DK168542B1 - Astaxanthine-producing Phaffia rhodozyma yeast cells, a process for preparing them, and their use - Google Patents

Description

in DK 168542 B3

The present invention relates to astaxanthin-producing Phaffia rhodozyma yeast strains, a method for producing astaxanthin-producing Phaffia rhodozyma cells or cell parts or astaxanthin. In addition, the invention relates to an animal feed containing astaxanthin-containing yeast cells or 5 yeast cell portions containing astaxanthin isolated therefrom, as well as to a method of feeding animals to the feed.

It is known that the red color of the flesh of anadromous fish such as salmon or sea trout is due to red pigments such as astaxanthin present in the food consumed by the fish. In natural environments, fish obtain their red color from seafood or other astaxanthin-containing organisms, but when grown in fish farming, fish do not have access to these natural pigment sources and therefore do not obtain the attractive red color unless red pigments are added to the feed.

15

Thus, astaxanthin isolated from seafood waste or synthetically produced, as well as other synthetic red pigments such as cantaxanthin, have been used as constituents of fish feed. However, the use of cantaxanthin in animal feed is prohibited in some countries, and the synthetic astaxanthin production as well as the method of isolating natural astaxanthin are quite expensive and often subject to seasonal variations.

Other natural astaxanthin sources are known, including the yeast Phaffia rhodozyma and certain microalgae such as the unicellular group of green algae Chlamydomonas 25 nivalis [Gert Knutson et al., "Pigmentation of salmon with astaxanthin from microalgae",

Norwegian Fish Breeding No. 3, pp. 4-6, 55 (1989)]. The astaxanthin produced by these organisms has been shown to transmit the desired red color to anadromous fish [Eric A. Johnson et al., "Phaffia rhodozyma as an astaxanthin source in salmonid diets", Aquaculture, 20, pp. 123- 134 (1980) and JP-A 57-206342].

30 However, the use of yeast cells in large quantities as nourishment for the fish is not desirable, as this feed is not sufficiently varied. On the other hand, the amount of astaxanthin produced by the organisms present in a nutritionally acceptable amount of yeast cells is not sufficient to achieve the desired staining, and the isolation of astaxanthin from yeast by known methods is quite expensive.

^ - - -----

DK 168542 B3 I

I I

In the Journal of General Microbiology, Vol. 115 (1979), pp. 173-183 (Johnson et al.), I

It is admittedly stated that Phaffia rhodozyma can produce 652 µg of astaxanthin I

I per g yesterday. However, this value is based on total carotenoid content and on

In Figure 5, an analytical method which, relative to the method used below, results in H

In high values. If the method of analysis is used as described below, 652 pg H corresponds

In effect to approx. 432 pg / g yeast. 1 Journal of Bacteriology, Vol. 59 (1985), pp. 243-255 I

Please note that in the aforementioned article, Phaffia rhodozyma is mentioned with an I

In astaxanthin content of 800-900 pg / g yeast. However, that content relates to I

In 10 carotenoid contents and corrected as above, the highest indicated astaxanthin-I

In content 540 pg / g yeast. IN

However, if it is possible to achieve a higher astaxanthin production from I

In these organisms, it would be possible to obtain a profitable astaxanthin production, I

In 15 that are not subject to seasonal conditions. IN

In the present invention, novel Phaffia rhodozymes provide

In yeast cells containing astaxanthin in sufficiently high concentrations that I

You enable the use of the yeast cells as or in feed for anadromous fish and H

In 20 other animals in which a staining of the animal's meat or animal product is

In desirable. The invention also provides an attractive method for I

In obtaining astaxanthin from astaxanthin-containing yeast cells, especially the aforementioned I

In yeast cells that have a high astaxanthin content. Important aspects of the invention are:

I based on a particular method for growing astaxanthin producing cells and / or I

In providing mutated strains that have an enhanced inherited ability to

In producing astaxanthin. IN

Thus, in one aspect of the present invention, a Phaffia rhodozyme relates to I

In yeast cell, which is the yeast strain deposited under landfill no. 225-87 I

In 30 CBS, or the yeast strain deposited under landfill no. 215-88 CBS, I

I or a mutant or derivative thereof, or a mutant or derivative thereof

In yeast strain deposited under landfill no. 225-87 CBS, which yeast cell I

You have retained the astaxanthin producing ability and which, when grown under

In conditions comprising an oxygen transfer rate of at least 30 mmol per IN

In 35 liters per liter. per hour on Difco YM medium at 20-22 ° C for 5 days in 500 ml shaker flasks with 2 L

B3 baffle plates containing 50 ml of the medium and subjected to orbital shaking at 150 rpm, the inoculum being 100 μΙ of a 4 day old culture in YM medium, produce astaxanthin at a rate of at least 600 µg per ml. g yeast solids determined by HPLC analysis using pure astaxanthin as standard on a yeast methanol extract prepared by subjecting a suspension of 0.2 g yeast solids to 20 ml methanol 5x1 minute disintegration at half-minute intervals, the disintegration is carried out at a temperature not exceeding 20 ° C in a glass ball mill containing 15 g of 0.4 mm diameter glass balls, the glass ball mill being equipped with a cooling jacket with ice water.

10

The culture and assay conditions mentioned above are listed to standardize the culture and test methods so that the result obtained will reflect the inherent astaxanthin producing ability of the yeast in question.

This method has proven by several attempts by the applicant to be a suitable and reproducible method which is easy to perform in practice. It should be noted that the method of determination is not the same as that used so far in the literature. The methods used in the literature so far, cf. for example, Eric A.

Johnson et al., "Astaxanthin formation by the yeast Phaffia rhodozyma". Journal of general microbiology 115, 1979, pp. 173-83, is based on the absorbance of a 1% 20 (w / v) solution in acetone in a 1 cm cuvette of 1600, whereas the value obtained by measuring the astaxanthin standard from Hoffmann-La Roche, is 2100. This value is based on the applicant's own measurements as well as information from Hoffmann-La Roche.

The known assay method further measures the yeast's total color content, whereas the above standard method used in the present application only measures the astaxanthin content. When comparing the values obtained by the above standardized method with the values given in the literature, it should be noted that the values used in the literature will be considerably higher than the true values. obtained by the above standardized method. Thus, the difference between extinction coefficients should be corrected by multiplying the total pigment content reported in the literature by 1600/2100 each time comparing the total pigment content with the literature.

35

DK 168542 B3 I

I I

In the above mentioned cultivation conditions are those which the applicant has found to be H

In reproducible and significant for the determination of the inherent astaxanthin product I

In grating ability. A more detailed explanation of cultivation and determination I

In the conditions used to determine yeast strains inherent astaxanthin-I

In 5 producing ability are listed in connection with the Examples. IN

In the yeast cell of the invention, the species belongs to Phaffia rhodozyma, being the H

In the only Phaffia species known at this time. IN

At 10 Phaffia rhodozyma is currently the only known yeast that you

You produce astaxanthin. Wild type P. rhodozyma is isolated from exudates from I

In deciduous trees, and an example of such a wild-type strain is deposited with I

In the American Type Culture Collection under landfill no. ATCC 24261. I

In 15 Vegetative P. rhodozyma cells form buds that heterobasidiomycete yeast. IN

In Clamydospores develops by bud firing, but promycelium and really I

In spore formation does not occur. The chlamydospores are relatively large spherical cells I

You have a higher fat content than vegetative cells. Attempts to cross different H

In strains hoping to observe dikaryotic mycelium and teliospore formation, H

In 20 failed. P. rhodozyma was therefore classified in the genus Deuteromycotina by

In the order of Blastomycetes (cf. M.W. Miller et al., "Phaffia, a new yeast genus in the I

In Deuteromycotina (Blastomycetes) ", in International Journal of Systematic M

In Bacteriology 26: 2, 1976, pp. 286-291). IN

In 25 Vegetative cells, ellipsoidal (3.6-7.5) x (5.5-10.5) µm are in liquid medium for I

Present individually, in pairs and in some cases in short chains or small groups. there

You do not develop an actual mycelium, but there may be a rudimentary I

In pseudomycelium present. Button Formation Occurs several times from the same I

At the point of the cell. P. rhodozyma has a strong cell membrane composed of I

In many layers, and a capsule material which gives the surface a granular appearance and

You are causing the aforementioned group formation. IN

I No sexual life cycle has been observed. During the Damydospores I

In development, vegetative cells are formed by bud firing. These cells cannot

I 35 is considered to be promycelia with spores as described for Aessosporon I

5 DK 168542 B3 (cf. JP van der Walt, "The perfect and imperfect states of Sporobolomyces salminicolor, J. Microbiol.Serol. 36, 1970, pp. 49-55). ), and their buds cannot be considered to be the haploid generation. It has not been possible in nuclear staining to demonstrate diploidization at any growth stage. Transmission electron micrographs have shown only a single nucleus during all growth phases (cf. MW Miller et al., op. cit.) P. rhodozyma is thus probably haploid, but this has not been proven.

10 After 2-4 weeks of growth on YM agar (Difco Laboratories Incorporated, Difco Manual: Dehydrated Culture Media and Reagents for Microbiology, 10th Edition, Detroit 1984), line cultures are orange to salmon-pink, depending on the strain.

P. rhodozyma has the special property of not growing at temperatures above 15 27 ° C. It ferments D-glucose, maltose, sucrose and raffinose, whereas D-galactose and melibiosis are not fermented. The most common carbon sources are assimilated; However, D-galactose, L-sorbose, melibiosis, lactose, glycerol and citrate are not assimilated. The yeast cannot grow in vitamin-free medium without the addition of biotin (M.W. Miller et al., Op.cit.). The most common nitrogen sources 20 are assimilated, including urea. Potassium nitrate and ethylamine are not assimilated. The yeast cannot grow on 50 wt% glucose yeast extract agar nor on 10 wt% sodium chloride yeast extract agar. Yeast acidification on chalk roofs is weak, and this also applies to gelatin melting. Casein hydrolysis, depolytic activity and growth in the presence of 0.1 μς cycloheximide per ml does not take place, whereas the yeast is capable of synthesizing starch-like compounds irrespective of pH. The G + C molar percentage is measured at 48.3 ± 0.18 (cf. Miller et al., Op. Cit.)

Growing under carbohydrate and / or nitrogen restricted conditions and when subjected to fat-batch fermentations, P. rhodozyma produces trehalose as 30 carbohydrate storage. This is a completely new observation made by the applicant and not previously reported.

P. rhodozyma produces a number of carotenoids, of which astaxanthin constitutes 83-87%, ($ -carotene 2-2.5%, echinenone 2-4% and phoenico-xanthin 5-7% according to the literature. In practice, the ratio of astaxanthin and total pigment produced

I DK 168542 B3 I

I 6 I

However, P. rhodozyma was found to vary considerably depending on the cultivation conditions

In the yeast cell thawings as well as the pigmentation method, and have

In general, it was found to be in the range of 50-80%. IN

Is I

I I

I 3-CAROTEN I

I 10 I

I ° I

I I

I I ASTAXANTHIN I

I 15 I

All hydroxylated pigments, including astaxanthin, have been described as I

In unbound, not as esters or other derivatives (Arthur G. Andrewes et al

I 20 "Carotenoids of Phaffia rhodozyma, a red-pigmented fermentation yeast", I

In Phytochemistry 15, 1976, pp. 1003-1007). Three optical isomers I exist

In astaxanthin informer: (3S, 3'S), (3R, 3'R) and (3S, 3R), each found in different I

In trans and cis configurations. It has been described that P. rhodozyma only I

In (3R, 3'R) -astaxanthin (Arthur G. Andrewes et al., "(3R, 3'R) -astaxanthin I

I 25 from the yeast Phaffia rhodozyma ", op.cit., Pp. 1009-1011).

