Agriculture
`
`ourna 0
`Science of
`FOod and
`
`A JOURNAL OF INTERNATIONAL RESEARCH
`
`Published by
`the Society of
`Chemical Industry
`
`I
`'-
`
`14 Belgrave Square
`London SW1
`
`
`
`
`
`Contents
`
`Vol 22 No 2I-February 1971
`49 A preliminary study of the agronomic factors afiecting the
`yield of extractable leaf protein
`D. 8. Adam” and G. N. Festsnster’n
`57 Efiect of pH on the growth, nodulation and nitrogen fixation
`of Centrosema pubescens and Styi'osanthes gracir‘is
`C. T. i. Odu, A. A. Fayemi and J. A. Ogunwafe
`60 Evaluation of whisky distillery by-products. V. Energy
`value of malt distiller's grains assessed by a modified com-
`parative slaughter technique with lambs
`A. E. Reveron. J. H. Topps, T. B. Miiier and G. Pratt
`65 Use of L-cysteine in bread baking—results ofa multi-genera~
`tion feeding experiment with breeding rats
`D. L. Frape, J. Wilkinson. L. G. Chubb, A. M. Buchanan and
`J. B. M. Coppock
`69 The ‘turbidity' test as a measurement of thermal denatura-
`tion of proteins in wheat
`E. E. Mrs-Dermot!
`'[2 Varietal differences in water-soluble gliadin fractions of
`Triticum aestivum and of Tritr'cum durum seeds
`. M. anetii. T. Petrucci, F. Poochiari, V. Sfiano and R. Aven'a
`1'5 Softening of gluten by wheat proteases
`D. G. Redman
`79 lsoelectric fractionation and some properties of a protease
`from soyabean seeds
`N. Catsimpooias. S. K. Funk, J. Wang and J. Kenney
`33 Chemical composition of developing carob pods
`W. N. L. Davies. P. i. Orphanos and J. Papaconstantr’nou
`Changes in polyphenolics on ripening of selected pear
`varieties
`A. S. Ranadr've and N. F. Heard
`90 Nitrate and nitrite nitrogen in fresh, stored and processed
`table beets and spinach from difierent levels of field nitrogen
`fertilisation
`C. Y. Lee. R. S. Shaiienberger, D. 1.. Downing, G. S. Stoewsand
`and N. M. Peck
`93 Composition and distinctive volatile flavour characteristics
`of the essential oil from Australian-grown ginger (Zingr'ber
`oflicfnaie)
`D. W. Conneh‘ and R. .4. Jordan
`96 Antimicrobial and preservative activity of garlic on fresh
`ground came} meat.
`I. Effect of fresh ground garlic seg-
`ments
`Kn. S. Ai-Dei‘aimy and M. M. F. Barairat
`99 Method of analysis of astaxanthin and its occurrence in
`some marine products
`G. Lambertsen and O. R. Braekkan
`102 Variation in the concentrations of higher alcohols, methanol
`and ethyl acetate in brandies
`Seryi Askew and D. B. tisr'e
`
`2%
`
`CODEN: JSFAAE
`
`Abstracts 343—664
`
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`This material may be protected by Copyright law {Title 17 US. Code)
`
`Method of Analysis of Astaxanthin and
`its Occurrence in some Marine Products
`
`
`by G. Lambertsen and O. R. Braekkan
`Governmental Vitamin Laboratory, Norwegian fisheries Research instiiate, P.0. Box 187, 5000 Bergen, Norway
`
`(Manuscript received i5 June, 1970)
`
`A metitodfor the determination ofasroxantl‘iin infree ona'erterrfiea’forms it based on silica gel chromato~
`graphy ofa lipid extract to obtain fractions ofdiester, monoester and free astaxantl‘tin. Thefroctions
`are reduced with borohydride and tire tetrahydroxy iii—carotene is measured by ultra-violet spectrophoto—
`metry at 450 and 476 am, The method has been applied to dtficrem crustacean .mmpfes‘ and Pmdflcn,
`to fish oils and to organs from rainbow trout. Values down to 0'1 ugig sample could be measured.
`The fractions were identified by thin-iayer chromatography.
`
`Introduction
`
`the
`is
`ASTAXANTHIN, 3,3'-dihydroxy-4,4’-diketo-,3~carotene.
`most commonly occurring pigment
`in marine organisms.
`The pink to orange-pink colouring of many echinoderms and
`crustaceans, the skin of several fishes, and the meat of many
`salmonid fishes, consists partly or wholly of astaxanthin
`and its esters. The comparative occurrence of carotenoids.
