Trgng lugng phan tir frung binh cua Carrageenan va oligosaccharid dugc xac dinh theo phuang phap do dg nhdt bing nhdt kl Ubeldt.. Tir ket qua thu dugc cho thiy trgng lugng phan tir tmng[r]
Trang 1Tieu ban: HOAT C H A T SINH HQC BI^N 677
NGHIEN ClTU tTNG DUNG K - C A R R A G E E N A N
OLIGOSACCHARID TlT RONG BIEN CO HOAT TINH SINH HOC
TRONG CHE BIEN VA BAO QUAN THl/C PHAJM
Tran Dinh Toai'*, Pham Hong Hai', Nguyen Van Thiet^
' Viin Hod hpc, Viin Khoa hpc vd Cong nghi Viet Nam
^Vien Cong nghi Sinh hpc, Viin Khoa hpc vd Cong ngh? Viit Nam
I M d DAU
Carrageenan (Car) la polysaccharide ciia galactose ggi la galactan Galactose frong mach galactan cd thi la a (l-3)-D-galactose (G), |3(I-4)-D-galactose (D) hoac P(l-4)-3,6-anhydro-D-galactose (DA)[1]
Carrageenan dong thdi la sulfat polysaccharide Ngoai mach polysaccharide chinh edn cd the CO cac nhom sulfat dugc gan vao d nhung vj tri va s6 lugng khac nhau tao nen cac sulfat polysaccharide va co ky hieu rieng nhu: X - , K - , i - , v - carrageenan [2]
Vi du, carrageenan tir rong H6ng Van Euc gelatinae dugc xac lap la K- carrageenan (hinh 1)
[3]-Hinh 1 K- carrageenan chiet iir rong Hong Van Eucheuma geletinae
Carrageenan de tan trong nude, cac dung djch carrageenan cd do nhdt rat Idn Chinh vi vay,
d ndng do rit nhd (1%), carrageenan ciing cd the tao gel Carrageenan de tuong tac lien kit vdi protein va cac polysaccharid khac [4] Chinh vi co nhimg tinh chat dac trung neu fren, Carrageenan dugc img dung trong nhieu ITnh vyc che bien thyc pham khac nhau Dac biet, carrageenan khdng the thieu dugc frong c6ng nghiep che bien sua [5] Carrageenan Oligosaccharide cd thi thu dugc bang each dimg enzyme thuy phan Carrageenan [6] Carrageenan cung nhu oligosaccharid ciia no cd boat tinh sinh hgc rat cao [7]
Do do, chung toi thiiy phan Carrageenan tao oligosaccharide va nghien ciiu img dung lam phu gia bao quan, chi bien thyc pham (biin tir tinh bgt gao)
H NGUYEN LIEU VA PHU'ONG PHAP NGHIEN c i r u
I.Nguyen lieu
K- carrageenan chiet tir rong H6ng Van Eucheuma geletinae [3]
Enzyme Phitopain do Trung tam "Ky nghe sinh hgc", Vi?n HLKH Nga cung cip
Trang 2678 Tuyen tjp Hgi nghi KHOA HQC T O A N Q U Q C V £ SINH HQC BI^N VA P H A T T R I ^ N B ^ N VLTNG
2 Phuong phap nghien ciiTi
- Phuang phdp xdc dinh trpng luang phdn tir m
Phuang phap do do nhdt de xac djnh trgng lugng phan tii trung binh cua Carrageenan va Carrageenan oligosaccharid theo he thiirc Mark - Houwink: [T]] = K.M"
- Phuang phdp pho xdc lap cdu true
Cac phuang phap ph6 cho phep xac lap cau friic cua carrageenan va oligosaccharid
Phuang phap ESI - MS (phuang phap sic ki long khii pho co bg kit ndi phun dien)
(Negative-ion electrospray ionisation-mass spectrometry) [9]
Pho cgng hudng tir hat nhan [10]
- Khdo sdt dp bin nen ep ciia banh tinh bpt
Ve nguyen tac, de xac djnh do ben cua mgt vat lieu ngudi ta su dung phucmg phap nen, ep hay keo vat do theo mgt chieu nao do tdi khi miu bj pha hiiy
D6i vdi bun, la mgt loai thyc phim nen dl xac djnh dg deo, dai ciia bun tbi hien nay chua
CO may nao thyc hien chiic nang nay, fren thyc tl ngudi ta danh gia dg deo, dai ciia bun bing lay phieu tham do cua nhurng ngudi tieu dimg san pham Do do, chiing tdi chi tao miu banh tinh bgt
