Top chi Cong nghe Sinh hoc 6(4): 489-496, 2008 TAO DOT BIEN NANG CAO HOAT TINH SINH TONG HOP POL Y-13HYDROXYBUTYRATE CEA VI KHUAN ALCALIGENES LATUS VN1 Pham Thanh Train Dinh Man', Yutaka Tokiwa2 Vien Ging nghe sinh hoc, Vien Khoa hoc va Cong nghe Viet Nam for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan 2Institute TOM TAT Poly-p-hydroxybutyrate (PHB) la mit loci plastic chiu nhigt co kha nang bi phan hily sinh hoc PHB dang duqc quan tam nghien tiro nhieu vi no co the giai quy6t &roc van nhiem moi throng chAt thai nhaa CO nguOn goc ter dAu moi gay Ngay nay, hau hat rththig nghien ciru filth vac tap ming vao vigc tach vi sinh vat sin sinh PHB tir nhting ngu6n khac va cai thign kha nang cita chimg Trong nghien ciru nay, tia UV ducic sir clang nhu tic nhan vat 1.S, anh hiring len chimg Alcaligenes latus VN1 da thu tilting dit bien cO hoat tinh sinh tong hop PHB cao Sau chiau tia UV di thu &roc 40 tren tong so 198 chimg co hoat tinh sinh PHB cao Nang suit tich lily PHB cita cac chung thu &roc cao han hen chung dai mit cach coy nein Nhung chimg khic cho nang suit tich lily PHB bang hoac thap han chimg be mg Phan ion cac chung dit bien sinh PHB cao thu ducic tai thiri diem 30 phut chign tia UV Tai thai diem hau het vi Ichufin bi diet Trong thing dit bien throe chin loc, chung dit bien A latus VN1-20 la tit nhAt, no co kha nang tich lay tai 55,6% PHB/trong luong kho to bao Nang suet sinh PHB chimg dit bien da cai thign ducrc khoang 40% so veil chimg dai San pham PHB thu duce len men chimg dit bien A latus VN1-20 tren mei truing co se/ sau 72 h da dtroc tech chit bang chloroform cho di tinh sach cao va chit luting tot dap Ung yen cau img dung ding nghigp Tie khoa: Alcaligenes latus, dOt ban, PHB, poly- fl-hydroxybutyrate, polymer phOn hr y sinh hoc MO DAU Nhaa c6 nguin goc tit dial moi dang dtroc dung ring rai cuic sing hang cue chung ta Tuy nhien, chung gay o nhiem moi truing nghiem boi tv nhien cac sin pham nhoa n6i chung can den hang ngan narri mai phan tiny duoc Them vao, str can kigt nguin dan m6 cang thitc day vigc can thiet phai tim cac nguon vat lieu mai de thay the Cac loaf polymer phan hily sinh hoc sinh to cac vi,sinh vat la giai phap tit nhat Poly-(3inydroxybutyrate (PHB) thugc Whom poly-hydrayalkanoates (PHAs) la cac polyester c6 tinh chat ttrcmg to nhua tong hop PHB dang doge quan tam nghien cau nhieu vi chimg co dac tinh deo din hei via tinh chju nhigt cao Dic bigt, PHB CO kha nang bj phan hay hoin toan boi vi sinh vat, khing co deic to va co the duqc sin xuit to nhing nguon c6 the tai tao PHB duqc tong hqp to nhien to bao chat dm vi khuan nhu the du tra carbon va nang !Pun spot pha sinh truing tinh cac to bao bj giai han mit hoic nhieu nhan to can thiet cite mai triremg nhu N, P, S, Mg, K, Fe hoac 02 nhung lai du thira nguOn C (Anderson, Dawes, 1990) Trong horn 250 loll vi khan dirge th8ng bao co kha nang tich lily PHB, Alcaligenes laws la vi Idmin co kha !tang tich lily PHB cao suit pha sinh truing Idling dai hOi so giai han dinh cluing (Wang, Lee, 1997) Daniel va doing tic gia (2003) gay at biin vii muc dich nang cao kha nang tich lily PHB tren Azospirillum brasilense bang sic nhigt, say Icho khong va bang chign tia UV Hikmet va ding tic gia (2003) nghien ciru tao dit bien