Simple Method for the Isolation of Astaxanthin from the ... - NCBI

APPLIED AND ENVIRONMENTAL MICROBIOLOCY, June 1978, p. 1155-1159

0099-2240/78/0035-1155$02.00/O Copyright i 1978 American Society for Microbiology

Vol. 35, No. 6

Printed in U.S.A.

Simple Method for the Isolation of Astaxanthin from the Basidiomycetous Yeast Phaffia rhodozyma ERIC A. JOHNSON, TOMAS G. VILLA, MICHAEL J. LEWIS, AND HERMAN J. PHAFF* Department of Food Science and Technology, University of California, Davis, California 95616

Received for publication 27 February 1978

A method is described for the quantitative and, possibly, large-scale extraction of astaxanthin from the yeast Phaffia rhodozyma. The method utilizes extracellular enzymes produced by the bacterium Bacillus circulans WL-12, which partially digests the yeast cell wall and renders the carotenoid pigments extractable by acetone or ethanol. Complete recovery of astaxanthin from heat-killed P. rhodozyma cells was obtained after growing B. circulans WL-12 on these yeast cells for 26 h and then extracting the yeast-bacterium mixture with acetone. A bacteria-free lytic system, which gave quantitative extraction of astaxanthin from P. rhodozyma, was obtained by concentrating the culture broth from the growth of B. circulans WL-12 on P. rhodozyma cells. Hydrolytic enzyme activities detected in this concentrate included f8-(1 -+ 3)-glucanase, ,B-(1 -* 6)-glucanase, a-(l -+ 3)-glucanase, xylanase, and chitinase. The lytic system was found to work most efficiently at pH 6.5 and with low concentrations of yeast. WL-12, both obtained from the culture collection of the Department of Food Science and Technology, University of California, Davis, Calif. P. rhodozyma was maintained on slants of yeast extract-malt agar (Difco Laboratories, Detroit, Mich.) and B. circulans on yeast nitrogen base agar (Difco) buffered at pH 6.5 with 0.1 M sodium phosphate buffer, containing 0.5% (wt/vol) xylan (Sigma Chemical Co., St. Louis, Mo.) as the carbon source. Preparation of CW. P. rhodozyma was grown as described below, harvested by centrifugation, and washed twice with water. Yeast cell walls (CW) were then prepared by the method of Benitez et al. (3). Growth of organisms. P. rhodozyma was grown for 50 h in a 60-liter fermentor at 22°C with an air flow rate of 40 liters/min and vigorous stirring (400 rpm). The growth medium (50 liters) contained 20 g of cerelose (CPC Int., Englewood Cliffs, N.J.) and 5 g of yeast extract (Difco) per liter of broth. Yeast growth was assayed by determining the dry weight; the cells were centrifuged from a known volume of medium, washed in distilled water, and dried in an air oven at 100°C to a constant weight. All WC and CW concentrations are expressed on a dry weight basis. B. circulans WL-12 was grown under two sets of conditions. In the first experiment, it was grown for 40 h in 2.8-liter Fernbach flasks which contained 1.0 liters of yeast nitrogen base medium buffered at pH 6.5 with 0.1 M sodium phosphate buffer. WC or CW of P. rhodozyma were used as sole carbon sources at a concentration of 2 g/liter. In the second experiment, 2 liters of an actively growing bacterial culture (3.7 x 10'0 cells/ml) in the WC medium was inoculated directly into a 40-h fermentor culture of P. rhodozyma cells. Before inoculation of B. circulans, the pH of the broth was adjusted to 6.5 with 2 M sodium phosphate MATERIALS AND METHODS buffer (final molarity = 0.08 M) and heated to 800C Organisms. The organisms used in this study were for 5 min. This procedure inhibited further yeast P. rhodozyma (UCD-FST 67-210) and B. circulans growth and inactivated extracellular enzymes pro1155

