- Tgp chi Khoa hoc - Cong nghe Thdy sdn So 2/2014
THONG BAO KHOA HQC
TAN DUNG BA DAU TOM TlT QUA TRINH C H £ BIEN B O T DAM GIAU CAROTENOID BANG PHU^ONG PHAP KET HOfP HAI ENZYME PROTEASE D£ THU HOI CHITIN VA CHITOSAN
RECOVERY OF CHITIN AND CHITOSAN FROM SHRIMP HEAD WASTE OF CAROTENOID-RICH PROTEIN EXTRACTION PROCESS
BY USING COMBINING TWO PROTEASES
Pham Thi Dan Phif^g', Trang Si Trung' Ngay nhan bai' 29/11/2013, NgSy phan bi?n thong qua 22/5/2014, Ngsly duy?t dang: 02/6/2014
T O M T A T
Ba ddu torn tie qud trinh thu nhdn che phdm dgm gidu carotenoid bdng phuang phdp xu ly kel hap liai enzyme protease dirpc tgn dung de thu hoi chitin vd chitosan. Ket qud phdn tich cho thdy bd ddu tom con chira hdm lugmg protein vd khodng thdp dudi 10% vd 16,8% vd cdn tiep luc loai protein vd khodng de dgt yeu cdu ve chat Itrcmg cda chitin ky thugt.
Bdddu lom dugc tiep tuc xu ly bdng NaOH 2% trong!2 gia de khu protein vd xu ly HCI 4% trong 3 gia de khir khodng Sdn phdm chitin - chitosan duac ddnh gid cd chdt lupng tinh sgch cao vui hdm lugng protein vd khodng ihdp duai 1%, chitosan co do nhdt cao han 1000 cps.
Tic khoa. bd ddu tom, dam gidu carotenoid, thu nhdn, chilin, chitosan A B S T R A C T
Shrimp head waste from the production process of the carotenoid-rich protein by new method combining two proteases have been used for extraction of chilin and chitosan Analytical results showed that shrimp waste head content low amounts of protein and minerals of 10% and 16.8% respectively bul needed Jicrther treatment in order reach the commercial quality of chilin Shrimp head waste was treated with 2% NaOH for 12 hrs to remove protein and 4% HCI treatment for 3 hrs to remove the minerals. Resultant chitin and chitosan have high purity with low residual protein and minerals of less than 1% and high viscosity of 1000 cps.
Keywords- shrimp head waste, carolenoid - rich protein, extraction, chitin, chitosan
I. OAT V A N 0 £ Trong nhung nam g i n day. mOt s6 nghidn cuu Sir dung dau tdm dS sdn xuSt bOt dgm gidu da dup'c trien khai de su di^ng co hidu qua nguon phe carotenoid Id mdt hui^ng di mai, nham tdng cufrng li^u dau torn tir cac nha mdy che bien thuy sdn de hi$u qud cua vide sir dung ngufin nguydn lidu c6n eh^ bl^n chitin, chitosan va protein (Tnjng vd es, 2011).
Igi trong che bien thuy sdn vd nhu d u v4 protein va O^c bi^t, dau tom da duoc nghien cuu che bien san chit mdu phyc vu cho ngdnh che bien thyc phim. pham b0t dgm gidu carotenoid ldm phu gia trong ch4 Vi$t Nam Id mpt trong nhung nudc ch4 biln t6m biln thyc pham bang phuong phap thuy phdn ket hpp ldn nhit the gidi, san luong torn hdng n i m udc dgt hai enzyme protease (Alcalase vd Flavourzyme). Vdi 473.000 t i n , do d6 lup'ng d i u tfim logi thdi trong qud phuang phdp ndy, hi^u suat thu hoi va chit lupng bpt trinh e h l biln udc khoang 97.000 tan/ndm (Anh vd dgm gidu caratenoid thu nhdn tir diu tdm du^e ndng cs, 2011). Ddy Id ngu6n nguyen li^u cdn Igi tO cfing eao ddng k l so vdi phuang phdp su dyng enzyme dan, nghidp che biln tfim can dugc nghien ci>u d l thu ddc bidt Id ve hdm lupng pnatein, carotenoid vd miii, nhdn cdc san phim gid trj gia tang, vj cOa ehl phim thu dupc {Phup'ngva Luyen, 2013).
