Phgm Thi Phucmg vd Dig T^p chi KHOA HQC & CONG NGHE 168(08): 2 3 - 2 7
NGHIEN c u t ! M O T SO TINH CHAT CHU'C NANG CUA CHITOSAN KHOI LU'gfNG PHAN TtT THAP TAO RA T f CHITOSAN THONG THlTdNG
Pham Thi Phuong*, Nguyen VSn Binh, Luu Hong Stm, Vu Thi Mai Truang Dai lioc Ndng Ldm - DH Thai Nguyen
TOM
T A TMyc dich cua nghien cuu nay la tong hgp va danh gia mot so tinh chat chiic nang cua chitosan khoi lugng phan tii thip (CTSLMW) t^o ra tii chitosan thong thudng, Chitosan khli lugng phan tii thap dugc tong hgp theo phucmg phap cua Liu et al, (2006) sau cac khoang thdi gian thiiy phan khac nhau thu dugc chitosan co khoi lugng phan tii dao dpng tfr 9,5 - 10,4 kDa, miic dp diacetyl hda thay doi khong dang ke khoang 97 ± 0,5%. Cac mang tao ra tir CTSLMW deu co tinh tan va tinh hiit am ldn hem 50%, chitosan co khoi lugng phan tii 10,4 kDa co tinh tan va tinh hiit am thap nhat. Chitosan co khoi lugng phan tii 10,4 kDa co kha n5ng khang nam moc Peniciliium expansum (P. expansum) tdt nhat (nong dp tic che tdi thieu nam moc P. expansum la 0,5% tren moi trudng long va 1% tren moi tnrdng ran). Nghien ciiu anh hudng ciia thdi gian khang nam moc P expan.sum cho thay chitosan co khoi lugmg phan tu 10,4 kDa c6 thdi gian xu ly ngan nhat la 22 gid, cr nong dp lie che tdi thieu thap nhat la 0,75%.
Til" khda: chitosan, chitosan khoi lucmg phdn tir thdp, Peniciliium expansum, nong do iec chi toi thieu, khdng khuan
MO DAU
Chitosan la mgt polysacharide dugc hinh thanh tir qui trinh diacetyl hda chitin. til nude ta viec sin xuit chitin, chitosan cd ngudn goc til vd tdm, mai muc mang lgi hieu qua kinh te cao va gdp phin giai quygt d nhiem mdi trudng cho nganh chg bien thuy san. Chitosan cd kha nang khang vi sinh vat, kha nang phin hiiy sinh hpc va khdng dpc dugc iing dung trong nhigu ITnh vuc nhu, cdng nghiep, y tg, che bien vi bio quan ndng san [8].
Chitosan cd kha nang uc chg su phat trien ciia nam mdc Aspergillus niger d ndng do 0,1 - 0,5 mg/ml, d ndng do 0,2 - 0,5 mg/ml chitosan cd tac dung gay ra su rd ri protein va cac chat hap phu tia uv cua Aspegillus mger.
Mat khac chitosan ndng dp 3 - 5 mg/ml cd kha nang lic che su phat trien cua nim mdc Aspergillus paraciticus vi ngan can sir hinh thanh aflatoxin [9]. Theo Munoz et al (2009) [13] cho thiy chitosan cd kha nang ire chl nam moc Collectotrtchum spp tren ca chua va nho, cd tic dung lam giam dang kg tdn thuang tren qua ca chua xii ly a nong do I - 2,5%o. Tic gia nay cung cho rang viec sir dyng
Tel: 0962 075082. Email: [email protected]
chitosan ciing cd hieu qua Iam giam benh than thu trgn ca chua va qua mong. Theo Rhoades
& Roller (2000) [14] cho ring nam men bi loai bd hoan toan khi tang thgm 0,3 g chitosan trgn mdi lit nude ep tao ddng chai tiet trimg luu trii d 7"C, sd lugng vi khuan lactic tang nhung thip hon so vdi ddi chung. Chitosan d ndng do 0,2 - 1 g/l nude ep tio co the lie chg sg tang trudng ciia mgt sd vi sinh vat gay hu hdng nude ep tao [I]. Nghien ciiu boat tinh khang khuan ciia chitosan phu thudc vao khdi lugng phin tit cho thiy CTSLMW dugc cho la cd kha nang khang khuan tdt han so vdi chitosan thdng thudng. Do CTSLMW cd kha nang tan trong nuoc tdt hem dan dgn phan img tdt ban vdi cac vi tri hogt dgng ciia vi sinh vgt [7]. Ket qua nghign ciiu cua Gerasimenko et al (2004) [10] cho i^ng khdi lugng phin tir tang lim giim hoat tfnh khang E.coli ciia chitosan.
