Extractability of astaxanthin in a mixed culture of a carotenoid over-producing mutant of Xanthophyllomyces dendrorhous and Bacillus circulans in two-stage batch fermentation

Process Biochemistry 37 (2002) 1235– 1245 www.elsevier.com/locate/procbio

Extractability of astaxanthin in a mixed culture of a carotenoid over-producing mutant of Xanthophyllomyces dendrorhous and Bacillus circulans in two-stage batch fermentation Tony J. Fang *, Joh-Ming Wang Department of Food Science, National Chung Hsing Uni6ersity, 250 Kuokuang Road, Taichung 40227, Taiwan, Republic of China Received 13 September 2001; received in revised form 2 December 2001; accepted 4 December 2001

Abstract The productivity and extractability of astaxanthin in a mixed culture of Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) NCHU-FS501, an astaxanthin over-producing mutant, and Bacillus circulans CCRC 11590 was evaluated in a 1.5 l fermentor using a two-stage batch fermentation technique. The first stage was for X. dendrorhous cultivation. The second stage was the mixed fermentation of the red yeast and B. circulans. The highest lytic enzyme activity of B. circulans was found at 24 h with yeast nitrogen base (YNB) as the nitrogen source and incubation at 30 °C in a pure culture. The induction of B. circulans lytic enzyme activity was influenced by the cell wall concentration of X. dendrorhous. The fastest induction was observed with addition of 2.5 g/l cell wall. The cultivation time in the first stage of fermentation significantly affected the extractability of carotenoid in the second stage of fermentation. Minimum extractability was found when X. dendrorhous was cultivated for 96 h in the first stage. Glucose concentration of 45 g/l in YNB supported high X. dendrorhous growth and astaxanthin production, with astaxanthin production of 9010 mg/l was observed during the first stage. Although the red yeast produced more astaxanthin with peptone as nitrogen source during the first stage, YNB supported the highest extractability of carotenoid and lytic activity of B. circulans in the second stage of fermentation. The optimal pH and temperature for pigment extraction in the mixed culture were pH 6.5 and 30–34 °C, respectively. Under these conditions, about 97% of the total pigment could be extracted after a 48 h incubation time. When the crude enzyme was recycled in the mixed culture, lytic enzyme activity of 20 units/ml was obtained after one incubation cycle (48 h), with 69% of total carotenoids extracted. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Phaffia rhodozyma; Xanthophyllomyces dendrorhous; Two-stage batch fermentation; Astaxanthin; Mixed-cultures

1. Introduction Astaxanthin (3,3%-dihydroxy-b, b%-carotene-4, 4%dione) is a commonly encountered keto-carotenoid in certain algae, many invertebrates and fish [1]. The use of astaxanthin as colourant in aquaculture, especially as feed supplement in farmed trout, salmon and prawns, is necessary to obtain the natural red – pink colours since they are not capable of de novo synthesis of carotenoids [2]. In addition, astaxanthin was also found to have antioxidation [3 – 5] and antitumor activity [6,7]. While the positive effects of astaxanthin on farmed fish * Corresponding author. Tel.: + 886-4-2286-1505; fax: + 886-42287-6211. E-mail address: [email protected], [email protected] (T.J. Fang).

and crustaceans have been recognized for years, the potential benefits of this pigment to human health are only now being revealed. Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) is a red-pigmented fermenting yeast discovered in 1976 [8]. The red yeast is strikingly different from other pigmented yeasts in that it synthesizes and accumulates the carotenoid astaxanthin as principal carotenoid pigment [9]. Several investigations, including the one in our laboratory, have reported the isolation of astaxanthin over-producing mutants and diphenylamine resistant mutants [10 –16]. The production of astaxanthin by X. dendrorhous (or P. rhodozyma) in batch, fed-batch and continuous cultures has been reported by our laboratory as well as by other researchers [17 –19]. Disruption of the cell wall of yeast biomass prior to addition to animal diet is essential for intestinal absorp-

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tion of the pigment. Related investigations have described several chemical, physical, autolytic and enzymic methods of cell wall disruption [20– 22]. Okagbue and Lewis [21,23] described a mixed culture of X. dendrorhous and Bacillus circulans for yeast-wall hydrolysis. Since these two microorganisms differ in growth conditions, further studies were needed to improve the efficiency of cell wall disruption by the mixed culture technique. This investigation examines the feasibility of using a two-stage batch culture as a fermentation system for astaxanthin production, as well as the extraction in a mixed culture of astaxanthin over-producing mutant of X. dendrorhous and B. circulans.