Yeast lipids are sometimes defined in an operational sense as being readily extractable from whole yeast cells (WC) by neutral solvent mixtures (e.g., chloroform-methanol) or as being bound and extractable in neutral solvents only after acid hydrolysis of the yeast cell (10). The carotenoid pigments of yeasts belong to the latter category. Unfortunately, carotenoids are quite sensitive to acids, and such a treatment may lead to considerable pigment destruction (4). Generally, their quantitative recovery from yeast cells first requires severe mechanical treatment of the yeast, such as passage through a French pressure cell or treatment in a Braun homogenizer (11). These methods are capable of treating only small volumes of cells, and they are expensive and laborious for the large-scale isolation of carotenoid pigments. Astaxanthin (3,3'-dihydroxy-4,4'-diketo-,B-carotene) is a pigment commonly found in marine and freshwater animals. Recently it has also been isolated from a yeast, Phaffia rhodozyma (1). The large-scale isolation of astaxanthin from this yeast is desirable because of its potential use as a pigment source in fish diets (5). Because this compound is labile to acids and bases, we sought a gentler method for its extraction. We describe here a method of using the extracellular enzymes produced by Bacillus circulans WL-12 to treat the yeast and allow complete extraction of astaxanthin by acetone.

1156

APPL. ENVIRON. MICROBIOL.

JOHNSON ET AL.

duced by P. rhodozyma. Bacterial growth was mea- taxanthin was estimated in the centrifuged acetone solution as described by Johnson et al. (5). sured by direct cell counting in a Levy chamber. Inoculum preparation of B. circulans. Because B. circulans relied upon the hydrolysis of insoluble RESULTS substrates (WC or CW) as carbon sources, it was necessary to build up the volume of inoculum graduExcretion of hydrolases by B. circulans ally. The culture was transferred from a slant to 1 ml WL-12. As indicated above, B. circulans was of WC or CW medium. This was then sequentially transferred to volumes of medium of 5 ml, 10 ml, 50 able to grow in WC or CW medium because the bacterium secreted different hydrolases which ml, and 1 liter. Culture broth concentration and assay of en- supplied the microorganism with the necessary carbon sources. After 40 h of bacterial growth in zyme activities. The broths obtained from B. circulans grown in shake flasks were freed of debris by shake flasks in WC medium, recognizable yeast centrifugation at 10,000 x g for 10 min at 4°C. The cells were still present, even though observation broths were then concentrated approximately 10-fold by phase-contrast microscopy revealed a severe by ultrafiltration in a pressure cell (Amicon Corp., degradation of the CW. Growth for the same Lexington, Mass.) with UM-10 membranes. The conof time in CW medium produced, in centrate was diluted in the cell with 2 volumes of period contrast, complete dissolution of P. rhodozyma water, and the mixture was reconcentrated. Samples obtained from fermentor culture were cen- CW. The WC and CW broths were concentrated, trifuged as described above, and the supernatants were dialyzed, and assayed for hydrolytic activities 3)- and f8-(1 freeze-dried. The residues were taken up in 15 ml of (Table 1). In both media, f3-(1 0.05 M sodium succinate buffer (pH 6.0) and dialyzed 6)-glucanases were, by far, the major enzyme for 20 h against the same buffer at 4°C. activities detected, although the concentration Substrates for enzyme assays were prepared by of f8-(1 3)-glucanase in the CW broth was dissolving (or suspending) 0.5% (wt/vol) substrate in significantly lower than in the WC broth. In 0.1 M sodium succinate buffer (pH 6.0). The substrates contrast, the CW lytic system showed a higher used were laminarin (Nutritional Biochemicals Corp., Cleveland, Ohio), pustulan (Calbiochem, San Diego, chitinase activity but slightly less xylanase activ3)-glucan, carboxy- ity. No protease, a-mannanase, fB-(1 4)-gluCalif.), xylan (Sigma), a-(1 4)-glucanase activities were methyl chitin, yeast mannan, soluble starch (Rascher canase, or a-(1 and Betzold, Inc., Chicago, Ill.), and bovine serum detected in either of the culture broths. albumin (Miles Laboratories, Elkhardt, Ind.). Yeast Digestion of P. rhodozyma with isolated 3)-glucan, and carboxy-methyl chitin lytic systems. The WC and CW lytic systems mannan, a-(l were obtained from the carbohydrate collection of this were equally effective, under the experimental laboratory. Sodium azide was added at a concentration conditions tested, in their ability to render the of 0.02% to prevent bacterial growth. Standard enzyme astaxanthin solvent extractable (Table 2). reactions were performed as described by Villa et al. yeast the WC system was easier to prepare Because which of enzyme (13). One enzyme unit is the amount catalyzes the release of 1 nmol of reducing sugar than the CW, the former was used in subsequent equivalent (determined according to the method of experiments. Autoclaving or boiling P. rhodozyma before Nelson and Somogyi [7, 12]) expressed as D-glucose, D-xylose, or N-acetyl-D-glucosamine per min at 30°C. incubation with the WC lytic system did not Protease activity was determined according to Kunitz promote faster digestion and pigment release. (6). The yeast suspension tended to gel when boiled, Extraction and assay of astaxanthin. The sedi- and some of its pigment was destroyed. The WC ment obtained by centrifugation in a Sharples centri- lytic system had an optimum pH for pigment fuge consisted of bacterial and yeast cells. Approxi- release of 6.5 (Fig. 1), and its effectiveness demately 20 times its volume of acetone was added to the pellet, and the mixture was throughly stirred. For creased rapidly at lower or higher pH values. comparison, P. rhodozyma was also disrupted in a The concentration of yeast in the incubation Braun homogenizer and extracted with acetone. As- mixture influenced the efficiency of astaxanthin -*

