U.S. patent application number 10/140283 was filed with the patent office on 2003-03-13 for astaxanthin over-producing strains of phaffia rhodozyma, methods for their cultivation and their use in animal feeds.
Invention is credited to Jacobson, Gunnard K., Jolly, Setsuko O., Sedmak, Joseph J., Skatrud, Thomas J., Wasileski, John M..
Application Number | 20030049241 10/140283 |
Document ID | / |
Family ID | 21961945 |
Filed Date | 2003-03-13 |
United States Patent
Application |
20030049241 |
Kind Code |
A1 |
Jacobson, Gunnard K. ; et
al. |
March 13, 2003 |
Astaxanthin over-producing strains of phaffia rhodozyma, methods
for their cultivation and their use in animal feeds
Abstract
Phaffia rhodozyma strains are described which produce greater
than 3,000 ppm astaxanthin based on dry yeast solids when
cultivated in a volume of nutrient medium of at least about 1,500
liters and containing in excess of 4 percent, preferably in excess
of 6 percent, dry yeast solids. These and other strains are
cultivated by an improved fermentation method comprising extending
the maturation phase of the fermentation by one or more various
techniques including exposing the yeast cells to a low-intensity
light, slow feeding the cells with a rapidly metabolized energy
source, e.g. glucose, and replacing the rapidly metabolized energy
source with a slowly metabolized energy source, e.g. gylcerol. The
cells of these strains are incorporated into animal feeds,
particularly feeds for salmonid fishes, to impart or enhance the
red pigmentation of these animals and products made from these
animals.
Inventors: |
Jacobson, Gunnard K.; (Brown
Deer, WI) ; Jolly, Setsuko O.; (Glendale, WI)
; Sedmak, Joseph J.; (Brookfield, WI) ; Skatrud,
Thomas J.; (Menomonee Falls, WI) ; Wasileski, John
M.; (Brookfield, WI) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W., SUITE 600
WASHINGTON
DC
20005-3934
US
|
Family ID: |
21961945 |
Appl. No.: |
10/140283 |
Filed: |
May 8, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10140283 |
May 8, 2002 |
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09372991 |
Aug 12, 1999 |
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6413736 |
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09372991 |
Aug 12, 1999 |
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08967034 |
Nov 10, 1997 |
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6015684 |
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08967034 |
Nov 10, 1997 |
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08557714 |
Nov 13, 1995 |
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5922560 |
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08557714 |
Nov 13, 1995 |
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08049825 |
Apr 19, 1993 |
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5466599 |
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Current U.S.
Class: |
424/93.51 ;
435/41 |
Current CPC
Class: |
C12N 1/145 20210501;
C12R 2001/645 20210501; C12P 23/00 20130101; A23K 20/179 20160501;
C12N 13/00 20130101; A23L 5/44 20160801 |
Class at
Publication: |
424/93.51 ;
435/41 |
International
Class: |
A01N 063/04; A01N
063/00; C12P 001/00 |
Claims
What is claimed is:
1. A strain of Phaffia rhodozyma which produces at least about
3,000 ppm astaxanthin based on dry yeast solids when cultivated
under suitable conditions in a volume of nutrient medium of at
least about 1500 liters and containing in excess of 4 weight
percent yeast solids.
2. The strain of claim 1 in which the dry yeast solids are in
excess of 6 weight percent.
3. The strain of claim 2 in which the volume of nutrient medium is
at least about 30,000 liters.
4. The strain of claim 3 which produces at least about 4,000 ppm
astaxanthin based on dry yeast solids.
5. The strain of claim 1 selected from the group consisting of the
strains deposited as ATCC-74218, ATCC-74219, ATCC-74220 and
ATCC-74221, and its mutants and derivatives which have retained the
ability to produce astaxanthin at a level of at least about 3,000
ppm based on dry yeast solids when cultivated under suitable
conditions and in a volume of nutrient medium of at least about
1500 liters and containing in excess of 4 weight percent yeast
solids.
6. An improved fermentation method for the production of
astaxanthin from a strain of Phaffia rhodozyma, the method
comprising cultivating cells of the strain under suitable
conditions in a nutrient medium containing a rapidly metabolized
energy source that is maintained over at least part of the
cultivation at a level such that the strain experiences (i) a
growth phase during which its cells increase rapidly in number and
produce astaxanthin, and during which the energy source is fed to
the strain at a rate such that it does not accumulate in the medium
in excess of a predetermined level, followed by (ii) a maturation
phase during which the increase in cell numbers slows but the
astaxanthin production continues at a rate at least as great as the
rate during the growth phase, the improvement comprising extending
the maturation phase.
7. The method of claim 6 in which the maturation phase is extended
by exposing the strain to a light source.
8. The method of claim 7 in which the light source provides light
to the strain at less than about 100 watts per kiloliter of
fermentation broth.
9. The method of claim 8 in which the exposure is essentially
continuous from the start of the maturation phase until the
termination of the fermentation.
10. The method of claim 9 in which the light source provides light
to the strain at a wavelength between about 250 and 700
nanometers.
11. The method of claim 6 in which the maturation phase is extended
by reducing, at the close of the growth phase, the feed rate of the
rapidly metabolized energy source to the strain.
12. The method of claim 11 in which the feed rate of the energy
source is reduced to no more than about 50% of the rate at which
the energy source was fed to the strain at the close of the growth
phase.
13. The method of claim 12 in which the energy source is
glucose.
14. The method of claim 6 in which the maturation phase is extended
by replacing, at the start of the maturation phase, the rapidly
metabolized energy source with a slowly metabolized energy
source.
15. The method of claim 14 in which the rapidly metabolized energy
source is glucose, and the slowly metabolized energy source is
selected from the group consisting of glycerol; polymerized forms
of glucose, sucrose and other polysaccharides found in molasses and
corn syrup; monohydric alcohols; and carboxylic acids.
16. The method of claim 15 in which the slowly metabolized energy
source is fed to the strain with the rapidly metabolized energy
source during the growth phase of the fermentation.
17. The method of claim 15 in which the slowly metabolized energy
source is fed to the strain after the cessation of the rapidly
metabolized energy source feed.
18. Astaxanthin made by the method of claim 6.
19. Astaxanthin made by the method of claim 7.
20. Astaxanthin made by the method of claim 11.
21. Astaxanthin made by the method of claim 14.
22. A Phaffia rhodozyma yeast cream made from a strain of claim
1.
23. A dried product made from the yeast cream of claim 22.
24. The dried product of claim 23 comprising an edible antioxidant,
an edible oil, and edible emulsifier.
25. The dried product of claim 24 in which the concentration, based
on the weight of the dried product, of the antioxidant is between
about 0.02 and about 2 weight percent, the oil between about 0.1
and about 10 weight percent, and the emulsifier between about 0.1
and about 10 weight percent.
26. The dried product of claim 25 in which the antioxidant is
selected from the group consisting of ethoxyquin, vitamin E,
ascorbyl palmitate, butylated hydroxyanisole, and butylated
hydroxytoluene.
27. The dried product of claim 26 in which the oil is selected from
the group consisting of safflower oil, corn oil, sunflower oil,
soybean oil and blends of two or more of these oils.
28. The dried product of claim 27 in which the emulsifier is
selected from the group consisting of soy lecithin and sorbitan
esters.
29. The dried product of claim 23 in which a substantial majority
of the yeast cells are unbroken.
30. The dried product of claim 29 prepared by spray drying or drum
drying the yeast cream.
31. The dried product of claim 23 in which a substantial majority
of the yeast cells are broken.
32. The dried product of claim 31 in which the yeast cells are
milled.
33. An animal feed comprising the dried product of claim 23.
34. An animal feed comprising the dried product of claim 24.
35. The animal feed of claim 34 comprising between about 0.1 and
about 10 weight percent of the dried product, based on the weight
of the animal feed.
