TẬN DỤNG PHẾ PHẨM KHÓM CẦU ĐÚC (HẬU GIANG) CHO QUÁ TRÌNH TRÍCH LY ENZYME BROMELAIN pptx

T Phn p, Thy s Sinh hc: 26 (2013): 162-170

162

TẬN DỤNG PHẾ PHẨM KHÓM CẦU ĐÚC (HẬU GIANG) CHO QUÁ TRÌNH
TRÍCH LY ENZYME BROMELAIN
Nguyn 
1
, Nguyn Minh Thy
2
  Dim Trang
3

1
Vingh Sinh hi hc C
2
c ng di hc C
3
H Thc phi hc C
Thông tin chung:
 18/01/2013
20/06/2013

Title:
Extraction of bromelain
enzyme from pineapple waste
Từ khóa:





Keywords:
Ammonium sulphate,
bromelain, Cau Duc (Hau
Giang), pineapple peaked-
shoots, vacuum drying
ABSTRACT
This research aimed to study the effect of some factors such as: (i)
precipitating agents (acetone, ethanol, ammonium sulfate); (ii)
ammonium sulfate concentrations (60, 70, 80%) and precipitation
temperatures (27
o
C and 4
o
C and (iii) drying methods (freeze-drying,
vacuum drying and by H
2
SO
4
) on bromelain preparation extracted from
pineapple peaked-shoots.
The research results showed that 82,86, 71,19 and 66,19% enzyme
activity recovery when precipitated by acetone, ethanol and ammonium
sulfate, respectively. The appropriate concentration of ammonium sulfate
and temperature for bromelain precipitation were 80% (v/v) and 27
o
C,
respectively. Vacuum drying (at 35
o
C for 9 hours) was the most effective
method for obtaining white bromelain powder with the moisture content

below 8%; 74,74% activity recovery compared with the total activity in
the crude extract.
TÓM TẮT
             
          
sulfate); (ii) ammonium         
(27
o

o

H
2
SO
4



           
          

o
ai
(35
o


enzyme 74,74%.

1 GIỚI THIỆU

      
enzyme thc vt ch
     u ng dng
trong y h  c phm. Trong y hc,
 
T Phn p, Thy s Sinh hc: 26 (2013): 162-170

163
trng m
   t qu, tr  ng, gi 
triu chng d   can thi  s t
ng c   u hiu trong
vic chm ch
chng hp thu thuc
(Tochi et al., 2008)   p thc
ph  c s d   
m th sa (Rabelo
et al., c thu nhn ch yu
t h Bromeliaceae c
t trong ph ph phm ca
di, v  n ph ph phm
m mt t l ln cng d
li    bin, khong 70% (Nguyn
 gii nhi
        ch
bromelain t ph ph phm bng nhi 
   t ta b   
y phun (Devakate
et al      
  c c nh kim loi (immobilized

metal affinity membrance) (Huali et al., 2008),
chit hai pha lng-lng (Ravindra et al., 2008),
nhiu ch ph     i. Tuy
 Vit Nam, mc nhi
sng da ln, vi ph
ph phm to ch phi ng dng
p thc phu (Li
Th Ng
c
sn ca Tnh Hu Giang. K
 bi  u sn ph 
c,
m    n tht qu 
trong ch bin ch chim kho
l   ph phm c  a enzyme
bromelain vt li Th
Ng
 i s  n c  n
xu   bi  ng ph ph phm t
 ng trng
 b t hoc
  bin rau
qu, phn ln ph ph
ng. Tn dng t
ngun ph phm s     n
gim thiu m     ng,
 kinh t chc
v hiu qu cho hong ch bin thc phm
   c phm. Do vy, m 
c

o qun b
 dng. Ho
li l ng dng thc t trong
thc phm, m ph    i
nhun cao cho sn xut c
2 PHƢƠNG PHÁP NGHIÊN CỨU
2.1 Phƣơng tiện nghiên cứu
  c thc hin t  
nghi   c phm, Vi 
c Sinh hcng
i hc C
Chồi ngọn trái khóm ( 1 c thu
nhn t a
Ti V Thanh, tnh Hu Giang.