In context, "astaxanthin" is used for trans as well as cis configurations of I

In astaxanthin. IN

The pigment in the individual P. rhodozyma cells is not visible when the cells are examined

I 30 in a microscope, suggesting that the pigment may be dispersed throughout the cell. The ten

However, it is also possible that the pigment is concentrated in certain parts of the cells. I 1

Astaxanthin is an oxidized carotenoid and therefore belongs to the xanthophyl group. I I

Similar to other carotenoids, astaxanthin is composed of 8 isoprene units. Do you know

The astaxanthin biosynthesis, which is catabolite repressed, isopentenyl pyrophosphate is formed from acetyl-CoA as shown below:

^ C £ Trv * CoA

5 / ^, C \ s * M, C \ * Η, ςν S *

0 C ♦ HfC-CoA HM0-COA

*, i ,,. _ Synthase / \ Reductase / \ 2HjC-C-SCeA ^ -Ch, -! -—-> h, C Ch, -Ch,

0-C 0 -C COOH mC-Ch, CCOH

SCod SCeA

ACL'lYL-CoA ^ ce ^^ cetYL-CqA 3S-HMGH3oA 3R- MEVALONIC ACID Mevalon kinase l "1> ° M>

C Pyrophosphamevalonate-νς 'Phosphcmevalonate _ \ X

/ \ decarboxylase / \ kinase / v m, c c «j <- ch? <-: - m, c PO-PO-CM, PO-PO-CH, COCH PO-CM, coch 15

ISOPENTENYL 3R- MEVALONIC ACID 3R- MEVALONIC ACID

PYPOFHGSPHAT 5-PYROPHOSPHATE. 5-ΡΗ06ΡΗΑΊΤ

Isopentenyl pyrophosphate, in three prenyltransferase reactions, forms geranyl-geranyl pyrophosphate via geranyl pyrophosphate and farnesyl pyrophosphate as shown below: λ ^ OPP_k λ ^ ΟΡΡ DIMETHYL ALL YL-isopcnu ™ .. r \ pp 25 Λ / \ Λχ ° "

IN GERANYL PYROPHOSPHATE

L-IPP

30 A / \ Ay \ A ^ 0PP - STEROIDER i

| -l PP FARNESYLPYROPHOSPHATE

Ayr \ A / \ A / \ A ^ 0PI>

geranylgeranylpyrophosphate

35

DK 168542 B3 I

Condensation of two-molecule geranylgeranyl pyrophosphate forms the phyto as, I

via dehydrogenation steps and cyclization, astaxanthin forms from β-carotene. IN

The latter part of biosynthesis has been unambiguously determined, but Andrewes et al. IN

5 (op.cit.) Has suggested the metabolic pathway shown on the basis of pigment-I

the repositioning in P. rhodozyma: I

9 DK 168542 B3

V "X PHYTOEN

phytofluene

ζ- CAROTEN

<^ ΑΑΑ4Λ4, Α / γν £! Χ

neurosporene

ζ ^ ^ ΛΛλΛΛ / γν / yl ^

0-ZEACAROTEN ^ y-CAROTEN

jX / VA? A ^ ^ / \ YXT / ^ \ ^^ / ^

J β-CAROTEN

yy \ yKy \ ^ YSY \ ^^^

\ Λ I ECHINENON

, 3-HYDR0XYECH1NEN0N

° l)> C ^ AyvAyv \ J ^ / vi ^^

*> \ A J PHOENICOXANTHIN

^ J ASTAXANTHIN

DK 168542 B3 I

I The enzyme system that converts geranylgeranyl pyrophosphate to astaxanthin, H

You do not know and therefore do not know why P. rhodozyma produces (3R, 3'R) - H

In astaxanthin and whether there are some regulatory steps during this part of the biosynthesis.

I On the other hand, the conversion of acetyl-CoA to isopentenyl pyrophosphate in I

In 5 isoprene-producing organisms different from P. rhodozyma and de

In enzymes taking part therein have been described in detail. You know

In less about the enzymes that convert isopentenyl pyrophosphate to geranylgeranyl

In pyrophosphate (isopentanyl pyrophosphate isomerase and prenyltransferase (J.W. H

In Porter, S.L. Spurgeon (eds.), "Biosynthesis of isoprenoid compounds". New York, H

I 1981-1983). IN

In the protein content of P. rhodozyma varies from 25 to 50% of yeast solids depending on the protein

I of the cultivation conditions. This is a relatively low protein content. Unlike I

In this, the fat content is extraordinarily high (14-27%). It is believed that H

In the 15 nucleic acid, 8% is similar to other yeasts and that the amino acid composition

You are similar to the composition of other known yeasts such as Saccharomyces cerevisiae and H

Thus, you have a very low content of certain amino acids, for example methionine and I

In cysteine (cf. Gerald Reed and Henry j. Peppier, Yeast Technology, 1973, p. 329, I

In published by The AVI Publishing Company, Inc.). This and the total yeast aggregation- H

In 20 sentences, which include a high amount of nucleic acid, the yeast renders unsuitable as

In animal nutrition, when the yeast is the sole source of nutrition, as indicated in I

In the above. Thus, the yeast will not be a suitable main nutritional component for fish I

In or other animals without the addition of certain amino acids and other nutritional sources. IN

I 25 The total amount of astaxanthin produced from wild-type P. rhodozyma when H

It is grown under the usual known conditions, sufficient to impart

In the yeast cell a red color but is not sufficient to isolate astaxanthin I

In from the yeast cells economically profitable. IN

I 30 None of the Phaffia rhodozyma species described in the literature have an inherent I

In astaxanthin producing capacity of more than 600 µg per g yeast solids when ana- I

You are lysed in accordance with the standard methods listed above, see Table 2 I

I and Table 6 of the Examples. However, according to the present invention, it has shown I

In itself, it is possible to obtain yeast cells which are inherently capable of producing I

B3 η astaxanthin in an amount of at least 600 pg per day. g of yeast solids, preferably at least 700 pg. g of yeast solids, in particular at least 1000 Mg. yeast solids, in particular at least 1500 Mg. yeast solids, and in particular at least 2000 m9 per hectare. g yeast solids, the cultivation and evaluation being carried out by the above standard methods. These 5 yeast cells can be prepared from naturally occurring Phaffia rhodozyma by mutagense. Preparation of the present Phaffia rhodozyma yeast cells exhibiting the high inherent astaxanthin producing ability explained above comprises treating a Phaffia rhodozyma yeast cell with a mutagen and selecting a resultant mutant which, when cultured under the above 10 conditions, are capable of producing astaxanthin in an amount of at least 600 µg per day. g of yeast solids determined by the above procedure.

The mutagenesis can be performed as a single mutagenesis, but it has been found advantageous to perform two or more consecutive mutagenesis, as it has been found that the inherent ability to produce astaxanthin can be enhanced at each mutagenesis step. The starting yeast cell subjected to mutagenesis is usually a yeast cell which, when grown under the above conditions, produces astaxanthin in an amount of less than 300 μς per day. g yeast solids determined by the above method, but it is clear that a normal candidate for the mutagenesis treatment will be a naturally occurring yeast cell which has as high inherent astaxanthin production as possible. Such yeast cells are usually yeast cells belonging to the genus Phaffia, especially yeast cells belonging to the species Phaffia rhodozyma, as mentioned above.

The mutagenesis treatment can be carried out using any suitable mutagen (in this context, the term "mutagen" is to be understood in its broadest sense as encompassing, for example, not only agents having a mutagenic effect, but also treatment having a mutagenic effect such as UV radiation). Examples of suitable mutagens are ethyl methanesulfonate, UV-irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, nucleotide base analogs such as bromouracil and acridines, but any other effective mutagen is expected to be suitable for the treatment.

In accordance with conventional mutagenization techniques, the mutagenization is followed by an appropriate selection of the cells having the highest

DK 168542 B3 I

astaxanthin. As astaxanthin is a pigment, this selection may be H-

is conducted relatively easily by normal visual means such as simple observation I

of single colonies. An alternative method is to analyze cultures prepared H

from single colonies, for example, using the standardized culture conditions and assay conditions explained above.

A strain prepared by the mutagenization method of the invention, and H

which shows a particularly high astaxanthin productivity, was deposited on April 6, 1987 H

at the Central Agency for Mold Cultures, Oosterstraat 1, PO Box 273, NL-3740 I

10 AG Baarn, The Netherlands (CBS) under 225-87, and a strain which is a traveling isolate of CBS I

225-87 (see Example 1) was deposited on March 23, 1988 with CBS under I

Deposit. 215-88, and one aspect of the present invention relates to these I

yeast strains as well as mutants thereof which have substantially conserved or I

enhanced the astaxanthin producing ability of these strains. IN

The invention also relates to a process for the preparation of astaxanthin-containing I

Phaffia rhodoxyma yeast cells or cell parts or astaxanthin derived from these H

yeast cells or cell parts. This approach is peculiar in that H

astaxanthin-producing Phaffia rhodoxyma yeast cells are grown under aerobic H

20 conditions in a medium containing carbohydrate sources, assimilable nitrogen and

phosphorus sources, micronutrients and biotin or desthiobiotin at a temperature I

in the range of 15-26 ° C to obtain a biomass containing astaxanthin in I

an amount of at least 600 pg per g yeast solids determined by the above I

method, and optionally one or more of the following steps is performed in arbitrary

25 order: I

The cells are harvested from the culture so as to obtain a yeast cream, I

the cells are opened, for example, by destroying the cell walls at I

by mechanical, chemical and / or enzymatic treatment and / or

The cells are subjected to sonication, autolysis, osmolysis and / or plasmolysis, I

optionally with the addition of suitable agents such as detergents, acids, bases, I

enzymes, autolysis enhancers, osmolysing agents such as salts,

and / or plasmolysing agents, I

the cells are homogenized to obtain a homogenate, I

The cells, cell fragments or homogenate are dried, preferably at a water content of not more than 12% by weight, preferably not more than 10%, astaxanthin is extracted from the cells, cell fragments or homogenate.

5

The amount of astaxanthin stated above, 600 µg per day. g yeast solids, is higher than. any astaxanthin concentration described in the literature. Although Johnson et al. up. cit describes an astaxanthin content of 295 µg per ml. g yeast solids, this value includes not only the astaxanthin content but in fact the total pigment content of the yeast cell. This pigment content was further measured using an absorbance value of 1% (w / v) solution in acetone in 1 cm cuvette of 1600, which is lower than that measured by the present applicants (2100) so that the value obtained described by Johnson et al. corresponds to a maximum of 295 x 1600/2100 = 225 µg total pigment (not only astaxanthin) per g yeast solids. This 15 pigment content from the literature should be compared with the total pigment content of the yeast strains of the present invention, which pigment content is 1176 pg / g yeast solids from strain CBS 225-87 and approx. 1340 pg / g of yeast solids from the strain CBS 215-88 or even higher, for example at least 2000 pg / g of yeast solids.

20

The high astaxanthin concentration in the yeast cells of the invention can be achieved partly by using particular culture conditions, which will be described below, and partly by selecting a yeast strain with a high inherent astaxanthin productivity, preferably a yeast strain as described above, and it is especially preferred to combine the particular cultivation conditions and the use of particular astaxanthin-producing yeast strains.

The cultivation is preferably carried out as fat-batch fermentation under conditions where alcohol is practically not formed. As mentioned above, the culture temperature is in the range 15-26 ° C. Below 15 ° C, growth tends to be too slow to be acceptable for industrial production, and above 26 ° C the viability of the culture is severely impaired. The preferred temperature range is 20-22 ° C.