36. The animal feed of claim 34 comprising between about 1 and
about 3 weight percent of the dried product, based on the weight of
the animal feed.
37. The animal feed of claim 35 formulated for an animal selected
from the group consisting of salmonid fishes, crustaceans and sea
bream fishes.
38. A method of feeding animals, the method comprising providing
the animals with the feed of claim 23.
39. The method of claim 38 in which the animals are selected from
the group consisting of salmonid fishes, crustaceans and sea bream
fishes.
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to astaxanthin. In one aspect, the
invention relates to astaxanthin produced by yeast cells while in
another aspect, the invention relates to methods of producing and
cultivating mutant strains of Phaffia rhodozyma yeast cells that
produce astaxanthin in excess of the typical Phaffia rhodozyma
yeast cell found in nature. In yet another aspect, the invention
relates to using products made from these yeast cells as a dietary
supplement in various animal feeds.
[0002] A distinct red color is of prime importance to customer
acceptance of certain food products, particularly aquatic food
animals such as salmon, sea bream, trout, shrimp, lobster and many
other marine animals. The oxygenated carotenoid astaxanthin
(3,3'-dihydroxy-6,6-carotene-4,4'-d- ione) is responsible for the
red color of these aquatic animals. In addition to being
responsible for the characteristic color of these animals,
astaxanthin plays a critical nutritional role in the life of these
marine animals (Torrissen, 1989. Proc. Third Int. Symp. on Feeding
and Nutr. in Fish, Toba August 28-September 1, Japan, pp. 387-399,
Meyers and Chen, 1982. World Aquaculture Society, Special
Publication No. 3, pp. 153-165). These references are incorporated
herein by reference. This carotenoid is also useful for adding
pigmentation to the flesh and products of other animals, and to
other foodstuffs, e.g. poultry and eggs, various dairy products,
snack foods, and the like.
[0003] Astaxanthin is the most abundant carotenoid present in the
aquatic world. Aquatic animals, like terrestrial animals, generally
cannot synthesize astaxanthin or any other carotenoid, although
many of these animals accumulate caroteniod compounds that are
present in their diets. Some of these animals, such as crustaceans,
can interconvert some carotenes to oxygenated forms of carotenoids
(called xanthophylls) of which astaxanthin is the predominant
compound formed. However, salmonid fishes and red sea bream
accumulate dietary astaxanthin even though these fish cannot
convert any other carotenoid compound to astaxanthin. Therefore,
the astaxanthin present in salmonid and sea bream fish, and in
products produced from these fish, must be derived directly from
dietary sources.
[0004] Plants, including marine microalgae and special yeasts such
as Phaffia rhodozyma, are the primary source of carotenoid
compounds in the world. As noted above, carotenoids are not
biosynthesized de novo by animals. However, animals in general
require certain carotenoids from which they benefit directly or
indirectly, and these carotenoids are obtained from dietary
sources. Examples of substances essential to most animals that are
derived from certain carotenoids are vitamin A and rhodopsin. In
the marine world, animals that are low on the food chain, such as
crustaceans, eat microalgae and other carotenoid containing
organisms from the plant world, and convert the carotenoid
compounds present in large part to astaxanthin by natural metabolic
processes. The astaxanthin is then stored in the body of these
astaxanthin producing animals.
[0005] Wild grown salmonid fishes and red sea bream obtain their
astaxanthin from the crustaceans and other astaxanthin containing
organisms that make up an important part of their diet. In the case
of pen-grown salmonids and red sea bream, the feeds used to produce
these fish must be supplemented with astaxanthin in order to
provide a dietary source of this important natural constitutent of
these fishes. Currently, synthetic astaxanthin is added to feeds
prepared for production of salmonids and red sea bream in
aquaculture to provide a source of this carotenoid compound. In
some cases, synthetic canthaxanthin (an oxygenated carotenoid
compound that is very closely related to astaxanthin) is used in
place of astaxanthin in feeds for salmonids and red sea bream, but
this compound does not function as well in these fishes as the
naturally predominant astaxanthin.
[0006] Natural sources of dietary astaxanthin are in great demand
by the aquacultural industries. Natural sources of dietary
carotenoids that have been investigated for farmed fish include
krill, crawfish, crustacean processing by-products, algae and
higher plants. However, these natural sources tend to be too
expensive and of limited availability and reliability to be
commercially viable.
[0007] The red yeast, Phaffia rhodozyma, has received great
attention from industry as a natural source of astaxanthin since it
was isolated from tree sap, and the red color identified as
astaxanthin (Niller, Yoneyama and Soneda. 1976. Int. J. Syst.
Bacteriol. 26:286-291, Andrewes, Phaff and Starr. 1976. Phytochem.
15:1003-1007) Phaffia rhodozyma was first demonstrated to pigment
salmonid fishes in 1977 (Johnson, Conklin and Lewis. 1977. J. Fish.
Res. Board. Can. 34:2417-2421, Johnson, Villa and Lewis. 1980.
Aquaculture. 20:123-134). The potential advantages of Phaffia
rhodozyma as a source of carotenoid pigments for aquaculture are
that it is a natural product rich in essential nutrients (e.g.
protein, lipids and B-vitamins) and that it contains astaxanthin
(Johnson, Villa and Lewis. 1980. Aquaculture. 20:123-134). However,
natural isolates of Phaffia rhodozyma produce so little astaxanthin
(typically 100 to 300 parts per million (ppm)) that they are not
practical or economical pigment sources for aquaculture (Torrissen,
Hardy and Shearer. 1989. Reviews in Aquatic Science 1:209-225,
Johnson and An. 1991. CRC Crit. Rev. Biotech. 11:297-326). If
Phaffia strains are to be an economically feasible feed additive
for coloring aquatic animals, or any other potential foodstuff
(animal or otherwise), then astaxanthin over-producing strains must
be developed. Each of the references cited in this paragraph are
incorporated herein by reference.
[0008] Mutants of naturally occurring "wild-type" Phaffia have been
described in the literature, allegedly capable of generating higher
levels of astaxanthin than the wild-type yeasts (International
Publication No. WO 88/08025 International Application No.
PCT/DK88/00068); EPO Publication No. 0 438 182 A1 (EPO Application
No. 91900682.3); EPO Publication No. 0 454 024 A2 (EPO Application
No. 91106436.8); International Publication No. WO 91/02060
(International Application No. PCT/US90/00558); EPO Publication No.
0 474 347 A1 (EPO Application No. 91306489.5); and EPO Publication
No. 0 427 405 A1 (EPO Application No. 90311254.8), all of which are
incorporated herein by reference). These strains reportedly produce
higher levels of astaxanthin than the wild-type isolates under
specific conditions. However, these mutant strains produce higher
levels of astaxanthin only at relatively low biomass
concentrations. At relatively high biomass concentrations, these
mutant strains produce only low levels of astaxanthin which are not
high enough to be practical.
[0009] Thus far, none of the reported Phaffia strains are capable
of producing astaxanthin efficiently enough to compete economically
with synthetic astaxanthin. Commercially viable strains have to
produce astaxanthin at substantially higher levels than strains
reported in the literature. To develop an economically viable
astaxanthin production process, a strain should produce in excess
of 3,000 ppm, preferably in excess of 4,000 ppm, astaxanthin based
on dry yeast solids at more than 4 wt %, preferably more than 6 wt
%, dry yeast solids (dys) in a large volume of nutrient medium,
e.g. 1,500 liters (1) or more. As here used, "wt % dry yeast
solids" or simply "dry yeast solids" are washed solids determined
by the method described in Official Method of Analysis, A.O.A.C.
14th Edition (1984) Sections 10.215-10.225, which is incorporated
herein by reference.