Hình 1: Nguyên liệu thí nghiệm (chồi ngọn
trái khóm)
2.2 Phƣơng pháp nghiên cứu
2.2.1 Khng c
kt ta ethanol, aceton, ammonium
n hiu su
t chi ngn c
Ch  c nghi     t
   c b    ch chit.
Dch chinh  4
o
C vi t
5300 g ( loi b
c   t ta d 
v    a acetone (tinh khit),

ethanol (99,8%), ammonium sulfate (70%),
c bi t l 2 aceton : 1 dch
er ,1957), 4 ethanol : 1
d  i Th Ng   
T Phn p, Thy s Sinh hc: 26 (2013): 162-170

164
NH
4
)
2
SO
4
       472 g
(NH
4
)
2
SO
4

, 1994).
T 1,5 kg chi ngn c
nghic 750 ml d 
n bng nhau. Phn 1
(250 ml) cho t t 118 g mu 
 nhi
nh (27
o
a, ra

ta bng acetone l    a bng
H
2
SO
4
. Ph       
ln nhi (0-4
o
 
acetone (-20
o
C  t  nhi  -20
o
C
t t
t phn 2, phn 3 (250 ml) sau khi
n nhi (0-4
o

-20
o
  nhit
 -20
o
C trong 2 gi    
Drauz et al. 
h  nhi
thp (<0
o
C) trong sut ta). Sau

  a xut hi     
s
2.2.2 Khng ca n
 kt ta
n hiu sut thu hi ho
bromelain
  c b    i 2
 
  kt t
o
C.
Sa bng H
2
SO
4
m 
4
o
C.
2.2.3 Khng c
n hoa enzyme

c b  th
y, bao
gm s-40
o
C), sy tron
m (5
o
     

o
C). Bt
t ta
v 27
o
C,
thi gian cho mua
y ta b cp.
      Lng enzyme
c (g) (s dn
t  oa enzyme
bromelain b    (1974)
ci tin    ng protein bng
u sut
thu hi ho   bromelain (%). So
u qu kinh t ct ta
y.
 X t qu: Kt qu c x ng

3 KẾT QUẢ THẢO LUẬN
Kt qu c t  ban
u cho thy ngt lc protease
cao nh         
t lc trong chi ngn gp 9,92
l     p 4,38 ln trong v
t qu  vi nhi
c    c v s hin din
ca pro    phn c 
et al., 2008; Li Th Ng
2009).

3.1 Ảnh hƣởng của các tác nhân kết tủa
đến kết quả thu nhận bromelain thô từ
chồi ngọn
      
phn chi ngn c    
kt ta vp, nhm
chc h
    t qu thu nhn enzyme
      t t 
     Bng 1. Ho 
enzyme thu hc sau khi sa tng
a mang lng nhau.
Kt qu th hin cho thy hiu sut thu hi
hoa bt cao
nht (82,86%) so v   
amonium sulfate (66,52%). Theo Devakate et
al.        
protein thu h c khi kt t  n
    n 40-60% (w/v)
ammonium sulfate  nhi 4
o
C. V
 loi b c phn
protein tng
 ammonium sulfate th t phn
protein b kt ta. M    lnh
 hn ch c s mt hoa
bromelain khi kt t  u sut thu hi
ho
T Phn p, Thy s Sinh hc: 26 (2013): 162-170

165
Bảng 1: Ảnh hƣởng của tác nhân tủa đến quá trình trích ly enzyme bromelain
Tác nhân tủa
HSTH hoạt tính
enzyme (%)
Hoạt tính riêng
(U/mg)
Khối lƣợng bột
enzyme (g)
Chi phí sản xuất**
20000 TU (VNĐ)
Ammonium sulfate (SA)