-s

TABLE 1. Activities in lytic enzyme preparations on various polysaccharides obtained by growing B. circulans for 40 h on WC or CW' Substrate

WC prepn

CW prepn

Associated enzyme activity

,B-(1 3)-Glucanase (EC 3.2.1.6) 34.0 61.5 ,B-(1 6)-Glucanase (EC 3.2.1.75) 68.5 69.7 a-(1 3)-Glucanase (EC 3.2.1.59) 2.3 2.1 fi-(1 4)-Chitinase (EC 3.2.1.14) 7.7 2.0 CM-chitin' 3.4 ,B-(1 4)-Xylanase (EC 3.2.1.32) 5.4 Xylan and Methods. in Materials as defined milliliter units per The results are expressed in b CM-chitin, carboxy-methyl chitin. Laminarin Pustulan a-(1-s 3)-Glucan

ASTAXANTHIN RECOVERY FROM P. RHODOZYMA

VOL. 35, 1978

TABLE 2. Comparison of the abilities of the WC and CW lytic systems to affect the extractability of astaxanthin from WC' Time of treatment (min)

Astaxanthin (ug) extracted from yeast cells treated with:

cw

WC

0.3 0.3 0 3.8 3.9 50 5.4 5.3 100 6.4 6.2 180 7.1 7.1 240 a Assays were carried out with 112 mg of cells plus 15 ml of lytic system. Samples of 3 ml were taken at the indicated times, centrifuged, and extracted with acetone. The activities of these enzyme solutions are shown in Table 1. 100

c 0

t;

2 x 0

-

0

50

N.40

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was grown in a 60-liter fermentor for 40 h, heated at 800C, and then inoculated with 2 liters of an actively growing culture of B. circulans. Assay of the astaxanthin in the yeast by mechanical disruption in a Braun homogenizer revealed that the pigment content of the yeast decreased from 513 ug to 439 yig/g during the heat treatment. The concentration of yeast at the time of inoculation with the bacterium was 5.2 g/liter. Growth of B. circulans, as reflected by an increase in cell number, began approximately 8 h after inoculation (Fig. 2) and continued until 20 h when a constant number of bacteria was obtained. Pigment extractability was apparent after 14 h and proceeded nearly to completion by 20 h. The extractability of astaxanthin lagged slightly behind bacterial growth but began before the release of nondialyzable carbohydrate. The pH of the fermentation broth decreased before pigment release but increased during the period in which the astaxanthin became extractable. After harvest by centrifugation, the yeast-bacterium cell mass was extracted with acetone, and the pigments were transferred to petroleum ether and concentrated by rotary evaporation at 30°C to a thick, red oil. The crude extract showed a visible absorption spectrum identical

9

.2 w

I a. /5

65

s5

5

pH

FIG. 1. Influence ofpH and yeast concentration on astaxanthin extractability. The incubation was done for 3 h and contained 0.5 ml of WC lytic system and 0.5 ml of a suspension of P. rhodozyma containing 5.6 mg (C), 11.5 mg (0) or 23 mg (0) of yeast. The ordinate expresses the percentage of total astaxanthin extracted from the incubation mixtures. The pigment was not released from the cells incubated without entyme.

extraction (Fig. 1). At the lowest yeast concentration (5.6 mg/ml), all of the pigment became extractable after 3 h of incubation at the optimum pH. At the highest yeast concentration (23 mg/ml), only 25% of the astaxanthin became extractable. At the two highest yeast concentrations, astaxanthin extractability was improved by longer incubation with the lytic system (not shown). Large-scale digestion of P. rhodozyma and extraction of astaxanthin. To determine the effectiveness of the bacterial lytic system on a larger scale than shake flasks, P. rhodozyma

E I

(A

3 a;,

I

x a

I

"I..