[0010] Besides the need to develop a suitable strain of Phaffia
rhodozyma for commercial astaxanthin production, methods for
cultivating Phaffia rhodozyma also need to be developed which
maximize astaxanthin production in large fermentors. Only limited
literature sources are available dealing with the growth of Phaffia
and production processes of astaxanthin in large fermentors
(International Publication No. WO 88/08025 (International
Application No. PCT/DK88/00068), and EPO Publication No. 0 454 024
A2 (EPO Application No. 91106436.8) both of which are incorporated
herein by reference). These reported fermentation processes give
significantly lower yeast solids and astaxanthin levels than that
required for an economically viable commercial scale fermentation
process.
[0011] Not only do the various elements of producing astaxanthin
from yeast cells require improvement, but so do the elements of
astaxanthin absorption and deposition in animal flesh and animal
products. Carotenoid absorption and deposition in fish are affected
by various factors: genetics, size, age, sex, duration of pigment
feeding, environmental factors, and diet composition (Torrissen,
Hardy and Shearer: 1989: Reviews in Aquatic Science, 1:209-225,
which is incorporated herein by reference).
[0012] Various reports are available concerning the formulation of
Phaffia or carotenoid products (International Publication No. WO
88/08025 (International Application No. PCT/DK88/00068), EPO
Publication No. 0 474 347 A1 (EPO Application No. 91306489.5), EPO
Publication No. 0 454 024 A2 (EPO Application No. 91106436.8), and
Japanese Patent Applications 57-206342, 2238855 and 90 JP-285090
are exemplary, all of which are incorporated herein by reference).
In these reports, Phaffia products were formulated in order to
protect the pigments during drying as well as during subsequent
processing into fish feed. Various antioxidants and stabilizers
were added to Phaffia preparations for those purposes.
[0013] The body temperature of salmonid fishes is equal to the
temperature of the water in which they inhabit, e.g. generally 0 to
14 degrees Centigrade (C). This means that the body temperature of
these fishes can be, and occasionally is, lower than 10 C.
Astaxanthin in Phaffia is concentrated in oil droplets. Phaffia oil
contains about 13% palmitic acid (16:00) with a melting point of 64
C, and about 32% oleic acid (18:1n9) with a melting point of 16 C.
Because of these high melting point fatty acids, Phaffia oil
solidifies near 10 C. This makes it difficult for fishes to
incorporate Phaffia astaxanthin at water temperatures below 10 C at
which Phaffia oil is solidified.
SUMMARY OF THE INVENTION
[0014] In one embodiment of the present invention, we have
developed and isolated novel strains of Phaffia rhodozyma which
produce greater than 3,000 ppm, often more than 4,000 ppm,
astaxanthin based on dry yeast solids at more than 4 wt %, often
more than 6 wt %, dry yeast solids when grown under suitable
conditions and in a working volume of nutrient media of at least
about 1,500 l. These Phaffia strains can produce astaxanthin
economically on a commercial production scale for the aquacultural
and food industries. Certain of these Phaffia strains were
deposited with the American Type Culture Collection of Rockville,
Md., on Apr. 6, 1993 under numbers ATCC-74218 (UBV-AX1), ATCC-74219
(UBV-AX2), ATCC-74220 (UBV-AX3), and ATCC-74221 (UBV-AX4).
[0015] In another embodiment of this invention, we have discovered
methods for the cultivation of Phaffia rhodozyma cells which
produce high yeast solids and high levels of astaxanthin.
Generally, these methods include providing special fermentation
conditions that promote the growth of the organism in terms of an
increase in cell numbers during a growth phase, and in terms of an
enhanced and steady accumulation of astaxanthin in the cells during
a maturation phase. More specifically, these methods include
exposing the yeast cells during the growth and maturation phases of
fermentation to a source of light, and/or extending and stimulating
the astaxanthin formation during the maturation phase by
controlling the feed rate of fermentable sugar, e.g. glucose, or by
providing the yeast cells with energy sources that are slowly
metabolized, e.g. glycerol.
[0016] In another embodiment of this invention, we have discovered
that the Phaffia strains of this invention need not be disrupted to
make astaxanthin available to salmonids, sea bream, crustaceans,
and other animals. Astaxanthin in undisrupted cells is more stable
during drying, storage and feed preparation processes.
[0017] In yet another embodiment of this invention, we have
discovered that a novel formulation of Phaffia product increases
the astaxanthin deposition in fishes. This formulation is a blend
of undisrupted Phaffia cells, vegetable oil or a blend of vegetable
oils with a low solidification point, an emulsifier, and an
antioxidant.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 describes the genetic history of three of the Phaffia
mutant strains of this invention.
[0019] FIGS. 2-9 are photomicrographs showing the appearance of
various mutant strains.
[0020] FIG. 10 describes the weight gain of rainbow trout over the
course of a feeding trial.
[0021] FIG. 11 describes the pigmentation of rainbow trout over the
course of the same feeding trial as that of FIG. 10.
DETAILED DESCRIPTION OF THE INVENTION
[0022] Mutagenesis
[0023] Astaxanthin over-producing yeast strains can be obtained by
consecutive mutagenesis followed by suitable selection of mutant
strains which demonstrate superior astaxanthin production.
Preferably, the yeast cells are of the genus Phaffia, and more
preferably of the species Phaffia rhodozyma. Starting wild type
yeast cell strains include those on deposit with the various
culture collections throughout the world, e.g the ATCC and the
Centraalbureau voor Schimmelcultures (CBS). Typically it is
necessary to perform two or more consecutive rounds of mutagenesis
to obtain desirable mutant strains.
[0024] 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.
[0025] In those mutagenesis protocols employing a chemical mutagen,
typically the mutagen is introduced into a buffered volume of
cells, thoroughly mixed, and then the tubes are incubated at room
temperature for 1 to 12 hours (hr) with continuous gentle
agitation. After the incubation, the reaction mixture is plated
directly onto agar medium plates. The composition of the agar can
vary, but typically the composition includes glucose (or other
carbon source), yeast extracts (such as those available from
Universal Foods Corporation), agar, and a screening agent that
inhibits pigment formation and/or cell growth. The cells are
distributed evenly across the agar surface, usually by shaking with
sterile 5 millimeter (mm) glass beads. The plates are then inverted
and incubated at about 21 C for 6-10 days under illumination which
is preferably a continuous low-intensity light. Similar growing
techniques are employed for those mutagenesis protocols in which
the mutagen is a nonchemical.
[0026] Screening for High Astaxanthin-Producing Mutants
[0027] Initial Screening on Plates and in Shake Flasks
[0028] After the incubation period, those yeast colonies that
appear better pigmented to the human eye than the mutagenized
parent strain are collected and subcultured onto new agar plates
(often of the same composition as those used to grow the
mutagenized parent strain). After an additional incubation period
of 6-10 days under like conditions, the new isolates that continue
to be heavily pigmented are inoculated into flasks, e.g. 50 ml
flasks containing 10 ml of a blend of glucose and yeast extract.
The isolates are then typically incubated for another 6-10 days
under similar conditions to the previous incubations but this time
on a shaker operating at about 200 revolutions per minute (rpm).
After this incubation period, an aliquot (e.g. 0.2 ml) is taken and
the total carotenoid content and astaxanthin content determined.
Those strains exhibiting enhanced astaxanthin producing
capabilities are candidates for additional screening in a small
batch fermentation, and are typically stored by freezing in an
appropriate medium, e.g. 15 vol % glycerol at minus 80 C, until
required for the next mutagenesis or screening. This storage
technique can be used after any of the screening steps or for
simply maintaining production strains.