The fermentation, or at least a portion thereof, is usually carried out in a medium comprising suitable macro and micronutrients for the cells, such as molasses or

DK 168542 B3 I

sucrose as a carbohydrate source and nitrogen sources such as corn molding liquid, diammo- I

nium sulfate, ammonium phosphate, ammonium hydroxide or urea, phosphate B

sources such as ammonium phosphate and phosphoric acid, and added micronutrient I

fer or mineral salts such as magnesium sulfate, zinc sulfate and biotin or B

5 desthiobiotin. Molasses or sucrose are preferably added to the medium separated from B

the other components in accordance with conventional methods B

by yeast production. When the medium comprises molasses, it has been found that the yeast cell B

growth is affected by the sugar concentration or other growth promoters therein in I

fermenter. This effect is not observed when the medium comprises corn mold B

10 liquid or solid. It may therefore be advantageous to regulate the fermentation, B

so that the sugar concentration (expressed as the total concentration of glucose B

and sucrose) in the fermentor is not more than 8 g / l, preferably not more than 5 g / l, and especially not more than B

1 g / l

The culture is aerated throughout the fermentation, ie. it is grown under aerobic B

conditions. By the term "aerobic conditions" is meant that the oxygen supply should B

be sufficient that there will be essentially no oxygen limiter B

during fermentation. B

According to a particular aspect of the present invention, as indicated by B

above, the astaxanthin concentration in the obtained biomass is increased by performing B

cultivation under selected conditions. These conditions include a cultivation, B

which comprises a growth phase under conditions which are substantially sufficient- B

just under essentially all growth conditions, and a subsequent growth condition

25 boundary phase. The growth restricted phase is preferably established by providing B

conditions where, under continued aeration, the culture medium lacks at least one B

growth factor, so that the production of astaxanthin is increased during the subsequent B

phase. B

30 The growth-restricted phase is generally understood to be a phase in fl

the majority of Phaffia rhodoxyma cells have ceased to grow. This B

phase, of course, exists when the medium lacks at least one growth factor, but B

is also observed during the latter part of the carbohydrate addition period when the amount of B

of cells present in the fermentor are much larger than the fermentor's B air

Capacity. B

DK 168542 B3 is

It is not known why the subsequent growth restricted phase has the surprising effect of significantly increasing astaxanthin production, but it is believed that astaxanthin precursors are produced during the growth phase and that the 5 subsequent growth restricted phase provides conditions that promote end production of astaxanthin, possibly oxidizing conditions. excess oxygen which becomes available when growth is terminated. At any rate, it seems essential that the aeration be continued during the subsequent growth-restricted phase. The duration of the subsequent growth-restricted phase is preferably at least 16 hours, such as 16-24 hours, with shorter durations tending to reduce the extra-achievable effect, while no significant effect can be obtained by extending the growth-restricted phase to more than 24 hours.

Expressed in a functional way, the conditions of the growth-restricted phase should be adapted to increase astaxanthin production at least 1.2 times the output obtained without the subsequent phase, such as at least 1.3 times the output obtained without the subsequent phase. preferably 1.4 times the output obtained without the subsequent phase and most preferably at least 1.5 20 times the output obtained without the subsequent phase.

The yeast cell that is subjected to the particular culture with the subsequent growth phase is a Phaffia rhodoxyma yeast cell of the invention which has an inherent and improved ability to produce astaxanthin, typically a yeast cell obtained by mutagenization as explained above. With these yeast cells with an inherently increased astaxanthin production, the astaxanthin concentration in the biomass obtained when the particular culture method comprising a growth restricted phase is used can be at least 600, preferably at least 800, especially at least 1000 pg. g of yeast solids, in particular at least 1500 pg. g yeast solids, for example, at least 2000 pg per day. g of yeast solids, and most preferably at least 3000 pg. g yeast solids determined as set forth above.

Normally, and as used above, the total pigment content and astaxanthin content in the yeast cells or yeast cell portions are indicated as pg / g yeast solids. However, it may turn out that other ways of listing the total pigment and astaxan ____ __

I DK 168542 B3 I

I 16 I

Thin content may be appropriate. For example, it may be useful to:

Indicate the total pigment content and astaxanthin content as pg / ml of the suspension

In retirement, in which it is present, for example, in the culture medium. Thereby you will

You do not need to determine the yeast solids weight of the yeast cells from which I

In the astaxanthin or total pigment, isolate. Thus, astaxanthin and / or I

In the total pigment content of the yeast cells is easily determined during the fermentation or I

In the cultivation of the yeast cells. · I

Following the culture described above to obtain yeast cells having a high I

In astaxanthin content, the culture may be subjected to the aforementioned successive I

In treatments to isolate the yeast cells and / or prepare them for their post-I

In the following application, such as by destroying the cells and / or astaxanthin, you can

You extract from the cells. IN

The following important examples of these treatments are described in more detail:

The cells can be destroyed by subjecting them to increased pressure and then releasing I

In the print. I 1

The cells can be subjected to the increased pressure and release of the pressure by becoming I

Transported through a system comprising a valve homogenizer, wherein it

Increased pressure builds up in front of the valve homogenizer. The valve homogenizer I

Typically, you include an aerojet through which the cell suspension is passed during high I

In pressure, and an obstruction element that the jet strikes substantially after passage I

In 25 through the valve. Examples of cell destruction valves are described in APV I

In Gaulin Technical Bulletin No. 74, March 1985 (APV Gaulin International SA, P.O. I

In Box 58, 1200 AB Hilversum, The Netherlands), to which reference is made. As an example of an I

In suitable cell destruction homogenizer there may be mentioned an APV Gaulin MC4 homologue.

In a generator with a CR cell type destruction valve described in above

In the said publication. The homogenizer is connected to a heat exchanger, through I

In which the suspension comprising damaged cells passes from cell destruction I

In the valve. For example, the cell suspension pressure in front of the valve may be approx. 400- I

In 1200 bar, such as, for example, approx. 700 bar. This treatment may, for example, I

Repeat 3 times with intermediate cooling of the homogenate in the heat exchanger. IN

I 35 I

17 DK 168542 B3

As explained above, it is necessary that the cells be destroyed or otherwise processed when used in animal feed, the utilization of the astaxanthin content greatly depends on the cell content available for the digestive system of the animal concerned. Thus, essentially no staining effect is obtained when fish are fed with feed containing undamaged astaxanthin-containing cells.

The damaged yeast cells can be subjected to ultrafiltration or evaporation to concentrate the damaged cells. The ultrafiltration can be carried out, for example, in a laboratory unit system available from the Danish sugar mills, for example a system 37 comprising three filtration units with a total filter area of 0.88 m2 of an RC70 ultrafiltration membrane. Another method of concentrating the damaged cells is to perform vacuum evaporation of water from the cell suspension.

The damaged cells can be dried by spray drying or drum drying. Prior to drying, carriers such as sodium caseinate, antioxidants and / or emulsifiers are preferably added. For example, spray drying can be carried out by subjecting a homogeneously mixed slurry of the damaged cells and optionally a carrier such as sodium caseinate, preferably in the form of an aqueous solution, to spray drying. Suitably, spray-drying can be carried out by mixing the aqueous sodium caseinate solution with the yeast slurry to obtain a sodium caseinate concentration of approx. 2-10% (w / v). The resulting mixture is then allowed to stir under a nitrogen atmosphere before being pumped into a spray drying tower, in which it is subjected to drying at a temperature of, for example, 150 DEG-200 DEG C., such as ca. 180 ° C to reduce the water content of the yeast cell material, for example to a maximum of 10% by weight. The yeast cell material is then subsequently atomized 1 by means of a spray wheel. The powdered yeast material resulting from the spray drying treatment is suitably isolated by means of a cyclone and optionally sieved and packaged thereafter. An example of a suitable spray drying equipment is an EAK-1 type spray mist from Anhydro. Alternatively, the damaged yeast cells can be subjected to drum drying, for example in a closed drum drying equipment at a temperature of 150-200 ° C.

Since the astaxanthin is very easily decomposed at high temperatures, is

DK 168542 B3 I

18 I

it is important that the damaged yeast cells be subjected to high temperatures in such a short time

as possible. Furthermore, since astaxanthin is oxygen sensitive, the drying should preferably be I

is carried out under non-oxidizing conditions, for example in an inert atmosphere I

such as water vapor (which may be the water vapor evaporated from the yeast suspension

5), nitrogen and / or carbon dioxide. IN

Prior to drying, the damaged cells are optionally mixed with suitable emulsifiers such as · I

sorbitan monostearate or antioxidants, butyl hydroxytoluene (BHT), butyl hydroxy-I

anisole (BHA), vitamin E, ascorbic acid, (II) sulphate or (II) phosphate esters of I

10 ascorbic acid or ascorbyl palmitate. IN

Dried, damaged cells can be used immediately as constituents of animal feed, I

as will be explained below. IN

The astaxanthin content of the yeast cells can be extracted from these using I

various extraction agents and extraction procedures - so as to ensure that I

a substantially total extraction of astaxanthin from the yeast cells is obtained. In the I

In most cases, the extraction must be done in broken cell material. It ruined you

Thus, cellular material that may be dry or wet can be extracted with an I

20, such as petroleum ether, which may conveniently be used

is used in the case of wet cell material, the petroleum ether forming a phase,

that is separate from the water phase. Other suitable organic solvents are acetone I

or alcohols such as methanol or ethanol, ethers, ketones and chlorinated hydro-I

carbons. In the extraction, astaxanthin is dissolved in the organic solvent. IN

The astaxanthin can be obtained by removing the solvent from the solution, such as at I

evaporation in a falling film evaporation system before drying. A concentrate of I

However, astaxanthin in the organic solvent may also be useful

for certain purposes. A concentrate can be used per se for the preparation of I

feed or food or the concentrate may be diluted and used in it

30 in the preparation of feed or foodstuffs, for example I

by impregnating feed or food ingredients with the solution or I

by using the solution (or concentrate) to color the food ingredient

parts such as oils or fats. IN

19 DK 168542 B3

The astaxanthin can also be extracted from yeast cells using carbon dioxide under supercritical conditions. The carbon dioxide may optionally be used with suitable mixers, such as organic solvents, especially solvents of the type mentioned above, or solvents such as chloroform 5 or acetonitrile, or glacial acetic acid. The yeast cells subjected to supercritical extraction may be wet or dry whole yeast cells or broken, for example homogenized yeast cells.

A preferred method of isolating whole astaxanthin-containing cells from the culture is to filter the yeast flow, for example, on a filter press or a rotary drum filter, so as to obtain a filter cake, for example, with a dry matter content of approx. 25-35%. The filter cake may conveniently be extruded into strands, for example in strands having a diameter of approx. 0.5-2.0 mm in an extruder equipped with a perforated plate so as to obtain strands consisting of yeast particles. The strands 15 are preferably extruded directly into hot air in a fluid bed where they are dried. The evaporation in the fluid bed is preferably controlled such that the temperature of the yeast particles is kept below 50 ° C such as at 30-40 ° C and the process is completed when the water content is reduced to below 10% by weight, preferably below 8% determined by yeast. - the solids content (the procedure is described in the examples). The drying may alternatively be carried out in a tray dryer under the same conditions as in the fluid bed. The dried whole cell material can then be comminuted in a ball stirrer mill, such as a Coball® mill, after which it is subjected to extraction.

According to a particular process, dried whole cell material, for example obtained as described above, can be mixed with an oily phase such as an edible oil or edible fat such as soybean oil or fish oil or other organic solvent such as a solvent of the type mentioned above. The temperature is preferably in the range 20-30 ° C. The mixture obtained from the cell material and the oily phase or the organic solvent can be ground in a mill, such as a ball mill, for example a ball stirrer mill, such as a Coball® mill, to destroy the cells and release astaxanthin from the cells. The resulting suspension can itself be used in feed, or the oily phase containing the astaxanthin can be separated from cell debris before use. The separation is conveniently performed by centrifugation in a fast-running centrifuge; the same principle used in separating bacteria from beer wort. Another

20 I

DK 168542 B3 I

Of course, the option is to mix destroyed, dried cell material obtained at H

the above-mentioned methods, with an oily phase in a similar manner to

extract the astaxanthin in the oily phase and perform the separation as I

described above. The oily phase can be used to dye feed on I

5 in the same manner as described above.