2. Materials and methods

2.1. Microorganisms The astaxanthin over-producing mutant of X. dendrorhous NCHU-FS501 was obtained by N-methyl-N%nitro-N-nitrosoguanidine treatment (MNNG, Sigma Chemical Co., St. Louis, MO) of X. dendrorhous CBS6938. B. circulans CCRC 11590 was obtained from Culture Collection and Research Center, Food Industry Research and Development Institute (Taiwan, Republic of China). The red yeast was maintained on slants of yeast malt agar (YM agar, Difco) at 4 °C. B. circulans CCRC 11590 was maintained at 4 °C on slants of a medium containing phosphate-buffered (KH2PO4, 0.05 M, pH 6.5) yeast nitrogen base agar (YNBA, Difco). X. dendrorhous and B. circulans were stored in 40% glycerol and YM broth (1/1, v/v) and 40% glycerol and YNB broth (1/1, v/v) at −80 °C, respectively.

2.2. Culti6ation media and conditions Flask cultures were shaken at 150 rpm in an orbital shaker incubator (S304R, Firstek Scientific, Taiwan) at 22.5 °C in 500-ml Hinton flasks. The seed culture of X. dendrorhous NCHU-FS501 was prepared by inoculating the yeast from a fresh slant into a 500-ml Hinton flask containing 100 ml YM broth, and incubating for 48 h in a rotary shaker (22.5 °C, 150 rpm). The yeasts were then centrifuged, washed twice with distilled water, and re-suspended in 100 ml YM broth. The seed culture of the B. circulans was prepared in the same way except that the culture was grown in YNB broth at 30 °C for 20 h. For the preparation of cell wall-induced lytic system, B. circulans CCRC 11590 was grown in Hinton flasks (250 ml), containing 50 ml of YNB broth with 5 g/l cell wall of strain NCHU-FS501 and buffered at pH 6.5 with 0.05 M phosphate buffer. The ability of B. circulans to produce cell wall lytic enzyme in various nitrogen source was tested in YNB broth supplemented with casein, peptone, tryptone, urea, (NH4)2SO4,

NH4NO3, NH4H2PO4, and KNO3. Additional nitrogen source, when used, were included at an equimolar level equivalent to 7 g/l total nitrogen. The medium (pH 6.5) for two-stage batch fermentation contained 15 g/l glucose, 5 g/l peptone, 7 g/l yeast nitrogen base (YNB) and 0.05 M KH2PO4.

2.3. Preparation of cell walls of X. dendrorhous Cell walls of X. dendrorhous NCHU-FS501 were prepared from 48 h-grown cultures in YM broth. Cell slurries were mixed with glass beads (0.45–0.50 mm) and broken in a carbon dioxide-cooled Braun mechanical cell homogenizer (B. Braun, model MSK, Germany). Cell walls of X. dendrorhous were washed four times with Tris buffer (0.1 M, pH 8.5) followed by four times with chilled distilled water [23]. Cell wall suspension was then lyophilized and stored at 0 °C.

2.4. Fermentation equipment and conditions Two-stage batch cultivation was conducted in a bench-top fermentor (B. Braun, Model Biostat B, Germany) containing 1.5 l YNB broth. Constant stirring (400 rpm) and aeration rate (3.6 vvm) were maintained during the fermentation period. Foam was suppressed, when necessary, by the addition of an anti-foamer (KM-72, Shinetu Chemicals Co., Ltd, Tokyo). For two-stage batch fermentation of the mixed-cultures, an inoculation (10% of the working volume) of the yeast was transferred from the seed culture to the fermentor and incubated at 22.5 °C for up to 96 h. During the second period of the two-stage batch fermentation, a 10% inoculum of B. circulans was made at 24, 48, 72 and 96 h during the first-stage fermentation. After B. circulans was added, the fermentation temperatures were then maintained at 22.5, 26, 30 and 34 °C, respectively. The pH of the mixed cultures was controlled at 6.0, 6.5 and 7.0 using 6 N H2SO4 and 6 N NaOH. The incubation time of the mixed cultures in YNB was 48 h in addition to the cultivation time of the first-stage.

2.5. Recycle of the lytic enzyme complex After the mixed cultures were grown in this two-stage batch cultivation for 120 h (72 h for the first stage and 48 h for the second stage), the mixed cell cultures were centrifuged (4 °C, 13 000 rpm, 10 min). Then NaN3 (0.4 mg/ml) was added to the supernatant to inhibit the growth of the cells. After the pH was adjusted to 6.5, the lytic activity of the supernatant (enzyme solution) was determined. Recycle of lytic enzyme complex was tested by adding 1.5 l of enzyme solution to a batch of 72-h X. dendrorhous cells, which was grown in 1.5 l of YM broth at 22.5 °C, centrifuged and washed twice. The mixture was then incubated at 30 °C for 48 h in a

T.J. Fang, J.-M. Wang / Process Biochemistry 37 (2002) 1235–1245

rotary shaker (100 rpm). Samples were taken every 12-h for the determination of residual sugar, carotenoid, lytic activity and extractability of carotenoid.

