Processes for extracting carotenoids and for preparing feed...

Details Processes for extracting carotenoids and for preparing feed... Processes for extracting carotenoids and for preparing feed...

2002-06-17

2004-11-16

Bhat, N. (Department: 1764)

Food or edible material: processes, compositions, and products

Processes

Extraction utilizing liquid as extracting medium

C426S431000, C426S478000, C426S250000, C426S253000, C426S540000

Reexamination Certificate

active

06818239

ABSTRACT:

This invention relates to an extraction process for extracting carotenoids from carotenoid sources such as biomasses, which may be naturally-occurring or may be cultivated for the purpose. Such carotenoids find commercial application as pigments, anti-oxidants and food supplements, both for human and animal consumption.
Salmonids reared in the wild acquire a pink coloration resulting from the ingestion of micro-crustacea containing the red pigment astaxanthin as a natural part of their regular diet. When salmonids are reared in captivity, this is astaxanthin is artificially added to the feed in order to produce the desired pink coloration of the salmonid flesh. The amount of astaxanthin required to achieve this coloration is typically about 60 mg per kilogram of fish feed. Such pigments are also used to increase the skin colour of farmed fish such as sea bream and in shrimp farming.
Both synthetic and natural sources of astaxanthin have been used for salmonid pigmentation. The main source currently used is synthetic due to the high cost of natural-source formulations. Natural-source formulations, have included the direct incorporation of astaxanthin-rich biomass such as the yeast
Pfaffia rhodozyma
, the alga
Haematococcus pluvalis
, and crustacean products directly in the salmonid feed. However, said direct incorporation has not proved cost effective vis-à-vis the synthetic product due to the inability of the fish's digestive system to effectively extract the astaxanthin from the biomass cells; i.e. the low bioavailability of this source.
Other commercially important carotenoids including lycopene and &bgr;-carotene are also currently more expensive when extracted from natural sources than their synthetic counterparts.
Given the economic importance of astaxanthin and other carotenoids, numerous methods of optimizing the production of carotenoid-rich biomasses and improving carotenoid-extraction therefrom have been attempted.
The known natural sources of astaxanthin include shellfish, krill, the yeast
P. rhodozyma
, the green algae
Haematococcus pluvialis
and certain bacterial strains. Natural sources of lycopene include tomatoes and tomato waste, and carrots are a natural source of &bgr;-carotene.
Due to the large volume of shrimp processing waste available this has seemed a promising source of astaxanthin. However the concentrations are low and the extraction methods attempted have proved expensive.
U.S. Pat. No. 3,906,112 describes the fine grinding of shrimp waste and the mixing and heating of the resulting powder with an oil in order to extract some of the astaxanthin into the oil.
U.S. Pat. No. 4,505,936 describes a similar process but where the chitinous shell is removed and the proteinaceous tissue is acidified before heating with oil.
More recent efforts have used supercritical CO
2
to perform the extraction, and U.S. Pat. No. 5,210,186 describes the use of boiling lye to form an alkaline extract.
The common disadvantage to all these methods is that, given the low concentration of astaxanthin present and the high cost of extraction, the astaxanthin extracted is not cost-effective relative to the synthetic alternatives.
Culturing and extracting products via the fermentation of bacteria is well known in industry, and thus considerable effort has been extended in the direction of strain development to this end. U.S. Pat. No. 5,607,839 describes a bacterium belonging to a newly discovered genus that can produce carotenoids including astaxanthin. Similar strain development efforts have also been performed for microalgal sources. Nevertheless, both these sources are still far from commercialization due to the low yields obtained.
Much commercial effort has been expended in recent years on the strain development of the astaxanthin-producing yeast,
P. rhodozyma
. This work has succeeded in producing high yields of astaxanthin by superior strains of
P. rhodozyma
. For example, U.S. Pat. No. 5,599,711 describes a strain of this yeast capable of producing astaxanthin in the thousands of ppm range. However, despite the high yields achieved, this yeast has not proved cost-effective due to the following reasons:
1. When
P. rhodozyma
is added directly to the feed, the bioavailability is low due to its hard cell-wall which hampers the extraction of the astaxanthin molecule by the digestive system of the fish within the digestive time cycle. Attempts to mill or otherwise rupture the cell wall to enhance this bioavailability have only marginally improved the results.
2. The tight binding of the astaxanthin within the cell is the reason behind the relatively expensive nature of the methods for chemically extracting this material from the
P. rhodozyma
. For example, U.S. Pat. No. 5,356,810 discloses the extraction of astaxanthin from dried, unruptured
P. rhodozyma
cells using glacial acetic acid.
There is also extensive literature concerning the extraction of carotenoids in general.
U.S. Pat. No. 5,830,738 describes the use of enzymes to decompose cellular walls so as to extract carotenoids trapped in plant cells.
U.S. Pat. No. 5,789,647 described the use of compressed gases such as butane and propane together with organic entraining agents in order to extract carotenoids from natural materials.
U.S. Pat. No. 5,773,075 describes the use of high-temperature and pressure to extract carotenoids from plant material. However, all these methods are expensive and are thus unsuitable for extracting carotenoids from biomasses in a commercial application.
In accordance with the invention described below, a carotenoid is extracted from a carotenoid source using a mixture of water, a water immiscible solvent and a water soluble co-solvent.
U.S. Pat. No. 4,439,629 discloses extracting &bgr;-carotene from algae by treating the algae at 50 to 100° C. with calcium hydroxide to saponify the chlorophyll present and then extracting the &bgr;-carotene with a solvent such as methylene chloride, hexane or high boiling petroleum ether.
U.S. Pat. No. 4,871,551 describes extracting astaxanthin from Haematococcus algae by extraction with solvents such as oils, aromatics (e.g. benzene), halogenated hydrocarbons (e.g. methylene chloride), or alkanes (e.g. hexane).
EP-A-612725 describes extraction of &bgr;-carotene from halophilic algae of the genus
Dunaliella
including
D. salina, D. parva, D. tertiolecta, D, primolecta
and
D. peircei
. An aqueous suspension of the biomass is emulsified with edible oil (soya bean oil, peanut oil, or sunflower seed oil) at elevated temperatures and the mixture is subjected to membrane ultra-filtration.
WO 98/03480 describes the extraction of &bgr;-carotene from algae (
Dunaliella
), vegetables (carrots), or fungi (Blakeslea) by extraction with a water immiscible solvent such as ethyl acetate, butyl acetate, hexane, or vegetable oil, followed by crystallisation and washing of the crystals with a poor solvent for &bgr;-carotene, such as ethanol or ethyl acetate.
WO 98/50574 describes extraction of a carotene present as crystals in a biomass by disrupting the cells of the wet biomass, possibly by using a solvent such as octanol, optionally adding a water immiscible solvent which may be oil, hexane or ethyl acetate, and removing debris, to leave solid carotene floating over liquid. The solid upper layer may be washed with water and then with a poor solvent for the carotenoid, such as methanol, ethanol, isopropanol or acetone to remove lipid.
Extraction of &bgr;-carotene from natural sources using as solvent one of acetone, methyl-ethyl ketone, methanol, ethanol, propan-2-ol, hexane, dichloromethane and super-critical carbon dioxide is described in U.S. Pat. No. 5,714,658, as is the use of a mixture of an acetic acid ester, such as ethyl acetate, and an edible oil. This however is a combination of two water immiscible solvents.
An extraction protocol for use in an assay for the content of astaxanthin in fish feed is described in “Analytical Methods for Vitamins and Carotenoids in Feed” Ed: P. Hofmann, H. E. Keller, J. Schierle and W. Schüep, Dept. of Vitamin Researc

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