`including astaxanthin, has been reviewed by Goodwin}
`Modern methods of isolation and characterisation, and new
`and revised knowledge of the different forms of carotenoids
`have been reviewed by Liaaen-Jensen & Jensen? The
`increase of fish farming,
`in particular of trout and salmon
`species, has led to an increased interest
`in the problems
`concerning pigments in fish feed, their uptake and possible
`in viva conversions.
`In Norway. pigmentation of the flesh of
`trout and salmon is usually achieved by feeding offal from the
`shrimp industry but methods of catching smaller planktomc
`crustaceans (euphausiids and copepods) as a feed for this
`purpose are also being investigated.3 The introduction of
`canthaxanthin as a synthetic carotenoid pigment should also
`beconsidered in an analytical procedure.“
`The absorption spectrum of astaxanthin in ultra-violet (u.v.)
`light is characterised by a broad, single peak with maximum
`at 480 nm.
`fi-Carotcnc has a well—defined spectrum with
`two sharp peaks at 450 and 476 nm but these absorption
`details are lost with the introduction of two conjugated
`carbonyls, as
`in astaxanthin. The proposed method is,
`therefore, based on a borohydride reduction of the astaxan~
`thin fractions followed by u.v. spectrophotometry. Astaxan-
`thin is alkali-labile, so that sapOnificatiOn and chromato—
`graphy on alumina must be avoided. The method involves
`dIreCt separation of the astaxanthin and its mono- and di-
`csters on columns of silica gel. The astaxanthin fractions
`and their reduction products have been identified by thin-
`Iill’er chromatography {t.l.c.).
`
`Extraction
`
`Experimental
`
`Fresh material was frozen and ground in a meat grinder, and
`"“3315 were ground in a small mill. The products were
`extracted with acetone followed by ethyl ether, until colourless.
`The extracts were evaporated in vacuum to dryness if little
`Water was present, or,
`if necessary, were re-extracted with
`Elihld ether and again evaporated. The lipid residues were
`5solved In hexane and stored in the freezer.
`
`Column chromatography
`rags-:5 g of lipids were separated on colomns of 20 X 200
`(Woel The columns'werc filled with a slurry of 20 g silica gel
`wait
`it],
`for partition chromatography‘, containing ~20%
`51‘} in hexane and kept free of air during chromatography.
`J' Sci- Pd Agric, 1971, Vol. 22, February
`
`The main triglyceride fraction was eluted with pure hexane.
`The astaxanthin fractions were eluted in hexane containing
`increasing concentrations of ethyl ether. The astaxanthin
`diester was eluted by 10—15% ether in hexane, the mono-
`esters by 20-30% and free astaxanthin by 50—60% ether.
`The columns had rather weak adsorption capacity, and in a
`few cases overloading occurred; re—chromatography of the
`three fractions was then carried out on new columns. The
`fractions were evaporated in vacuum, and the residues were
`taken up in a few ml of hexane.
`
`Thin-layer chromatography of the astaxanthin fractions
`The fractions from column chromatography were tested for
`purity and identity by t.].c. Pre—coated aluminium plates of
`silica gel (Merck, 20 .Y. 20 cm, 025 mm layer) were used
`straight from the box, and 10—50 pl of the fractions and of
`the original extract were applied to the plate. The elution
`solvent was 25% isopropyl acetate in benzene. Well-defined
`oblong spots were obtaincd with Rt values of 090-096 for
`the diesters, 0’75 080 for the monoesters and 0-35—0145
`for free astaxanthin. Known corresponding fractions from
`a Cotonou lipid. extract were usually run on each plate as
`standards. Canthaxanthin,
`if present, gave an Rf value
`between the mono— and dicsters of astaxanthin.
`
`Reduction of the fractions
`
`Aliquots of each fraction were transferred to ethanol and
`reduced with a few grains of potassium borohydride at room
`tempcraturc.
`It was necessary to complete the reduction in
`leSs than 15 min, to avoid saponification of the astaxanthin
`esters. A pure yeIIOw iii-carotene colour showed that the
`keto groups were
`reduced to hydroxy groups. ViSual
`observation was generally sufficient, as found subsequently by
`t.].c. Surplus borohydride was filtered off if necessary.
