Tp ch Khoa hc Trng i hc Cn Th Phn B: Nng nghip, Thy sn v Cng ngh Sinh hc: 36 (2015): 21-28

TI U HA QU TRNH TRCH LY POLYSACCHARIDE V TANNIN

TRONG NM LINH CHI (Ganoderma lucidum)

Phm Bo Trng1 v Nguyn Minh Thy2

1
Cao hc CNTP K19, Trng i hc Cn Th
2
Khoa Nng nghip & Sinh hc ng dng, Trng i hc Cn Th

Thng tin chung:
Ngy nhn: 15/08/2014
Ngy chp nhn: 26/02/2015
Title:
Effects of temperature and
time on extraction of
polysaccharide and tannins
from red lingzhi (Ganoderma
lucidum)
T kha:
Nm linh chi, nhit , thi
gian, polysaccharide, tannin
Keywords:
Lingzhi mushroom,
temperature, time,
polysaccharides, tannins
ABSTRACT
The effects of temperature (70130oC) and extraction time (1590
minutes) on the polysaccharide and tannin content of red lingzhi were
studied. Optimizing extraction process was realized based on the
experimental design of times and temperatures. The results showed that
temperature of 130C and extraction time of 30 minutes produced the
highest polysaccharide content (684.1 14.5 mg/l). Meanwhile, the highest
tannin content (630.9 18.2 mg/l) achieved by implementing the extraction
process at temperature of 120C for 45 minutes. A response surface model
to describe the combined temperature and time effects on the extraction of
polysaccharide (or tannin) content was presented.
TM TT
Nghin cu c thc hin trn c s kho st nh hng ca nhit
(70130oC) v thi gian trch ly (1590 pht) n hm lng
polysaccharide v tannin trong nm linh chi . Ti u ha quy trnh
trch ly c thc hin da trn b tr th nghim v thi gian v nhit .
Kt qu nghin cu cho thy nhit 130oC v thi gian trch ly 30 pht
cho hm lng polysaccharide trong nc linh chi cao nht (684,1 14,5
mg/l). Trong khi , hm lng tannin cao nht (630,9 18,2 mg/l) t
c khi thc hin qu trnh trch nhit 120oC v thi gian 45 pht.
M hnh b mt p ng din t nh hng ca kt hp nhit v thi
gian trch ly n hm lng polysaccharide (hoc tannin) c xc nh.

1 GII THIU trong tt c cc loi dung mi th nc c xem l

Nm linh chi (ganoderma lucidum) cung cp
mt lng ng k cc cht c hot tnh sinh hc
nh polysaccharide (giu -glucan), tannin,
triterpenoid, steroid, saponin Trong ,
polysaccharide c xem l nhm cht rt quan
trng bi v chng c kh nng chng li t bo
ung th (Sakai v Chihara, 1995) v tannin l
nhm cht c kh nng chng oxy ha rt cao.
Vic trch ly cc hot cht sinh hc c trong nm
linh chi ph thuc vo rt nhiu yu t nh loi
dung mi, nhit trch ly, thi gian trch ly
loi dung mi tt nht cho qu trnh trch ly bi v
nc khng c hi, khng d chy, gi thnh r v
c th ng dng trong sn xut thc phm mt
cch d dng (Herrer et al., 2006). Ty thuc vo
iu kin nhit c th trch ly c cc nhm
cht khc nhau bao gm cc hp cht phn cc
c trch ly nhit thp v cc hp cht khng
phn cc c trch ly nhit cao (trn 100oC).
Trch ly nhit di 100oC s xy ra qu trnh
ha tan cc phn t polysaccharide mch ngn v
tannin ha tan, ngc li trch ly trn 100oC s xy

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Tp ch Khoa hc Trng i hc Cn Th Phn B: Nng nghip, Thy sn v Cng ngh Sinh hc: 36 (2015): 21-28

