Stability and changes in astaxanthin ester composition from Haematococcus pluvialis during storage*

Chinese Journal of Oceanology and Limnology Vol. No. 6, P. 8-89, 2 http://dx.doi.org/.7/s4--25- Stability and changes in astaxanthin ester composition from Haematococcus pluvialis during storage* MIAO Fengping ( 苗凤萍 ), 2, GENG Yahong ( 耿亚洪 ), LU Dayan ( 卢大炎 ), ZUO Jincheng ( 左进城 ), **, LI Yeguang ( 李夜光 ) Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 474, China 2 Laboratory of Coastal Zone Bioresources, Yantai Institute of Costal Zone Research, Chinese Academy of Sciences, Yantai 264, China College of Life Science, Ludong University, Yantai 26425, China Received May 9, 22; accepted in principle Jul. 2, 22; accepted for publication Aug. 4, 22 Chinese Society for Oceanology and Limnology, Science Press, and Springer-Verlag Berlin Heidelberg 2 Abstract In this paper, we investigated the effects of temperature, oxygen, antioxidants, and corn germ oil on the stability of astaxanthin from Haematococcus pluvialis under different storage conditions, and changes in the composition of astaxanthin esters during storage using high performance liquid chromatography and spectrophotometry. Oxygen and high temperatures (22 25 C) significantly reduced the stability of astaxanthin esters. Corn germ oil and antioxidants (ascorbic acid and vitamin E) failed to protect astaxanthin from oxidation, and actually significantly increased the instability of astaxanthin. A change in the relative composition of astaxanthin esters was observed after 96 weeks of long-term storage. During storage, the relative amounts of free astaxanthin and astaxanthin monoesters declined, while the relative amount of astaxanthin diesters increased. Thus, the ratio of astaxanthin diester to monoester increased, and this ratio could be used to indicate if astaxanthin esters have been properly preserved. If the ratio is greater than.2, it suggests that the decrease in astaxanthin content could be higher than 2%. Our results show that storing algal powder from H. pluvialis or other natural astaxanthin products under vacuum and in the dark below 4 C is the most economical and applicable storage method for the large-scale production of astaxanthin from H. pluvialis. This storage method can produce an astaxanthin preservation rate of at least 8% after 96 weeks of storage. Keyword : Haematococcus pluvialis ; astaxanthin ester; storage stability; storage life; high performance liquid chromatography (HPLC ) INTRODUCTION Natural astaxanthin, a natural antioxidant, is usually found in algae, especially in Haematococcus pluvialis, and aquatic animals. The antioxidant activity of natural astaxanthin is several times higher than vitamin E (Naguib, 2) and times higher than other carotenoids, including zeaxanthin, lutein, ß-carotene, and canthaxanthin (Miki, 99; Shinidzu et al., 996). Pre-clinical research has suggested that astaxanthin is a suitable candidate for development as a therapeutic agent for cardiovascular oxidative stress and inflammation (Pashkow et al., 28), because it can inhibit the formation of oxidized low-density lipoprotein (LDL) in humans (Iwamoto et al., 2) and eliminate the lipid peroxidation caused by rofecoxib in cellular membrane models (Mason et al., 26). Astaxanthin also has neuroprotective effects against retinal damage because of its antioxidant activity (Nakajima et al., 28). In addition, astaxanthin has been widely used in aquatic animal feeds, as a functional human food and in cosmetics. About tons of astaxanthin is required annually to feed the salmonids produced globally by aquaculture (Bjerkeng, 28). At present, more than 95% of the astaxanthin used worldwide is chemically synthesized, * Supported by the Yunnan Provincial Sciences and Technology Department, China (No. 27AD9), the National Natural Science Foundation of China (No. 27268), and the Ministry of Science and Technology of China (No. 2AA6585) ** Corresponding author: yeguang@wbgcas.cn