`
`Thin-layer chromatography of the reduced astaxanthin fractions
`if necessary for
`further
`identification, aliquots of the
`reduced fractions were transferred to hexane and spotted on
`pro—coated silica gel plates as described previously. An
`elution solvent of dioxanc—hexane (20 : 80 by vol.) gave the
`best separation of these fractions, with R; values of ~0'90
`for the dihydroxy di-fatty acid esters, 060—040 for the
`trihydroxy mono-fatty acid esters and 0* 10 for tetrahydroxy-
`fi-carotene. Dihydroxy-B-carotene (reduced canthaxanthin)
`gave an R: value of 085.
`
`Spectrophotometry of reduced astaxanthin
`Absorption curves of original and reduced astaxanthin
`fractions in ethanol, shown in Fig.
`l, were recorded on
`a Unicam SW00 spectrophotometer. Accurate measure
`ments of absorption values, at wavelengths 450 and 476 nm,
`
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`

`100
`
`Lumber-ism d’t Broekkan: Analysis and Occurrence of Astaxantnin in some Marine Products
`
`given by reduced fractions were taken on a Zeiss PMQII
`spectrophotometer, and were used for calculation.
`£113;
`values of pure flcarotcne were reduced by the molecular weight
`ratio
`between
`Jii-carotene
`and
`tetrahydroxy—B-carotene
`(0-8935), inverted and multiplied by 10I5 to give microgram
`per gram of sample. Pure B—carotene (Hoffman La Roche),
`0-2 mg in 100 ml ethanol, was used as a standard. The
`calculation factors were:
`
`106
`--
`_
`1%
`forEimtasonm) —m “ 448
`,
`10‘5
`t/,
`.— _____ -
`for Eicn1{476nrt1) _ 2140 x 0-8935 H 520
`The two calculated values were averaged to give astaxam
`thin (diester, mono-ester or free) in gigjig of sample.
`
`Results and Discussion
`
`In Fig. 1. the recorded absorption Curves for astaxanthin,
`reduced astaxanthin (tetrahydroxy—fi-carotene) and Jti-carotene
`(Hoffman La Roche)
`in
`ethanol, are compared. The
`same ,S—carotene was used for the determination of the basic
`absorption values. Astaxanthin has a broad absorption
`curve with maximum at 480 nm on which most spectrophoto-
`metric methods have been based.5 ‘7 Astaeene, tetraketoufi-
`carotene, formed from astaxanthin in an alkaline medium,
`has a similar absorption curve, and this has also been used for
`astaxanthin determinations.
`Reduction of ketOscarotenoids has been used in structural
`
`investigations? The reduction is usually carried out with
`sodium or potassium borohydride. Reduced astaxanthin
`has the same chromophore as ,B-carotene, and thus shows the
`same two peaks in the absorption curve (Fig.
`l). The ratio
`
`wavENUMeER, cm"
`
`2t;
`20
`30
`
`18
`
`0 -5
`
`D- it
`
`Lu0
`Z4:mK
`Otom
`4
`
`0-2 -—
`
`
`
`u _
`
`
`
`325
`
`I. 1? 5
`£00
`WAVELENGIH . nm
`
`5 5|]
`
`1 Absorption curves for astoxantht’n, retrahydroxy-fi—carotene
`FIG.
`and B—carotene in ethanol
`
`Tetrahydroxy-fiucarotcnc; — —- - - ficarotene; ‘‘‘‘‘ astaxanthm
`
`between the two peaks gives a measure of purity and identity;
`this absorption curve provides a better basis for a repro.
`duciblc speetrophotometric method than the curve of astaxam
`thin or astacene.
`In the analyses, the ratio of 51%
`l V
`i‘ffl'ltjfiénm):
`4516;1(450nm, was
`found to vary between the limits of
`0-83 and 086 for acceptable determinations. For higmy
`Oxidised products,
`i.e. shrimp meals,
`the ratio may f3"
`below this limit, giving erroneously high results.
`It is usually
`the non-esterified astaxanthin which is susceptible to oxidative
`damage. When the total quantity of astaxanthin falls
`below I ,u-gg’g sample, the extinction ratio is greater than 0‘ 85,
`indicating reduced precision at this low level.
`
`As the amount of pigments present may vary considerably
`both in quantity and form,
`it was necessary to identify all
`samples both before and after reduction. The chromato.
`graphic methods reported by Egger,8 which were applied to
`keto-carotenoids of plant materials, were modified and fennd
`to give the best results. Solvent systems,
`isopropyl acetate;
`benzene and dioxaneihexane, used with pre-coated silica
`gel thin-layer plates, were most useful for keto and hydroxy
`forms. respectively.
