B GIO DC V O TO I HC NNG LM TP. H CH MINHB MN CNG NGH SINH HC

***000***

KHA LUN TT NGHIP

NUI TRNG V XC NH THNH PHN AMINO ACID CA MT S LOI NM BO NG (Pleurotus spp.) BNG K THUT SC K LNG CAO P (High performance liquid chromatography - HPLC)

Ngnh hc: CNG NGH SINH HC Nin kha: 2001-2005 Sinh vin thc hin: ON BO QUC

Thnh ph H Ch Minh Thng 9/2005

B GIO DC V O TO I HC NNG LM TP. H CH MINHB MN CNG NGH SINH HC

***000***

KHA LUN TT NGHIP

NUI TRNG V XC NH THNH PHN AMINO ACID CA MT S LOI NM BO NG (Pleurotus spp.) BNG K THUT SC K LNG CAO P (High performance liquid chromatography - HPLC)

Gio vin hng dn:TS. L NH N

Sinh vin thc hin:ON BO QUC

Thnh ph H Ch Minh Thng 9/2005

LI CM TCHN THNH CM N: Ban Gim Hiu Trng i hc Nng Lm Thnh ph H Ch Minh. B mn Cng Ngh Sinh Hc B mn Bo v Thc vt Qu thy c truyn t kin thc cho chng ti trong sut qu trnh hc ti trng. TRN TRNG BIT N Thy L nh n tn tnh hng dn, to mi iu kin gip chng ti hon thnh kha lun ny. Thy Bi Cch Tuyn, Thy Bi Minh Tr to iu kin cho ti thc tp ti Trung Tm Phn Tch Th Nghim Ha Sinh. C Trn Th Dung, Thy Lu Phc Li, Thy Nguyn Vn Cng, Anh Mai Huy Hong gip ti c trang thit b cng nh phng th nghim hon thnh kha lun. Cc anh ch Trung Tm Phn Tch Th Nghim Ha Sinh, i Hc Nng Lm TP. H Ch Minh nhit tnh gip ti han thnh kha lun. Anh L Vit Ngc Vin ht nhn Lt, anh Hong trng i hc Khoa Hc T Nhin Thnh ph H Ch Minh cung cp ging nm bo ng cho ti thc hin kha lun. Cc thnh vin lp Cng Ngh Sinh Hc 27 ng vin, gip ti trong thi gian thc tp.

ii

TM TT

on Bo Quc, i hc Nng Lm Thnh ph H Ch Minh. Phn tch amino acid trn nm bo ng Pleurotus spp. bng sc k lng cao p - high performance liqud chromatography (HPLC) thi gian t 14/2/2005 n 18/8/2005. Hi ng hng dn: TS. L nh n Kha lun c thc hin trn i tng nm bo ng Pleurotus spp.. y l loi nm rt ph bin trn th trng Vit Nam. V th, thnh phn dinh dng ca chng, c bit l thnh phn amino acid rt c quan tm. thc hin phn tch amino acid trn nm, chng ti tin hnh trng nm ti Trung tm Cng Ngh Sinh Hc v s dng thit b l sc k lng cao p (HPLC) ca Trung tm Ha L u thuc Trng i Hc Nng Lm Thnh Ph H Ch Minh Nhng kt qu t c: Nui trng c 4 dng nm Pleurotus spp. trn c cht l mn ca c b sung thm cm go, v mt s cht khc. Phn tch v nh lng c 17 amino acid trong 2 loi nm Pleurotus flabellatus v Pleurotus pulmonarius. T chng ti tin hnh so snh thnh phn amino acid ca chng vi mt s rau qu khc, vi mt s nm khc v so snh gia chng.

iii

MC LCCHNG Trang ta Li cm ta ................................................................................................................ i Tm tt .................................................................................................................... ii Mc lc .................................................................................................................. iii Danh sch cc ch vit tt ..................................................................................... vi Danh sch cc hnh ............................................................................................... vii Danh sch cc bng ............................................................................................. viii 1. M U ..................................................................................................... 1 1.1. t vn .......................................................................................... 1 1.2. Mc ch ............................................................................................ 2 1.3. Yu cu ................................................................................................ 1.4. Ni dung thc hin ............................................................................ 2 2. TNG QUAN ............................................................................................. 3 2.1. Nm trng .......................................................................................... 3 2.1.1. Khi nim v nm ........................................................................ 3 2.1.2. Sinh sn v chu trnh sng ........................................................... 5 2.1.3.. c im bin dng v sinh l ................................................. 7 2.2. Nm bo ng...................................................................................... 8 2.2.1. Gii thiu ..................................................................................... 8 2.2.2. Mt s loi nm bo ng c nui trng VIt Nam .............. 9 2.2.2.1. Nm bo ng Pleurotus florida ........................................ 10 2.2.2.2. Nm bo ng Pleurotus sajor-caju ................................... 10 2.2.2.3. Nm bo ng Pleurotus pulmonarius ............................... 11 2.2.2.4. Nm bo ng Pleurotus abalonus ..................................... 11 2.3. Phng php sc k ......................................................................... 12 2.3.1. c im chung ca phng php sc k ................................. 12 2.3.2. C s ca phng php sc k .................................................. 12 2.4. Mt s phng php sc k trong phn tich amino acid ................. 13iv

TRANG

2.4.1. Sc k giy (Paper chromatography) ......................................... 13 2.4.2. Sc k lp mng ( Thin layer chromatography _TLC) ............. 14 2.4.3. High Performance Liquid Chromatography (HPLC) ................ 15 2.4.3.1. Pha tnh .............................................................................. 15 2.4.3.2. Pha ng ............................................................................ 16 2.4.3.3. B dng c HPLC ............................................................. 16 2.5. Cc bc phn tch amino acid ....................................................... 17 2.5.1. Qu trnh thy phn (Hydrolysis) .............................................. 17 2.5.2. Qu trnh to dn xut (Derivatisation) .................................... 19 2.5.3. Phn tch trn HPLC ................................................................. 20 2.5.4. Phn tch v tnh ton d liu .................................................... 21 2.4.4.1. nh tnh ............................................................................ 21 2.4.4.2. nh lng......................................................................... 21 2.6. Phn tch amino acid trn nm bo ng .......................................... 23 3. PHNG PHP NGHIN CU ............................................................ 23 3.1.Thi gian v a im thc hin ....................................................... 23 3.1.1. Thi gian thc hin ................................................................... 23 3.1.2 .a im ..................................................................................... 23 3.1.3.Vt liu nghin cu ..................................................................... 23 3.2. Ha cht v dng c ........................................................................ 23 3.2.1. Ha cht ..................................................................................... 23 3.2.2. Dng c v thit b ..................................................................... 24 3.3. Phng php nhn ging v trng nm ........................................... 24 3.3.1. Mi trng ................................................................................. 24 3.3.2. Phng php .............................................................................. 24 3.2.2.1. Chun b mi trng PGA ............................................... 24 3.2.2.2. Chun b mi trng ht ................................................... 24 3.3.2.3. Chun b mi trng gi mi ........................................... 25 3.2.2.4. Chun b mi trng nui trng ........................................ 25 3.2.2.5. Tm tt quy trnh ............................................................... 26 3.3.3. Phn tch amino acid .................................................................. 26 3.3.3.1.Thy phn mu ................................................................... 26v

3.3.3.2. To dn xut: ..................................................................... 27 3.3.3.3. Phn tch HPLC ................................................................. 28 3.3.3.4. Phn tch v tnh ton kt qu ........................................... 28 4.. KT QU V THO LUN ................................................................ 29 4.1. Kt qu ............................................................................................. 29 4.1.1. Kt qu trng nm...................................................................... 29 4.1.1.1. S tng trng ca h si trn cc mi trng ................. 29 4.1.1.2. S hnh thnh qu th ........................................................ 30 4.1.2. Kt qu phn tch amino acid .................................................... 32 5. KT LUN V NGH ...................................................................... 35 5.1. Kt lun ............................................................................................ 35 5.2. ngh............................................................................................. 35 6. TI LIU THAM KHO ........................................................................ 36

vi

DANH SCH CC CH VIT TTACN:Acetonitril HPLC: High performance liquid chromatography TLC: Thin layer chromatography PITC: phenylisothiocyanate PTC: phenylisocarbamyl TEA: Triethylamine.

