TAP CHI SINH HOC, 2012, 34(2): 213-223 N G H I E N CUtJ ANH Hl/CfNG CUA NONG DO M U 6 l LEN SINH T R l / O N G VA KHA N A N G T I C H LUY ASTAXANTHIN CUA VI T A O HAEMATOCOCCUS
PLUVIALIS L A M C O SCT B l T d C DAU C H O QUI TRINH NUOI CAY 2 PHA Lu-u Thi TSm, Dinh Due Hoang, Dinh Thj Nggc Mai,
Ngo Thi Hoai Thu, Hoang Thj Lan Anh, fiang Dilm Hong*
Vien Cdng nghe sinh hgc, '*'[email protected]
TOM TAT: Vi tao luc Haematococcus pluvialis la mpt nguon cung c3p sdc t6 astaxanthin tu nhien - mot ]o?ii sac to dugc su dung rpng rai trong nuoi trong thiiy san, cong nghi?p thuc phim va dugc ph3m, Trong bai bao nay, chiing toi nghien ciru anh hudng ciia nong do muoi NaCl len sinh trudng va tich iuy astaxanthin ciia loai vi lao H. pluvialis Flotow phan lap lai tinh Hoa Binh. Thi nghiem duac ti^n hanh Iheo mo hinh nuoi cay hai pha, trong do d pha mgt tao dugc nuoi cSy dudi di^u ki?n t6i uu di dat mat do le bao cue dai, sau do chuyen sinh khoi tao vao pha thir hai s6c mu6i vdi n6ng do NaCl ciia moi trudng la 0,8%, 1,5% va 2,5%, Ket qua cho thSy, nong do NaCl cao trong moi trirdng gay ra sy uc che sinh trudng va tang cudng su tich Iuy astaxanthin trong te bao. O nong do NaCl 2,5%, te bao tao chuyen sang giai doan bao xac sdm nhat, vdi ham luang astaxanthin tSng nhanh tir 10 pg/te bao len 48 pg/te bao sau khi chuyen sang pha hai 15 ngay. Ngoai ra, cac ket qua xac djnh ham lugng lipid tong so ciing cho thay, sir lich iCiy astaxanthin va lipid Irong te bao H. pluvialis xay ra dong thdi. Ham lugng lipid dat cao nhat la 19
•t 1,3% va 26 ± 0,12%, tuong ling d cong thirc thi nghiem soc muoi 1,5% va 2,5% sau khi chuyen sang pha hai 10 ngay.
Tit khoa, Haematococcus pluvialis, lipid long so, nuoi cay 2 pha, soc muoi, tich Iuy astaxanthin.
MO' BAU
Astaxanthin (3, 3'- dihydroxy P) la dan xuat ciia p-carotcnoid dugc sir dung phd bien trong nuoi trdng thiiy san (thuc an bd sung cho ca hoi, ca canh), cong nghiep Ihiic phara (chat raau ty nhicn), dugc pham (chat chdng gxi hda, tang cudng klia nang mien djch, phdng chdng ung thu) va thyc pham chirc nang [13, 17].
Astaxanthin cd the dugc tong hop d thyc vat, vi kliuan, ragt vai loai nam va tao l u c . Trong sd do, loai vi tao luc nude nggt Haematococcus pluvialis dang thu but dugc sy quan tam nghien ciru boi klia nang tich Iuy astaxanthin cao, cd the len tdi 4-5% trgng lugng khd [24],
Astaxanthin dugc tich Iuy d te bao tao H pluvialis trong sudt qua trinli chuyen tir giai doan sinh dudng (te bao chuyen dpng, edmau xanh) sang giai doan bao xac (te bao hinh cau va kliong chuyen dgng), 0 dieu kien mdi trudng tdi uu, cae te bao tao H. pluvialis duy tri d trang thai sinh dudng va astaxanthin dugc tich Iuy d muc do lh3p. Khi gap cae dieu kien mdi trudng bat lgi nhu: thieu hyt nito, photpho, ty le C/N cao hoac dd man, cudng do anh sang, nhiet do cao, qua trinh tich Iuy astaxanthin se dugc tang cudng nhu mpt phan ung bao ve ciia eo the [2, 12, 23].
Nhieu nghien ctru da dugc tien hanh nham tim ra phuong phap tdi uu cho nudi trdng vi tao H. pluvialis vdi ham lugng cao astaxanthin.