ra qu trnh ha tan cc phn t hemicellulose khong 8-10% (Hnh 1) (c s sn xut nm linh

(Sattler et al., 2008) v tannin khng ha tan. chi Ct Sn, 42 i Cn - Bo Lc, Lm ng).
Nghin cu ca Askin et al. (2007) cho rng vic
Thit b s dng cho nghin cu bao gm: my
tng nhit trch ly trn 100oC s lm tng ng
nghin ct (2 li dao), b iu nhit (waterbath),
k hm lng polysaccharide trong nm linh chi
ni p sut. Bnh trch ly chu nhit trn 100oC
, tuy nhin nhit trch ly khng c vt qu
(bnh trung tnh Schott Duran, c, chu c p
200oC do qu trnh phn hy cc hp cht hu c
sut v chn khng t 1 1,5 bar - ISO 4796-1,
xy ra, c bit l polysaccharide. Bn cnh ,
chng sc nhit 30 K v nhit ti a 140oC)
tannin cng l nhm cht c nhit phn hy
c s dng.
khng n nh ty thuc vo loi tannin v iu
kin mi trng. Mc tiu m ti hng ti l
chn nhit v thi gian trch ly ti u nhm thu
c hm lng hot cht sinh hc cao nht, trong
trng tm l 2 nhm cht polysaccharide v
tannin c th ng dng hiu qu trong sn xut
cc sn phm c lin quan.
2 PHNG PHP NGHIN CU
2.1 Chun b mu v b tr th nghim Hnh 1: Nm linh chi dng nguyn tai v
ct mng
Nm linh chi ct mng c m trong
Bng 1: T hp cc nghim thc thc hin
Nhit trch Thi gian trch ly (pht)
ly (oC) 15 30 45 60 75 90
70 x x x x x x
80 x x x x x x
90 x x x x x x
100 x x x x x x
110 x x x x - -
120 x x x - - -
130 x x - - - -
Du (X) l cc nghim thc c thc hin, du () l cc nghim thc khng thc hin do iu kin thit b
Th nghim c b tr hon ton ngu nhin Hm lng tannin c nh lng theo g/l da
theo t hp gia nhit trch ly 70130oC (cch vo phn ng oxy ha kh KMnO4 vi ch th
nhau 10oC) v thi gian trch ly 1590 pht (cch indigocarmin (Muller v Allan, 1992).
nhau 15 pht) (Bng 1). Cn chnh xc 10 gr nm 2.3 Phn tch thng k
linh chi nghin mn v cho vo ti vi c kch
thc l lc < 0,5 mm. B sung 500 ml nc ct Phn tch thng k (STATGRAPHIC) c s
(t l nm linh chi v nc c chn trong nghin dng chn m hnh ph hp cho cc d liu thu
cu ng thi ca nhm tc gi) v cho tt c vo thp. M hnh (1) c xut (gi tr Y) trong
bnh trch ly. Thc hin cc th nghim theo nhit trng hp ny:
v thi gian b tr. Y = bo + bnXn + bnnXn2 + bnmXnXm (1)
2.2 Phn tch mu
Trong : bo l h s; bn, bnn v bnm l cc h s
Cc hp cht c hot tnh sinh hc trong nm bc 1, bc 2 ca phng trnh hi quy; Xn, Xm l
linh chi c phn tch bao gm polysaccharide cc gi tr ca bin c lp.
(giu -glucan) v tannin.
chn c cc iu kin ti u, phn tch
nh lng polysaccharide (g/l) theo phng hi quy a bin c p dng din t s thay i
php phenol - acid sulfuric c (Foster et al., hm lng cc cht c hot tnh sinh hc
1961), o hp thu dung dch ti bc sng 488 (polysaccharide v tannin) ca nm linh chi c
nm so vi ng chun glucose (Harshal v xc nh l bin ph thuc vo nhit v thi
Priscilla, 2011). gian trch ly c ghi nhn (k hiu l X1 v X2

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Tp ch Khoa hc Trng i hc Cn Th Phn B: Nng nghip, Thy sn v Cng ngh Sinh hc: 36 (2015): 21-28