tren B megatherium Y6, B subtilis K8, B sphaericus X3 va B firmus G2 bang chit acriflavin va 5-bromourasil Nhieu nghien tiro khic cling thong bao su sinh PHB thay dei nhimg chiinp dit bien (Ojumu et al., 2004) Do do, tic nhan dit bien ducx xem nhu co tie'm nang vigc cai thign nang suit PHB o vi Trong nghien dm chting fel da dung tic nhan dit Wen vat ly la tia UV de nang cao kha nang tich lily PHB cite cang vi khan A laws VN I 489 Tap chi Cong nghe Sinh hpc 6(4): 489-496, 2008 VAT LIEU VA PHIJONG PHAP Vi sinh vet Chung vi khan A latus VN1 phan lap tar dat nhiem au Viet Nam c6 kha nang tich luy PHB lchoang 40% tong lucmg kho dm brio Nuoi cay Cac nuoi cay tren thach dia, nuoi cay lac, nhan giong va len men duqc den hanh tren mei truang co ser (/1): Glucose 30 g; Peptone 10 g; Cao men g; Na2Mo04.2H20 0,5 mg; Na2W04.2H20 0,5 mg; MnSO4 0,5 mg; Fe SO4.7H20 10 mg; CaC12.2H20 20 mg; NaC1 0,1 g; MgSO4.7H20 0,2 g; (N114)2SO4 g; KH2PO4 0,2 g; K2HPO4 1,6 g; pH = (Lee, Chang, 1995) Nuoi city len men: Qua trinh len men duqc thuc hien binh len men (Bioflo 3000, New Brunswick Scientific Co., Edison, NJ) chira 2,51 mei truerng Nhiet de duqc trl 30°C, toe de khuay 300 vonf/phirt, clang thoi Ichi 1,0 wm pH = 7,0 ± 0,1 duqc dieu khien tu dung suet qua trinh len men Sau 20, 24, 28, 44, 48, 52, 68 va 72 h len men: xac djnh OD, tong luqng kho to bio, ham lucmg PHB, Chiang khir Phtrang phip phin tich Sinh truing caa vi khuaAn duqc xac djnh bang met de quang hoc (OD) djch nuoi 7, = 620 nm Sinh khzii thu &Km tar ly tam 20 ml djch nuoi (10000 yang/phut, 4°C, 10 phut) dupe say kho 105°C den ltryng killing dai va can (can phan tich dien to AB 204, Mettler Toledo, They st) pH cuoi cua djch nuoi dirge xac djnh bang may pH Chpn loc dQt biEn throng tinh biing dinh tinh Dot bien throng firth duqc tech tren moi truang thach c6 bo sung 1% thutic nhuem Nile blue A (Sigma) Nuoi nhung dia cay in 30°C Sau nuoi ay, quan sat cac khuiti lac dueri anh sang cue tim (235 nm) Nhang Idiutin lac duong firth PHB c6 mau yang huynh quang chuyen sang da cam, nhung Ichuen lac am tinh c6 mau trang (Hilcmet et al., 2003) Chpn !pc thit Min throng tinh bring djnh lsrpng Cac got bien duang tinh chop IQc tar thach dia duqc nuoi tren mei truing djch the co se/ 48,h in 30°C, lac 200 yang/phut Cuoi giai doan nuoi cay tian hanh OD xac djnh ham luqng PHB, cac mau duqc ly tam (15 phut, 6000 vong/phat) Sinh khoi duqc dra lin vii nuerc khir ion vi duqc say kho chlin kitting Te bao kho duqc thiry phan NaOH 5N in 95°C h va duqc trung haa bin HC1 (Belma et al., 2002) Ham lucmg PHB to bao duqc phan tich bang sac ki long cao ip HPLC (high-performance liquid chromatography) so yen duerng chuan PHB Chili rat va tinh sach san pluim Mqt gto bao kho duqc then yeti 10 ml (acetone:con 90° viii tSr 1:1), lac 20 phut Sau d6 hoa vio 100 ml chloroform va &Km chuyen sang he thong soxhlet, chiet rut 65°C 16 h loci lipit va nhimg phan tar Ichong phai PHB Loc, thu PHB hem tan chloroform Sau da chloroform duqc cho bay hoi PHB tinh sach thu &Km bang each kit taa vai (Belma et al., 2002) Xdc djnh ciiu frac viz thIc aim If h6a es& pluint Cau trac cua san phim tech chiet dirge xac djnh thong qua phe hong ngoai (IR) Ham hem glucose dirge phan tich bang kit Glucose C2 ten may so rnau