I HC QUC GIA TP H CH MINH TRNG I HC BCH KHOA KHOA K THUT HA HC*

LUN VN TT NGHIP I HCNGHIN CU QU TRNH KH CAFFEINE TRN L CH TI NGUYN LIU TRONG CNG NGH SN XUT CH XANHSVTH:NHM MINHMSSV:60301674CBHD:ThS. LI QUCTB MN:CNG NGH THC PHMTP H Ch Minh, 01/2008NHIM V LUN VNNHN XT CA GIO VIN HNG DANNHN XET CUA GIAO VIEN PHAN BIN

Con xin cm n cha m nui nng, dy d con nn ngi. Cha m lun ng h, ng vin con trong sut thi gian qua.LI CM N

Em xin chn thnh cm n Thy Li Quc t. Thy tn tnh hng dn em hon thnh tt lun vn.Em cng xin t lng bit n su sc n cc Thy C ca trng i hc Bch khoa Thnh ph H Ch Minh, c bit l cc Thy C trong b mn Cng ngh Thc phm. Thy C truyn dy cho em nhng kin thc v cng qu bu, cng nh h tr em rt nhiu em thc hin bi lun ny thnh cng.Cui cng, ti xin cm n cc bn trong lp lun ng vin, gip ti vt qua mi kh khn.

TM TT LUN VNHin nay, ch xanh c tiu th rng ri trn th gii bi v ch l mt ngun thc phm cha nhiu cc cht c li cho c th chng ta. Tuy nhin, vi s" ngi mc bnh v tim mch, vic s dng ch c th lm tng nguy c v tim mch, do trong ch c cha nhiu caffeine. Do , chng ti tin hnh nghin cu qu trnh kh caffeine trn l ch ti nguyn liu trong cng ngh sn xut ch xanh. Phng php chng ti chn l kh caffeine bng phng php Swiss water. Qua nghin cu, chng ti thu c nhng kt qu nh sau:v qu trnh kh caffeine: hiu sut kh caffeine l 90%, tn tht cht kh ha tan phi caffeine l 5% v tng lng polyphenol trong l ch sau kh caffeine tng 6.935% khi nhit kh l 90c, t l ch/nc l 3.75g/100ml nc v thi gian kh l 7 pht.v qu trnh hp ph caffeine ca than hot tnh bo ha cht kh ha tan phi caffeine: hiu sut hp ph caffeine l 22%, mc tn tht cht kh ha tan phi caffeine l 3.4% v mc tn tht polyphenol tng l 11 - 12% khi qu trnh tin hnh vi t l than hot tnh l 4% (w/v), nhit dch nc ban u l 70c, thi gian hp ph l 100 pht.MC LC mcTrangTrang baiNhim v lun vnNhn xt ca gio vin hng dnNhn xt ca gio vin phn binLi cm niiTm tt lun vniiiMc lcivDanh mc bngviiDanh mc hnhXDanh mc cc t vit ttxiiLi m uxiiiCHNG l.TNG QUAN11.1. Gii thiu chung v cy ch11.1.1 c tnh thc vt ca cy ch11.1.2 Thnh phn ha hc ca l ch21.2. Caffeine101.2.1 Gii thiu chung v caffeine101.2.2 Nhng tc ng sinh l ca caffeine i vic th111.3. Catechin151.3.1 Gii thiu chung v catechin151.3.2 Nhng tc ng sinh l ca catechin i vic th171.4. Dc tnh ca cy ch181.5. Mc ch ca vic kh caffeine trong l ch181.6. Cng ngh kh caffeine191.6.1 Phng php kh caffeinebng dung mi191.6.2 Phng php kh caffeinebng nc211.6.3 Phng php Swiss water25- iv -

- iii -

1.6.4 Phng php kh caffeine bng C2 siu ti hn29CHNG2. Nguyn liu v phng php nghin cu372.1. Phng php nghin cu372.1.1 Mc ch nghin cu372.1.2 Ni dung nghin cu372.2. Nguyn liu392.2.1 L ch nguyn liu392.2.2 Than hot tnh392.2.3 Ha cht s dng392.3. Cc thit b dng trong nghin cu392.4. Quy trnh cng ngh kh caffeine402.4.1 Quy trnh chun b dch trch cn bng cc cht kh ha tan trong l ch402.4.2 Quy trnh chun b than hot tnh bo ha khng cha caffeine422.4.3 Quy trnh chun b dch trch cn bng cha t caffeine432.5. Cc phng php phn tch432.6. Phng php tnh ton432.6.1 Qu trnh kh caffeine432.6.2 Qu trnh hp ph caffeine t dch trch cn bng bng than hot tnh bo ha 44CHNG 3.KT QU V BN LUN463.1. Kho st nguyn liu l ch ti463.2. Kho st nh hng ca t l chmc nqu trnh kh caffeine473.2.1 Hiu sut kh caffeine473.2.2 Tn tht cht kh ha tan phi caffeine493.2.3 Hm lng polyphenol tng ca l ch sau kh caffeine513.2.4 Kt lun533.3. Kho st nh hng ca nhit n qutrnhkh caffeine533.3.1 Hiu sut kh caffeine543.3.2 Tn tht cht kh ha tan phi caffeine553.3.3 Hm lng polyphenol tng56- V -

- V -

3.3.4 Kt lun583.4. Kho st nh hng cathi gian nqu trnhkh caffeine583.4.1 Hiu sut kh caffeine583.4.2 Tn tht cht kh ha tan603.4.3 Hm lng polyphenol tng623.4.4 Kt lun643.5. Kho st nh hng ca nhit dch qua than hot tnh n qu trnh hp phcaffeine663.6. Kho st nh hng ca t l than:nc n qu trnh hp ph caffeine ca thanhot tnh683.6.1 Hiu sut hp ph caffeine683.6.2 Tn tht cht kh ha tan693.6.3 Hm lng polyphenol tng703.6.4 Kt lun713.7. Kho st nh hng cathi gian hpph n qutrnh hp ph caffeine ca thanhot tnh723.7.1 Hiu sut hp ph caffeine723.7.2 Tn tht cht kh ha tan733.7.3 Tn tht hm lng polyphenol tng753.7.4 Kt lun76CHNG 4. KT LUN V KIEN ngh78TT LIU THAM KHO80PH LC 85Ph lc 1: Cc phng php phn tch85Ph lc 2: Xy dng ng chun caffeine:95Ph lc 3: Xy dng ng chun xc nh hmlng polyphenol tng98Ph lc 4: Xy dng ng chun xc nh hmlng ng kh99- vi -

- # -

Bng 1-1: Hm lng cc cht trong l ch ti2Bng 1-2: Thnh phn ha hc ca tm ch ti v cc l ch non3Bng 1-3: S phn b nc trong bp ch4Bng 1-4: Lng ng ha tan trong bp ch (tnh theo phn trm cht kh)6Bng 1-5: S phn b" proteine trong bp ch7Bng 1-6: Thnh phn catechin ca bp ch10Bng 1-7: Tnh cht vt l ca caffeine11Bng 1-8: Hm lng caffeine trong bp ch11Bng 1-9: S d ha caffeine ngi tiu th 5mg caffeine/kg th trng 14Bng 1-10: Thnh phn thp cc chttanin trong l ch ti16Bng 1-11: nh hng ca cu trc l n qu trnh kh caffeine24Bng 1-12: So snh tc kh caffeine nhng loi vi sinh vt khc nhau28Bng 1-13: Tnh cht ca C2 siu ti hn29Bng 1-14: nh hng ca kch thc nguyn liu n qu trnh kh caffeine31Bng 1-15: nh hng ca nhit v p sut n qu trnh kh caffeine32Bng 1-16: nh hng ca cht ng dung mi n qu trnh kh caffeine34Bng 1-17: nh hng catc dngCO2 nqutrnh kh caffeine35Bng 3-1: Kt qu kho st nguyn liu gia hai ln mua khc nhau46Bng 3-2: Hiu sut kh caffeine cc t l ch/ nc khc nhau47Bng 3-3: nh hng ca t l ch/nc n tiu hao cht kh ha tan tng49Bng 3-4: nh hng ca t l ch/nc n tn tht cht kh ha tan phi caffeine 49 Bng 3-5: nh hng ca t l ch/ nc n hm lng polyphenol tng ca l chsau kh caffeine51Bng 3-6: nh hng ca t l ch/nc n s thay i hm lng polyphenol tngca l ch sau kh52Bng 3-7: nh hng ca nhit n hiu sut kh caffeine54DANH MC BNG

DANH MC BNG

Bng 3-8: nh hng ca nhit n tiu hao cht kh ha tan tng55

Bng 3-9: nh hng ca nhit n tn tht cht kh ha tan phicaffeine55Bng 3-10: nh hng ca nhit n hm lng polyphenol trong l ch sau khcaffeine56Bng 3-11: nh hng n nhit n s thay i polyphenol tng56Bng 3-12: nh hng ca thi gian n hiu sut kh caffeine59Bng 3-13: nh hng ca thi gian n s tiu hao cht kh ha tan tng ca l chsau kh caffeine60Bng 3-14: nh hng ca thi gian n tn tht cht kh ha tan phi caffeine ca lch.sau kh caffeine60Bng 3-15: nh hng ca thi gian n hm lng polyphenol tng ca l ch saukh caffeine62Bng 3-16: nh hng ca thi gian n s thay i lng polyphenol tng ca l chsau kh63Bng 3-17: nh hng ca nhit n hiu sut hp ph caffeineca than hot tnhbo ha ch't kh ha tan66Bng 3-18: nh hng ca t l than hot tnh n hiu sut hp ph caffeine68Bng 3-19: nh hng ca t l than hot tnh n tn tht cht kh ha tan tng.. 69Bng 3-20: nh hng ca t l than n tn tht cht kh ha tan phi caffeine69Bng 3-21: nh hng ca t l than hot tnh n tn tht polyphenol tng ca qutrnh hp ph70Bng 3-22: nh hng ca thi gian n hiu sut hp ph caffeine72Bng 3-23: nh hng ca thi gian n s tiu hao cht kh ha tan tng73Bng 3-24: nh hng ca thi gian n tn tht cht kh ha tan phi caffeine74Bng 3-25: nh hng ca thi gian n tn tht polyphenol tng75Bng 0-1: Cch ly ha cht trong phn tch ng kh90Bng 0-2: Thi gian lu v din tch peak ca cc mu chun caffeine:95Bng 0-3: hp thu ca cc mu chun cc nng khc nhau98

Bng 0-4: hp thu ca cc mu chun glucose cc lng khc nhau99

- # -

- ix -

Hnh 1-1: Bp ch tm v 2 l non2Hnh 1-2: Cu trc ca caffeine10Hnh 1-3: Cng thc cu to ca cc catechin17Hnh 1-4: Quy trnhkh caffeine bng dung mi20Hnh 1-5: Quy trnhkh caffeine bng nc22Hnh 1-6: M hnh tng th ca qu trnh kh caffeine bng vi sinh vt27Hnh 1-7: M hnh kh caffeine bng SCF30Hnh 1-8: nh hng ca thi gian trch ly nqu trnh kh caffeine33Hnh 2-1: Quy trnhcng ngh kh caffeine40Hnh 2-2: Quy trnh to dch trch cn bng cht kh ha tan tng (so vi thnh phn cachng trong l ch ti)40Hnh 2-3: Quy trnh to than hot tnh phi caffeine boha cht kh ha tan42Hnh 2-4: Quy trnh to dch trch phicaffeine c nng cc chtkhha tan cn bngvi thnh phn ca chng trong l ch ti43Hnh 3-1: nh hng ca t l ch. nc n hiu sut kh caffeine48Hnh 3-2: nh hng ca t l ch. nc n tn tht cht kh ha tan phi caffeine49Hnh 3-3: nh hng ca t l ch.nc n hm lng polyphenol tng ca l ch51Hnh 3-4: nh hng ca t l ch/nc n s thay i polyphenol tng ca l ch52Hnh 3-5: nh hng ca nhit n hiu sut kh caffeine54Hnh 3-6: nh hng ca nhit n tn tht cht kh ha tan phi caffeine56Hnh 3-7: nh hng ca nhit n hm lng polyphenol tng ca l ch sau kh57Hnh 3-8: nh hng ca thi gian n hiu sut kh caffeine59Hnh 3-9: nh hng ca thi gian n tn tht cht kh ha tan phi caffeine61Hnh 3-10: nh hng ca thi gian n hm lng polyphenol trong lchsaukh63Hnh 3-11: nh hng ca nhit dch ban u n hiu sut hp phcaffeine cathanhot tnh 67DANH MC HNH