82 CHIN. J. OCEANOL. LIMNOL., (6), 2 Vol. and has a lower antioxidant activity compared with natural astaxanthin (Lim et al., 22). Therefore, the production of natural astaxanthin from mass cultures of astaxanthin-rich Haematococcus pluvialis is becoming increasingly important in the microalgalbiotechnology industry. Astaxanthin is sensitive to light, oxygen and temperature, so investigating the stability of astaxanthin under different conditions, and finding an economical and efficient storage method for natural astaxanthin, is critical. Several methods have been suggested to improve the stability of astaxanthin (Gouveia and Empis, 2; Cinar, 24; Cysewski and Lorenz, 24; Xu, 26; Chen et al., 27; Kittikaiwan et al., 27; Yuan et al., 28; Raposo et al., 22). It has been reported that storing astaxanthin with other compounds to protect against temperature and light, such as β-cyclodextrin, hydroxypropyl-βcyclodextrin, and chitosan, improves astaxanthin stability (Cinar, 24; Xu, 26; Chen et al., 27; Yuan et al., 28). Gouveia and Empis (2) reported that H. pluvialis retained 9% of the initial carotenoid content after.5 years of storage in dry algal meal under vacuum conditions (Gouveia and Empis, 2). The information obtained from previous studies shows the importance of studies into the effect of storage conditions on the stability of astaxanthin in H. pluvialis. However, previous studies have been limited to establishing economical, efficient, and applicable storage and preservation methods for the large-scale production of natural astaxanthin from H. pluvialis. Currently, little is known about how astaxanthin mono- and diesters, which account for 75% and 5% of the total astaxanthin content, respectively, change during storage. In this study, the stability of astaxanthin from Haematococcus in algal meal of broken cysts and frozen fresh intact cysts was investigated under different conditions during long-term storage of 96 weeks. The composition of astaxanthin esters during storage and the change in ester composition were also analyzed using high performance liquid chromatography (HPLC). The purpose of this study is to understand how the stability of astaxanthin esters from H. pluvialis is influenced by physical and chemical factors during storage, and help to establish economical and effective storage methods for the large-scale production of astaxanthin from H. pluvialis. 2 MATERIAL AND METHOD 2. Preparation of samples The alga H. pluvialis strain WZ26 (ACWBG-H26) was grown in a 5 m 2 circular pond in a greenhouse in medium BG- (Miao et al., 25). The cysts were harvested with centrifuge SS6 (Zhangjiagang, China), broken by a vacuum bead beater QM-QX (Nanjing, China) and dried with a vacuum drier YZG6 (Changzhou, China). 2.2 Measurement of astaxanthin content The astaxanthin content was measured by the spectrophotometric method described by Boussiba and Vonshak (99). The harvested cells were resuspended in a solution of 5% KOH in % methanol and heated in a 7 C water bath for 5 min. The mixture was centrifuged and the resulting pellet was extracted with dimethylsulfoxide (DMSO) after adding five drops of acetic acid, and heated at 7 C for 5 min. The centrifugation and DMSO extraction was repeated if necessary until the cell debris was totally white. The concentration of astaxanthin in the extracts was measured based on the absorption % coefficient A =2 22 in DMSO at 492 nm. cm 2. Analysis of astaxanthin esters The astaxanthin ester content in the algal powder and frozen fresh cysts was determined by HPLC in accordance with Miao et al. (26). The Agilent series HPLC (Palo Alto, CA, USA) was equipped with a diode array detector (DAD). The column was a 25 4.6 mm YMC analytical column (5 μm C8- reversed phase), including a 4.6 mm i.d. precolumn, which was held in the column oven at 2 C. The mobile phase consisted of a 6 min gradient from 8:7 to 98:2 (acetone/water), with a flow rate of.8 ml/min. The injection volume was. μl and the DAD scan range was 8 nm. The content of astaxanthin esters was determined by the external standard method using free astaxanthin as the standard. 2.4 Short-term storage experiment The algal powder was stored in unsealed aluminumplastic bags, and the samples were separated into two groups with vitamin C (.5% w/w) added to one group. Both groups were stored at 22 25 C for 9 days in the dark. Triplicate measurements were made for each treatment. The astaxanthin content was determined at Day,, 6, and 9. 2.5 Long-term storage experiment The storage treatments and temperatures were as follows: frozen (-8 C) fresh intact cysts under

