HOA HOG-CONG NGHE THlfC PHAM
TOI iTU HOA DIEU KIEN TRICH LY ANTHOCYANIN Tl/ LA CAM
(Peristrophe Bilvalvis (L.)
• MAI HUYNH CANG - D 6 THj THUY TIEN - N G U Y I N THI TU
T 6 M T A T :
Muc tieu chinh cua dl tai la toi tfu h6a dieu kien ttich ly anthocyanin tit U Cam (Peristrophe bivalvis (L.) Mer.), va danh gia kha nang khang oxy h6a cua anthocyanin ttong dich chiet. Cic dieu kien toi tfu de trich ly anthocyanin d quy mo phong thi nghidm: trich vfii li le dung moi/nguyen Ueu la 40/1 (ml/g), tai 43.5''C, frong thdi gian 63 phiit. Ham lu'dng anthocyanin thu dtfdc khoang 1,236 mg/g. Dung m6i suf dung la nu'dc - acid acetic: 100/0.1 (ml/ml).
TCf khoa: La Cam, anthocyanin, Peristrophe Bilvalvis (L).
LDgtvandl
Cay la Cam (Peristrophe bivalvis (L.) Merr) hay con gpi la cay nhuom mau la each goi cua ngu'di Viet Nam de chi loai cay iTa am va ifa bong ram, thu'dng phan bo' rai rac d hau het cac tmh phia BSc nu'dc ta, Anthocyanin trong la Cam du'dc xac dinh bao gom 2 loai khung chinh la perlagonidin va pyranopeonidin [1], Anthocyanin la nhii'ng goc giucozit do goc difdng glucose, galactose,... ke't hdp vdi go'c aglucon qua lien ke't glycoside, thuoc nhom flavonoid, la sac to' trong khong bao thtfc vat tan trong ntfdc chiu trach nhi6m ve mau do sang, tim hoac mau xanh cua hoa, vo, hat, qua va la [2], Anthocyanin la dai dien cho nhiing chat chong oxy hoa quan trpng, cd vai trd tich ctfc trong phong cho'ng va giam thieu nguy cd mac benh tim mach va ung thtf, khang viem va cd nhieu hoat tinh cho'ng lai benh tat khac [3]. Athocyanin khong c6 doc tinh va khong c6 ba't ki gidi ban toi da nao cho cac iJng dung trong thtfc pham.
Hien nay, viec su' dung cha't mau ttf nhien trong thtfc pham dang dtfdc tfa chupng, dem lai nhieu tfu
diem hdn so vdi sif dung chat mau tong hdp, vi ngoai viec tao mau, chung con an toan va c6 gia tri dinh dtfdng cao. Do do, chat mau anthocyanin dtfdc su" dung rpng rai trong cac boat dong san xuat cong nghiep [4]. Anthocyanin da dtfdc cha'p nhan lam chat mau siJ dung trong thu'c pham d Hoa Ky trong danh muc chat mau ntfdc ep trai cay (21 CFR 73,250) hoac chat mau ntfdc ep rau qua (21 CFR 73.260). Tai chau Au, anthocyanin dtfdc xep vao cac chat mau c6 nguon gd'c ttf nhien [5]. Muc tieu cua nghien ctfu la tim ra dieu kien toi tfu de trich ly anthocynin til la Cam.
2. Dd'i tu'dng va phtfdng phap nghien eu'u 2.1. Nguyen lieu, hoa chat vd thie't bi Nguyen lieu: La Cam ttfdi sau thu hoach dtfdc, njfa sach, say kho va xay nho vdi kich thtfdc
< 0.5mm. Bao quan trong bao bi hiit chan khong roi baoquan65"C.