Kang et al, (2005) [11] da so sanh kha nang tich liiy astaxanthin d vi tao lye nay khi nudi quang ty dudng va dj dudng, Ket qua cho thay, khi nudi dj dudng (sii dung acetate nhu ngudn C), hara lugng astaxanthin thdp hon 3 - 4 lan khi nudi cay quang ty dudng trong mdi truong thieu nito dugc bd sung bicarbonate (HCO/) hoac CO2 lien tyc va chieu sang d cudng do cao, Cac ket qua nghien ciru khac ciing cho thay, khi mgt yeu td mdi trudng cd tac dyng lam tang cudng hoac duy tri sinh trudng sinh dudng ciia te bao tao H. pluvialis, se khdng cd vai tro kich thich te bao tao tich liiy astaxanthin va nguoc lai, tac nhan cd tac dung kich thich qua trinh sinh tdng hgp astaxanthin cao se lie che sinh trudng cua tao. Vi vay, quy trinh nudi cay 2 pha, trong dd, pha dSu thiet lap cae dieu kien tdi uu cho sinh trudng cua cae te bao sinh dudng nhara dat mat do cao nhat va pha II, vdi cac dieu kien thuan lgi cho viec tich liiy astaxanthin, duoc coi la hieu qua va phii hgp cho viec san xuat sinh khdi tao giau astaxanthin [18],
Hien nay, d Viet Nam, nudi trdng vi tao
Luu Thi Tam et al.
H. pluvialis mdi dang bat dau vdi mgt sd it cac nghien cim dugc cdng bd [9, 10], Trong bai bao nay, anh hudng cua ndng dg mudi len sinh trudng va kha nang tich liiy astaxanthin ciia vi tao H. pluvialis da dugc nghien ciru, Cac ket qua thu dugc se la ea sd budc dau cho qui trinh nudi trdng 2 pha loai vi tao nay de cd the thu dugc sinh khdi tao giau astaxanthin cho nhieu irng dung khac nhau trong tuong lai.
PHUONG PHAP NGHIEN ClTU Vat lieu
Chung vi tao H. pluvialis Flotow phan lap tai tinh Hda Binh (nam 2009) do phdng Cdng nghe tao, Vien Cdng nghe sitih hgc cung cap.
Tao dugc luu gift va nhan gidng so cap trong mdi trudng C, d 25°C, cudng dp chieu sang 1,5 klux vdi chu ky sang tdi la 12: 12 gid, Thanh phSn mdi trudng C va RM theo cdng bd ciia Dang Diem Hdng va nnk. (2010) [10].
Phifong phap
Tao H. pluvialis dugc nudi trong mdi trudng C, dudi cac dieu kien tdi uu cho sinh trudng (25°C, cudng dp anh sang 1,5 klux, chu ky sang' tdi la 12: 12 gid). Sau 5 ngay nudi (cac te bao chii yeu d trang thai sinh dudng, chiem 80- 90% tdng sd te bao), djch nudi cay dugc ly tam d 6000 vdng/5 phiit. Loai bd phan djch tren, thu can te bao va bd sung mdi trudng RM cd chira cac nong dd mudi khac nhau de tien hanh thi nghiem,
Nghien cihi anh hudng ciia ndng dg mudi len sinh trudng ciia tag H. pluvialis: Thi nghiem dugc tien hanh trong radi truong RM d binli tam giac 250 mL vdi ndng dp NaCl khac nhau: 0%;
0,2%; 0,4%; 0,6% va 0,8%. Mat dp te bao tao ban dau trong cac cdng thite thi nghiem la 6 >=
10'' te bao (TB)/ml, Tao dugc nudi d cac dieu kien tdi uu cho sinh trudng nhu md ta d tren.
Mdi cdng thiic dugc lap lai 3 lan.
Nghien ciiu anh hudng ndng dp mudi len kha nang tich Iuy astaxanthin ciia tao H. pluvialis (thi nghiera nudi cay 2 pha sir dyng tac nhan sdc raudi): Sau khi chgn ra dugc ndng do NaCl thich hgp cho qua trinh chuyen pha nhanh tir te bag sinh dudng sang dang bao xac va tich Iuy astaxanthin, chiing tdi su dyng ndng dp raudi nay de nghien cuu tiep anh hudng ciia
chiing len kha nang tich Iuy astaxanthin d tao H. pluvialis. Thi nghiem nay dugc thuc hien theo quy trinh nudi ciy 2 pha: d pha I tao dugc nuoi d dieu kien toi uu, kich thich tao sinh trudng nhanh va dat mat do ti bao cao nhk trgng 10 ngay. Sau dd, dich tao nudi trong pha I se dugc ly tam va thu te bao. Can te bao dugc hda trong mdi trudng RM mdi cd bd sung NaCl d ndng do 0,8; 1,5 va 2,5% (pha n). Lac deu va chia djch tao vao cac binh tam giac 500 va 1000 ml tuong img vdi 350 va 500 ml djch tao/binh. Cac binh nudi tao dugc dat trong dieu kien nhu md ta d tren. Pha 11 trong thi nghiem nudi cay 2 pha sit dung tac nhan soc mudi, trong 24 gid dau, tao dugc lay 8 gid/lan de quan sat sy thay ddi hinh thai te bao dudi kinli hien vi quang hgc. Nhudm te bao bang Nile Red de quan sat kha nang tich liiy lipid va soi dudi kinh hien vi huynh quang.