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statistical analysis system. The results were also analyzed using analyses of variance and Duncan’s test at the 5% significance level [26].

2.6. Analytical methods Viable count determinations were made in replicate on Nutrient Agar (Difco) for B. circulans and on Potato Dextrose Agar (Difco, pH 3.5) for X. dendrorhous. The plates for B. circulans and for X. dendrorhous were incubated at 30 °C for 24 h and at 20 °C for 72 h, respectively. Residual sugar concentration in the culture medium was determined with 3,5-dinitrosalicylic acid [24]. Qualitative determination of lytic activity of B. circulans was based on reduction in turbidity of suspensions of X. dendrorhous cell walls. The preparation and reaction of lytic activity was as described by Okagbue and Lewis [23]. The decrease in turbidity was calculated by the following equation: %Decrease = [(Abs0 − Abst )/Abs0]× 100 where, Abs0 denotes the initial absorbance at 600 nm of the reaction mixture, and Abst is the value at time t. The amount of enzyme that caused a 50% reduction in absorbance (600 nm) in 60 min was defined as 20 lytic units/ml. The dimethyl sulphoxide (DMSO) method as described by Sedmak et al. [25] was used to rupture X. dendrorhous prior to extraction of the carotenoids into hexanes:ethyl acetate 50%:50% (v/v) (HPLC grade). The concentration of astaxanthin was estimated by measuring the absorbance at 480 nm with a Hitachi U2000 spectrophotometer (Hitachi Instruments, Japan). The extinction coefficient [25], and astaxanthin concentration was determined as described by Fang and Chiou [17]. The astaxanthin produced by the red yeast was measured quantitatively by HPLC methods [17]. Samples for HPLC analysis were diluted in hexanes:ethyl acetate 50%:50% (v/v) and then made to 0.1% with glacial acetic acid and filtered (0.22 mm) prior to injection. The eluting solvent was ethyl-acetate:methanol:water=5:18:2 (v/v/v) with a flow rate of 0.5 ml/min and the eluant was monitored at 474 nm. Extractability of carotenoid was calculated for any sample by employing the formula [23]:

3. Results and discussion

3.1. Parameters influencing lytic enzyme acti6ity of B. circulans The lytic enzyme activity of B. circulans influenced by various nitrogen sources is shown in Fig. 1. This organism was cultivated in a medium containing 5 g/l cell wall of X. dendrorhous, 0.05 M KH2PO4, and nitrogen sources at an equimolar level equivalent to 7 g/l total nitrogen. After 24 h of incubation at 30 °C and 150 rpm in 250-ml flasks, YNB supported the highest enzyme activity (60 units/ml) produced by B. circulans, followed by urea and peptone with lytic enzyme activities of 35 and 25 units/ml, respectively. Similar lytic enzyme activities were observed in media containing

%Extractability=[FEC (mg/ml)/TC (mg/ml)] × 100 where, FEC denotes the free extractable carotenoid that was determined as the amount extractable by the direct acetone treatment of cell pellet, total carotenoid (TC) was extractable in acetone only after DMSO treatment [25]. The formation of osmotically fragile cells of X. dendrorhous in the mixed culture was monitored by taking 50 ml sample, treated with crystal violet and safranine O, diluting them 20-fold with distilled water, then viewed under a phase-contrast microscope (model AFT-II, Nikon, Japan). Results were analyzed by a

Fig. 1. Influence of different nitrogen sources on lytic enzyme activity of B. circulans CCRC 11590.

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incubation, which was the highest activity among the temperatures tested in this investigation (Fig. 2A). After 48 h of incubation, similar activities were found in the groups of 30 and 25 °C. B. circulans grown at 40 °C showed the lowest lytic enzyme activity compared to the other groups (Fig. 2A), although the optimal temperature of this crude lytic enzyme was found to be 40 °C (data not shown). Previous studies have shown that the optimal temperature for X. dendrorhous growth and total volumetric astaxanthin formation was 22.5 °C [17]. In order to overcome the different growth conditions of X. dendrorhous and B. circulans, a two-stage batch fermentation was performed.

