TC.DD&TP9(1)-20I3
NGHIEN CUtJ HOAT TINH KHANG VI SINH VAT CUA AMINOREDUCTONE*
Trdn Thi Thu Tran^, Vu Thu Tran^, Ldm Xuan Thanh'
•Nghien cim nay dupc t^i Up bdi Quy Phat trien Khoa hpc va Cong ngh? Qu6c gia (NAFOS- TED) ma so 106.99-2011.22.
Phan ling Maillard mpt phan img vo ciing quan Irpng ddi vdi thyc pham vi anh hudng tryc liep ldi chat lupng cua san phAm thyc pham. Trong nghien ciru nay, hogt tinh khang vi sinh vat cua aminorcductone (AR) - san pham dupc lao thanh trong giai doan dau ciia phan iing Maillard duac khao sal va so sanh vdi hoat tinh ciia mpt so khang sinh thong dyng {amikacin (AN), ciprofloxacin (CIP), meropcnnem (MEM) and levonoxacin (LVX)). BSng phuang phap khuech tan tren dTa thach nghien ciru da xac dinh AR co kha nang irc che manh me tdi sy phat trien cua cac 7 chung vi sinh vat thir nghi?m kha tuang dong vdi cac chat khang sinh. Vdi 2,5 mg AR, vdi dirdng kinh vong tron khang khuSn dao dpng tir 15±0 mm (Bacillus cereus ATCC® 13061 ^M) tdi 28,25±0,35 mm (Staphylococcus aureus ATCC® 25923"^"). Do AR ket hpp vdi khang sinh co kha nang iic che sy phat iri^n lai ciia mpt so khuin lac khang khang sinh nen AR co the la hgp chat tiem nang trong san pham thuc phSm co Igi cho sire khoe va ho trp ihuoc khang sinh trong dieu tri mpt so benh do vi sinh vat.
TlT kh6a: phdn img Maillard. aminoreductone. hogt tt'nh khdng vi sinh vgt.
hinh thanh trong giai doan dau cua qua L D A T V A N D E
Phan img Maillard la phan u:ng phd biin gitra dudng khir va nhom amin cua axit amin va protein, xay ra trong qua trinh chl bien va bao quan Ihyc pham [1].
Day la phan ung vd cimg quan Irpng tao nen huang vi dac trung cho san pham thyc pham, ben canh do, phan ung cung gay ra mpt so van de iam giam gia tri dinh dudng cua thyc pham va tao thanh mpt so chat khong mong muon hay mdt sd san pham khong cd lgi cho siic khoe con ngudi. Vi anh hudng tryc tiep tdi chat lugng cua san pham thuc pham, nghien Cliu ve phan u'ng Maillard ludn la moi quan tam hang dau cua cac nha cong nghe thuc pham tren the gidi.
Aminoreductone (AR) la mgt trong nhiing san pham cua phan img Maillard
trinh gia nhiet [6]. AR da dugc sir dung nhu mpt thong so quan trgng de phal hien va kiem soat phan ling Maillard trong thyc pham. Chinh vi le dd, nghien cim ve cac dac tinh va c a che hinh thanh cua AR dat ra hudng tiep can mdi cho cac nha khoa hgc. Nhiing nghien cim budc dau cho thay AR co dac linh chdng oxy hoa cao va CO kha nang khang chimg vi khuan gay benh dudng rudt va ung thu da day Helicobacter pylori [6]. Chi'nh vi le do, chiing toi gia thiet rang AR cd the co kha nang anh hudng tdi cac tac dpng sinh ly cua mgt so vi khuan gay benh khac nua.
Trong no Iyc tim kiem cac nguon thay the cho thuoc khang sinh va lam sang 16 cac dac linh chiic nang cua AR, nghien ciiu hien tai tap trung vao nghien curu kha Dai hpc Bach Khoa Ha Npi
Email: [email protected]
^TS. Vien CNSH - CNTP, DHBKHN
¥ G S . T S . Vi?n CNSH - CNTP, DHBKHN
Phan bien I: GS.TS Nguyen Thj Hien- Hpi Dinh dudng Viel Nam Phan bien 2: TS Vu Thj Hi6n-
Vipn Dinh duang Ngay nhan bai: 11/2012 Ngay dang bai: 15/04/2013 30
TC.DD&TP 9 (1)^2013 nang khang vi sinh vat gay benh va gay
hu hong trong thuc pham ciia AR. san pham dugc hinh thanh trong qua trinh chS bien thuc pham.
n . DOI TLTgfNG VA PHU'ONG PHAP NGHIEN CU'U
2.1. Hoa chat
Moi trudng Mueller-Hinton (MHB) dugc mua lir Becton. Dickinson (My).
cac dia khang sinh thuong mai dat lieu chuan dudng kinh 0=^ 6mm bao gom:
amikacin (AN: 30pg/ dia), ciprofloxacin (CIP: 5pg /dTa). Meropenem (MEM:
lOpg /dTa) va levofloxacin (LVX, 5pg/
dia) dugc mua tir Cdng ty Nam Khoa, Viet Nam (Takeuchi va dong sy, 2006).
