CCH XC NH RU ETHYLIC V CARBON DIOXIDE TRONG DCH LN MEN PGS.TS.

Bin Vn Minh Trng HSP - i hc Hu Trong qu trnh ln men, khi phn hy ng trong iu kin k kh nm men Saccharomyces cerevisiae chuyn hydrogen t NADH (Nicotinamide adenine dinucleotide) n acetaldehyde to thnh ru ethylic ti to NAD+, sn phm to thnh l ru ethylic v carbon dioxide (CO2). Trong bi vit ny chng ti mun gii thiu cch xc nh ru ethylic v CO 2 c mt trong dch ln men. 1. Ha cht v dng c - Dung dch ko mch nha 15% hay ng glucose 10%; - Dung dch tartaric acid 1%; - Dung dch NaOH 10%; Dung dch Ba(OH)2 10%; Iod (I2) dng tinh th; - Dung dch lugol (Iodine): Ho tan 1g Iod trong 3-5 ml nc ct, thm 2g KI (Kali iodide), khuy cho tan ht, thm nc ct cho 300 ml. Bo qun trong l ti. - Dung dch H2SO4 m c; K2Cr2O7 dng tinh th; - Men bnh m (Yeast bread) dng bt (hnh 1, 2 - c tnh cht minh ha); - Dng c th tinh (bnh tam gic, ng nghim, pipete ); - T m; - Hai ng nghim lng nhau: 1 ng to v mt ng nh (ng nh gi l ng phao, hnh 3); bnh ln men Smith (hnh 4).

Hnh 1: Men bnh m dng bt

Hnh 2: Hnh dng t bo nm men bnh m S. cerevisiae di knh hin vi quang hc (x100).

2. Cc bc tin hnh a. Chun b dch ln men xc nh ru ethylic v CO2 c mt trong dch ln men, ta phi chun b trc dch ln men theo mt trong hai cch sau: Cch 1: Cho vo bnh tam gic dung tch 250cc - 200 ml dung dch ng glucose 10%, cho thm 2-3 git tartaric acid 1% acid ha mi trng. y nt bnh tht cht (nt c vi cao su).

Cch 2: Ko mch nha 20gam, nc ct 200ml v 0,5gam (NH4)2SO4 un nng, va khy cho tan ri lc qua bng cho sch. Khi lc xong th rt vo bnh tam gic dung tch 250cc. Nt bng v qun giy ming bnh ri em hp kh trng. Hp autoclave th hp 1,1 atm trong 30 pht, hp cch thy th hp 4 gi. Hp xong ly ra ngui. Trc khi lm th nghim thm 1gam men bnh m dng bt vo 200ml dch ln men. t vo t m 350C trong 1- 2 gi. iu kin qu trnh ln men ru s xy ra. b. Xc nh kh CO2 ca dch ln men ru 1) o th tch kh CO2 trong ng phao hay bnh Smith: Cch 1: o th tch kh CO2 trong ng phao (hnh 3). ng th nghim (a): y ng phao (loi ng nghim nh c chia vch) dch ln men va mi trn nm men ri p ngc trong ng nghim ln hn cng cha dch ln men ny. Ch khng cho khng kh lt vo ng phao khi p , nt cht ming bnh bng nt bng, ri t vo t c nhit 350C trong khong thi gian 15-20 pht. Kh CO2 sinh ra lm cho mc nc trong ng phao tt dn xung. Tnh th tch kh c hnh thnh trong mt n v thi gian. ng i chng (b): Lm tng t nh trn, song thay dch ln men bng dch khng c nm men. So snh th tch kh chon ch hai ng th nghim v i chng sau khong thi gian xc nh.

Hnh 3

Hnh 4

Cch 2: Th nghim ln men ru trong bnh Smith (Hnh 4) Cho vo bnh cu dung tch 500cc 250ml dung dch ng 10%, thm vo 1g men n bt m dng bt, lc tht u. y bnh bng nt cao su c ng thy tinh un cong xuyn qua np, u kia ca ng thy tinh c nhng chm vo ng nghim p ngc cha y nc. t bnh th nghim nhit 350C, sau 10-15 pht quan st thy bt kh thot ra lm mc nc trong ng nghim tt dn xung. Gii thch: Trong qu trnh ln men, CO2 sinh ra s to thnh nhng bt kh nh tch t li lm ct nc tt dn. c s ml kh CO 2 chon ch trong bnh ln men Smith. Th tch kh chon ch cng nhiu, cng nhanh th chng t kh nng ln men ethylic cng mnh (hnh 4). 2) Xc nh kh CO2 trong dch ln men bng phn ng nh tnh. Theo mt trong 2 cch sau: Cch 1: Phn ng vi Ba(OH)2