CO hinh fru lam vat lieu va tir do xacdinh do bin nen ep Mlu banh cd hinh fry, vdi dudng kinh:
d = 6,5cm, chieu dai: 1 = 12cm M6i mgt miu dugc cit ra lam hai mau nhd cd chieu dai: 1, = 6cm Mgt mau dugc nen theo chieu dgc, mgt miu dugc nen theo chilu ngang
Do ben nen ep nay dugc xac djnh tai Trung tam ky thuat 1 thugc Tong cue tieu chuin do ludng chat lugng tren may nen ep van nang Insfron (My) vdi t i c do ep la 15mm/pbiit
- Xdc dinh cdc chi tiiu vi sinh ciia bun
xac
"^ Cac miu bim sau khi dugc cho them cac chit bao quan vdi ham lugng khac nhau dugc
djnh cac chi tieu vi sinh tai Trung tam ky thuat 1 thugc Ting eye Tieu chuin Do ludng Chit lugng
Cac chi tieu vi smh bao gIm: ting sd vi khuin hilu khi, tong nam men va n i m moc tone
Coliforms [U] ' ^
- Phuang phdp xir ly s6 liiu
Ket qua thyc nghi?m dugc xu ly theo phuang phap binh phuang toi thilu tren may tinh dl tim phuang trinh h6i quy m6 ta tuang hgp kit qua nhan dugc Phan tich thong ke dugc thuc hi?n theo cac tieu chuan Student va Fisher [12]
III KET QUA NGHIEN ClTU
1 Nghien cihi thu nhan oligosaccharid
De thu nhan Carrageenan oligosaccharid, tiln hanh thiiy phan Carrageenan bing dung dich enzyme a nhiet dg 60 C Trgng lugng phan tir frung binh cua Carrageenan va oligosaccharid dugc xac dinh theo phuang phap do dg nhdt bing nhdt kl Ubeldt
Vdi cac gia trj K va a dac frung da bilt, theo he thiic Mark - Houwink: [ri] = K M" xac
! ' " ' ; , ^ J ^ ^ ^ 9 " g lugng phan tu frung binh cua Carrageenan va Carrageenan oligosaccharid la M
- 173,8 (kDa) va M = 27,7 (kDa) tuang irng
Tir ket qua thu dugc cho thiy trgng lugng phan tir tmng binh cua oligosaccharid nh6 ban so
\ a i trgng lugng phan tu trung binh cua Carrageenan ban diu Dilu do chimg to thuy phan da cit ngan mach polysaccharid ciia Carrageenan
Trang 3Tieu ban: HOAT C H A T SINH HOC BI^N 679
- Khdo sdt cdu true cua Carrageenan oligosaccharid bdng phuang phdp pho
Pho khoi luang
Do Carrageenan ciing nhu oligosaccharid deu la polysaccharid da dien tich nen ph6 kh6i ciia chiing rat phirc tap bao g6m nhieu dinh va cac dinh lai chen sat nhau Ket qua phan tich sy phan manh ciia chiing trong ph6 MS ciia Carrageenan oligosaccharid (binh 2) dugc the hien d bang 1 Tir dd co the m6 ta sy phan manh theo sa do fren hinh 3
Bang 1 Su> phan manh cua Carrageenan oligosaccharid
Manh Oligosaccharid
[Galp4S - H2O - K""]"'
[Galp4SAnGalp - C2H4O2 - K"]"'
[Galp4SAnGalp - H2O - K"]"'
[Galp4SAnGalp + C4H3O3 - K*]"'
[AnGalpGalp4SAnGalp - C2H2 - K " ] '
[Galp4SAnGalpGalp4SAnGalp - C2H4O3 - K^]''
[Galp4SAnGalpGalp4SAnGalp - C2H4O - K^]"'
m/z
241
341
385
467
503
611
647
Display Repx>rt - Selected Window Selected Analysis
lAo 3ba xo SJL AA al.iU.iS>»7rv ••
M50 T r ^ i (teport V « (At 0|K2)
Hinh 2 Pho MS cua Carrageenan oligosaccharid
Tir kit qua tren ta thay nhung manh c6 tin suit cao fren ph6, the hifn rang viec phan manh ciia chung tuin theo ca che phin manh ciia cacbohydrat da dugc c6ng b6 frudc day [13] Sy phan minh cua chiing chii yeu xay ra d lien ket glycosit giira cac monomer Day la nhirng vi tri