3.2. Two-stage fermentation with different culti6ation time of X. dendrorhous

Fig. 2. Effect of temperature (A) and cell wall concentration of X. dendrorhous (B) on the formation of lytic enzyme by B. circulans CCRC 11590.

casein, peptone, tryptone and YNB after 48 h of incubation (Fig. 1). Johnson et al. [27] indicated that b-(1 “ 6) and b-(1“ 3)-glucanases were the most important enzymes in X. dendrorhous cell wall lysis. High lytic enzyme activity was found when B. circulans was grown in media containing cell walls of X. dendrorhous, xylan and CM-chitin. In this study, the results demonstrate that the lytic activity of B. circulans could be supported by casein, peptone and tryptone, in addition to YNB. The influence of X. dendrorhous cell wall concentration on the lytic enzyme formation by B. circulans CCRC 11590 is given in Fig. 2B. The fastest induction of lytic enzyme activity was found when 0.25% cell wall was added, with 65 units/ml detected. The results showed that the higher the cell wall concentration, the slower is the induction of this enzyme produced by B. circulans, although similar lytic enzyme activities were detected after 48 h of fermentation among groups supplemented with different cell wall concentrations (Fig. 2B). Temperature was an important factor that influenced the lytic enzyme activity of B. circulans CCRC 11590. When the cells were grown at 30 °C, lytic enzyme of 60 units/ml were detected after 24 h of

When 10% of B. circulans CCRC 11590 was added to a 24 h X. dendrorhous NCHU-FS501 culture, the temperature and pH was adjusted to 30 °C and 6.5, respectively. After inoculating the bacteria, the mixed cultures were cultivated for an additional 48 h. The TC production (mg/ml) in the pure culture of the red yeast was growth-associated and increased during the exponential growth phase of X. dendrorhous (Fig. 3), which is similar to a previous study [17]. Lytic activity was detected after B. circulans was added; a maximal level was reached after 36 h and remained fairly stable up to 48 h of the second stage. Digestion of the yeast cell walls indicated by extractability of carotenoid, was associated with production of the lytic enzyme complex. More than 90% of the total yield of carotenoid synthesized by X. dendrorhous was extracted after 48 h in the mixed culture. Fig. 3 also reveals that the residual sugar in the fermentation broth was low at the time of inoculation with B. circulans. Fleet and Phaff [28] indicated that the lytic enzymes (1,3-b-D-glucanase and 1,6-b-D-glucanase) were strictly inducible, and this induction was inhibited by the high concentration of glucose [29]. Table 1 shows the effect of different cultivation periods of X. dendrorhous in the first stage on pigment extractability of the red yeast, and on the lytic activity of B. circulans CCRC 11590 in this twostage mixed culture fermentation. Significant differences were found in TC, total astaxanthin, carotenoid extractability and lytic activity of yeast cells cultivated for 24, 48, 72 and 96 h. The extractability of carotenoid and lytic activity were significantly lower (PB0.05) when X. dendrorhous was cultivated for 96 h than when it was grown for 24, 48 or 72 h (Table 1). X. dendrorhous was apparently most resistant to hydrolysis when cultivated for 96 h, showing only 8.7% carotenoid extractability. The extractability values for the cells grown for 24, 48 and 72 h were 97.0, 97.4 and 97.1%, respectively (Table 1). A related investigation has

y2760 y3310 x3820 x4190

y3370 x4060 x4890

2630 3200 4090 4240

y3250

7.86 7.95 7.93 8.09

174 201 265 274

4.71 4.44 5.61 5.78

Total astaxanthin (mg/l)

TC (mg /l)

Astaxanthin yield (mg/g glucose)

Total astaxanthin (mg/l)

Final pH

Viable counts (log CFU/ml)

Second stage

First stage

y8.7

x97.1

x97.4

x97.0

Extractability of carotenoid (%)

y8.6

x49.6

x51.3

x51.3

Lytic activity (units/ml)

5.61 4.03 3.63 3.25

7.93 8.07 8.00 7.95

4090 6350 8000 9010

265 248 240 234

Astaxanthin yield (mg/g glucose)

x7580 w10

x8920 w11

070

y6270

580

z3570 y7210

Total astaxanthin (mg/l) z4060

TC (mg/l)

Total astaxanthin (mg/l)

Final pH

Viable counts (log CFU/ml)

Second stage

First stage

y73.3

x98.2

x99.4

x97.1

x48.6

x47.0

x48.0

x49.6

Extractability of carotenoid Lytic activity (%) (units/ml)

The total cultivation time for the two-stage mixed cultures was 120 h with 72 h of cultivation time for the red yeast in the first stage. Data are means of replicate determinations. Values in the same column with the same subscript letter were not significantly different at 5% confidence level.