Sinh trudng ciia tao dugc danh gia thong qua mat do te bao, mat do quang d budc song 680 nm (ODesonm), ham lugng sac to (chlorophyll a, astaxanthin) va ham lugng protein ndi bao [14, 21, 22]. Ham lugng chlorophyll a, astaxanthin va ham lugng protein ngi bao dugc xac djnh theo cdng bd cua Dgng Dilm Hdng va nnk. (2010) [ 10].
Nhudra te bao tao bang Nile Red theo quy trinh ciia Doan & Obbard (2011) [6]: Bd sung 5 pi dung djch Nile Red (9 - diethylamino - 5H benzo [a] phenoxazine-5-1) cd ndng do 0,1 mg/ml trong aceton vao 5 ml djch tao. Hon hgp dugc vortex nhe va u trong tdi 10 phiit d nhiet dp phdng. Sau dd, cac te bao tao da nhudm bang Nile Red se dugc quan sat dudi kinh hien vi huynh quang vdi budc sdng kich thich tir 450- 490 nm.
Tach chiet lipid tdng sd va xac djnh ham lugng lipid tong sd theo phuong phap cua Bligh
& Dyer (1959) (I] vdi mgt sd cai ti^n de phii hgp vdi dieu kien phdng thi nghiem cua Viet Nam: can 0,1 g sinh khdi tao khd cho vao coi chay sir, bd sung them 0,2 g Na2S04 va nghien bdn hgp nay thanh bot mjn. Sau dd bd sung them 10 ml hon hgp dung mdi chloroform : methanol (ty le 2:1 (v/v)) va ngam trong 1 gid d nhiet dd phdng. Lgc hon hgp qua giay lpc Whatman sd I va thu djch trong. Ba sinh khoi dugc tiep tuc chiet vdi chloroform tir 2 - 3 lan
214
TAPCHiSINHHOC. 2012, 34(2) 213-223 de thu tdi da lipid trong smh khoi tao, Djch
chiet dugc trgn d^u va chuyen sang pheu chiet, Sau dd, bd sung them 10 mi dung dich NaCI 0,9% va de tTnh d nhiet do phdng qua dera. Ldp dung moi hiiu co phia dudi chira cac thanh phSn lipid dugc thu nhan. Sau dd, dung mdi dugc bay hoi d dO^C va lam khd trong desiccator. San pham duoc hda tan trong n-hexan, lgc bd can va
lam bay hoi n-hexan de thu hoi lipid, Sd lieu thi nghiem dugc xii ly biing phan mem Excel va xit ly thdng ke ANOVA d rauc y nghTa P < 0,05.
KET QUA VA THAO LUAN
Anh hudng cua ndng do NaCl len sinh trirdng cua tao H. pluvialis
- ^ D / C - • - 0 , 2 0 % - * - 0 , 4 0 %
Hinh 1. Mat do te bao ciia tao H. pluvialis d cae ndng dg NaCI khac nhau sau 35 ngay nudi
Hinh 2. Su thay ddi ciia ham lugng chlorophyll a (chi a) ciia H. pluvialis a cac nong dp NaCl
khac nhau
Hinh 3. Sy thay ddi cua ham lugng astaxanthin eua H. pluvialis a eac ndng dp NaCl khac nhau
- • - l ) . 2 % - * - 0 / l %
Hinh 4. Su thay ddi cila ty le astaxanthin /chi a cua H. pluvialis a cac nong dp NaCl khac nhau
Hinh 5. Sy thay ddi ciia ham lupng protein noi bao ciia H. pluvialis d eac ndng do NaCl
khac nhau
Hinh 6. Su thay ddi ham lupng lipid tdng so ciia tao H. pluvialis d cdng thirc ndng do NaCl 0,8%
Anh hudng ciia ndng dp NaCI len sinh bao (MDTB) dugc trinh bay d hinh 1, Ket qua trudng ciia tao H. pluvialis thdng qua raat do te cho thay, ndng do NaCl ed anh hudng ro ret len
Luu Thi Tam el al.
sinh trudng cua tao H. pluvialis. Trong 7 ngay dau ticn, d eac ndng do mudi khac nhau tao vin sinh trudng phat trien binh thudng, khdng cd sy sai khac cd y nghTa thdng ke giira cac cdng thirc thi nghiem, Sau 7 ngay nudi, sinh trudng cua tao bat dau cd sy sai khac giira cac cdng thiic thi nghiem cd ndng do mudi khac nhau. Tao sinh truong tot nhat d cdng thuc doi chiing (khong bo sung mudi) vdi mat do te bao dat cue dai la 35 >^ IO'' TB/ml sau 12 ngay, tiep den la d cong thuc 0,2%, 0,4%o, 0,6% va 0,8% NaCl tuong iing vdi mat dp te bao dat cue dai la 24; 20; 19 vii \b "• IO"* TB/ml. Ci cong thuc ddi chung, tao duy tri chu yeu d dang te bao sinh dudng, cd mau xanh, 2 roi. Chung loi da khdng quan sat thay cd sy thay ddi hinh thai tc bao tu dang "sinh dudng sang dang bao xac, Ngugc lai, d cdng thiic thi nghiera vdi 0,8%i NaCl, trang thai te bao tao chu yeu d dang bao xac va cd tich Iuy astaxanthin ben trong te bao.