Unlike most traditional extraction procedures, as stated. IN

above is done on broken cell material, it has been found that glacial acetic acid with I

success can be used to extract astaxanthin from whole, unbroken yeast cells. Thus, astaxanthin can be extracted from whole yeast cells with a solution of

agent comprising glacial acetic acid, the extraction being preferably carried out at an H

temperature above the freezing point of the solvent, for example in the range of 20-100 ° C, I

preferably in the range 20-80 ° C, and especially in the range 20-60 ° C. It's supposed to be you

possible to obtain a more selective astaxanthin extraction when the extraction is carried out

15 at the lower temperatures, with the accompanying extraction of fat or other I

extensible components will be limited at these low temperatures. IN

The glacial acetic acid concentration in the solvent is preferably in the range of 5-100, 10-

70. The extraction with glacial acetic acid results in an extraction of the pigment I of the cells

of approx. 70-90%, ie essentially the entire pigment and astaxanthin I yeast cells

20 contents are found in the glacial acetic acid extract. In addition, the extract usually contains I

30-35% yeast solids. The yeast subjected to extraction with glacial acetic acid is I

conveniently in the form of dried yeast, for example, yeast which has been filtered and subsequently extruded into a fluid bed in which it is dried, thus

traded yeast cells will not be destroyed during extraction treatment I

25 (unless the extraction treatment involves vigorous mechanical treatment of I

the yeast cells). This will facilitate the subsequent separation of the extract which I

contains the pigment from the yeast cells, in comparison with extraction of broken I

or homogenized cells which due to their relatively small size in I

Comparison with undamaged cells to a large extent tends to

30 block the pores in the filter used. The glacial acetic acid extraction is illustrated in I

Example 8. Extraction of wet yeast cells with glacial acetic acid may also be I

applicable. IN

The extracted astraxanthin as well as the dried whole cell material is kept

35 preferably under oxygen deficiency so as to protect the astaxanthin from I

21 DK 168542 B3 decomposition. Thus, the astaxanthin-containing yeast cells or the extracted astaxanthin are preferably protected by antioxidants such as butyl hydroxyanisole (BHA), butyl hydroxytoluene (BHT), vitamin E or ascorbic acid, (II) sulfate or (II) phosphate esters of ascorbic acid or ascorbyl acid or emulsifiers such as monoglycerides or sorbitan esters and conveniently kept under hermetic conditions.

The invention also relates to an animal feed which is characterized in that it comprises Phaffia rhodozyma yeast cells according to claim 1 or yeast cell parts containing 10 astaxanthin in an amount of at least 600 Mg per day. g yeast solids determined as described in claim 1, eg yeast cells according to claims 1-2 or parts thereof or yeast cells or yeast cell parts prepared by the method according to any one of claims 3-8, together with other feed ingredients, where the astaxanthin-containing yeast cells or yeast cell parts are at most 10% by weight of the total dry matter of the animal feed composition, preferably at most 5% and especially at most 3%, the other components being selected from protein and carbohydrate sources such as fish meal, whey, blood meal, vegetable flour, fats such as fish oil and vegetable oils. , vitamins and minerals. These values are calculated on the basis of the final feed with which the animals are to be fed. It is also possible to prepare feed premixes having a higher concentration of 20 yeast cells. Optionally, the yeast cells or yeast cell portions or astaxanthin are mixed with emulsifiers capable of making the astaxanthin dispersible in water.

In addition, the astaxanthin-containing yeast cells or yeast cell parts can be protected from oxidation by the aforementioned anti-oxidants and / or emulsifiers, and / or the animal feed can be packed in airtight and possibly evacuated containers.

25

The astaxanthin-containing dried yeast cells can also be packed per se for use as a feed ingredient where the final feed mixture is prepared at the site of application, or the yeast cells are administered per se to the animals which are otherwise fed normal or adapted feed mixtures.

The yeast cells or yeast cell portions are conveniently and usually mixed with other nutrient components which are preferably selected from protein and carbohydrate sources, fats or oils and micronutrients such as vitamins and minerals.

Casein, albumin, wheat gluten, fish meal, concentrated fish waste products 35 (fish lime and blood meal) can be cited as examples of protein sources. As

22 I

DK 168542 B3 I

Examples of carbohydrate sources include gelatinized starch extruded

wheat molasses, vegetable flour and cornstarch. The fat components in the feed can

for example, be fish oil and cod liver oil and / or vegetable oils such as H

corn oils. The minerals can be selected, for example, from inorganic or simple H

5 organic compounds of calcium, phosphorus, sodium, potassium, chlorine, magnesium, H

copper, manganese, zinc, cobalt and selenium. Examples of vitamins include:

vitamin Bi2, proline, vitamin A, vitamin D, vitamin E, vitamin K, thiamine, ascorbine-I

acid, riboflavin, pyrodoxin, panthotenic acid, niacin, biotin, choline and inositol. IN

io Astaxanthin, which has been extracted from yeast cells, for example by means of an I

any of the above methods, preferably from yeast cells of op

the yeast or yeast cells produced by the method of the invention, H

can be used with the feed ingredients described above and also together

but with other food or nutrient components and mixed with other I

15 pigments. Thus, astaxanthin extracted from yeast cells is suitable alone or I

together with other pigments for use in edible oils, butter, margarine, H

shortening, mayonnaise, patées, soup, snacks, surimi-based products, H

desserts, ice cream, confectionery, baked goods and beverages. When the astaxanthin I

used in feed consisting mainly of water or water phases, H is mixed

Preferably the astaxanthin with an emulsifier as described above, which makes H

the astaxanthin dispersible in the aqueous phase without tending to crystallization and without I

the need to add an oily phase to dissolve the astaxanthin. H

The invention further relates to a method of requiring animals to obtain an H

25 reddish pigmentation of their meat and / or products produced by the animals, H

which method is characterized in that an H is administered to the animals

feeds containing Phaffia rhodozyme yeast cells according to claim 1 or cell parts H

containing astaxanthin, in an amount of at least 600 pg per day. g yeast solids determined I

by the method of claim 1, wherein the animals are preferably fish, especially salmon or H

30 sea trout, cows for coloring their butter or poultry for coloring their H

æggebloommer. IN

The amount of feed containing the astaxanthin or astaxanthin-containing I

yeast cells administered to the animals will depend on the animal species and I

35 of the pigmentation effect which it is desired to achieve by means of I

23 The astaxanthin. It is clear that the principle to be followed is that the animal should have a normal recommended daily amount of macro and micronutrients and in addition astaxanthin in a form and amount which will result in the desired pigmentation of the animal's flesh or the subject concerned. animal product. In some cases, the 5 amount of astaxanthin to be administered will be seasonal dependent; Thus, for example, it would not normally be preferable to administer astaxanthin or other carotenoids to cows to achieve a pigmentation of the butter in the summer, the butter pigmentation being usually considered appropriate when the cows graze.

The amount in which the feed containing the astaxanthin or the ten astaxanthin-containing yeast cells or cellular parts is administered to the animals may also in some cases be seasonal dependent. For example, in the case of fish, such as salmon or sea trout, the amount of feed consumed by the fish in winter may be relatively low, which is in contrast to the amount consumed by the fish in summer. However, a suitable amount of feed administered to the fish may be approx. 1.5% of the fish body weight per day, which is in line with the recommendations of the California State Department of Fish and Game.

When poultry is fed by the method set forth above to pigment the egg yolks produced by the poultry meat and / or poultry meat, the feed may consist of conventional poultry feed components, an example being a feed consisting primarily of protein and carbohydrate sources such as soybean meal. , soybean protein, cellulose, starch and fat sources, such as soybean oil, vitamins such as a general vitamin mix and minerals, such as a mixture of the common mineral components for poultry as well as calcium sources for the eggshells, where the calcium sources are preferably calcium carbonate and calcium hydrogen. A small amount of sodium chloride may also be present. The feed can be administered in a traditional dose.

The invention is further illustrated by the following examples:

DK 168542 B3 I

MATERIALS AND METHODS

Maintaining Cultures H

5 Phaffia rhodozyma cures are maintained in two ways: I

1) On inclined agar (YM agar). The oblique agar cultures are incubated for a week at · I

20 ° C and kept at 4 ° C for one month, re-cultured in YM medium before I

new oblique agar cultures are produced. H

2) Cryoprotection at -80 ° C. Derived from cryo vials or I

oblique agar is inoculated into 50 ml of YM medium in 250 ml shake flasks. IN

The shake flask is incubated on an orbitshaker (150 rpm) H

15 at 20 ° C for 4-5 days. The yeast cells are allowed to settle and the liquid I

decanted from. The precipitate is mixed with glycerol to a concentration of

20%, poured into cryo vials and stored in freezer at -80 ° C. IN

Determination of yeast solids content

5 ml of yeast cell culture is centrifuged in a weighed Sarstedt tube (which has been dried to H.

constant weight at 110 ° C) at 10,000 x g for 5 minutes and washed twice in 1

demineralised water. The liquid is removed by decantation and the weight of the tube with I

the cells are measured after drying to constant weight at 110 ° C to give yeast

Cell weight (Y g). Yeast solids content (YDMC) is then calculated as: I

YDMC (g / l) = Y / 5.00 x 1,000 I

The content indicated is the mean of two determinations. IN

30

Spectrophotometric determination of total pigment content

The total pigment content of a methanol extract is determined photometrically by means of I

by Xma, and Beer's law as described by B.H. Davies, "Carotenoids," in T.W. Goodwin I

35 (ed.), Chemistry and Biochemistry of plant pigments, New York, 1976, 2nd ed., P. I

The spectrophotometer used is a Shimadzu UV visible recording spectrophotometer UV 260. Pigment content is calculated using formulas 1, 1a, 2, 2a below and the extinction coefficients from Table 1 below.

TABLE 1

Extinction coefficients for the astaxanthin standard in various solvents prepared as described for the standard solution above 10

Absorption glV

Solvent maximum 1 cm

Acetone 475 2105

Methanol 472 2100 Ethanol 476 2100

Glacial acetic acid 482 1856 ε1% = adsorbance of 1% (w / v) solution in a 1 cm cuvette limb 20

Pigment extraction analysis method 1

Ca. 30 ml of the yeast culture was transferred to Sarstedt tubes and centrifuged for 5 minutes at 10,000 x g. The yeast cells were washed in demineralized water and suspended for about 25 minutes. 20 ml of methanol. To a Bead Beater glass ball mill (Biospec Products Inc., U.S.A.) in which the rotor was covered with 0.4 mm diameter glass balls (about 15 g glass balls), the methanol suspension was added to fill the remaining free ball mill volume. Disintegration treatment was performed by running the mill 5 times for 1 minute at 30 second intervals, keeping ice water in the cooling jacket to ensure that the temperature of disintegration treatment was kept below 20 ° C. Immediately after the disintegration treatment, part of the homogenate was transferred to Sarstedt tubes and the yeast solids were determined as described above, but without centrifugation. A known amount (b g) of the homogenate was transferred to a 10 ml volumetric flask and solvent was added up to 10 ml.

Absorbance was measured in this solution.

DK 168542 B3 I

The total pigment content in pg per g of yeast solids is determined by: I

,. E x 10 ml I

x __ X 1,000,000 (1) I

Elcn> x b * D I

E = absorption at Xmax in the solvent used in a 1 L

cm cuvette H

®lcm = Absorbance of IV (w / v) solution in a 1 cm cuvette I

b = g extract H

D = mg of yeast solids / g of extract I

20 Pigment Extraction and Analysis · Method 2 I

A predetermined amount of the yeast culture (a ml) was transferred to Sarstedt tubes and H

centrifuged for 5 minutes at 10,000 x g. The yeast cells were washed in demine-H

water and suspended for approx. 20 ml of methanol. For a Type I gas ball mill

25 Bead Beater (Biospec Products Inc., U.S.A.) in which the rotor was covered with I

glass balls having a diameter of 0.4 mm (about 15 g of glass balls), methanol suspension was

the pension added to fill the remaining free ball mill volumes. Disintegra- I

Treatment was performed by running the mill 5 times for 1 minute at intervals I

of 30 seconds, holding ice water in the cooling jacket to ensure that disintegrate

The temperature of the treatment was maintained below 20 ° C. Immediately after disinte- I

After the grating treatment, the liquid was transferred to a 50 ml volumetric flask. The glass balls I

Wash in the mill with 4 x 8 ml of methanol. The fractions are collected and mixed and I

methanol is added up to 50 ml. The methanol extract is filtered before pigment analysis. IN

The yeast solids in the culture are determined as described above. IN

35 I

27 DK 168542 B3

The total pigment content per ml sample is determined by: E x 50 ml X '= _ x 10,000 (2) 5 1%

Elcm * a X '= μς pigroent / ml sample a = volume sample in ml 10 E = absorption of the solvent used in a 1 cm cuvette 1%

Elcm = absorbance of 1% (w / v) solution in a 1 cm cuvette 15

The total pigment content per g of yeast solids is determined at 20 X 'Y = _ x 1,000 (2a)

YDMC

Y s / µg pigment / g yeast solids 25 YDMC = g yeast solids / 1 culture liquid HPLC analysis for astaxanthin determination - Method 1 30 HPLC data:

Equipment: 35 Pillars: LKB Ultropac Precolumn, Lichrosorb RP 18 7 μητι

DK 168542 B3 I

4x30 mm, H

LKB Ultropac Column, Lichrosorb RP 18 5 μπη 4x250 mm I

Detector: LKB 2151 variable wavelength monitor (variable I

5 wavelength monitor) H

Integrator: Waters 740 Data Module I

Controller: LKB 2152 HPLC Controller

Pumps: LKB 2150 HPLC pumps I

Autosampler: LKB 2157 autosampler with variable loop I

Manual injection: Rheodyn 20 μΙ loop I

Solvents: I

A: 860 ml of acetonitrile + 100 ml of water + 40 ml of I

Formic acid H

B: 960 ml of ethyl acetate + 40 ml of formic acid I

All solvents are of HPLC grade I

Flow: 1.0 ml / min. IN

Gradients: 0-100% B 20 minutes, linear gradient I

100-0% B 10 minutes, linear gradient I

30 I

Detector: 471 nm I

Temperature: Ambient temperature I

29 DK 168542 B3

Standard Solution: 5 mg pure astaxanthin (melting point 220-222 ° C, absorbance of 1% volume / weight acetone solution in 1 cm cuvette 2100) from Hoffmann-La Roche (hereinafter referred to as the astaxanthin standard) is weighed and dissolved in 500 ml acetone.