[0029] Screening in a Small Batch Fermentation
[0030] Following selection of appropriate astaxanthin producing
mutants at the 50 ml flask level, mutants are then usually screened
for performance in 2 l fermentors (e.g. Omni-Culture Fermentors,
Virtis Corp., Gardiner, NY) containing approximately 1.5 l of
nutrient medium. In a typical screening procedure at this level,
250 ml flasks containing 30 ml of a blend of glucose and yeast
extract are each inoculated with 100-250 .mu.l of mutant yeast
cells taken from a frozen stock of the strain of yeast cells that
is to be tested. These flasks are then shaken at 200 rpm at 20-22 C
under continuous light for 3 days. Aliquots of these cell cultures
(15-20 ml) are then used to inoculate into the fermentors.
[0031] The working volume in these 2 l fermentors is usually 1.5 l
of an enriched glucose yeast extract medium supplemented with
various vitamins and minerals in assimilable form. Typical of these
vitamins and minerals are ammonium sulfate, potassium phosphate,
magnesium sulfate, zinc sulfate, ferric ammonium sulfate, copper
sulfate, inositol, pyridoxine hydrochloride, thiamine, calcium
pantothenate, biotin, and the like. The combinations and
concentrations of these materials, including the glucose and yeast
extracts, can vary to convenience. If desired, an antifoam agent
and/or other additives can also be incorporated into or used with
the medium.
[0032] The fermentor with its medium is sterilized by autoclaving.
The pH of the media is usually maintained between 4.5 and 7, and
the temperature between 15-24 C. The medium is usually sparged with
filter sterilized air (e.g. 2 liters per minute (1/min)), and it is
continuously agitated (e.g. 700 rpm). Fermentations typically last
4-6 days, and they are sampled periodically for cell growth and
astaxanthin production. Strains showing appropriate levels of
growth and astaxanthin production are candidates for screening at
the fed-batch fermentation level.
[0033] Screening in a Fed-Batch Fermentation
[0034] Following identification of astaxanthin over-producing
mutants in small batch fermentations, mutants are further screened
for fast growth rate, high solids production and high astaxanthin
production levels in 14 l, 20 l, 250 l and 2,000 l fermentors.
Typically, strains are propagated in fermentors over a pH range of
about 4.5 to 7 controlled with aqueous ammonia, sodium hydroxide or
both at a temperature range of 15 to 24 C. Agitation and aeration
are adjusted as necessary to maintain desired growth rates. The
media with which the fermentors are charged are compositionally
similar to those used in the small fermentors, i.e. they contain
glucose (or other suitable carbon source), yeast extracts,
vitamins, minerals and other additives, all in assimilable form,
although the exact recipe may and probably will vary from that used
for the small fermentors. Antifoam agents and other processing aids
can be used as desired.
[0035] The culture can be conducted as a batch fermentation until a
desired cell density is obtained, or the glucose (or other suitable
carbon source) supply can be started immediately. This supply is
adjusted as necessary to obtain the best yeast growth rate and
astaxanthin production. Additional nitrogen can be supplied to the
fermentation in the form of ammonia or urea.
[0036] Cultivation of Phaffia rhodozyma
[0037] The Phaffia strains of this invention initially show rapid
growth which gradually slows as they approach a high cell
concentration, e.g. greater than 6 wt % biomass solids. In a
typical fermentation, the rate of astaxanthin formation is
initially slower than the growth rate of the Phaffia cells, but
ultimately (during the later phase of the fermentation) the rate of
astaxanthin formation exceeds the cell growth rate. We have termed
this later phase the "maturation phase". We have found that the
rate of astaxanthin production is highest during the early stages
of the maturation phase.
[0038] In order to obtain better astaxanthin-producing
fermentations, we have developed three approaches to sustain or
enhance the high rate of astaxanthin production that occurs during
the early stages of the maturation phase. During this enhanced
maturation phase, the astaxanthin concentration is increased over
the standard maturation phase by at least 2-fold, often 3-fold or
more. The first technique is to expose the yeast cells to a light
source, preferably a continuous, low-intensity light, during the
maturation phase, particularly from its inception to that point in
the phase at which the rate of astaxanthin production begins to
significantly decline.
[0039] An and Johnson (Antonie van Leeuwenhoek. 1991. 57: 191-203,
which is incorporated herein by reference) indicated that high
intensity light inhibits the growth and carotenoid production by
Phaffia. Evans, et al. (EPO Publication No. 0 427 405 A1, EPO
Application No. 90311254.8) indicate that Phaffia strains do better
in daylight than in darkness. Both studies used Phaffia grown on
agar plates. Like An and Johnson, we found that continuous
fluorescent light (regular lab lighting) reduced pigment yields of
Phaffia in shake flasks, and like Evans we found darkness to be
inhibitory of carotenoid development in some Phaffia strains.
However, we also found that a low-intensity, preferably continuous,
fluorescent light, i.e. less than about 100, preferably less than
about 25 watts per kiloliter fermentation broth, actually promotes
carotenoid production in some Phaffia strains grown in liquid
media. These strains include those on deposit with the American
Type Culture Collection bearing numbers ATCC-74218, ATCC-74219,
ATCC-74220 and ATCC-74221. We have also found that better
astaxanthin production is obtained when these strains are provided
with such a source of light during fed-batch fermentation. The
light is not limited to any particular wavelength, but light of 250
to 700 nm, particularly visible light, is preferred. In those
circumstances in which the fermentor is constructed of metal or
another opaque material, the light can be made available through
one or more glass (or similarly translucent material) ports while
the nutrient medium containing the yeast cells is continually
circulated past t h e port(s). Fermentation broth can also be
pumped in a return circuit through a light transparent tube and
returned to the fermentor. In both these cases a high-intensity
light, e.g. a light that delivers more than 15 w/kl can be used
since only a relatively small surface area is exposed to the
light.
[0040] Second, the yeast cells are provided with a controlled
amount of a rapidly, relative to a polysaccharide, metabolized
energy source, e.g. glucose or sucrose. During the growth phase,
the yeast cells are fed this energy source, typically glucose, as
rapidly as possible with the proviso that it does not accumulate in
the nutrient medium to any substantial extent. This rate will vary
with the yeast cell strain, the composition of the nutrient medium,
the fermentation protocol, and similar factors. The accumulation of
the energy source in the medium is generally a signal that the
growth phase is ending and the maturation phase is beginning.
[0041] After the desired yeast solids is achieved and prior to the
accumulation of the energy source, the feed rate of the energy
source is reduced to at least about 50%, preferably at least about
40%, more preferably at least about 30%, of the maximum feed rate
during the growth phase. During this period of reduced energy
source feed, the yeast cells continue to produce astaxanthin, but
the growth in the number of yeast cells, i.e. the growth in the
cell density, is restricted. This period is allowed to continue
until the cells no longer produce astaxanthin at an economically
desirable rate.
[0042] To maximize the accumulation of astaxanthin during the
maturation phase, the energy source feed rate can be reduced in a
step-wise or continuous manner. For example, with the onset of the
maturation phase, the energy source feed rate can be reduced by 50%
and after a certain period of time, typically 12 to 24 hours, it
can be reduced another 50%.
[0043] The third technique is to feed the yeast cells a slowly,
relative to glucose, metabolized energy source during the growth
and/or maturation phases. These energy sources or maturation
extenders include most materials that can be slowly metabolized by
a yeast cell. Glycerol; polymerized forms of glucose, sucrose, and
other polysaccharides typically found in molasses and certain corn
syrups; various alcohols such as methanol, ethanol, etc.; and
various organic acids such as succinic, glutamic, maleic; etc., are
exemplary of these extenders.
