferably esters of ascorbic acid with linear Ci₄-ie alkanols, more preferably ascorbyl palmitate.

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

19. Algal oil for human consumption comprising a compound of formula (I),

wherein A is CH(R³), and

20. The algal oil according to claim 19 additionally comprising esters of ascorbic acid with linear C12-20 alkanols, preferably esters of ascorbic acid with linear Ci₄-ie alkanols, more preferably ascorbyl palmitate.

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

comprising alpha-tocopherol and/or gamma-tocopherol.

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

wherein A is CH(R³), and

wherein R³, R⁴ and R⁶ are independently from each other H or Ci-₄-alkyl, and wherein R⁵ is H or OH or Ci-₄-alkyl or Ci-₄-alkoxy, with the proviso that at least two of R⁴, R⁵ and R⁶ are not methyl, when R³ is H and one of the substituents R¹ and R² is methyl and the other of the two substituents R¹ and R² is C12-21 -alkyl.

23. The edible oil according to claim 22, 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.

24. 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

wherein A is CH(R³), and

25. The method according to claim 24, wherein 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.

26. 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

wherein A is CH(R³), and

27. The method according to claim 26, wherein 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.