Extraccion astaxantina

Technical report

Analysis

of Total Astaxanthin
Haematococcus

in algae meal prepared from
pluvialis.

(TR.1002.001)

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The method used at Aquasearch production facility in Kona, Hawaii, to determine the total astaxanthin content of Haematococcus pluvialis algae meal is described and discussed. This method is able to determine total astaxanthin in Haematococcus pluvialisalgae mea/ within a 5% error margin.

INTRODUCTION

Astaxanthin is a red carotenoid pigment (xanthophyll) that Haemafococcus pluvialis cells accumulate The method described herebelow is used on a routine basis at in response to stress conditions. Aquasearch production facility in Kona to control astaxanthin content in Aquaxan HD algae meal. It consists of two steps: an astaxanthin extractionphase in DMSO (dimethyl sulfoxide) and a measure of astaxanthin light absorption by spectrophotometry at 489 nm. Total astaxanthin measurements reported on Aquasearch’s certificates of analysis and guaranteed on product specifications are based on this method.
METHODOLOGY

Supplies and Equipment
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Precision waterbath Disposable plastic pipettes Glass cuvetteSpectrophotometer Production lab computer Vortex mixer Weight scale balance Aluminium boats 15 ml sample tubes Pipettor with DMSO 1.O ml pipette
For further details, confacf:

Aquasearch Inc.,
73-4460 Queen Kaahumanu Highway, Suite 110. Kailua-Kona. HI 96740, USA Tel: 606-326 9301, Fax: 606-326 9401

Technicai reDor/
Lab tape Sharpie marker Latex gloves Astaxanthin analysis data sheet Amber coloured jarsDisposable clear tubes with caps

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Precision Samples analysed from early May ‘98 through the first week in August ‘98 indicate an average Oherror of 2.94 (n = 65) using this method. Procedure A. Pre-labelling 1) Label 2 15ml tubes for each sample to be analysed (eg. 1A, 1B, 2A, 28, etc). 2) Label 2 aluminium boats for each sample to be analysed (eg. IA, 1B, 2A, 2B, etc). 3) Label 2amber coloured jars for each sample to be analysed (eg. 1A, 1B, 2A, 28, etc). B. Extraction

1) Turn on weight balance, place uncapped 15ml tube in a small cup and zero out the readout
(zero). Take sample and weigh 0.02 gr - 0.03 gr into 15ml tube. You can use a small plastic disposable pipette to make weighing easier. Record wt. on data sheet in “WEIGHT OF SAMPLE gr” column. Cap tube.
2)Repeat step # 1 for all samples.

3) Add 1ml DIH20. Add 9ml DMSO. Vortex well. Place in 70 “F waterbath for 30 minutes. 4) Remove from waterbath, wipe tubes dry with paper towel and centrifuge 3-5min. 5) Remove tubes from centrifuge, and pour supernatant into numbered amber jars. Supernatant should be poured with the highest angled portion of the pellet abreast of the amber jar. This is extractprocess #I. Record in a “EXTRACTIONS” column a checkmark for each extraction completed.
6)

Repeat step #5 for all tubes.

7) Add IOml DMSO to each tube & cap tube after DMSO has been added. Vortex well. Place in waterbath for 30 minutes and repeat this process until the 41hextraction is completed or unless advised that all of the pigment has been removed (sometimes as much as 5 extractions).8)

Amber coloured jars should be stored in refrigerator until you’re ready for the next extraction. IOml DMSO to each tube. These disposable tubes are the dilution tubes. Next remove lml of the sample from the amber jar. Add the Iml of your sample to the disposable clear tube containing 1Oml DMSO.

9) Prepare disposable clear plastic tubes by labelling each tube the same as the 15ml tubes.Add

10) Ensure that the sample you remove corresponds to the appropriate numbered dilution tube. Mix well by inverting 3 times. C. Spectrophotometric readings 1) Turn on spectrophotometer at least 15 min. before making measurements. 2) Ensure your ABS WAVELENGTH is 489nm (see Appendix A). Blank the spectrophotometer with glass cuvette filled with DMSO.

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@Aquasearch Inc., Revised:...