[0044] In one embodiment of this technique (and like the reduced
energy source feeding technique), these extenders are fed to the
yeast cells at the time energy source (e.g. glucose) accumulation
is detected in the nutrient medium, and they are fed in an amount
and in a manner such that the astaxanthin production of the cells
continues at a desirable rate. As this astaxanthin production rate
slows, the amount of extender fed to the cells is reduced
accordingly. The exact amounts and the rate of extender fed will
vary with the strain, fermentation protocol, and like
considerations.
[0045] In another embodiment of this technique, these slowly
metabolized extenders can be fed to the yeast cells during the
growth phase as part of or in addition to the energy source feed.
In this circumstance, the yeast cells will first deplete the
rapidly metabolized energy source, e.g. glucose, and as their rate
of consumption of this energy source slows, this signals that the
growth phase is ending and the maturation phase is beginning. At
this time, the addition of the energy source to the nutrient medium
is stopped and as the remaining rapidly metabolized energy source
in the medium is exhausted, the yeast cells will begin to
metabolize the remaining slowly metabolized energy sources. These
remaining slowly metabolized energy sources can be supplemented, as
desired, with additional materials as described above.
[0046] The use of maturation extenders, in either format, will
extend this period in a manner similar to the other two extension
techniques. In addition, these techniques can be used in any
combination with one another, and typically the continuous light
technique is used in combination with one of the feed
techniques.
[0047] The early stages of the maturation phase continues until the
astaxanthin production rate slows (as measured in the production of
astaxanthin per unit volume per unit time, e.g. mg/l/hr). This
usually occurs 2 to 4 days after the end of the growth phase. The
rate is, of course, a continuum and at some point after it begins
to decrease from the maximum, the early stages of this phase are
over and continuation of the maturation phase becomes economically
undesirable and it is terminated.
[0048] Yeast Cell Recovery and Product Preparation
[0049] Phaffia yeast is manufactured as a pure culture fermentation
product. Fermentation raw materials are selected from a number of
vendors based on availability and suitability for the process. The
major components for the growth of Phaffia rhodozyma include high
dextrose equivalent (DE) corn syrup (or equivalent source of
fermentable sugars), yeast extract, ammonia, inorganic phosphates,
and magnesium sulfate. In addition to these major components, a
number of micronutrients, such as trace metals and vitamins, are
required. All of these nutrients are commonly used in commercial
fermentation processes, including the manufacture of yeast for feed
and food. Each component is optimized to generate the maximum yield
of Phaffia yeast astaxanthin. Foaming during the fermentation is
controlled through the addition of food approved defoaming
agents.
[0050] The fermentation is conducted in pure culture fermentors
which have been cleaned and sterilized prior to use. All nutrients
added to the fermentors during the fermentation process are
sterilized before use.
[0051] The production of Phaffia yeast described in this invention
consists of a fed-batch fermentation. Other fermentation techniques
can be used if desired. The addition of nutrients during the
fermentation is optimized to achieve rapid production of both the
Phaffia yeast biomass and astaxanthin.
[0052] During the fermentation a number of conditions are
controlled and maintained. These include the acidity of the medium,
the fermentation temperature, and the degree of aeration.
[0053] The fermentation is usually terminated when the astaxanthin
production falls below a commercially desirable level, and it is
terminated typically by transferring the culture (i.e. fermentation
broth) from the fermentor by any conventional means to a
refrigerated storage vessel to await concentration by
centrifugation or filtration. Continuous centrifuges or filtration
units are used for the preliminary concentration of the
fermentation broth. The fermentation broth is passed through these
units, and the yeast concentrate (i.e. yeast cream) is transferred
to a refrigerated yeast cream storage tank.
[0054] An edible antioxidant, such as ethoxyquin, vitamin E,
butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), or
ascorbyl palmitate; an edible oil such as a vegetable oil, e.g.
corn oil, safflower oil, sunflower oil, soybean oil, etc.; and an
edible emulsifier, such as soy bean lecithin or sorbitan esters,
can be added to the yeast cream at this stage. As here used,
"edible" means a material that is approved for human or animal
consumption. This serves to protect the astaxanthin during
processing and shipment and also improves the pigment deposition in
fish flesh.
[0055] The oil, emulsifier and antioxidant can be added at any
convenient time during Phaffia yeast processing. In one embodiment,
the oil, or blend of oils, emulsifier(s), and one or more
antioxidants, are blended with one another, and then the blend is
mixed with the broth concentrate (yeast cream), dried, and packaged
for shipping. In another embodiment, the oil, emulsifier and
antioxidant are added to the yeast cream after it has been dried.
In yet another embodiment, one or two of the additives are blended
with the yeast cream prior to drying, while the remaining
additive(s) are blended with the yeast cream after it has been
dried. The oil or blend of oils comprises between about 0.1 and 10
wt %, the emulsifier(s) between about 0.1 and 10 wt %, and the
antioxidant between about 0.02 and 2 wt % based on the total weight
of the final dry product.
[0056] Prior to drying, the cream is standardized to a suitable
astaxanthin content usually 3,000, 4,000 or 5,000 milligrams per
kilogram of the product (mg/kg) through the addition of feed yeast
(Yeaco.TM.-20 produced by Universal Foods Corporation or
equivalent).
[0057] The standardized product is then dried by any suitable
means, e.g. spray drying, drum drying, etc., and eventually
packaged, typically in either 20 or 25 kg, heat-sealed, polylined
boxes. The packaging is designed to protect the product from light,
oxygen, and moisture.
[0058] In one embodiment of this invention, the yeast cream is
dried using conventional equipment and techniques, such that a
substantial number, preferably a substantial majority, of the cells
are not disrupted by the process, i.e. such that the cells retain
the bulk of their spherical shape. We have found that unbroken
yeast cells provide astaxanthin to consuming animals as, or nearly
as, efficiently as broken cells.
[0059] In another embodiment of this invention, the yeast cream is
dried in a manner that promotes disruption of the cells such that
most of the cells in the dried product are broken or fragmented,
i.e. the cells no longer retain their substantially spherical
shape. If such broken cells are desired and not provided by the
drying process, then the cells can be subjected to a milling step
or enzymatic treatment in which the cells are broken.
[0060] The following examples are illustrative of certain specific
embodiments of this invention. Unless indicated to the contrary,
all parts and percentages are by weight.
Specific Embodiments
[0061] Pigment Extraction and Astaxanthin Analysis
[0062] Astaxanthin content is determined as reported by Sedmak,
Weerasinghe and Jolly in Jolly. 1990. Biotechnol. Techniques
4:107-112, which is incorporated herein by reference.
[0063] Yeast cells are washed twice in 12.times.100 mm test tubes
with 4 ml of deionized water. After washing and decanting the
water, the test tubes containing the washed cell pellets are
inverted to drain the water. The insides of the tubes are then
wiped with tissue paper to remove most of the water from the tube.
To each tube is then added 0.5 ml of dimethyl sulfoxide (DMSO)
(Sigma Chemical Co., St. Louis, Mo.) preheated to 55 C. The tubes
are vortex agitated for 20-30 seconds (sec) and 0.1 ml of 0.02 M
sodium phosphate (pH 7.0) then is added to partition the
carotenoids into 1 ml of subsequently added organic solvent. For
compatibility with high performance liquid chromatography (HPLC)
analysis of astaxanthin, 1 ml of hexanes:ethyl acetate (50:50
volume basis, HPLC grade) is used. The tubes are then vortex
agitated for 30-40 sec to mix the aqueous and organic phases. The
phases are then separated by centrifugation for 3 min in a desk-top
clinical centrifuge. The organic phase is removed and the pigmented
carotenoid content of the organic phase determined from the
absorbency at 480 nm (A.sub.480). The total carotenoids are
calculated using the extinction coefficient 1 E 1 cm 1 % = 2 ,
150
[0064] (based on determinations using authentic synthetic
astaxanthin).
