PROt`~FORTl~RFc~OVFRYOFc~ OlDP~ lTFROMAl~AE
The present invention r~lates broadly to the recovery of astaxanthin carQtenoid pigment frQm algae. ~lore particu-larly, the inventiorl relates to the recovery of astaxan-thin carotenoid from ~iaematococcu6 pluvial is .
S BA~K(:RnuND C)F TH~ NTION.
As known, astaxanthin is a red-viQlet crystalline carote-noid pi~ment f~und in natural origin especial ly in the shells Qf crustaceans, yeasts~ and accumulates in Haema-tococcus pluvialis algae.~t is largely used as a pigment-ation supplement compo6ition, generally for incorparation in feeds and particularly in animal feeds such as various types of fis~ ~ince the cell wall6 of this type of algae are rigid and flexible, and therefore not easily disinte-grated. the valuable pigment i8 available only after its recovery from the respective algae.
According to the PCT Pu~lication No. 89~06910, the me~hod involves a grinding of encysted Haematococcus after a prior drying, at cryogenic temperatures, such as liquid nitrogen usually follGvved by t~le addition of an antioxi-dant in order to inhi~it the degradation of the pigment.
The algae become impervious to many types of chemical and physical 6tresses al1d accordingly will with6tand conven-tional ~rinding ~ec~miques.
Anot~1er method 6uggested for proce66ing of 31aematococcus pluviaIis, applica~le mainly on a laboratory scale, involves a vigorous vorteYing with gla66 ball6.

- 2 - 2177752 Ano~her suggested method was to perform an enzymatic pre-treatment which has the purpose of weakening the algae walls, preferably followe~ by glass balls grinding. The disadvantaee of thi6 method i6 that it requires a 6ePara-S te washing and an additional drying step, to remove theenzyme residue from the final product. However,a complete recovery of astaxanthin pigment was not achieved in any of the abQve olentianed methods A recent French Patent Application No. 2~703,692 claims a 10 method for Qbtaining carQtenoid6 from HaematocQccu6 algae. The methvd involves freeze drYing the encysted IIaematQcoccus cells at a temperature below -50C fQllowed by the addition of sodium chloride and comminuting the ce~s, in Qrde~ to obtain particles with an average size 15 below about 10 Il. The carotenoids are f~rther obtained froM tile co~minuted E~aematococcus algae by extraction using known solvents, such as:methylene chloride, hexane, benzene, acetone, etc. .4lthough, this method migllt give posilive results on a small scale, it is doubtful w~ether 20 i t would be applicable on a large scale, due to the required stringent conditions. Furthermore, the use of organic solvents for this purpose seems to be quite unat t rac t ive .
I t is an object of t~le ~resent invention to provide a new 25 process for the recovery of astaxanthin carotenoid pig-ment From ~aematococcus pluvial i s algae. It i6 another .
object of the present invention to provide a new process fQr an efficient rupture of algae cell walls without using strlngent conditions. It is yet another object of t~le pr~sent invention, to provide a simple proces6 for an efficient rupture of algae cell walls, which can be easily implemented on an industrial scale.
BRl~F l~ESC~II'TION OF l~h INVENl'iON.
Thc invention relates to a process for the recov~ry of astaxanthin carotenoid pigment from Haematococcus pluvi-alis algae, which comprises the steps of: comminution of t~le algae in a liquid medium bY rupturing t~le cell walls algae containing said carotenoid and dispersing the resulted pigment f~om said algae, ~eing characteri~:ed by the fact t~at said comminution is carried out in a ~igh pressure apparatus consisting of two consecutive units:
a pressure vessel and an interaction chan7ber, whereby a slurry of said algae containing above 1% solids and generall~ bet~eerl 5~; to ~a% by weight, enters into said pressure vessel maintained at a pressure of at least 2,000 psi and preferably in the range o~ 3,0~Q to 65000 psi and is conveyed into said interaction cl~amber maintained at a pressure of above l0,00a psi, and prefe-rably in the range of 13000 to 18000 psi said interaction chamber containing at least one channel, through which the slurry is passed at an increased velocity to effect a tur~ulent jet causing a rupture of the wal~s of the a~gac ' cells and a substantially decrease in the particles si~e of t}le algae, which impinge the disintegrated particles within a low pressure zone, whereby the astaxanthin carotenoid pigment is released from the comminuted algae and is 6ubsequently recovered. This recovery may be carried out either by drying the slurry containing the pigment, or by extraction of the pigment with an organic solvent. In the latter case one may also conceive to disintegrate the algae walls in the presence of an organic solvent which will subsequently e2~tract tlle pigment. The particles si~e after the rupture of the algae cell walls decrease to about 20 to ~5 ,~L.
It was found that by using this process, more than 9s% of the algae cell walls are disintegrated in a very short period of time. AS a result,a high release of the pigment is achieved, as indicated by a very high extraction efficiency of the astaxanthin pi~ment, which is two to tl~ree times higher than the amount obtained by the known convent ional rnethods .
BRllE~ ~ESCRIPTlQN QF Til~ FIGIIUF`C
Figure I, represents a f lowsheet of the process for the recovery of astaxanthin carotenoid from llaema-tococcus pluvialis according to the present i nvent i on .
Figure 2, illustrates the percentage of the rupture as a 2t 77752 function of the concentration of tha algae in the slurry entering into the apparatus.
DETAII,ED DE~CRIPTION OF THE INVENTION.
According to the present invention~ the rupture of the 5 algae cell walls is based upon performing a turbulent jet on the 1 iquid mixture containing the algae in a high pressure app~ratus. The turbulent jet is produced along a common boundary as formed by the mixture. In order to achieve a better efficiency of breaking the algae walls, it is preferred to recycle at lea~t a substantial portion of tlle mi~ture through said chamber at the pres~ure prevailing in the respective unit at t~e corresponding velocity .
The mixture fl~wing through the channel~ develops a high velocity, being under a high pressure, linear shear cavi-tation and impact forces. In this manner, the brokcn particles of the cell walls became smaller and are dis-persed through the liquid medium. The solid ~articles of the biomass containing the pigment are separated out from 20 the dispersion followed by extraction of the E~igment.
Alternatively, the entire biomass may be dried.
The extent of the algae cell walls breakage and the release of the carotenoids can be evaluated according to tlle colour imparted to the liquid medium, or quantitati-25 vely determined by a microscopic or spectroscopic exami-nat ion .