DANH MC HNH

Hnh 3-12: nh hng ca t l than hot tnh n hiu sut hp ph ca qu trnh68- X -

- X -

Hnh 3-13: nh hng ca t l than hot tnh n tn tht cht kh ha tan ca qu trnhhp ph caffeine70Hnh 3-14: nh hng ca t l than n tn tht polyphenol tng ca qu trnh71Hnh 3-15: nh hng ca thi gian n hiu sut hp ph caffeine ca than hot tnh.... 72Hnh 3-16: nh hng ca thi gian n tn tht cht kh ha tan ca qu trnh74Hnh 3-17: nh hng ca thi gian n tn tht polyphenol tng ca qu trnh76Hnh 0-1: Din tch peak ca cc chun caffeine cc nng khc nhau96Hnh 0-2: ng chun caffeine96Hnh 0-3: ng chun polyphenol tng bng phng php Prussian blue98Hnh 0-4: ng chun phn tch ng kh tnh theo glucose99T vit tt, k hiuNgha

DNSDinitrosalicylic

ECEpicatechin

ECGEpicatechin gallate

EGCEpigallo catechin

EGCGEpigallocatechin gallate

FDAHip hi Thc phm v Dc phm ca M

HPLCPhn tch sc k lng cao p

ROLc thm thu ngc

SCFCht lng siu ti hn

SC-C2CO2 siu ti hn

SW1Dch cn bng cc cht kh ha tan

SW2Dch cn bng cht kh ha tan phi caffeine

TCVNTiu chun Vit Nam

DANH MC CC T VIT TAT

- 12 -

- # -

LI M UCh l mt dng thc ung truyn thng ca ngi Trung Quc ni ring v c dn Chu ni chung. Ngi ta s dng khng ch v mc ch gii kht m cn bi nhng dc tnh ca n. Trong ch c nhiu cht chng oxi ha v c s dng nh l mt dc phm cha nhiu bnh nh: cha ung th, tiu chy, cha cc chng khng tiu, chng cao cholesterol trong mu... Ngy nay, vic tiu th ch, c bit l sn phm ch xanh, ngy cng c tiu th rng ri trn th gii.Chnh v vy, din tch trng ch cng nh sn lng ch Vit Nam ngy cng tng nhanh. Cy ch c xy dng thnh mt trong mi chng trnh trng im pht trin nng nghip trong K hoch pht trin kinh t x hi ca Nh nc Vit Nam n nm 2010.Tuy nhin, cng nh coffee, trong ch c cha mt lng ln caffeine. y l mt thnh phn kch thch h thn kinh trung ng, tc ng ln tim mch v gy nh hng xu n mt b phn ngi tiu dng. Do , vic pht trin mt sn phm ch xanh khng cha caffeine l mt nhu cu cn thit nhm p ng nhu cu ca mt b phn ln ngi tiu dng nhy cm vi caffeine. Chnh v vy, chng ti tin hnh thc hin ti: Nghin cu qu trnh kh caffeine trn l ch ti nguyn liu trong cng ngh sn xut ch xanh. ti c nhng ni dung chnh nh sau: Tm hiu v ch xanh, caffeine v nhng tc ng ca n n c th c ngi. Tm hiu v cc cng ngh kh caffeine. La chn phng php kh caffeine trn l ch ti. Kho st cc yu t" nh hng n qu trnh kh caffeine. nh gi nh hng ca qu trnh n hiu sut kh caffeine, mc tn tht cht kh ha tan v polyphenol tng trn l ch.CHNG 1. TNG QUAN1.1. Gii thiu chung v cy chCy ch c tn gi khoa hc l Camellia sinensis (L) (hay Thea sinenss L.) (1935 - Hi ngh Quc t v thc vt). Cy ch c xp trong phn loi thc vt nh sau [3]:NgnhHt knAngiospermae

LpSong t ipDicotylednae

BChTheales

HChTheaceae

ChiChCamellia (Thea)

LoiChc. sinensis

1.1.1 c tnh thc vt ca cy ch 31Thn v cnh: cy ch c thn thng v trn, phn nhnh lin tc thnh mt h thng cnh v chi. Ty theo chiu cao, kch thc ca thn v cnh m cy ch c chia thnh 3 loi: cy bi, cy g nh v cy g va. Thn cnh v l to thnh tn cy ch; tn ch mc t nhin c dng vm u. Da vo kch thc gia thn chnh v cnh, ngi ta chia cy ch thnh ba loi tn. C th ni tn l l mt tiu chun quan trng chn ging ch. Vi nhng cy ch c tn to, rng th cy c im sinh trng nhiu, cho bp nhiu v sn lng cao.Hoa ch: Hoa ch bt u n khi cy ch t 2-3 tui. Hoa mc t chi sinh thc nch l. Cy ch l mt loi thc vt c hoa lng tnh, trng hoa c 5 - 9 cnh mu trng hay pht hng. B nh c ca hoa c 100 - 400 ci, trung bnh c 200 - 300 ci. Cc bao phn ca hoa ch gm hai na bao, chia lm 4 ti phn. Ht phn c hnh tam gic, mu vng nht, khi chn chuyn sang mu hong kim.Qu ch: qu ch l loi qu c 1 - 4 ht, c hnh trn, tam gic hoc hnh vung. Qu ch thng mc thnh tng chm ba, ban u c mu xanh ca chi. Khi tng trng, qu ch cng dn v chuyn sang mu nu v nt ra. Ht bn trong qu c th c dng gieo trng.L ch: l ch mc cch nhau trn cnh, mi t c mt l. Hnh dng v kch thc ca l ch thay i ty theo tng ging. Vi ging ch Trung Quc th l di 4 -14cm v rng 2 - 2.5cm c mu xanh nht v bng. Cn vi ch Assam th l ch di 8 - 20cm v rng 3.5 - 7.5cm, mng v mm. Trong khi , ch Nam Indo c kch thc trung bnh gia hai ging ch: Trung Quc v Assan. C th ni l ch l mt b phn quan trng trong vic khai thc cy ch v c ng dng trong vic ch bin cc sn phm ch thng mi nh: ch xanh, ch en v cc dng thc phm thng mi khc.1 Tm

L non 1

L non 2

Hnh 1-1: Bp ch tm v 2 l non.Trn th gii hin nay, cc nh khoa hc chia cy ch thnh ba ging chnh da vo c im sinh thi v thc vt ca cy ch (c bit l da vo kch thc ca l ch): Ch Trung Quc: Camella sinensis (L). Da vo kch thc l, Sealy (1958) phn bit ch Trung Quc thnh 2 loi: F. macrophylla (Sieb.) Kitamura v F. parviolia (Miq.) Ch Assam: Camellia assamica. Ch Nam Indo: Camellia assamca.1.1.2 Thnh phn ha hoc ca l chThnh phn ha hc ca cy ch v l ch:Bng 1-1: Hm lng cc cht trong l ch ti [46]Thnh phnHm lng (% khi lng cht kh)Thnh phnHm lng (% khi lng cht kh)

Proteine20-30Cht bo4-7

Amino acid1 - 5Acid hu c< 3

Alkaloide3-5Khong4-7

Polyphenols20-35Cht mu< 1

Carbonhydrate35-40Vitamin A, E, c,2

B1,B2,P...

Thnh phn ha hc ca l ch ph thuc vo rt nhiu yu t" nh ging cy, thi tit, kh hu, t ai v iu kin canh tc [6]. Bn cnh cc yu t" th dng, t ai, kh hu v iu kin canh tc th thnh phn ha hc gia cc l ch trn cng mt cy ch cng khc nhau. Do , trong cng nghip ch bin ch xanh v cc loi ch khc, cc nh sn xut ch quan tm n thnh phn ha hc ca tm ch v cc l ch non.

Bng 1-2: Thnh phn ha hc ca tm ch ti v cc l ch non [52].Thnh phn% khi lng cht khThnh phn% khi lng cht khThnh phn% khi lng cht kh

Polyphenol25 - 30Chlorophylls v cc cht mu0,5 - 0,6Pectin5-6

Epigallocatechingallate8 - 12Khong5-6Lignin5-6

Epicatechingallate3-6Caffeine3-4Proteine14 - 17

Epigallo catechin3-6Theobromine0,2Lipid3-5

Epicatechin1 -3Theophylline0,5Cc hp cht bay hi0,01-0,02

Catechin1 -2Amino acid4-5

Gallocatechin3-4Acid hu c0,5 - 0,6

Flavonols vflavonolglucosides3-4Monosaccharide4-5

Polyphenolic acids and depsides3-4Polysaccharide14-22

Leucoanthocyanins2-3Cellulose v hemicellulose4-7

CHNG 1: TNG QUAN

CHNG 1: TNG QUAN

CHNG 1: TNG QUAN

- 1 -

- 2

- 3

1.1.2.1 Nc: L thnh phn ch yu ca l ch (70-85%).Nc c vai tr quan trng trong s sinh trng v pht trin ca sinh vt. Nc l mi trng gip ha tan cc cht ha tan, tham gia cc phn ng thy phn v oxi ha kh bn trong cc t bo l. I vi cy ch, nc tham gia hu ht cc phn ng trong qu trnh sinh trng v pht trin ca cy ch, s vn chuyn in t, c cht qua mng t bo cht v cc phn ng sinh tng hp cc cht trong ch.Cng nh cc c th sng khc, hm lng nc gim dn t l n thn cy v vi tng l cng khc nhau. Hm lng nc tng dn t cc t bo c s n cc phn ngoi ca l. Khi tui th ca cy v l ch cng cao th hm lng nc trong l ch cng thp.Bng 1-3: S phn b" nc trong bp ch [52].Cc b phn ca bp chHm lng nc (%)

Tm76.00

L 175.60

L 275.60

L 374.26

Ngoi ra, hm lng nc trong cc bp ch c t ba l tr ln cng thay i nhiu tu thuc vo thi gian thu hi, in kin thi tit v rt nhiu cc yu t" khc. [52]1.1.2.2 CarbonhvdrateCc carbonhydrate trong l ch bao gm cc loi ng n gin v cc loi polysacchraride, trong ch yu l cc polysacchraride.

CHNG 1 : TNG QUAN

CHNG 1 : TNG QUAN

Cc loi ng n gin c tm thy trong l ch yu l glucose, fructose, sucrose, raffinose v stachyose. Trong l ch, hm lng ca cc loi ng ha tan l rt t v ch chim khong 1 - 2% (so vi khi lng cht kh) [1]. Trong khi , hm lng ca cc polysacchrarie trong l ch c th ln n 10 - 15% (so vi khi lng cht kh)

-4

-#

CHNG 1: TNG QUAN

CHNG 1: TNG QUAN

Lng ng ha tan trong l ch lun thay i v ph thuc vo nhiu yu t". Nu xt theo mc non gi ca l v v tr ca l trn bp ch th l cng gi, lng ng kh v sucrose cng nh tng lng ng ha tan cng cao.