ma nang lugng lien ket ciia chiing thap ban so vdi nhimg lien ket con lai frong phan tir
Pho cpng huang tir hat nhdn
Phi cgng hudng tir hat nhan cua san phim thuy phan dugc trinh bay fren cac hinh 4, 5
Trang 4680
M2-CARAGEENXN.OLIGOSACCHARIDE-D20-1H M2-CJUIAGEEKXH.OLIGOSACCHARIDB-D20-C13CPD
Ml^
UWJ _.,,
%
Hinh 4 Ph6 1H- NMR cua Carrageenan
'^ Oligosaccharid
rtiiiiWi<>iiiii«wm<imni
Hinh 5 Pho 13C-NMR cua Carrageenan
Oligosaccharid
Phan tich cac tin hieu phi NMR, xac djnh do chuyen djch hda hgc cua cac mau Oligosaccharid nhu sau:
Bang 2: Do chuyen dich hda hgc 13C NMR va 1H NMR ciia m i u Oligosaccharid
P(1,3)-D-galactose
p(1,4)-3,6 anhydro
galactose
C1 H1 103,03 5,043d 95,72 5,527d
C2 H2 70,13 4,031i 70,43 4,625br
C3 H3 79,34 4,397dd 79,73 4,942d
C4 H4 74,46 5,255d 78,87 5,043d
C5 H5 75,31 4,228br 77,31 5,063s
C6 H6 61,82 4,228br 70.01 4,560br
Qua vifc phan tich va so sanh cac dir lieu ve ph6 cgng hudng tir hat nhan cua Carrageenan [3] va Oligosaccharid cho thay qua trinh thuy phan khong lam thay d6i don vj ciu tnic ma chi pha vd cac lien ket glycosit lam cat ngan mach polymer ciia Carrageenan
2 Nghien cuu sir dung Carrageenan oligosaccharid lam phu gia bao quan, che bien thuc pham (bun lam tir tinh bot gao)
- Khdo sdt tdc dung ciia Carrageenan oligosaccharid lam tdng dp ciing cua banh tinh bpt
Tac dung ciia carrageenan oligosaccharid lam tang do cung cua banh tinh bgt dugc the hi?n
d do ben nen dgc, nen ngang cua banh tinh bgt
Cac ket qua xac djnh lyc nen dgc va lyc nen ngang tac dgng tdi miu banh tinh bdt dl danh gia tac dung ciia phu gia oligosaccharid dugc ghi frong bang 3
Ket qua tien hanh xu ly s6 lieu tim phuang trinh hoi quy md ta quan hg giira lyc nen dgc
vdi nong dg chat phu gia>' = f(x) va quan be giira lyc nen ngang vdi nIng dg chat phy gia z =
<p (x) cho thiy cac quan he nay cd dang phuang trinh tdng quat cua mgt parabol:
A
Cac he s6 bo, b|, b2 dugc xac djnh theo phuong phap binh phuong toi thilu
Ti s6 tuong quan dac frung cho do chat che cua quan he phi tuyln dugc tinh nhu sau:
Trang 5Tieu ban: HOAT C H A T SINH HOC BI^N 681
0 CO gia tri tir 0 den 1 va 9 cang Idn thi quan he phi tuyen tim dugc cang co do tin ciy cao Cac sd lieu trong bang 3dugc xir ly tren may tinh theo phuang phap da neu cho kit qua nhu sau:
P h u a n g trinh boi quy m6 ta sy phu thugc vao ham lugng oligosaccharid ciia lyc nen dgc va lyc nen ngang deu co dang phi tuyen nhu sau:
- P h u a n g trinh boi quy d6i vdi lyc nen dgc:
ya = 1358,7-I-2107x2 - 2932,1x2^ Vdi: 9 = 0,967
- Phuang trinh hdi quy ddi vdi lyc nen ngang:
Zd= 1192,2-1-604x2-^-299,5x2^ Vdi: 9 = 0,99
Cac sd lieu thyc nghiem va tinh toan ciing dugc dua vao bang 3
Bang 3 K i t qua thyc nghifm va tinh toan cac thdng sd dg cii'ng cua banh tinh bpt khi su>
dung c h i t phu gia oligosaccharid
STT
1
2
3
4
5
6
So
Sanh
oligosaccharid,
% 0,05
0,1 0,2 0,3 0,4 0,5
Han the (0,5%)
Lyc nen dgc, N
ya
1425
1587
1661
1701
1745
2144
1530
A
y"
1456
1540
1662
1727
1732
Lyc nen ngang, N
Zd
1217
1267
1318
1402
1482
1812
1190
A
Za
1223
1256
1325
1400
1482