5

For HPLC anaphysis, 20 μΙ of that sample is injected into the HPLC chromatograph. HPLC Assay for Astaxanthin Determination - Method 2 HPLC Data:

Equipment: Pillars: Supelco column 15 Supelco LC 18 - DB, particle size 5 μ Column dimensions 4.6 x 250 cm

Detector: LKB 2151 variable wavelength monitor 20

Integrator: Waters 740 Data Module

Controller: LKB 2152 HPLC Controller 25 Pumps: LKB 2150 HPLC pumps

Autosampler: LKB 2157 autosampler with variable loop

Manual injection: Rheodyn 20 μΙ loop 30

Solvents: A: 400 ml tetrahydrofuran 400 ml methanol 200 ml 0.02 M glycine buffer pH = 2.6 B: 1000 ml tetrahydrofuran 35

DK 168542 B3 I

30 I

I All solvents except the buffer are of I

In HPLC grade. The buffer is sterile filtered through an I

In 0.22 mm filter before use I

I Detector: 480 nm I

In Temperature: Room temperature

In Gradients and flow I

I 10 I

I Resolution% Time Flow I

In the mean min. ml / min.

I BO 0-11 1-0 I

I B 0-90 11-21 1-0 I B 90 21-29 1.5

I B 90-50 29-31 15 I

I B 50 31-32 10

I B 50-0 32-35 1-0 I

I BO 35-39 10 I

I 20 _ _ 1

Standard solution: Weigh out 5 mg of astaxanthin standard and dissolve in 500 ml of I

In tetrahydrofuran. IN

I 25 I

I For the HPLC analysis, 20 μΙ of the sample in question is injected into the HPLC chromatograph. IN

In Sarstedt tubes I

I 30 Polypropylene centrifuge tubes equipped with a polypropylene plug of type 55533 from I

In Hounisen's Laboratory, Aarhus, Denmark. IN

In Chemicals I

In 35 Laboratory scale chemicals were of analytical grade. IN

31 DK 168542 B3

Chemicals used in fermentations were of food grade.

Glucose and sucrose concentrations were analyzed using kits (Order No. 139041) from Boehringer Mannheim.

5

Medium for shake coal cultivation and agar plates YM (Yeast Morphology) medium from Difco Laboratories Incorporated (Difco Manual: Dehydrated culture media and reagents for microbiology, 10th edition, Detroit, 10 1984).

cryovials

Polypropylene tubes with a volume of 2.0 ml of type 363401 from Tecnunc, 15 Roskilde, Denmark.

EXAMPLE 1

Mutagenization 20 In each case, the mutagenization treatment was performed to achieve a 1-5% survival rate in the treated culture. Suitable mutants were selected by visually comparing the intensity of the red color from the mutants when plated as single colonies on agar plates.

25 UV radiation

An exactly cloudy 4 day old culture of ATCC 24261 grown in YM medium at 20-22 ° C was diluted in a 0.9% NaCl solution to concentrations of ΙΟ1, ΙΟ-1.5, 10 ', respectively. 2, 10-2.5 and 10'3, and 0.3 ml of each of these dilutions were plated on agar plates to obtain agar plates of 100-300 colonies. The plates were then subjected to ultraviolet radiation at 254 nm for 30 seconds at a distance of 20 cm from the radiation source (Vilbert Lourmat VL 30 LC) and then grown at 20-22 ° C for 10 days, after which the color of the resulting 35 colonies was compared.

32 I

DK 168542 B3 I

EMS (Ethyl Methane Sulfonate) Treatment I

2x15 ml of a 4 day old culture of ATCC 24261 grown in YM medium I

5 on a shaking table at a temperature of 20-22 ° C, was centrifuged for 15 minutes at 1

1250 x g in an MSE Major centrifuge and the pellet was suspended in 2x15 ml sterile I

0.9% NaCl solution in 200 ml centrifuge tube. One of the cell suspensions was used as a control. One gram of EMS (Serva 28755) was added to the other cell suspension.

pensions. After treatment for 30 minutes at 20 ° C, 150 ml of cold, sterile 0.9% I was obtained

10 NaCl solution added. The yeast cells were washed twice in sterile 0.9% NaCl and suspended in 0.9% NaCl. The yeast cell suspension was then diluted and

plated on agar plates in the same manner as described above in connection with I

UV treatment. One of the isolated mutants was deposited with CBS (Centraaal-I

office for Schimmelcultures) on April 6, 1987 under landfill no. 224-87.

(The strain is not covered by claim 1)

Treatment with N-methyl-N'-nitro-N-nitrosoguanidine I

Ca. 25 mg of N-methyl-N'-nitro-N-nitrosoguanidine (Aldrich Chemie BDR) was added to an I

20 10 ml tared measuring glass fitted with glass stopper. Water was added up to one I

total volume of 10 ml and the N-methylhyl-N'-nitro-N-nitrosoguanidine was dissolved in

therein by shaking. 10 x 1 ml stock solution was obtained from this solution

equipment. IN

25 2x20 ml of a 4 day old culture of CBS 224-87 (the mutant obtained in I)

by the aforementioned EMS treatment) grown in YM medium on a shaking I

table at 20-22 ° C, transferred to Sarstedt tubes and subjected to centrifuge twice

miter. The pellet was suspended in 1.5 ml of 0.9% sterile NaCl solution and 1 ml of I

the stock solution prepared above was added. After incubation for 1 hour at I

At 20-22 ° C, the yeast cells were washed 5 times in 10 ml of cold, sterile 0.9% NaCl solution, I

whereby the N-methyl-N'-nitro-N-nitrosoguanidine was removed. The yeast cells became I

then diluted and plated on agar plates in the same manner as described in

above for UV treatment. One of the isolated mutants was deposited with CBS I

on April 6, 1987 under Deposit No. 225-87. IN

35 I

33 DK 168542 B3

Travel insulation CBS 225-87 has been insulated as follows. From a freeze-dried glass, yeast cells are suspended in YM medium and incubated for 5 days at 20-22 ° C. The culture is plated on 5 YM plates and incubated for 10 days at 20-22 ° C and new colonies isolated. One of the colonies was deposited with CBS on March 23, 1988 under landfill no. 215-88.

EXAMPLE 2 10

Determination of astaxanthin content in wild-type Phaffia rhodozyma strains and in mutants prepared in Example 1

The yeast cell culture and the astaxanthin assay described in this example, on the one hand, constitute the conditions under which yeast cells are grown and, on the other hand, the conditions under which astaxanthin is determined in the applicant's aforementioned standard method for determining the yeast strain's inherent astaxanthin producing ability. These are the same standard conditions as those mentioned in the claims.

20

Shake flask culture 100 μΙ of a 4 day old culture of ATCC 24261 grown in YM medium on a shaking table of 20-22 ° C was inoculated into 50 ml of YM medium contained in a 500 ml shake flask with 2 baffle plates. The culture was grown on a shaking table with shaking in circular motion at 150 rpm. per minute for 5 days at 20-22 ° C and an oxygen transfer rate of 30 mmol / l / hour, whereby the yeast cell culture reached a density of 0.6%.

30

pigment Analysis

The astaxanthin content of the extract was identified by the following three methods.

DK 168542 B3 I

34 I

1. An acetone, methanol and ethanol extract, all of which were prepared as I

described for the methanol extract preparation above was subjected to spectro-I

photometric scanning, and the values listed in Table 1 were obtained. IN

2. To one half of a 10 ml ethanol extract prepared in the same manner I

as above, was set approx. 50 mg of potassium borohydride to reduce astaxanthin, and I

the mixture was stirred for 30 minutes. The absorptions of the extract and of the I

potassium borohydride treated sample was measured at varying wavelengths of I

spectrophotometer. The free astaxanthin showed a wide peak at 480 nm, and it I

10 reduced astaxanthin showed two peaks at 450 and 476 nm, corresponding to those I

values listed in the literature. IN

3. The retention time of the astaxanthin-containing extracts by HPLC under standard I

conditions as defined above were compared to the retention time of the I

15 standard solution defined above, i.e. the peaks of the astaxanthin-containing I

sample according to the invention obtained by HPLC under standard conditions became I

compared to the peaks of the standard solution obtained by HPLC. Retention I

the times turned out to be identical. IN

The mutant strain of the invention (CBS 225-87) as well as the known deposited I

astaxanthin producing P. rhodozyma strains were grown and analyzed on I

the same way as described above. The total pigment content of the strains and I

astaxanthin content is listed in Table 2 below. IN

Total pigment analysis was performed according to the method: Pigment extraction and analysis - I

Method 1. Astaxanthin was analyzed by HPLC analysis for astaxanthin determination

method - method 1

30 I

35 I

35 DK 168542 B3 TABLE 2

Strain µg of total µg of astaxan pigment / g thin / g of yeast dry of yeast dry substance CBS 5905 = ATCC 24202 = OCD 67-210 332 254 CBS 5908 = ATCC 24203 s UCD 67-383 318 252 CBS 6938 303 204 10 CBS 6954 <50 <100 ATCC 24201 = OCD 67-203 229 143 ATCC 24203 = UCD 67-383 338 164 ATCC 24228 = UCD 68-653C 254 107 ATCC 24229 = UCD 67-202 287 142 ATCC 24230 = UCD 67- 385 247 132 15 ATCC 24261 = UCD 67-484 449 286 CBS 224-87 885 570 CBS 225-87 (strain of the invention) 1176 706

The values given are the mean of 4 independent measurements. It should be noted that the mutant strains of the invention exhibit a significantly increased astaxanthin content.

EXAMPLE 3 (REFERENCE) Fermentation

The fermentations were carried out as fat-batch fermentations under carbohydrate restriction in carefully washed and sterilized 4 m 3 Bubble Column fermenters with a stationary aeration system consisting of perforated trachea.

The fermentors were equipped with pH electrodes, inlets for pH regulators and antifoam agents, and alcohol detectors for measuring alcohol in the exit air. The fermentor sheaths were thermostated.

The starting wort had the following composition: 20 g / l molasses, 0.6 g / l diammonium sulfate, 0.8 g / l diammonium hydrogen phosphate, and 0.125 g / l magnesium sulfate.