' ThS. Ph^m Thj Dan Phupng: Khoa C6ng ngh$ thuc phSm - Tnr&ng D?i hpc Nha Trang ' PGS. TS. Trang ST Trung- Tnr&ng Dai hgc Nha Trang
TRUdNG OAI HOC NHA TRANG • 37
Tgp chi Khoa hoc - Cong nghe Thuy sdn So 2/2014
Tuy nhien, hidn nay bd diu torn tip qua trinhc h l biln bpt dgm gidu carotenoid bdng phuang phap ket hffp Alcalase va Flavourzyme chua duac nghidn cuu de tan dyng de thu nhgn chitin, chitosan.
Chitin, dgc bidt la chitosan Id nhung polyme smh hoc CO nhieu irng dgng trong nfing nghidp, cong nghe thuc pham, moi truo'ng, cong nghp smh hpc (Hirano, 1996, Kumar, 2000). Theo Trung vd cs (2012).
hdm lup'ng chitin trong dau tom dat khoang 9,3%.
Do do, luang ba thai ndy Id nguon nguydn lieu rat phil hap de thu nhgn chitin vd chitosan, nhdm ndng cao hieu qua cua cfing nghp xir ly diu tfim de san xuat bfit dam gidu carotenoid Tren t h l gidi, viec thu nhgn da san pham tir diu torn da duac quan tam nghidn ciru (Chakrabarti, 2002;
Klomklao va cs. 2009). Trong bdi bdo ndy, chung tfii xin trinh bay ket qua nghien cuu tan dung ba tfim tir qua trinh che bien che pham dam giau carotenoid bing phuang phdp kit hpp hai enzyme protease d l thu h6i chitin vd ehtosan.
II. NGUYiN VAT LIEU vA PHlTCrNG PHAP NGHIEN CIJU
1. Nguyen vgt lifu
Ba ddu tom dupc thu hoi tir quy trinh thu nhgn che pham dgm giau carotenoid bang phuong phap ket hap Alcalase va Flavoursyme tgi Phong thi nghiem - Trudng Dai hpe Nha Trang theo quy trinh nghien cipu cua (Phuo'ng. P.T.D & Luyen TT, 2013).
Alcalase va Flavourzyme dupc mua tu Cong ty Novozymes (Dan Mgch). Alcalase 2,4 L cd hoat tinh Id 2,4 AU/g va Flavourzyme co hoat tinh 500 LAPU/g. Hoa chat sir dyng trong nghien cuu Id cdc logi hoa chdt tinh khiet.
2. PhiFO'ng phap nghien CLFU
2.1. Xac dinh che do khii protein vd khir khoang d§
thu nhan chitin tu' ba dau tom
Ba dau tfim the chan trang lay tir qua trinh thu nhdn c h l pham dam gidu carotenoid bdng phuang phdp ket hap enzyme Alcalase va Flavourzyme Sau do tiep tyc tien hanh tdch protein bang kiem loang va khCr khoang bdng acid loang de san xult chitin.
2.1.1. Xac dmh nfing dp NaOH su dung vd thdi gian de khu protein con Igi trong bd dau torn
Ba ddu tfim sau thu hoi, dup'c khu protein bdng NaOH^vfi'i cac nfing dfi khac nhau: 1, 2, 3%, irng vdi mfil nfing dp bd tom duoc khCp d cdc thdi gian khac nhau: 6, 12, 18 h. Che dp khu protem thich hpp duac chpn dya tren viec ddng gid ham luong protein cfin lai trong bd dau tfim
2.1.2. Xdc djnh ning 6p HCI su dyng vd thdi gian ah khir khodng cfin igi trong ba dau tfim
Ba d i u tdm sau qud trinh khLr protein, tilp tgc tiln hdnh khu khoang bang HCI vdi ede ning d^
khde nhau: 3. 4, 5%; ung vdi moi ning dfi ba torn dup'c khi> d cde thdi gian khde nhau: 3, 6, 12h.
C h l dp khir khodng thich hp'p dirp'c chpn dya trgn vipc ddnh gid hdm luang khoang con Igi trong ba d i u tfim.
2.1.3. Deacetyl chitin de thu nhgn chitosan Chitin tir ba d i u tfim s§ duac deacetyl d 65°C trong thdi gian 20 gid vdi NaOH 50% de thu nh§n chitosan (Trung vd cs, 2006). Chitin vd chitosan thu dup'c dup'c xdc djnh cdc chi tieu ehit lupng ca bdn, 2.2. Phuvng phcip phim tich
Hdm lup'ng I m va khoang dupc xac djnh theo phuang phdp chuan cua AOAC (1990). Hdm lirpng protein trong d i u tfim dup'c xac djnh theo phuong phap cua Boyer (1993). Hdm luang lipid dupc xac dinh theo phuang phap Folch vd es (1957). Ham luang chitin duac xac dmh bang phuang phdp cua Erika vd cs (2006). Ham luang protein trong chitin vd chitosan duac xdc dmh theo phuang phap microbiuret cua Ruth va cs {1964). Dp tan dupc xdc djnh theo phuang phdp cua Rao vd cs (2007). Dp nhdt bieu kien cua dung dich chitosan (1%, w/v trong acid acetic 1 %) duac do bdng nhfil ke Brookfield vd do dgc do bdng may do dp due HACH. Dfi deacetyl cua chitin dupc xde dinh theo phuang phap quang pho ciia Tan vd cs (2008).
2.3. Xir 1^ thong ke
S6 lieu bao cdo Id gia tn tmng binh cua 3 lln lgp lgi. Ket qud duac phdn tieh thong kd bdng phin mem Minltab va Microsoft Excel. Gid tri cua p < 0,05 duac xem la cfi ^ nghTa ve mat Uiong ke.
III. K t T QUA NGHIEN CLTU VA T H A O L U ^ N Ba diu torn tir quy trinh xu 1^ kit hpp hai enzyme protease de sdn xuat dgm giau carotenoid duoc xde djnh thdnh phan hoa hpc ca ban. Kit qud duyc trinh bdy d bang 1
Bang 1. Thdnh p h i n hoa hoc co- ban ciia ba torn Thinh phin h6a h^c
Ham lup'ng am Ham luyng protein' Hdm lup'ng khoang*
Hdm lugng lipid*
Hdm luyng chitin*
Ham l(r?ng (%) 79,1 ± 2,8 7,5 ±1,9 16.8 ±2,6 2,9 ±1.0 65,8 ± 3,3
•Kk qui tinh theo hdm lur^g chit kho tiiy$t d6i Trong bd dau tfim chira chii yeu la chitin vdi 65,8%, tiep theo la khodng (16,8%) va protein (7,5%).
38 • TRLfONG OAI HOC NHA TRANG
1 gp chl Khoa hgc - Cong nghe Thuy sdn So 2/2014
Thdnh phan chii yeu cila ba d i u tfim Id chitin. Tuynhidn, de dgt yeu cau ve chit luang eiia chitin ky thudt (ham lugng khodng va protein con lgi trong chitin < 1 % (Rao va cs, 2007) thi can tiep tyc logi phan khoang va protein cdn Igi. K i t qua eiJa qua trinh khif protein cdn lgi vd khi> khoang dupc trinh bay d hinh 2 vd hinh 3.
Ketqud cho thiy khi sir dyng NaOH 1% thi kha ndng loai protein d nong dp nay thap, luong protein cdn trong ba cao rat nhilu so vdi hai nong dfi NaOH 2% vd 3%, d cae dieu ki$n thdi gian xii ly khac nhau (hinh 1). 6 thdi gian xu ly Id 6h thi vdi ning dfi NaOH 1% lup'ng pnDtein cfin trong ba Id 4.2%, trong khi d ndng dp NaOH 2% va 3% thi lup'ng protein cdn lai tuang irng la 3,2% va 2,4%. Khi tang thdi gian xu ly tip 6h len 12h thi ham lup-ng protein cdn lai trong bd giam manh tir khoang 3% (NaOH 2% va 3%) xuong dgt tdi muc khoang 1 %, dgt yeu cau chit lugng eOa diitin ky thugt. Tuy nhien, Mil kdo ddi thdi gian xir ly len 18 h thi protein cdn lgi d ca hai nong dfi NaOH 2% va 3% lai giam rat it. Dilu ndy cd the do lugng protein con Igi nay cd lien ket chat che vdi chitin nen khd taeh ra, k l ca XLF ly dilu kidn NaOH 3% vd keo dai 18h (Trung va cs, 2010). Nhu vay, nong dp NaOH 2% vd XCP I;^ trong thdi gian 12 h id dilu kidn thich hgp de tach lugng protein cdn Igi tong ba de sdn xult chitin. K i t qua ndy phu hgp vdi nghidn eiru cua Stevens (2001) khi xir ly phe lidu torn de Uiu nhgn chitin vdi ning dp NaOH tir 2% den 3%.