VAT LI$U VA PHUONG PHAP NGHIEN CUU Vat li^u nghien cii'u
Chitosan do Viet Nam sin xuit cd do diacetyl hda DD > 85%, khdi lugng phan tii khoang 30 kDa. Nam mdc P expansum do Vien Vi sinh vat va Cdng nghg Sinh hpc, Dgi hgc Qudc gia Hi Not cung cip.
23
Ph^m Thi Phuong vd Dtg Tap chi KHOA HQC & CONG NGHE 168(08): 23-27 Phuang phap nghien ciiu
Phuo-ng phdp tdng hpp CTSLMW CTSLMW duac tdng hgp theo phuang phap ciia Liu et al (2006) [12] Can 10 g chitosan cho vao 190 ml axit axetic 5% li d 50 °C trong 25 h, 37 h, 45 h, sau dd ly tam 5000 vong/phiit trong 20 phiit, sau khi li tam chitosan dugc thgm NaOH 4M cho din khi pH ciia chitosan tir 7 - 9, Riia kit tiia bing nude may va lim khd d 50 °C. Do nhdt ciia chitosan dugc do bang may do dp nhdt. Khdi lugng phin tii chitosan dugc tinh theo cdng thiic cua Mark Houwink ([q]= k.m°), vdi K=
1,64. IO"'". DD'* va a = -1,02 . 10"^ + 1,82.
Mlic dp diacetyl hda cua chitosan dugc xac dinh bing phuong phip do mat dp quang (OD) theo Berth (2002) [3] DDA = (161,1 .A.V - 0.0218m)/(3,3615m - 42,1.A.V) (%), miic do diacetyl hda tinh theo cdng thiic: DA
= 100-DDA(%).
Phuomg phdp chuan bf mdng
Phuang phap do mang dugc tien hanh theo phuong phip ciia Aguirre - Loredo et al (2014) [2]. Do 80 ml dich ldng CTSLMW vao dia petri phi 15 va Iam khd d 50 "C trong 72 h trong tii say (dp am tucmg ddi ~ 30%) sau dd raang dugc bdc ra khdi dia petri va giit trorg binh hiit im cd dg am tuong ddi li 50 ± 5% d nhiet do phdng 23 ± 3 °C, trong 7 ngay trudc khi md ta cac tinh chit ciia ming.
Phucmg phdp xdc dinh tinh tan ciia mdng trong nu&c
Dp tan trong nude cua ming dugc dinh nghTa la ty lp chit khd hda tan trong nude cua mang dugc giai phdng sau khi ngim trong nude cit.
Do tan trong nude S cua mang dugc xac dinh theo phuang phap ciia Aguirre - Loredo et al (2014) [2] can cac manh mang khd cd kich thudc 4 cm^ sau do ngam trong 30 ml nude cit d 25 °C, khuiy dgu theo chu ky trong mgt gid. Cac mau dugc Igc bing giiy lpc. Phin khong tan cua ming dugc siy khd de xac dinh khdi lugng cudi ciing. Do tan dugc xac dinh bang ty le khdi lugng chat khd sau khi say tren khdi luang miu ban dau.
Phuffngphdp xdc dinh tinh hdt am cua mang Tfnh hiit im cua mang dugc xac dinh gian tigp qua ty le phan tram lugng nude bay hai qua mang trgn tdng lugng nude ban dau sau mgt khoang thdi gian xac djnh.