tng ng). Kim nh s tng thch ca d liu gian di hn (60 n 90 pht). Tuy nhin, hm
theo m hnh v thc nghim c thc hin. lng ny tng c ngha thng k trong khong
xc nh chnh xc gi tr cc i (gi tr ti u) nhit trch ly c thc hin (70-90oC).
ca phng trnh hi quy a bin c th p dng nhit ny xy ra qu trnh trch ly cc phn
tnh cht o hm, tm im dng ca m hnh, lp t polysaccharide ha tan m ch yu l
bng bin thin bao gm cc im dng v cc polysaccharide peptide vi khi lng phn t
im quan tm nhm tm ra im cc i ca khong 5105 Da. Phn t polysaccharide peptide
phng trnh (gi tr ti u l thuyt). c cu to t cc phn t polysaccharide v cc
amino acid, trong phn polysaccharide bao gm
3 KT QU V THO LUN
glucose, galactose, arabinose, xylose v mannose,
3.1 nh hng ca nhit v thi gian cc phn t lin kt vi nhau thng qua lin kt -
trch ly n hm lng polysaccharide glucoside. Khong 17 loi acid amin lin kt vi
3.1.1 Nhit trong khong 70100oC cc phn t polysaccharide (Chan et al, 2006).
Chnh cc gc acid amin c tnh phn cc lm cho
Kt qu thng k cho thy khi tng nhit cc phn t ha tan d dng hn khi tng nhit .
trch ly t 70-100oC th hm lng polysaccharide Do y l qu trnh trch ly cc phn t cht tan
cng tng c ngha (Bng 2). Khi thi gian trch nn thi gian trch ly ko di cng lm tng hm
ly tng t 15 n 60 pht th hm lng lng polysaccharide ha tan cho n khi t n
polysaccharide trong dch cng tng, hm lng hm lng ti u.
ny cng khng tng thm khi trch ly vi thi
Bng 2: Hm lng polysaccharide (mg/l) trong dch linh chi theo nhit (70100oC) v thi gian
trch ly (1590 pht)
Nhit Thi gian trch ly (pht) Trung
trch ly (oC) 15 30 45 60 75 90 bnh
70 250,2 260,2 310,6 383,0 392,2 416,0 330,4a
80 249,8 259,6 322,3 415,3 407,4 447,3 350,3b
90 256,1 293,3 459,1 458,5 459,0 446,9 395,5c
100 391,2 420,4 466,9 477,6 483,1 470,7 451,6d
Trung bnh 286,8a 308,4b 389,7c 433,6d 435,4d 445,2d
Ghi ch: Cc ch trong cng mt ct hoc mt hng biu th s khc bit c ngha ca cc trung bnh nghim thc (
tin cy 95%)

Tng quan gia hm lng polysaccharide vi Y = 380,52 - 0,0001 X23 - 0,0200 X22
nhit v thi gian trch ly c th c din t + 3,9037 X2 (3)
theo phng trnh 2.
Trong Y l hm lng polysaccharide
Y = 1474,08 + 0,0015 X13 0,0001 X23 - 0,0051 (mg/l), X2 l thi gian trch ly (1590 pht).
X1 X2 - 0,0002 X1X22 + 0,8421 X1X2 25,946 X1
2

29,306 X2 (2) Ly o hm Y ca phng trnh 3, cho Y=0,

Trong Y l hm lng polysaccharide
(mg/l), X1 l nhit trch ly (oC) v X2 l thi
gian trch ly (pht).
M hnh b mt p ng (Hnh 2) cho thy
nh hng ng thi ca nhit v thi gian
trch ly n hm lng polysaccharide (mg/l) trong
dch trch.
xc nh thi gian trch ly ti u ng vi
nhit ti hn 100oC, c th thay X1 = 100oC vo
phng trnh 2 v thu c phng trnh 3.
tm c 2 im dng X21=-198,8 v X22 = 65,5
(pht). Da vo kt qu xt du (th hin Bng 3)
cho thy thi gian v nhit trch ly tng ng l
65,5 pht v 100oC, hm lng polysaccharide t
c cao nht. Nhit trch t 70 n 100oC ch
c th ly trch c cc phn t polysaccharide
mch ngn. Sau thi gian trch ly 65,5 pht th hm
lng polysaccharide s khng tng do qu trnh
trch ly hu nh xy ra hon ton v thi gian
trch ly ko di c th gy ra s phn hy cc
polysaccharide mch ngn c cu trc khng bn
vng v hu qu c th lm cho hm lng
polysaccharide gim.