vii

DANH SCH CC HNHHNH .......................................................................................................... TRANG Hnh 2.1. Cc trc t bo nm ................................................................................ 3 Hnh 2.2. Chu k sng ca nm m...................................................................... 6 Hnh 2.3. Pleurotus pulmonarius ......................................................................... 11 Hnh 2.4. Pleurotus abalonus ............................................................................... 11 Hnh 2.5 Pleurotus sp .......................................................................................... 11 Hnh 2.6. Pleurotus floridanus ............................................................................. 11 Hnh 2.7. M hnh sc k giy ............................................................................. 14 Hnh 2.8. M hnh sc k lp mng ..................................................................... 14 Hnh 2.9. B dng c HPLC ................................................................................ 16 Hnh 2.10. Thy phn protein bng HCl .............................................................. 18 Hnh 2.11. To dn xut amino acid bng PITC .................................................. 20 Hnh 2.12. Kt qu phn tch amino acid trn Pleurotus sp bi sc k giy trn ..... 22 Hnh 4.1. Sinh trng trn mi trng ht ca Pleurotus floridanus, Pleurotus flabellatus, Pleurotus flabellatus .......................................................................... 31 Hnh 4.2. Pleurotus abalonus trn gi m .................................................................. 31 Hnh 4.3. Bo t Pleurotus abalonus ................................................................... 31 Hnh 4.4. Bo t m Pleurotus floridanus...................................................................................... 31 Hnh 4 .5. Bo t m Pleurotus abalonus ...................................................................................... 31 Hnh 4.6. Qu th nm Pleurotus floridanus ........................................................ 32 Hnh 4.7. Qu th nm Pleurotus abalonus ......................................................... 32 Hnh 4.8 Qu th nm Pleurotus flabellatus ........................................................ 32 Hnh 4.9 Qu th nm Pleurotus pulmonarius .................................................... 32

viii

DANH SCH CC BNGBNG ......................................................................................................... TRANG Bng 2.1. Nhit thch hp i vi mt s nm bo ng .................................. 10 Bng 2.2. Cc dng sc k c bn ........................................................................ 13 Bng 4.1. Thi gian lan t y thit b ca cc dng nm ................................... 29 Bng 4.2. c tnh qu th ca cc dng nm ..................................................... 30 Bng 4. 3. Thnh phn amino acid trn Pleurotus pulmonaius, Pleurotus flabellatus .. 33

ix

1

Phn 1. M U1.1. t vn Hin nay, nm bo ng ang dn tr thnh mt trong nhng khu phn chnh trong ba n ca ngi dn. Bn cnh mi v thm ngon, giu dinh dng, rt d ch bin, nm cn c xem nh l mt loi rau sch v c gi tr dc liu. Sn lng nm bo ng tng ln rt nhanh trong vi thp k qua. Theo thng k ca Chang v ctv (1999) ch trong vng 36 nm tr li y tng sn lng nm trng tng gp 21 ln t 350.000 nm 1965 tng ln 7,5 triu tn, trong nm bo ng Pleurotus spp. tng t 169000 tn nm 1986 ln 876000 tn nm1997. Gn y, Vit Nam cng ang pht trin cng ngh trng nm trong nc. V th, ngoi vic khai thc nm hoang di em v trng, nc ta cn nhp khu nhiu loi nm bo ng khc t nc ngoi nh Trung Quc, i Loan, Nht Bn. Trong s , c mt s loi c bit rt r v thnh phn dinh dng, mt s loi cn li thnh phn ny vn cha c cng b chnh thc, c bit l thnh phn amino acid. V th, vic xc nh thnh phn amino acid trn nm bo ng i vi Vit Nam l iu cn thit c th nh gi gi tr sinh hc, cng nh so snh s khc nhau gia chng. T xa n nay, vic xc nh amino acid c thc hin trn rt nhiu cng c khc nhau nh: sc k giy, sc k lp mng. Phn tch amino acid trn cc cng c phn tch truyn thng ny th i hi nhiu thi gian v rt phc tp. Ngy nay, vi s pht trin ca k thut phn tch, vic xc nh thnh phn amino acid trong mu sinh ha c n gin hn rt nhiu, bi hu ht cc cng c c t ng ha v c ni trc tip b phn x l s liu. Hin nay, trn th trng ang tn ti rt nhiu k thut phn tch amino acid nh: sc k lng cao p, HPLC (high performance liquid chromatography), sc k trao i ion (ion exchange chromatography), in di mao qun (capillary electrophoresis). Trong phm vi cho php, chng ti ch thc hin Phn tch amino acid bng sc k lng cao p trn mt s loi nm ang c th trng Vit Nam. T , vi kt qu thu c chng ti s tin hnh so snh vi cc i tng khc cng nh s khc nhau gia chng v d liu v thnh phn amino acid ca tng loi nm bo ng s c lu li lm c s cho nhng nghin cu sau ny.

2

1.2. Mc ch So snh s thnh phn amino acid gia cc loi nm bo ng ang c trn th trng Vit Nam. Khi , chng ti s chn ra mt loi ph hp so snh thnh phn amino acid khi trng trn cc c cht khc nhau 1.3. Yu cu To c mu c tinh sch cao. Trnh hin tng lm thay i tnh cht mu nh oxy ha, halogen ha, hay lm mu b hy hon ton trong qu trnh thy phn. S dng thit b HPLC (high performance liquid chromatography) ca Trung tm Phn Tch Ha L phn tch thnh phn amino acid ca cc loi nm k trn 1.4. Ni dung thc hin Thu thp v nhn ging cc loi nm ang c trn th trng. Nui trng cc loi nm ny trn mi trng mn ca c b sung mt s vi lng. Tin hnh thu qu th Xc nh thnh phn amino acid trn cc loi nm bo ng bng sc k lng cao p (HPLC).

3

Phn 2. TNG QUAN TI LIU2.1. Nm trng 2.1.1. Khi nim v nm Hin nay, s loi nm trng c ch chim mt phn rt nh trong s nm n thin nhin. Ngoi c im chung l qu th hay tai nm c kch thc ln (a s dng tn d), chng cn n ngon v rt giu dinh dng [5]. Nm rt phong ph v a dng, bao gm c nhng loi n c v khng n c, nhng loi cho qu th ln c th thy bng mt thng v nhng loi phi s dng knh hin vi mi quan st c [5]. Ban u nm c xp vo gii thc vt. Tuy nhin, quan im ny ngy nay khng cn thuyt phc na v nhiu nh phn loi hc ngh xp nm vo mt gii ring, gi l gii nm. S d xp Nm thnh mt gii ring m khng xp vo gii Thc vt v nm khng c lc lp, khng c sc t quang hp, khng c i sng t dng nh thc vt, khng c s phn ha thnh r, thn, l, khng c hoa, phn ln khng cha cellulose trong thnh t bo [1].

Thnh t bo

Lysosome

Mng t bo cht

Nhn

B my golgi Ti th

Ribosome

Mng li ni cht

Hnh 2.1. Cc trc t bo nm (Nguyn Ln Dng, 2001)

4

Nm n c cu to ch yu l h si nm. Cc si nm n c dng ng trn. Cc ng ny u c vch ngn. Si nm cn gi l khun ty (hypha), h si nm cn gi l khun ty th (mycelium). Khong cch gia hai vch ngn gi l t bo. T bo nm c nhng c trng ring, khc bit vi t bo ng vt v thc vt. Trc ht l thnh t bo. Di knh hin vi in t c th quan st thy thnh t bo cu to bi nhiu lp. Nu nh thnh t bo ca thc vt cu to ch yu bi cellulose th thnh t bo ca nhng nhm nm khc nhau li c cu to bi nhng thnh phn khc nhau. Hu ht cc loi nm m u c thnh t bo cu to ch yu bi kitin- glucan. Thng qua cc l trn vch ngn, cht nguyn sinh c th di chuyn d dng trong si nm.[1]. Trong t bo si nm khi quan st di knh hin vi quang hc phng i t 1000 n 1500 ln (Hnh 2.1) c th thy Mng t bo Nhn t bo B my golgi Ribosome Lysosome Ti th Mng li ni cht. Si nm c th pht trin t bo t hay t mt on si nm. Bo t ny mm theo nhiu hng khc nhau, si nm phn nhnh nhiu ln, to nn mt h si chng cht v thng c mu trng. Ring nm m ni chung thng c ti 3 cp si nm. Si nm cp mt (s sinh), si nm cp hai (th sinh) v si nm cp ba (tam sinh). Si nm cp mt lc u khng c vch ngn v c nhiu nhn, dn s to thnh vch ngn v phn thnh nhiu t bo n nhn trong si nm. Si nm cp hai c to thnh do s phi trn ca hai si nm cp mt. Khi nguyn sinh cht trong hai si nm s phi trn vi nhau. Hai nhn vn ng ring r lm cho t bo c hai nhn. Ngi ta cn gi si nm loi ny l si nm song nhn (dycaryolic hyphae). Si nm cp ba l do si nm cp hai pht trin thnh. Cc si nm n y lin kt cht ch vi nhau v to thnh qu th [1].