I DK 168542 B3 I

I 36 I

In sulphate, which was all cooked in a fermentor for 30 minutes with I

In an appropriate amount of water (30 I in the propagation fermentor of 100 I and 2000 I in I

In the production fermentor pi 4 m3) before the fermentor in question was inoculated. IN

In the medium fed to the fermentor in the fat-batch fermentation came from two I

In 5 different holdings, ie. a chemical stock containing 10 kg I

In diammonium sulfate, 5.6 kg of diammonium hydrogen phosphate and 80 L of water; and an I

stock of molasses containing 450 kg of molasses and 1000 l of water which had become 1

In the autoclave. 0.1 mg of desthiobiotin and 1.6 kg of magnesium sulfate were fed directly to I

In the fermentor before the rest of the medium was added. All the chemicals were from foodstuffs

In 10 part quality. The molasses was sugar beet molasses from the Danish sugar factories. IN

In the aeration during fermentation was 8.4 m3 per minute. Contraspum 210 I

I (Zschimmer & Schwartz) was used as antifoaming agent and as pH-I

Sulfuric acid was used in the regulating agent. IN

I 15 I

Yeast cells of the strain ATCC 24261 were propagated by transfer from an I

In sloped agar for a tube of 2 cm diameter containing 5 ml YM-I

In medium in which the cells were grown for 4 days on a shaking table under sufficient I

In aeration at a temperature of 20-22 ° C, the culture was transferred to 2-1 L

In 20 Erlenmeyer flasks containing 1 L of YM medium. After incubation for 3 days on an I

In shaking tables and under sufficient aeration at a temperature of 20-22 ° C, I became

In 1 L of culture transferred to a 100 L fermentor containing 30 L of starting watch. The Culture I

You were subjected to batch cultivation at 20-22 ° C until a yeast dry I was obtained

In substance content of 1 g / l. Then, the nutrient addition was started and fat batch I

The fermentation was carried out at 20-22 ° C. After 2 days of growth, 30 L of culture was I

I transferred under sterile conditions by means of a peristaltic pump to 4 m3-I

In the fermentor, which contained 200 I of starting watch. The culture was subjected to batch cultivation

Stir at 20-22 ° C until a yeast solids of 1 g / l was obtained in the culture. Then you

In, the molasses addition was started and continued for 38 hours, after which the molasses retention I

In the 30 th was exhausted. The chemicals were added proportionally to the molasses during I

For the first 24 hours. The fat-batch fermentation was carried out at a temperature of 20 I

At 22 ° C. The amount of molasses in the molasses stock was adjusted so that no I

Yeast content of more than approx. 4% in the fermented herb. IN

37 DK 168542 B3

The molasses addition to the fermentor during fat-batch fermentation was adjusted to achieve a specific growth rate of the yeast cells of μ = 0.15 hour'1 and was further regulated by the ethanol concentration in the herb, which should be less than 0.1% by volume. Thus, the ethanol concentration was often measured and when it was found to be too high, the molasses addition rate was reduced until again an acceptable ethanol content was obtained.

The aeration of the fermented herb was continued for 16 hours at 20-22 ° C without any nutrient addition.

The composition of the yeast cells as well as their total pigment content and astaxanthin content were measured at time 0, ie. just before the nutrient addition to the 4 m3 fermenter was started, after 38 hours of fermentation and growth, and after 16 hours of aeration of the fermented herb. The total pigment content and astaxanthin content were determined as described in Example 2, and the composition of the yeast cells was determined by conventional techniques. The total nitrogen content was thus determined by Kjeldahl analysis, the trehalose content was determined as described in Journ. Am. Chem. Soc. 72, 1950, p. 2059, and the phosphoric acid content was determined as described in Water and Wastewater, 20 American Public Health Association, Inc. pp. 199 (1960). Ethanol analysis was performed by Boidin's method of determining small amounts of alcohol (cf. Annal, de la brasserie et de la distillerie, 1924-25, p. 177). The results are listed in Table 3 below.

Total pigment analysis was performed according to method 1. Astaxanthin analysis by HPLC was performed according to method 1. In 30 35

DK 168542 B3 I

TABLE 3 I

Fed-batch fermentation of ATCC 24261 I

m

Timer O 18 24 31 38 45 54 -

μς total pigment / g H

yeast content 181 218 284 415 471 579 I

µg astaxanthin / g H

yeast solids content H® ^ 30 ^ 00 ^ 50 H

% w / w yeast solids content 0.08 0.48 0.95 2.75 3.06 3.15 3.29 H

% v / v ethanol 0.00 0.00 ° · 00 ° · 10 ° '00 ° '00 I

pH 4.74.4 4.04.04.57.88.5

V w / w N in yeast solids- H

15 content 6'2 6.1 5.7 I

V w / w P-, Otr in yeast solids- H

content 2.7 2.6 2.4

V w / w trehalose in yeast * H

substance content - - 2.6 4.4 11.4

EXAMPLE 4 (REFERENCE) I

In a manner similar to that described in Example 3, fat-batch I was obtained

fermentations with strain ATCC 24261 performed, the only difference being that I

The starting watch volume was 1000 I, inoculum in the 4 m 3 fermenter was 6x11 ATCC I

24261, which had been propagated as set forth above, and the chemical stock I

consisted of 0.5 kg of diammonium sulfate, 2.8 kg of diammonium hydrogen phosphate and 80 l

In water, the stock of molasses consisted of 250 kg of molasses and 1000 l of water. Under I

the fat-batch fermentation, the nutrients were fed to the fermentor in the first I

30 hours, after which the nutrient addition was stopped and the culture was subjected to aeration for 72 hours.

The results are given in Table 4 below. IN

39 DK 168542 B3 TABLE 4

Fed-Batch Fermentation of ATCC 24261 5 -: ---

Hours O 65 137 µg total pigment / g yeast solids content 149 379 561 µg astaxanthin / g yeast solids content 89 231 369 10 * w / w yeast solids content 0.05 3.5 3.75 '/ v / v ethanol - 0.0 pH 4.5 7, 8 9.1 V w / w N in yeast solids content - 4.1 4.5 15 V w / w P2Os in yeast solids content - 1.9 2.2 V w / w trehalose in yeast solids content 2.6 13.2 12.8 EXAMPLE 5

Experiments with the mutant strain CBS 225-87 of the invention were carried out in the same manner as described in Example 4, with a starting watch volume of 1000 I, with inoculum being 6x11 CBS 225-87 propagated by the method described in Example 3, and with a chemical stock consisting of 5 kg of diammonium sulfate, 2.8 kg of diammonium hydrogen phosphate and 80 l of water. No alcohol was formed during the fat-batch fermentation and the nutrients were added between hours 0 and 57, after which the culture was subjected to aeration without nutrient addition. The yeast cell composition at 30 hours 80 was 6.5% nitrogen in yeast solids, 2.5% phosphorus pentoxide in yeast solids and 6.6% trehalose in yeast solids. The total pigment, astaxanthin and yeast solids content were determined during the fat-batch fermentation to give the values listed in Table 5 below:

DK 168542 B3 I

40 I

TABLE 5 I

Batch Batch Fermentation of CBS 225-87 I

ς

Fermentation Solids- Total pigment Astaxanthin

content i i i i i

sample yeast sample yeast H

Hours g / 1 »jg / ml µg / g pg / ml µ9 / 9 I

1.4 0.9 640 I

22 11.2 8.1 720 I

33 18.4 12.5 680 I

37 20.0 15.6 780 11.0 S50 I

40 20.8 16.5 790 8.8 420

57 23.8 24.0 1010 16.2 680 I

15 gi 24.7 24.1 980 17.0 690 64 24.3 2S, 5 10S0 17.3 710 80 24.6 36.6 1490 23.6 960

20 I

The total pigment and astaxanthin content in ng / ml has been calculated from that I

analyzed yeast solids content and pg / g values of total pigment and astaxanthin. IN

EXAMPLE 6

25 I

The pigment and astaxanthin content of CBS 215-88 and P. rbodozyma mutant strains

DBT 406 and DBT 403, wild-type strains CBS 5905 and ATCC 24261 were determined. IN

The mutant strains DBT 406 and DBT 403 are derived from the wild-type strain ATCC 24230. I

30 Shake coal cultivation I

From a scry agar, the yeast cells were inoculated into YM medium and incubated for 2 days at H

20-22 ° C. 1 ml of culture was inoculated into 50 ml of YM medium contained in 250 ml of I

shake flasks with 4 baffle plates. The culture was grown on a circular shaking table

35 shaking at 150 rpm. per minute for 5 days at 20-22 ° C. IN

41 DK 168542 B3

Quantitative determinations of total pigment were performed according to method 2. Astaxanthin determination was performed according to method 2.

S Examples of calculations (for CBS 215-88):

The total pigment content of the methanol extract was determined by spectrophotometric analysis. The absorption of the 50 ml extract minus the absorption of methanol was measured to E = 1.189. The sample volume was 29 ml. The total pigment content of the yeast extract was calculated according to formula (2) as follows: X '= 1.160 x 50/2100/29 x 10,000 f.g / ml = 9.52 pg / ml The yeast solids content was determined to be 6.7 g / l. The total pigment content per g yeast solids are determined by formula (2a) as follows: Y = 9.52 / 7.1 x 1,000 pg / g = 1340 pg / g

The astaxanthin concentration in the methanol was determined by HPLC analysis 20 to 6.4 pg / ml corresponding to: 6.2 pg / ml / 7.1 g / l = 880 pg astaxanthin / g yeast solids

All strains were grown and analyzed in the same manner as described above.

The total pigment and astaxanthin content of the strains is given in Table 6.

30 35

I DK 168542 B3 I

I I

I TABLE 6 I

I Strain Yeast solids content Total pigment Astaxanthin I

I 9/1 pg / mi pg / g pg / n »i pg / g I

I CBS 5905 5.3 1.38 260 0.84 160 I

I ATCC 24261 5.5 2.10 380 1.38 250 I

I DBT 406 2.2 5.84 2650 3.4 1540 I

In DBT 403 1.6 5.04 3150 3.3 2050. IN

I 10 CBS 215-88 7.1 9.52 1340 6.2 880 I

It is noted that DBT 406 and DBT 403 have a high content of astaxanthin per day. g I

In yeast, however, these strains grow poorly, as evidenced by yeast solids

In the contents / l. IN

EXAMPLE 7 I

In 20 Fed batch fermentation of the CBS 215-88 mutant strain of the invention I

I was carried out using corn molding liquid solids and sucrose as

In carbohydrate sources in the same 4 m3 fermentor as described in Example 3. I

The Start Tour consisted of: I

In 10 10 kg of corn casting liquid solids I

In 12 kg of sucrose I

In 10 kg of diammonium sulphate I

In 3 kg of potassium dihydrogen phosphate I

In 1 kg of magnesium sulfate I

0.05 g of biotin I

In 300 ml anti-foam Contraspum 210 I

I 1000 I water I

I was sterilized at pH 4.6 by steam injection at 95 ° C for one hour and after cooling I

At 35 to 22 ° C, 6x1 of CBS 215-88 propagated as above was added as I

43 DK 168542 B3 inoculum. After 35 hours of aeration (4.2 m 3 / min), addition of sucrose solution (0.30 g / L) was initiated. The rate of addition was 2.3 l / h. The aeration was increased to 8.4 m3 per hectare. minute and the pH regulator started (set point 4.0).

When the sucrose concentration in the medium was reduced to ca. 1 g / l after 28 hours, 5 the sucrose addition was increased to 7.3 l per liter. hour and kept at this speed for 24 hours. Thereafter, the sucrose addition was stopped and the aeration rate was reduced to 4.2 m per minute and continued for 72 hours. Total pigment and astaxanthin were determined during the fat-batch fermentation to give the values listed in Table 7.