No.6 MIAO et al.: Changes of astaxanthin esters during storage 8 Table Effect of antioxidant on astaxanthin stablility in H. pluvialis Astaxanthin content (%, DW) Time (d) Broken cyst powder, 22 C Broken cyst powder added with vit. C, 22 C.82±.7.8±.45.42±.9.7±.6 6.2±.2.±. 9.245±.9.75±.5 vacuum conditions (I) and under normal conditions (II); algal powder stored under vacuum conditions at 22 25 C (III), 4 C (IV) and -8 C (V); algal powder with added vitamin C was stored under vacuum conditions at 22 25 C (VI); algal powder with added corn germ oil was stored at 22 25 C (VII) and 4 C (VIII); algal powder mixed with corn germ oil and vitamin E was stored at 22 25 C (IX) and 4 C (X). A vacuum packaging machine was used to seal the samples stored under vacuum conditions, while the samples stored under normal conditions were not sealed and exposed to the air. For the samples mixed with corn germ oil or corn germ oil and vitamin E, test tubes with covers were filled up with these materials to remove the air, and were sealed without vacuuming. All samples were stored in the dark. For the broken cyst powder, vitamins C and E and corn germ oil were added at.5% (w/w), 2% (w/w), and 67% (w/w), respectively, and mixed uniformly. Three replicates were used for each treatment, and the astaxanthin contents were measured after,, 2, 4, 8,, 52, 72, and 96 weeks. RESULT. Changes in astaxanthin content during shortterm storage During the short-term storage experiment, the astaxanthin content decreased significantly ( P <.) when the samples were exposed to the air. Surprisingly, the astaxanthin content of the samples with added antioxidant (vitamin C) decreased even faster than the control (no antioxidant) ( P <., Table )..2 Composition and content changes in astaxanthin esters during long-term storage The HPLC chromatograms of the astaxanthin esters from H. pluvialis after and 96 weeks of storage are shown in Fig., and the sizes of the numbered peaks are shown in Table 2. The HPLC chromatograms of the astaxanthin esters suggest that the area of each astaxanthin ester decreased, and the ratio of astaxanthin diester to monoester increased after 96 weeks of storage. The order of the changes in the HPLC chromatograms for the different treatments, from low to high change, can be divided into five groups. The first group includes frozen fresh intact cysts stored under vacuum conditions (I), frozen fresh intact cysts exposed to the air (II) and algal powder stored under vacuum conditions at -8 C (V). The HPLC pattern for this group is consistent with Fig.a. The second group is the algal powder stored under vacuum conditions at 4 C (IV) and is shown in Fig.b. The third group includes the algal powder stored under vacuum conditions at 22 25 C (III) and algal powder with added vitamin C stored under vacuum conditions at 22 25 C (VI), as shown in Fig.c. The fourth group includes algal powder mixed with corn germ oil, sealed and stored at 4 C (VIII) and algal powder mixed with corn germ oil and vitamin E, sealed and stored at 4 C (X), shown in Fig.d. The fifth group includes algal powder mixed with corn germ oil, sealed and stored at 22 25 C (VII) and algal powder mixed with corn germ oil and vitamin E, sealed and stored at 22 25 C (IX) (Fig.e). These results indicate that at higher temperatures, greater changes were observed in the HPLC chromatograms, and the addition of corn germ oil and antioxidants also led to greater changes in the HPLC chromatograms. The changes in relative astaxanthin content in the H. pluvialis over time with different storage conditions are shown in Table. The astaxanthin contents of the different treatments, from highest to lowest concentration, were as follows: V, II, I, IV, III, VI, VIII, X, VII, and IX (Table 4). It can be seen that as more unfavorable factors (such as higher temperatures, and addition of vitamin C, vitamin E and corn germ oil) were included, lower astaxanthin preservation rates were achieved, and more changes in the HPLC chromatograms were observed. The relative amounts of free astaxanthin,

84 CHIN. J. OCEANOL. LIMNOL., (6), 2 Vol. 2 a 4 6a 8a 8b 5 5 2 5a 5b 5c 6b 7b 9 8c 8d a b 2 4a 4b 5 6 78 2a 2 9 2b 22 2 4 2 8 6 4 2 b 2 4 5a 5b 5c 6a 6b 7b 8b 8a 9 b 2 7 6 8 5 2a 4b 2 Absorbance (mau) 25 2 5 5 c 4 5c 6a 6b 7b 8 8b 9 b 2 4b 4a 8 7 2a 2 b 2 4 2 d 8 6 4 2 2 4 5b 5c 6a 6 8d b 2 8 6 7 2a 4b 2 9 22 2 5 25 2 5 5 e 4 5b 6a 6b 7b 8a 8b 2b 22 2 4 5 6 Retention time (min) 8d a b 2 4b 5 8 9 2a Fig. The HPLC spectrum of astaxanthin esters in H. pluvialis The compounds corresponding to the numbers representing the peaks of HPLC spectrum were shown in Table, and the number with letters represents the isomers of astaxanthin ester. a. Algal powder at the beginning of the experiment; b. Algal powder stored under vaccum at 4 C for 96 weeks; c. Algal powder stored under vacuum at 22 C for 96 weeks; d. Algal powder mixed with corn germ oil, sealed and stored at 4 C for 96 weeks; e. Algal powder mixed with corn germ oil, sealed and stored at 22 C for 96 weeks.