quang hoc = 505 nm trung binh (Me) can san , Kited luqng phan pham duqc xac djnh bang sic IcS, loc gel GPC (Gel permeation chromatography) so von 0,5% PHB chuan (Sigma) Tgo apt ban KET QUA VA THAO LUAN Hai mum ml to bao chimg dai nuoi qua dem (met de' Idioang 5.108 bhol ml) duqc cho vao dia petri 90 mm va det chiuig duai tia UV sang ngan (245 nm) tar don VL-6LC UV Wilber-Lourmat, Cedex, Phap) 60 phut Vi khuan song sat duqc xac dinh car sau 10 phut, (let bien duqc thu tS, le gay chit 99,99% (Hikmet et al., 2003) PHB la met vet lieu polymer ten tai duai dang hat nhu the du tit carbon va nang luqng to bao vi khan (Reddy et al., 2003) Trong thuc to ngotti cac chang tu nhien, chang tai to hqp, lived to can san xuat PHB tar cac chung dot bien (Gang et al., 2004) 490 Tap chi Cong nghe Sinh hoc 6(4): 489-496, 2008 A latus &Qv Iva chqn de nfhien cfru vi no co the san sinh PHB de clang chiet rut yeti nang suit cao (Wang, Lee, 1997) a Tao dat ban Anh sang UV (245 nm khoang cach 20 cm) chieu len huyen phit cua 20 ml chi:mg dai duqc nu8i cay qua dem (mat de khoang 5.108 to bao/m1) Hinh KhAn lac tren thach dia sau 10 phut dueri anh sang UV, xic djnh sv sting sot cua vi khuan tren mai trirong thach dia co se Nhang ket qua quan sat duqc (Hinh 1, 2, Bang 1) cho thay duai tic gong cua anh sang UV, so linyng vi khuan (Lich nuoi gian nhanh cheng theo theri gian Sau 20 phin dueri anti huOngcua tia UV, 99,94% vi khuan da bi diet TO theri diem nhting khan lac don song sot ten thach dia duqc tach de Iciem tra hog firth so veri chi:mg dai ca sb sau thbi gian chi& tia UV (Theri gian chi& UV x de pha loang dich vi khuan) Thad gian (phut) khan sau thbi gian Tit 14 song sot (%) 100 Wit tobao (CFUlm') 109 Bang TS', la song sot cua vi chit' anh hied/ng cua tia UV 107 105 103 10 2, 765 20 0,0061 30 0,000508 10 20 30 AO 50 00 40 0,000114 Th.:Asian chau UV (Omit) 50 0,000048 60 0,000008 101 Hinh Mat dg to bao vi khan sau theri gian chit, anh huerng cua tia UV 491 Tv chi Cong nghe Sinh hqc 6(4): 489-496, 2008 Sabi) chip lQc dOt bien co the bing dinh tinh PHB Vi khuin c6 kha &trg song sot dtrai,so tic d'Ong dat tia UV duqc chuyen sang nuoi cay teen m8i truing thoch ca sa c6 bo sung 1% Nile blue A Sau quan sat dirai anh sang cut tim (245 nm), nhang khuan lac co matt yang huYnh quang chuyen sang da cam duqc Lich, bb qua nhung khan lac mau T6ng s6 198 khuin lac co hoat tinh sinh PHB da dirqc thu nhan va tiep tic chop lac bang dinh lugmg Chan loc dOt bien au the hen chung dai bing djnh luvng PHB Cac chung dr boat tinh ainh tong hqp PHB duqc chan Jac va tiep tic nuoi cay tren mai truing djch the de xac djnh kha nang tich lay PHB nqi bao Ket qua phan tich sau thay phan to bao bang NaOH va tren HPLC cho thay chi co 40 so 198 chung sinh PHB thu duqc nghien dm chi so sinh twang va kha nang tich liiy PHB cao hen chiing dai mit tech CO y nghia Nhang chimg khic the , hien nang suet tich lay bang hoac thap hen chung b8 mg Phan 16n cac chung dOt bien sinh PHB yeti rang suit cao hen chung dai da thu dugc tai thii diem 30 phut chieu tia UV thai diem hau het vi khuan da chet Lach va ding tic gia (1990), ding thong bao sr sinh PHB tang chung det bien Bacillus (Reddy et al., 2003) Trong nghien ciru dm ha, chimg dijt bien B subtilis B8 va B firmus