5

2.1.2. Sinh sn v chu trnh sng Kh nng sinh sn l mt c im quan trng ca nm. Mt tai nm rm trng thnh c th phng hng t bo t. Nh vy, nm pht trin rt nhanh v phn b rng. Bo t ca nm ph bin c hai dng: v tnh v hu tnh. vi nm, nm mc lan truyn ch yu nh bo t v tnh, chng gy nhim khp ni di dng ht bi nh li ti, mu sc khc nhau ty loi. Ring i vi nm n, bo t sinh ra bn di cu trc c bt gi l m nm hay tai nm. M nm thng c cung nng ln cao c th nh gi a bo t i xa. Bo t ny mm cho li h si nm mi [3]. Ngi ta quen gi h si nm giai on tng trng (hay dinh dng) l tn dinh dng, phn bit vi qu th nm (hay c quan sinh bo t hu tnh ca nm) l tn sinh sn. Hu ht nm trng l nm m, c quan sinh sn c c cu to c bit l tai nm. Tai nm ch yu gm m v cung nm. M thng c dng nn hay phu, vi cung nh gia hay bn. Mt di m ca nhm ny cu to bi cc phin mng xp st vao nhau nh hnh nan qut. mt s trng hp, phin nm cn ko di t m xung cung (chn nm), nh nm bo ng (Pleurotus) [3]. Bo t m (basidiospore) to ra b mt phin, trn cu trc c bit gi l m (basidium). Ring nm u (Coprinus), khi tai nm trng thnh m nm s chy thnh dch nc en mang theo cc bo t m; cn cc bo t khc s rng v bay theo gi. m c to thnh t cc u ngn si nm. T bo ny phng to v bn trong hai nhn ng ring l, s nhp li thnh mt nhn. Qu trnh ny gi l qu trnh th tinh [3]. Nhn th tinh s phn chia v cui cng to ra 4 nhn con. Bnh thng mi nhn s c khi sinh cht y vo mt ci gai nh (xut hin trn m) to ra mt m bo t, nhng i khi mt m bo t c th cng lc hai nhn nh nm rm (t l chim n 1/4) hoc c bit nm m (Agaricus bisporus) ch sinh ra hai m t, mi bo t cha hai nhn. Cc t bo m hp li thnh lp trn b mt ca phin, c gi l lp th tng (hymennium) v v vy m bo t cng thnh lp ph trn trn b mt phin. iu ny thy r nm rm, khi trng thnh phin nm chuyn sang mu l do mu ca bo t.

6

Ngi ta phn bit nm ng m (homobasidiomycetidae) v nm d m (hemibasidiomycetidae) l da vo cu trc ca m. Nm ng m c m l mt t bo ng nht, cn nm d m hay tin m chia lm 4 phn, mi phn to ra mt m bo t. m bo t l bo t hu tnh, khi rng s bay i khp ni. Khi gp iu Qu th Hnh thnh qu th Si nm song nhn Phin nm

Qu trnh dung hp

Si m

Gim phn hnh thnh bo t m

Hp t

Si nm th cp (song nhn)

Bo t m Si dng Si nm s cp (n nhn)

Hnh 2.2. Chu k sng ca nm m ( Peter H. Raven v ctv, 1992) kin thun li ny mm v cho li h si nm. H si nm thng ch c mt nhn nn c gi l si nm s cp (primary mycelium). i vi nm d tn (heterothallic), phi c s phi hp gia hai si nm s cp pht sinh t hai bo t c c tnh di truyn khc nhau mi hon thnh si nm th cp (secondary mycelium). Trong khi nm ng tn (homothallic) ch cn h si s cp t bo t ny mm cng c th t phi cho ra h si nm th cp. T h si nm th cp cha hai nhn, nm pht trin thnh mng si, lan khp ni trn c cht ht cht dinh dng. Trong trng hp b t khc, cc si nm t lm lnh vt thng v ti lp li h si, tng t nh cy trng trong gim hay chit cnh. nhng iu kin nht nh, nh m , nhit thch hp, h si nm s bn li v to thnh hch nm. Hch nm s tip tc pht trin cho qu th trng thnh.[3].

7

nm m, s phi hp nguyn sinh cht gia hai si nm cp mt xy ra rt sm, si nm song nhn l hnh thi ch yu ca si nm. Qu th l do cc si nm song nhn lin kt li to thnh. Si nm song nhn v mt di truyn uc biu th l (n+n) khc vi si nm n nhn l n v hp t l 2n [1]. mt s loi cn c mt dng bo t khc gi l hu bo t hay bo t mng dy (Chlamydospore), th d nh nm rm. Hu bo t ging nh mt bo t thu nh vi y nguyn sinh cht v hai nhn nhng c c hn v c vch tng i dy bao bc. C th xem y l mt dng sng tim sinh ca nm v sau bo t ny c th ny mm v cho li h si nm th cp [3]. 2.1.3. c im bin dng v sinh l Nm ch yu sng d dng, ly thc n t cc ngun hu c (ng vt hoc thc vt). Ngoi tr nim khun thay i hnh dng t bo nut ly thc n (tng t ng vt), cn li hu ht cc loi nm u nm ly dinh dng qua mng t bo h si (ging r cy thc vt). Nhiu loi nm c h enzym phn gii tng i mnh, gip chng c th s dng cc dng thc n phc tp, bao gm cc i phn t nh (cellulose, hemicellulose), cht m (protein), cht bt (amidon, polysaccharide), lignin.Vi cu trc si, t nm len li su vo trong c cht (rm, r, mt ca v nhiu c cht khc) rt ly thc n em nui ton b c th nm (tn dinh dng hay tn sinh sn) [3]. Da theo cch thc dinh dng ca nm c th chia thnh 3 nhm: Hoi sinh: c tnh chung ca hu ht cc loi nm, trong c nm trng. Thc n ca chng l xc b ng thc vt. Nhm nm ny c h enzym tiu ho tng i mnh, phn gii c nhiu loi c cht. Chng c kh nng bin cc cht ny thnh nhng cht c thnh phn n gin c th hp th c. Tuy nhin, cng c nhng trng hp nm khng th phn gii c cht m phi nh vo cc vi sinh vt khc (vi khun, nm mc, x khun) tin hnh trc mt bc[3]. K sinh: Bao gm ch yu cc loi nm gy bnh. Chng sng bm vo cc loi sinh vt khc (ng vt, thc vt, cc loi nm khc). Thc n ca chng chnh l cc cht ly t c th k ch, lm suy yu hoc lm tn thng k ch. Mt s nm n c th sng trn cy cn ti nhng i sng thc s vn l hoi sinh, nn c xp vo nhm trung gian, gi l nhm bn k sinh (trng hp nm mo).

8

Cng sinh: y l nhm c bit, ly thc n t c th ch nhng khng lm cht hoc gy tn thng k ch, ngc li cn gip chng pht trin tt hn. V vy cc loi ny i vi k ch c mi quan h mt thit vi nhau. Do , vic nui trng tr nn phc tp hn, thng ging nm c cy cng lc vi vic trng cy (th d nm Tuber hoc Boletus). 2.2. Nm bo ng 2.2.1. Gii thiu Nm bo ng (cn gi l nm s, nm bnh c, oyster mushroom) gm nhiu loi thuc: Chi Pleurotus H Pleurotaceae B Agaricales Lp ph Hymenomycetidae Lp Hymenomycetes Ngnh ph Basidiomycotina Ngnh nm tht-Eumycota Gii nm Mycota hay Fungi Nm bo ng c ti 50 loi khc nhau. Tuy nhin, s loi c nui trng c khng nhiu, khong 10 loi [2]. Nm bo ng c nui trng rng ri trn th gii. chu u nm bo ng c trng Hungary, c, , Php, H Lan. Chu nm bo ng c trng Trung Quc vi sn lng rt cao (khong 12 nghn tn mi nm) gi khong 2.2 triu ng Vit Nam. Ngoi Trung Quc, nm ny cn c trng Nht Bn, Vit Nam, Hn Quc, Thi Lan n [2]. Nm bo ng khng ch n ngon m cn c gi tr dinh dng cao. Trong nm cha y cc amino acid. Ngoi gi tr dinh dng, nm bo ng cn c gi tr dc liu. Nhiu nghin cu cho thy nm bo ng cng mt s nm n khc c tc dng chng ung th. Bng phng php khuch tn vo thch nhm nghin cu ca Trng i hc Khoa Hc T Nhin cho thy nm bo ng Pleurotus sajor - caju dng bn cu lch c tc dng c ch 2 chng Vi khun Gran dng S. aureus v B. subtilis v 2 chng Gram

9

m E. coli v Pseudomonas aeruginosa [5]. Ngoi ra, theo nghin ca nh khoa hc Trung Quc Ph Lin Giang nm bo ng cn c kh nng lm gim lng cholesterol trong mu [3]. Mt trong nhng c im quan trng ca nm l h enzym. Nh enzym cellulase v mt s enzym khc, nm bo ng kh nng chuyn ho cellulose thnh nhng cht c bn gp phn gii quyt nn nhim mi trng do cht thi nng nghip to ra nh b ma, rm r. Ngoi ra, nm cn c kh nng phn hy thuc tr c Antrazin nh vo s gia tng cc enzym P 450 ca h Cytochromeoxxygenase v peroxydase (Sannia v ctv, 1990) [3] . Nm bo ng c c im chung l tai nm dng phu lch, phin nm mang bo t ko di xung chn. Cung nm gn gc c lng mn. Tai nm cn non c mu sc ti, nhng khi trng thnh tr nn sng hn. Qu th nm bo ng pht trin qua nhiu giai an, da theo hnh thi tai nm m tn gi cho tng giai on khc nhau. Dng san h: qu th mi to thnh dng si mnh hnh chm. Dng di trng: m xut hin di dng khi trn. Dng phu: m m rng trong khi cung cn gia Dng bn cu lch: cung ln nhanh mt bn so vi v tr trung bnh. Dng lc bnh: cung ngng tng trng, nhng m vn pht trin. Nm bo ng c chu trnh pht trin cng nh cc loi nm m khc, bt u m bo t hu tnh, ny mm cho h si t dinh dng (s cp v th cp) v kt thc bng vic hnh thnh c quan sinh sn l tai nm. Tai nm sinh ra m bo t v chu trnh li tip tc [3] . 2.2.2. Mt s loi nm bo ng c nui trng VIt Nam. Hin nay, nm bo ng c trng rng ri Vit Nam c min Bc ln min Nam v kh hu nc ta rt thun li cho vic pht trin loi nm ny. Hin nay, nm bo ng c trng vi nhiu chng loi (khong 7 loi) tp trung nhiu Lt v Thnh ph H Ch Minh. Hu ht cc dng nm c nui trng nc ta l cc ging c nhp t nc ngoi. Tuy nhin vn c mt s t c khai thc ngay trn a l ca Vit Nam sau m v nui trng trn iu kin nhn to.