No.6 MIAO et al.: Changes of astaxanthin esters during storage 85 Table 2 Relative content of astaxanthin esters in H. pluvialis after 96 weeks of storage No. Compound Relative content (%) Control I II III IV V VI VII VIII IX X Astaxanthin 2.68 6.98 6.5 5.62 4.79 7.72.89 7.57 8.42 6..457 8 5a ME C8:4 2.845 2.86 2.85 9.6 6.29 4 2.2.59.78 9.47 8.5 5.482 4 5b ME C8:4.669 7.4.69.55.969 7.45.562 5.5.526 2.55 7.577 4 5c ME C8:4.56 5.495. 8.68 4.9.8.655 9.955 7.559 4.49 5.628 6 6a ME C8: 2.52 2 4.29 6 4.422 8.85 9 4.895 7 9.5 2 8.5. 2 4.24 5.724 9 4.267 6b ME C8:.895.52 9.29 2 8.874 4 5.28 4 6.67 6.96 2.47 5 4. 5 2.6 8 4.49 7 ME C8:2 5.798 6 2.9 9 2.9 2.7 2 7.465.9 7.96 6 5.99 2 5.927 8 5.92 5 6.8 5 7b ME C8:2.98 4 4.726 7 4.54 5 9.26 6 4.7 9 2.96 8 2.4 6.9 4 5.779 6.65 7 6.9 8 ME C8:2 5.585 6.646 8.66 6 2.75 2 6.666 7.99 5 2.6 9 2.4 4 5.45 2 2.4 5.629 8a ME C8:.987 2.974.945 2 2.448 7 5.67 9.582 7 7.86 6.892 4.62 7.97 4.876 8b ME C8:..72 4.945 2 8. 2.425 7 8.942 9 2.2 5 2.4 9.765 2.45 4 5.489 8 9 ME C6:.685 7 2.72 2.478 2 2.92 7 5.55 5 2.988 7 4.65.24 9 4.656 7.87 6.79 6 8c ME C8:.878 8.882.79 8.95 6.924 2.427 5 2.2 4.742 7. 8.927.887 4 8d ME C8: 2.898 6 2.49 4 2.2 2 8.4.9 6.89 8 2.56 2.29 4.758 6.664 2.87 6 2 ME C6:.7.96.68 6 2.57.6.98 6.587 2.894.547 4.26 5.59 ME C7:.574 6.677 9.682.55 2.826 6.82 9 4.485 2. 4 2.47 4.49 4 2.67 4a DE C8: /C8:2.22 9 2.57 8 2.5 5 4.82 9.67 2.45 8 6.662 6 4.8.625 8 4.74 8.687 5 4b DE C 8:2 /C8:.985 9.622 9.62 4.86 9.766.2 5 2.762 4 2.54 2.98 2.524.994 6 5 DE C 8:2 /C8:.68.24 4.272 5 2.546 4 2.45.494.886 4.872 9 2.44 4.42 5 2.449 7 6 DE C 8:2 /C6:.67 7 2.22 9 2.49.89 5 5.56 7.9 6.929 7 7.264 4.78 6.85 5 4.77 7 DE C 8: /C8: 2.58 4 2.6 2.6 6.598 7 4.64 8 2.466 6.7 2 9.74 4.7 2 8.65 7 4.5 8 DE C 8:2 /C6: 2.7 2.72 5 2.2 2 5.46 5.65.7 6 7.54 9 9.48 7 5.52 2 8.6 8 5.56 9 DE C 8: /C8:.22 4.945 4.99 6 6.92 2.495 2.259 6.5 6.7 7 2.7 5.459 2.679 2 2a DE C6: /C8: 2.44 9.886 7.846 9 2.89 4.96 6 2.288 5.78 5 7.28 7 4.27 9 6.746 7 4.29 9 2 DE C 6: /C6:.77.2 7.78 9 4.59 6.487.89 9 4.9 4 5.972 7.678 9 5.98.586 7 2b DE C 6: /C8:.4 4.78 2.65 4.976 4.58.72.257 2.55 6 2.485 6.947 4.42 8 22 DE C 8: /C8:.69.248.25.9 8.98 4.476 6.997 7 2.477.557 6 2.288 5.499 2 DE C 8: /C6:.567 2.82 4.7 7.5 2.26 8.59 6.254 7.87 2.58 2.6 4.867 2 ME: astaxanthin monoester; DE: astaxanthin diester; I: Frozen fresh intact cysts under vacuum conditions were stored at -8 C; II: Frozen fresh intact cysts under normal conditions were stored at -8 C; III: Algal powder was stored under vacuum conditions at 22 25 C; IV: Algal powder was stored under vacuum conditions at 4 C; V: Algal powder was stored under vacuum conditions at -8 C; VI: Algal powder with added vit. C was stored under vacuum conditions at 22 25 C; VII: Algal powder with added corn germ oil was stored at 22 25 C; VIII: Algal powder with added corn germ oil was stored at 4 C; IX: Algal powder mixed with corn germ oil and vit. E was stored at 22 25 C; X: Algal powder mixed with corn germ oil and vit. E was stored at 4 C. astaxanthin monoesters and diesters under different storage conditions after 96 weeks are shown in Table 2. Table 2 shows a decreasing trend in the relative amounts of free astaxanthin and astaxanthin monoesters but an increasing trend in the relative amounts of astaxanthin diesters, which can be seen more clearly in Fig.. The data shown in Table 4 also demonstrates that as the amount of astaxanthin esters changed, the ratio of astaxanthin diester to monoester increased.. Effect of temperature, corn germ oil, and antioxidant on astaxanthin stability The effects of temperature, corn germ oil and antioxidants on astaxanthin stability during shortterm and long-term storage are summarized in Fig.2. During the long-term storage study, algal materials were stored at different temperatures (-8 C, 4 C and 22 25 C) under vacuum conditions for 96 weeks. Storage at -8 C gave the highest astaxanthin