Bng 1-4: Lng ng ha tan trong bp ch (tnh theo phn trm cht kh) [1].L chng kh (% so vi khi lng cht kh)Sucrose (% so vi khi lng cht kh)Tng s

L th nht0.990.641.63

L th hai1.150.852.00

L th ba1.401.663.05

Tuy hm lng ca cc loi ng kh trong l ch thp nhng n c vai tr cc k quan trng trong vic to hng v cho sn phm ch xanh. qu trnh sao trong cng ngh sn xut ch xanh, di tc dng ca nhit, cc ng kh ny b caramel ha hoc tham gia vo phn ng Maillard to hng, v ngt cho sn phm ch xanh v gip iu ha v cho ch [3], [1]. Thc nghim cho thy rng: s tc dng tng h gia glucose hay fructose vi cc acid amin - phenilalanine v tannin c to thnh cc aldehyde bay hi c mi thm ca hoa ti, mi mt ong [1].1.1.2.3 Cc hp cht cha nitrogenI.I.2.3.I. Protene v amino acidCc hp cht proteine chim khong 25 - 30% khi lng cht kh ca l ch ti [3]. Trong cng ngh sn xut ch xanh, proteine c vai tr quan trng trong vic iu ha v ca ch [31. Bn cnh , nhng bp ch cha nhiu proteine s h tr kh nhiu cho qu trnh v ch. Proteine gip qu trnh v ch c thc hin d dng, l ch xon hn v c ngoi hnh p, nh , lm tng gi tr cm quan ca ch xanh thnh phn sau ny [3].Bn cnh , hm lng proteine c ngha quan trng i vi vic hnh thnh cht lng ca ch bi chng l ngun cung cp cc acid amin t do trong sut qu trnh ch bin. Di tc dng ca nhit, proteine b thy phn to thnh cc acid amin v cc acid amin ny tham gia vo cc phn ng Maillard to hng v cho sn phm ch xanh [1]. Hm lng proteine trong l ch ph thuc vo ging ch, thi v thu hi, tui l, iu kin sinh trng, cch bn phn...Bng 1-5: S phn b" proteine trong bp ch [6].B phn ca bpProteine (tnh theo % khi lng cht kh)

Tm29.06

L th nht26.06

L th hai25.96

L th ba24.94

v thnh phn acid amin c trong l ch, nhng phng php sc k trn giy tm thy 17 loi nh sau: alanine, serine, threonine, arginine, histidine, lysine, tyrosine, phenylalanine, proline, aspartic...Acid amin c tc dng tt vi cht lng ch xanh, gp phn to hng v v mu sc nc pha ch. Trong s cc acid amin trn c 3 loi acid amin quan trng l: theanine (50%), acid glutamic (12%). Cc acid amin ny c tc dng sinh l tt vi con ngi v tham gia vo s hnh thnh hng thm ca ch.1.1.2.3.2. AlkalodeAlkaloide l nhm hp cht vng hu c c cha nitrogen trong phn t. Phn ln cc alkaloide l nhng cht khng mu, c v ng v t ha tan trong nc. Trong l ch, ngi ta tm thy cc alkaloide ch yu l caffeine, theobromine v theophyline. Trong , caffeine chim khong 2 - 5% lng cht kh; theobromine v theophylline vi hm lng nh hn rt nhiu so vi hm lng ca caffeine, chim khong 0.33% khi lng cht kh [6]. Tuy vy, vai tr ca theobromine v theophylline trong dc tnh ca cy ch quan trng hn so vi caffeine. Nguyn nhn l do so vi caffeine th theobromine kch thch h thn kinh trung ng c yu hn nhng lm li tiu tt hn [11.Hm lng caffeine trong l ch bin i theo ging, thi v, bin php canh tc v trn b phn khc nhau ca cy ch. Trong qu trnh ch bin, hm lng caffeine rt t bin i v thng lin kt vi tannin to thnh tannate caffeine c v d chu v mi thm [3].1.1.2.4 Tinh duCHNG 1: TNG QUAN

CHNG 1: TNG QUAN

Tinh du l mt hn hp cc cht bay hi tn ti trong cc c quan ca cy ch. Hm lng tinh du trong l ch ti v trong ch thnh phm rt thp, chng ch chim - #

- 7

khong 0.02% cht kh. Mc d vy, tinh du c ngha ht sc quan trng cho sn phm ch xanh v chng nh hng n hng v ca ch thnh phm [1 ]. Tinh du ca cy ch c hnh thnh trong qu trnh sinh trng, pht dc ca cy ch v trong qu trnh ch bin ch. Hng thm l mt ch tiu quan trng nht nh gi cht lng ch thnh phm. Hng thm ca ch thnh phm c to ra t ba ngun sau y: [3] Tinh du trong bp ch. Sn phm c mi trong s chuyn ha ca catechin v acid amin. Cc sn phm ca qu trnh caramel ha trong qu trnh ch bin ch.Bng phng php sc k kh, ngi ta pht hin trong l ch ti c khong 69 cu t hng khc nhau [1]. Theo ti liu ca nhiutc gi, trong chtiv ch thnhphm c cha cc cu t tinh du sau y: [1] Nhm cc acid hu c gm cc acid: acetic, propionic, salicilic, n-butyric, isobutyric, isovalerianic, capronic, caprilic, hexenic panmitinic, phenylacetic, benzoic... Nhm ru: isoamylic, isobutylic, n-butylic, hexanol, n-octanol, pentanol, benzilic, benzyletylic, isopentanol, metyletyletanol, phenyletanol, metylbutanol, acetylpirol... Nhm aldehyde: n- v iso-butaral, n-valerianal, metylacetal, hexenal, benzal, hexanal, vanilin, paraoxybenzealdehyd... Nhm cc hp cht khc gm: phenol, cresol, geraniol, linalol, citral, citronelol, tecpineol, acetonphenol, metylacetat, acrolein, metylsalicilat...Trong s cc thnh phn trn, cc aldehydelthnh phn cut quan trng catinh du ch. i vi cht lng ch, cc aldehyde bayhi gi vai tr quantrng v nhhng trc tip n hng thm ca ch thnh phm v bn thn mt s" aldehyde c mi thm. Hn na, cc aldehyde cn tham gia vo s to thnh cc phc cht c mi thm c o ca ch, tham gia vo s hnh thnh mu sc v v nc ch [1 ].1.1.2.5Sc t"Cng vi hng v v ca ch th mu sc ca nc pha ch l mt ch tiu cht lng c cc nh ch bin ch quan tm. Trong cng ngh ch bin ch xanh, mu sc ca nc pha ch yu do cc sc t" c trong nguyn liu v cc sc t" to ra trong qu trnh ch bin. Trong l ch ch yu cha nhiu sc t" thuc h chlorophyll v carotenoids. Trong cng ngh sn xut ch xanh, chlorophyll l sc t quyt nh n muca nc ch pha. Bn cnh , trong l ch cn c sc t" anthocyanidins. anthocyanidins. Trong l ch, hai sc t" ny tn ti di hai thnh phn chnh l elphenidin v cyanidin, y l hai thnh phn hon ton khng c li cho sn phm ch xanh v chng lm nh hng xu n mu ca nc ch.1.1.2.6 EnzymeTrong l ch c hai nhm enzyme quan trng l nhm enzyme oxi ha kh v nhm enzyme thy phn. Trong , nhm enzyme oxi ha kh vi i din l enzyme polyphenol oxydase hin din trong cc t bo biu b ca l ch. i vi cng ngh sn xut ch xanh, enzyme ny hon ton khng c li v c tiu dit hon ton trong giai on dit men.Bn cnh enzyme polyphenol oxydase, trong l ch cn tn ti mt h gm nhiu enzyme khc. Trong , cc enzyme ch yu l: Proteinase: xc tc cho qu trnh thy phn proteine trong qu trnh lm ho. sn phm to ra ca phn ng thy phn l cc peptide v cc acid amin t do. Nhng sn phm ny s tham gia vo cc phn ng Maillard trong qu trnh sao v to ra hng v cho ch xanh. Chlorophyllases tham gia qu trnh thy phn chlorophyll.1.1.2.7 PolyphenolCHNG 1: TNG QUAN

CHNG 1: TNG QUAN

Polyphenol l cc hp cht to v cht cho sn phm. Trong l ch, thnh phn polyphenol quan trng nht l flavanoid. Trong , thnh phn chnh ca flavanoids c tm thy trong l ch ti l catechin v flavonol. Hm lng ca catechin tng dao ng trong khong 20 - 30% khi lng cht kh ca l ch. Khi tin hnh kho st s phn b" ca ca cc catechin trong cc b phn ca bp ch, ngi ta pht hin rng, cc catechin c nhiu nht trong cc phn non ca cy ch nh: tm, l th nht, l th hai. Cc bp ch b su bnh v cc l gi th cha rt t catechin.

Bng 1-6: Thnh phn catechin ca bp ch (tnh bng nig/g tannin trong bp ch) [6]:CatechinLCnh

1234

(-) EGC122.10152.27206.42232.36222.08

(+) GC00048.9668.70

(-) EC45.5347.8445.1655.7965.62

(-) EGCG580.54577.93431.14284.13292.03

(-) ECG138.53132.27103.0980.5887.98

Tng s"886.69910.31786.11701.82737.40

1.2. Caffeine1.2.1 Gii thiu chung v caffeineCaffeine l mt hp cht t nhin c tm thy trong l, ht v tri cy ca trn 63 loi nng sn t nhin v l mt phn ca mt s" nhm cc hp cht c bit n nh methylxathine. Caffeine l mt cht gy nghin ch ng v thng c s dng nh mt loi dc phm v c tnh cht ca mt cht kch thch thn kinh n ha v c bi tit ra ngoi trong vng vi gi sau khi c hp th vo c th.Caffeine l mt cht thuc h alkaloide. dng tinh khit, caffeine l mt cht bt mu trng v c v ng. Cng thc ha hc ca caffeine l C8H10N4O2, danh php IPAC l 1,3,5-trimethylcayhine v c cng thc cu to l:Hnh 1-2: Cu trc ca caffeine

Bng 1-7: Tnh cht vt l ca caffeine [10]:Tnh cht vt lTnh cht

Trng thiKhng mi, mu trng, xp v l dng bt

Khi lng phn t (g)194.19

Nhit nng chy (C)236

Trng lng ring (g/cm3)1.2

S bay hi (%)0,5

Kh nng ha tan trong nc (%)2.17

T trng ca hi6.7

Caffeine l mt cht c tnh kim yu, khng c tnh acid. Khi tc dng vi acid to thnh cc mui bn.Ch l mt trong nhng nguyn liu cha nhiu caffeine. Theo nhiu nghin cu, cc cht dinh dng trong t c th lm thay i thnh phn caffeine trong l ch. Khi trong mi trng t thiu phosphorus th hm lng caffeine c th gim 20%, cn khi trong t thiu potassium th li c th lm tng 12% lng caffeine. Vic thm nitrogen trong phn bn lm tng caffeine ln n 40%. Bn cnh , hm lng caffeine trong l ch phn b" cng khng ng u trong cc b phn ca bp.Bng 1-8: Hm lng caffeine trong bp ch [3]V tr ca bpLng caffeine (% khi lng cht kh)V tr ca bpLng caffeine (% khi lng cht kh)