Phuomg phdp xdc dinh nong dp uc che toi thieu cda chitosan trong moi tru&ng long Bao tir nam mdc P. expansum dugc thu bang nude mudi sinh 1^ vi pha loing tdi mat do 10*
bao tir/ml. Dich chitosan ban diu cd ndng do 2% dugc pha loang trong mdi trudng PDB vdi ndng do giam dan 1%, 0,75%, 0,5%, 0,25%. Cho 5 ml chitosan d cac ndng dg tren vao cac dng nghiem, mau kiem chiing chiia 2,5 ml PDB va 2,5 ml dem acetate. Bd sung 100 pi dich bio tii nim moc vao cac dng nghigm chiia chitosan va miu kiem chiing.
Sau dd dem nudi gong tu lac d 30 "C trong 24 gid. Rut 100 pi dich trong cac dng nghiem trang dgu Ign dia thach chira mdi tnrdng PDA va nudi trong tii am 30°C sau 24 - 48 h quan sit va xac dinh nong dp lie che [6].
Phucmg phdp xdc dinh kha ndng uc chi toi thieu cua chitosan trong moi tru&ng ran Dich bao tu nam mdc dugc chuan bi nhu trgn.
Chitosan dugc pha loang frong dung dich axit axetic l%i d cac ndng dp 1%, 0,75%, 0,5%, 0,25%. Hilt 100 pi dung dich chitosan d cac ndng dp trgn trang dgu len trgn bg mat dia thach chiia mdi tnrdng PDA, dg khd sau dd hiit 100 pi dich bao tit nam mdc trang ddu Ien trgn bg m§t thgch, nudi trong bi am d 30 °C sau 24 - 48 h, quan sat va xac dinh ndng dp ire che [15].
Phuong phdp xdc dinh dnh hu&ng ciia thdigian khdng ndm moc P. expansum cda chitosan Chitosan dugc chuan bi tuong tu nhu xac dinh ndng dp lie chg tdi thieu nam mdc P.
expansum trong mdi trudng long. Cic ong
nghiem chua chitosan va bio tir nim moc
dugc nudi lac d 30 "C trong tu lie trong cac
khoang thai gian 0, 14, 18, 22 va 26 gid. Liy
100 pi dung dich chitosan sau cac klioang thdi
gian tren ciy trang tren dia petri chiia moi
Phgm Thj Phuong vd Dtg Tap chi KHOA HQC & CONG NGHE 168(08): 2 3 - 2 7 trudng PDA, nudi trong to i m 30 °C sau 24 - 48
h quan s i t va xac djnh nong do lie c h l [12].
K E T Q U A V A T H A O L U A N Nghien ciru t 6 n g h o p C T S L M W Ket qua x a c dinh khdi lugng p h i n ttr va miic do diacetyl hda ciia chitosan d u g c trinh bay trong b a n g 1.
Bang 1. Khoi luong phdn tie vd miic dp diacetyl hoa C6ngthfi-c ^ ^ ! ! T . ^ " " ; f ? 9 * a c e t y l
phan tu-(kDa) hoa (%) Chitosan 25h 10,4 97,53 Chitosan 37h 9,9 97,27 Chitosan 45h 9,5 96,84 Tat c i cac m a u chitosan tao ra deu cd m i u trang nga, cd khdi lugng phan t u dao dpng tir 9,5 den 10,4 kDa va m u c do diacetyl hda ciia chitosan thay ddi khdng dang ke khoang (97 ± 0,5%i). Kgt qua p h u h g p vdi ket q u i cita Chen e t a l (2002) [4].