TABLE 7

Batch Batch Fermentation of CBS 215-88 15 __________: -—-

Hours of sucrose supply 28 52

Hours of aeration after completion of sucrose application ® 20 µg total pigment / ml culture medium 14.1 37.7 43.4 µ9 astaxanthin / ml culture medium - 23.4 29.9 pH 4.0 4.0 4.0% w / w N in yeast solids content - - 7.69% w / w P205 in yeast solids content - - 3.72 30 V w / w trehalose in yeast solids content - - 4.4 35

DK 168542 B3 I

The yeast cells were separated from the medium by centrifugation in a De Laval I

0A5M centrifuge and washed with water 2 times. The yeast cells were separated from I

the yeast flow by filtration in a FILTROX filter, type VARJOX 40/40 cm, and I

the filter cake with 26.3% solids was extruded through a 1 mm sieve in a laboratory I

5 fluid bed dryers (GLATT, Haltingen-Binzen Bd.) And dried at 30 ° C for 90 minutes. IN

The dried yeast (91.6% solids) contained H

1360 μς total pigment / g yeast solids content

10 1080 μg astaxanthin / yeast solids content I

EXAMPLE 8 I

Downstream processing I

IS I

Yeast cells obtained by the method of Example 3 were isolated from the I

fermented herb by centrifugation in a De Laval OA5M centrifuge. The cells became I

washed twice with water and a yeast cream with 13% yeast solids was obtained. IN

The pH of the yeast flow was adjusted to 4.0 by the addition of sulfuric acid, and I

20 sodium benzoate was added to a concentration of 0.2% (w / v) in yeast I

In the cream. During treatment, the isolated yeast cells were kept in a nitrogen I

In atmosphere so as to avoid significant oxidation of the astaxanthin in yeast

In the cells and the temperature was kept at approx. 10 ° C. IN

In 25 the yeast flow was sent three times through a system consisting of an APV-Gaulin I

In MC4 homogenizer equipped with a cell destruction valve and a heat exchanger,

In which the yeast flow was subjected to disintegration at a pressure of 700 bar, whereby I

In the temperature was increased by 10-15 ° C, and subsequently cooled in the heat exchanger to 1

At a temperature of 15 ° C. The yeast flow was circulated in the system with an I

At 30 speeds of 2501 pr. hour. IN

The astaxanthin content of the disintegrated cells was determined as follows:

In 0.5 ml homogenized yeast cream was weighed and transferred to a Sarstedt tube and I

In the shake with 5 ml of acetone. The sample was centrifuged, transferred to a 10 ml volumetric flask I

I 35 and washed three times with acetone with intermediate centrifuges and I

45 DK 168542 B3 quantitative transfers. The total pigment content was determined by Method 1 and the astaxanthin content was determined by HPLC Method 1 and the content was related to the total dry matter content of the sample as determined by the method explained in the Materials and Methods section above. By comparing the 5 extractable astaxanthin content in the yeast flow homogenized as described in this example with the content of the yeast flow determined by Method 1, where the cells were completely disintegrated, the degree of disintegration in this example was determined to account for more than 90% of the total cells.

To the disintegrated cells thus kept in nitrogen atmosphere, 7% sodium caseinate was added at a temperature of ca. 45 ° C with stirring. The yeast cell homogenate in admixture with sodium caseinate was then subjected to spray drying in an Anhydro spray tower in which the input temperature was 180 ° C. The yeast cell mass was atomized using a spray wheel and the temperature of the outlet air from the spray tower had a temperature of approx.

90 ° C. The resulting yeast powder was collected using a cyclone. The water content of the yeast powder was less than 10% by weight.

EXAMPLE 9 20

Extraction of total pigment with glacial acetic acid 20 g of non-destroyed Phaffia rhodozyma yeast cells (which had been filtered and subsequently extruded in a fluidized bed where they had been dried) with a dry solids content of 95% and containing 523 μm astaxanthin per ml. g of yeast solids was introduced into a column of 12 cm in length and an inner diameter of 2.4 cm.

The column was equipped with a jacket in which water with a temperature of 75 ° C was circulated. At the bottom of the column, a small amount of acid washed sand (a sand filter) was placed on a cotton pad. The yeast cells were extracted with 5 x 100 ml glacial acetic acid at a temperature of 75 ° C, and the amount of astaxanthin in each of the extracts as well as in the extracted yeast cell material (comprising about 100 ml glacial acetic acid remaining in the column) was determined. The extracted yeast cell material had been evaporated to dryness (resulting in 16.16 g of material) before the astaxanthin assay was performed. The results are listed in Table 8 below.

35

DK 168542 B3 I

I 46 I

I TABLE 8

In Total astaxanthin- H

In yeast cell contents H

I 5 before extraction 20 g x 523 // g / g 10460 Mg I

I Astaxanthin content in I

In IQ 1. Extract 100 ml x 55.1 µ ^ / κΐ 5570 Mg I

In 2nd extract 100 ml x 14.6 Mg / ml 1460 µg I

In 3rd extract 100 ml x 4.4 Mg / ml 440 µg M

In 4th extract 100 ml x 3.1 Mg / ml 310 µg m

I Total astaxanthin- I

Extract content 7780 µg I

I Astaxanthin content i

In extracted dry- I

In yeast material 16.16 g x 22.5 µg / g 363.6 µg I

I 20 I

I Total astaxanthin- I

In content released from I

In yeast cells at the extraction 8143.6 µg I

I 25 I

Thus, in 77.9% (8143.6 / 10460 x 100%) of the yeast cells' astaxanthin content, I

In released by the extraction. IN

EXAMPLE 10 I

In Destruction of Yeast Cells by Homogenization in a Ball Mill I

In Phaffia rhodozyma yeast cells which had been dried in a fluid bed and contained

In 35 336 Mg of astaxanthin / g of yeast solids, was suspended in soybean oil in a concentrate.

47 DK 168542 B3 tion of 40% by weight. The suspension was pumped to a ball mill (CoBall® mill type MSZ-12) containing zirconium balls (0.1-1.5 mm) and with a ball content of 70-75%. The stroke of the rotor was 13 m per second. second. Samples were taken after each run and the astaxanthin content of the samples was analyzed by HPLC. The temperature of the ball mill was maintained at 40-50 ° C.

Similarly, a suspension of the above dried yeast cells in 75% water was dried in a ball mill. In this treatment, the rotor speed was 15 m per second. second.

10

The results are given in Table 9, where the astaxanthin content is stated as pg / g of yeast solids TABLE 9 15 60% soybean oil 75% water First run 221 179 20 Second run 308 192

Third Driving 333 27®

In comparison, only 23 µg of astaxanthin per ml was obtained. g of yeast solids when the dried yeast was treated with soybean oil or water without simultaneous homogenization.

In the experiment it is shown that approx. 3 runs in the ball mill are sufficient to achieve a substantially total destruction of the yeast cell.

30 35

DK 168542 B3 I

I 48 I

EXAMPLE 11 I

In Feeding fish

In 5 fish feeds with varying astaxanthin content were prepared. The fish feed became you

Made from commercial fish feed Ecoline 16 from Danish Trout feed, Brande, I

In Denmark, which is a mixture of fish meal, soy flour, fish oil, extruded wheat,

In lecithin and vitamins in the form of a premix. Different amounts of astaxanthin were given

You added this feed. The astaxanthin was obtained from P. rhodozyma yeast cells which were I

I have been grown in the same manner as described in Example 3 and which had become I

In the spray dried. The spray-dried yeast cells were prepared from 28 kg of homogenized I

In yeast cream which had been mixed with 0.475 kg of sodium caseinate dissolved in 2.7 kg

In water at approx. 50 ° C. 0.068 kg GRINDTEK MOR 50 containing 2 g of ascorbyl palmitate I

I and 1 9 soybean tocopherols were emulsified in the sodium caseinate solution. IN

The sodium caseinate solution (containing antioxidants) was mixed with the I

In homogenized yeast cream and the mixture was spray dried as described in I

In Example 8. The spray dried product (92.6% solids) contained approx. 674 pg I

In astaxanthin per g yeast solids. The spray dried product was added to it

In divided fish feed so that the varying astaxanthin concentrations were obtained

In 20 tions in the fish feed (feed A-D), as seen in Table 10 below. IN

A fish feed containing synthetic astaxanthin (feed E) was used as I

In control. The feed E contained astaxanthin in an amount equal to 40 ppm. IN

In 25 Feeds A-E have the following composition:

I 30 I

I 35 I

49 DK 168542 B3 TABLE 10 Fish feed

5 A B C D E

Astaxanthin Fg / g feed 4.4 12.8 20.4 39.2 40

Synthetic astaxanthin Dry content V 91.94 91.49 91.56 91.88 88.81 10 Ash% 9.12 9.00 8.79 8.45 7.03

Cellulose V 1.46! / 45 1'60 2.05 1.71

Protein% 43.62 43.20 43.86 43.81 43.15

Fat v 17.59 17.21 18.62 20.00 21.19

Phosphorus g / kg H.58 10.89 11.25 10.80 9.96 Nitrogen-free extract V 20.15 20.53 18.69 17.57 15.73

Ca. 60 rainbow trout, each weighing approx. 400 g, were used in these experiments with each of the fish feed types A-E. The fish were kept in cages and fed ad libitum. The water 20 had a temperature in the range of 2.5-14 ° C, with the lower temperatures being in the latter part of the fish feeding experiment.

Extraction of astaxanthin from fish 25 The equipment used is the equipment normally used in laboratory experiments.

A skinless rainbow trout was cut and 15 g of meat was weighed into a centrifuge tube (100 ml). 15 ml of tetrahydrofuran was added as an extractant.

The meat was further comminuted in an ULTRATURAX mixer and subsequently centrifuged. The tetrahydrofuran tract was transferred to a 50 ml volumetric flask. The residue was washed with 10 ml of tetrahydrofuran for 2-3 minutes on a sonication bath and centrifuged, and the tetrahydrofuran phase was transferred to the flask to which additional tetrahydrofuran was added up to 50 ml. 10 ml of the 50 ml were evaporated under a nitrogen atmosphere at a temperature of 40 ° C. evaporative

DK 168542 B3 I

the residue was redissolved in 1 ml of mobile phase and filtered through a 0.45 µm filter before

further analysis. IN

HPLC Analysis I

• I

Column LiChrosorb RP-18, 5 µm, 250 x 4.6 mm I

Mobile phase: 40 ml formic acid I

60 ml of water I

384 ml of ethyl acetate I

516 ml of acetonitrile I

Flow: 10 ml per mine. IN

Injection: 20 μΙ loop, Rheodyne 7120 I

Pump: Waters 510 I

Detector: Waters 481, UV Spectrophotometer, 471 nm I

15 Integrator: Waters 740 I

Determination of the color of the fish

The color of the fish meat was determined by the L * a * b * color determination method under I

20 using a Minolta Chroma Meter II. The L * value denotes the light component, I

a * value (assuming values from -60 to +60) denotes the green / red compo- I

nent (the negative values denote the green component and the positive values I

denotes the red component), and the b * value (assuming values from -60 to 1

+ 60) denotes the blue / yellow component of the color. Only the a * value is listed in Table 11. I

25 I

Rainbow trout meat without skin was homogenized in a blender to obtain an I

homogeneous mass which was placed in small petri dishes (with a height of 1 cm and a diameter of 3.5 cm), so as to fill this whole volume. The surface of

the mass in the petri dish was smoothed and covered with a glass plate and then I

30 ready for analysis. IN

The data obtained for the fish feeding trial are given in Table 11 below:

35 I

51 DK 168542 B3 TABLE 11

Analysis of the fish feed for 16 days j µg astaxanthin / fish color of fish g fish weight (g) meat (a *)

Feed A 0.50 380 1.45 0.60 434 1.12 0.65 252 0.08 0.60 580 0.48 0.65 392 -0.02

Feed B 1.45 343 1.53 0.15 385 0.05 0.20 298 -0.52 1.00 388 0.52 0.60 317 -0.33

Feed C 0.75 471 0.00 0.85 290 1.20 0.4S 328 0.23 0.30 300 -0.38 0.75 359 -0.38

Feed D 0.70 258 -0.20 0.45 643 0.72 1.00 369 2.05 0.70 450 0.50 0.30 382 -0.10

Feed E 0.25 374 -0.80 0.60 339 0.12 0.80 405 0.98 0.65 579 0.36 1.20 534 1.18

52 I

DK 168542 B3 I

TABLE II (continued) I

Analysis of the fish feed for 23 days I

µg asthma color of I

xanthine / the fish's fish- H

g fish weight (g) meat (a *) M

Feed A 0.65 668 0.76 I

0.60 571 1.00 I

0.40 535 0.26 I

0.50 469 -0.50 I

0.20 386 0.06 I

Feed B 0.40 348 0.02 I

0.40 325 0.16 I

0.75 500 1.32 I

0.75 355 0.16 I

0.50 406 0.18 I

Feed C 1.25 356 1.25 I

1.05 464 1.88 I

1.05 317 1.98 I

0.25 301 -1.44 I

0.50 329 -0.44 I

Feed D 0.50 287 -0.14 I

0.65 380 1.34 I

0.45 436 0.62 I

1.65 449 3.38 I

1.85 409 3.26 I

Feed E 2.00 487 4.08 I

0.50 429 0.82 I

0.35 673 2.53 I

1.20 441 3.16 I

1.15 404 0.90 I

53 DK 168542 B3 TABLE 11 (continued)

Analysis of the fish feed for 30 days µg astaxia of xanthine / fish fish - g fish weight (g) meat (a *)