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Bng 2.1. Nhit thch hp i vi mt s nm bo ng (L Duy Thng, 1999) Nhit thch Loi nm hp cho t tng trng ( 0C) Pleurotus floridanus Pleurotus sajor - caju Pleurotus pulmonarius Pleurotus abalonus Pleurotus flabellatus 25-30 25-30 22-28 27-32 20-28 25 25 25 25 20-25 Nhit thch hp cho ra nm ( 0C)

2.2.2.1. Nm bo ng Pleurotus floridanus (Hnh 2.6) y l loi thch nghi vi nhit kh rng t 8 - 30oC. Qu th mu trng sa. C tnh khng tp nm, tp khun cao. Si nm c hat tnh mnh i vi vic phn gii cellulose. Sn lng trn n v nguyn liu tng i cao. Nm ny c th thch nghi vi nhiu loi c cht khc nhau: b ma, mn ca, rm, tru. Khi trng trn tru, nng sut c th t c t ra qu th u tin l 114g nm ti trn 360 g nguyn liu khi c b sung ure, KH2PO4, cm go [4]. 2.2.2.2. Nm bo ng Pleurotus sajor - caju (nm s phng v hay nm s dng phu) Qu th c hnh thnh nhit tng i cao so vi cc loi nm khc. Gn y, nm ny ang dn chim th trng v qu th chc, n ngon v rt thch hp i vi kh hu Vit Nam. Ngoi ra, dch chit t nm ny c th c ch mt s loi vi khun: S. aureus v B. subtilis, E. coli v Pseudomonas aeruginosa [5]. Ngoi cc c cht thng thng, gn y nm ny cn c trng trn c cht l cy mai dng chng s xm ln ca chng gp phn gii quyt nn mi trng. Loi nm ny c nhp t Nht Bn v c nui trng thnh cng trn mn ca u tin Thnh ph H Ch Minh.

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Hnh 2.3. Pleurotus pulmonarius

Hnh 2.4. Pleurotus abalonus

Hnh 2.5 Pleurotus sp.

Hnh 2.6. Pleurotus floridanus

2.2.2.3. Nm bo ng Pleurotus pulmonarius Phn ln c nhp t nc ngoi (Hnh 2.3). Nhng gn y, GS. L Xun Thm v cng s (1999) pht hin c mt loi khc Daklak - Ty Nguyn, sau a v Lt v hon thin quy trnh trng trn mn ca [7] . 2.2.2.4. Nm bo ng Pleurotus abalonus Nm ny c ngun gc t i Loan. L mt loi nm a nng nn rt thun li pht trin nui trng trong iu kin kh hu ca Vit Nam [1]. Nm ny c c im l qu th rt to (Hnh 2.4), ng knh m nm c th t n 35 cm [2]. Ngoi ra cn mt loi khc cng ang c trng Vit Nam nh Pleurotus eryngii, Pleurotus flabellatus. V mi y (2005) Thc s C c Trng trng thnh cng 2 loi nm bo ng mi: Pleurotus citrinopileatus Singer Pleurotus (qu th mu vng) v Pleurotus djamor (qu th c mu hng)[7].

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2.3. Phng php sc k - Phn tch amino acid 2.3.1. c im chung ca phng php sc k Phng php sc k c pht trin vo u tin bi Mikhail Semyonovich Tsvet (1901) trong qu trnh nghin cu v chlorophyll. n nm 1952, Archer John Porter Martin and Richard Laurence Millington Synge ot gii Nobel ha hc v sc k phn tch. T k thut sc k ngy vng c pht trin cao hn cho n ngy nay [24]. Sc k l phng php phn tch, phn li, phn tch cc cht da vo s phn b khc nhau ca chng gia hai pha ng v pha tnh. Khi tip xc vi pha tnh, cc cu t ca hn hp s phn b gia pha tnh v pha ng tng ng vi tnh cht ca chng (tnh hp ph, tnh tan). Trong cc h thng sc k ch c cc phn t pha ng mi chuyn ng dc theo h sc k. Cc cht khc nhau s c i lc khc nhau vi pha tnh v pha ng. Trong qu trnh pha ng chuyn ng dc theo h sc k ht lp pha tnh ny n lp pha tnh khc, s lp i lp li qu trnh hp ph, phn hp ph. H qu l cc cht c i lc ln vi pha tnh s chuyn ng chm hn qua h thng sc k so vi cc cht tng tc yu hn vi pha ny. Nh vy m ngi ta c th tch cc cht qua qu trnh sc k [10]. 2.3.2. C s ca phng php sc k Phng php sc k da vo s phn b khc nhau ca cc cht gia pha ng v pha tnh. C nhiu nguyn nhn a n s phn b khc nhau gia nhau ca cc cht, nhng chnh s lp i lp li hin tng hp ph - phn hp ph ca cc cht khi dng pha ng chuyn ng qua pha tnh l nguyn nhn ch yu ca vic tch sc k. Hu ht cc phng php sc l u c da theo nguyn l trn, v hiu qu ca tng phng php ph vo s chn la gia pha ng pha tnh [8]. 2.3.3. Phn loi qu trnh sc k Trong phng php sc k pha ng phi l cc lu th cn pha tnh l cc cht trng thi lng hoc rn. Da vo trng thi tp hp ca pha ng, ngi ta chia sc k thnh hai nhm sc k kh v sc k lng. Da vo c ch trao i ca cc cht gia pha ng v pha tnh m ngi ta li chia cc phng php sc k thnh nhm nh hn.

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Ty theo trng thi tp hp ca cc pha, loi tng tc v s hnh thnh sc k m ngi ta phn bit cc loi sc k nh bng 2.2. Bng 2.2. Cc dng sc k c bn [9]Cch b tr Dng sc k Kh Kh- hp ph Kh -lng Lng Lng - rn Lng - lng Lng - nha trao i Lp mng Ry (sc k gel) Lng Lng Lng Lng Lng Rn Lng Rn Rn Lng Ct Ct Ct Lp mng Ct Hp ph Phn b Trao i ion Hp ph Theo kch thc phn t Kh Kh Rn Lng Ct Ct Hp ph Phn b Pha ng Pha tnh pha tnh C ch trao i cht

2.4.. Mt s phng php sc k trong phn tch amino acid Phn tch amino acid l phng php xc nh thnh phn amino acid cng nh nh lng chng trong mu protein hay peptide. Phn tch amino acid c th s dng sc k giy (paper chromatography), sc k lp mng (thin layer chomatographyTLC), sc k kh (gas chromatography), sc k trao i ion (ion exchange chromatography), sc k lng cao p (high performance liquid chromatography), in di mao qun (capillary electrophoresis) [15]. 2.4.1. Sc k giy (Paper chromatograhhy) - Qu trnh phn tch c thc hin trn giy sc k hoc giy lc (Hnh 2.7). - Dng vit ch, v mt ng mnh trn gc ca giy (1,5 - 2 cm). nh nhng du nh dc theo ng va k. - Dng pipett ht lng mu vi th tch nht nh cho vo nhng du va mi nh. kh, tip tc cho vo mu th 2 trn u tube khc nhau. Lm kh. - t chng vo bn sc k (cho dung mi ngp khong 1cm giy sc k). - bn ni mt trnh nh sng trc tip, khong 1 gi.

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- Ly ra, dng vit ch k ng nh du ni m dung mi di chuyn. Lm kh. - Phun ninhydrin vo ton b mt giy. kh. Tnh gi tr Rf. [24] - i vi sc k giy dng cho phn tch amino acid, cc nh phn tch cn dng sc k cun trn v sc k giy trn. 2.4.2. Sc k lp mng ( Thin layer chromatography _TLC) Trong phng php ny, pha tnh l mt lp mng cht hp ph c gn vo mt gi mang thng l bn thy tinh v pha ng i qua lp mng ny bng lc mao dn. Trong TLC mt chiu, cc thnh phn trong mu c tch ri nhau bng mt ln chy sc k vi mt dung mi no , cn TLC hai chiu l mt k thut sc k c kh nng tch mnh bng hai ln chy sc k vi hai chiu vung gc nhau v vi hai h dung mi khc nhau [7]. Quy trnh phn tch amino acid trn TLC [23] - Ly tm TLC, dng vit ch v mt ng mnh cch mp di ca tm TLC khong 2 cm, nh du trn ng bt ch va c v. Ch nh du tng vt u nhau v dn nhn trc c th khng nhn ln khi pht hin. - Nh trn mi nh du mt git dung dch amino acid mu, lm kh tm TLC. - Thm dung mi cho vo mt beaker rng iu chnh lng dung mi c th ph ton b chiu ngang ca tm TLC ( ch ph khong 1cm chiu cao ca lp mng).