NghiSn ctii] tinh tan va tinh hiit a m cua m a n g Tinh tan cua m a n g la mdt d i e tinh quan trgng cua mang sinh hpc trong thuc phara, nd quygt dinh su phan hiiy sinh hgc cua mang, Tuy nhien tinh tan cua m a n g trong n u d e cao cd thg gay b i t Igi cho cac m i n g nay khi sii dung trong mdi trudng cd dp am tucmg doi cao [2],
Bang 2. Tinh lan vd tinh hiit dm cda mdng Khoi lupng Tinh tan Tinh hut am ph3n tif (kPa) (%) (%)
10,4 9,9 9,5
75 66 60
75,4 79,8 83,5 Kgt qua bang 2 chitosan cd khdi lugng phan tli 10,4 kDa cd tinh tan va tinh hut i m t h i p n h i t do chitosan cd khdi lugng phan tir cao cd dp nhdt cao nen m a n g tao ra day h a n do do co tinh tan va tfnh hiit am t h i p h o n M a t khac viec khdi lugng phan tii ciia chitosan giam Iam tang tfnh linh hoat cua cac phan tii chitosan trong mang, d i n d i n lam tang tfnh tan trong n u d e cua m i n g . K i t qua phii h g p vdi kgt qua cua Clasen et al (2006) [5].
N g h i e n ciru n o n g dp lie c h e toi thieu n a m m l c P. expansum c u a C T S L M W t r o n g m 6 i t r i r d u g long va m o i t r i r d u g r a n K i t q u i nghign ciiu n o n g do lie che tdi thieu n i m m d c P. expansum cua C T S L M W tren
mdi trudng Idng va ran d u g c trinh bay d bang 3 va b i n g 4.
Bang 3. Nong do ire che toi thieu ndm moc P.
expansum ciia CTSLMW tren moi tru&ng long Khoi lirimg
p h i n fu' (kDa) 10,4
9,9 9,5 Bang 4. Nong, expansum ciia Khli lirtmg p h i n to- (kDa)
10,4 9,9 9,5
0,25 + + +
Nong dg (Vo) 0,5 0,75 1
+ So lie chi toi thiiu ndm moc P CTSLMW trin moi tru&ng rdn
0,25 + + +
Nong do (%) 0,5 0,75 1
+ + + + + + + +
"+ " Xudt hiin khudn lac, "~" Khong xudt hien khudn Igc "
Ket qua b i n g 3 va 4 cho t h i y chitosan cd khdi lugng phan tir 10,4 kDa cd kha nang khang nam mdc P. expansum tdt nhat d ndng do ire chg tdi thigu la 0,5% tten mdi trudng long va 1% tren mdi trudng ran. Ket qua nghien ciiu phii hgp vdi kgt qua cua Fang et al (1994) [9]
cho rang chitosan nong dp 0,2 - 0,5 mg/ral cd t i c dung gay ra su rd ri protein va cac chat h i p phu tia u v ciia Aspegillus niger. M a t khac khi khdi lugng p h i n tir tang l i r a tang kha nang k h a n g n i m radc ciia chitosan. Ket qua nghien ciiu phu hgp vdi kgt qua cua Jeon et al (2001) [11] bao cao rang chitosan cd khdi lugng p h i n tii (I den 10 kDa) cd hieu qua k h a n g k h u i n tang khi tang khdi lugng p h a n tir.
N g h i e n ciiu a n h h u ' d n g t h o i gian k h a n g n a m m o c P. expansum c u a C T S L M W Anh h u d n g ciia thdi gian xir ly chitosan dugc trinh bay d bang 5, 6, 7.
Bang S. Anh hu&ng cua thai gian xu ly chitosan khoi luong phdn tu 10,4 kDa len chungnam moc
P. expansum Thoi gian
(h) 0 14 18 22 26
0,25 + + + + +
Nong do (Vo) 0,5 0,75
+ + -1- + + + +
+
1 + + +
Pham Thi Phuong v^ Drg Tap chi KHOA HOC & CONG NGHE I68(US):2J-27 Bang 6. Anh hu&ng ctia th&i gian xir ly chitosan
khoi luong phdn tu 9,9 kDa din chungnam moc P. expansum
N i n e do ( % ) ^ Thori gian
(h) 0,25 0,5 0,75
Bang 7. Anh hu&ng ciia ih&i gian xii ly chitosan khoi lup'ng phdn tu 9,5 kDa din chung nam moc
P expansum Thfri gian
(h)
Nong dp (%) 0,5 0,75
deacetyl hda thay ddi khdng dang ke khoang 97 ± 0,5%. Chitosan cd khdi lugng phan tii 10,4 kDa cd tfnh tan va tinh hiit am thip nhit, kha nang khang tdt nhat ddi vdi nam mdc P.