I#A

.x i

-

1a

c0- 4

0

z 0

.

E

am .U

0; 10

20 Time (hours)

FIG. 2. Growth of B. circulans WL-12 (0) and astaxanthin extracted (@) from P. rhodozyma in fermentor culture. Changes in pH (U); concentration of nondialyzable carbohydrate (O). See text for details.

1158 JOHNSON

ET AL.

with that of astaxanthin obtained from the yeast by mechanical fracture (Ama = 478 nm in acetone). The yield of astaxanthin from 257 g of yeast was 113 mg, which represented 100% extraction, compared with a value obtained from samples subjected to mechanical disruption. The acetone-extracted yeast-bacterium residue was dried to a white powder and weighed 210 g. The synthesis of extracellular enzymes by B. circulans was followed in the fermentor culture (Fig. 3). Increases in the activities of ,8-(1 -* 6)glucanase and xylanase began between 8 and 14 h of bacterial growth. A slight increase of a-(1 * 3)-glucanase activity in this time period was also detected. The 13-(1-- 6)-glucanase activity increased steadily until 22 h and then reached a constant value. The xylanase activity reached a constant level after only 18 h of incubation. Increases in ,f-(1 -* 3)-glucanase and chitinase activities were noticeable after 15 h of incubation and continued to increase in activity until harvest. The growth of B. circulans and digestion of P. rhodozyma were also followed by phase-contrast microscopy. The CW appeared partially digested after 22 h. Nevertheless, most of the cells maintained their normal morphology.

APPL. ENVIRON. MICROBIOL.

DISCUSSION The majority of carotenoid-containing yeasts belong to the Teliomycetes (Basidiomycotina, or their imperfect counterparts), and their CW are thought to be considerably more complex than those of ascomycetous yeasts (2). Because the tough CW is believed to be the barrier which prevents the thorough extraction of carotenoids (11), it is necessary to weaken this barrier to allow solvent penetration and consequent carotenoid extraction. This hypothesis is supported by the fact that spheroplasts of yeast or mechanically disrupted cells are amenable to carotenoid extraction. The enzymatic breakdown of the CW is preferred because it is gentle and is unlikely to cause destruction of the carotenoid

pigments.

The potent hydrolytic activities of B. circulans were confirmed in this study with the yeast, P. rhodozyma, which is thought to be related to the Basidiomycotina. When cultured on the CW of this yeast, B. circulans produced extracellular enzymes which completely degraded the CW. Although WC were not completely digested, their loss of integrity was apparent both under the microscope and because high centrifugal forces were required to sediment the yeast after digestion. The rise in nondialyzable carbohydrate and pH supports this observation. The WC lytic system did not show an ini 1240_ creased rate of degradation of autoclaved yeast cells over intact, thawed cells. It would be expected that autoclaving would partially disrupt cellular integrity and render the yeast more susceptible to enzymatic attack. The autoclaved yeast suspension became quite viscous during autoclaving and gelled upon cooling. It is possi0 ble that the gel prevented the enzymes from digesting the CW. B. circulans WL-12 produced a number of carbohydrases when cultured on CW or on WC. The highest activities detected includedfl-(l -:Z 30 / and fl-(1 -* 6)-glucanases. These enzymes 3)0 have been previously implicated in the digestion .E 30 (9). In addition we observed the synthesis U1-_ / ofof CW xylanase, a-(1 -* 3)-glucanase, and chitinase, although their contribution to P. rhodozyma CW digestion is unknown. The xylanase may be important in the degradation of CW, since basidiomycetous yeasts frequently have CW or capsules containing xylose (e.g., Cryptococcus albi10 20 dus [8]). In fact, we have found that isolated CW from P. rhodozyma do contain a small, yet Time (hours) FIG. 3. Excretion of extracellular enzymes by B. significant, proportion of xylose (E. A. Johnson, circulans in fermentor culture. The substrate con- T. G. Villa, M. J. Lewis, and H. J. Phaff, unpubsisted of heat-killed P. rhodozyma cells (5.2glliter of lished data). The cell concentration treated with the lytic broth). Symbols represent ,f-(1---6)-glucanase (0), ,8(1- 3)-glucanase (0), xylanase (A), a-(1---3)-glucan- system was critical since, at high yeast concenase (O), and chitinase (-) activities. trations, very little astaxanthin became extract-