D7 thu duqc c6 nang suit tang gap doi so vai chimg dai Trong so cac chung d8t bien duang tinh duqc ching toi chin lac dila tren nang suit PHB cao chung dai, chung dOt bien A latus VN1-20 la tot nit& (Hinh 3) N6 co kha nang tich lily tai 55,6% PHB/trang lugmg kho to bao Chung duqc lila chan de len men thu hOi PHB Trong chung dai chi tich lay duqc 39,3% PHB/trong lacing kho to bao 0.8 0.6 00 tt 04 0.2 dai 13 20 39 55 120 122 139 142 195 Chung Hinh Sinh tnrerng VA kha nang tich by PHB cua chung dOt bien &rang tinh cao nhat so v6i chOng Nu6i cay len men Nuoi cay len men co dieu khien la phuong phap chung nhat duqc dimg de nghien dal dOng hac sinh truang to bao va su too Minh sin pham; moi quan he gift sinh truong to bao va dieu kien mOi tivang (Wang, Lee, 1997) Chang toi da tien hanh nuoi cay len men chimg dOt bien A latus VN1-20 (Hinh 4) de, nghien thu dOng hac ctia qui trinh sinh PHB c6 dieu khien pH dieu kien tucmg to vai chin dai Sinh trtrong cua vi khuan, so tich lily PHB, so tieu thu glucose 492 duqc xac dinh de tau duqc sr thay deri suet qua trinh len men Deng huc qui trinh len men cita chiing dOt bien A latus VN1-20 va chung dai duqc the hien (Hinh 6) Nhung ket qua thu duqc cho thiy c6 sr tang sinh truing cua chimg dOt bien so veri chinig dai Trong truing hqp chung dOt bien, sinh truing nhanh da duqc quan sat, to bao dot tin pha tinh sau 28 h Sinh truing chimg dai la cham to lac bit diu len men cho tai 28 h Sau do, sinh throng tang va dot tai pha tinh 44 h Sinh twang toi da cua chung (tat bien Tgp chi Cling nghe Sinh hgc 6(4): 489-496, 2008 dat 15,42 g/I a thai diem 28 h, veri chfing dai la 10,14 g/1 sau 48 h Sinh twang cua chting dai cham va kern han Sau 72 h len men ham luang glucose lai mei tatting van cao Dieu dr nghia la nhu cau del hal co chat cho len men dm chimg dai la khong cao Viec tang sinh khoi cita vi sinh vat hien ty le nghich veri su giam uguon carbon mei true:111g Doi veri chung dOt bien it han g/I glucose lai cuoi caa qua trinh len men Khi glucose mei traang can kiet sir len men phai dimg lai ngay, neu tiep.tuc thi sin pham PHB se bj phan hit), trer nguon carbon va nang lugng cho sinh truang cua vi khuan Vi vay truang hap chting drat bien, qua trinh len men nen dung lai sau khoing 30 h nuoi cay Sin pham PHB a chung dai dat tkda 0,415 g/g sau 52 h nuoi cay Vii chimg got bien sin phim PHB tang len va dot raj da 0,583 g/g sau 28 h nuoi cay Ket qui cho thay ca the sinh tong hap PHB dadtrac thiet lap cong a ching dOt bien Nang suit sinh PHB a thing (Vat bien da dirge cii thien khoing 40% so veri chung dai Hinh Qua trinh len men thu nhan PHB cua chOng dert bier' VN1-20 VN1-20 Hinh San phArn PHB tinh sach ta, chung dOt Wan Chung dai Chung adt hien 3C0 18 16 C") 14 12 tq to 7; = a e 18 16,E —259 14 12 10 ; o 2C0 E 193 a KO E 93 20 40 60 11161 gfMn eilfn won fba -e—00Vv IA 80 60 80 libidsilan by mom 0$ -6- PH8 (9f9) • Glucose du (rngld1) Hinh Eking hoc qua trinh len men dm chUng dai va chung dOt bien VN1-20 493 Tgp chi Cong nghe Sinh hpc 6(4): 489-496, 2008 dinh hap thu 1725,42 tuang Ong viii nhem chitc cho phep khang (firth san pham thu duqc la PHB Ket qui tich chiet PHB Him luqng PHB tinh sach thu duqc sau len men chimg dOt hien A latus VNI-20 (Hinh 5) so vai tong sinh kh6i