86 CHIN. J. OCEANOL. LIMNOL., (6), 2 Vol. Table The changes of astaxanthin relative content in H. pluvialis with time under different storage conditions No. Astaxanthin relative content (%) week week 4 weeks 8 weeks weeks 52 weeks 72 weeks 96 weeks I.±.67.±2.2.±.5 98.2±.68 98.99±. 99.±6.6 94.95±2.2 86.5±.7 II.±.67 99.±. 96.97±.5 96.6±. 92.59±4.7 96.97±2.6 95.62±.7 89.9±2.69 III.±.8.±5.8 99.58±2.8 99.67±7. 78.8±. 58.25±8.92 67.8±.5 5.8±.67 IV.±.8.±2.58.±4.25.±2. 9.±5.67.±. 97.8±.42 79.58±.25 V.±.8 95.8±.8.±.7.±.58.±4.8.±.75.±.5 89.92±4.7 VI.±.69 92.7±.5 95.7±.85 9.8±.9 7.9±.2 5.52±2.7 66.8±. 45.59±4.74 VII.±2.2 98.9±2.5 87.2±2.5 88.4±.76 66.94±.6 9.65±5.2 28.22±.8 9.68±.54 VIII.±2.2 99.6±4.57 99.6±2.42 9.99±4.4 82.8±.88 65.2±.88 5.75±.88 26.8±2.55 IX.±2.2.±2. 85.5±2.42 85.22±2.42 59.8±.9 8.98±.48 5.±.48 8.6±.54 X.±2.2 99.7±6.5 9.67±2.28 87.±.67 75.67±4.57 59.54±.48 47.98±.6 2.64±.8 I X: Same as in Table 2. Table 4 The relationship between decrease of astaxanthin content (%) and change of the ratio of astaxanthin diesters to monoesters 5 a Storage condition Decreasing of astaxanthin content (%) Di-/mono-esters I.47.8-8 4 22 II..9 III 48.92.95 IV 2.42.54 V.8.27 VI 55.7.99 VII 9.2.96 VIII 7.92.64 IX 9.4.58 X 78.6.6 Astaxanthin preservation rate (%) 5 5 b c Algal meal Algal meal+corn germ oil 4 22 Algal meal Meal+vit.E. I X: Same as in Table 2. preservation rate, while the amounts of astaxanthin decreased by.5% at 4 C and 4.9% at 22 C when compared with storage at -8 C (Fig.2a). The astaxanthin content decreased significantly when the algal materials were mixed with corn germ oil. The preservation rates of astaxanthin in the samples with added corn germ oil decreased by 67.2% at 4 C and 8.5% at 22 C, respectively, compared with the control group without (corn germ oil treatment) after 96 weeks of storage (Fig.2b). During the short-term storage experiment, the addition of vitamin C decreased the astaxanthin preservation rates by 69.4% (Table ). During the 4 22 Temperature ( C) Fig.2 Effect of temperature, corn germ oil and antioxidant on the astaxanthin stability a. Effect of temperature on the astaxanthin stability; b. Effect of corn germ oil on the astaxanthin stability; c. Effect of vit. E on the astaxanthin stability. long-term storage experiment, the addition of vitamin C reduced the astaxanthin preservation rates by.75% (Table ), while the addition of vitamin E reduced the astaxanthin preservation rates by 7.2% at 4 C and.6% at 22 C (Fig.2c), when compared with the controls without added vitamins C or E.

No.6 MIAO et al.: Changes of astaxanthin esters during storage 87 4 DISCUSSION 4. Effect of oxygen on the stability of astaxanthin The astaxanthin content of the algal powder decreased by 77.6% when it was exposed to the air during the short-term storage experiment (9 days of storage at 22 25 C) (Table ). In contrast, no significant changes were observed in storage under vacuum conditions at 22 25 C for up to 8 weeks. During the long-term experiment, the astaxanthin content decreased by 48.92% after 96 weeks of storage (Table, III) under vacuum conditions at 22 25 C. These results suggested that astaxanthin is destroyed by oxygen, which is consistent with previous studies (Cysewski and Lorenz, 24). 