Qua nhimg kit qua thyc nghiem thu dugc cho thay rang oligosaccharid thyc sy la chit phy gia chi biln va bao quin co tic dyng lam tang do cirng cua banh tinh bgt diing de lam bun Dieu nay co thi giii thich nhu sau: oligosaccharid cd mach ngan, de tan frong nude nen de dang phan tan lam tang sy tuong tie vdi tinh bgt tao cho tinh bgt co dg cung cao ban, ben chac hon
- Cdc chi tieu vi sinh
D I danh gia khi nang bao quan ciia carrageenan va oligosaccharid, chiing tdi tien banh thi
nghiem tren cic miu bun, dugc them cac chit phu gia khac nhau trong qua trinh sin xuit Ket
qua so sinh hieu qua Ore che sy phat trien ciia vi sinh vat cua cac chit phy gia dugc ghi frong bing 4
Tir cac so lieu thyc nghi?m thu dugc cho thay rang chi co nhom vi khuin hieu khi phat triln manh theo thdi gian Sau 3 ngay, t6ng s6 te bio d?it tdi 10 CFU/g frong tat ca cac mau thi
nghiem Nam men va moc bj uc che nhieu hon, sd te bao chi vugt qua 10* CFU/g Coliforms hau
nhu bj lie chi hoan toan Nhu vay, carrageenan oligosaccharid cung nhu ban the va chitosan deu
cd tic dung ire chi vi sinh vat gin tuong ty nhu nhau
Trang 6682 Tuyen tap Hpi nghi KHOA HQC T O A N Q U O C V £ SINH HQC BI^N VA P H A T T R I 6 N B £ N VCTNG
Bang 4 Sy phat tri§n ciia vl sinh v^t trong thyc phim khi bao quan
1 Oligosaccharid Carrageenan Han the Chitosan
sau 24 gio' Tong vi khuan h i i u khi, CFU/g
Tong n i m men + moc, CFU/g
Tong Coliforms, MPN/g
8,5.10^
5,1.10' Khong phat hien
8,6.10^
4,6.10"
Khong
6,7.10^
3,8.10"
Khong
2,6.10^ 2,3.10" Khong sau 48 g i d
Tong vi khuan hieu khi, CFU/g
Tong nam men + moc, CFU/g
Tong Coliforms, MPN/g
1,3.10' 2,9.10^
Khong phat hien
8,8.10' 1,7.10^
0,36
1,3.10' 2,6.10^
Khong
5,9.10^ 2,8.10^ 0,36 sau 72 gio
Tong vi khuan hieu khi, CFU/g
Tong nam men + moc, CFU/g
Tong Coliforms, MPN/g
1,5.10' 2,4.10^
Khong phat hi^n
9.10' 2,7.10^
0,36
1,4.10' 2,6.10^
0,36
9.10' 2,6.10^ 0,36
TV KET LUAN
Tir cac ket qui nghien ciiu, chiing t6i rut ra cic ket luan sau:
1 Thiiy phan kh6ng lam thay d6i ciu true, chi cd tic dung phi vd cac lien kit glycosit lam cat ngan mach cua Carrageenan Sau khi thuy phin, Carrageenan oligosaccharid thu dugc co kh6i lugng phin tir tmng binh (27,7 kDa) giim so vdi khii lugng phin tii trung binh ciia Carrageenan ban diu (173,8 kDa)
2 Carrageenan oligosaccharid co tie dung lam tang do ciing ciia banh tinh bgt diing d l lam biin Ham lugng t6i uu oligosaccharid nay dl tang do ciing cua tinh bgt cd gia tri la 0,2%, frong khi ham lugng t6i uu cua phu gia Carrageenan cung nhu bin the la 0,5%
3 Nhu vay, Carrageenan oligosaccharide co t h i diing lam phu gia che biln va b i o quan thay the co hieu qui va kh6ng dgc hai so vdi b i n the trong sin xuit cac sin phim lam tir tinh bgt (bun)
V TAI LIEU THAM KHAO
1
5
Thanh Thj Thu Thuy (2002) Stmcture of sulfated polysaccharides in aqueous solufron Doctoral thesis submitted to Kyoto Institute of Technology, Japan
Christian W Kasbauer, Dietrich H Paper, Gerhard Franz (2001) Sulfated
y0-(l-^4)-galacto-ohgosachharides and thefr effect on angiogenesis Carbohydrate Research, 330
427-430
Trin Dinh Toai, Nguyin Xuan Nguyeri, Pham Hong Hii, Nguyen Bich Thuy, Trin Thj H6ng (2006) Carrageenan tir rong biln: san xuit va ling dung Nxb KH & KT Ha Ngi^
100 frang