`Astaxanthin contents of different crustacean products are
`given in Table I. These species and products are recognised
`sources for the natural astaxanthin colouring of salmonid
`fishes. The use of shrimp-industry offal, consisting mainly
`of boiled prawns (Pandaitts bot-cans) minus the tail meat, and
`of planktonic species of euphausiids and copepods, has
`gained wide application in the feeding programme in fish
`farms. Results show that the content varies considerably
`from one type of product to another, the values being in the
`range 10—100 lttglt'g wet weight. Astaxanthin content in the
`fat is considerable; oils extracted from a planktonic copepod
`{Calories .finmart‘hicns), and from krill and prawns showed
`concentrations of 500—1100 nglg. Analysis of the distri-
`bution between esters and the free form showed extensive
`
`TABLE I
`Asmxanthin content of diflerent crustacean products
`
`Sample
`Astaxanthin,
`ugfg
`Dicster Monoestcr
`
`
`'X, Astaxanthin as
`
`Shrimp
`(Pasiphaea sp.)
`
`Krill (euphausiids)
`mixed sample: A
`B
`
`Cat'anns finmar-
`chit-us T
`
`Stomach content‘k
`of herring,
`mainly Cataract
`
`Lobster shells,
`boiled
`
`Prawn offal,
`botled
`
`i4- 9
`
`77-4
`22 ’4
`
`46 - 5
`
`90-8
`
`35 - 3
`
`66-0
`
`Prawn oil
`(Panda-ins borealfs)
`
`|095
`
`69
`
`48
`33
`
`3 T
`
`55
`
`34
`
`67
`
`79
`
`[7
`
`49
`36
`
`3 8
`
`26
`
`26
`
`28
`
`18
`
`Free
`
`i4
`
`3
`3|
`
`2 5
`
`l9
`
`40
`
`5
`
`3
`
`Krill oil
`(euphausiids)
`Cdiaries oil
`40
`33
`2?
`520
`(C'. firtmart‘in’cns)
`—.—.—._.—.—.—.—_.—i—FF
`
`51
`
`43
`
`6
`
`7'27
`
`”—4..-
`
`T3-s% Fat
`* Represents 4% of whole herring
`
`J. Sci. Fd Agric., 1971, Vol. 22, Februat'i'
`
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`

`Lambertsen & Braekkan: Anaiyst's and Occurrence of Astaxanrhin in some Marine Products
`
`l0l
`
`TABLE I]
`
`TABLE IV
`
`Astaxanthin content of fillet and roe of rainbow trout, and of cod roe
`
`Sample
`Commercial, adult fillet: A
`B
`
`Roe
`
`Fillet from feeding expt.
`(10 weeks)
`
`A. Shrimpmeal
`
`B. Shrimp offal, 2%
`
`C. Shrimp offal, 25%
`
`D. Herring with stomach
`filled with Catrina:
`
`E. Synthetic canthaxanthin
`added
`
`Cod roe, unripe
`
`Cod roe, ripe
`
`Astaxanthin,
`pgfg wet wt.
`0‘56
`019
`
`Form of
`astaxanthin
`Free
`Free
`
`0- 86
`
`Free
`
`0'0
`
`0- 12
`
`1-19
`
`015
`
`0‘38
`0 - 06
`
`S -56
`
`0-30
`
`——
`
`Free
`
`Free
`
`Free
`
`Canthaxanthin
`Free
`
`Diester, 56%
`Monoester, 21 %
`Free, 23 %
`
`Esters, 45%
`Free, 55%
`
`In Table IV analyses of fillet and roe from rainbow trout
`(Sail-no gait-(inert) and of cod roe are given. These samples
`were analysed prior to pigmentation studies at present
`in
`progress.
`In particular, it may be noted that only the samples
`of cod roe contained astaxanthin in esterified form, and that
`the unripe ovaries had eight times as much pigment (by wt.)
`as did the mature ovaries. This may be a result of the
`uptake of water during the final stages of maturation.9
`Samples from rainbow trout showed no evidence of esters of
`astaxanthin; this was confirmed by t.l.c. This corresponds
`to the early observation of Steven,10 and is contrary to the
`findings of Thommen & Gloor.11 The feeding experiments
`given in Table IV show that shrimp offal is a suitable source
`of pigment for the rainbow trout. Synthetic canthaxanthin
`also gave pigmentation, and the pigment was mainly de-
`posited in the flesh as shown by t.].c.