Ty le caroteniod/chlorophyll a la mpt chi sd the hien trang thai sinh ly ciia te bao lao va ty le nay cfing la thdng sd tdt de danh gia kha nang tich liiy astaxanthin cua Haematococcus pluvialis [7, 15], Vi vay, mac du H. pluvialis cd chua ca chlorophyll a- va b nhung chung tdi da lua chpn nghicn cim su thay ddi ham luong chlorophyll a va ty le astaxanthin/chloropliyil a la nhirng thdng sd dac trung cho sinh trudng va tieh Iuy astaxanthin ciia loai vi tao luc nay Ham lupng chlorophyll a, astaxanthin, ty le astaxanthin/chl a va protein ndi bao d cac cdng thuc thi nghiem cd ndng do mudi kliac nhau dugc chi ra d hinh 2, 3, 4 va 5, Tir cac ket qua dugc chi ra d hinh 2, 3, 4 va 5 nhan thay, hara lugng sac td chlorophyll a va astaxanthin cd xu hudng tang va tang manh sau 15 ngay nudi cay, Hara lugng astaxanthin dat cao nhat d cdng thite ddi chung vdi gia tri la 1182 pg/1 sau 35 ngay nudi, tiep dd la 479, 448, 391 va 283 yg/l tuong img d cac cdng thiic thi nghiem cd ndng do NaCl la 0,2%), 0,4%i, 0,6%i va 0,8%, Dieu nay cd the giai thich la do tao sinh trudng manh d cdng thirc ddi chiing, mat do te bao tang eao dan den ham lugng astaxanthin tdng sd trcn don vj the tich nudi la cao. Tuy nhicn, ty le astaxanthin/chl a lai cho ket qua ngugc lai. C) cdng thtic cd ndng dg NaCl la 0,8%, ty le astaxanthin/chl a dat gia trj cao hon 0,5 sau 15 ngay, trong kin cac cdng thuc nong dg NaCl
khac ty le nay deu nhd hon 0,5 (hinh 4). Theo Kobayshi et al, (1997) [12] _ khi ty le astaxanthin/chl a > 0,5 cho thay te bao tao chii yeu d trang thai bao xac va cd su tich Iuy astaxanthin. Nhu v^y, ndng do 0,8% NaCl giiip te bag tao H. pluvialis chuyen giai doan tir cac te bao dang sinh dudng sang dang bao xac nhanh hon, Dieu nay rat quan trgng khi ap dung cho quy trinh nudi cay 2 pha ddi vdi loai vi tao luc nay.
Ham lugng protein ndi bao cd xu hudng giam dan theo thdi gian nuoi d tat ca cac cong thiic thi nghiem. Ket qua sau 35 ngay nuoi, ham lupng protein ngi bao cdn lai deu nhd hon 100 pg/te bao, Ci cdng thiic ddi chung, ham lugng protein ndi bao giam 1/2 lan so vdi gia Irj ban dau. Trong khi dd, bam lupng nay chi giam 1/4 lan so vdi gia trj ban dau d cae cdng thiie eo bo sung NaCl (hinh 5).
Cung vdi sy thay ddi ve ham lugng sac to va protein ngi bao thi ham lugng lipid tdng so cung cd su bien ddi trong qua trinh nuoi. Su thay ddi ham lugng lipid tdng sd ciia tao H. pluvialis d cdng thuc thi nghiera cd bo sung 0,8%i NaCl dugc trinh bay d hinh 6. Ket qua cho thay, ham lugng lipid tdng sd cd xu hudng tang dan trong qua trinh nudi. Khi cac te bao chuyen sang giai doan bao xac va tich Iuy astaxanthin thi dong thdi cd su tang ve ham lugng lipid tdng so, 0 thdi diem ban dau, ham lupng lipid chiem 13,54 ± 0,23%) trgng lugng khd-TLK va sau do tang dSn len 21,34 ± 0,35%. TLK d ngay thir 31.
Ket qua nay hoan toan tuong ty vdi eac ket qua ciia cac tac gia khac da cdng bd tren the gidi [4, 5, 16, 19].
Nhu vay, viec bd sung NaCl vdi cac nong do khac nhau da anh hudng len sinh truong va kha nang tich liiy astaxanthin d tao H. pluvialis.
6 nong do NaCl thap (0,2 va 0,4%.), sy sinh trudng ciia tao chua bj anh hudng nhieu Tuy nhicn, khi tang ndng do mudi len 0,6 va 0,8%, sinh trudng cua tao bj itc che va qua trinh chuyen pha tir te bao sinh dudng sang dang bao xac dien ra nhanh hon, dac biet la d ndng dp NaCI 0,8% (hinh 7). Vi vay, chung tdi chgn ndng do mudi nay lam tac nhan cam ung de tien hanh thi nghiem nudi cay 2 pha d tao H. pluvialis.