Baeza R I.; Carp D J.; Porez O E.; Pilosof A M R (December 2002) icCarrageenan -Protein hiteractions: Effect of -Proteins on Polysaccharide Gelling and Texfriral Properties
Lebensmittel- Wissenschaft und-Techiwlome Volume 35, Number 8, pp 741-747(7)
Tijssen R L M, L S Canabady-Rochelle, M Mellema (Jun 2007) Gelation upon long
storage of milk drinks with carrageenan J Dairy Sci (Vol 90, Issue 6, Pages 2604-11)
Trang 7Tieu ban: HOAT C H A T SINH HQC BigN 683
6 Haijin M., Xiaolu J.; Huashi G A K-carrageenan derived oligosaccharide prepared by
enzymatic degradation containing anti-tumor activity, Joumal of Applied Phycology
Volume 15, Number 4, July 2003, pp 297-303(7)
Huamao Yuan and Jinming Song Preparation, structural characterization and in vitro
antitumor activity of ^ap/?a-carrageenan oligosaccharide fraction from Kappaphycus
striatum Joumal of Applied Phycology Volume 17 Number 1 / January 2005 pp 7-13
8 Vu Nggc Ban Giao trinh thyc tap Hoa ly (1989) DH KHTN-Ha Ngi
9 Ekeberg D, Knutsen S H, Sletmoen M (Aug 2001) Negative-ion elecfrospray
ionisafron-mass specfrometry (ESI-MS) as a tool for analysing stmctural heterogeneity in
kappa-carrageenan oligosaccharides Carbohydr Res (Vol 334, Issue 1, Pages 49-59)
10 Gutierrez A M., Kumosinski T F Farrell H M., Jr (1998) K- Carrageenan interaction with
bovine and caprine caseins as shown by sedimentation and nuclear magnetic resonance
specfroscopic techniques J Agric Food Chem., 46, 4987-4996
11 Cac Tieu chuan Viet Nam: TCVN 5165-90: X i c djnh tong s i vi khuin hilu khi; TCVN
7137: 2002 Xac djnh ting nam men va nim mic; TCVN 7135:2002 Xac djnh tong
Coliforms
12 AxnaaapoBa C Jl., Kaijiapoa B B OnTHMHsauHa 3KcnepHMeHTa B XHMHH H xHMHMecKOH
TexHO.nornH, 1978 Hsfl "Bbicmaa mKona", MocKsa
13 Yu G, Guan H loanoviciu AS Sikkander SA Thanawiroon C Tobacman JK Toida T
Linhardt RJ (2002) Structural studies on kappa-carrageenan derived oligosaccharides
Carbohydr Res; 331:433 AO
S T U D Y O N T H E U S E O F T H E B I O A C T I V E K - C A R R G E E N A N
O L I G O S A C C H A R I D F R O M R E D S E A W E E D S AS AND A D D I T I O N A L
S U B S T R A T E F O R T H E F O O D P R O C E S S I N G AND P R E S E V A T I O N
Tran Dinh Toai, Pham Hong Hai, Nguyen Van Thiet
SUMMARY
Carrageenans, a common cell wall polysaccharide, are a family of linear sulphated
polysaccharides exfracted fi-om red seaweeds Carrageenans are large, highly flexible molecules
which curl forming helical structures This gives them the ability to form a variety of different
gels at room temperature They are widely used in the food and other industries as thickening
and stabilizing asents Carrageenan oligosaccharides has this high effect thanks to thefr smaller
molecule weight as compared to the one of Carrageenan
In this study, we demonstrate that k-carrageenan oligosaccharides were prepared by
enzymatic digestion of kappa-carrageenan from red seaweeds Hong Van Euchema gelatinae
And recently il is shown that Carrageenans and carrageenan oligosaccharides are an effectively
additional substrate for the processing and preservation of food derived from rice starch