expansum a ca mdi trudng nudi cay ran v i mdi trudng nudi c i y Idng (ndng dp lic che tdi thilu trgn mdi trudng Idng la 0 , 5 % v i 1% tren mdi trudng r i n ) . Nghien ciru anh hudng thdi gian khang n i m mdc P. expansum cua C T S L M W trong mdi trudng Idng cho thay chitosan cd khdi lugng phan tu 10,4 kDa cd kha n i n g k h i n g nam m d c tdt n h i t trong khoang thai khi gian xii ly 22 gid, d ndng dp 0,75%,
"+ " Xuat hiin khudn Igc, "-" Khong xuat hiin khudn tgc Tir k i t qua bang 5, 6, 7 cho t h i y chitosan cd khdi lugng p h i n tii 10,4 kDa cd thdi gian khang nam m o c P. expansum ngan nhat la 22 gid, nong dg ire che tdi thigu thap nhit l i 0,75%. Thdi gian x u ly tang lam tang kha nang khang n i m m d c cua chitosan d i l u nay cd t h i la do khi t i n g thdi gian x ^ ly d i n mpt mirc n h i t djnh du d l qua trinh h i p thy chitosan Ign t l bao n i m mdc dgt trang thai can b i n g , dn dinh khi dd tg bao nam moc bi ton thuong n h i l u n h i t . Luc lign kgt manh giiia chitosan va tg bao cd t h i dan d i n pha vd mang t l b i o hoac cd the l i y di nhirng ion kim loai c i n thilt cho hogt dgng trao ddi chat dan d i n roi loan trong q u i trinh trao ddi chat cua t l bao vi sinh v i t [5] hogc chitosan t h i m t h i u qua m i n g t l b i o , chiing d l dang tuong tac vdi cac nhdm phosphoryl ciia thanh p h i n phospholipit t l bao lam cho te bao mdi tgo thanh khdng cd mang ho^c m a n g t l bao tgo thanh r i t m d n g g i y nen su rd ri c i c hgp chat npi bao [9].
KET L U A N
C T S L M W d u g c tdng h g p theo phuong phap ciia Liu et al (2006) [12] cd k h l i lugng phan tii dao dpng tir 9,5 d i n 10,4 kDa, mirc dO
TAI LIEU T H A M K H A O l.Abd A, J., Niamah A, K. (2012), "Effect of chitosan on apple juice quality". International Journal of Agricultural and Food Science, 2, pp.
153-157.
2. Aguirre - Loredo R. Y., Rodriguez-Hernandez and Chavari'i'a-Hernandez (2014), "Physical properties of emulsified films based on chitosan and oleic acid", CyTA - Journal of Food, 12(4), pp. 3 0 4 - 3 1 2
3. Berth G,, Dautzenberg H. (2002), "The degree of diacetylation of chitosans its effect on the chain conformation in aqueous solution", Carbohydrate Polymers, 47, pp. 39 -51.
4. Chen X G., Zheng L., Wang Z„ Lee C. Y., &
Park H, J. (2002), "Molecular affinity and permeability of different molecular weight chitosan membranes", Journal of Agricultural and Food Chemistry, 50, pp 5915-5918.
5. Clasen C„ Wilhehns T., Kulicke M. K (2006),
"Formation and characterization of chitosan membranes", Biomacromolecules, 7, pp. 3210-3222.
6. Tran Bang Diep, Nguyen Duy Lam, Tran Mmh Quynh and Tamikazu Kume (2005), "Radiation - induced enhancement of antifungal activity of chitosan on fruit - spoiling fringi during postharvest storage" Jeari — conf. 2001 - 2005, pp. 1 7 - 2 6 .