TAP CHI SINH HOC, 2012, 34(2); 213-223 Ddi chirn" l).2"„ Nut 1 4 \ ( l 0.(."„Xafl ".S"., NaCI
Hinh 7. Hinh thai te bao tao H plmialis a cae nong do NaCl khac nhau sau 35 ngay nudi d cap dp binh tara giac 250 ml dugc ehup bang kinh hien vi quang hgc vdi do phdng dai x400
Hinh 8. Mat do td bao ciia H. pluvialis trong thi nghiem nudi cay 2 pha vdi pha II su dung tac nhan sdc mudi d ndng do 0,8%o NaCl
Hinh 9. Su thay ddi ham lugng astaxanthin eua tao H pluvialis trong thi nghiem nudi cay 2 pha
vdi pha II sdc mudi
Thi nghiem nuoi cSy 2 pha d tao H, pluvialis vdi pha II s6c muoi
Quy trinh nudi cay hai pha trong dd pha ragt nudi cay tag duoi dieu kien tdi uu de dat raat do te bao cue dai, sau dd chuyen sinh khdi tao vao pha thir 2 vdi cac dieu kien thuan lgi cho viec tich Iuy astaxanthin da dugc chimg ramh ia md hinh hieu qua di thu dugc sinh khdi H. pluvialis giau astaxanthin [18]. Trong thi nghiem nudi c§y 2 pha vdi pha II sdc mudi nay, chiing tdi da ti6n hanh theo diing quy trinh tren. Trude het, tao dugc nudi cay dudi dieu kien tdi uu cho den khi tao bat dSu di vao pha can b5ng vdi mat do tg bao eye dai la 28 ^ lO'' TB/ml va ham lugng
Hinh 10. Sy thay ddi ham lugng lipid tdng sd ciia tao H. pluvialis trong thi nghiem nudi cay 2
pha vdi pha II sdc mudi chlorophyll a dat 400 ± 10 pg/l sau 10 ngay nudi cdy. Ti^p theo, tao dugc chuyen sang dieu kien soc mudi dl kich ihich t6 bao chuyen sang giai doan bao xac cd tich Iuy cao astaxanthin.
Theo cdng bo cua Boussiba & Vonshak (1991) [3], khi tao H. pluvialis gap dieu kien bat lgi nhu ndng do raudi cao thi te bao tao se cd ca ch6 ty bao ve bang each ngimg sinh trudng hoan loan, d bao chuyen tir trang thai sinh dudng, cd mau xanh, chuyen ddng sang dang bao xac, mau dd va tich Iuy lugng Idn astaxanthin trong te bao. Trong nghien cuu nay, chung tdi cung quan sat thay sy thay doi hinh thai cita te bao tao khi cam ung bang ndng dp
Luu Thi Tam et al NaCl 0,8% (hinh 7). Su thay ddi ve mat dd tg
bao trong 2 pha ciia thi nghiem dugc trinh bay d hinh 8.
Ket qua chi ra d hinh 8 cho thay, trong 10 ngay ciia pha 1, mat dp te bao cd xu hudng tang nhe va dat gia tri cao nhat la 31 x IO"' TB/ml d ngay thii 5 sau nudi. Sau 10 ngay nudi, NaCl se dugc bd sung vdi ndng dd 0,8%i vao mdi trudng nudi d6 kich thich t6 bao tao tich liiy astaxanthin Luc nay, mat do te bao tao trong pha II cd sy sai khac ro ret giira cdng thirc ddi chimg va thi nghiem. O cdng thiic ddi chimg, mat do te bao cd xu hudng duy tri on djnh trong qua trinh nudi. Ngugc lai, d cdng thirc thi nghiem, khi cam ung bang ndng dp mudi cao se ire che sinh trudng cua tag, mat dd te bao cua cdng thite thi nghiera lai giam 2,5 lan so vdi gia
tri mat do te bao d§u pha II sau 22 ngay nudi.
Su thay ddi ve ham lugng sac td astaxanthin va hara lugng lipid tong sd trong nudi cay d pha II dugc trinh bay d hinh 9 va 10. Ket qua chi ra tren hinh 9 va 10 da cho thay, ham lugng astaxanthin/te bao ciia cdng thirc ddi chung va thi nghiera ed xu hudng tang dan trong ca pha 1 va pha II, Tuy nhien, khi cd su dyng tac nhSti sdc mudi, ham lugng astaxanthin/te bao tang dot bien. Sau 6 ngay sdc mudi, ham lugng sac td astaxanthin tang tir 5 pg/TB len 37 pg/TB.
Ham lugng lipid tdng sd eua tag d pha 1 cd xu hudng tang nhe tit 13,54 ± 0,23% TLK d 0 ngay dgn 14,92 ± 0,34% TLK d ngay thir 10. Sau khi chuyen tao sang pha II dudi dieu kien sdc mudi, ham lugng lipid tdng sd tang nhanh, tu 14,92 ± 0,34%, TLK len 17,14 ± 0,26%, TLK sau 6 ngay.