4.2 Effect of temperature, corn germ oil, and antioxidants on astaxanthin stability Astaxanthin is sensitive to changes in temperatures (Gouveia and Empis, 2), and the preservation rate of astaxanthin decreased significantly with increasing temperature (Fig.2a). This result is consistent with a previous study conducted by Gouveia and Empis (2). In previous studies, the abundance of unsaturated fatty acids in corn germ oil has played a role in protecting astaxanthin from oxidation in the presence of oxygen (Chen and Meyer, 982; Cysewski and Lorenz, 24). However, the results of this study show that corn germ oil did not protect astaxanthin from degradation, and in fact increased the instability of astaxanthin. This may occur if the astaxanthin performs as the antioxidant instead of the corn germ oil, protecting the corn germ oil from oxidation during long-term storage, resulting in the degradation of the astaxanthin. Similarly, the addition of antioxidants did not protect astaxanthin from oxidation, but in fact increased the instability of the astaxanthin. In both the short-term and long-term storage experiments, adding an antioxidant significantly reduced the preservation rates of astaxanthin in algal meal. Gouveia and Empis (2) reported that when the microalgal biomass ( Chlorella vulgaris and H. pluvialis ) was stored at room temperature in the dark, the carotenoid content of H. pluvialis powder with added vitamin C was reduced to 65.2% of the original after.5 years of storage, while the control was reduced to 66.66%. Similarly, only 9.% of the original carotenoid content of C. vulgaris powder with added vitamin C remained after storage while 7.6% of the control remained. To explain why the addition of an antioxidant does not improve the stability of astaxanthin, but enhances its oxidation and degradation, we suggest that because astaxanthin has a much greater antioxidant activity than vitamins C or E (Miki, 99; Shinidzu et al., 996; Naguib, 2), it acts as a protector during long-term storage, which enhances its degradation. 4. Change in astaxanthin monoesters and diesters during storage In well-matured Haematococcus cysts, astaxanthin monoesters and diesters comprise 75% and 5%, respectively, of the total astaxanthin. In this study, the relative amounts of astaxanthin diesters increased while the astaxanthin monoesters decreased during storage. With the decrease in astaxanthin ester content, the ratio of astaxanthin diesters to monoesters increased. These results show that astaxanthin diesters are more stable than monoesters during storage, so the ratio of astaxanthin diester to monoester may be used to indicate if the astaxanthin esters have been preserved properly. If the ratio of astaxanthin diester to monoester is around.2, it means that the astaxanthin esters have been well preserved. If the ratio is substantially higher than.2, this suggests that the astaxanthin has not been preserved properly, and the decrease in astaxanthin content could be higher than 2% (Table 4). 