Lactose monohydrat da dugc mua tir Cong ty SIGMA, My. n-Butylamine va aga-aga dugc lay tir Wako Pure Chemical Industries (Osaka, Nhat Ban). Tat ca thuoc thii khac deu thupc loai tinh khiet.
Nudc Milli-Q hoac nudc tiet trimg dugc sii dung trong tat ca cac thi nghiem.
2.2 Tach chiet AR
AR dugc tach chiet tir dung dich mau lactoza va butylamine, qua trinh tinh che dugc chuan bj theo phuong phap ciia Shi- mamura va dong sy (2004) [3]. Lactoza (262mM) va butylamine (1,16M) dugc hoa tan trong dung dich dem phot phat 1,28 M (pH 7.0). Dung dich mau (10ml) dugc gia nhiet d 1 OO^C trong 15 phut,va dugc iam lanh ngay lap tire trong nudc da. Dung dich sau gia nhiet dugc chiet 3 ian bang lugng thS tich gap doi dung dich ethyl acetate. Dung dich ethyl acetate dugc bay hoi va say chan khong. San pham say dugc hda tan trong 10ml dung dich methanol 20% va dugc dua qua cdt chuAn bi mau Sep-Pak Plus C18 cartridge (Waters Corporation, Milford, MA,My) (trudc do da dugc boat hoa bang 5ml ethanol va can bang bang nudc Milli-Q
hay nudc cat) de loai bo het thanh phan melanoidin mau nau. Dung dich trong suot thu dugc dugc boc hoi, co chan khong de thu dugc AR linh che.
2.3 Chung vi khuan va dieu kien nuoi cay
7 chiing vi sinh vat gay benh va gay hu hong trong thyc pham trong bp suu tap chiing gidng cua Hoa Ky gom co Staphy- lococcus aureus subsp. aureus ATCC®
25923''^". Salmonella enterica subsp. en- terica serovar Typhimurium ATCC®
14028™, Bacillus cereus ATCC®
13061™. Bacillus subtilis ATCC®
11774™, Escherichia coli ATCC®
25922™, Enterococcus faecalis ATCC®
29212™. Listeria innocua ATCC®
33090'^'^ da dugc mua tir cong ly Vi- comtec, Viet Nam. Tat ca cac chiing da dugc nuoi cay Iren cac dia thach Mueller- Hinton (MHB) bo sung vdi moi trudng thach 1,4% va ii trong tii am d 37°C trong dieu kien hieu khi trong 24 gid [5].
2.4 Phuo'ng phap khuyech tan tren dia thach
Phuang phap khuech tan tren dTa thach dugc sii dung de xac dinh kha nang irc che vi sinh vat ciia AR [6]. DTa giay lpc tieu chuan dudng kinh 6mm co chiia 2,5 mg AR dugc dat tren cac tim thach MHB ma trudc do da dugc cay san vi sinh vat vdi ham lugng khoang 108 CPU/ dia. Sau do cac dTa dugc ii trong tii am d 37'^C trong dieu kien hieu khi trong 24 gid. Sau 24 gid vdng Iron khang khuan dugc do va ghi lai theo mm va dudng kinh dugc xac dinh bang gia Iri trung binh cua it nhat hai lan thi nghiem lap. Bon loai dTa khang sinh thuong mai dat lieu chuan nhu:
amikacin (AN: 30pg/ dia), ciprofloxacin (CIP: 5pg/dia), meropenem (MEM: lOpg /dia) va levofloxacin (LVX, 5pg/ dia) ciing dugc sir dung trong thi nghiem [6].
Kha nang lie che vi sinh vat dugc the hien 31
T C . D D & T P 9 ( 1 ) - 2 0 1 3 bang so sanh duong kinh cua vong tron
khang khuan [6].
III. K f e j Q U A
3.1 K h a nang lie che su- p h i t trien ciia vi sinh vat cua AR
Bdng 1: Khd udng irc che vi sinh vgt cua AR
Kha n i n g khang vi sinh v|it cua AR d6i voi 7 chiing vi sinh vat chuan da duoc kiSm tra bang p h u a n g phap khuech tan tren dia thach. KSt qua d u o c thS hien trong bang 1.