10 ngay 14 ngay 1 S ngay 22 ngay 0 Tiga)
^
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^^^ft^Ki^l
^^K^'^^^^^^^^^l
Hinh 11. Hinh thai te bao tag H. pluvialis chup dudi kinh hien vi quang hgc (hang tren) va kinh hien vi huynh quang sau khi nhudm Nile Red (hang dudi) (do phdng dai 1000 lan) d cdng
thuc ddi chtxng va cdng thirc 0,8% NaCl trong bmh tam giac 250 mL Sy thay ddi ham lugng lipid ben trong te
bag tao H. pluvialis cung dugc phat hien nhanh bang phuang phap nhudm le bao bang Nile Red.
Chat nhudm Nile Red se di qua mang te bao va lien ket \di cac the lipid trung tinh ngi bao va phat huynh quang mau vang khi dugc chieu bang anh sang huynh quang d dai budc sdng ttr 450^90 nm (hinh 11), KSl qua chi ra d hinh 11
da cho thay, d cdng thiic ddi chimg, the lipid nhd, tap trung d mep ngoai ciia te bao, the hien qua cac the bat mau vang, Trong khi dd, d cong thuc thi nghiem, cac the lipid to tap trung d ca ben trong te bao viing quanh nhan, bat mau vang dam
Tuy nhien, khi tien hanh sdc mudi d ndng dp 0,8%) NaCl thi qua trinh carotenoid hda diln
TAP CHI SINH HOC, 20! 2, 34(2): 213-223 ra ch§m, eac te bao chuyen sang giai doan bao
xic nhanh nhung qua trinh tich Iuy astaxanthin bfin trong te bao keo dai. Sau 22 ngay, sic td dd ciia cac t6 bao H. pluvialis d cdng thiic thi nghi?m soe mudi chi tap trung quanh viing nhan. Ket qua nay ciing hoan toan phii hgp vdi nghien ciru ciia Sarada et al. (2002) [20] da cdng bd. Tac gia da chi ra rang, thdi gian nudi
giong ban dau (tuoi tao) la ySu to quygt djnh kh__di ddng qua trinh sinh tdng hgp astaxanthin d mau cam ling bdi sdc mudi. Khi djch tao mdi dugc hoat hda (4-8 ngay nudi) rSt min cam vdi NaCI, trong khi dd, nSu djch tao nudi trudc dd 12-16 ngay thi co kha nang chdng chju tot hon vdi sdc mudi va vi vay, thdi gian tich liiy astaxanthin keo dai den 20 ngay.
- * - D « ; - • - 1 , 5 % NaCl
Hinh 12. Su thay doi mat dg te b^o tao H. pluvialis a cac ndng do NaCl khac nhau
Hinh 13. Sy thay ddi ham lugng ehi a cua tao H. pluvialis a cac ndng do sdc mudi khac nhau
~OIC-m-l^% NaO -*-2,S%Naa Hinh 14. Sy thay doi ham lugng astaxanthin
ciia tag H. pluvialis a eac nong do sdc mudi khac nhau
Hinh 15. Su thay ddi ham lugng lipid tdng sd cua tao H. pluvialis a cac ndng do sdc muoi
khac nhau Cung theo tac gia Sarada et al. (2002) [20],
khi s6c muoi d tao H. pluvialis can cd chu I^
cam ung dai hon hoac ket hpp vdi nhieu tac nhan cam img khac mdi tich Iuy lugng Idn carotenoid ben trong te bao. De lam ro hon van de nay, chiing tdi da tien hanh thi nghiera sdc mudi lan 2 vdi ndng dg mudi cam ung la 1,5 va 2,5% ket hgp vdi nhi?t do cao (> 30°C). Ket qua thi nghiem dugc trinh bay d hinh 12, 13, 14 va 15.
Ket qua chi ra d hinh 12 da cho thay, mat do te bao d cdng thite ddi chung tiep tuc tang, tir 33 X iC* len 47 X lO" TB/ml va chung tdi khdng quan sat thay cd su thay doi trang thai te bao tu
te bao d^ng sinh duong sang dang bao xac sau 15 ngay nuoi. Trong khi dd, gia trj mat do te bao d mdi trudng cd bd sung NaCl vdi cac ndng dg khac nhau lai cd xu hudng giam r5 ret, dac biet la d ndng do mudi 2,5%). Sau 15 ngay, mat dp te bao d cdng thiic nay giam hon 2 lan so vdi gia trj ban dau, tir 33 x lO" xudng 14 x 10^ TB/
ml, te bao d trang thai bao xac va tich liiy mgt lugng Idn astaxanthin ben trong te bag. Tuy nhien, te bao bj mat dan sac td va bj chet khi nudi d ndng do muoi cao trong thdi gian dai,
Khi sir dung ndng do mudi cao ket hgp vdi nhiet dp cao thi qua trinh chuyen giai doan tir te 219
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bao sinh dudng sang te bao dang bao xac diln ra rat nhanh trong vdng 8 gid. Den ngay thir 5, chiing tdi quan sat thay dich tao chuyen sang mau nau dd va tich Iuy mgt lugng Idn sac td dd ben trong te bao ciia chiing. Ket qua ve su thay ddi ham lugng sac td (chlgrgphyll a va astaxanthin) dugc trinh bay d hinh 13 va hinh 14, Hara lugng chlorophyll a d eac cdng thiic cd xu hudng tang sau 5 ngay nudi, sau dd ham lugng nay giam dan sau 15 ngay sdc raudi (hinh 13) Rieng d cdng thiic ddi chung, trong giai doan tir ngay thii 10 den 15, ham lugng chlorophyll a giam trong khi mat do te bao van tang. Dieu nay dugc giai thich la do sy giam ham lugng chlorophyll a trong te bao tao