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- Cho tm TLC vo beaker, mu c t pha di. - Khi dung mi di chuyn c khong 85 % ca tm mng th ly tm mng ra dng vit ch v mt ng mnh ngang vi ng dung mi va di chuiyn n. chng kh hon ton. - Cho bng mng vo t ht, nh ninhydrin vo c th thy mu. Lm kh. .Khi amino acid s xut hin mu trn bng mng. nh du tng vt bng bt ch ngay tm. Tnh gi tr Rf ( retardation factor). - Lu gi tr Rf tnh ton nhng amino acid cha bit. Ngoi ra, amino acid c c phn tch trn sc k trao i ion thng qua pht hin sau khi b tch qua ct, sc k kh,, in di mao qun, HPLC. Tuy nhin, i vi phn tch amino acid, sc k lng cao p (HPLC) thng c s dng nht bi v n c nhiu u im hn nh: thi gian ngn. phn tch cao, dn xut n nh v c th pht hin thng qua UV (Ultraviolet).

2.4.3. High Performance Liquid Chromatography (HPLC) HPLC c ra i vo u nhng nm1970 l mt trong nhng cng c phn tch u tay ca cc nh phn tch. HPLC da vo pha ng (dng lng) phn tch cc thnh phn trn ln vo nhau. Nhng thnh phn ny u tin c ha tan vo trong dung mi v sau di tc dng ca p sut cao , chng s b li ko di chuyn qua ct sc k. Trong ct, hn hp s c phn tch thnh nhng cu t ca chng. S tng tc gia cht tan vi pha ng v pha tnh c th ph thuc vo s chn la khc nhau c dung mi v pha tnh. Kt qu HPLC s thu c nhiu cu t khc nhau m cc k thut sc k khc khng thc hin c v n d dng phn tch s lng ln cc cht ho hc trn ln vo nhau. 2.4.3.1. Pha tnh Ty theo mu phn tch m ta c c m hnh pha tnh khc nhau. Liquid - Solid Da trn phn cc. Nhng cu t no c phn cc mnh s lin kt ch vi pha tnh hn nhng cht c phn cc yu hn. V th nhng cht t phn cc s b li cun ra khi ct nhanh hn.

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Size - Exclusion Da trn kch thuc phn t ca thnh phn c phn tch. Pha tnh cha cc ht xp bn trong c nhiu l nh. Nhng thnh phn c kch thc ln hn ht s b loi ra ngoi v c tch ra u tin. Nhng cu t c kch thc ph hp s b hp thu v l v c tch ra sau. Normal phase (pha bnh thng) Pha tnh phn cc cao hn phn cc ca dung mi pha ng . Reverse Phase (ngc pha) pha tnh c phn cc thp, pha ng c phn cc cao hn.

Ion-Exchange c tin hnh da trn s thay th nhng ion trong mu vi nhng ion tri du trong pha tnh. Khi pha tnh s gi li mu v pha ng di chuyn qua ct s th ch cho ion mu v y mu ra khi ct. 2.4.3.2. Pha ng Ty thuc vo m hnh ca HPLC m pha ng c th thay i. i vi pha bnh thng, th dung mi thng l khng phn cc cn i vi m hnh ngc pha th th thng s dng c s pha trn gia nc v mt vi dung mi hu c phn cc nh acetonitrile. i vi m hnh size - exclusion, yu cu i vi pha ng l ngn cn mu gn kt trn b mt ca pha tnh.

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2.4.3.3. B dng c HPLC Bm: Bm trong HPLC uc xem l mt trong nhng thnh phn rt quan trng trong h thng sc k lng. Bi v h thng sc k lng i hi phi c t l dng n nh lin tc gn lin vi injector, ct v detector. C hai loi bm c bn - Bm p sut lin tc. - Bm dng lin tc. Ct: Hin nay c rt nhiu loi ct ang c s dng. Tuy nhin ty thuc vo loi mu, tnh cht ca mu m ta c loi ct v kch thc ct khc nhau. i vi sc k ngc pha th ct C-18 v Pico-Tag column, 3.9 mm x 15 cm thng c s dng nht. Detector: Mt s detector thng c s dng: - Detector Ultraviolet/visible: thng c s dng pht hin cc cht hu c. - Detector chui diode. - Detector hunh quang. V mt s detector khc nh detector in ha hc,detector dn in H thng d liu. chuyn d liu phn tch, detector thng c ni trc tip vi mt my vi tnh. Khi d liu c chuyn thnh nhng peak trn sc k v c tnh ton. 2.5. Cc bc phn tch amino acid Phn tch amino acid c thc hin thng qua cc bc sau y [17]. - Chun b mu - Thy phn protein thnh nhng amino acid n. - To dn xut. - Phn tch amino acid trn h thng sc k - Phn tch d liu v tnh ton. Trc khi tin hnh phn tch amino acid, qu trnh chun b mu cng ng vai tr rt quan trng v nu khng chun b k s rt d b nhim trong qu trnh phn tch. - Mu c t trong mt tube sch. - Tt c mu c lm kh trc khi phn tch. Trong qu trnh tin hnh phi eo gng tay , tuy nhin trnh cc bi trn gng tay bm vo mu.

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2.5.1. Qu trnh thy phn (Hydrolysis) phn tch amino acid trong mu protein, mu phi c thy phn to thnh nhng amino acid n. Thy phn bng acid l phng php thng thng nht thy phn protein trc khi tin hnh pnn tch amino acid. Phng php thy phn bng acid c th to

Hnh 2.10. Thy phn protein bng HCl [17] ra s phn ct khc nhau i vi mt vi amino acid- ph hy hon ton hoc mt phn. Mt s kh khn khi thy phn bng acid. - Trytophan b ph hy hon ton - Serine v threonine b ph hy mt phn - Methionine c th b oxy ha - Cysteine thng c pht hin nh l cystine (nhng cystine thng c pht hin rt km bi v chng b phn hy mt phn). - Asparagine v glutamine tng ng b chuyn thnh acid aspartic v acid glutamic. Nh vy, nu k thut thy phn khng tt c th gy ra nhiu kt qu khng chnh xc. Mt s phng php thy phn thng c s dng trong HPLC. Phng php 1: Dng HCl 6N v phenol (t 0.1- 1 %) Phng php thng c s dng nht l dng HCl c cha phenol. Thm phenol vo ngn cn phn ng halogen ha tyrosine.Dung dch thy phn: HCl 6N cha t 0,1 n 1 % phenol. Phng php tin hnh: - Cho mu vo tube thy phn v lm kh (mu c lm kh nc khng pha long acid).

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- Thm dung dch thy phn vo mu vi t l nht nh. - Lm lnh tube cha mu. - Mu thng c thy phn 110oC, trong 24h trong chn khng hoc trong kh tr ngn cn qu trnh oxy ha. Thi gian thy phn c th thay i ty theo mc ch phn tch. - Sau khi thy phn lm kh mu trong chn khng loi b acid tha. Phng php 2: Dng Mercaptoathanesulfonic (MESA) 2.5M. - Mu c lm kh trong mt tube trc khi thy phn. - Tube cha mu ny cho vo mt tube ln hn cha dung dch thy phn. - Tube thy phn ny c tin hnh trong chn khng lm bay hi dung dch thy phn. Tube thy phn c gia nhit t 170oC n 185oC khong 12 pht. - Sau khi thy phn tube c lm kh trong chn khng loi b acid dung dch tha [17]. 2.5.2. Qu trnh to dn xut (Derivatisation) [17] Nhng amino acid s khng c pht hin nu chng khng c to dn xut. Hin nay c rt nhiu phng php to dn xut, s chn la bt k mt k thut no ph thuc vo nhy, thi gian phn tch v chnh xc ca phng php. Ni chung mt na trong s k thut phn tch amino acid l k thut sc k trao i ion km theo postcolumn (ninhydrin hay o phthaladehyde). K thut pht hin bng postcolumn ch yu cu vi lng mu nh (khong 5-10 g protein trong mt ln phn tch). K thut phn tch amino acid lin quan n to dn xut precolumn cng c ng dng rt rng ri v yu cu l HPLC ngc pha. K thut ny rt nhy nn i hi lng mu phn tch rt nh ch khong 0.5-1 g. Phng php pht hin bng ninhydrin postcolumn Phng php sc k trao i ion vi ninhydrin postcolumn l mt trong nhng phng php c ng dng nhiu nht trong k thut nh lng amino acid. Sau khi amino acid c phn tch trn sc k s c pht hin thng qua phn ng ca chng vi ninhydrin s to mu vng hay mu tm. Nhng amino acid thng cho mu tm s c pht hin bc sng hp th l 570 nm. Nhng amino acid cho mu nh l proline thng c pht hin bc sng 440 nm. Ngoi

