Extracting Astaxanthin of Phaffia rhodozyma with Lytic Enzyme Produced by Bacillus circulans A 1.383

1 No.1 Vol. 6 Journal of Chemical Engineering of Chinese Universities Feb. 6 13-915(6)1-147-5 1, 1, 1, 1,,, (1., 516;., 515) ph 5. 33mL( 13.8U g 1 ) 371r min 1 16.5h 98% Q556.9TQ95.9TQ96. A Extracting Astaxanthin of Phaffia rhodozyma with Lytic Enzyme Produced by Bacillus circulans A 1.383 JIAN Hua-li 1, ZHU Ming-jun 1, WU Zhen-qiang 1, LIANG Shi-zhong 1, YAO Shuo-ying, LIANG Shu-wa, XIA Feng-geng (1. The College of Biological Science and Engineering, South China University of Technology, Guangzhou 516, China;. Guangzhou Microbe Research Istitute, Guangzhou 515, China) Abstract: The optimal reaction conditions of lytic enzyme produced by Bacillus circulans A 1.383 and used for extracting astaxanthin from Phaffia rhodozyma cells were studied by single factor experiment and uniform design. The results show that when the reaction temperature and ph for extraction of astaxanthin are 37 and ph 5., respectively, and when 33 ml(13.8u g 1 dry yeast) lytic enzyme is added, over 98% of the astaxanthin could be extracted from phaffia rhodozyma cell within 16.5 h. Also, the investigation of the influence of light irradiation and heat on the decolorization of astaxanthin shows that astaxanthin is stable to heating but quite sensitive to light. Meanwhile, the astaxanthin extracted by using the enzyme from Bacillus circulans A 1.383 is more stable to light and heat than that obtained by using mechanical or chemical treatments. Key words: lytic enzyme; Bacillus circulans; astaxanthin; extract; stability 1 [1] [~5] B. circulan A1.383 4-9-35-3-1 (C111)(3ZE131) (1977-) E-mailfesliang@scut.edu.cn

148 6.1 P. rhodozyma ATCC 667 B. circulans A 1.383...1 [6] 7.9 U ml 1 ( r min 1 15 min).. [3] (decrease in turbidity)[(abs Abs t )/ Abs ]1 Abs (initial absorbance at nm of the reaction mixture)abs t t (absorbance of the reaction mixture at time t) 3pH = 6.5 min 5 ( lytic units)..3 TA() [3] 5 ml 3 ml 3mol L 1 HCl 3 min 5 ml 1min :: =5:18:(v/v/v).5 ml min 1 3 4 nm FEA() %=[FEA(µg g 1 )/TA(µg g 1 )]1..4 () [7] (9~1) 3 5mL 53 W 1 cm ( lx) 47 nm 47 nm 3 3.1 ph ph 5 ml( 18.5 U) 1.5 g( 87.4 µg g 1 ) 3 h 1 ph 5.5 3. ph 5.5 5 ml 4. 4.5 5. 5.5 6. 6.5 7. 7.5 8. 8.5 ph 1 ph Fig.1 Influence of ph on extractability of astaxanthin 3 h ( ) 35

1 149 5 3 35 45 5 55 Temperature / 15 5 3 35 45 5 55 Enzyme amount(lytic activity:7.9u ml 1 ) / ml Fig. Influence of temperature on extractability of astaxanthin 3 Fig.3 Influence of enzyme amount on extractability of astaxanthin 3.3 1 1.5 g ph 5.5 35 3 h ( 3) 3 ml 6 1 18 4 3 36 4 48 54 Time / 4 3.4 Fig.4 Influence of time on extractability of astaxanthin 3 ml( 187U) ph 5.51.5 g 35 6 h 4 3.33.4 3.5 [8] U * 16(16 1 ) 1 DPS ( ) Y = 5.854497.53694X 1 18.173557X 4 +4.75877X 1 +.8136X 3 +.14485X 4.1671X 1 X.349486X 1 X 3 +.17463X 1 X 4 +.4184X X 3.4148X X 4.4519X 3 X 4 Eextractability of astaxanthin / % 1 Table 1 Uniform design and result for reaction conditions of lytic enzyme Serial number ph Temperature / Enzyme amount / ml Time / h X 1 X X 3 X 4 Y 1 1(4.5) 5(3) 6(7) 8(7) 64.4 (4.5) 1(45) 1(45) 16(51) 65.1 3 3(5) 15() 1(1) 7(4) 37. 4 4(5) 3(4) 7(3) 15(47) 85.8 5 5(5.5) 8(39) 13(48) 6(1) 76.1 6 6(5.5) 13(54) (15) 14(45) 68.5 7 7(6) 1(18) 8(33) 5(18) 67.8 8 8(6) 6(33) 14(51) 13(4) 79.5 9 9(6.5) 11(48) 3(18) 4(15) 44.7 1 1(6.5) 16(63) 9(36) 1(39) 56.4 11 11(7) 4(7) 15(54) 3(1) 68.5 1 1(7) 9(4) 4(1) 11(36) 86.5 13 13(7.5) 14(57) 1(39) (9) 41.8 14 14(7.5) (1) 16(57) 1(33) 79.4 15 15(8) 7(36) 5(4) 1(6) 36.1 16 16(8) 1(51) 11(4) 9(3).5