15 25 35 45

Glucose conc. (g/l)

Table 2 Effect of glucose concentration in YNB broth on pigment extractability of X. dendrorhous and lytic activity of B. circulans CCRC 11590 in the two-stage mixed cultures

The total cultivation time for the two-stage mixed culture regarding cultivation times of P. rhodozyma for 24, 48, 72 and 96 h were 72, 96, 120 and 144 h, respectively.Data are means of replicate determinations. Values in the same column with the same subscript letter were not significantly different at 5% confidence level.

24 48 72 96

Cultivation time (h) for the yeast

Table 1 Effect of cultivation time of X. dendrorhous NCHU-FS501 on pigment extractability of the red yeast and lytic activity of B. circulans CCRC 11590 in the two-stage mixed cultures

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demonstrated that cells in the stationary phase are most resistant to extra-cellular hydrolytic enzyme activity [30]. Okagbue and Lewis [21] reported that digestibility of the cell walls of X. dendrorhous depended on the age of the culture. According to their results, stationary phase cells were most resistant to hydrolysis. This finding correlates with our current results.

3.3. Effect of glucose concentration Table 2 summarizes the effect of glucose concentration on pigment extractability of the two-stage mixed culture. Volumetric TC and volumetric total astaxanthin rose steadily at the second stage of the mixed culture as glucose concentration increased and reached the highest concentration at 45 g/l glucose, which was significantly higher than those groups using 35, 25 and 15 g/l glucose as the carbon source (P B 0.05). Yamane

et al. [19] indicated that astaxanthin production by X. dendrorhous was enhanced by an initial high carbon/nitrogen (C/N ratio) present in the medium, but cell growth was inhibited by a high glucose concentration. A previous report showed that the highest volumetric astaxanthin production was yielded with glucose as the carbon source [17]. However, Johnson and Lewis [31] indicated that higher astaxanthin contents were found when X. dendrorhous was grown on D-cellobiose, maltose, mannitol and sucrose. In this two-stage mixed culture cultivation, extractability of carotenoid was significantly lower with 45 g/l glucose than with 35, 25 and 15 g/l glucose as the carbon source (Table 2). No significant difference of lytic activity was found amongst the media containing various glucose concentrations. Okagbue and Lewis [23] used single stage mixed culture of X. dendrorhous and B. circulans for evaluating lytic activity. In their system, the range of

Fig. 3. Effect of inoculating B. circulans CCRC 11590 on pH, residual sugar, growth, TC, extractable carotenoid and lytic activity of the two-stage mixed culture fermentation with 24-h culture of X. dendrorhous NCHU-FS501 in the first stage. Symbols: ( – "– ) pH; (– – ) residual sugar; ( – –) log(CFU/ml) of B. circulans CCRC 11590; ( – – ) log(CFU/ml) of X. dendrorhous NCHU-FS501; (– “ – ) TC; (–  –) extractable carotenoid; ( – – ) lytic activity. The vertical arrow indicates the time for initiation of the second stage fermentation.

7.85 8.12 8.23 7.93

4.33

7.44 6.48 5.61

5760 6040 4090

4460

x5520 x5500 z3570

x6450 x6450 z4060

378 397 265

y4490

y5150

Total astaxanthin (mg/l)

289

Astaxanthin yield (mg/g glucose)

TC (mg/l)

Total astaxanthin (mg/l)

Final pH

Viable counts (log CFU/ml)

Second stage

First stage

x97.1

y1.9

y1.6

y1.7

x49.6

y13.6

y10.1

y10.2

Extractability of carotenoid Lytic activity (%) (units/ml)

The total cultivation time for the two-stage mixed cultures was 120 h with 72 h of cultivation time for the red yeast in the first step. Data are means of replicate determinations. Values in the same column with the same subscript letter were not significantly different at 5% confidence level.

Ammonium sulfate Casein Peptone YNB

Nitrogen source

Table 3 Effect of nitrogen source on pigment extractability of X. dendrorhous and lytic activity of B. circulans CCRC 11590 in the two-stage mixed cultures

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Table 4 Effect of temperature after inoculation of B. circulans CCRC 11590 in YNB broth on pigment extractability of X. dendrorhous and lytic activity of B. circulans CCRC 11590 in the second stage of the two-stage mixed cultures Temperature (°C)

TC (mg/l)

Total astaxanthin (mg/l)

Extractability of carotenoid (%)

Lytic activity (units/ml)

22.5 26.0 30.0 34.0

x4370

x3890

z0.1

y6.5

x4450

x3690

y62.3

x49.6

x4060

x3570

x97.1

x46.7

x4310

x3430

x96.9

x48.4

The total cultivation time for the two-stage mixed cultures was 120 h with 72 h of cultivation time for the red yeast in the first stage. Data are means of replicate determinations. Values in the same column with the same subscript letter were not significantly different at 5% confidence level.

glucose concentration at 10– 20 g/l appeared to be critical for good yields of astaxanthin and yeast cell wall modification. In the two-stage mixed culture system, up to 35 g/l glucose supported a good yield of volumetric carotenoid and astaxanthin, and also good extractability of carotenoid. Since the lytic enzyme was strictly inducible, and this induction was inhibited by high concentration of glucose [29], the effects of high levels of the sugar on the mixed culture can be explained by catabolite repression by residual sugar, or inhibition of oxidative metabolism.