63.559
Acetone



124.888
Ethanol



118.893
 lch chun) c

Hoa

       
  i ammonium sulfate. S  t
 do  nhi thp, s kt hp gia
        c trong

enzyme kt hp li v     n
nh c      
Gortner (1957); Su et al. (1967) nhiu dung
c s
dng cho vic kt ta bromelain. M 
t t
trong dung d i ra khi ta d
y do nhi b
thp, vn
trong bt enzyme sau khi s  i s hin
din ca ammonium sulfate trong ch phm
n kh 
ca bromelain.
Khng b 
c khi ta b
khi ta b ba
   c tin  i m ng
d
         
      
      
 c khi ta bng ammonium sulfate
(11,90 U/     
enzyme ta b    
acetone (14,15 U/mg). V    

        
   m hng s  n,
ng thc hp dn gi
t protein 
kt ta protein. M     
        i
m lt v
t     protein t li vi
a xung. Tuy v
        ng
u ki ng
tm khng
b  i
ammonium sulfate  n t ta
protein rt tt.
Khi tinh ta bromelain b
h        c
 nhau. Ta bng ammonium sulfate d
ti    
thc hin ngay  nhi  ng
Th Thu (2004) khi s d
 kt ti ti
nhi l hn ch s mt hoa

 kt t
    nh d  n nhi 
khong 0-4
o
c
gi lnh -20

o

r dn s bin
 i s   a
ethanol trong dung d   t trong
nhng y      
l   a bng dung    
tng th a d 
  t nhiu so v ng dch
  u (ta bng acetone th  
n so vng d
vi ethanol th       
      y ta ra khi
dung dc bin tit ta
vn.
 sn xut
 hoi
t tc thc
hin. V  nh kinh t    a
b         a
b      
    p 1,95 ln so vi ta
b      p
T Phn p, Thy s Sinh hc: 26 (2013): 162-170

166
1,90 lt ln na cho thc
s bt li na ca bng dung
i nhng kt qu nhc 
   thy rng s dng ammonium

  kt t     phn
chi ngn c      
nghi        
thc hi c h
thu so vi.
3.2 Ảnh hƣởng của nồng độ ammonium
sulfate và nhiệt độ kết tủa đến hoạt tính
của enzyme bromelain thô
     kt ta
       
dung d    ng loi protein
    b kt ta  nhng nng
      
  u qu c   
ammonium sulfate (t 60 -  
kt ta (4
o
  
o
 n ho   
ng protein ca bc th hin 

   cp, nhi   u t quan
trng       
 c bit khi s d  t
tt ta
bromelain b
khi kt tn kh 
ng protein trong dung
dch. Kt qu thu nhc cho thy khi kt

ta dch chi ngn bng ammonium sulfate 
nhi  4
o
      
        
nhi 27
o
C  bt k n mu
nhi lnh (4
o
t ta protein
   n ch s bi  n ra tt
 ctv. (1994)
u v ng ca nhi kt
t        
ng
nh rng ho  a ch ph h ly 
nhi 4
o
 nhi 27
o
C.
ng thi kt qu ra rng
khi n  mu     
    ng
 n 80% ammonium
sulfate cho kt qu thu hi ho
ng protein tt nht  c hai mc nhit
 
o


o
t qu t cao nht ti
n 80% mui  nhi 4
o

qua thc nghim cho tht ta 
nhi 4
o
c hi
  
o
  t
b nh nhi ng  4
o
t
t

Hình 2: Hiệu suất thu hồi hoạt tính và hàm
lƣợng protein khi kết tủa ở những nồng độ
ammonium sulfate và nhiệt độ khác nhau
m thc 1: 
o
C; Nghim thc

o
C; Nghim th
o
C;
Nghim th

o
C; Nghim thc 5: 70% SA

o
C; Nghim th
o
C
Hiu sut thu hi hoa
bng ammonium sulfate  n   
 27
o
C cho kt qu 
u kt ta  nhit
 4
o
C  n 
tt nht khi kt ta  nhi   
o
C).
       