H. pluvialis Idn han so vdi su tang mat do.
Ngoai ra, cdng cd thg ham lugng chlorophyll b vSn tang cao trong trudng hgp nay nen tao van tang mat do tS bao. Ngugc lai, vdl xu hudng thay ddi eua chlorophyll a, ham lugng astaxanthin cua te bao tao H. pluvialis tang manh, tir 10 pg/TB d ngay thii nhit len 39 va 48 pg/TB d ngay thu 15, tuong dng vdi cdng thiic ndng do mudi l,5%o va 2,5%), lan lugt 0 ndng do mudi 2,5%o, tao chuyen sang giai doan bao xac sdm, chi trong vdng I ngay. Nhu vay, sy kel hgp giiia ndng do mudi cao va nhiet do cao la nhan td gidi ban, giiip tag H. pluvialis chuy6n pha nhanh va cam ling tich liiy lugng Idn astaxanthin ben trong te bao.
10 nga\ 15 ngiiy 24 gid 5 nguy 10 ngay
• • • ^
Hinh 16. Hinh thai ik bag tao H. pluvialis chup dudi kinh hien vi quang hpc (hang tren) va kinh hi6n vi huynh quang sau khi nhudm Nile Red (hang dudi) (do phdng dai 1000 lan) d cdng thiic
ddi chimg, cdng thirc 1,5 va 2,5% NaCl trong binh tara giac 500 va 1000 ml
TAPCHISINHHOC, 2012. 34(2); 213-223 Sy tich Iuy astaxanthin trong te bao
H pluvialis dudi dieu kien sdc mudi da dugc trinh bay trong cac nghien ciru cua Boussiba &
Vonshak (1991), Cifuentes ct al. (2003), Kobayashi et al. (1997) [3, 5, 12] Mac du thi nghiemcua chimg tdi dugc tien hanh dya theo cong bd cua Cifuentes et al. (2003) [5] nhung n6ng dp NaCl thich hgp nhit d^ kich thich su lich liiy astaxanthin eua chiing tao H. pluvialis Flotovi' ma chung tdi phan lap dugc tai Hda Binh la cao hon so vdi ket qua ciia nghien cim cua cac lac gia trcn da cdng bd (2,5% NaCl so voi 0,S% NaCl). Vi vay, viec ap dung cac phuong phap nghicn ciru khac nhau ddi vdi chung lao dja phuong nen dugc cai biSn hgp ly de thu dugc cac ket qua tdt va tin cay
Golyal (2007) [8] da chi ra rang sdc muoi la nguycn nhan gay ra sy tang ham lugng cua lipid npi bao va glycerol d tao Duiialtella tertiolecia.
Trong thi nghiem nay, chung tdi cung tim hieu moi lien he giiia qua trinh chuyen pha te bao tir sinh dudng sang giai doan bao xac, tich liiy astaxanthin va qua trinh tich liiy lipid tong so ben trong te bao. Ket qua ve sy thay ddi ham lugng lipid tdng sd cua tao H. pluvialis a cac ndng dg NaCI kliac nhau dugc trinh bay d hinh 15. Ket qua chi ra d hinh 15 cho thay, qua trinh tieh Iuy lipid tdng sd va astaxanthin xay ra ddng thdi, O cdng thirc ddi chimg va cdng thiic cd ndng dp raudi 1,5%), hara lupng lipid tdng sd dao ddng trong khoang 15 ± 0,35% - 19 ± l,3%o TLK va khdng cd su kliac biet nhieu gitia cac ngay nudi, Ngugc lai, ham lugng nay tang manh d cdng thirc cd ndng do muoi 2,5%o (tang tir 15
± 0,35% len 26 ± 0,12% TLK) sau 10 ngay sdc mudi, Tuy nhien, khi keo dai thdi gian sdc mudi len 15 ngay thi ham lugng lipid tdng sd lai giara di. Dieu nay cd the do tao bi chet khi nudi d nong do mudi cao, dan tdi ham lupng lipid tdng so tren trgng lugng khd te bag giam,
Hinh 16 cho thay, eac the lipid tap trung nhieu d vimg mang xung quanh te bao va co kich thudc khdng deu, Tuy nhicn, khi nhudra te bag H. pluvialis chimg tdi nhan thiy, rang muc dp bat mau vang vdi chat nhugra Nile Red tang dan kin te bao chuyen sang giai doan bao xac va bat diu tich liiy astaxanthin, nhung sau dd giam din khi te bao chuyen sang dang bao xac hoan toan. DICU nay ggi y rang, cd the trong qua trinh
chuyen pha sang giai doan bao xac, thanh phin lipid cGng cd sy bien ddi ve lugng giira triacylglyccrol, phospholipid va cholesterol. K^t qua nay cuiig tuong ddng vdi k^t qua dinh lugng lipid tdng sd theo phuong phap cua Bligh
& Dyer (1959) [1].