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ninhydrin, o phthalaldehyde (OPA) cng c s dng trong phn tch postcolumn. Bc sng pht hin ca phng php ny thng vo khong 348 - 450 nm. i vi phng php precolumn th tc nhn to dn xut thng c s dng l phenylisothiocyanate (PITC). PITC phn ng vi amino acid nhit phng trong vng 5-10 pht (Pierce Chemical company, 1999) to thnh phenylisocarbamyl (PTC) v c th c pht hin bc sng 254 nm. V th to dn xut amin acid vi PITC thng qua sc k ngc pha (reversed-phase HPLC) c th c s dng phn tch nh lng amino acid vi detector UV. Vi phng php ny, gii hn pht hin cho phn ln nhng amino acid l khong 1pmol. tuyn tnh tng ng (response linearity) thu c t 20 n 500 pmol. thu c kt qu tt, lng mu ln hn 500 ng trc khi thy phn l tt nht. Ngoi s dng cc thnh phn phn ng nu trn, cc hp cht sau y cng c s dng pht hin amino acid trong cc mu sinh ha. o - phthaladehyde (OPA).(dn xut sau khi qua ct) Hydroxysuccinimidyl carbamate (AQC) (dn xut tin ct) Dimethylaminoazobenzenesulfonyl chloride (DABS-Cl) (dn xut tin ct) 9-Fluornylmethyl choloformate (FMOC-Cl) (dn xut tin ct) 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) (dn xut tin ct)

Hnh 2.11. To dn xut amino acid bng PITC [10].

2.5.3. Phn tch trn HPLC [17] u tin, chun amino acid c phn tch trc vi cc nng khc nhau lm d liu cho cng vic tnh ton sau ny. Mu thng c bm vi th tch nht nh.

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Ty theo phng php precolumn hay postcolumn m ta c th s dng pha ng, pha tnh, ct cng nh iu chnh bc sng pht hin v detector ph hp. Khi thi gian pht hin cng khc nhau. Nu l PTC-aa thng c phn tch trn ct cilica C18, hoc Pico -Tag ca sc k ngc pha; pha ng thng l acetonitrile; bc sng pht hin 254nm v tt c cc aa thng c pht hin khong 12 pht.khng k thi gian ra ct hay thi gian thm to cho ng nn tr li. 2.5.4. Phn tch v tnh ton d liu [10] 2.5.4.1. nh tnh pht hin c cc amino acid, phng php thng thng l da vo thi gian lu ca chun c thc hin chy sc k trc. sau , thi gian lu ca mu c so snh vi chun ny v t o xc nh tng amino acid tng ng vi tng din tch peak nh lng. 2.5.4.2. nh lng Thnh phn ca tng amino acid c th hin bng phn trm v s mol (mol%) so vi tt c nhng phn khc trong mu protein hay peptide. Chng hn nh nu protein c 5 nmol Ala tng s tt c cc amino acid trong protein l 200 nmol k c Ala th phn trm s mol ca Ala l 2.5. Kt qu thu c s to nn mt profile ring bit i vi tt c cc amino acid trong mt protein hay peptide c th. 2.6. Phn tch amino acid trn nm bo ng Thnh phn amino acid trn nm bo ng c tin hnh phn tch t rt lu, nht l khi k thut sc k ra i. .Nm 1962, Zakia Bano v cng s, vin nghin cu cng ngh thc phm Mysore, n tin hnh phn tch trn sc k giy trn. V ng pht hin trong nm ny cha n 17 amino acid. Trong hin hin y 10 amino acid cn thit [15].

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Hnh 2.12. Kt qu phn tch amino acid trn Pleurotus sp bng sc k giy trn (Zakia Bano, 1962) L: lycine IL:Isoleucine Ph Al: Phenylalanine Glu: Glutamic acid Thr: Threonine Ser: Serine Gly: Glycine Asp: Aspartic acid Arg: Agrinine, Pep: Peptide Lys:Lysine, His: Histidine Cys: Cysteine

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PHN 3. PHNG PHP NGHIN CU3.1.Thi gian, a im thc hin v vt liu nghin cu 3.1.1. Thi gian thc hin Kha lun c thc hin t ngy 14/2/2005 n ngy18/8/2005. 3.1.2. a im Trung tm Cng Ngh Sinh Hc, i hc Nng Lm Thnh ph H Ch Minh. Phng Ha L, Trung tm Phn Tch Th nghim Ha Sinh, i hc Nng Lm Thnh ph H Ch Minh. 3.1.3. Vt liu nghin cu Ging nm Pleurotus floridanus Pleurotus sajor caju Pleurtus abalonus Pleurotus pulmonarius Pleurotus flabellatus Ngun cung cp i hc Khoa Hc T Nhin TP H Ch Minh i hc Khoa Hc T Nhin TP H Ch Minh i hc Khoa Hc T Nhin TP H Ch Minh Vin ht nhn Lt (L Vit Ngc) Vin ht nhn Lt (L Vit Ngc)

3.2.

Ha cht, dng c v thit b 3.2.1. Ha cht HCl 6N Phenol PITC (phenylisothiocyanate). Methanol Ethanol Nc kh ion TEA (Triethylamine) Chun hn hp 17 amino acid ACN (Acetonitril). Glucose Saccharose KH2PO4 MgSO4 Sodium acetate HNO3 m c EDTA

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3.2.2. Thit b v dng c Thit b T cy v trng T sy T mt Ni hp kh trng My HPLC Thit b thi kh tr Ct Pico-Tag Dng c ng nghim a petri 9 cm Bnh tam gic Bnh qu l Pipett 0.1-10 l Pipett 10-100 l Filter 0.20 m

3.3.

Phng php nhn ging v trng nm Mi trng ht Cm go CaCO3 La Mi trng gi mi Khoai m Cm go KH2PO4 MgSO4 CaCO3 Mi trng gi th Mn ca Cm go KH2PO4 MgSO4

3.3.1. Mi trng Mi trng thch (PGA) Khoai ty Glucose Agar

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3.3.2. Phng php 3.2.2.1. Chun b mi trng PGA - Khoai ty bc v, ra sch. Cn 200 g, un si, lc ly nuc. - Thm 20 g agar, cho thm nc ct 1 lt, un tan agar. - Cho vo ng nghim (khong 1/5 so vi chiu cao ng nghim), y nt bng. - Hp kh trng 121oC, 1.2 atm, trong 20 pht. - Ly ra, nghing chun b cho cy chuyn. - 5 dng nm bo ng sau khi em v phng th nghim, c tin hnh cy chuyn sang mi trng thch nghing PGA.. 3.2.2.2. Chun b mi trng ht - La c un si, nt nanh, trn vi CaCO3 (2%) , cm go (10%). - Cho vo bnh tam gic, hoc bnh thy tinh, hp kh trng 121 oC, 1,2 atm, trong 90 pht. - Khi t nm pht trin y ng nghim, chng s c cy sang mi trng ht. em trong bng ti. Ty theo mi loi m thi gian pht trin y bnh s khc nhau. Trong qu trnh , chun b mi trng gi mi. 3.3.2.3. Chun b mi trng gi mi - Cng m c ct khong 15 20 cm, ty theo kch thc ca ti ny lng, ngm khong 24 gi, trn vi mt s ha cht khc nh KH2PO4, MgSO4 vi t l nht nh ( 2% i vi MgSO4 v 1 % i vi KH2PO4). - Hp kh trng 121oC , 1,2 atm trong 90 pht. - Khi t nm pht trin y bnh tam gic, to ra mu trng mn trong bnh, cy sang mi trng gi mi. 3.2.2.4. Chun b mi trng nui trng - Mn ca c sn lc loi b rc v nhng mnh c kch thc ln. - Ngm trong nc vi 5% trong 24 gi B sung cm go, bt bp, cho vo ti ny lng hoc bnh thy tinh. - Hp kh trng trong 90 pht, 1atm, 121oC 2 ngy sau hp kh trng tip nh ln th nht.