ThMKbiBlAl^a)
Hinb 1. Anh bcrong cua thM gian vk nang il$ NaOH \& 1^
d i n h i m log'ng protein cdn l^i trong m i a chitin {xir ly a nhiet df phdng)
Cdc gia tri trung binh cua cpl co cac ky tif (a, b, c) khac nhau Ihi khac bifl coy nghm vi mat thong ke (p<O.OS)
K i t qua khu khodng cua ba cho thay trong khodng thdi gian xir l>? tir 3 h d i n 9 h thl ning dO HCI cd anh hudng rat Idn; kha nang khu khoang ciia HCI d ning dfi 3% thap han rat nhilu so vdi d cdc ning dfi xir 1^ HCI 4% va 5% (hinh 2). Khi nong dfi HCI tdng tir 3% ldn 5% thi lugng khoang cfin lgi trong bd giam tO g i n 4,5% xuong cdn khodng 1,2%
trong thdi gian Id 3 h. 6 nong dp HCI su dyng Id 4%
thi ham lugng khoang cdn Igi trong m i u chitin giam xuong < 1 % neu thdi gian xu ly la 6 h. Trong thdi gian xif ly Id 6 h thi hdm lugng khoang con Igt trong chitin khi XCP ly d nong dp HCI 4% vd 5% thl khde bipt khdng cd )/ nghTa thong ke (p > 0,05). Keo ddi thdi gian xir ly den 9 h thi n l u sir dyng nong dfi HCI 3% cung chua dgt dugc ham lugng khodng edn lgi trong chitin <1 % ma phai can ning dp HCI su dyng tif 4 - 5%. Tdm Igi, d l dgt ydu cau chitin ky thu§t (hdm lifgng khodng phdi dudi 1%) thi che dp tdch khoang thich hgp dupc chpn Id XLF ly bdng HCI 4%
trong 6 h, d nhidt dp phfing.
K i t qua ddnh gia chat lugng chitin vd chitosan thu hoi tir ba d i u tfim trong qua trinh thu hoi c h l phim DGC tif dau tdm cho thiy chl^n, chitosan cd tmh sgch cao (hdm lugng khoang vd protein cfin Igi
< 1 %) vd chitosan cfi dp nhoi cao (>1000 cps) va dfi dye thap (bang 2).
r^
lii IL ru
Hinli 2. Anh buong cua thai gian va nong dg HCI i v l y Oin b^m lu^nig kbofing cdn I^i trong man cbitin
( I U [;f a nhif t d$ phdng)
Cac gid tri tnmg binh co cdc ky tu (a. b. c) khac nhau thl khde bi4l co y nghia ve mgl ihdng ke QX0.05)
Bang 2. Chi tieu chat luong co ban cua chitin vd chitosan tiiu hoi tCp ba dau tdm trong qua trinh thu nhan che pham dam giau carotenoid
STT 1 Chi«u Chitin
1 2 3 4
MSu sac DO Sm (%) Hdm li/gng khoang (%) Ham lu-o'ng Protein {%) Chitosan
1 2 3 4 5 6 7 8
Mau sSc Bp Sm (%) Ham iifO'ng khoang (%) Hdm IU(?ng protein (%) Do nhdt (cps) Do deacetyl ho^ {%) Do tan (%) Dp aye (NIU)
Kit qua phfin tich*
Hong nhat 9,7 0,94 0,98 Trang sdng
9,4 0,87 0,89 1234 85,8 99,8 19,2
* Kit qu§ Unh Iheo Mm lut^g ch^t khd luy^t ^6i v6 Ctrac mb 'A cdm quan sa b^
TRUONG OAI HOC NHA TRANG • 3 9
Tap chi Khoa hoc - Cong nshe Thuy san So 2/2014
^^
Ba dau tom^
5 10 15 20 25 30 Gdcqaet2e(d9)
Hinh 3. Pbo nhieu xa tia X cua chitosan tbu nh^n tir ba dau torn
Dfi ran cua chitosan thu nhdn tir bd diu tfim dugc xdc djnh bdng phuang phdp nhilu xa «a X (X-ray diffraction) va dugc trinh bay d hinh 4. Pho nhieu xg ciia chitosan co mpt peak khodng 20 dp ciia goc quet 26. Op cao ciia peak tuang doi thip t h l hien dp rdn tuong doi thap ciia chitosan.
Hinh 4. Chitin (Irai) v i chitosan (phai) thu nhSn tfr ba d^u tdm
Nhu vdy bd diu tdm tir qud trinh sdn xudt chl pham dgm gidu carotenoid se dugc xis ly vdi NaOH 2% trong 12 gid de khu protein va khir khoang bang HCI 4% trong 6 gid de thu nhan chitin ky thugt vdi ham lugng protein va khoang con lai dudi 1% (hinh 5)
Khir protein bang NaOH^
RCra trung tinh
Khir khoang bang HCP
Deacetyl hod trong NaOH ddc"
Chitosan
Hinh 5. Stf do qui trinh Ihu nhSn cbitin - chitosan tir ba ddu torn
'Bdddu lom lir qua trinh sdn xuat bpi ilam giau carolenoid bang phmmg phdp xii ly ke! hpp hai enzyme protease;
^Nong do NaOH 2%. Ihm gian xir ly 12 b, nhiel dp phong.