[0065] The astaxanthin content is then determined by HPLC. Samples
for HPLC analysis are diluted in hexanes:ethyl acetate (50:50
volume basis), and then made to 0.1% acetic acid with glacial
acetic acid and filtered prior to injection. The column is an
Alltech (Deerfield, Ill.) Nucleosil 100 silica 5 micron,
250.times.4.6 mm stainless steel column with an Alltech guard
column. The eluting solvent is hexanes:ethyl acetate (50:50 volume
basis) without glacial acetic acid. The eluant is monitored at 476
nm. The percent astaxanthin is then calculated from the area under
the curve at the position of a synthetic astaxanthin standard.
[0066] Astaxanthin concentration is then calculated as follows:
Total carotenoid in .mu.g/ml of fermentation
broth=(A.sub.480/2,150).times- .10.sup.4.times.dilution factor
Astaxanthin in .mu.g/ml of fermentation broth=(Total carotenoid in
.mu.g/ml of fermentation broth.times.% Astaxanthin determined by
HPLC).div.100
Astaxanthin in .mu.g/g of yeast=Astaxanthin in .mu.g/ml of
fermentation broth.div.g Dry yeast solids/ml
EXAMPLE 1
Development of Mutant Strains
[0067] Phaffia rhodozyma mutants ATCC-74218 (UBV-AX1), ATCC-74219
(UBV-AX2), ATCC-74220 (UBV-AX3) and ATCC-74221 (UBV-AX4) were
developed from wild type strains by repeated mutagenesis as
described in the section entitled Mutagenesis. FIG. 1 illustrates
the genetic history of UBX-AX2, UBX-AX3 and UBV-AX4. UBV-AX1 was
isolated after seven mutagenizations.
EXAMPLE 2
Cultivation of Novel Strains
[0068] The data in Tables 1, 2 and 3 demonstrate that the Phaffia
strains of this invention grow to high levels of cell solids and
produce high levels of astaxanthin in working volumes of various
sizes. In some of the literature, astaxanthin is not distinguished
from total carotenoids, and thus the actual level of astaxanthin is
considerably lower than that reported. The astaxanthin values in
the Tables below are based on HPLC quantifications of
astaxanthin.
[0069] 14 l Fermentation
[0070] About 30 g (dys basis) of actively growing culture in 1.5 l
of appropriate growth medium are inoculated into a 14 l
Virticulture fermentor (Virtis Corporation, Gardiner, N.Y.). A
typical formulation for the set broth is:
1 INGREDIENT AMOUNT (g) YEAST EXTRACT 75 AMMONIUM SULFATE 25
MONOBASIC POTASSIUM PHOSPHATE 15 MAGNESIUM SULFATE 5 INOSITOL 0.65
PYRIDOXINE 0.35 ZINC SULFATE 0.15 THIAMINE HYDROCHLORIDE 0.1
CALCIUM PANTOTHENATE 0.08 FERRIC AMMONIUM SULFATE 0.05 CUPRIC
SULFATE 0.01 BIOTIN 0.0005 TAP WATER 5 l ANTIFOAM 5 ml pH 5.5
[0071] Start-up conditions are 8 standard liters per minute (SLPM)
aeration and 500 rpm agitation. The pH is controlled at 5.5 with
750 ml 1:3 dilution of reagent grade ammonium hydroxide,
NH.sub.4OH. Food grade antifoam is added as required. The culture
is fed 1750 g glucose (Cerelose.TM., CPC International,
Summit-Argo, Ill.) as a 50% by weight solution at a rate such that
the glucose concentration is less than 5 grams per liter (g/l),
preferably between 0.1 and 2.5 g/l throughout the fermentation.
Dissolved oxygen is controlled by agitation and airflow to between
20% and 90% saturation. Results of such fermentations with UBV-AX3
and 4 are presented in Table 1:
2TABLE 1 Astaxanthin Production in 14 l Fermentor Time Solids
Astaxanthin Strain (hrs) (%) (.mu.g/g dys*) UBV-AX3 115 8.6 6660
UBV-AX4 115 7.7 4670 *dys: Dry Yeast Solids
[0072] 2000 l Fermentation
[0073] About 3.6 kg (dys basis) of actively growing culture in 120
l of appropriate growth media are inoculated into a 2000 l IF2000
New Brunswick Scientific fermentor (New Brunswick Scientific
Corporation, Edison, New Jersey) containing about 700 l of the
broth described below:
3 INGREDIENT AMOUNT YEAST EXTRACT 6 kg MONOBASIC POTASSIUM
PHOSPHATE 4 kg MAGNESIUM SULFATE 4 kg DEXTROSE 3 kg SODIUM CHLORIDE
700 g FERROUS SULFATE 55 g CALCIUM PANTOTHENATE 25 g ZINC SULFATE
15 g CITRIC ACID 5 g MANGANOUS SULFATE 3 g COPPER SULFATE 1 g
BIOTIN 400 mg COBALT SULFATE 300 mg THIAMINE HYDROCHLORIDE 200 mg
BORIC ACID 80 mg TAP WATER 700 l ANTIFOAM 200 ml pH 5.5
[0074] Start-up conditions are 700 SLPM aeration and 100 rpm
agitation. The pH is controlled at 5.5 with anhydrous ammonia. Food
grade antifoam is added as required. The culture is fed 800 kg of a
45% by weight solution of unrefined high dextrose corn syrup
(Cargill Incorporated, Eddyville, Iowa) at a rate such that glucose
does not accumulate in the fermentation broth to more than 5 g/l,
preferably to 0.1-2.5 g/l. Dissolved oxygen is controlled by
agitation and airflow to between 20% and 70% saturation. The volume
of the broth upon termination of the fermentation is approximately
1,500 l. The results of such a fermentation with UBV-AX2 are
presented in Table 2.
4TABLE 2 Astaxanthin Production in 2,000 l Fermentor Time Solids
Astaxanthin Strain (hrs) (%) (.mu.g/g dys*) UBV-AX2 136 10.9 5321
*Dry Yeast Solids
[0075] 40,000 l Fermentation
[0076] About 160 kg (dys basis) of actively growing culture in
4,000 l of appropriate growth media are inoculated into a 40,000 l
fermentor. A typical formulation for the set broth is:
5 INGREDIENT AMOUNT (kg) YEAST EXTRACT 81 MONOBASIC POTASSIUM
PHOSPHATE 68 MONOBASIC SODIUM PHOSPHATE 14 MAGNESIUM SULFATE 80
DEXTROSE 50 FERROUS SULFATE 0.650 CALCIUM PANTOTHENATE 0.470 ZINC
SULFATE 0.150 CITRIC ACID 0.060 MANGANOUS SULFATE 0.035 COPPER
SULFATE 0.035 SODIUM MOLYBDATE 0.020 BIOTIN 0.0035 COBALT SULFATE
0.0035 THIAMINE HYDROCHLORIDE 0.0018 BORIC ACID 0.0023 TAP WATER
13,500 ANTIFOAM 4.1 pH 5.5
[0077] Start-up conditions are 8,000 SLPM aeration and 40 rpm
agitation. The pH is controlled at 5.5 with anhydrous ammonia. Food
grade antifoam is added as required. The culture is fed 17,000 kg
of a 42% by weight solution of unrefined high dextrose corn syrup
(Cargill Incorporated, Eddyville, Iowa) at a rate such that glucose
does not accumulate in the fermentation broth to more than 5 g/l,
preferably to 0.1-2.5 g/l. Dissolved oxygen is controlled by
agitation and airflow between 20% and 70% saturation. Results of
such a fermentation with UBV-AX1 are presented in Table 3:
6TABLE 3 Astaxanthin Production in 40,000 l Fermentor Time Solids
Astaxanthin Strain (hrs) (%) (.mu.g/g dys*) UBV-AX1 157 7.8 5060
*Dry Yeast Solids
EXAMPLE 3
Strain Descriptions: Colony Morphology
[0078] Colony morphology of the novel strains were observed on 2-4
week old streak plates which contained 2% glucose, 0.5%
Tastone.TM.-154 (Universal Foods Co. Yeast Extract) and 1.5%
agar.