3.4. Effect of nitrogen source Table 3 shows the effect of nitrogen source on pigment extractability of the two-stage mixed culture. The red yeast produced significantly higher amounts of volumetric carotenoid and astaxanthin when casein and peptone were used as nitrogen source (P B 0.05). It has been reported that changing the nitrogen source had no significant effect on b-carotene production in pure cultures of X. dendrorhous [32]. However, X. dendrorhous produced significantly more astaxanthin when the microorganism was grown on casein hydrolysate as nitrogen source in batch culture [17]. Similar results were observed when X. dendrorhous was grown on yeast carbon base with MES buffer and peptone as nitrogen source [33] or when X. dendrorhous NCHU-FS301 was grown on media containing peptone, beef extract, casein hydrolysate and nutrient broth [16]. In this investigation, although X. dendrorhous NCHU-FS501 produced significantly more astaxanthin when grown in casein and peptone as nitrogen source in the mixed culture (Table 3), YNB supported the highest extractability of carotenoid and lytic activity of B. circulans in the second stage of fermentation.

3.5. Effect of temperature and pH Previous study shows that the optimum temperature for cell growth and total volumetric pigment formation by X. dendrorhous over-producing mutants was 22.5 °C [17]. Johnson and Lewis [31] indicated that the opti-

mum temperature for X. dendrorhous UCD67-210 growth and pigment formation was 20–22 °C, and Meyer and Du Preez [18] reported a similar result for X. dendrorhous strain J4-3. Although Johnson and Lewis [31] indicated that the maximum growth temperature for X. dendrorhous was 27.5 °C, there was little growth of X. dendrorhous strain NCHU-FS501 at 25 °C [17]. In this two-stage mixed culture cultivation, X. dendrorhous was first cultivated at 22.5 °C for 72 h in the first stage. After inoculation with B. circulans, temperatures were shifted to 26.0, 30.0 and 34.0 °C and cultivated for an additional 48 h. Since the temperature used in the first stage was suitable for growth and astaxanthin production by the red yeast, no significant difference (P\ 0.05) was found regarding pigment formation between the groups (data not shown). However, very low lytic enzyme activity was found at 22.5 °C during the first stage of fermentation. In addition, no significant difference in lytic enzyme activity was found (P\ 0.05) among groups incubated at temperatures above 26 °C (Table 4). Okagbue and Lewis [23] reported that only a relatively narrow range of temperature (20–27 °C) supported good yield and extractability of astaxanthin in a one-step mixed culture of X. dendrorhous and B. circulans. Since X. dendrorhous was unable to grow at temperatures above 30 °C [17], the two-stage mixed culture cultivation system can overcome the different requirement of growth temperatures of the red yeast and the bacillus. For evaluating the effect of pH on the extractability of carotenoid in this two-stage mixed culture system, X. dendrorhous was cultivated at 22.5 °C in the first stage for 72 h. After inoculation with B. circulans, the temperature was shifted to 30.0 °C and the pH adjusted to 6.0, 6.5 and 7.0, respectively, followed by an additional 48-h incubation. Over 90% of extractability was found at the pH ranging from 6.0– 7.0, with pH 6.5 showing the highest extractability (97.4%) (Fig. 4). Previous results showed that X. dendrorhous produced the highest volumetric carotenoid at pH 5.0 and 6.0 with the highest specific growth rate at pH 6.0 [17]. Okagbue and Lewis [23] also indicated that in their single step mixed culture system of X. dendrorhous and B. circu-

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lans, over 90% extractability was found in the pH range 6.8 – 7.4. Fig. 5 shows phase-contrast micrographs of X. dendrorhous cells in the first stage (Fig. 5A) and during the second stage of the mixed culture after 12 h (Fig. 6B) and 24 h (Fig. 5C) of incubation. The development of osmotic fragility was accompanied by change in the shape of the X. dendrorhous cells. After 12 h incubation in the mixed culture of the second stage, some dumbbell-shaped cells were found (Fig. 5B). Additional 12 h incubation of the mixed culture caused the red yeast cells and spheroplast cells to lyse (Fig. 5C).