(1994), khi kt ta bromelain t ng
ammonium sulfate     t  n
  t lun rng  n  70%
ammonium sulfate cho hiu sut thu hi hot
t. S t kt qu 
do trong chi ng     
ng protein cao gp nhiu ln so vi 
phy  n
mui th t t
b       n ra

 n 80% hu h
u b kt t
Kh     a bt enzyme
c khi kt ta bromelain  nhng nng
T Phn p, Thy s Sinh hc: 26 (2013): 162-170

167
       
kt qu th hin   y ho 
 a mu kt ta  n  60%
c kt
ta v mui  c nhi
  
o
     gi   
n  60% mu  n l  
trong dung dch b kt t   
 mu ng protein tp
b kt ta s m ho
ca mu. Ho   a mu bromelain
kt ta  nhi 4
o
u kt ta
 nhi  27
o
C. Nhi     
     t trong cu t ca
u, s  c
gian c      
s mt ho  a enzyme. Nguy 

  . (1994) cho rng s kt ta
bromelain bng ammonium sulfate  nhi
thn ngh
th hn ch t mt hoa enzyme
t ta  nhi 27
o

 hoa enzyme
bromelain.

Hình 3: Hoạt tính riêng enzyme khi kết tủa ở
những nồng độ SA và nhiệt độ khác nhau
Nghim th
o
C ; Nghim thc 2: 70%

o
C ; Nghim th
o
C ; Nghim
thc 4 
o
C ; Nghim thc 5 
o
C ; Nghim th
o
C
Kh ng b   c khi ta
vi SA    
th hin  

Hình 4: Khối lƣợng bột enzyme thu đƣợc khi
kết tủa ở các nồng độ chất tủa và nhiệt độ
khác nhau
m th
o
C ; Nghim thc

o
C ; Nghim th
o
C ;
Nghim th
o
C ; Nghim thc 5: 70%

o
C ; Nghim th
o
C
Kh ng ca b   c
 hiu khi m c kt ta vi nng
      i
ammonium sulfate  n 
kt ta protein c  
ch
  a enzyme v  a nhiu
protein kh      
nhau, nh 
ra bt ta vi ammonium sulfate 

nh     ng bt
  c khi kt ta  nhi  4
o
C
nhi   t ta  nhi  27
o
C do 
nhi  27
o
C kh   bi  a
enzyme vn di     
u.
3.3 Ảnh hƣởng của phƣơng pháp sấy đến
hoạt tính bột enzyme bromelain thô
Nhi i gian su t quan
trng n hoa ch phm
y  nhi  t
hoa enzyme do protein b bi
y  nhi thi gian
si s hin
din ca      t   
protease s di   gim ho  a
enzyme. Kt qu  t
 hiu sut thu hi ho
gi  u sy b   
0
2
4
6
8

10
12
1 2 3 4 5 6
Nghiệm thức
Hoạt tính riêng (U/mg) .
0
2
4
6
8
10
12
14
1 2 3 4 5 6
Nghiệm thức
Khối lượng bột enzyme (g) .
T Phn p, Thy s Sinh hc: 26 (2013): 162-170

168
  hin
 a mu sy b   
    s dng H
2
SO
4

(Bng 2).
Bảng 2. Ảnh hƣởng của phƣơng pháp sấy đến chất lƣợng, khối lƣợng và thời gian sấy bột enzyme
bromelain thô
Phƣơng pháp sấy

Hiệu suất thu hồi hoạt
tính enzyme (%)
Hoạt tính riêng của
enzyme (U/mg)
Khối lƣợng bột
enzyme (g)
Độ ẩm
(%)
Thời gian sấy
(giờ)





9
Sy bng H
2
SO
4





17






15
 lch chun) c 
       
hoc 85,73% so vi
      a bt enzyme
t 11,96 (U/mg). S    35
o
C
trong 9 gi  c 74,74% hot  
v       a b t
10,07 (U/mg). V
ng H
2
SO
4
 nhi 4-5
o
C trong
17 gi     c thu h 
ho     t 9,88 (U/mg).
y, hoa bromelain 
khi s ca
y thp (-40
o

c c       
  c. Theo Tr   
s li chng sn phm tt
       c ca sn

phm bn m trc tip t trng
n sang trng
ng  iu kit thp. Devakate et
al   u qu ca s  
sch kt
qu   c 95% ho  c gi li
 i vi
sy phun.
S 
         
bo qun trong th     n
t). So vi s
c ti   nhi    u
(35
o
a bromelain sau khi sy
thp sy bng
H
2
SO
4
 nhi  4
o
  t qu  t
    m  th   u
   n nhit (nhit
 t
o
 nhi khong 35
o