L th I3.39L th V1.70

L th II4.20L gi0.76

L th III3.40Cung ch0.36

L th IV2.10

1.2.2 Nhng tc ng sinh l ca caffeine v c th

Trong SCJ cc cht alkaloide c trong l ch ti, tr caffeine, theobromine v thyophyline ra, cc cht cn li c hm lng khng ng k. Tc dng ca caffeine, theobromine v thyophyline vi c th con ngi c th hin ch yu ba im: Th nht, caffeine kch thch h thn kinh trung ng, lm cho tinh thn minh mn, tng cng s nhn bit ca b c [1]. Mt s" gi thit cho rng caffeine ni ring v cc methylxanthine ni chung hm lng trung bnh s phn ng ln h thn kinh bng vic ngn chn s tip nhn adenosine ca t bo thn kinh. phn ng li, cc t bo thn kinh s tng v s" lng. Vic tng ny lm tng kh nng tip nhn cc adenosine cho t bo thn kinh, do , to cm gic kch thch cho h thn kinh trung ng, v lu di, vic tn ti mn tnh ca caffeine trong c th lm tng s lng ca cc v tr c quan tip nhn adenosine v lm cho kh nng chu ng ca c th vi caffeine tng ln [22]. Khi , kh nng kch thch ca caffeine i vi c th s gim. Bn cnh , caffeine kch thch qu trnh tit ra seroronine v no, gp phn lm tng s minh mn ca b c [221. Mt nh hng khc ca caffeine i vi h thn kinh trung ng l lm gim nhng cn au u. Nhng cn au u thng xuyn xut hin ch yu l do s gin mch ca cc mch mu thn kinh. iu ny c hn ch bi phn ng ca methylxanthine trn cc c quan tip nhn adenosine khi caffeine c s dng [22]. Tc dng th hai ca caffeine l vic kch thch c nng v tng cng hot ng ca tim. Nh tc dng ny m c th chng c s mt mi, tng cng hiu sut lm vic v sc do dai [1]. Caffeine v cc methylxanthine nh hng ln chc nng ca tim bng hai cch: th nht l lm bin i trc tip nhng s i lp ca tim v mch mu, th hai l nh hng n h thng dn truyn thn kinh v cc h thng trung ng th yu [22]. ngi s dng cc sn phm cha caffeine, p sut mu di chuyn trong c th tng nh v ch tr v hm lng ban u sau 3 - 4h [22]. Vic tng p sut mu kch thch hot ng ca c tim, tng kh nng hot ng ca tt c cc c quan, tng kh nng hp th v dn truyn oxy ca mu [22]. Bn cnh , caffeine gy ra s tng epinephrine v norepinephrine trong huyt tng, nh hng n hm lng ca ion Ca2+ ca t bo v s phn b" ca n trong t bo, lm thay i nhp tim, thay i in l ca tim, tng tc dn truyn mu. Kt qu ca nhng tc ng sinh l trn l lm tng hot ng ca c tim, gip c th chng li s mt mi. iu ny l hon ton khng tt i vinhng ngi c bnh v tim mch. Vic tng hot ng ca c tim v tc dn truyn mu lm tng huyt p v nguy c v ng mch. Bn cnh , vic s dng cc thc ung cha hm lng caffeine cao s lm tng hm lng homocysteine trong huyt tng v y l mt nguy c cho tim mch. Theo nhiu nghin cu y khoa, liu lng ca homocysteine trong huyt tng tng s tc ng mnh hn n tim mch v d dng kt hp vi hm lng cholesterol trong huyt thanh [22]. Tc dng th ba ca caffeine i vi c th l kch thch hot ng ca thn. Nh gip c th nhanh chng thi ra ngoi nhng sn phm tha ca qu trnh trao i cht [1]. Caffeine lm tng cng tc mu n thn, tng tc lc ca thn v tng cng s tit renin thn [221. Bn cnh , caffeine gip tng cng s trao i cht, s hp th oxy trong c th. Bn cnh nhng tc ng trn ca caffeine i vi thn th tc dng quan trng nht ca caffeine i vi c th l li niu [1].Ngoi ra, theo nhng nghin cu mi y ca cc nh khoa hc, caffeine c nh hng n h thng d dy, h hp v c bp. i vi h thng d dy, caffeine kch thch s tit ra cc cht ca cc tuyn, kch thch s tit hormone l lch, nh hng n tnh linh ng ca d dy v rut. Chnh iu ny, m theo kho st th c khong trn 60% ngi s dng caffeine c cc triu chng nh kh tiu, chng nng, au d dy v nhng triu chng gy ra bi s y hi v to bn [22]. Cn i vi h thng h hp, caffeine kch thch h h hp, to s thoi mi cho c v kh qun. Do , caffeine thng c s dng trong y khoa nh mt cht tr h hp hay mt th thuc cha bnh suyn [22].Bn cnh , caffeine cng nh hng n qu trnh trao i cht c bn. Caffeine lm tng tc trao i cht v thng c s dng cc vng lnh. Chnh v tc dng ny ca caffeine m trong mt thi gian di, n c coi nh l mt cht gy bo ph.Caffeine hu nh khng b oxi ha hay bin i di nhng tc ng vt l, chng d dng vt qua cc mng t bo cht. Vic hp th caffeine ca c th tu theo pH ca dung dch cha caffeine hay pH ca cc c quan trong c th. Theo nghin cu, th sau 20 pht, phn trm caffeine c hp th d dy l 9% pH = 2.1; 14% pH = 3.5 v 22% pH = 7. Qua c th thy, vic tng pH lm tng kh nng hp th caffeine trong c th [22].Do nhng tc ng c li ln c hi ca caffeine i vi c th con ngi, nn ngi tiu dng quan tm n tnh cht kch thch ca caffeine, n qu trnh d ha v bi tit ca c th i vi caffeine.Bng 1-9: S d ha caffeine ngi tiu th 5mg caffeine/kg th trng [22]Thi gian nhn xt(gi)Tc d ha(Hg/pht/kg)Tng lng c d ha(ng/kg)Tng lng cht tch lu c dha (ng/kg)Tng lng cht c gi li trong c th (|0,g/kg)

n ng 0- 111.16456454294

1 - 37.286715123427

3-56.982923412598

5-84.886032011738

8-123.89224123816

12-241.1744486742

Ph n 0- 117.3102110213979

1 - 36.780618273173

3-56.172725542446

5-84.682833821618

8-123.17324114886

12-241.07364850150

Ph n lc nga thai 0- 17.24344344497

1 - 33.43918254106

3-54.654113663687

5-83.561119773076

8-123.581227892264

12-241.6114439331120

So vi caffeine th theobromine kch thch h thn kinh trung ng c yu hn nhng tc dng li niu th tt hn rt nhiu. Chnh v vy, trong thc t ngi ta s dng cc ch phm ca theobromine v cc dng thc phm cha theobromine nh mt cht cha mt s" bnh. V d nh mui ca theobromine-sodiumsalicilate c dng tng cng tc dng co bp ca mch mu trong tim v kch thch thn nhm lm li tiu tinVic tiu th trung bnh caffeine ngi dao ng t 80 - 400mg/ngy. Vi lng tiu th nh vy lm cho hm lng caffeine trong huyt tng dao ng t 5 - 20|aM. Theo s. Gokulakrishnan, ch vi liu lng ca caffeine nh trn cng nh hng n gic ng (c v cht lng v s lng) [25].Theo mt vi nghin cu mi, caffeine c th gy ra s t bin, s c ch vic sa cha ADN v c ch enzyme adenosine monophosphodiesterase. Ngoi ra, trong qu trnh mang thai n gy ra cc d tt ca bo thai. Caffeine cng gy ra cc chng long xng, lm gim s m c ca nguyn liu [48].1.3. Catechin1.3.1 Gii thiu chung v catechinCHNG 1: TNG QUAN

CHNG 1: TNG QUAN

Catechin trong ch l mt hn hp gm nhiu loi khc nhau nh: catechin, epicatechin, epigallo catechin, gallocatechin, epicatechin gallate, epigallocatechin gallate, gallocatechin gallate. th rn, cc catechin l cht kt tinh hoc v nh hnh, tan trong nc, khng mu, v c v cht ng mc khc nhau. Chng d dng b oxi ha v to hp cht phc tp vi nhiu cht khc nhau nh methylxanthine. Epigallocatechin gallate l thnh phn polyphenol ch yu trong ch, chim khong 12% khi lng cht kh ca l ch [11.- 16

- 15

Bng 1-10: Thnh phn t hp cc cht tanin trong l ch ti [ 1 ]:Tn chtHm lng (% tng lng polyphenol)(% tng lng catechin)

Bp ba l (Lin X)Ch xanhBp ba l (Vit Nam)

L-epicatechin1.334.45.54

D,L-catechin0.401.7-

L-epigallo catechin12.0016.016.09

D,L-gallocatechin2.007.99.98

L-epicatechin gallate18.1010.312.73

L-epigallocatechin gallate58.1049.155.64

L-gallocatechin gallate Cc cht mu vng hc Flavonols v cc cht mu khc1.406.5

Tng s"98.6095.999.98

CHNG 1: TNG QUAN

CHNG 1: TNG QUAN

Cng thc cu to ca cc loi catechin quan trng trong ch:

Hnh 1-3: Cng thc cu to ca cc catechin: (a) EGCG; (b) ECG; (c) EC; (d) catchinCatechin l thnh phn ha hc quan trng nht trong l ch ti cng nh trong cc loi ch thnh phm. N quyt nh n cht lng ca ch. Catechin khng nhng to nn v v dc tnh ca ch m cn tham gia vo cc qu trnh bin i ha hc di tc dng ca cc enzyme oxi ha kh to ra hng v v mu sc nc pha c trng cho mi loi ch. Trong cng ngh sn xut ch xanh, ngay ti qu trnh u tin ca cng ngh c qu trnh dit men. Ti giai on ny ta tin hnh v hot hon ton enzyme polyphenoloxydase, nn catechin trong ch hu nh khng b bin i ng k trong sut cc qu trnh ch bin cn li. Chnh nh cc enzyme b v hot hon ton trong qu trnh dit men, nn trong giai on v ch v sao ch, cc catechin b oxi ha v bin i ch yu di tc dng ca nhit v m. Do , sn phm thu c l ch xanh c v cht mnh, c hu v ngt, c mu nc xanh ti hoc xanh vng v c hng thm c trng. Ni chung, hm lng polyphenol trong l ch ti hoc trong ch xanh thnh phm cng cao th cht lng ca ch xanh cng tt. Bn cnh , chng ta cng cn ch n hm lng ca cc hp cht vi t l thch ng mi c li v to nn c cht lng ton din ca ch [ 1 ].1.3.2 Nhng tc ng sinh l ca catechin vi c thi vi c th con ngi, catechin c nh hng tt n c th. Trc ht, catechin c tc dng gii kht, cha mt s" bnh ng rut v c tc dng cm mu. Nhm cht catechin trong thnh phn polyphenol ca ch cn c kh nng lm tng tnh co dn, nng cao nng lc khng ca thnh vi huyt ca c th ng vt, nh lm gim nhanh hin tng sung huyt ca c th. Hn na, catechin c tc dng tng cng s tch ly v ng ha vitamin c trong c th, gip phng nga bnh hoi huyt. Do , catechin c hot tnh ca mt vitamin p v hot tnh ca n cao hn c hot tnh ca vitamin p ly t la mch. Ch phm catechin ch c dng nh mt ngun cung cp vitamin p cho c th ngi. Theo ti liu ca Lin X th ngi ln cn 200mg v tr em cn lOOmg trong mt ngy. Hin nay, ngi ta thit lp c phng php thu nhn ch phm catechin t l ch gi v c dng cha cc bnh c hin tng chy mu trong c th do nhim c cc mao qun, cha bnh huyt p cao v t thp tim. c bit, theo kt qunghin cu ca Lin X, ch phm catechin c tinh khit cao (82%) c tc dng cha bnh nhim phng x v c hot tnh cao ch thua km natri gallate.Cc th nghim tin hnh trn chut bch cho thy rng, catechin ch c th tng cng mnh m sc khng ca vi huyt qun ca chut bch, hn na cn nng cao c nng lc ca cc m tng tr vitamin c. Cc kt qu th nghim i vi bnh nhn lm sng cng cho thy, khi nng lc khng ca vi huyt qun gim xung, ch cn cho bnh nhn mi ngy ung 100 - 200 mg catechin th s nhanh chng lm cho tnh thm thu ca thnh vi huyt qun ca bnh nhn t n trng thi bnh thng [1].1.4. Dc tnh ca cy chC th ni, ch l mt thc ung truyn thng ca ngi Trung Hoa v Nht Bn. N c s dng rng ri bi nhng dc tnh ca n. V ngy nay, ch c tiu th ph bin trn th gii. Trong thnh phn ha hc ca ch cha nhiu catechin v caffeine. Do , theo cc mc 1.3 v 1.4 (c trnh by trnh by trn) th ch l mt thc phm rt c li cho sc kho. Theo nhiu nghin cu ca cc nh khoa hc v y khoa, ch c tc dng cha kh nhiu bnh: cha ung th v chng tia cc tm, bnh tiu chy v kit l, chng khng tiu, cm nhim h tit niu, cha tr vim gan dng hong m cp tnh, cha au bng do giun a v au rng, cha chng cao cholesterol trong mu, cha chng bo ph, cha chng suyn, cha phong nhit au u, cha cho ngi ang b bnh lao, cha cm, khng khun, bo v rng, gim nguy c tiu ng.1.5. Muc ch ca vc kh caffeine trong l chNh trnh by mc 1.2, 1.3 v 1.4, r rng ch l mt sn phm rt tt cho sc kho chng ta. Ch c nhiu tc dng gip gim cc nguy c bnh tt. Tuy nhin, ch c cha caffeine, l cht tc dng ln c th chng ta theo hai hng. Caffeine c nhng tc dng c li, gip tng cng s tnh to v minh mn ca b c, chng li s mt mi... Tuy nhin, vi mt s" ngi, caffeine li gy ra nhng tc dng khng tt. Caffeine gy ra nhng cn au u, nhng chng mt ng, tng kh nng hot ng ca c v tng lng hot ng ca tim, gy ra chng cao huyt p... Do , vi mt s" ngi th nhu cu v mt sn phm khng cha hoc cha mt lng rt nh caffeine l rt cao. y l nhm ngi c nhu cu s dng sn phm ch nh mt dc liu, mt thc ung gii khtdo nhng c tnh sinh hc quan trng ca chng, nhng khng mun nhng tc ng c hi i vi sc kho, c bit l nhng ngi c bnh cao huyt p, c chng mt ng.1.6. Cng ngh kh caffeine [45]Nguyn tc ca cc phng php kh caffeine l da trn s ha tan chn lc ca caffeine trong cc loi dung mi khc nhau, t trch ly caffeine khi nguyn liu.Hin nay, trn th gii c bn phng php kh caffeine trong ch xanh ni ring v trong cc loi thc phm dng rn cha caffeine ni chung. Cc phng php ny c phn loi da trn vic la chn dung mi hay tc nhn cho qu trnh kh caffeine. Nh chng ta bit, caffeine l cht phn cc yu, nn cn chn mt dung mi hay tc nhn thch hp cho qu trnh tch caffeine khi l ch. Hin nay, cc phng php c s dng l: phng php s dng dung mi hu c, phng php dng nc, phng php Swiss water, phng php dng C2 siu ti hn.1.6.1 Phng php kh caffeine bng dung mi1.6.1.1 Nguyn tcCHNG 1: TNG QUAN