Bwifng kinh v6ng trdn khang khuan (mm)*
STT Cliijng vi sinh vat
AR AN LVX MEM CIP (2,Smg/dTa) (30(ig/<na) (5(tg/dTa) (lOjig/dla) (5^g/dIa) 1 S. aureus (+) 28,25±0.35 29,75±0,35 31±0,7mm 32,75±0,35 31±0 2 S. typhymurium (+) 23,65±0,21 27,65±l,20 35,75±0,35 33,65±l,20 35,6S±1,62 I SiO 27,75±0,35 25,4±0,56 10,I5±0,49 27,5±0,7 B. cereus (+)
4 5 6 7
B. subtilis (+) E coli (-) E. faecalis (+) L. innocua (+)
17±0,7 20,9±0,07 19,75±0,35 23,25 ±0,35
35,5±0,7 29±0,35 18,5±0,7 28±0
3 5,3 5± 1,2 34,7S±0,53 26,9±0,14 27,25±0,35
37,5±0,7 32,75±0,17
17,9±1,55 26,65±0,21
38,S±0,7 38,25±0,17
27,5±0,7 27,25±0,35
* Dirdng kinh cua moi dia gidy lgc Id 6 mm vd kit qud Id gid tn trung binh cua it nhdt 2 ldn thi nghiem lap; AN. Amikacin; LVX. Levofloxacin: MEM, meropenem: CIP. Ciprofloxacin;
AR (2,5 mg) the hien kha nang iic che vdi hau het cac chiing vi sinh vat thir nghiem vdi dudng kinh vong Iron khang khuan dao ddng tir 15±0 mm ldi 28,25±Q,35 mm. AR th^ hien hoat tinh cao nhat doi vdi chung Staphylococcus aureus ATCC® 25923™ (dudng kinh vdng Iron khang khuan 28,25±0,35 mm) va thap nhat doi vdi chung Bacillus cereus ATCC® n06U'^ (dudng kinh vong Iron khang khuanl5±0 mm). K^t qua nghien cim cho thdy ph6 khang khuan ciia AR kha rgng, khong chi iic che cac vi sinh vat gram (+) ma doi vdi vi sinh vat gram (-) nhu E.coli, AR cung c6 kha nang lie ch€ ro ret vdi dudng kinh
vong tron khang khuan cua E.coli la 20,75±0,25 mm.
AR la hgp chat cd kha nang oxi hoa manh, nd de dang bi oxi hda bdi Cu"' (Shimamura va ddng su, 2004), no ciing de dang bi bien ddi thanh nhiing san pham phan huy khac. De kiem nghiem hoat tinh cua AR, h6n hgp AR va Cu"*
(10|ig) ciing dugc thu nghiem trong nghien ciiu nay. Ket qua cho thay kha nang khang khuan ciia hon hgp AR va Cu'"^ giam so vdi dTa giay lpc chi chua AR (hinh 1). Dieu nay khang dinh rang AR, chii khdng phai san pham phan hixy cua nd CO tac dung iic che sy phat trien cua vi sinh vat.
32
T C . D D & T P 9 ( I ) - 2 0 1 3
Hinh 1: Hinh dnh vong tron khdng khudn cda AR vd hdn hffp AR vd Cu2+ do! v&i cdc chiing vi sinh vgi.
3.2 So sanh hoat tinh cua AR vdi mot so loai k h a n g sinh
Nghien ciiu nay ciing khao sat hoat tinh cua mot so loai khang sinh thudng diing de dieu tri cac benh do vi sinh vat va so sanh vdi AR. Ket qua so bp cho thay hoat tinh cua AR 2,5 mg kha luong d6ng vdi AN, LVX, CIP, MEM d ndng dp thu nghiem. Trong trucmg hgp ciia chung vi khuan B. cereus, mot so khuan lac khang khang sinh van phat Irien trong
vong Iron khang khuan ciia chat khang sinh MEM (binh 2). Khi bo sung AR vao giay lpc c6 chira khang sinh boat tinh khang khuan ciia hon hgp khang sinh va AR dugc nang cao ro ret. Khuan lac khang khang sinh khong con phat trien trong vdng Iron khang khuan (Hinh 2).
Ket qua tren ciing chiing to rang AR khong nhiing khong irc che boat dpng cua cac hgp chat khang sinh ma con co the ket hgp vdi khang sinh trong dieu trj benh.