15 6 R =.9996F = 477.35 P =. S =.88 Table Factor Regression coefficient of each factor in the regression model and the prominence checkout Regression Partial Prominence t-checkout coefficient correlation level (P) X 1-97.536946 -.6435 1.59854.178 X 4-18.17355733 -.6665 1.78686.131 X 1 4.75877586.6554 1.7356.14315 X P 3.81395.98967 13.8.4 X 4.144851754.67846 1.8475.1 X 1 X -.1676788 -.99586 1.91365. (X 3 )>(X 4 )>ph(x 1 ) X 1 X 3 -.3494865991 -.9916 14.85769. X 1 X 4.17463561.75413.9665.76 X (X ) X 3.4184151.99717 6.53988. X X 4 -.41483619 -.995 14.435.3 X 3 X 4 -.451919978 -.9975 8.919. Y >1 Y1 Y1 X 1 X X 3 X 4 5 5 ( 3) 9 1,3 98 1 3 Y(/ )=1 X 1 (ph)=5.4x (/)=36.7X 3 (/ml)=33.x 4 (/h)=16.56 3 Table 3 The result of checking experiment Serial number Temperature / Enzyme amount ml Time / h Extractbility of astaxanthin / % Forecast value Determined value 1 6.9 36.1 3. 35.55 1 98.1 6.8 37. 38..1 1 9.3 3 5.4 36.7 33. 16.56 1 98.8 4 8. 6. 19.43 4.7 1 9.3 5 6.3 34.54 37.9 39.35 1 91.6 3.6 ( 5) 6h 7.6% 14.7% 35.1% ( 6) [9] 15.8 84. 5 48 [1] Degree of color decrease / % 15 1 95 9 85 75 7 65 lytic Lytic enzyme HCl treating bead Bead grinding 1 3 4 5 6 Time / h 5 Fig.5 The heat stability of astaxanthin extracted by different methods ( Each astaxanthin is incubated at Degree of color decrease / % 11 1 9 7 5 3 1 lytic Lytic enzyme enzyme HCl treating bead Bead grinding -1 1 3 5 6 7 Time / h 6 (lux ) Fig.6 The light stability of astaxanthin extracted by different methods (under lux)

1 151 9-13- ph () 4 ph 5. 33 ml( 13.8 U g 1 )37 16.5 h 98% β - [1] Miller M W, Yoenyama M, Soneda M. Phaffia, a new yeast genus in the Deuteromycotina (Blastomycetes) [J]. Int J Syst Bateriol, 1976, 6: 86-91. [] Okagbue R N, Lewis M J. Mixed culture of Bacillus circulans WL-1 and Phaffia rhodozyma on different carbon source: yeast-wall lytic enzyme production and extractability of astaxanthin [J]. Biotechnol Lett, 1983, 5: 731-736. [3] Okagbue R N, Lewis M J. Influence of mixed culture conditions on yeast-wall hydrolytic activity of Bacillus circulans WL-1 and on extractability of astaxanthin from the yeast Phaffia rhodozyma [J]. J Appl Bacteriol, 1985, 59: 43-55. [4] Tony J Fang, Joh-Ming Wang. Extractability of astaxanthin in a mixed culture of a carotenoid over-producing mutant of Xanthophyllomyces dendrorhous and Bacillus circulans in two-stage batch fermentation [J]. Process Biochemistry,, 37(11): 135-145. [5] Akiko Nishi, Kazuhiko Ohbuchi, Masaaki Hamachi, Chieko Kumagai. Breeding of a constitutive-producing strain of Streptomyces rochei PHA-34 that produces the lytic enzyme for Phaffia rhodozyma and extraction of astaxanthin using the enzyme [J]. Seibutsu-kogaku, 1999, 77(): -65. [6] JIAN Hua-li(), ZHU Ming-jun(), WU Zhen-qiang, et al (). Study on the fermentation conditions of Bacillus circulans A 1.383 producing lytic enzyme ( Al.383 ) [J]. Food and Fermentation Industries(), 4, 3(1): 1-5. [7] Kloostermaniv J, Enfors S O. An integrated approach to the recovery of intracellular products from yeasts by bead milling and precoat filtration [J]. The Chemical Engineering Journal, 1988, 37: 47-54. [8] YANG Nian-lin(), QU Hai-bin(), CHENG Yi-yu(). An optimization method for extraction process of Coptis Chinensis with uniform design and regression analysis() [J]. J Chem Eng of Chinese Univ (), 4, 18(1): 16-13. [9] Yoshida M, Nishi A, Ohbuchi K, et al. Screening of the lytic enzyme for the red yeast Phaffia rhodozyma cell wall and extraction of astaxanthin [J]. J Seibutsu-Kogaku Kaishi, 1997, 75(4): 9-38. [1] Schroeder W A, Calo P, Declercq M L, et al. Selection for carotenogenesis in the yeast Phaffia rhodozyma by dark-generated single oxygen [J]. Microbiology, 1996, 14: 93-99.