Hoa chdt: ethanol, i.sopropanol, iod, kali iodua, 1-a naphthol, acid sunfuric, acid acetic, natri hydroxit, sdt(III) clorua, acid clohydric, chloroform deuxua'txtf tif Trung Quoc,
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TAP CHi CONG THKtfNG
Thie't bi: May do pH S220 (METTLER TOLEDO, Thuy Si), may UV-vis (Thermo Spectronic My), bep khuay ttf gia nhiet (Scilogex, My), can phan tich DJ 620-S (DJ, Dtfc), may ly tam EBA 200 (Hettich, Dd'c),
2.2. PhUt^g phdp phdn tich
Xac dinh do am cua nguyen lieu la Cam bang phtfdng phap say kho de'n kho'i Itfdng khong doi [7]
va am dp cua cao thanh pham cuoi ciing bang phtfdng phap sa'y den khoi Itfdng khong doi d 75''C;
do tro cua nguyen Heu dtfdc xac dinh theo TCVN 5253:1990; Dmh tinh cac thanh phan alkaloids, carbohydrate, glycosid, flavonoid, phenol, tannin, axit ainin, protein, saponin, sterol, terpenoids, quinon, oxalat [8J de xac nhan stf hien dien cua cac chat chuyen hoa thtf cap trong dich chie't la Cam va trong cao thanh pham,
Xdc dinh ham luong anthocyanin theo phuong phdp pH vi sai: Ham Itfdng anthocyanin trong cao kho tuyet doi dtfdc xac dinh theo phtfdng phap pH vi sai (Giusti va Wrolstad, 2000) va dtfdc tinh quy theo cyanidin-3-glucoside. Cyanidin-3-glucoside dtfdc chpn vi day la dang pho bien cua anthocyanin trong ttf nhien, Dich chie't thu dtfdc d cac thi nghiem dtfdc xac dinh ham Itfdng anthocyanin du'a trdn nguydn tac anthocyanin thay doi mau theo pH, Tai pH = 1 cac anthocyanin ton tai d dang oxonium hoac fjavium c6 dp hap thu ctfc dai, tai pH = 4,5 thi chiing lai d dang carbino! khong mau. Phtfdng phap pH vi sai cho phep xac dinh ham Itfdng anthocyanin tong so chinh xac va nhanh chong, tham chi khi c6 stf hien dien cua cac hdp chat can thiep khac [10].
Mau dtfdc pha loang trong 2 dung dich dem: dem kali clorua 0,025 M (pH 1.0) va dem natri axetat 0,4 M (pH 4.5). Ta't ca cac phep do do hap thu bang may do quang pho dtfdc thtfc hien d btfdc sdng 500nmva700nm[10J,
Xac dinh anthocyanin theo cong thtfc:
_ AxMWxDFxWOO V
" " e x / ^ m^iilOO-wjxW-^
Trong do: A la dp ha'p thu cua anthocyanin vdi:
- ( A A V I S . *^7aanm)pH4 5
MW = 449.2 g/mol la khdi Itfdng phan tu' cua cyanidin-3-glucosdie, DF la dp pha loang; V la the tich dich chie't; 1 la be day cuvet (cm);
e = 26900 !a he so hap thu phan til ciia cyanidin-3- glucosdie (l.mol-l.cm-1); mNL la khoi Itfdng nguyen lieu (g); w la am dp nguyen heu; a la itfdng anthocyanin (g).
2.3. Phumgphdp thUc hien
La dm dtfdc lam sach de loai bo nhtfng bui ban va tap cha't, ban chd' tdi mtfc toi da vide tap nhidm gay sai sd' trong qua trinh thtfc hien thi nghiem. Sau do, tie'n hanh sa'y kho nguyen lieu (am dp 3,293%) d may say khay. Sau qua trinh say, la Cam dtfdc xay nho (kich thtfdc ray 0,5 mm) de giam kich ihtfdc nguyen lieu, tang dien tich tie'p xiic giiJa nguyen lieu va dung moi, giiip tang hidu qua trich ly, Tie'n hanh tach cholorophyll d dieu kien toi tfu da nghien ctfu tH trtfdc (dung moi: con 96, ti le dung moi va nguyen lidu la 30:1, thdi gian: 97 phut, nhidt dp: 49"C). Sau dd, trich ly anthocyanin vdi dung moi 3 cac dieu kien khac nhau de khao sat dieu kien chie't toi tfu. Lay dich trich ly dem loc qua gia'y loc, dinh mtfc va do quang d btfdc song thich hdp de xac dinh ham Itfdng anthocyanin c6 trong dich chie't. Cuoi cung, khao sat cac dieu kidn de tao san pham bdt mau.