4.4 Stability of astaxanthin esters in frozen fresh cysts of H. pluvialis In laboratory conditions, it is common for the harvested cysts of H. pluvialis to be stored in the freezer (-8 C) for a period before pigment extraction and analysis. This study showed that the rate of astaxanthin loss was less than 5% of the original after being stored frozen for 72 weeks and less than 5% after storage for 96 weeks either under vacuum or being exposed to the air. No obvious changes were observed in the HPLC chromatograms. It is believed that the thick cell walls of the cysts and the ice coating isolates the astaxanthin from oxygen, thus providing a double layer of protection. Storing the frozen fresh cysts in the dark is a simple and reliable method, which is more convenient for laboratory work. 4.5 Appropriate conditions and storage time for preserving astaxanthin in H. pluvialis Because of the chemical nature of astaxanthin,

88 CHIN. J. OCEANOL. LIMNOL., (6), 2 Vol. light is a major factor that affects the stability of astaxanthin (Gouveia and Empis, 2; Kittikaiwan et al., 27). Previous experiments showed that astaxanthin degraded quickly under intensive light. Therefore, astaxanthin must be stored in the dark. Table 4 shows that to achieve an astaxanthin preservation rate of at least 8%, algal powder should be vacuum-sealed and stored in the dark. The storage life is 96 weeks when stored at 4 C, and weeks when stored at room temperature. Frozen fresh cysts can be stored in the dark for more than 96 weeks either under vacuum or exposed to the air. 4.6 Preservation methods suitable for large-scale production of natural astaxanthin from H. pluvialis Nitrogen-filling and micro-encapsulation have been shown to significantly improve the stability of various forms of astaxanthin (Gouveia and Empisv, 2; Cysewski et al., 24; Chen et al., 27; Kittikaiwan et al., 27), and are suitable for storing natural astaxanthin products. However, compared with vacuum-packing, which is widely used in the food industry, nitrogen-filling and microencapsulating are more complicated, and vacuum is required for nitrogen-filling. Packing algal powder from H. pluvialis or other natural astaxanthin products under vacuum and storing it below 4 C in the dark is the most suitable method for storage of large-scale astaxanthin products from H. pluvialis. 5 CONCLUSION The following conclusions are drawn from this study: ) Oxygen and high temperatures (22 25 C) could significantly decrease the stability of astaxanthin esters in H. pluvialis. Corn germ oil and antioxidants [ascorbic acid (.5%, w/w) and vitamin E (2%, w/w)] could not protect astaxanthin from oxidation, but degrade it. We suggest that astaxanthin plays the role of antioxidant instead of vitamin C and tocopherol during long-term preservation, and is itself degraded. 2) During long-term storage, the absolute amounts of free astaxanthin, astaxanthin diester and monoesters are all decreased. The HPLC chromatograms of astaxanthin esters change significantly, with the relative amounts of astaxanthin monoesters declining, while the relative amounts of astaxanthin diesters increases. The ratio of astaxanthin diester to monoester increases, suggesting that the astaxanthin diesters are more stable than free astaxanthin and astaxanthin monoesters. ) Packing algal powder from H. pluvialis or astaxanthin products under vacuum and then storing them below 4 C in the dark will provide an astaxanthin preservation rate of at least 8% after 96 weeks storage. These storage conditions are the most economical and applicable method for storing largescale production of astaxanthin from H. pluvialis. 