CHNG 1: TNG QUAN

Phng php ny da trn nguyn tc s dng mt dung mi hu c cho qu trnh kh caffeine. Theo Katx (1987), nhng yu cu khi chn mt dung mi cho qu trnh kh caffeine l kh nng ha tan chn lc vi caffeine, tnh an ton, gi c, d dng tch dung mi ra khi nguyn liu, d thu hi dung mi, khng c v an ton vi mi trng. Vi nhng yu cu nh vy th nhng dung mi thng c dng l Methylene chloride v ethyl acetate. Trong , hin nay, methylene chloride c s dng nhiu nht do c tnh chn lc cao tch caffeine m khng tch cc cu t hng v cc cht to v trong l ch [45]. Bn cnh , methylene chloride bc hi nhit 40c trong khi l ch ti thng c sy kh cc giai on ch bin v sau nhit t 80 - 90c. iu ny lm cho trong sn phm ch cn li mt lng nh caffeine. Theo FDA (hip hi Thc phm v Dc phm ca M), qu trnh kh caffeine bng methylene chloride cho kt qu kh tt. Hu ht cc sn phm kh caffeine th c hm lng methylene chloride cn li thp hn 0. lppm.- 20

- 19

1.6.1.2 Phng php thc hinQuy trnh kh caffeine trong ch bng dung mi c bt u bng qu trnh dit men, lm sch v lm m l ch. Cc qu trnh ny c thc hin bng cch xng hi vo l ch. L ch ti c lm ho v chuyn vo thit b trch ly. Dng hi nhit 110c c dn truyn vo thit b trch ly t pha di ca thit b. L ch sau khi c lm m vi dng hi s c b mt l d thm thu hn v caffeine c trch ly d dng khi dung mi tip xc vi caffeine. Dng hi i qua thit b trch ly trong khong 0.5 gi. Dng hi ra khi thit b c thu hi, ngng t v cha trong cc thit b cha. Sau qu trnh xng hi, dung mi sch c bm vo thit b trch ly vi t l 4:1. Qu trnh kh caffeine c thc hin trong khong thi gian 10 pht. Sau qu trnh kh caffeine, dung mi c tho ra khi thit b, cn l ch sau c em loi dung mi v ta c sn phm l l ch ti kh caffeine [45].

Hnh 1-4: Quy trnh kh caffeine bng dung mi So vi cc dung mi khc, methylene chloride c hiu qu cao trong qu trnh kh caffeine. Nhng methylene chloride tim n nhiu nguy c khi c s c xy ra nh: gy ra s chng mt, au u nu ht vo nng cao. Do , bn cnh methylene chloride, ta c th s dng mt s" dung mi khc nh ethyl acetate. Ethyl acetate c th tch caffeine t l ch mt cch hiu qu, nhng c nhc im l n trch ly c nhng thnh phn ha hc c li trong ch. Theo cc nghin cu qu trnh kh caffeine ch xanh vi ethyl acetate th mt lng khong 30% EGCG v nhng hp cht chng oxi ha khc b trch ly cng vi caffeine [10]. Hn na, ethyl acetate cng l mt cht c tuy mc gy c thp hn methylene chloride.Bn cnh , mc d chloroform l mt cht c, nhng trong mc ch ca phng th nghim, choloroform vn c s dng nh l mt dung mi tt nht trch ly caffeine [10].Phng php kh caffeine bng dung mi hu c cho thy nhng u im nh r tin, thit b n gin, d thc hin, tnh ha tan chn lc cao vi caffeine. Chnh nhng u im ny m trong nhng nm trc y, phng php kh caffeine bng dung mi hu c c p dng rng ri v ph hp vi nhng c s sn xut nh. Tuy nhin, ngy nay do nhng nhc im ca phng php nh nh hng n tnh an ton ca nguyn liu, nh hng n mi trng v kh thu hi dung mi nn phng php ny ngy cng t c s dng v c thay th bng cc phng php khc hiu qu hn.1.6.2 Phng php kh caffeine bng nc1.6.2.1 Nguyn tcCHNG 1: TNG QUAN

CHNG 1: TNG QUAN

CHNG 1: TNG QUAN

Phng php ny dng nc nng trch ly caffeine t l ch. y l phng php c s dng rng ri v khng s dng bt k mt dung mi no gy c n cht lng ca sn phm cng nh nh hng n mi trng. Phng php ny da trn kh nng ha tan caffeine trong nc trch ly caffeine khi l ch v to ra sn phm ch xanh t cha caffeine. Tuy nhin, phng php ny khng c tnh chn lc cao i vi caffeine, mc tn tht ca nhng thnh phn ha tan, cc cht thm v hng v trong l ch cao [45].-21

- 22

- #

Hnh 1-5: Quy trnh kh caffeine bng nc.Qu trnh khcaffeine bng nc nng c thc hin theo qui trnhnh sau: trcCHNG 1: TNG QUAN 1.6.2.2 Phng php thc hin

ht lch ti clm sch nhm tch ht cc tp cht, loi b cc lch h hng,khng t yu cu ra khi nguyn liu ban u. Sau , l ch c ngm trong nc nng trong mt khong thi gian nht nh thc hin qu trnh tch kh caffeine khi l ch. Qu trnh ngm trong nc nng y c thc hin nhm hai mc ch: dit men v kh caffeine. Sau mt khong thi gian nht nh, l ch c ly ra khi thit b trch ly. Hn hp nc bao gm rt nhiu cht kh ha tan, trong c c caffeine c em tch caffeine bng phng php trch ly lng-lng vi mt dung mi khc. Dch nc cn li cha cc cht kh ha tan, cc polyphenol v cc cht thm c cho ti hp th vo trong l ch.1.6.2.3 Cc yu t' nh hngQu trnh khcaffeine bng nc chu nh hng ca cc yu t" nh: nguyn liu(kchthc, cu trcnguyn liu, thnh phn ha hc), cc thng s" cngngh ca qutrnh (nhit , thi gian, t l dung mi/ c cht), thit b v phng php thc hin.nh hng ca nguyn liu n qu trnh kh caffeine: Kch thc ca l ch: khi l ch c ct, nghin nh th hiu sut kh caffeine tng ln do cu trc t bo b ph v, lm tng din tch tip xc ca l vi nc.

m ca nguyn liu: nguyn liu cng m th t bo l cng gin v hiu sut kh cng cao.

CHNG 1 : TNG QUAN

CHNG 1 : TNG QUAN

nh hng ca cu trc l ch ln qu trnh kh caffeine: khi tin hnh kh caffeine trn l ch v v ch kh th tn tht catechin ca qu trnh kh l rt ln [35]. iu ny c gii thch nh sau: qu trnh v v sy c lm ph hy t bo l, lm tht thot lng catechin. Khi l ch c v, t bo l v mng t bo b tn thng v ph hu, lm cho catechin v caffeine thot ra b mt l. Trong trng hp ny, catechin v caffeine d dng ha tan vo trong nc. Mng t bo l cng b ph hy khi sy kh sau v do , lm tng kh nng khuch tn ca catechin vo trong nc. Do , mc tn tht catechin tng tng dn theo th t l ch nguyn vn < l ch v < ch kh. Nh vy, s h hng ca l l mt yu t" quan trng nh hng n kh nng trch ly ca catechin tng [35].- 23

-28

-29

Bng 1-11: nh hng ca cu trc l n qu trnh kh caffeine [35]:Mu chCaffeine (mg/g cht kh)EGCG (mg/g cht kh)Catechin tng (mg/g cht kh)