[0079] UBV-AX1: Well isolated colonies are 4-5 mm in diameter. Some
colony size heterogeneity exists within the culture. Smaller
colonies range in size from 1-3 mm. Some of these smaller colonies
can be darker in color than the rest of the colony population.
Colonies edges are entire; but some colonies will show irregular
edges. This becomes typical after 3-4 weeks. Colonies are convex to
slightly umbonate. Color is red orange, darker at the raised
center. Colony finish is matt. Where crowded some colonies appear
wrinkled, especially on older plates. After 3-4 weeks colony color
is dark red. Lighter colonies, yellow to orange, can be
occasionally seen in lawns of cells. These lawns can have a grainy
appearance.
[0080] UBV-AX2: Well isolated colonies are 4-5 mm in diameter. Some
colony size heterogeneity is present, with smaller colonies
occasionally seen. Colony edges are entire. Colonies are convex to
slightly umbonate and glossy in appearance. Color is red-orange,
darker at the raised centers, especially where crowded. Occasional
irregularities can be seen at some colony edges. After 3-4 weeks
colonies are still glossy and dark red. Lawns of cells remain
smooth and glossy. The lack of lighter colored colonies indicates
that the genes for pigmentation in this strain are very stable.
[0081] UBV-AX3: Colony morphology can be heterogeneous, especially
after 6 days growth in glucose/yeast extract broth. After 10 days
on glucose/yeast extract plates, the colonies range in size from
1.5 to 3 mm in diameter. Color ranges from red to orange. Smaller
colonies are typically darker red than the larger colonies.
[0082] UBV-AX4: After two weeks on glucose/yeast extract plates,
colonies are 2-3 mm in diameter. The colony edges are entire,
although a small number of colonies may show scalloped edges.
Colony color is a dark red-orange. The colonies are glossy, and
have a conical profile, especially where crowded. After 3 weeks,
the colony profile shows flattening at the base. The color of the
colony center is darker red than the perimeter. Colony edges remain
entire.
EXAMPLE 4
Strain Description: Microscopic Appearance
[0083] Microscopic appearance of cells were observed on the novel
strains grown in 2% glucose, 0.5% Tastone.TM.-154 for 6 days.
[0084] UBV-AX1 (FIGS. 2 and 3): Cells are single or in pairs with
occasional short chains. Cells are ellipsoid to oviform. Cells are
reddish-tan. Some cells are devoid of cytoplasm with lipid
droplets. Cells with granular, slightly pigmented forms are seen.
Some pleomorphic shapes are also seen. Occasional apiculate shaped
cells are present.
[0085] UBV-AX2 (FIGS. 4 and 5): Cells are single or in pairs. Cells
with two buds at the same end of a mother cell are common. Cells
are oblate ellipsoidal to globose. Many cells are obviously
pigmented reddish brown. Cells with granular appearing, pigmented
cytoplasm are fairly common. Occasional enlarged, deeply pigmented,
rounded "chlamydospore-like" structures are seen. The structures
lack the thickened cell walls of chlamydospores.
[0086] UBV-AX3 (FIGS. 6 and 7): Cells are single, in pairs, short
chains or small clumps of 3 to 6 cells. Pleomorphic forms are seen.
Many cells are obviously pigmented. The cytoplasm in these cells
appear granular.
[0087] UBV-AX4 (FIGS. 8 and 9): Cells are single or in pairs or
short chains. Pleomorphic forms are seen. Some cells have
reddish-brown granular cytoplasm. Some cells appear to have
red-crystalline structures present. Lipid droplets are very obvious
in some cells.
EXAMPLE 5
Strain Description: Cell Sizes
[0088] Table 4 shows cell sizes of the novel strains which were
grown in 2 % glucose and 0.5% Tastone.TM.-154 for 6 days.
7TABLE 4 Cell Sizes of Certain Phaffia Strains Strain Cell Size
Range (.mu.m) UBV-AX1 4.2 .times. 6.3 to 8.4 .times. 12.6 UBV-AX2
4.2 .times. 5.3 to 9.5 .times. 10.5 UBV-AX3 4.2 .times. 6.3 to 9.4
.times. 11.5 UBV-AX4 4.2 .times. 5.2 to 9.4 .times. 10.4
EXAMPLE 6
Enhancement of Astaxanthin Production by Exposure to Low Intensity
Light
[0089] Table 5 reports that darkness is inhibitory to pigment
production in shake flasks. Phaffia was grown in shake flasks with
2% glucose and 0.5 t yeast extract at 200 rpm at 21 C for six
days.
8TABLE 5 Enhancement of Astaxanthin Production by Exposure to Low
Intensity Light Astaxanthin (.mu.g/ml culture broth) Strain
Continuous 10 ft-C Light Dark UBV-AX1 30.1 19.2 UBV-AX2 43.8 17.3
UBV-AX3 41.6 16.1
EXAMPLE 7
Hydrolyzed Corn Syrup
[0090] Hydrolysed corn syrup (DE=95) and a partly hydrolysed corn
syrup (DE=63) are mixed to produce a feed material which contains
glucose and significant levels of various higher sugars such as
maltose, maltotriose and higher polymerized sugars. These sugars or
polysaccharides are not metabolized until the glucose is depleted.
A 75:25 mixture of these two corn syrups produces a feed of about
78% glucose and 10% maltose (about DE=87).
[0091] The data in Table 6 shows that UBV-AX1 produces
significantly higher levels of astaxanthin in a 20 l fermentor with
a 75:25 mixture of a hydrolysed corn syrup (DE=95) and a partly
hydrolysed corn syrup (DE=63) than with a hydrolyzed corn syrup
(DE=95) alone.
9TABLE 6 Astaxanthin Production Levels with DE = 87 and DE = 95
Corn Syrup Feed Yeast Astaxanthin Corn Syrup Solids (%) (.mu.g/g
dys*) DE = 87 5.9 6,712 DE = 95 5.9 5,892 *Dry Yeast Solids
EXAMPLE 8
Glycerol Effect on Astaxanthin Production During Maturation
[0092] For most Phaffia strains astaxanthin synthesis continues
after growth has stopped. Astaxanthin is therefore considered by
some to be a secondary metabolite in Phaffia. We have found that
the addition of glycerol to Phaffia stimulates carotenoid synthesis
during the non-growth phase after the primary carbon source
(glucose) has been utilized. HPLC analysis indicates that if
glucose and glycerol are present together, the glucose is
preferentially utilized and the glycerol is used only after glucose
depletion. As seen in the following tables, inclusion of glycerol
along with glucose greatly enhances the astaxanthin yield of our
Phaffia strains. These strains grow very poorly if glycerol is the
only carbon source.
[0093] Table 7 shows that the astaxanthin production level of
Phaffia rhodozyma is greatly increased by glycerol in shake flasks
with 2% glucose and 0.5% yeast extract in continuous low intensity
light for six days.
10TABLE 7 Glycerol Effect on Astaxanthin Production in Shake Flasks
Astaxanthin (.mu.g/ml culture broth) Strain Without Glycerol With
1% Glycerol UBV-AX1 30.0 37.6 UBV-AX2 42.8 49.2 UBV-AX3 54.2 71.3
UBV-AX4 42.0 56.7
EXAMPLE 9
Slow Feeding During Maturation
[0094] The data of Table 8 shows the effect on maturation with and
without a continued feed of the energy source. Fermentations were
conducted in 20 l Chemap fermentors with UBV-AX1 and the standard
fermentation format substantially as described above. In Case 1,
the fermentation was ended as the growth rate of cells declined. In
Case 2, feeding of the energy source was terminated as cell growth
rate declined and the cells were allowed to mature for 72 hours. In
Case 3, the feed rate of the energy source was reduced to 25% of
the maximum feed rate as the yeast growth rate declined and the
cells were allowed to mature for 72 hours with slow feeding.