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Tp ch Khoa hc Trng i hc Cn Th Phn B: Nng nghip, Thy sn v Cng ngh Sinh hc: 36 (2015): 21-28

Hnh 2: Biu b mt p ng th hin tng quan gia nhit v thi gian trch ly n hm
lng polysaccharide (mg/l) trong khong nhit 70-100oC
Bng 3: Bin thin ca hm lng
polysaccharide theo thi gian
X2 (pht) -198,8 15 65,5
Y 0 + 0
Y (mg/l) -402,6 434,2 549,8
Trong khong nhit trch ly 70100oC v
thi gian trch ly 1590 pht, hm lng
polysaccharide c tnh bng cch thay gi tr X1
(70100) v X2 (1590) vo phng trnh 2.
Phng trnh tng quan gia hm lng
polysaccharide c tnh theo phng trnh 2 v
hm lng polysaccharide thc nghim c h s
tng quan kh cao (R2=0,90), cho php s dng
m hnh tnh ton c lng hm lng
polysaccharide theo nhit (70100oC) v thi
gian trch ly (1590 pht).
3.1.2 Nhit trong khong 110-130oC
Trong khong nhit t 110 n 130oC, hm
lng polysaccharide tng quan vi nhit v
thi gian trch ly c din t theo phng trnh 4
(h s tng quan R2=0,942).
Y = 1072,94-5,99 X1-68,65 X2+0,63 X1X2 (4)
Trong Y l hm lng polysaccharide
(mg/l), X1 l nhit trch ly (oC) v X2 l thi
gian trch ly (pht).
Kt qu th hin trn m hnh b mt p ng
(Hnh 3) cho thy nhit 110oC, khi tng thi
gian trch ly t 15 n 60 pht th hm lng
polysaccharide tng rt t. Tuy nhin, hm lng
90 polysaccharide li tng rt mnh khi trch ly
- nhit 120 v 130oC khi thi gian trch ly tng t
496,9 15 n 60 pht. Nguyn nhn tng cao hm lng
ny c th do nhit cao to iu kin tt cho
qu trnh trch ly thm phn ln cc phn t
hemicellulose (Sattler et al., 2008). Nhng phn t
ny c cu trc phn nhnh nn c th b thy phn
thnh cc phn t c khi lng nh hn (Yu et
al., 2008) khi x l nhit cao. Do vy nhit
trn 120oC, ngoi trch ly cc phn t ha tan
nh polysaccharide-peptide, cn c th ly trch
thm hemicellulose trong thnh t bo (Sattler et
al., 2008). ng thi qu trnh trch ly nhit
cao s lm cho kch thc phn t ca cc hp cht
polysaccharide nh hn. Kt qu nghin cu ca
Matsunaga et al. (2013) cho rng nhit 135oC
kch thc ca polysaccharide trong khong 0,5-6
m (2 chim t l cao nht) v khi tng nhit
ln 170oC th kch thc ca polysaccharide s dao
ng trong khong 0,5-3 m (1 chim t l cao
nht). Thnh phn polysaccharide ha vo trong
dch trch ly nm linh chi nhit cao phn ln
l -glucan vi cu trc phn t rt nh (Askin,
2007). Trong khong nhit trch ly 110130oC
v thi gian trch ly 1560 pht, hm lng
polysaccharide d on c th c tnh bng cch
thay gi tr X (110130) v Y (1560) vo
phng trnh 4.

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Tp ch Khoa hc Trng i hc Cn Th Phn B: Nng nghip, Thy sn v Cng ngh Sinh hc: 36 (2015): 21-28

Hnh 3: Biu b mt p ng th hin tng quan gia nhit v thi gian trch ly n hm
lng polysaccharide (mg/l) trong khong nhit 110-130oC