L ch bnh thng3.450.2113.6

L ch v1.231.769.1

Ch kh2.317.043.5

nh hng ca cc thng s" k thut: nh hng ca nhit n qu trnh kh caffeine: khi nhit tng th hiu sut ca qu trnh kh caffeine l ch cng tng theo. C th ni, nhit l thng s" quan trng nht nh hng n phng php kh caffeine bng nc nng ca l ch. Theo H Liang et al, nhit ca qu trnh kh khng nn nh hn 100c [35]. nh hng ca thi gian trch ly v t l ch/nc: khi tng thi gian trch ly th hiu sut kh caffeine tng theo. Theo H Liang et al, thi gian trch ly v t l ch/nc th t nh hng n qu trnh kh caffeine [35]. Hiu sut kh caffeine trong thi gian 3-5 pht gn nh tng ng vi nhau, v cao hn r rt trong trng hp kh caffeine trong thi gian 1 pht. Do , theo H Liang et al, thi gian kh caffeine khng nn thp hn 3 pht v t l ch:nc khng nn thp hn 1:20 ( w/w) [35]. nh hng ca qu trnh kh caffeine n hm lng catechin tng: hm lng catechin tng ca l ch kh caffeine v nguyn liu ban u th khc nhau khng ln. Kt qu cho thy: hn 95% catechin tng c gi li sau qu trnh x l. Hn na, hai thnh phn chnh ca catechin trong l ch l EGCG v EGC th khng thay i trong sut qu trnh kh caffeine bng nc nng.So vi phng php kh caffeine bng dung mi hu c th phng php kh caffeine bng nc nng c mt s" u, nhc im sau: v u im, r rng phng php ny cho sn phm c an ton cao, hiu sut kh caffeine cao, t tn tht cht kh do c qu trnh ti hp thu, t tn tht nhit dokhng c qu trnh xng hi. Do t s dng nhit trong sut qu trnh kh caffeine nn sn phm t c s bin i v sn phm c cht lng cao hn [45]. Tuy vy, phng php kh caffeine bng nc nng cng cho thy nhng nhc im nh s tht thot cc hp cht catechin, cc cht ha tan v mt s" cht thm trong ch.1.6.3 Phng php Swiss water1.6.3.1 Nguyn tcPhng php Swiss water c thc hin da trn nguyn tc l dng than hot tnh kh caffeine trong dch trch to ra dch trch cn bng, t , to ra c mt dch trch bo ha cht kh nhng khng cha hoc cha rt t caffeine. Sau , dch trch ny s c dng kh caffeine thay v dng nc thng [45].1.6.3.2 Phng php thc hinPhng php Swiss water l mt dng ca phng php kh caffeine bng nc. Phng php ny da trn l thuyt v s ha tan ca caffeine trong nc bo ha cht kh. Vi dch nc bo ha cht kh ha tan (khng tnh caffeine), th dch nc s khng ha tan thm cc thnh phn khc trong l ch, c bit l catechin v cc cht ha tan m ch ha tan caffeine. L ch lc u c trch ly bng nc nng. Khi , cc thnh phn cht ha tan v caffeine ha vo trong nc v to ra mt dch trch cn bng v cc cht ha tan, cc catechin, caffeine. Tip theo, caffeine c ly ra khi dch trch bng cch cho qua ct than hot tnh. Dng nc ra khi ct hp ph l dng nc bo ha cc cht ha tan nhng khng cha caffeine. Dng nc ny c c tnh l khng th trch ly thm cc cht ha tan v cc catechin na nhng vn cn kh nng trch ly caffeine. iu ny cho php cc m tip theo c hiu sut kh rt cao m vn duy tr c cc cht ha tan trong l ch nguyn liu. Dng nc trch ly sau li c cho qua ct than hot tnh bo ha cht kh nhm loi caffeine ra khi dch trch. Qu trnh c th tip tc cho cc ln trch tip theo [45].So vi phng php kh caffeine bng nc nng bnh thng th phng php Swiss water pht huy nhng u im ca phng php trn. Bn cnh , n khc phc nhng nhc im ca phng php dng nc nng nh: phng php ny trnh c vic tn tht cc cht kh ha tan, cc catechin c trong nguyn liu l ch v to ra sn phm c hng v tt hn. Phng php ny c xem l kh hiu qu cho qu trnh kh caffeine trong l ch ti.Ngy nay, vic s dng mng lc membrane hay lc than hot tnh trong vic tch caffeine ra khi dch trch tr nn khng cn hiu qu kinh t v thn thin vi mi trng [48]. Mt hng pht trin mi trong vic tch caffeine ra khi dch trch l vic s dng cng ngh enzyme v vi sinh vt [481- Vi sinh vt c cho vo dch kh caffeine trong dch trch Swiss water v to ra mt dch trch bo ha cht kh ha tan nhng vn khng cha hoc cha rt t caffeine. Mt s"ging vi sinh vt c nghin cu cho qu trnh ny nh peudomunas, aspergillus, penicillium....Qu trnh kh caffeine sinh vt nhn nguyn thy v sinh vt nhn tht v ang c nghin cu. Con ng tng th kh caffeine vi sinh vt c th hin nh sau:[25]Catlne I

Con ng demethyl ha [Demethylase enzymes (in mammals CYP450Con ng oxi ha [Xanthine oxidase enzyme]

Hnh 1-6: M hnh tng th ca qu trnh kh caffeine bng vi sinh vt [25]. sinh vt nhn tht: i vi nhiu ging cy trng, vic kh caffeine xy ra trong sut qu trnh demethyl v sn phm cui cng to thnh l xanthine. Phn ng demethyl c xc tc bi nhng enzyme demethylase nh N-l demethylase, N-7 demethylase v N-3 demethylase. Sau , xanthine c chuyn thnh C2 v NH3 bi s ph hy vng purine. Tri li, mt s" ging, con ng kh caffeine ng vt l kt qu ca s hnh thnh methyl xanthines v methyl uric acids bi nhng enzyme cytochrome P450 nh CYP1A2, CYP3A4, CYP2E1 xanthine oxidase v N-acetyl transferase. Nhng xanthine ny c methyl ha v nhng acid uric hnh thnh trong qu trnh kh caffeine c bi tit ra ngoi c th [251- Cn i vi nm, sn phm u tin ca qu trnh kh caffeine c tm thy l theophyline. Nhng xanthine dimethyl v monomethyl khc c to thnh nh mt sn phm ca qu trnh kh caffeine. Tuy nhin, con ng phn gii ca cc xanthine nm cha c bit mt cch r rng [251- sinh vt nhn nguyn thy, con ng kh caffeine cng cha c tm hiu y . vi khun (tiu biu l ging Pseudomononas), caffeine c chuyn thnh theobromine v paraxanthine bi enzyme demethylase. Vic demethyl to ra cc xanthine vi 7-methylxanthine nh l mt sn phm trung gian. Sau , cc xanthine, mono v dimethyl xanthine b oxi ha thnh acid uric v i vo con ng phn hy purin. Serratia marcescens, con ng phn hy th tng t nh Pseudomonas sp, ngoi tr s hnh thnh sn phm trung gian methyl uric acid [25].Trc y, vic kh caffeine bng vi sinh vt cha c nghin cu nhiu v caffeine vn c bit n nh mt cht c vi vi khun. Trong mi trng sinh sng ca vi sinh vt, nng caffeine ln hn 2.5mg/ml s c ch s pht trin ca mt s" loi vi khun. V vy, khi caffeine thng c s dng nh mt cht chng vi sinh vt ging nh chloramphenicol. Nhng nghin cu v vic kh caffeine trong cc dng thc phm khc nhau bng vi sinh vt ch thc s c nghin cu k t nm 1970. V k t n nay, mt vi thnh tu t c trong vic s dng caffeine nh l mt c cht cho s pht trin ca vi sinh vt. vi vi khun, nhng loi c kh nng kh caffeinec bit n l Pseudomonas v Serratia genus. Fe2+ c xem l co-factor, n xc tc cho s to thnh enzyme demethylase. Chnh v vy, s c mt ca Fe2+ (0.04%) trong mi trng lm cho vic kh theobromine ca Pseudomonas tng gp 10 ln [25]. Cn i vi cc loi nm mc, kh nng s dng caffeine nh mt c cht ca qu trnh trao i cht cng c ghi nhn. Vic kh caffeine c thc hin mt vi loi nm nh Stemphylium sp, Pnicillium sp v aspergillus sp. A tamari, A niger, A fumigaus v p commune v kt qu cho thy s pht trin ca cc loi khi s dng caffeine nh l ngun cung cp nitrogen. A tamar v p commune th cho thy c kh nng kh caffeine tt (60%) trong khi mt vi loi khc l thp hn 20% [25].Tc kh caffeine mt s" loi vi sinh vt quan trng c cho bng 1-12.Bng 1-12; So snh tc kh caffeine nhng loi vi sinh vt khc nhau [251Vi sinh vtNng caffeine ban u (g/l)Ngun (g/l)Qu trnh kh caffeine (%)TC kh caffeine (g/gi)

Klebsiella v0.5Glucose (1)100% trong 10h0.05

Rhodococcus

Serratia marcescens0.6-100% trong 72h0.008

Stemphyllium sp.0.19Sucrose (30.1)100% trong 29h0.0053

Pseudomonas putida5-95% trong 50h0.095

Aspergillus tamarii1.2Sucrose (28.4)67% trong 48h0.0536

Pnicillium commune1.2Sucrose (28.4)61 % trong 48h0.0521

Nh bng trn ta thy, vic kh caffeine vi khun th tt hn nm mc. V caffeine cng l cht c vi vi sinh vt nn nng caffeine ban u trong qu trnh ln men c ngha quyt nh. nm mc, s tn ti ca cc ngun nitrogen bn ngoi s c ch hon ton qu trnh kh caffeine. S tn ti ca urea v ammonium sulphate th ngn cn qu trnh kh caffeine ca A. niger v Pnicillium verrucosum. Tuy nhin, tri vi nm mc, s tn ti ca cc ngun nitrogen hu c bn ngoi th khng ngn cn qu trnh kh caffeine vi khun. pH ban u ca mi trng cng l mt thng s" quan trng nh hng n qu trnh kh caffeine. pH ca mi trng nn c tnh axit (4 3.2) i vi Penicillium roqueforti v l baz i vi Aspergillus sp.So vi phng php Swiss water kt hp vi than hot tnh trong vic kh caffeine th phng php Swiss water kt hp vi vi sinh vt cho kt qu kh caffeine cao hn v hon ton sch vi mi trng do khng dng bt c dung mi no [481- Trong phng php dng than hot tnh th ta phi s dng dung mi trong vic ra gii caffeine khi than hot tnh cn phng php ny th hon ton khng dng bt c mt dung mi no.1.6.4 Phng php kh caffeine bng C2 siu ti hn1.6.4.1 Nguyn tcPhng php ny da trn nguyn tc: dng CO: trng thi siu ti hn, mt trng thi trung gian gia trng thi lng v trng thi kh, kh caffeine trong l ch ti. Phng php kh caffeine bng CO2 siu ti hn c xem l mt phng php rt thn thin vi mi trng.1.6.4.2 Phng php thc hinPhng php kh caffeine bng CO2 c thc hin nh sau: kh CO2 c nn p sut cao v gia nhit ln nhit cao. Khi , CO2 chuyn sang trng thi siu ti hn. trng thi siu ti hn, CO2 tr thnh mt dung mi c kh nng ha tan chn lc mt s" cht, c bit l caffeine. vi CO2, gi tr nhit ti hn l 31.3c v p sut ti hn l 80atm. trng thi siu ti hn, CO2 va c nhng tnh cht nh mt cht lng va c nhng tnh cht ca mt cht kh [45].Bng 1-13: Tnh cht ca CO2 siu ti hn:Trng thi ca CO2T trng (g/cm3)Kh nng khuch tn (cm2/s) nht ng hc (g/cm.s)

Kh1 X 10'31 X 10 11 X 10 4

Lng1.05 X 1061 X 102

Lng siu ti hn3 X 10'11 X 10'31 X 10'4

M hnh kh caffeine bng C2 siu ti hn (SCF): [27]

Hnh 1-7: M hnh kh caffeine bng SCF: 1, bnh cha CO2; 2, thit tng cng in; 3, thit bi gia nhit ;4, bm CO2; 5, thit b iu khin; 6, bnh cha ng dung mi; 7,bm ng dung mi; 8, thit b gia nhit; 9, bm tun hon; 10, thit b trch ly; 11, thit b tch;12, thit b tch 2; V, van ; V2, van 2; BPR Qu trnh bt u vi vic chun b l ch, tch tp cht v tch m. m ca nguyn liu chun b cho qu trnh kh caffeine nn l 30 - 35%. Nguyn liu c t vo mt thit b trch ly. Mt h thng bm bm kh CO2 qua thit b gia nhit n nhit 40 - 80c v tng p sut ln khong 120 - 180bar. Dng CO2 siu ti hn khi s di chuyn vo trong thit b trch ly. Ti thit b trch ly, caffeine c trch ly bi CO2 siu ti hn v dng CO2 giu caffeine ra khi thit b trch ly [451- Sau , caffeine c tch khi CO2 siu ti hn v thu gom li dng tinh khit. Hin nay, c hai cch tch caffeine t CO2 siu ti hn. Cch th nht l ha lng CO2 bng vic h nhit ca CO2 siu ti hn n khong 20 - 25c. Khi CO2 chuyn t trng thi siu ti hn sang trng thi lng v khng cn kh nng ha tan caffeine. Nh vy, caffeine c tch ra khi dng CO2 v thu nhn. Cch th hai tch caffeine t CO2 siu ti hn l: bng vic h thp p sut. p sut c h thp n khong 58 - 65at. p sut thp, caffeine cng c tch ra khi CO2 do lc ny CO2 chuyn t trng thi siu ti hn sang trng thi kh bnh thng [45].1.6.4.3 Cc yu t nh hdngQu trnh kh caffeine bng CO2 siu ti hn chu nh hng ca cc yu t" nh: nguycn liu (kch thc, cu trc nguycn liu, thnh phn ha hc), cc thng s" cng ngh ca qu trnh (nhit , p sut, lu lng CO2 siu ti hn, thi gian lu, t l CO2/ l ch), cht ng dung mi, thit b v phng php thc hin.nh hng ca nguyn liu: Kch thc nguyn liu: Kch thc l ch l mt thng s" quan trng trong qu trnh trch ly. Kch thc ca l ch nh hng n s khuch tn ca cc cht tan vo dung mi v ngc li. N quyt nh n din tch b mt tip xc ca mu v dung mi. So vi mu khng c ct nh th l ch xanh trong nghin cu ca Hyong Seok Park [27] c ct n kch thc 235.5|im cho kt qu kh caffeine nhiu hn 15 - 17% da trn thnh phn ban u ca caffeine trong l ch ti [27]. iu ny c gii thch l do khi kch thc ca cc phn t l ch tng ln th khong cch m cc phn t cht chit phi di chuyn t bn trong n b mt tip xc tng. iu ny lm cho kh nng trch ly thp hn. Hn na, khi kch thc ca cc phn t l ch nh th din tch b mt tip xc ln v cho php cc cht tan d dng tip xc vi dung mi. Tuy nhin, khi ta ct l ch thnh nhng phn c kch thc nh hn th chnh cc phn t catechin cng d dng b trch ly v iu ny s nh hng n hm lng catechin cn st li trong ch xanh thnh phm [27].Bng 1-14: nh hng ca kch thc nguyn liu n qu trnh kh caffeine:Kch thc nguyn liu% cht chit c tch da trn thnh phn ban u (%, w/w)