2.4. Bo tri thi nghiem
2.4.1. Khdo sdt thdnh phan, ddc tinh hoa ly ciia nguyen lieu Id Cam
La Cam nguydn lieu dtfpc phan tich cac chi so do am, do tro, dinh h'nh cac thanh phan alkaloids, carbohydrate, glycosid, flavonoid, phenol, tannin, axit amin, protein, saponin, sterol, terpenoids, quinon, oxalate, xac dinh ham Itfdng chat tan dtfdc trinh bay ct muc 2.2.
2.4.2. Thi nghiem khdo sdt dnh hudng cua cdc dieu kien trich ly de'n hdm luong anthocyanin
Cac thi nghiem dtfdc bo' tri hoan toan ngSu nhidn kieu luan phidn ttfng bid'n mot yeu to theo thtf ttf la: loai dung mdi (ntfdc cat, ethanol 99,5% va acid acetic 0.1 %), ti Id dung mdi/nguyen lieu (50/1, 75/1, 100/1, 150/1 ml/g), nhidt dp chiet (khoang 30"C, 40»C, 50"C, 60"C, 70"C), thdi gian chie't (30, 60, 90 phut) va toe dp khuay (100, 200 va 300 vong/phut). Kd't qua Itfa chon khi khao sat cac yd'u to' trtfdc dtfdc su" dung de khao sat cho yd'u to sau.
Dieu kien ban dau la 100ml dung moi/ Ig nguyen lieu vdi thdi gian chie't 60 phut tai nhiet dp phong (khoang 30"C), dich chiet khong c6 tac dong khul'y, Chi tidu theo doi la ham Itfdng anthocyanin.
Ba ye'u to ty le dung moi/ nguyen lieu, nhiet do chie't va thdi gian chid't dtfdc bd' tri theo kieu true quay tam (Rotatable Central Composite Design) de xac dinh anh htfctng ciia cac yeu to' va td'i tfu hda quy trinh chid't anthocyanin bang phtfdng phap dap tfng be mat RSM, Cao mau thanh pham dtfdc xac dinh am dp, dinh tinh alkaloids, tannin, glycosides,
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HOA HOC-CONG NGHE THlfC PHAM
resins, flavonoid, sterol, saponin, carbohydrates, phenol, terpenoids.
2.5. Xd ly sd'lieu:
Mdi thi nghiem dtfdc thtfc hidn lap lai 3 lan, dtfdc tinh toan va ve do thj bang ph^n mem Microsoft® Office Excel 2013. Phan tich tho'ng ke ANOVA va trac nghiem LSD dtfdc su' dung de so sanh anh htfdng ciia cac ye'u to', dtfdc xif ly bang phan mem Statgraphics Centurion XV 9 (Statgraphics Technologies, Inc., USA) va stf hd trd cua phan mem thong kd JMP 10, Dp tin cay 95%
dtfdc ap dung cho ta'tca cac phep tho'ng ke.
3. Ket qua va thao luan 3.1. Khdo sdt nguyen lieu vd thdnh phdn hoa hoc cua Id Cdm
La Cam ttfdi chtfa bam Itfdng ntfdc cao c6 dp am la 75.324%, do tro la 3.293%, 6 dp am trdn, cac vi sinh vat (vi khuan, na'm md'c) van cd the' phat trien dtfdc neu bao quan d nhiet dd phdng trong moi trtfdng am. Do dd, muon bao quan trong thdi gian dai, nen sa'y kho roi dtfng nguyen lieu trong bao bi hut chan khdng roi bao quan d 5^*0. Mat khac, viec say kho la cam va bao quan d dang hot kho van la giai phap thuan tidn, giup chu dpng dtfdc nguon nguyen lieu trong san xua't. Qua ke't qua dinh tinh thanh phan hoa hpc cua nguyen lieu cho tha'y cd stf xua't hidn cua tanin, alkaloid, flavonoid, cacbohydrat va glycoside, Quines. Khi thay doi pH ttf 1 - 14, mau sac dich chie't bang dung moi ntfdc thay dd'i (PTinh 1). Trong moi trtfdng axit (pH <
7), cac anthcyanin c6 mau do cam, duy d mtfc pH 4, anthocyanin khong ben va chuyen thanh mau cam. 0 pH = 7 - 11, cac anthocyann ton tai dtfdi dang base quinon ndn cd mau tim. Ttf pH = 12 14, anthocyanin c6 sir chuyen doi ttf xanh dtfdng dam den xanh la
nhat (hdi nga vang) vi c6 stf chuyen doi ttf dang quinoidal anhydro thanh dang chalcon.