3.6. Recycle of lytic enzyme complex Lytic enzymes of cell-free culture liquid of the twostage mixed culture could be recycled for treatments of unmodified red yeast cells to facilitate astaxanthin recovery. Two successive treatments are shown in Fig. 6. For the first cycle, the initial lytic activity and residual sugar were 41.6 U/ml and 0.65 g/l, respectively. After 48 h of incubation, extractability of carotenoid was 68.8%. Residual sugar was increased to 2.0 g/l, probably due to the lysis of the yeast cell walls [28]. Lytic activity was decreased to 20 U/ml after the first cycle. The results also show that after the first cycle, lytic activity was reduced. The extractability of carotenoid and lytic activity after the second cycle were found to be 18.5% and 11 U/ml, respectively.

Fig. 5. Phase-contrast micrograph of a 72-h culture of X. dendrorhous NCHU-FS501 in the first stage (A) and cells in the mixed culture after 12 h (B) and 24 h (C) of cultivation in the second stage.

4. Conclusions

Fig. 4. Effect of pH of the mixed culture on extractability of astaxanthin. The total cultivation time for the two-stage mixed cultures was 120 h with 72 h of cultivation time for the red yeast in the first stage.

Results of the two-stage batch mixed cultures of X. dendrorhous NCHU-FS501 and B. circulans CCRC 11590 indicated that this system could provide a useful way to treat the cell wall of the yeast. The suitable incubation time, temperature and glucose concentration for X. dendrorhous cultivation in the first stage was 72 h, 22.5 °C and 45 g/l, respectively. During the second stage of the mixed culture, YNB supported the highest extractability of carotenoid and lytic enzyme activity of B. circulans CCRC 11590. The optimal temperature

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Fig. 6. Effect of first recycled (A) and second recycled (B) crude lytic enzyme after mixed cultivation on pigment extractability of X. dendrorhous. The crude lytic enzyme activity in the beginning of incubation for the first recycled and the second recycled experiments were 41.6 and 19.5 units/ml, respectively. Symbols: ( – “ –) TC; (– –) extractable carotenoid; ( – – ) lytic activity; ( –  – ) residual sugar; ( –  – ) extractability of carotenoid.

and pH for carotenoid extraction in the mixed culture was 30 –34 °C and pH 6.5, respectively. Within 48 h, over 96% of the TC could be extracted during the second stage of this two-stage mixed fermentation under optimal conditions. The recycling of lytic enzyme complex could be useful to facilitate astaxanthin recovery, or absorption from animal feed.

Acknowledgements We gratefully acknowledge the support for this research by the National Science Council, Taiwan, Republic of China (NSC89-2214-E-005– 011).

References [1] Johnson EA, Villa TG, Lewis MJ. Phaffia rhodozyma as an astaxanthin sources in salmonids diets. Aquaculture 1980;20:123 – 34. [2] Johnson EA, An G-H. Astaxanthin from microbial sources. Crit Rev Biotechnol 1991;11:297 – 326. [3] Naguib YMA. Antioxidant activities of astaxanthin and related carotenoids. J Agric Food Chem 2000;48:1150 – 4. [4] Terao J. Antioxidant activity of b-carotene-related carotenoids in solution. Lipids 1989;24:659 – 61. [5] Yamashita E. Chromatographic analysis of functional components in astaxanthin from Euphausia superba. Food Develop (Japan) 1992;37:38 – 40. [6] Chew BP, Park JS, Wong MW, Wong TS. A comparison of the anticancer activities of dietary [beta]-carotene, canthaxanthin and astaxanthin in mice in vivo. Anticancer Res 1999;19:1849 – 53.