CaffeineEGCGEGCECGEC

236.5 |im97.370.881.376.647.8

543.7|im95.673.680.879.038.1

5.5 - 17.4 nim80.647.564.250.931.7

(Qu trnh c thc hin 300bar v 70c, tc dng C2 l 11.5g/pht trong 120 pht, ethanol 95% c s dng nh mt cht ng dung mi vi lng 4.6g/100g CO2).

Cu trc nguyn liu. Thnh phn ha hc: m, thnh phn cc cht ha tan...nh hng ca cc thng s"cng ngh:Bng 1-15: nh hng ca nhit v p sut n qu trnh kh caffeine [281iu kin trch lyPhn trm cht tch so vi thnh phn banu (%, w/w)

Nhit (C)p sut (bar)CaffeineEGCGEGCECGEC

5030062.653.282.460.633.3

6030081.657.482.666.738.5

7030092.864.278.272.524.0

8030092.869.582.371.250.0

7015077.750.562.054.620.0

7020090.566.772.372.635.5

7025092.169.075.678.037.6

(Qu trnh kh caffeine c tin hnh vi l ch xanh vi kch thc 543.7|im, tc

dng C2 l 8.5g/pht, thi gian 120 pht, ethanol 95% (v/v) c s dng nh cht ng dung mi 4.6g/100g CO2). nh hng ca nhit n qu trnh kh caffeine: khi p sut trch ly c gi 300bar, hiu qu kh caffeine tng khi nhit tng. Hiu sut kh caffeine l cao nht nhit 80c (92.8%). Tuy nhin s tht thot cc catechin l rt ln (khong trn 70%). iu ny c gii thch: khi nhit tng th tc chuyn ng ca cc phn t tng. Cc phn t chuyn ng nhanh hn v d dng di chuyn vo trong dung mi [28]. nh hng ca p sut n hiu sut kh caffeine: khi nhit khng i, p sut tng lm cho kh nng tch caffeine cng tng theo. iu ny c cho l do s tng kh nng ha tan ca SC-CO2 bi vic tng mt phn t trong dng dung mi CO2 siu ti hn. Khi p sut trch ly trn 200bar nhit 70wc, hn 90% caffeine v trn 60% EGCG c tch [281- nh hng ca thi gian trch ly n qu trnh kh caffeine: thi gian trch ly tng th hiu sut kh caffeine cng tng theo. Nhng i chung vi vn l t l catechin b trch ly khi l ch cng tng theo.

*7T7.1030160240320400480Extractor! Time (min/

Hnh 1-8: nh hng ca thi gian trch ly n qu trnh kh caffeine.nh hng ca cht ng dung mi: nh hng ca nc nh mt cht ng dung mi n hot tnh ha tan ca C2 siu ti hn c nghin cu. y, cht ng dung mi c hiu l cht thm vo dung mi chnh (CO2 siu ti hn) to thnh mt h dung mi nhm tng cng kh nng ha tan caffeine ca CO2 siu ti hn. Nh chng ta u bit, SCF l mt cht khng phn cc hay phn cc yu nn SCF thng c s dng trong vic trch ly cc cht t hoc khng phn cc. Do , kh nng ha tan ca caffeine trong CO2 siu ti hn l rt thp. Chnh v vy, vic b sung thm cht phn cc vo SC-CO2 nh mt cht ng dung mi tng phn cc ca SC- CO2 v to thnh mt hn hp gm SC-CO2 v cht ng dung mi. Hn hp ny c s dng trch caffeine t l ch. Trong , nc v ethanol (95% v/v) c nh gi l cc ng dung mi tt m khng nh hng n an ton thc phm[27]. Khi tng hm lng nc trong h dung mi th c thnh phn caffeine v catechin trong l ch cui cng u gim. Kt qu ny cho thy rng: vic tng phn cc ca SC-CO2 thc y qu trnh trch ly caffeine v catechin khi l ch [27].Bng 1-16: nh hng ca cht ng dung mi n qu trnh kh caffeine [27]:ChtThnh phn dch trch

ngmg/g l ch xanh khmg/kg tng lng CO2 s dng

dungmi(g/100gC02)CaffeineEGCGEGCECGECCaffeineEGCGEGCECGEC

140.214385.626.34.8

23.719.211.93.4934.8178.3111329.9

36.246.237.96.11.057.5429.135257.35.5

416.972.154.111.90.6157669.450211132.

531.110074.615.73.5289930.66931465

628.763.155.012.05.4267585.751111250.

735.077.655.415.51.7326720.85141441

840.390.465.019.11.02.6374838.860417716.39.324.8

nh hng ca ethanol nh mt cht ng dung mi: cng tng t nh nc, ethanol lm tng phn cc ca SC-CO v kh nng trch ly caffeine ca SC- CO. Tuy nhin, so vi nc th ethanol cho hiu qu trch ly caffeine cao hn v s tn tht catechin th thp hn. iu ny c cho l: do ethanol t phn cc hn nc nn hn hp gm SC-CO2 v ethanol th t phn cc hn hn hp SC-CO2 v nc. V caffeine t phn cc hn catechin nn vi hn hp SC-CO2 v ethanol th c hiu qu trch ly caffeine tt hn v tn tht catechin thp hn [27]. Bn cnh , ha tan ca ethanol trong SC-CO2 th cao hn so vi nc, cho nn tng cng kh nng trch ly caffeine ca hn hp SC-CO2 v cht ng dung mi. Vvy, hn hp COi/ethanol c xem l mt h dung mi tt cho qu trnh kh caffeine trong l ch xanh [27]. nh hng ca tc dng C2 n kh nng kh caffeine ca CO2 siu ti hn: theo cc kt qu nghin cu, tng lng caffeine v catechin c tch khi l ch tng khi tng tc dng CO2. Khi tng tc dng lm cho thi gian lu ca CO2 trong thit b trch ly ngn v d dng li ko cc phn t cht tan theo dng SC- CO2 [27]. Hn na, tng cng s trch ly cc cht chit, vic s dng tc dng CO2 qu nhanh trong thit b trch ly l khng th bi s tn ti ca lp mng ngoi bao quanh l ch [27].Bng 1-17: nh hng ca tc dng CO2 n qu trnh kh caffeine [27]:iu kin trch lyPhn trm cht tch so vi thnh phn ban u (%, w/w)Kh nng trch ly ca CO2 siu ti hn (mg/kg CO2)

TcThi

gian

dnglu

CaffeineEGCGEGCECGECCaffeineEGCGEGCECGEC

C02ca

(g/ph)C02

(pht)

5.512.9080.146.067.746.08.9475.1958.4791.179.7.7

8.58.3492.864.278.272.524.356.4865.09813.

11.56.1795.673.680.879.00271.3732.8591.183.4

14.54.8993.277.086.580.938.209.8608.15415.

1451.147.7

51.7716.

2383.119.8

69

So vi phng php kh caffeine bng dung mi v nc nng th phng php kh caffeine bng CO2 siu ti hn c nhng u im sau: ta c th iu khin c khnng ha tan ca caffeine trong C2 siu ti hn (SCF) bng cch iu chnh nhit v p sut; tnh tr ca CO2; tnh chn lc ca CO2 i vi caffeine cao; m bo c hng v ca sn phm; tch dung mi d dng; d thu hi caffeine; sn phm c tinh khit cao v khng gy c nh phng php dng dung mi. Tuy nhin phng php ny tn ti kh nhiu nhc im so vi cc phng php khc: p sut vn hnh kh cao, gi thnh thit b cao; ha tan ca caffeine trong SCF thp v lng catechin b tn tht kh nhiu lm cho phng php ny t c s dng trong vic kh caffeine trong l ch. V vy cn phi c nhng nghin cu thm trong tng lai ci thin tnh chn lc caffeine ca qu trnh kh trc khi phng php ny c p dng i vi l ch tiCHNG 1: TNG QUAN

CHNG 1: TNG QUAN

[28].