3.2. Anh hU^g cua dieu kien trich ly de'n hdm liding anthocyanin
Hinh 2 cho tha'y, loai dung moi khac nhau se thu dtfdc ham Itfdng anthocyanin khac nhau. Ham Itfdng anthocyanin thu dtfdc ttf dung mdi acid acetic 0,1% la cao nha't va dung mdi ethanol 99,5% cho ham Itfdng anthocyanin la tha'p nha't. Vi anthocyanin la hdp cha't kha phan ctfc nen se tan td't trong cac dung moi phan ctfc. Dung mdi cang phan ctfc se chie't dtfdc cang nhieu anthocyanin. Kd't qua ANOVA cho tha'y yeu to' loai dung mdi cd anh Hinh 1: Mdu sdc djch chief Id Cdm thay doi theo pH
|fi 1 2 2 9 10 11 12 13 14
Hinh 2: Anh hifdng cua logi dung mdi ten hdm tifdng anthocyanin
Hinh 3: Anh hifdng cua fl le dung moi/ nguyen lieu len hdm lifdng anthocyanin
hi T
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if III I I 1 1
Hinh 4: Anh hifdng cua nhiet do den hdm Iifdng anthocyanin
Hinh 5: Anh hifdng cua thdi gian den hdm Iifdng anthocyanin
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i 100/1 I5(V1 ii/Dgn>en Ufo
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TAP CHi CONG THtrONG
htfdng tdi ham Itfdng anthocyanin cd y nghia d mtfc dp tin cay 95%. Ki^m tra sif khac bidt gitfa cac loai dung moi bang phtfdng phap LSD tha'y stf khac biet gitfa 3 loai dung moi. Vay nen, ta chon dung mdi axit acetic 0,1% trong cac thi nghiem tiep theo de thu dtfdc ham Itfdng anthocyanin la cao nha't.
Hinh 3 cho tha'y vdi ti le dung moi/ nguydn lieu
= 75:1 (v:w) cho ham Itfdng anthocyanin cao nha't (0,885 mg/gNL) cdn ti le 150:1 cho ham Itfdng tha'p nha't (0,679 mg/gNL), Ttf ti le 50:1 dd'n 75:1 ham Itfdng anthocyanin thu dtfdc tang Nhtfng ttf 75:1 de'n 150:1 thi ham Itfdng anthocyanin thu dtfdc lai giam dan. Vdi dieu kien chie't d nhiet dp phong (30°C) trong thdi gian 1 gid thi toan bo khd'i nguydn lieu dtfdc tid'p xiic hoan toan vdi dung mdi. Do dd, Itfdng anthocyanin khud'ch tan vao dung moi la cao nha't, Kd't qua ANOVA cho tha'y, ye'u to' ti le dung mdi cd anh htfdng de'n ham Itfdng anthocyanin cd y nghia d mtfc dp tin cay 95%. Ket qua LSD cho tha'y gitfa tile 50:1 va 125:1 khong cd stf khac bidt va ti le 75:1 va 100:1 it c6 stf khac biet, nhtfng ti le 75:1 cho ham Itfdng anthocyanin nhieu hdn. Do do, chpn tl Id 75:1 cho cac thi nghiem sau de vtfa tie't kiem dung moi vtfa thu dtfdc hieu qua chie't cao nha't.
Hinh 4 cho tha'y vdi dieu kien chid't trong 1 gid, ttf 30''C dd'n SO^C ham Itfdng anthocyanin tang dan, ttf SO^C de'n 70"C cd xu htfdng giam. Nhiet do cho ham Itfdng anthocyanin cao nha't la 50'*C (1,099 mg/gNL) va tha'p nhat la 70"C (0,472 mg/gNL). 6 nhiet dp cao (ttf 70"C), anthocyanin de bi bie'n tinh va ma't dan nen d dieu kien chie't 70"C thu dtfdc ham Itfdng anthocyanin la thap nha't. Kd't qua ANOVA cho tha'y, ye'u to' nhidt dd cd anh htfdng de'n ham Itfdng anthocyanin, cd y nghia d mtfc dp tin cay 95%. Ke't qua LSD chi ra stf khac biet gitfa cac nhiet dp chie't. Do do, ta chon 50"C la nhiet dp chie't td'i uXi trong cac thi nghiem tiep theo.
