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. Biochem. 72, 248-254. 2. Devakate R.V., V.V.Patil, S.S.,T.Waje, B.N.Thorat. 2009. Purification and drying of bromelain. In: Separation and purification Technology. 64, 259264. 3. Dixon M. and Webb E.E. 1975. Enzyme, NewYork, Academic press. 4. Drauz.K, Groger.G, May.O. 2011. Enzyme catalysis in organic synthesis. Willey-VCH. 5. ng Th  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 systems containing PEO-PPO-PEO block copolymers, J. Chromatogr., B 807, p. 61-68. T Phn p, Thy s Sinh hc: 26 (2013): 162-170 170 15. Ravindra Babu, B., Rastogi N.K. and Raghavarao K.S.M.S. 2008. Liquid-liquid extraction of bromelain and polyphenol oxidase using aqueous two-phase system. Chemical Engineering and Processing: Process Intensification, 47, 83-89. 16. Soares, Paulo A.G., A.F.M. Vaz, M.T.S. Correia, A. Pessoa Jr, M. G. Carneiro-da-Cunha. 2012. Purification of bromelain from pineapple wastes by ethanol precipitation. In: Separation and Purification Technology. Elserver. 17. Tochi B.N, Wang Z, Xu S-Y and Zhang W, 2008, Therapeutic application of pineapple protease (bromelain): Review, Pakistan journal of nutrition, 7(4): 523-520. 18. Trt k h thng sc. . 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. 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