`
`Ackn0wledgment
`The authors are indebted to Miss. I. Marthinussen for
`technical assistance.
`
`References
`
`the
`
`‘The comparative biochemistry of
`Goodwin, T. W.,
`carotcnoids,’
`1952, (London: Chapman & Hall Ltd.)
`Liaaen-Jensen, S., & Jensen, A., Progr. Chem. Farr, 1965, 8, 133
`Wiborg, K. F., & Bjorke, H., Hikers Gang, 1966, p. 819
`Schmidt, P. J., at Baker, E. (1,1. ma. Res. Ed Cm, 1969, 26,
`351'
`Fox, D. L., 8:. Hopkins, F. 5., Comp. Brat-hem. Physiol., 1966,
`19, 261'
`Lee, W. L., Comp. Biochem. Physiol., 1966, 19, 13
`Hsu, W-J.._ Chichester, C. 0., & Davies, B. H., Comp. Biochem.
`Physiol., 1970, 32, 69
`Egger, K., Playrochemisrry, 1965, 4, 609
`Braekkan, O. R., Fi'sicDir. Skr., Ser. Tekn. under-s, 1958, 3, (7)
`Steven, D. M., J. exp. Biol, I948, 25, 369
`Thommen, H ., & Gloor, U., Naturwissenschafren, 1965, 52,161
`
`
`
`7‘99.”r-‘P‘P‘P?!"
`
`RIMFROST EXHIBIT 1101
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`RIMFROST EXHIBIT 1101 page 0004
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`page 0004
`
`Astaxanthin content of some shrimp meals
`‘__________.————
`Sample
`Astaxanthin, ugfg
`% as diester
`...——-——-————
`stria! meals:
`A
`B
`C
`
`8-9
`3‘9
`0-0
`
`82-0
`82-8
`_
`
`IndIJ
`
`unkno
`
`wn, of foreign origin:
`A
`B
`C
`
`Vacuum dried
`.._—————
`
`24-5
`16-3
`12-8
`
`76 2
`
`34.9
`39.
`86-8
`
`Til-0
`
`TABLE III
`Astaxanthin content of different fish oils
`___.__i——I—————
`
`Astaxanthin
`
`ugig
`
`‘
`
`% Astaxanthin as
`
`Diester Monoester
`
`Free
`
` .
`
`Oli sample
`
`Ca clin:
`p
`
`A
`B
`C
`D
`
`Mackerel:
`A
`B
`
`Ocean perch
`Polar cod
`
`94-3
`39-5
`6-6
`5-7
`
`11 -3
`6-3
`
`0 - 8
`18-9
`
`59
`53
`(100)
`(100)
`
`36
`36
`
`(100)*
`52
`
`26
`33
`tr.
`tr.
`
`25
`28
`
`__
`28
`
`15
`14
`Ir.
`tr.
`
`39
`36
`
`_
`20
`
`11'. ; trace
`* Concn. too small for separation
`
`It appears that, in the
`differences, and no particular trend.
`live animal, the pigment is present as its fatty acid diester,
`and that active lipolytic enzymes cause hydrolysis to the
`monoester and to free astaxanthin after death of the animals.
`Thus, the prawn oil which was extracted from freshly caught
`samples, had 79% diester and only 18 and 3% monoester
`and free astaxanthin, respectively.
`It can be assumed that
`the free form is less stable towards oxidation than the esters
`and may therefore disappear more quickly.
`Some analyses of shrimp meals are given in Table II.
`Shrimp meal is a normal
`industrial product of the shrimp
`industry. The values show that the astaxanthin content is
`generally low or even absent. A vacuum-dried meal shows
`a much higher value, indicating that loss is due to the pro-
`cessing method. Apart from the diester, only small amounts
`of monoester were found, and free astaxanthin seemed com-
`Dletely absent. Even the vacuum-dried shrimp meal showed
`a concentration of astaxanthin of the order of non-dried
`Ofial (Table 1), indicating a very low stability of the pigment
`during drying
`'Results of analysis of some red-coloured fish oils are
`given in Table 111. All the species are known to feed on
`Plant-{tonic copepods.
`It may be noted that two of the capelin
`0|IS_had fairly high contents of astaxanthin, making them
`feaSible as feed supplements with regard to pigmentation.
`The skin, meat and liver of these species and of herring show
`little astaxanthin content, and the pigment of the oils is
`Certainly derived from the stomach content (Table I).
`
`J. Sci. Fd Agric., 1971, Vol. 22, February
`
`