ITinh 5 cho tha'y khi tang thdi gian ttf 30 phiit len 60 phut thi ham Itfdng anthocyanin tang nhtfng khi tang thdi gian chid't Idn 90 phut thi Itfdng anthocyanin giam. Ham Itfdng anthocyanin cao nha't thu dtfdc d thdi gian 60 phiit (1.203 mg/gNL).
Stf oxy hoa dtfdc xem ia mot tac nhan anh htfdng de'n ham Itfdng anthocyanin, Theo thdi gian, Itfdng anthocyanin dtfdc hda tan vao dung mdi it hdn so vdi Itfdng anthocyanin hi oxy hoa. do do Itfdng anthocyanin giam dan. Trong bang ANOVA, P=0.0122 (< 0,05) ndn ye'u to' thdi gian cd anh htfdng de'n ham Itfdng anthocyanin la c6 y nghTa vdi mtfc dp tin cay la 95%. Ke't qua LSD chi ra stf khac
bidt gitfa 60 phut vdi 2 mtfc thdi gian cdn lai. Do do, ta chon 60 phut la dieu kidn chie't cho cac thi nghiem hep theo.
Hinh 6: Anh hifdng eua foe do khuay den hdm Iifdng anthocyanin
1.203
• "^ »•"» ens
Toe do khuay (voug/ phul)
Hinh 6 cho tha'y td'c dp khua'y cang tang thi ham Itfdng anthocyanin thu dtfdc cang giam. Ham Itfdng anthocyanin thu dtfdc cao nha't khi khua'y la 0,216 mg/gNL (d dieu kidn khua'y 50v/p) tha'p hdn rat nhieu so vdi ham Itfdng anthocyanin thu dtfdc khi khdng khua'y (1,203 mg/gNL). Khi khua'y, lam tang Itfdng oxy trong khdng khi lan vao trong dung mdi.
Toe dp khua'y cang cao thi oxy hoa tan trong dung mdi cang nhieu, dSn dd'n Itfdng anthocyanin bi mat di do stf oxy hda cang nhieu, gay nen hidn ttfdng giam ham Itfdng anthocyanin. Do do, yd'u to' khua'y khdng ap dung trong qua tnnh chie't anthocyanin ttf la Cam.
Du'a vao kd't qua cac thi nghidm khao sat sd bo cac yd'u to anh htfdng de'n ham Itfdng anthocyanin, ta chon tam nhtf sau: ti le dung mdi: nguydn lieu:
75:1 (v:w), nhiet dd chiet: 50"C, thdi gian chie't: 60 phut. Ttf cac yeu to da chpn. ma tran thi nghiem td'i tfu dtfdc thid't lap theo kieu quay tam nhtf Bang 1.
Bang 1. Bang ma hoa bien trong toi Uu Yeu to
Ty le dung moi; nguyen lieu (v:w) Nilia do chief ("C) Tiidi gian chiet (phut)
1 501
40 30
0 1 +1
76:1 50 60
125:1 60 90
Ttf Hinh 7, ta thay P=0.004 (<0.05) va RSq=0.96 (>0.8) cd nghia la md hinh dtf doan tuye'n tinh dtfdc xem la td't, phii hdp de suf dung. Do dd, phtfdng trinh dtf doan ham Itfdng anthocyanin la:
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HOA KOC-CONG NGHE THlTG PHAM
Hinh 7: Bleu dd dif dodn hdm Iifdng Athocyanin
Bang 2. Bang doi chieu dieu kien td'i lAi va thuc te
n r
0,2 0,4 0.6 0.8 1 1.2 1.4 Y Predicted P=0,004 RSq=0,96RMSE=0.1127
y = -0.24xj' - 0.365x/ + 0.02Ix,X2 -O.llSxj-0.183x2 +1.148
Trong dd: X] la nhidt do ("C); X2 la ti l e ; X3 la thdi gian (phiit), Phtfdng trinh hoi quy nay dtfdc dung nhtf mdt md hinh d e dtf doan dtfdc h a m Itfdng anthocyanin vdi cac dieu kien nhiet dd, ti Id va thdi gian ttfdng tfng (Bang 2).