T.J. Fang, J.-M. Wang / Process Biochemistry 37 (2002) 1235–1245 [7] Jyonouchi H, Sun S, Iijima K, Gross MD. Antitumor activity of astaxanthin and its mode of action. Nutri Can 2000;36:59 – 65. [8] Miller MW, Yoneyama M, Soneda M. Phaffia, a new yeast genus in the Deuteromyotina (Blastomycetes). Int J Syst Bacteriol 1976;26:286 – 91. [9] Andrewes AG, Phaff HJ, Starr MP. Carotenoids of Phaffia rhodozyma, a red-pigmented fermenting yeast. Phytochemistry 1976;15:1003 – 7. [10] Ramirez J, Nunez ML, Valdivia R. Increased astaxanthin production by a Phaffia rhodozyma mutant grown on date juice from Yucca fillifera. J Ind Microbiol Biotechnol 2000;24:187 – 90. [11] An G-H, Schuman DB, Johnson EA. Isolation of Phaffia rhodozyma mutants with increased astaxanthin content. Appl Environ Microbiol 1989;55:116 –24. [12] Bon JA, Leathers TD, Jayaswal RK. Isolation of astaxanthinoverproducing mutants of Phaffia rhodozyma. Biotechnol Lett 1997;19:109 – 12. [13] Chumpolkulwong N, Kakizono T, Nagai S, Nishio N. Increased astaxanthin production by Phaffia rhodozyma mutants isolated as resistant to diphenylamine. J Ferment Bioeng 1997;83:429 – 34. [14] Meyer PS, Du Preez JC, Van Dyk MS. The effect of monoterpenes on astaxanthin production by a mutant of Phaffia rhodozyma. Biotechnol Lett 1994;16:125 –8. [15] Fang TJ, Cheng YS. Isolation of astaxanthin over-producing mutants of Phaffia rhodozyma and their fermentation kinetics. Chin J Microbiol Immuno 1992;25:209 – 22. [16] Fang TJ, Cheng Y. Improvement of astaxanthin production by Phaffia rhodozyma through mutation and optimization of culture conditions. J Ferment Bioeng 1993;75:466 –9. [17] Fang TJ, Chiou T-Y. Batch cultivation and astaxanthin production by a mutant of the red yeast, Phaffia rhodozyma NCHUFS501. J Ind Microbiol 1996;16:175 –81. [18] Meyer PS, Du Preez JC. Effect of culture conditions on astaxanthin production by a mutant of Phaffia rhodozyma in batch and chemostat culture. Appl Microbiol Biotechnol 1994;40:780 – 5. [19] Yamane Y, Higashida K, Nakashimada Y, Kakizono T, Nishio N. Astaxanthin production by Phaffia rhodozyma enhanced in fed-batch culture with glucose and ethanol feeding. Biotechnol Lett 1997;19:1109 – 11.

1245

[20] Johnson EA, Villa TG, Lewis MJ, Phaff HJ. Simple method for the isolation of astaxanthin from the basidiomycetous yeast Phaffia rhodozyma. Appl Environ Microbiol 1978;35:1155 –9. [21] Okagbue RN, Lewis MJ. Mixed culture of Bacilllus circulans WL-12 and Phaffia rhodozyma on different carbon source: yeastwall lytic enzyme production and extractability of astaxanthin. Biotechnol Lett 1983;5:731 – 6. [22] Okagbue RN, Lewis MJ. Autolysis of the red yeast Phaffia rhodozyma: a potential tool to facilitate extraction of astaxanthin. Biotechnol Lett 1984;6:247 – 50. [23] Okagbue RN, Lewis MJ. Influence of mixed culture conditions on yeast-wall hydrolytic activity of Bacillus circulans WL-12 and on extractability of astaxanthin from the yeast Phaffia rhodozyma. J Appl Bacteriol 1985;59:243 – 55. [24] Miller G. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426 – 8. [25] Sedmak JJ, Weerasinghe DK, Jolly SO. Extraction and quantitation of astaxanthin from Phaffia rhodozyma. Biotechnol Tech 1990;4:107 – 12. [26] Helwing JF, Concil K. SAS/STAT user’s guide, version 6, vol. 1, 4th ed. Cary NC: SAS Institute Inc, 1989. [27] Johnson EA, Villa TG, Lewis MJ, Phaff HJ. Lysis of the cell wall of the yeast Phaffia rhodozyma by a lytic enzyme complex from Bacillus circulans WL-12. J Appl Biochem 1979;1:273 –82. [28] Fleet GH, Phaff HJ. Lysis of yeast cell wall: glucanases from Bacillus circulans WL-12. J Bacteriol 1974;119:207 – 19. [29] Esteban R, Nebreda AR, Villa TG. Synthesis and regulation of Bacillus circulans WL-12 1,3-b-D-glucanase. J Gen Microbiol 1984;130:2483 – 7. [30] Duetch CE, Parry JM. Spheroplast formatting in yeast during the transition from exponential phase to stationary phase. J Gen Microbiol 1974;80:259 – 68. [31] Johnson EA, Lewis MJ. Astaxanthin formation by the yeast Phaffia rhodozyma. J Gen Microbiol 1979;115:173 – 83. [32] Nelis HJ, De Leenheer AP. Microbial production of carotenoids other than b-carotene. In: Biotechnology of vitamins, pigments and growth factors. London: Elsevier, 1989:43 – 80. [33] Meyer PS, Du Preez JC, Kilian SG. Selection and evaluation of astaxanthin-overproducing mutants of Phaffia rhodozyma. World J Microbiol Biotechnol 1993;9:514 – 20.