- Cho vo ng nghim 5ml dch ln men sau 2h, thm 1ml dung dch Ba(OH)2 10%. Dng kp g cp ng nghim v un nh trn ngn la n cn. lng. Nu c mt ca CO2 s to thnh kt ta mu trng do s to thnh BaCO 3 theo phn ng: CO2 + Ba(OH)2 BaCO3 + H2O ng i chng: Lm tng t nh trn, song thay dch ln men bng dch khng c nm men. Quan st kt ta hai ng th nghim v i chng. Cch 2: Th nghim lm c nc vi trong Ly bnh ln men ra (dch ln men sau 2h), chun b trc mt ng nghim ng nc vi trong Ca(OH)2. cn thn m vi cao su v cho ngay vo ng nghim cha nc vi trong. Kh ln men thot qua ng cao su lm c nc vi trong, chng t kh to ra trong dch ln men ru l CO 2. Ca(OH)2 + CO2 CaCO3 + H2O ng i chng: Lm tng t nh trn, song thay dch ln men bng dch khng c nm men. Quan st c hai ng th nghim v i chng. c. Xc nh ru ethylic c trong dch ln men C th lm theo mt trong hai cch sau: Cch 1: nh tnh ru ethylic bng phn ng to mu vi Potassium Dichromate (K2Cr2O7): - Cho vo ng nghim 2ml dch ang ln men ru, thm 2 ml H2SO4 m c, thm vi tinh th K2Cr2O7 lc u. Nu trong dch ln men c mt ru ethylic s xut hin mu xanh lam, do phn ng ca ru ethylic vi K2Cr2O7 theo phng trnh: 2 K2Cr2O7 + 8 H2SO4 + 3CH3CH2OH 4KCr(SO4)2 + 3CH3COOH + 11 H2O ng i chng: Lm tng t nh trn, song thay dch ang ln men bng dch khng c nm men. So snh s i mu hai ng th nghim v i chng. Cch 2: Ly 1ml dung dch ang ln men trn cho vo ng nghim; cho tip vo ng nghim 3 git dung dch lugol v 10 ml dung dch NaOH 10%, un nh ng nghim trn n cn. Ta ngi thy mi rt c trng ca iodofo rm* bay ra chng t dch ln men trong ng nghim c cha ru ethylic. CH5OH + 4I2 + 6NaOH CH3I + 5NaI + HCOONa + 5H2O *CH3I l iodoform c mi rt c bit. 3. Nhng im cn lu - Khi s dng men bnh m phi dng loi cn hn s dng, cc ht men mu crem khng dnh nhau, khng chuyn mu. - Cc ng phao phi rt y dch ln men, khi p ngc sao cho khng c bt kh. Nu khng c ng phao chia vch th ta t lm ly bng cch ct mt di giy chia vch ri dn bn ngoi ng phao. - Khi thc hin phn ng nh tnh ru ethylic bng phn ng to mu, phi lun ghi nh ha long 2ml H2SO4 m c vo 2ml dch ln men, khng c lm ngc li. - Cng ln men ru c th xc nh da vo lng CO 2 thot ra hoc hm lng ethanol c to ra t mt th tch mi trng xc nh, trong mt thi gian nht nh. Thng thng ngi ta dng phng php xc nh theo hm lng CO2 v n d dng xc nh hn hm lng ru ethylic.

Ti liu tham kho 1 Nguyn Thnh t, Thc hnh vi sinh hc, Nxb GD, H Ni, 2000. 2 John William Henry Eyre, (2009). A Laboratory Guide for Medical, Dental, and Technical Students, http://www.gutenberg.org/. 3 http://www.kingarthurflour.com/blog/2010/10/11/baguettes-take-a-walk-on-thewild-yeast-side/.

METHOD TO DETERMINE THE ETHYL ALCOHOL AND CARBON DIOXIDE IN FERMENTING JUICE ABSTRACT To know an exact wine fermentation, the presence of ethyl alcohol and CO2 must be determined in fermenting juice. To determine the presence of CO2 in wine fermentation, the volume of CO2 is measured using the tube floats or by reaction with Ba(OH)2 to create the precipitation of CaCO3. To determine ethyl alcohol in fermenting juice, the reaction of ethyl alcohol with K2Cr2O7 to create green solution is usually used, otherwise we can use the smell reaction with iodoform. --------------------------------CCH XC NH RU ETHYLIC V CARBON DIOXIDE TRONG DCH LN MEN TM TT Mun bit chnh xc dung dch ang ln men l ln men ru, ta phi xc nh s c mt ca ru ethylic v CO2 c mt trong dch ln men. Xc nh kh CO2 ca dch ln men ru ta o th tch kh CO 2 trong ng phao hay dng phn ng vi Ba(OH)2 to kt ta CaCO3 mu trng. Xc nh ru ethylic c trong dch ln men thng ta thc hin phn ng to mu xanh lc vi s c mt ca K2Cr2O7, hay s dng phn ng to mi ca iodoform.