0~~~~~~

ASTAXANTHIN RECOVERY FROM P. RHODOZYMA

VOL. 35, 1978

able. The limited extractability of astaxanthin is probably due to a decreased hydrolysis of CW. The pH of incubation was also important. The optimum pH of 6.5 for enzymatic hydrolysis and astaxanthin extraction coincided with the optimum pH for growth of B. circulans. The enzyme excretion by the bacterium appeared to follow a definite pattern during the 26h fermentation. A rapid increase in laminarinase was detected, which corresponded with the release of astaxanthin. The potency of this gentle and simple method for large-scale preparation of astaxanthin was evident when 257 g of P. rhodozyma was inoculated with B. circulans. In only 22 h, all of the astaxanthin became extractable from the yeast. ACKNOWLEDGMEN'TS We thank M. Miranda for capable technical assistance. T. G. Villa is indebted to the Commission of Cultural Exchange between Spain-U.S.A. for financial support during the course of this work. This research and a traineeship for E. A. Johnson were sponsored by NOAA, Office of Sea Grant, Department of Commerce, under grant number NOAA-04-6158.

LITERATURE CITED 1. Andrewes, A. G., H. J. Phaff, and M. P. Starr. 1976.

Carotenoids of Phaffia rhodozyma, a red-pigmented fermenting yeast. Phytochemistry 15:1003-1007. 2. Bartnicki-Garcia, S. 1968. Cell wall chemistry, morphogenesis and taxonomy of fungi. Annu. Rev. Microbiol.

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22:87-108. 3. Benitez, T., T. G. Villa, and I. Garcia Acha. 1975. Chemical and structural differences in mycelial and regeneration walls of Trichoderma viride. Arch. Microbiol. 105:277-282. 4. Davies, B. H. 1976. Carotenoids, p. 38-165. In T. W. Goodwin (ed.), Chemistry and biochemistry of plant pigments, vol. 2. Academic Press Inc., New York. 5. Johnson, E. A., D. E. Conklin, and M. J. Lewis. 1977. The yeast Phaffia rhodozyma as a dietary pigment source for salmonids and crustaceans. J. Fish. Res. Board Can. 34:2417-2421. 6. Kunitz, M. 1947. A modified method for the determination of amino acids. J. Gen. Physiol. 30:291-298. 7. Nelson, N. J. 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153:375-380. 8. Phaff, H. J. 1971. Structure and biosynthesis of the yeast cell envelope, p. 135-210. In A. H. Rose and J. S. Harrison (ed.), The yeasts, vol. 2. Academic Press Inc., New York. 9. Phaff, H. J. 1977. Enzymatic yeast cell wall degradation, p. 244-282. In R. E. Feeney and J. R. Whitaker (ed.), Food proteins. Advances in Chemistry Series, no. 160. American Chemical Society, Washington, D.C. 10. Rattray, J. B. M., A. Schibeci, and D. K. Kidby. 1975. Lipids of yeasts. Bacteriol. Rev. 39:197-231. 11. Simpson, K. L, C. 0. Chichester, and H. J. Phaff. 1971. Carotenoid pigments of yeasts, p. 493-515. In A. H. Rose and J. S. Harrison (ed.), The yeasts, vol. 2. Academic Press Inc., New York. 12. Somogyi, M. 1952. Notes on sugar determination. J. Biol. Chem. 195:19-23. 13. Villa, T. G., M. A. Lachance, and H. J. Phaff. 1977. On the structure of the fi-(1 -+ 3)-glucan component of the cell wall of baker's yeast. FEMS Microbiol. Lett. 1:317-319.