- 36

- 35

CHNG 2. NGUYN LIU V PHNG PHP NGHIN CU2.1. Phng php nghin cu2.1.1 Mc ch nghin cuNghin cu vic kh caffeine trn l ch tinguyn liu bng phng php Swisswater, trong , chng ti kho st 2 qu trnh: Qu trnh kh caffeine bng nc bo ha phicaffeine. Qu trnh hp ph caffeine t dch trch bngthan hot tnh bo hachtkhhatan phi caffeine.Kho st nh hng ca cc thng s" cng ngh n qu trnh kh caffeine nh: nhit , t l cknc, thi gian.Kho st nh hng ca cc thng s" cng ngh n qu trnh hp ph caffeine ca than hot tnh bo ha cc cht kh ha tan phi caffeine.2.1.2 Ni dung nghin cu2.1.2.1 Kho st nguyn liu Mc ch:nh gi cht lng ca l ch ti v mc ng nht ca nguyn liu gia cc ln mua khc nhau.Cc i lng xc nh:Xc nh m, hm lng cht kh ha tan, caffeine, hm lng tro, lng tro ha tan trong nc v lng tro khng ha tan trong nc, hm lng polyphenol tng, ng kh.2.1.2.2 Kho st qu trnh trch l caffeine bng phng php Swiss water Kho st nh hng ca t l ch:nc n qu trnh kh caffeine:Kho st nh hng ca t l ch:ci n qu trnh kh caffeine. Trong , chng ti theo di cc thng s" sau: hiu sut kh caffeine, tn tht cht kh ha tan phi caffeine v s thay i lng polyphenol tng trong l ch.S" nghim thc: 3 nghim thc X 3 ln lp = 9 th nghim.Kho st nh hng ca nhit n qu trnh kh caffeine:Kho st nh hng ca nhit n qu trnh kh caffeine. Trong , chng ti theo di cc thng s" sau: hiu sut kh caffeine, tn tht cht kh ha tan phi caffeine v s thay i lng polyphenol tng trong l ch.S" nghim thc: 3 nghim thc X 3 ln lp = 9 th nghim.Kho st nh hng ca thi gian n qu trnh kh caffeine:Kho st nh hng ca thi gian trch ly n qu trnh kh caffeine. Trong , chng ti theo di cc thng s" sau: hiu sut kh caffeine, tn tht chtkhha tanphicaffeine v s thay i lng polyphenol tng trong l ch.S" nghim thc: 6 nghim thc X 3 ln lp = 18 th nghim.2.1.2.3 Kho st qu trnh hp phu caffeine t dich trch cn bngbngthanhoattnh bo ha cht kh ha tan phi caffeine Kho st nh hng ca nhit n qu trnh hp ph ca than hot tnh:Kho st nh hng ca nhit dch nc ban unqu trnhhpph cathan hot tnh bo ha. Trong , chng ti theo di cc thng s"sau: hiusuthp phcaffeine ca than hot tnh bo ha, tn tht cht kh ha tan phi caffeine v tn tht lng polyphenol tng.S nghim thc: 6 nghim thc X 3 ln lp = 18 th nghim.Kho st nh hng ca t l than hot tnh:Kho st nh hng ca t l than hot tnh n qu trnh hp ph ca than hot tnh bo ha. Trong , chng ti theo di cc thng s" sau:hiusut hpphcaffeineca qu trnh, tn tht cht kh ha tan phi caffeine v tnthtlng polyphenol tngtrong dch ch sau hp ph.S" nghim thc: 3 nghim thc X 3 ln lp = 9 th nghim.Kho st nh hng ca t l than hot tnh:Kho st nh hng ca thi gian n qu trnh hp ph ca than hot tnh bo ha. Trong , chng ti theo di cc thng s sau: hiu sut hp ph caffeine ca qu trnh, tn tht cht kh ha tan phi caffeine v tn tht lng polyphenol tng trong dch ch sau hp ph.S" nghim thc: 6 nghim thc X 3 ln lp = 18 th nghim.2.2. Nguyn liu2.2.1 L ch nguyn liuLoi ch bp c thu mua t Lm ng.2.2.2 Than hoat tnhLoi bt mn, xut x Trung Quc.2.2.3 Ha cht s dungDung dch acid acetic 99.5%, xut x: Trung Quc.FeC3, dng bt, xut x: Trung Quc.K3Fe(CN)6, dng bt, xut x: Trung Quc.HC1 m c, xut x: Trung Quc.Acid gallic, dng bt, xut x: Nht.Acid Phosphoric 85%, xut x: Trung Quc.NaOH, dng bt, xut x: Trung Quc.Bromothymol blue, dng bt, xut x: Trung Quc.DNS, dng bt, xut x: Merck.Formalin, dng lng, xut x: Vit Nam.Gum arabic, dng bt, xut x: Trung Quc.KNa Tartrat, dng bt, xut x: Trung Quc.Methanol (HPLC), dng lng, xut x: Merck.Phenolphtalein, dng bt, xut x: Trung Quc.Acetonitrile (HPLC), dng lng, xut x: Merck.Acetonitrile, dng lng, xut x: Trung Quc.KH2PO4, dng bt, xut x: Trung Quc.Caffeine, dng bt, xut x: Trung Quc.2.3. Cc thit bi dng trong nghin cu Thit b sy m hng ngoi SCALTEC_c.Cn phn tch 4 s"l SARTORIS_c, cn phn tch 1 s" l. L nung LENTON_Anh, bn nh siu m.CHNG 2: NGUYN LIU V PHNG PHP NGHIN cu

CHNG 2: NGUYN LIU V PHNG PHP NGHIN cu

CHNG 2: NGUYN LIU V PHNG PHP NGHIN cu

My phn tch HPLC, my quang ph so mu hng ngoi.- # -

-40-

-39-

H thng ct nc, h thng lc nc RO, my khuy t khng gia nhit.2.4. Quy trnh cng ngh kh caffeine

Hnh 2-1: Quy trnh cng ngh kh caffeine.

2.4.1 Quy trnh chiran bi dich trch cn bng cc cht kh ha tan trong l ch

La chn

Lng ch/ lOOml nc: 75g/100ml Nhit : 90c Thi gian: 20 phtNu

Lc

Hnh 2-2: Quy trnh to dch trch cn bng cht kh ha tan tng (so vi thnh phn cachng trong l ch ti.

2.4.2 Ouv trnh chun bi than hoat tnh bo ha khng cha caffeine

Than hot tnh (50g)

CHNG 2: NGUYN LIU V PHNG PHP NGHIN cu

CHNG 2: NGUYN LIU V PHNG PHP NGHIN cu

-42-

-43-

Ngm ln 1Ngm ln 2Ngm ln 3Dung dch SW1 (lOOOml)Dung dch SW1 (lOOOml)Dung dch SW1 (lOOOml)

Ngm ln 4Dung dch SW1 (lOOOml)

Hnh 2-4: Quy trnh to dch trch phi caffeine c nng cc cht kh ha tan cn bng vi thnh phn ca chng trong l ch ti.2.5. Cc phng php phn tch2.4.3 Ouv trnh chun bi dich trch cn bng cha t caffeine

Xc nh hm m (xem ph lc 1, mc 1).Xc nh hm lng tro tng (xem ph lc 1, mc 2).Xc nh hm lng tro tan v khng tan trong nc (xem ph lc 1, mc 3).Xc nh hm lng cht kh ha tan (xem ph lc 1, mc 4).Xc nh hm lng ng kh (xem ph lc 1, mc 5).Xc nh hm lng caffeine (xem ph lc 1, mc 6).Xc nh hm lng polyphenol tng (xem ph lc 1, mc 7).2.6. Phng php tnh ton2.6.1 Qu trnh kh caffeineHiu sut kh caffeine: Tin hnh xc nh lng caffeine c trong nguyn liu l ch ban u em kh; xc nh lng caffeine c trong nc trc v sau khi kh caffeine. T , nh gi hiu sut kh caffeine theo cng thc:Hk =^^[footnoteRef:1]100% m [1: Trong : ith: lng caffeine c trong nc sau qu trnh kh caffeine (g).]

mI: lng caffeine c trong nc trc qu trnh khcaffeine (g).mo: lng caffeine c trong l ch ti ban u (g).Hk: hiu sut kh caffeine (%).Tn tht cht kh ha tan phi caffeine: Tin hnh xc nh lng cht kh ha tan phi caffeine c trong nc trc v sau qu trnh kh caffeine (g); xc nh tng lng cht kh hatan phicaffeine c trong ch trc khi em kh caffeine (g).T ,xc nhtn tht chtkh ha tan phi caffeine ca nguyn liu theo cng thc:Hck = m2~m\ *100o/o m0 Trong : m2: lng cht kh ha tan phi caffeine c trong nc sau qu trnh kh caffeine (g).mI: lng cht kh ha tan phi caffeine c trong nc trc qu trnh kh caffeine (g).mo: lng cht kh ha tan phi caffeine c trong l ch ti ban u(g)-Hck: phn trm tn tht cht kh ha tan phi caffeine (%).Lng polyphenol tng b tn tht trong qu trnh kh caffeine: Tin hnh xc nh lng polyphenol tng c trong l ch trc v sau qu trnh kh. T , xc nh s thay i lng polyphenol tng nh sau:H =m2~m' *100% Trong : mI: lng polyphenol tng tnh theo acid gallic trong l ch trc qu trnh kh caffeine (g).m2: lng polyphenol tng tnh theo acid gallic trong l ch sau qu trnh kh caffeine (g).Hp: s thay i lng polyphenol tng (%).2.6.2 Qu trnh hp phu caffeine t dich trch cn bng bng than hoat tnh bo haHiu sut hp ph caffeine HhP: tin hnh xc nh lng caffeine c trong nc trc v sau khi qua than hot tnh. T , xc nh hiu sut hp ph caffeine theo cng thc:H = md 'ms * I 00% mdTrong mci: lng caffeine trong dung dch trc hp ph (g).ms: lng caffeine trong dung dch sau hp ph, (g).HhP: hiu sut hp ph caffeine ca qu trnh (%).Mc tn tht cht kh ha tan phi caffeine Httck: Tin hnh xc nh lng cht kh ha tan phi caffeine c trong nc trc v sau qua than hot tnh. T , xc nh c tn tht cht kh ha tan phi caffeine ca qu trnh:Httck =^*1000/0 m,Trong mi: lng cht kh ha tan phi caffeine trong dung dch trchp ph (g).m2: lng cht kh ha tan phi caffeine trong dung dch sau hp ph, (g).Httck: tn tht cht kh ha tan phi caffeine ca qu trnh (%).Mc tn tht hm lng polyphenol tng Http: Tin hnh xc nh lngpolyphenol tng c trong nc trc v sau khi qua than hot tnh. T , xc nh ctn tht lng polyphenol tng ca qu trnh:= m, -m2 *100% m,Trong mi: lng polyphenol tng tnh theo acid gallic c trong dungdch trc hp ph, (g).m2: lng polyphenol tng tnh theo acid gallic c trong dung dch sau hp ph, (g).Http: mc tn tht hm lng polyphenol tng (%).CHNG 3. KT QU V BN LUN3.1. Kho st nguyn liu l ch tiTin hnh xc nh thnh phn ha hc ca l ch ti c thu nhn hai thi im khc nhau. Kt qu kho st nh sau:Bng 3-1: Kt qu kho st nguyn liu gia hai ln mua khc nhau:Ch tiuMu 1(ngy 14/9/2007)Mu 2 (ngy 23/9/2007)

m (%)68.5572.40

Lng l non/ch nguyn liu (%)59.2858.41

Cht kh ha tan (tnh trn khi lng cht kh) (%)23.8539.02

Hm lng tro (tnh trn khi lng cht kh) (%)5.55.13

Hm lng tro tan trong nc (tnh trn khi lng cht kh) (%)2.382.46

Hm lng tro khng tan trong nc (tnh trn khi lng cht kh)(%)3.122.67

Hm lng ng kh tnh theo glucose (tnh trn khi lng cht kh)(%)4.18056.419

Hm lng polyphenol tng tnh theo acid gallic (tnh trn khi lng cht kh) (%)16.519.99

Hm lng caffeine (tnh trn cht kh)(%)2.542.935

CHNG 2: NGUYN LIU V PHNG PHP NGHIN cu

CHNG 3: KT QU V BN LUN

CHNG 3: KT QU V BN LUN

-44 -

-#-

-45-

Da vo bng 3.1, ta thy: Thnh phn cc cht trong l ch ti nm trong cc gi tr ca l thuyt. Tuy nhin, nguyn liu cung cp cho th nghim l khng n nh v t l cc thnh phn ha hc trong nguyn liu dao ng kh cao. Nguyn nhn l do:Th nht: thnh phn ha hc ca l ch ph thuc vo kh nhiu yu t"nh: ging, thi tit, iu kin canh tc, v tr lM Vit Nam, iu kin canh tc,ging v thi tit l khng n nh, c bit l trong thi im ma bo.Th hai: do vn vn chuyn ko di lm tn tht m, s pht trin ca vi sinh vt v su hi lm cho thnh phn ha hc ca ch ni chung v hm lng polyphenol tng ni ring thay i.Nh vy, vi ngun nguyn liu khng n nh nh vy, chng ti tin hnh xc nh li hm lng cc thnh phn trong nguyn liu sau mi ln th nghim. Thc hin th nghim theo tng m v ly mu theo nguyn tc chung ca TCVN nhm to ra mt mu chung ng nht ti mt thi im nht nh. T mu chung , ta xem thnh phn ha hc ca cc l ch l nh nhau. Ly mu theo nguyn tc ngu nhin trong mu chung cho cc phn tch ca th nghim.3.2. Kho st nh hng ca t l chrnc n qu trnh kh caffeineTrong th nghim ny, chng ti tin hnh qu trnh kh caffeine 90c, trong thi gian 5 pht v thay i t l chinc cc t l khc nhau nh gi nh hng ca t l ch:nc n hiu sut kh caffeine, tn tht cht kh ha tan phi caffeine, s thay i lng polyphenol trong nguyn liu sau kh caffeine. Chng ti tin hnh th nghim vi cc t l ch:nc l: 3.75, 5.0, 6.25g trn 100ml nc. Kt qu thu c nh sau:3.2.1 Hu sut kh caffeineKt qu thu c v nh hng ca t l ch:nc n hiu sut kh caffeine c th hin bng 3-2Bng 3-2: Hiu su't kh caffeine cc t l ch/ nc khc nhau.MuKhi lng muT l ci:ncHiu sut kh caffeine (%)

13.751:2672.3568+ 1.8107

25.001:2073.3226 + 3.9742

36.251:1663.7010 3.6948

_00J90 f 80 JS _2 70