Bng 4: Bin thin hm lng polysaccharide vi m hnh d on (hm lng polysaccharide l

theo nhit v thi gian trch ly 691,7 mg/l).
X1 (oC) 108,9 110 110 120 130 T phng trnh 4, ly o hm Y theo X1 v
X2 (pht) 9,5 15 30 30 30 X2. Khi Y(X1) = -5,99 + 0,63 X2 v Y(X2) = -
Y(X1) 0 + + + + 68,65 + 0,63 X1. Cho Y(X1) = 0 v Y(X2) = 0, ta
Y(X2) 0 + + + + c im dng X = 108,9 v Y = 9,5.
Y (mg/l) 420,22 423,79 433,54 562,64 691,74 Phng trnh tng quan gia hm lng
Da vo kt qu xt du hm s Bng 4, c polysaccharide d on theo m hnh v thc
th thy hm s Y l hm tng trong khong nhit nghim th hin h s tng quan cao (R2=0,939).
t 110-130oC, v vy hm lng polysaccharide Nh vy c th da trn m hnh c thit lp
s tng khi tng nhit hoc thi gian trch ly. c lng hm lng polysaccharide vi chnh
Tuy nhin, vic gia tng nhit s xy ra qu xc tng i cao trong khong nhit trch ly
trnh phn hy cc hp cht hu c, trong c c 110130oC trong thi gian t 1560 pht.
polysaccharide. Kt qu nghin cu ca Askin et 3.2 nh hng ca nhit v thi gian
al. (2007) cho rng khi tng nhit trn 200oC v trch ly n hm lng tannin (mg/l)
gi nhit trn 30 pht, hm lng cc cht hu c
Kt qu thng k cho thy trong cng mt thi
(polysaccharide) gim mnh. Kt qu thc nghim
gian trch ly, khi tng nhit t 70100oC th
cho thy hm lng polysaccharide cao nht
hm lng tannin lun tng v c khc bit ngha
(684,114,5 mg/l) khi thc hin qu trnh trch ly
o (Bng 5).
130 C trong thi gian 30 pht, kt qu ny ph hp
Bng 5: nh hng ca nhit v thi gian trch ly n hm lng tannin (mg/l)
Nhit Thi gian trch ly (pht) Trung
trch ly (oC) 15 30 45 60 75 90 bnh
70 179,2a 280,3cd 295,1de 352,9fg 321,8ef 329,6f 293,2A
80 226,ab 333,8f 321,5ef 345,5fg 456,8kl 442,9k 354,5B
90 248,1bc 323,9ef 387,0h 374,4gh 485,5lm 471,2kl 381,7C
100 333,1f 469,2kl 506,7mn 555,6o 596,1p 522,3n 497,2D
Trung bnh 246,7A 351,8B 377,6C 407,1D 465,0E 441,5F
Ghi ch: Cc ch thng khc nhau trong cng mt ct hoc 1 hng biu th s khc bit c ngha ca cc nghim
thc ( tin cy 95%)
Cc ch in hoa khc nhau trong cng mt ct hoc mt hng biu th s khc bit c ngha ca cc trung bnh nghim
thc ( tin cy 95%)

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Tp ch Khoa hc Trng i hc Cn Th Phn B: Nng nghip, Thy sn v Cng ngh Sinh hc: 36 (2015): 21-28

Trong cng mt nhit trch ly, khi tng thi
gian trch ly th hm lng tannin ch tng trong 75
pht u tin, sau khng tng na. Vi nhit
trch ly t 70 n 100oC v thi gian t 15 n 90
pht th tng quan gia hm lng tannin vi
nhit v thi gian trch ly c biu din theo
phng trnh 5 (R2=0,902).
Y = 1450,23 + 0,0013 X13 0,0039 X12X2
0,0005 X1X22 + 0,7342 X1X2 25,3506 X1
27,1415 X2 (5)
Trong : Y l hm lng tannin (mg/l), X1 l
nhit trch ly (oC) v X2 l thi gian trch ly
(pht).
M hnh b mt p ng (Hnh 4) cho thy hm
lng tannin thu c t qu trnh trch ph thuc
vo thi gian nhiu hn l nhit . Hm lng
tannin tng rt chm khi tng nhit trong
khong 70 n 100oC, trong khi hm lng ny
li tng rt nhanh khi thi gian trch ly tng trong
khong 15 n 90 pht. Tuy nhin khi thi gian
trch ly tng n mt lc no th hm lng
tannin khng tng c na v c chiu hng suy
gim (th hin qua cong ca b mt p ng).