- 6 - 2~77752 Accordlng to a most preferred embodiment, it is suggested to add an antioxidant reagent, such as butylated hydroxy-ani601e, in order to avoid any oxidation of the rigment.
The recovery of the pigment from the algae according to 5 the present invention was found to be mofe efficient than other known methods. In the following Table 1 the results of the pigment recovery from algae are 8iven, as compared wi th other known methods .

Table 1: ComParison of astaxanthin extraction.
he method used Free' mg astaxanthin~per dry weight of algae.

The presènt invent ion 19 Grinding in the presence of liquid nitrogen 17 Water extraction ~ heat 14 Enzymatic treatment 13 Vortexing with glass balls 9 ~ The astaxanthin released from the algae and quantitatively determined.

It may be concluded from the above Table that only the method using llquid nitrogen reaches an extent of release which is of the same order of magnitude as the process - 7 - 2l77752 .
according to tl~e present invention. However, the metllod UsinB liquid nitrogen shows the fo] lowing main disadvan-tages:
- it can not be casily applied on an industr~al scale, 5 - it requires a prior drying of the algae, an~
- it is much more eYpensive than the process according to the present invention.

DETAIIED ~RSCR~PTION OF T~R pl~T-~
As shown in the Figure 1, the algae biomass enter into the pressure vesse~ (Cell l). In this cell, the working pressure is maintained in the range of 2000 to 6000 psi and ca~lses a separation of the algae aggregates.According to a ~referred embodiment, the algae biomass is recyclcd in this cell at least three times, in order to increase the yield of pigment recovery.
The resulted slurry from Cell 1, is conveyed to the Interaction chamber ~Cell 2), where a rupture of the algae cell w~lls occ~lrs, resulting in a dispersion of thc pigment into the solution.
The working pressure in Cel~ 2 is between 10000 to 23000 psi and preferably between 13000 to 18000 psi. As in Cell 1, it is also most ~referable to recycle the resulted dispersion at least four times.