Tid'n hanh thtfc nghidm vdi c a c dieu kien dtf doan, thu dtfdc h a m Itfdng anthocyanin trung binh
Dieu kj$n chiet Nhiet do ("C) Tlie dung moi.
nguyen iieu (v w) Th6i gian (phut) Ham iuong athocyanin (mg/gNL)
Ly thuyet 4348580776 39.62616834:1 63.38824601
1.236
Chon thuc te 43.5 401 63
1.202±0.041
la 1,202 ± 0,041 (mg/gNL). sai so'be hdn 5 % so vdi h a m Itfdng dtf d o a n theo ly thuye't, Dieu nay chtfng td m o hinh nay cd y nghTa va phii hdp d e tach chid't anthocyanin trong la C a m , Khi d a n h giii chat Itfdng cao thanh p h a m thu dtfdc im do c a o m a u la 7,129%. van cdn stf hien didn cua ankaloids.tanins, glycosides, flavonoid, terpenoid va carbohydrat so vdi nguyen h d u ban dau. Vi the, cac nghien ctfu tid'p theo can nghidn cSu ve dieu kien tinh sach cao d e n a n g c a o h a m Itfdng anthocyanin.
4. Kd't luSn
B i e u kidn td'i tfu d e chie't anthocyanin ttf la C a m la su' dung dung mdi la ntfdc - axit acetic cd ti le la 100:1 (v:v) trong 63 phtit tai 43.5''C, vdi ti le dung mdi: nguyen h d u la 40:1 (v:w) •
TAI LIEU T H A M K H A O :
/, Bid Xuan Tinh, (2011), Nghiin ciiu thanh phdn hoa Iwc va tham do hoat tinh sinh Iwe cua chdt mau li/ Id Cdm (Peristrophe bivalvis), Dai hoc Da Ndng.
2. Gross J. 1987. Pigments in Fruits. London: Academic Press.
3. Farah S. Hosseinian, Wende Li, Trust Beta (2007). /Measurement of anthocyanins and otherphytochemicals in purple wheat. Food Chemistry 109 916 - 924.
4 Brasii. (2001). Ministerio da Saude. Agenda Nacional de Vigildncia Sanitaria - ANVISA. Resolu^ao da Diretoria Colcgiada n" 34. Aprova o regulamenio tecnico sobre uso de adilivos alimentares. Dtario Oficial da Unido, Brasilia.
5. Tim Langrish and Don Chiou (2008) Stabilization of Moisture Sorption in Spray-Dried Bioactive Compounds by Using Novel Fibre Carriers to Crystallize the Powders, international Journal of Food Engineering, 4(1).
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8. Srlyeta Chakraborty. Souvagyalaxml Sahoo. Bhagat A., et al. (2017). Studies On Antimicrobial Activity.
Phytochemical Screening Tests. Biochemical Evaluation OfClltorea Ternatea Linn. Plant Extracts. Zenodo.
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Ngay nh^n bai: 20/10/2019
Ngay phan bi$n danh gia va suTa chffa: 30/10/2019 Ngay cha'p nh^n dang bai: 10/11/2019
Thong tin lac gla:
1, TS. IMAI HUYNH CANG 2, D 6 THI THUY TIEN 3, NGUYEN THI TU
BO mdn Cong ngh^ Hoa hoc, Tri^cfng Dai h^c NOng Lam TP. H 6 Clu Minll
OPTIMIZATION EXTRACTION CONDITION OF ANTHOCYANIN FROM
Peristrophe Bilvalvis (L.) Merr Leaf
• DR, MAI HUYNH CANG
• DO THI THUY TIEN
• NGUYEN THI TU
Department of Chemical engineering a n d processing Nong Lam University of Ho Chi IVlinh City ABSTRACT:
The objective of research is optimisation conditions for anthocyanin extraction from the Peristrophe bivalvis (L.) Mer leaf. Optimum conditions for anthocyanin extraction in laboratory scale were ratio of 40/1 (ml/g), 43.5''C and 63 minutes for ratio of solvent/material, extraction temperature and extration time, respectively. The solvent extraction was H20 - acid acetic:
100/0.1 (ml/ml).
Keywords: Peristrophe bivalvis (L.) Mer leaf, anthocyanin.
218 Sd21 -Thdng 11/2019