Hnh 4: B mt p ng th hin nh hng ca nhit v thi gian trch ly n hm lng tannin

(mg/l) trong khong nhit 70-100oC
Gi tr cao nht trn b mt p ng c th tm
c bng cch thay gi tr X1 = 100oC vo
phng trnh 5 v thu c phng trnh 6.
Y = 215,17 + 7,2836 X2 0,05 X22
Trong Y l hm lng tannin (mg/l) v X2
thi gian trch ly (pht). Ly o hm Y ca
phng trnh 6. Cho Y=0, im dng X2 = 72,8
c tm thy.
Bng 6: Bin thin hm lng tannin theo nhit
v thi gian trch ly
X2 (pht) 15 72,8
Y + 0 -
Y (mg/l) 313,2 480,4
Kt qu xt du th hin Bng 6 cho thy ng
vi nhit 100oC, khi tng thi gian trch ly n
72,8 pht th hm lng tannin s cao nht (im
cao nht ca b mt p ng Hnh 4). Nu tip tc
tng thi gian trch ly th hm lng tannin s
khng tng v c chiu hng suy gim nhng
khng ng k.
Bn cnh , kt qu c trnh by Bng 7
(6) cho thy cc nghim thc 1, 5, 7, 8, 10 khng th
hin s khc bit ngha v mt thng k, iu
c ngha l khi tng nhit ln trong khong 110-
130oC s rt ngn c thi gian trch ly m vn c
th cho hiu sut trch tannin ti u (Adachi et al.,
2013). Kt qu thc nghim cho thy hm lng
tannin khng gim khi tng nhit trong khong
110-130oC, chng t tannin khng b oxy ha trong
90 qu trnh trch ly. Tannin c th b oxy ha khi c
mt ca oxy nhit tng i cao di tc
465,7 dng ca enzyme polyphenol oxydase (L Ngc
T, 2003). Tuy nhin, qu trnh trch ly thc hin
trong mi trng nc nn c th hn ch tip xc
tannin vi oxy nn c th lm gim ng k tin
trnh oxy ha. ng thi vic tng nhit cao s
gp phn v hot enzyme polyphenol oxydase, v
vy tannin thiu iu kin cn v qu trnh
oxy ha c th xy ra.

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Tp ch Khoa hc Trng i hc Cn Th Phn B: Nng nghip, Thy sn v Cng ngh Sinh hc: 36 (2015): 21-28

Bng 7: nh hng ca nhit v thi gian trch ly n hm lng tannin trong dch linh chi
T hp nghim thc
Nghim thc Hm lng tannin (mg/l)
[Nhit (oC) Thi gian (pht)]
1 100 - 75 (gn vi im ti u ca b mt p ng Hnh 6) 596,1d
2 110 - 15 401,7a
3 110 - 30 544,0c
4 110 - 45 532,1c
5 110 - 60 596,7d
6 120 - 15 428,1a
7 120 - 30 620,0d
8 120 - 45 630,9d
9 130 - 15 479,1b
10 130 - 30 615,2d
Ghi ch: Cc s liu trong bng l trung bnh ca 3 ln lp li, trong cng mt ct cc trung bnh nghim thc mang
ch s m ging nhau th khng khc bit c ngha thng k (p<0,05)
cc khong nhit v thi gian ny, hm water by using a macchinetta extractor.
lng tannin d on c tnh bng cch thay gi International Conference on Biology,
tr X1 (70100) v X2 (1590) vo phng Environment and Chemistry, 58, pp. 72-75.
trnh 5. Phng trnh tng quan gia hm lng 2. Askin R, Sasaki M, Goto M. 2007. Sub- and
tannin c c tnh v thc nghim c h s xc superitical fluid extraction of bioactive
nh tng quan kh cao (R2=0,901). Nh vy, c compound from Ganoderma Lucidum.
th da trn m hnh d on c lng hm Proceeding of International of Symposium
lng tannin trong khong nhit trch ly t 70 on Ecotobia Science, 07, pp. 575-577.
n 100oC vi thi gian t 1590 pht.
3. Atanassova M, Christova V. 2009.
4 KT LUN Determination of tannin content by
titrimetric method for comparision of
C th thit lp c tng quan tt gia different plant species. Journal of the
nhit v thi gian trch ly vi hm lng University of Chemical Technology and
polysaccharide (hoc tannin) 2 khong nhit Metallurgy, 44, pp. 413-415.
khc nhau (70100oC v 110130oC). Nhit
trch ly thp hn 100oC yu cu thi gian trch ly 4. Chan WK, Law HK, Lin ZB, Lau YL and
di hn c th t c gi tr hm lng Chan GC. 2007. Response of human
polysaccharide v tannin ti u. Tuy nhin khi dendritic cells to different
tng nhit cao hn 100oC th thi gian trch ly immunomodulatory polysaccharides derived
ngn m vn m bo c hiu sut trch ti u from mushroom and barley. International
(hm lng cao nht ca tannin v polysaccharide Immunology, 19, pp.891-899.
t c). Kt qu thc nghim cho thy nhit 5. Foster DS and Cornella TS. 1961.
trch ly 130oC trong 30 pht cho hm lng Colorimetric Method of Analysis.1961.
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