7. Dutta P K., Tripathi S , Mehrotra G, K., and Dutta J, (2009), "Perspectives for chitosan based antimicrobial films in food applications". Food Chemistry, 114(4), pp, 1173-1182.
8. Elsabee M. Z., Naguib H. P., Morsi R. E.
(2012), "Chitosan based nanofibers", Materials Science and Engineering C, 32, pp. 1711-1726.
9. Fang S, W„ Li C. F., and Shih D. Y. C. (1994),
"Antifiingal activity of chitosan and its preservative
Pham Thi Phucmg vd Dtg T^p chi KHOA HOC & CONG NGHE 168(08): 23 - 27 effect on low-sugar candied kumquat", Joumal of 13. Munoz Z., Moret A, and Garces (2009), food protection, 56, pp. m - m . "Assessment of chitosan for inhibition of
10. Gerasimenko D. V., Avdienko I. D (2004), r- rr . „ • i. » » j
"A , 1 , . • 1 «• i uiv-uivu ^ tiuv/T;, CoUetotrichum spp. on tomatoes and grapes . Antibactenal effects of water - soluble low - ^ „ . -!, .,, . „
molecular weight chitosans on diffirent Cm;, ^'''''ec/,.^, 28, pp. 3 6 - 4 0 .
microorganisms". Applied Biochemistry and 14. Rhoades J , Roller S. (2000), "Antimicrobial A/icra6io/ogy, 40, pp, 2 5 3 - 2 5 7 , actions of degraded and native chitosan against 11, Jeon Y L, Park P. J,, Kim S, K. (2001), spoilage organisms in laboratory media and
"Antimicrobial efect of chitooligosaccharides foods". Application Environment Microbiology, produced by bioreactor, Carbonhydrade polymer, gg^ pp_ 80-86. 15, Singburaudom N., Onuma P., Tida D. (2011),
"Antimicrobial Activity of different molecular 44(1), pp 71 - 7 6 .
12. Liu N., Chen X G , Park H. J , Lm C, G., Liu
C. S., Meng X H , Yu L. L (2006), "Effect of . , , .
MW and concentration ofchitosan on antibacterial ^^'S^t chitosans to mhibit some important plant activity of Escherichia coli". Carbohydrate pathogenic fungi", Kasetsart Jounal {Nat. Sci.), Polymer, 64, pp. 60-65. 45, pp 644 - 655.
S U M M A R Y
S T U D Y O N T H E F U N C T I O N A L P R O P E R T I E S OF C H I T O S A N L O W M O L E C U L A R W E I G H T F O R M E D F R O M N A T I V E C H I T O S A N
Pham Thi Phuong , Nguyen Van Binh, Luu Hong Son, Vu Thi Mai University of Agriculture and Forestry - TNU The purpose of this study was to synthesize and investigate some of the functional properties of chitosan low molecular weight (CTSLMW) forming from native chitosan. Chitosan low molecular weight was synthesized under Liu et al (2006) method, after different hydrolysis periods of time chitosans had molecular weight ranging from 9.5 to 10,4 kDa, deacetylation degree changing insignificantly at 97 ± 0,5% Films forming from CTSLMW have water solubility and water vapour permeability at over 50% and chitosan with molecular weight 10,4 kDa has the lowest water solubility and water vapour permeability Chitosan with molecular weight 10.4 kDa has the best inhibited Peniciliium expansum (P. expansum) growth at concentration of 0.5% in liquid midium and 1% in solid medium. Study on the effect of time inhibition on P. expansum mold showed that chitosan with molecular weight 10 4 kDa has the shortest processing time of 22 hours at minimun inhibitory concentration of 0.75%.
Keywords: chitosan, chitosan low molecular weight, Peniciliium expansum, minimum inhibitory concentration, antibacterial
Ngay nhgn bai:22/6/2017; Ngdy phan blfn:06/7/2017; Ngdy duyftddng: 31/7/2017 Tel: 0962 075082. Email: phamthuphuonghb@gmad a