11TABLE 8 The Effect of Slow Feed During Maturation Phase on
Astaxanthin Production Levels Feed during Yeast Astaxanthin
(.mu.g/ml Case Maturation Solids % fermentation broth) 1 No
maturation 7.0 175 2 Maturation 5.9 362 without feed 3 Maturation
with 7.9 561 slow feed
EXAMPLE 10
Evaluation of the Novel Strains as a Source of Pigment for
Salmonids
[0095] This example demonstrates that the novel strains of Phaffia
rhodozyma pigment salmonids flesh effectively when supplied in feed
as an additive. This example also shows that the strains of this
invention do not require disruption to make astaxanthin available
to salmonids (Binkowsky, Sedmak and Jolly. 1993. Aquaculture
Magaine. March/April:54-59, which is incorporated herein by
reference).
[0096] The Phaffia strain was grown in a 2,000 l fermentor in a
manner substantially described as above. Cells were concentrated to
15-22% solids by centrifugation and dried. The feed was processed
by Zeigler Bros. Inc., Gardners, Pa. The novel Phaffia rhodozyma or
synthetic astaxanthin (5% Carophyll Pink, Hoffmann-LaRoche) were
incorporated into their #1/8 Trout Grower pelleted food.
[0097] Rainbow trout (Onchorhynchus mykiss, Shasta strain) were
obtained from a local commercial trout grower. After acclimation to
laboratory conditions and an experimental control diet, the trout
were moved into separate experimental tanks (28 fish per tank). The
experimental tanks held approximately 250 liters of water (76
centimeter (cm) diameter; 56 cm standpipe) and had an average flow
rate of 6.5 liters of water per minute.
[0098] Collectively, the procedures and diets employed in the
experiments were designed to result in near-optimal growth of trout
at 10 C, the approximate temperature at which most
commercially-produced trout are raised. By the end of the
experiment, the remaining trout were approximately market size.
[0099] At 4, 8 and 12 weeks, 6 fish per tank were randomly removed
to determine growth rates and pigment levels. The fish were stunned
by a blow to the head; total length measured; weight after blotting
dry determined; and then each fish was cut across the isthmus
severing the ventral aorta. The fish were allowed to bleed out in
cool water and then two skin-on fillets were taken from each fish.
With the information acquired from each sampling, tank biomass was
recalculated and food rations adjusted so that each tank received
1.5% of its estimated biomass in food per day.
[0100] The fillets were processed to determine carotenoid content
per wet weight of flesh. About 15 g of flesh pooled from various
portions of a fillet were homogenized and 0.2 g samples of
homogenized flesh extracted with 1 ml of acetone for 30 minutes at
room temperature to release the flesh carotenoids. The carotenoids
were then partitioned into hexanes:ethyl acetate (1:1 by volume)
and the total carotenoid and astaxanthin content were determined as
described above in the section entitled PIGMENT EXTRACTION AND
ASTAXANTHIN ANALYSIS.
[0101] Pellets of food were crushed with a mortar and pestle. An
accurately weighed portion (0.05 g) of the crushed pellets were
treated with dimethylsulfoxide (DMSO) for 30 minutes at room
temperature to extract the carotenoids. The DMSO extracted
carotenoids were then quantified as described above in the section
entitled PIGMENT EXTRACTION AND ASTAXANTHIN ANALYSIS. This
procedure was used for feed analysis throughout the experimental
trials.
[0102] The growth and pigmentation of rainbow trout that were fed
diets containing our novel Phaffia yeast (one whole cells, the
other milled or disrupted cells) were compared both to rainbow
trout fed an unpigmented diet and to rainbow trout fed a diet
containing synthetic astaxanthin.
[0103] As seen in FIG. 10, the weight gain of all four groups of
fish was comparable with fish growing from approximately 240 g to
380 g during the 84 day trial period. This study indicates that
whole or disrupted Phaffia yeast added to the diet at the above
level gives the same or better palatability and digestibility as
the control fish food. Gross anatomical examination of fish at 84
days indicated that there were no differences between the four
treatment groups.
[0104] The flesh carotenoid content of rainbow trout fed the diet
devoid of astaxanthin (Diet D) was the same low level at 0 and 84
days of feeding indicating that the base diet for these studies was
indeed devoid of any carotenoid that might be deposited in the fish
flesh. After 84 days of feeding, the flesh carotenoid content of
the Phaffia and synthetic astaxanthin fed fish were the same (FIG.
11). All four groups of fish colored at comparable rates during the
84 day trial period.
EXAMPLE 11
Evaluation of a Novel Formulation of Phaffia Product for Pigmenting
Salmonids
[0105] This example demonstrates that a formulation of Phaffia
cells blended with ethoxyguin, lecithin and safflower oil increases
astaxanthin deposition in salmonid fish flesh.
[0106] Phaffia was grown in a 2,000 l fermentor in a manner as
substantially described above. Cells were concentrated to 15-22%
solids by centrifugation and dried. Ethoxyquin, lecithin and
safflower oil were added to the yeast cream prior to drying. The
feed was processed by Zeigler Bros. Inc., Gardners, Pa. The treated
and untreated Phaffia were incorporated into their #1/8 Trout
Grower pelleted food.
[0107] Rainbow trout (Onchorhynchus mykiss, Shasta strain) were
obtained from a local commercial trout grower. After acclimation to
laboratory conditions and an experimental control diet, the trout
were moved into separate experimental tanks (14 fish per tank). The
experimental tanks held approximately 650 l of water (120 cm
diameter; 56 cm standpipe) and had an average flow rate of 11
liters of water per minute.
[0108] Collectively, the procedures and diets employed in the
experiments were designed to result in near-optimal growth of trout
at 10 to 12 C, the approximate temperature at which most
commercially-produced trout are raised. By the end of the
experiment, the trout had grown from an average 300 g to an average
650 g. Fifteen fish were randomly sampled to determine base line
weight, length and flesh color data before separating fish into
experimental tanks. At 63 days 10 fish per tank and at 93 days 14
fish per tank were randomly removed to determine growth rates and
pigment levels in the same manner as that described in Example 10.
Similarly, the procedures of Example 10 were used to process the
fillets (to determine carotenoid content per wet weight of flesh),
and the pellets of food.
[0109] The growth and pigmentation of rainbow trout fed diets
containing Phaffia yeast with and without lecithin and safflower
oil were compared. Diet A contained Phaffia treated with 2,000 ppm
ethoxyquin. Diet B contained Phaffia treated with 2,000 ppm
ethoxyquin and 2.5% soy lecithin. Diet C contained Phaffia treated
with 2,000 ppm ethoxyquin, 2.5% soy lecithin and 2.5% safflower
oil.
12TABLE 9 Pigmentation of Fish Flesh at 93 Days Astaxanthin in Feed
in Flesh Diet (ppm) (ppm/wet wt) A 49 2.01 .+-. 1.32 B 49 3.02 .+-.
1.87 C 48 4.50 .+-. 1.40
[0110] As seen from the data in Table 9, after 93 days of feeding,
fish fed the diet containing Phaffia treated with ethoxyquin, soy
lecithin and safflower oil were pigmented best.
[0111] Although the invention has been described in considerable
detail through the preceding examples, such detail is for the
purpose of illustration. Many variations and modifications can be
made by one skilled in the art without departing from the spirit
and scope of the invention as described in the appended claims.
* * * * *