Nghiên cứu ứng dụng K-Carrageenan Oligosaccharid từ rong biển có hoạt tính sinh học trong chế biến và bảo quản thực phẩm

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Tieu 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]

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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.

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Tieu 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.

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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 =

Cac he s6 bo, b|, b2 dugc xac djnh theo phuong phap binh phuong toi thilu.

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Tieu 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

y"

1456
1540
1662
1727
1732

Lyc nen ngang, N

Zd
1217
1267
1318
1402
1482
1812
1190

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.

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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.

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)

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Tieu 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

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