KET LU^N
Ndng dg raudi NaCl cd anh hudng rd ret len sinh trudng va kha nang tich Iuy astaxanthin ciia lao H. pluvialis. Ndng dp mudi 0,6- 0,8% se kira ham sinh trudng va kich thich tao tich Iuy astaxanthin.
Viec bd sung NaCl 0,8% vao mdi trudng nudi trong pha II cua quy trinh nudi cay 2 pha tao H. pluvialis nhu la mgt tac nhan de cam ung su lich tich Iuy astaxanthin cho hieu qua khdng eao, Thdi gian chuyen pha tu te bao sinh duong sang dang bao xac keo dai hon 20 ngay, Khi tang nong dp NaCl len 2,5%o ket hpp vdi nhiet dd cao sS kich thich tag chuyen sang giai doan bao xac nhanh hon (trong vdng 8 gid) va bat dau lich liiy astaxanthin. CJ ndng do mudi 2,5%, hara lugng astaxanthin tang tu 10 pg/TB len 48 pg/TB sau 15 ngay sdc raudi Ket qua thu dugc nay la nhirng dir lieu cd gia tri khoa hgc budc dau phuc vy cho nhung nghien ciru tiep theo ve quy trinh nudi eiy bai pha de thu dugc sinh khoi tao H. pluvialis giau astaxanthin.
Qua trinh tich Iuy astaxanthin va lipid long so d tao H. pluvialis xay ra ddng thdi vdi qua trinh chuyen trang thai te bao tu sinh duong sang dang bao xac. 0 cdng thiic ddi chiing va cdng ihue cd ndng dp NaCl 1,5%, ham lupng lipid tdng sd dao dgng tir 15 + 0,35%o den 19 ± l,3%ti trgng lugng khd. Khi tang ndng do NaCI len 2,5%), lipid tdng sd dat gia trj cao nhat la 26
± 0,12% trgng lugng khd sau 10 ngay.
Lffi cam on: Cdng trinh dugc hd trg kinh phi tu de tai "Nghien ciin cdng nghe nudi vi lao Haematococcus pluvialis va cdng nghe chiet xuat astaxanthin" Bp Ndng nghiep va Phat trien ndng thdn thupc Chuong trinh Cdng nghe sinh hgc trong thiiy san nam 2010-2012 cho Phong Cdng nghe tao, Vien Cdng nghe sinh hgc.
TAI LIEU THAM KHAO
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STUDY ON T H E EFFECT OF SALT CONCENTRATION ON GROWTH AND ASTAXANTHIN ACCUMULATION OF MICROALGAE HAEMATOCOCCUS
PLUVIALIS AS THE INITL^L BASIS FOR TWO PHASE CULTURE OF ASTAXANTHIN PRODUCTION
Luu Thi Tam, Dinh Due Hoang, Dinh Thi Ngoc Mai, Ngo Thi Hoai Thu, Hoang Thi Lan Anh, Dang Diem Hong
Institute of Biotechnology, VAST SUMMARY
Microalga Haematococcus pluvialis Flotow is one of the natural sources of astaxanthin, a pigment widely used in aquaculture, food, pharmaceutical and nutraceutical industries. In the present study, we investigated the effect of high NaCl concentrations on growth and astaxanthin accumulation in the microalgae. Growth was observed under different salt concentrations (0,8%, 1 5% and 2,5% NaCl) according to two - phase culture mode. The obtained results have indicated that the high NaCl concentrations caused an increase in carotenoid content per cell and a decrease m the algal growth. The best carotenogenic condition by addition of salt was obtained at 2,5% NaCI under high temperature. Astaxanthin content per cell increased 4.8 folds in comparison to the initial value, from 10 pg.celf' to 48 pg.celf' after 15 days of salt stress. We observed that the accumulation of total lipid content was correlated with an increasing in astaxanthin content. The total lipid content of control was 15 ± 0,35% of dry cell weight whereas that of cell exposed to salt stress was 19% ± 1,3% and 26 ± 0.12% at 1.5% and 2.5% NaCl concentrations, respectively. This study will be an initial basis for the two phase culture process to obtain astaxanthin rich Haematococcus pluvialis biomass
Keywords: Haematococcus pluvialis, Astaxanthin accumulation, salt stress, total lipid, two phase culture.
Ngdv nhgn bdi: 14-2-2012