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- Cy ging t mi trng gi mi sang. - trong ti, khi no thy t pht trin y trng , ph kn bt phi, c nhng git nc li ti trn thnh ti th tin hnh ct ti bng dng c kh trng. - em ra nh nui trng, ti v n nm - Nm sau khi hi, c cn tnh trng lng ti, sau em sy kh, bo qun trong t lnh, chun b cho phn tch amino acid. - Tip tc ti v n nm. - Sau khi hi nm ln hai, nm lng nm cng c em cn trng lng ti, c cht em sy kh, tnh trng lng ca chng. - Tnh nng sut sn phm. - Tt c nm hi c em o ng knh tnh trung bnh. 3.2.2.5. Tm tt quy trnh [1, 2] - Khoai ty 200 g / lt Ging nm - Glucose 20 g / lt - Agar 20 g / lt - La nu (88 %) Mi trng ht Cy sang mi trng gi mi Mi trng gi mi - CaCO3 (2%) - Cm go (10%) - Cng m (87 %) - MgSO4 ( 2%) - KH2PO4 (1 %) - Cm go (10 %) - Mn ca (84 %) Gi th trng trong ti Ti n nm - Cm go (10 %) - Bt bp (5,5 %) - KH2PO4 (0,5 %)

Thu qu th

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3.3.3. Phn tch amino acid Mu nm sau khi thu hoch c em phn tch thnh phn amino acid. Chng ti tin hnh phn tch 2 trn 4 mu: Pleurotus pulmonarius, Pleurotus flabellatus. Qu trnh phn tch thnh phn amino acid trong 2 loi nm trn c thc hin theo quy trnh Thomas T. Andersen v cng s. 3.3.3.1.Thy phn mu - Cho 2 g mu c ct nh vo tube chu nhit v lm kh. - Thm vo 10 ml dung dch HCl 6N. - Loi b khng kh bng cch thi kh tr vo khong khng ca tube ng dung dch mu. - Thy phn 152oC trong 1h. - Ly ra, ngui, lc qua giy lc. - Cho vo l, dn nhn v trong t mt. Vi nhit cao v trong iu kin acid c nng cao (HCl 6N), cc protein s c phn ct thnh nhng amino acid n (Hnh 2.10). 3.3.3.2. To dn xut Khi phn tch thnh phn amino acid trn HPLC, mu phi c to dn xut vi PITC c th pht hin thng qua detector. Qu trnh to dn xut c thc hin nh sau: - Ht 2l mu cho vo bnh qu l c y nhn. - Lm kh bng hn hp dung dch ethanol, nc v TEA vi t l tng ng 2:2:1. Qu trnh ny c thc hin trong iu kin c thi kh tr loi b nhng acid cn tha li trong qu trnh thy phn cng nh gip cho PITC c th bt vi tng amino acid d dng hn. Bi v di tc ng ca phc hp ny c bit l TEA gip cho nhng amino s b kh 1 proton, khi PITC s gn vo. - Mu c to dn xut vi dung dch ethanol: nc: TEA: PITC = 7:1:1:1 khong 5 n 10 pht trong ti. Khi PITC s gn vo u amin ca cc amino acid (Hnh 2.11), Theo Molnar-Perl, (1994), phn ng gn ny rt bn, nu bo qun trong iu kin lnh chng s n nh t nht 6 thng).

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- Mu phn tch c em i thi kh vi kh tr. Bc quan trng nht trong qu trnh to dn xut l loi b nhng tc nhn sau khi dn xut. iu ny c thc hin trong iu kin chn khng thi kh tr hoc trch vi heptane (Thomas T. Andersen, 1995). Vi iu kin phng th nghim chng ti tin hnh bng phng php thi kh vi kh tr. Nu PITC khng c loi thi tt th n s lm h cng nh ph hy ct HPLC. - Mu c pha long trong 2 ml dung dch pha long trong mu v c lc qua b phn lc ng knh 20 m chun b cho phn tch HPLC. 3.3.3.3. Phn tch HPLC Qu trnh thc hin phn tch trn HPLC c thc hin vi: Buffer A: sodium acetate v TEA, pH 6,8. Buffer B: 60% acetonitile, nc 40% (v/v). Ct: Pico- Tag, 3.9 mm x 15 cm. Detector: Ultra-violet (UV). Tc dng: 1 ml / pht. Bc sng: 254 nm. Qu trnh thc hin: Ht 20 l dung dch chun 17 amino acid, bm v theo di. Lu li cc thng s cn thit tin hnh nh tnh cng nh Ht 20 l mu, tin hnh tng t nh i vi chun.

nh lng (thi gian lu, din tch peak,)

3.3.3.4. Phn tch v tnh ton kt qu Sau khi bm chun, ta s c nhng d liu thu t my tnh nh l thi gian pht hin peak, din tch ca tng peak th hin trn sc k . Cc thng s ny c lu li tnh hay nh lng cho mu phn tch. C rt nhiu cch nh lng amino acid n: Dng chun vi cc nng khc nhau, sau lp phng trnh hi quy gia din tch peak v nng vi h s tng quan xc nh. T din tch peak ca mu bit qua qu trnh chy ta c th tnh nng tng ng cho tng amino acid. y l phng php chnh xc nht tnh nng cc amino acid.

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Ch bm chun 1 ln, ghi li cc din tch peak thu c trn sc k sau khi phn tch kt thc. Sau lp t l gia din tch peak v nng ca chun v mu, t tnh c nng tng amino acid trong mu.

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PHN 4. KT QU V THO LUN4.1. Kt qu trng nm 4.1.1. S tng trng ca h si trn cc mi trng Sau khi chng ti thc hin nui trng theo quy trnh mc 3.2.2.5, chng ti t c mt s kt qu trnh by bng 4.1. Bng 4.1. Thi gian lan t y ca cc dng nm Dng nm Mi trng la Pleurotus pulmonarius Pleurotus flabellatus Pleurotus floridanus Pleurotus abalonus Nhn xt: Tt c cc loi nm u c thc hin u c tin hnh trn cc thit b ging nhau tng ng vi tng mi trng (mi trng la trong bnh tam gic, mi trng cng m trn ti ny lng) khi chuyn sang mi trng nui trng, chng ti thc hin Pleurotus floridanus, Pleurotus abalonus trn ti ny lng, ng thi vi hai loi Pleurotus flabellatus, Pleurotus pulmonarius chng ti thc hin trn nhng l bng thy tinh. V th, chng ti ch so snh trn hai mi trng u vi 4 dng nm cn mi trng nui trng chng ti phn ra tng cp so snh. Vi kt qu thu c (Bng 4.1), chng ti nhn thy rng: Thi gian tng trng trn mi trng ht ca Pleurotus abalonus l chm nht, k n l Pleurotus floridanus; Pleurotus flabellatus v Pleurotus pulmonarius c thi gian tng trng tng ng nhau. C 3 dng nm Pleurotus floridanus; Pleurotus flabellatus v Pleurotus pulmonarius u c h si t trng nhng Pleurotus floridanus th trng hn v h si khng tha nh hai dng cn li (Hnh 4.1) Khi cy sang mi trung gi mi, th kt qu cng ging nh trn mi trng ht, thi gian sinh trng ca Pleurotus abalonus vn l chm nht. V trn mi trng ny, th nm ny vn khc so vi cc nm cn li l trn u mi si nm c mu en, trong cha cc bo t.( Hnh 4.2 v Hnh 4.3). 15 ngy 15 ngy 18 ngy 30 ngy Mi trng gi mi 17 ngy 18 ngy 20 ngy 40 ngy Mi trng nui trng 25 ngy 27 ngy 30 ngy 40 ngy

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4.1.2. S hnh thnh qu th Bng 4.2. c tnh qu th ca cc loi nm Loi nm ng knh trung bnh qu th Pleurotus floridanus Pleurotus abalonus Pleurotus pulmonarius Pleurotus flabellatus. 8 cm 13 cm 11 cm 10 cm Nng sut sn phm (%) 70 62 60 56

Nng sut sn phm = Trng lng nm ti / Trng lng c cht kh V hnh thi Sau khi em ra nh trng, tui n nm, chng ti thu c hnh thi ca 4 dng nm nh sau - Pleurotus floridanus (Hnh 4.1) qu th mu trng, hi ph lng mn gc. cc qu th mc thnh tng chm. Khi em quan st dui knh hin vi c th thy c bo t m (Hnh 4.4) - Pleurotus abalonus (Hnh 4.2) qu th c mu trng c, rt dy, chc, trn b mt c nhng vy nh. Cung nm cng rt to. em quan st di knh hin vi quang hc, chng ti cng thy c bo t m tung i ln hn so vi Pleurotus floridanus (Hnh 4.5) - Pleurotus pulmonarius (Hnh 4.3) trng hi ng xm, hi x thy mt ngoi ca phin - Pleurotus flabellatus (Hnh 4.4) phin nm c mu xm m, cnh mng, rt ging vi Pleurotus pulmonarius. Nh vy , v hnh thi 4 dng nn ny hon ton khc nhau. Pleurotus abalonus c qu th to v dy nht, k n l Pleurotus floridanus; hai loi cn li v mt hnh thi tng i ging nhau, ch khc nhumu sc trn mt phin nm. V nng sut sn phm: nng sut sn phm c tnh bng trng lng nm ti/trng ng c cht kh. Theo bng 4.2 th nng sut ca Pleurotus floridanus cao nht. Cc dng cn li nng sut cng tng i cao. Phin nm mng, c mu

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Hnh 4.1. Sinh trng t nm trn mi trng la ca Pleurotus floridanus, Pleurotus flabellatus, Pleurotus flabellatus Thn cy m

Bo t n

Hnh 4.2. Pleurotus abalonus trn mi trng thn cy m b sung MgSO4 ( 2%), KH2PO4(1 %), Cm go (10 %)

Hnh 4.3. Bo t Pleurotus abalonus quan st trn h si t (bo t n)

Bo t m

Bo t m

Hnh 4.4. Bo t m quan st trn qu th ca nm Pleurotus floridanus

Hnh 4 .5. Bo t m quan st trn qu th nm Pleurotus abalonus

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Qu th

Qu th

Hnh 4.6. Qu th nm Pleurotus floridanus

Hnh 4.7. Qu th nm Pleurotus abalonus

trn ti ny lng cha mn ca c b sung: Cm go (10 %) Bt bp (5,5 %) KH2PO4 (0,5 %)

trn ti ny lng cha mn ca c b sung: Cm go (10 %) Bt bp (5,5 %) KH2PO4 (0,5%) )

Qu th Qu th

Hnh 4.8 Qu th nm Pleurotus flabellatus trn bnh thy tinh cha mn ca c b sung

Hnh 4.9 Qu th nm Pleurotus pulmonarius trn bnh thy tinh cha mn ca c b sung:

Cm go (10 %) Bt bp (5,5 %) KH2PO4 (0,5%)

Cm go (10 %) Bt bp (5,5 %) KH2PO4 (0,5%)

4.2. Kt qu phn tch amino acid Sau khi tin hnh phn tch, trn 2 dng nm Pleurotus pulmonaius, Pleurotus flabellatus trn HPLC, chng ti thu c thnh phn ca tng amino acid c lit k trong bng 4.3.