~ - 8 - 2 1 77752 As 6hown in Figure l, the astaxanthin pigment may be extracted (step 3A) from the dispersion followed by formulation (step 4A) and drYing (Step SA).
According to another embodiment, the ~io~ass slurry con-taining the pigment is directly conveyed to a formulation 5 step (4~, whereby an antioxidant is added in order to protect the oxidation and decay of the pigment.
According to another embodiment ~not shown in said Figure l), encapsulating agents, such as polysaccharides and gelatin are incorporated, thus recovering the desired 10 pigment in a particular form.
In the last step, the pigment is dried or crystallized according to the specific requirement, or its envisaged use .
Figure 2, illustrates in a graphic manner, the results 15 obtain~d in the Example presented be~ow. As can be observed, the percentage of the algae rupture is a funct ion of the concentrat ion of the algae in the slurry.
Thus, at a concentration of 8.5% w/w algae, the rupture percentage was 6~% after five passages compared with 94%
20 after a concentration of 2.5% ~Y/w also after the five passages . of course, the parameters of the metilod, wi 11 also depend and may varied according to the type of the algae used as well as on the pressure maintained at the Interaction chamber, as will be shown in Example 2.

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The invention will be Xereafter illustrated by the following Examples, being understood that these Examples are not limiting the scope of t~le invention, and are given only for a better understanding of the invention. A
5 person skilled in the art, after reading the present specification, will be in a position to insert slight modifications, without being outside the scope of the invention as covered by the appended Claims.

EXAMPLE 1 .

10 A slurry of red algae grown in an outside pond, havin~ a concentration of lO.8% (weight~volume), was utilized in this experiment.
The slurry was diluted to four dif~erent of solids concentrations: 2.5%, 5%, 7% and 8.5% (weight~volume).

15 Each portion (250 cc) was introduced into the pressure vessel (Cell 1) where a pressure of between 2500 to 3500 psi was maintained. In order to avoid oxidation, the process was carried out below an ambient temperature. The flow rate was maintained between 400 to 500 ml~min 20 The resulted solution was conveyed into the interaction chamber (Cell 2), where a ~ressure of between 13000 to 17000 psi was maintained.
The extent of algae breakaBe was determitled by a micro-sco}lic count of the integer algae in the measuring cell 25 (hematocryte).

~o 2 1 77752 The results obtained are summari~ed in the fol lowing Tal:, I e 2 .
. , TABI,E 2. Percentage of rup~ure as a functi~n of the concentration of the algae.
5 Concentrat ion % ruptured algae (% wei~ht/volume) A B C
2 . 5 19 83 94 8.5 23.5 58 68 A: one cyc 1 e .
B: two cycles.
C: five cycles.

5 T~le decrease in t~e extent of ruptllre at the concentra-tion of 8.5%, is explained bY tlle high density of the slurry, which caused clog~in6 o~ some channels.
~igure 2, illustrates graphically the ab~ve results.

EX~1PL~ 2.

20 An amount of 250 cc of a s~urry o!~ red algae, containing 36g/1 (on dry basis) was introduced into the cell 1, where a pressiJre in the range of 200l) ~o 4500 psi was maintained, and the slurry was recycl~d four times. Tlle temperature was maintained at about 250C, in order to avoid oxidation of the pigment, The resulted solution was .conveyed into the interaction chamber (cell 2), where a pressure of 11000 psi was main-5 tained.
The extent of algae breakage was determined by a micros-copic count of the integer algae of the solution in the measuring cell and was found to be 84%.
The same experiment was repeated, but the solution from lO the cell 1, was conveyed into the interaction chamber (cell 2), where a pressure of be~ween 13000 to 17000 psi was maintained.
The extent of algae breaking in this case, determined as ment ioned above was above 95%

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