kho dem chitt dat khoing 53% So sanh voiket,qui xac dinh ham lung PHB sinh kh6i bang sac Id long cao ap HPLC (58,3%) cho thy co su chenh 16ch Sai so li stn hao but san pham qui trinh tach chiet TO bio chung vi Ichuan d6t hien VN1-20 tich luy cac hat PHB duqc quan sat ro dual kinh hi6n vi din tin (Hinh 7) Xic dinh cAu true vi dic diem ISr boa ctia san pham Hinh Anh kinh hi6n vi Men tin to bao chung dOt lat qua hling ngoai (Hinh 8) cho ditty c6 Wen VN1-20 china tint PHB (X 25000 x) se x x ;,,xxsrxes.,,,, „ 22 Hinh Phi IR cOa san pham ?Non 2000 Mr nun-41w cm-1 3000 1000 1500 1000 500 PHB ChUng dgt hien Chung dai hal loo.00 100.00Col 50.00 50.00 0.00 20.00 10.00 0.00 lain) Mw 4,46679 x 105 Da Hinh Khoi luvng phan tin trung binh cua san pham PHB 494 Mw: 3,99455 x 105 Da Tap chi Cong nghe Sinh hoc 6(4): 489-496, 2008 Sau chiet rat can tach PHB has tan chloroform !way bao ye, polymer khoi bj phan hay San pham phai c6 luong phan tir trung binh (Mw) > 250000 Da mai &roc chap nhin cho img dtmg, cong nghiep Tinh chit IS, hoc PHB tan! d6 ket tinh, dO deo, deo dan h6i phu thu6c vao kh6i luong phan tir Kh6i lucmg phan tit cua sin pham thi chat anh huang bed chung vi sinh vat &roc sir dung, dieu kien nuoi cay va dO tinh sach cua mitt thu duqc PHB co khoi luong phan tir cao la hau ich cho ang dung ding nghiep (Chen et al., 2001) Khei luong phan ta trung binh caa san phim PHB tinh sach thu dtroc sau qua trinh len men cfrang dai va chung dOt bien khoang 4,47.105 va 3,99.105 Da, tuong img (Hinh, 9) So sanh vii kit& Wong phan fir caa PHB chuan (Sigma) la 5,3.105 Da, khoi Wong phan tir caa san phim PHB ma chung !el thu duoc tit Chung dOt bien kha cao dap ang yeu eau ang dung cong nghiep }CET LUAN Dung tic nhan vat ly tia UV chieu len to bao chung vi khuin A latus VN1 da thu nhin 40 chung d6t bien co hoot firth sinh PHB cao han chimg dai Da sa cac chung dOt hien sinh PHB cao deu thu dtroc tai Uteri diem 30 phut chi& tia UV Chang dOt bien A latus VN1-20 la tit nhit, n6 c6 Icha nang rich lay tai 55,6% PHB, tong chung dai chi tich Iuy duvc 39,3% PHB/ tong Ming kho to bap Sinh truing ciut chung dot bien dot tai da 15,42 g/I sau 28 h len men, vii chung dai la 10,14 g/1 sau 48 h Nang suit sinh PHB a chung dOt bien da cai thien duqc Ichoing 40% so vii chung dai Buerc ditu chimg tea da Lich chi& cong sin phim PHB c6 d6 tinh sach cao va chit Wong tot dap ling yen au ang dung cong nghiep Lai cAm an: Cong !rink claw Mac hien veil sa try kinh phi caa De tai cap Vin Cong nghesinh hpc 2007 va chitcmg trinh hop tic quac to "Research on Biomass Technology 2006" ta chac bed JICA, Nhat Ban TAI LIEU THAM KHAO Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role and industrial uses of bacterial polyhydroxyalkanoates Microbial Rev 54: 450-472 Belma Aslim, Zehra Nur Yuksekdag, Yavuz Beyatli (2002) Determination of PHB growth quantities of certain Bacillus species isolated from soil Turk Electron J Biotechnol special issue: 24-30 Chen Guogiang, Wu Qiong, Chen Jinchun (2001) Biosynthesis of polyhydroxyalkanoates Tsinghua Sci Technol 6(3): 193-199 Daniel Kadouri, Edouard Jurkevitch, Yaacov Okon (2003) Involvement of the Reserve Material Poly-fthydroxybutyrate in Azospirillum brasilense Stress Endurance and Root Colonization Appl Environ Microbial 69(6): 3244-3250 Gang Guk Choi, Hyung Woo Kim, Young Ha Rhee (2004) Enzymatic & Non-enzymatic degradation of PHB-PHV Copolyesters Produced by Alcaligenes sp MT16 J Microbial 42(4): 346-352 Hikmet Katircioglu, Belma Aslim, Zehra Nur Yuksekdab, Narime Mercan, Yavuz Beyatli (2003) Production of poly-p-hydroxybutyrate (PHB) and differentiation of putative Bacillus mutant strains by SDS-PAGE of total cell protein African J Biotechnol 2(6): 147-149 Lee SY, Chang HN (1995) Production of poly (3hydroxyalkanoic acid) Adv Biochem Eng Biotechnol 52: 27-58 Ojumu TV, Yu J, Solomon BO (2004) Minireview: Production of Polyhydroxyalkanoates, a bacterial biodegradable polymer African J Biotechnol 3(1): 1824 Reddy CSK, Ghai R, Rashmi, Kalia VC (2003) Polyhydroxyallcanoates: an overview Bioresour Technol 87(2): 137-146 Wang F, SY Lee (1997) Poly (3-hydroxybutyrate) production with high polymer content by fed-batch culture of Alcaligenes latus under nitrogen limitation App! Environ Microbial 63: 3703-3706 495 Tgp chi Cong nghe Sinh hoc 6(4): 489-496, 2008 IMPROVEMENT OF POLY-B-HYDROXYBUTYRATE PRODUCTION OF ALCALIGENES LATUS VN1 STRAIN BY MUTAGENESIS SELECTION Pham Thanh Tran Dinh Man', Yutaka Tokiwa2 'Institute of Biotechnology 21nstitute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan SUMMARY Poly-p-hydroxybutyrate (PHB) is a biodegradable plastic that has attracted much attention due to solution to the existing grave problems of plastic waste Today, most research efforts in this field concentrate on the isolation of PHB producing microorganisms from different sources and improvement of PHB production abilities of microorganisms This study was under taken by applying UV light mutagen on Alcaligenes latus VN1 to attempt to obtain mutant strains with higher PHB percentage yield Forty of the 198 obtained putative mutant strains showed significantly higher PHB yield, while other strains showed PHB yield equal to or less than the wild type strain Most of putative mutant strains have been selected at around 30 minutes of UV light treatment A PHB yield of 55.6% of total dried weight could be achieved from one mutant strain (A latus VN1-20) This strain has increased yield to about 40% in compare to the wild type strain The results after extracting of PHB product by chloroform showed that this bacterium was able to accumulate intracellular PHB with high molecular weight in basal medium after 72 hours of fermentation PHB product could be developed for industrial applications Keywords: Alcaligenes latus, biodegradable plastic, mutation, PHB, poly-fi-hydroxybutyrate • Author for correspondence: Tel: 84-4-37567103;E-mail: ptha04Avahoo.com 496 ...Tap chi Cong nghe Sinh hpc 6(4): 489-496, 2008 VAT LIEU VA PHIJONG PHAP Vi sinh vet Chung vi khan A latus VN1 phan lap tar dat nhiem au Viet Nam c6 kha nang tich luy PHB lchoang... ly tia UV chieu len to bao chung vi khuin A latus VN1 da thu nhin 40 chung d6t bien co hoot firth sinh PHB cao han chimg dai Da sa cac chung dOt hien sinh PHB cao deu thu dtroc tai Uteri diem... bio chung vi Ichuan d6t hien VN1- 20 tich luy cac hat PHB duqc quan sat ro dual kinh hi6n vi din tin (Hinh 7) Xic dinh cAu true vi dic diem ISr boa ctia san pham Hinh Anh kinh hi6n vi Men tin