C
vn gi c ho   i t  
nhi lnh (4
o
C).
Kh ng b     

y bng H
2
SO
4
. Sau khi st
 t th
rt nhiu so vi sn ph
 m 7,9%, sy bng H
2
SO
4
 m ca sn
phm cut 6,80%. S t v  m
     t v kh   nh
n s nh ca ch phm bromelain
trong thi gian bo qu  thc hi 
y b m bo qun an
      
chim thi gian ngn nht khong 9 gi, trong
khi sy bng H
2
SO
4

phi mt 17 gi i

i gian s)
     i m c khi
  y ph     n
-20
o
t b s n
c tt tic t 
c s dng ry
gii ca b     t
  i ca Merck (Bng 3) cho
thy hong trong 1 g bt enz
phm th     i
     a enzyme

vy, nu s d thy
u qu thy gii s 
i (Merck).

T Phn p, Thy s Sinh hc: 26 (2013): 162-170

169
Bảng 3: So sánh bột enzyme bromelain thành phẩm và bột enzyme thƣơng mại
Loại bromelain
Hoạt tính trong 1 g
enzyme (TU/g)
Hoạt tính riêng
(TU/mg protein)
m

1705
a

15,48
a

i (Merck)
1950
b
10,55
b
t nhng s  mc

4 KẾT LUẬN
  n din trong tt c 
phn ph ph phm c    
 t nhiu nht trong phn chi ngn ca

Kt ta bromelain chi ngn bng acetone
cho kt qu 82,86% hoc thu
hi. Kt ta bng ethanol 79,19% hot 
      u. Kt ta bng
ammonium sulfate hiu sut thu hi ho
t 66,19%.
N  kt
ta dch chi ng

o
C.
Thch

t qu 
hot.
S    35
o
C trong 9 gi thu
c bng,
 m < 8%, hiu sut thu hi ho 
t 74,74%.
TÀI LIỆU THAM KHẢO
1. Bradford, M.M. 1976. A rapid and sensitive
mocrogam quanlities of protein utilizing the
principle of protein dye biding, Anal.
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2. Devakate R.V., V.V.Patil, S.S.,T.Waje,
B.N.Thorat. 2009. Purification and drying of
bromelain. In: Separation and purification
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NewYork, Academic press.
4. Drauz.K, Groger.G, May.O. 2011. Enzyme
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Phm Thu Thy, Nguyn Th .
2004. g ngh enzyme. NXB Khoa hc K
thui. 16, 230-231.
6. Evens M. 2006. Extraction et formulation de la


7. Heinicke R.M and Gortner W.A. 1957. Stem
bromelain-A new protease preparation from

pineaaple 417 plants, Econ. Bot. 11, 225-234.
8. Huali N., S. Li, Y. Zhou, T. Chen, Z. He, S.
Su, H. Zhang, Y. Xue, L. Zhu. 2008.
Purification of bromelain using immobilized
metal affinity membranes. Journal of
Biotechnology 136S, 402459.
9. Kunitz M. 1974. Determination of proteolytic
activity by the casein digestion method. J. Gen.
Physiol. 30, 291.
10. Li Th Ng
to ch phm enzyme bromelain t ph ph
phm da, TKhoa hPn
-tp 7, s 2:203-ng i
hnghii.
11. Nguyu ph phm
da  
i hp,
i.
12. Nguy
 Thanh Mai. 1994. N
cn xu 
nghim thu cp B B91-07-03.
13. Pessoa Jr. A. and B.V. Kilikian. 2005.


14. Rabelo A.P.B, Tambourgi E.B, Pessoa Jr.A. 2004.
Bromelain partitioning in twophase aqueous
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