' Nong dp HCI 4%. then gian 6h, nhif I dp phong.
'Deacetyl hoa bdng NaOH 50% irong ihoi gian 20 ho nhi^t dp 65'C IV. KtT LUAN
Ba dau torn tir qud trinh ^ u nhan che pham dgm gidu carotenoid bdng phuang phap xii ly kit hgp hai enzyme protease chua hdm luong chitin cao (65,8%), Id nguin nguyen lieu rdt phii hgp d l thu nhan chitin vd chitosan. Chit lugng chitm.
chitosan thu dupc cfi ham lugng protein vd khodfig con lgi thap (<1%) vd chitosan cfi dp nhdt cao (>1000cPs).
TAI LIEU THAM KHAO liing Vifl
1 Pham Tlii Dan PliupTig, TrSn Thj Luyen, 2013. Chi6t nil chl pbim dam giau carotenoid tir dau tom the chan tring. Tap chi Khoa hpc - C6ng nghe Thiiy san, 1: !25-l31.
Tieng Anh
I. Anh. P, T, Dieu. T, T. M., Arthur. P, J. M,, Carolien, K..& Simon, R B , 2011. Towards eco-agro industnal clusters in aquatic production' Tlie case of shnmp processing mdustry in Vietnam, Joumal of cieaner production, 19- 2107-2118.
3. AOAC, 1990, Official method of analysis (Voi. 15th) Arlington, VA: Association of official analytical chemists.
4. Boyer R.F, 1993. Modem expenmental biochemistry. Benjamin/Cummings Senes in the Life Sciences and Chemistry, Califomia, Vol 2: 51-55.
5 Chakrabarti. R., 2002 Carotenoprotein from tropical brown shrimp shell waste by enzymatic process. Food Biotechnol. 16;
81-90.
6 Enka I.D.R. Waldo, M A.M. Inocencio H C, Javier H, Jaime. L.M, & Francisco M,G.. 2006 Detemiination of Chitin and Protein Contents dunng the Isolation of Chitin from Shrimp Waste. Macromo! Biosci, 6: 340-347.
7 Folch, J.. Lees, M , & Sloane Stanley. G, H,, 1957 A simple method for the isolation and purification of total lipids from animal tissues. J, Biol Chem, 226: 497-509,
40 • TRUONG DAI HOC NHA TRANG
Tgp chi Khoa hoc - Cong nghe Thdy sdn So 2/2014
8. Hirano. S., 1996. Chitin biotechnology application. Biotechnology Annual Review, 2: 237-258.
9. Klomklao, S., Benjakul, S., Visessanguan, W., Kishunura, H., & Smipson, B. K, 2009. Extraction of carotenoprotein from black tiger shrimp shells with the aid of bluefisb trypsin. Journal of Food Biochemistry, 33- 201-217.
10. Kumar, R M. N. V., 2000. A review of chitin and chitosan applications. Reactive and Functional Polymers, 46, 1-27.
11 Rao, M. S., Nyein, K A , Trung, T. S., & Stevens, W. F, 2007. Optimum parameters for producUon of chitm and chitosan fix)m Squilla (S. empusa). Joumal of Applied Polymer Science, 103 3694-3700.
12. Ruth. F.L& Gill. D.M.. 1964. AMicro-Biuret method for estimatmg proteins Anal Biochem, 9 40M10
13. Stevens, W F, 2001. Production of chilin and chitosan: Refinement and sustainability of chemical and biological processing.
Chitm and Chitosan in life science. In T. Uragami, K. Kurila & T Fukamizo (Eds.), Proceedmgs 8th Intemational Conference on Chitm and Chitosan and 4th Asian Pacific Chitin and Chitosan Symposium Yamaguchi. Japan, 293.
14. Tan SC, Khor E, Tan TK, Wong SM, 1996. The degree of deacetylalion of chitosan. advocatmg the first denvative UV spectrophotometry method of determination. Talanta, 45. 713-719,
15. Trung, T. S., Thein-Han, W. W., Qui, N. T, Ng, CH. and Stevens, W F, 2006. Functional characteristics of shrimp chitosan and its membranes as affected by the degree of deacetylation.Bioresour Technol. 97: 659-663.
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