6 ACKNOWLEDGEMENT The authors thank Dr. Liming LUO, Dr. Zhongkui LI and Dr. Ying SONG for reading and editing this manuscript. References Boussiba S, Vonshak A. 99. Astaxanthin accumulation in the green alga Haematococcus pluvialis. Plant Cell P hysiol., 2 : 77-82. Bjerkeng B. 28. Carotenoids in Aquaculture: Fish and Crustaceans. Birkhäuser, Basel. Chen H M, Meyer S P. 982. Effect of antioxidants on stability of astaxanthin pigment in crawfish waste and oil extract. J. Agri. Food Chem., : 469-47. Chen X L, Chen R, Guo Z Y, Li C P, Li P C. 27. The preparation and stability of the inclusion complex of astaxanthin with β-cyclodextrin. Food Chem., : 58-584. Cinar I. 24. Storage stability of enzyme extracted carotenoid pigment from carrots. Elec. J. Environ. Agricul. Food Chem., : 69-66. Cysewski G R, Lorenz R T. 24. Industrial production of microalgal cell mass and secondary products-species of high potential: Haematococcus. In: Richmond A ed. Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Blackwell Publishing Ltd, Oxford, U K. http://dx.doi.org/.2/9784799528. Gouveia L, Empis J. 2. Relative stabilities of microalgal carotenoids in microalgal extracts, biomass and fish feed: effect of storage conditions. Innov. Food. Sci. Emerg. Technol., 4 : 227-2. Iwamoto T, Hosoda K, Hirano R, Kurata H, Matsumoto A, Miki W, Kamiyama M, Itakura H, Yamamoto S, Kondo K. 2. Inhibition of low density lipoprotein oxidation by astaxanthin. J. Atheroscler Thromb., 7 : 26-222. Kittikaiwan P, Powthongsook S, Pavasant P, Shotiprut A. 27. Encapsulation of Haematococcus pluvialis using chitosan for astaxanthin stability enhancement. Carbohydr. Polym., 7 : 78-85. Lim G B, Lee S Y, Lee E K, Haam S J, Kim W S. 22. Separation of astaxanthin from red yeast Phaffi a rhodozyma by supercritical carbon dioxide extraction. Biocheml. Eng. J., : 8-87. Mason R P, Walter M F, Mcnulty H P, Lockwood S F, Byun J, Day C A, Jacob R F. 26. Rofecoxib increases susceptibility of human LDL and membrane lipids to oxidative damage: a mechanism of cardiotoxicity. J. Cardiovasc. Pharmacol., 47 (Suppl. ): S7-S4.

No.6 MIAO et al.: Changes of astaxanthin esters during storage 89 Miao F P, Li Y G, Geng Y H, Hu H J. 25. The effects of temperature on the biomass and the astaxanthin output of Haematococcus pluvialis. J. Wuhan Botany Res., 2 : 7-76. (in Chinese with English abstract) Miao F P, Lu D Y, Li Y G, Zeng M T. 26. Characterization of astaxanthin esters in Haematococcus pluvialis by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. Anal. Biochem., 52 : 76-8. Miki W. 99. Biological functions and activities of animal carotenoids. Pure Appl. Chem., 6 : 4-46. Naguib Y. 2. Antioxidant activities of astaxanthin and related carotenoids. J. Agric. Chem., 48 : 5-54. Nakajima Y, Inokuchi Y, Shimazawa M, Otsubo K, Ishibashi T, Hara H. 28. Astaxanthin, a dietary carotenoid, protects retinal cells against oxidative stress in-vitro and in mice in-vivo. J. Pharm. Pharmacol., 6 : 65-74. Pashkow M D F J, Watumull D G, Campbell M D C L. 28. Astaxanthin: a novel potential treatment for oxidative stress and inflammation in cardiovascular disease. Am. J. Cardiol., (Suppl.): 58D-68D. Raposo M F J, Morais A M M B, Morais R M S C. 22. Effects of spray-drying and storage on astaxanthin content of Haematococcus pluvialis biomass. World J. Microbiol. Biotechnol., 28 : 25-257. Shinidzu N, Goto M, Miki W. 996. Carotenoids as singlet oxygen quenchers in marine organisms. Fish Sci., 6 : 4-7. Xu X M, Chen X M, Jin Z Y. 26. Stability of astaxanthin from Xanthophyllomyces dendrorhous during storage. J. Food Sci. Biotechnol., 25 : 29-6. Yuan C, Jin Z Y, Xu X M, Zhuang H N, Shen W Y. 28. Preparation and stability of the inclusion complex of astaxanthin with hydroxypropyl-β-cyclodextrin. Food Chem., 9 : 264-268.