Feed A 0.80 410 1.12 0.60 448 0.78 0.50 409 1.50 0.55 483 1.40 0.65 352 0.40

Feed B 0.75 344 0.35 0.35 410 1.17 0.45 547 0.40 0.35 493 2.20 0.95 228 2.14

Feed C 1.10 517 3.30 0.95 405 1.48 1.55 381 2.26 0.75 330 1.48 0.95 413 1.80

Liner D 2.15 635 6.10 0.85 384 1.68 2.10 363 0.56 1.70 423 3.70 1.00 348 1.92

Feed E 1.15 390 1.84 2.10 427 4.00 3.00 433 4.14 0.40 337 0.02 0.55 342 0.85

DK 168542 B3 I

TABLE 11 (continued) I

Analysis of the fish feed for 43 days

µg asthma color of H

xanthine / the fish's fish- H

g fish weight (g) meat (a *) H

Feed A 1.00 429 2.50 0.60 300 -0.56

0.90 848 2.32 I

0.50 417 0.16 I

0.45 385 -0.54 M

Feed B 0.75 623 1.94 H

1.00 352 1.88 H

1.00 620 1.44 I

0.75 484 1.52 0.95 441 0.78

Feed C 0.75 604 1.30 I

0.90 480 1.84 I

1.20 540 2.96 I

2.20 444 4.78 I

1.35 414 0.74 I

Feed D 1.45 471 3.66 I

2.10 436 6.61 I

2.15 508 5.56 I

3.00 510 6.04

1.15 381 1.72 I

Feed E 1.20 512 2.40 I

3.00 452 4.84 I

4.30 634 8.04 I

1.75 474 4.43 I

3.70 517 5.46 55 DK 168542 B3 TABLE li (continued) 'i

Analysis of the fish feed for 72 days µg astaxis of xanthine / fish fishg fish weight (g) meat (a *)

Lining A 0, S5 409 1.08 0.70 628 4.02 1.95 1177 3.84 0.70 663 1.42 1.65 401 1.34

Feed B 1.05 386 2.24 0.90 666 2.46 0.30 507 2.46 2.05 585 4.90 1.20 518 1.52

Feed C 1.15 701 2.40 1.80 415 4.42 4.80 739 9.36 5.00 451 7.26 4.00 594 5.48

Feed D 1.15 444 2.32 2.05 514 6.98 4.25 493 8.40 4.80 612 8.24 4.25 633 6.66

Feed E 3.05 627 6.32 3.75 618 6.26 4.65 507 9.78 5.95 654. 8.56

The results show that the astaxanthin in each of the fish feed mixtures AE has been absorbed by the fish and that the fish meat achieves increasing red pigmentation with increasing amounts of astaxanthin in the feed (feed AD) and with increased feeding time (as observed at the a * value (indicating red color) component)).

I DK 168542 B3 I

I 56 I

In addition, the results suggest that the presence of astaxanthin in the feed does not

You influence the growth of the fish. IN

In Pisces was also assessed visually and generally turned out to have an attractive red I

In 5 colors. After 43 days of feeding there was no significant difference in pigment I

In the dice of fish fed with feed D and E (containing about 40 ppm I respectively

In astaxanthin produced according to the invention and 40 ppm synthetic astaxanthin). After . IN

In 72 days of feeding, no significant difference in pigment was observed

In the cage of fish fed with feed C, D and E (containing about 20 ppm I respectively

In 10 astaxanthin produced according to the invention, 40 ppm astaxanthin produced according to I

In the invention and 40 ppm synthetic astaxanthin). IN

I

I 15 I

In 1. Phaffia rhodozyma yeast cell, I

In which is the yeast strain deposited under landfill no. 225-87 CBS, or I

In the yeast strain deposited under deposit no. 215-88 CBS, or a mu- I

In a tant or a derivative thereof, or a mutant or a derivative of the yeast strain which I

I 20 is deposited under landfill no. 225-87-CBS, which has retained the I

In astaxanthin producing ability and which, when grown under conditions that I

You comprise an oxygen transfer rate of at least 30 mmol. I per hour at Difco I

In YM medium at 20-22 ° C for 5 days in 500 ml shaker flasks with two baffle plates I

Containing 50 ml of the medium and subjected to shaking by circular motion at I

At 25,150 rpm. per minute, inoculum being 100 μΙ of a 4 day old YM culture, I

You produce astaxanthin in an amount of at least 600 pg per day. g yeast solids determined I

I by HPLC analysis using pure astaxanthin as a standard of an I

In the methanol extract of the yeast prepared by subjecting a suspension of I

In 0.2 g of yeast solids in 20 ml of methanol 5x1 minute disintegration at intervals of one I

For 30 half minutes, disintegrating at a temperature not exceeding 20 ° C in an I

In a glass ball mill containing 15 g of glass balls with a diameter of 0.4 mm, I

In the glass ball mill is equipped with a cooling jacket with ice water. IN

The yeast cell of claim 1

Claims (12)

B3 characterized in that, when grown under the conditions set forth in claim 1, it produces astaxanthin in an amount of at least 700 µg per day. g of yeast solids, preferably at least 1000 pg. g of yeast solids, in particular at least 1500 pg. g of yeast solids and in particular at least 2000 pg. g yeast solids, as determined by the method of claim 1. Process for the preparation of astaxanthin-containing Phaffia rhodozyma cells or cell parts or astaxanthin, characterized in that astaxanthin-producing Phaffia rhodozyma yeast cells, as defined in claim 1, are grown under aerobic conditions in a medium containing carbohydrate sources, assimilable nitrogen and phosphorus sources. , micro-nutrients and biotin or desthiobiotin at a temperature in the range of 15-26 ° C so as to obtain a biomass containing astaxanthin in an amount of at least 600 µg per day. g yeast solids as determined by the method of claim 15 1, and optionally performing one or more of the following steps in arbitrary order: The cells are harvested from the culture so as to obtain a yeast cream, the cells are opened, e.g. destroying the cell walls by mechanical, chemical and / or enzymatic treatment and / or subjecting the cells to sonication, autolysis, osmolysis and / or plasmolysis, optionally with the addition of suitable agents such as detergents, acids, bases, enzymes, autolysis enhancers. , osmolysing agents such as salts, and / or plasmolysing agents, the cells are homogenized to obtain a homogenate, the cells, cell fragments or homogenate dried, preferably to a water content of not more than 12% by weight, preferably not more than 10% of astaxanthin extracted from the cells, cell fragments. or the homogenate. Process according to Claim 3, 30, characterized in that the culture is carried out as a fat-batch fermentation under conditions where substantially no alcohol is formed, and optionally where the total glucose and sucrose concentration is at most 8 g / l, preferably 5 g / l, and especially 1 g / l, preferably at a temperature of 20-22 ° C, wherein the fermentation or part thereof is carried out in a medium comprising molasses and / or sucrose and 58 I DK 168542 B3 In nitrogen sources such as diammonium sulfate, ammonium phosphate, ammonium hydroxide or urea, phosphorus sources such as ammonium phosphate and phosphoric acid and added micronutrients or mineral salts such as magnesium sulfate, zinc sulfate and biotin or desthiobiotin are added to the medium. separate from the other constituents. IN Process according to claim 3 or 4, characterized in that the cultivation comprises a growth phase under conditions which are substantially sufficient for substantially all growing conditions, and a subsequent growth restricted phase under conditions wherein In the aeration medium under continued aeration, at least one growth factor is depleted so as to increase the production of astaxanthin during the subsequent phase, with the subsequent growth restricted phase preferably having a duration of at least approx. 16 hours, such as 16-24 hours, with the subsequent growth-restricted phase preferably being adjusted to increase astaxanthin production at least 1.2 times the production obtained without the subsequent phase, preferably at least 1.3, such as In 1.4, and in particular 1.5 times the production obtained without the subsequent phase, with the subsequent growth restricted phase being preferably carried out substantially without any addition of any nutrient or micronutrient to the medium. IN 20 I Process according to claim 5, characterized in that the concentration of astaxanthin in the obtained biomass is I at least 700 pg. g yeast solids as determined by the method of claim 1 25 I Process according to any of claims 3-6, characterized in that the yeast cell being cultured is a yeast cell according to claim 1 I or 2, wherein the astaxanthin concentration in the obtained biomass is at least 800, especially at least 1000 µg. per. g of yeast solids, in particular at least 1500 pg. g yeast dry In 30 fabrics, for example at least 2000 pg per day. g of yeast solids, and most preferably at least I 3000 pg. g yeast solids as determined by the method of claim 1. I A method according to any one of claims 3-7, characterized in that the yeast cell after cultivation and, optionally, after the growth restricted phase, is subjected to one or more of the following treatments: I 59 DK 168542 B3 Destruction of the cells by exposure the cells at an increased pressure and then releasing the pressure and subsequently subjecting the destroyed cells to ultrafiltration or evaporation so as to concentrate the destroyed yeast cells, destroying the cells by subjecting the cells to an increased pressure and then releasing the pressure and subsequently spray drying or drum drying the cells. broken cells, | filtering to obtain a filter cake which is then extruded, and then the j extrudate is dried, for example in a fluid bed or by tray drying, J mixture of dried whole cell material which has been dried in one; fluid bed or by tray drying, with an oily phase such as an edible oil or edible fat, and grinding the mixture in a mill such as a ball stirrer mill for destroying the cells and releasing astaxanthin to the oily phase, extracting broken cells which are preferably homogenized with organic solvents such as petroleum ether, acetone or an alcohol such as methanol, ethanol or ίεομΓορβηοΙ, to obtain astaxanthin dissolved in the organic solvent and then possible removal of the solvent for example by evaporation; extracting whole cells with a solvent comprising glacial acetic acid, to obtain astaxanthin contained in the glacial acetic acid solvent, supercritical extraction of broken or whole, wet or dry carbon dioxide cells, optionally with organic solvents, for example of the above type, the treatments being preferably carried out under oxygen limited conditions such as in an inert atmosphere where, for example, the inert atmosphere is provided by water vapor and / or by nitrogen and / or carbon dioxide, the extraction of the entire yeast cells with the glacial acetic acid solvent preferably being carried out by a temperature above the freezing point of the solvent, for example in the range 20-100 ° C, preferably in the range 20-80 ° C, and especially in the range 20-60 ° C. 35 _____ ___ 1 I 60 I DK 168542 B3 I Animal feed, characterized in that it comprises Phaffia rhodozyme yeast cells according to claim 1 I or yeast cell parts containing astaxanthin in an amount of at least 600 µg per day. g II yeast solids as determined by the method of claim 1, eg yeast cells according to II 5 of claims 1 or 2 or parts thereof or yeast cells or yeast cell parts prepared by the II method of any of claims 3-8, together with other II feed constituents, wherein the astaxanthin-containing yeast cells or yeast cell constituents constitute II not more than 10% by weight of the total dry matter of the animal feed composition, preferably II not more than 5% and especially not more than 3%, the other components being preferably selected from II 10 protein and carbohydrate sources such as fish meal , whey, blood meal, vegetable flour, II fats such as fish oil and vegetable oils, vitamins and minerals. IN A method of feeding animals to obtain a reddish pigmentation of II their meat and / or of products made from the animals, II characterized in that a feed is administered to the animals containing II Phaffia rhodozyma yeasts according to claim 1 or cell parts containing II astaxanthin in an amount of at least 600 µg per ml. g yeast solids as determined by the method of claim 1, wherein the animals are preferably fish, especially salmon or sea trout, cows for coloring their butter or poultry for coloring their egg yolks. IN