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Hnh 4.9. Sc k chun amin acidASP: Aspartic acid; GLU Glutamate; SER: Serine; GLY: Glycine ; HIS: Histidine ARG: Arginine; THR: Threonine; ALA :Alanine; PRO: Proline; TYR: Tyrosine ; VAL: Valine ; MET: Methionine; CYS: Cysteine; ILEU: Isoleucine; LEU: Leucine; PHE : Phenylalanine; LYS: lysine

Hnh 4.10. Sc k ca mu nm Pleurotus flabellatusASP: Aspartic acid; GLU Glucine; SER: Serine; GLY: Glycine ; HIS: Histidine ARG: Arginine; THR: Threonine; ALA :Alanine; PRO: Proline; TYR: Tyrosine ; VAL: Valine ; MET: Methionine; CYS: Cysteine; ILEU: Isoleucine; LEU: Leucine; PHE : Phenylalanine; LYS: lysine

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Hnh 4.11. Sc k ca mu nm Pleurotus pulmonariusASP: Aspartic acid; GLU Gluatamate; SER: Serine; GLY: Glycine ; HIS: Histidine ARG: Arginine; THR: Threonine; ALA :Alanine; PRO: Proline; TYR: Tyrosine ; VAL: Valine ; MET: Methionine; CYS: Cysteine; ILEU: Isoleucine; LEU: Leucine; PHE : Phenylalanine; LYS: lysine

Theo bng ny, th c trong 2 dng u cha y 17 amino acid. Trong nm Pleurotus pulmonaius hm lng Leu chim vi t l cao nht, trong khi th cc amino acid nh Val, Asp, Cys, Met, Ileu, Lys, Glu chim vi t l rt thp, c bit l Glu chim t trng thp nht trong 17 amino acid c pht hin. Vi nm Pleurotus flabellatus cng tng t nh Pleurotus pulmonaius , Leu chim t l cao nht trong 17 amino acid; Val chim t l thp nht. Trong 17 amino acid ny, cha n 7 trong 8 amino acid thit yu (c in nghing trong bn) i vi ngi, ngoi tr Tryp cha c xc nh. Nhng amino acid ny c th con ngi khng th tng hp c m phi c cung cp t bn ngoi. Cc amino acid thit yu ny chim n 42 % trong tng s amino acid i vi Pleurotus pulmonaius v 44 % trong tng s amino acid ca nm Pleurotus flabellatus. T l ny cao hn so vi nm rm (38,2 %) v nm m (38,4 %) [Nguyn Ln Dng, 2002]. Khi c so snh vi thnh phn amino acid vi thc vt nh bp, u nnh th thnh phn amino acid ca nm bo ng vn cao hn.

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Bng 4. 3. Thnh phn amino acid trn Pleurotus pulmonaius, Pleurotus flabellatus S th t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Amino acid Leucine Glycine Serine Arginine Alanine Proline Histidine Tyrosine Threonine Phenylalanine Valine Asparagine Cysteine Methionine Isoleucine Lysine Glutamate Pleurotus pulmonarius (mg/g) 21, 15 8, 92 7, 74 6, 49 5, 93 4, 50 4, 42 3, 75 3, 67 2, 21 1, 88 1, 88 1, 79 1, 77 1, 31 1, 15 0, 25 Pleurotus flabellatus (mg/g) 17, 15 11, 39 7, 85 5, 26 4, 95 3, 08 5, 48 6, 78 2, 68 0, 97 0, 91 1, 63 2, 59 4, 49 13, 19 0, 89 2, 04

Nh vy, nm bo ng c th c xem nh l mt thc phm giu dinh dng cha gn nh y cc amino acid thit yu. Thnh phn amin acid ca n cao hn hu ht cc loi rau qu. Khi so snh gia 2 loi nm, thnh phn amino acid (aa) gia chng tng ng nhau (u cha 17aa). Nhng khi xt ring tng amino acid, th gia chng c s khc bit. Glu ca nm Pleurotus flabellatus cao gp 8 ln so vi Glu (ch pht hin dng vt: 0,25 mg) ca Pleurotus pulmonaius, v Ileu cng cao hn gp 10 ln. Tt c cc aa cn li c s khc bit khng ln lm.

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PHN 5. KT LUN V NGH

5.1. Kt lun. trng c 4 loi nm bo ng. Trong , c hai loi cha c trng Thnh Ph H Ch Minh: Pleurotus pulmonarius v Pleurotus flabellatus. Vi nng sut sn phm t c khong 55- 70%, trong iu kin nhit 28 20C, m 70%. Bng sc k lng cao p HPLC, chng ti pht hin v nh lng c thnh phn amino acid trn 2 loi nm Pleurotus pulmonarius v Pleurotus flabellatus. Hai loi ny cha y 17 amino acid, trong leucine chim t l cao nht 21,15% i vi Pleurotus pulmonarius v 17,15% i vi Pleurotus flabellatus. Trong nm Pleurotus pulmonarius th Glutamate chim t l thp nht, nhng i vi nm Pleurotus flabellatus (0,25%), Valine chim t l thp nht (0,91%). 5.2. ngh Sau khi phn tch amino acid, nn tin hnh trng trn cc loi c cht khc nhau sau phn tch tip amino acid xem s khc bit do nh hng ca c cht .

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PHN 6. TI LIU THAM KHOTING VIT 1. Nguyn Ln Dng, 2002. Cng ngh nui trng nm, tp 1. Nh xut bn nng nghip H Ni. 200 trang. 2. Nguyn Ln Dng, 2002. Cng ngh nui trng nm, tp 2. Nh xut bn nng nghip H Ni. 244 trang. 3. L Duy Thng, 2001. K thut trng nm, tp 1. Nh xut bn Nng Nghip. 4. Nguyn L Chung Sn, 2004, S dng sinh khi cy mai dng lm nguyn liu trng nm bo ng (Pleurotus sajor- caju ) gp phn kim sot s xm ln ca loi thc vt ngoi lai. Kha lun tt nghip c nhn cng ngh sinh hc, Trng i hc Khoa Hc T Nhin Thnh ph H Ch Minh. 5. Hunh Hu Tn, 2004, nghin cu s dng tru lm nguyn liu trng nm bo ng (Pleurotus florida). Kha lun tt nghip c nhn sinh hc, Trng i hc Khoa Hc T Nhin Thnh ph H Ch Minh. 6. Hong Trm Anh, inh Hong Anh Khoa, L Thy Anh, L Duy Thng, 2003. Nghin c khng khun ca nm bo ng (Pleurotus sajor- caju). Tp ch y dc hc. tr 201-204. 7. Lm Th Nhn, 2001. Ci tin k thut nh lng aflatoxin trong thc phm. Lun n thc s khoa hc chuyn ngnh ha phn tch, Trng i hc Khoa Hc T Nhin Thnh ph H Ch Minh. tr 16-19 8. Nam Phng, 2005. Trng thnh cng hai loi nm bo ng mi ti Vit Nam. Bo khoa hc ph thng s 25: tr 14. Ta son bo khoa hc ph thng, Thnh ph H Ch Minh. TING NC NGOI 9. Archer J.P. Martin 1952. The development of partition chromatography. Nobel lecture, December 10. Alexander Efimov, 2005. HPLC introduction. 11. Hans Neurath. The Protein - Composition, Structure, Function. 2nd edition. Volume 1. Academic Press, New York and London p. 2-39. 12. Royer Y. Stainer, Michael Doudoroff, and Microbial World. Prentice-Hall, Inc. p.88-90. Edward A. Adelberg. The

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13. Thomas D. Brock. Biology of microorganism. Pretice-Hall, Inc. p.684-681. 14. Arthur T. Henrici, M.D. A handbook for students of Bacteriology. Molds, Yeast, And Actinomycetes. John Wiley & Sons, Inc, New York. p.7-9.

15. Zakia Bano, K.S. Srinivasan, and H.C. Srivastava, 1963. Amino acid composition of the Protein from a Mushroom (Pleurotus sp.). Appl. Microbiol. 11. p.184-187 16. Bernard Wathelet, 1999. Nutritional analyses for proteins and amino acids in beans (Phaseolus sp.). Biotechnol. Agron. Soc. Environ, p 197200 17.