T "'^"Vl
he/^O
Hinh 2: Tuffng tdc cua NEM, AR vd MEM doi v&i chung B. cereus
TC.DD&TP9(1)-2013 IV. BAN LUAN
AR la san pham hinh thanh Irudc melanoidin, d6ng vai tro quan trpng nhu la chat chi thj ciia qua trinh gia nliicl. Tuy nhien, trong nghien ciiu ve cac dac linh chiic nang ciia san pham phan iing Mail- lard, nhi^u nlia khoa hpc mdi chi de cap d^n cac tac dung ciia sin pham hinh thanh trong giai doan cuoi ciia phan irng - Melanoidin nhu boat tinh chdng oxi hoa, hoat tinh khang vi sinh vat [2]. Chi mpt vai nghien cim ve hoat tinh ciia AR dugc nghi nhan [4]. Nghien ciru ciia chiing toi la nghien cim co ban ve lac dpng tryc liep ciia AR la mpt hgp chat tu nhien hinh thanh trong qua trinh che bien thuc pham CO tac dung doi vdi vi sinh vat gay benh va gay hu hong trong san pham thuc pham. Trong nghien ciiu nay, mgi kel qua so bg da chi ra rang AR cd tac dpng irc che mpt so chiing vi sinh vat thudng gap trong thyc pham hay vi sinh vat gay benh.
Va kha nang lie che cua AR len moi vi chimg vi sinh vat la khac nhau. Tai 2,5mg AR CO lac dung irc che hieu qua vdi tat ca cac chiing. Pho khang khuan ciia AR kha rgng, AR co the irc che ca vi sinh vat gram (+) va gram (-). Tuy nhien, co che ciia dac linh khang vi sinh vat ciia AR cho ldi nay van chua dugc iam sang to.
Melanoidin la hgp chat cao phan tir vdi cac goc anion co kha nang hap phu cac ion kim loai trong moi trucmg dinh dudng ciia vi sinh vat nhu la sat, kem.
dong..., la nhung hgp chat can thiet d6i vdi qua Irlnh chuyen hoa de duy tri su song cua vi sinh vat bao gom ca cac vi khuan gay benh [2]. Nghien ciru ciia chung toi lai chi ra rang, AR vdi khdi lugng phan tii thap (217 Da), lai co lac dung lie che sy phai trien cua vi sinh vat, day la ket qua vo cimg thii vi. Cung gidng nhu melanoidir, AR ciing Ihl hien hoat linh chong oxi hoa manh [4], di^u nay ggi
y rang, co che khang vi sinh v$t ciia AR ciing cd Ihl tuong ty nhu melanoidin \i nhd sy giam kha nang oxi hda khu trong moi trucmg sinh duang dan den roi loan chuyin hda cac chat qua thanh te bao vi sinh vat.
M^c dil CO che chinh xac ciia AR doi vdi kha nang iic che su phat irien cua vi sinh vat chua dugc iam sang td, de tai nay dang la hudng tiep can mdi cho cac nha nghien ciiu khoa hpc thyc pham vdi h-ien vpng tim ra mdt hgp chat tu nhien co the lhay ths cho thuoc khang sinh.
V. KET LUAN
Qua khao sat so bp cho thay AR co kha nang iic che mpt so chung vi sinh vat gay benh va gay hu hong trong thuc pham. Ngoai ra AR con co the ho trg cho mgl so loai khang sinh irc che sy phat trien cua vi sinh vat. De tai nay hien van dang tiep tyc khao sat hoat tinh khang khuan ciia AR bang nhieu phuang phap khac nhau de co the dua ra nhiing ket qua lam sang to dac tinh ciia AR nham tim kiem mgt hgp chat ty nhien hiru ich co the lhay the cho thuoc khang sinh, co the dung AR ket hgp de giam bdt lieu lugng khang sinh sii dyng.
TAI LIEU THAM KHAO 1 VanBoekelM. A.J. S. (1998). ^/ecr
of healing on Maillard reactions in milk. Food Chem., 62, 403-414.
2. Rufian-Henares, J. A. and Morales F.
J. (2008) Antimicrobial activity of melanoidins against Escherichia coli is mediated by a membrane-damage mechanism. J. Agric. Food Chem., 56, 2357-2362.
3. Shimamura, T., Ukeda, H. and Sawa- mura M. (2004). Relationship between the XTT reducibility and aminoreduc- tone formed during the Maillard 34
TC.DD&TP9(1)-2013 reaction of lactose: the detection of
aminoreductone by HPLC. Food Sci. Tech- nol. Res. 10,6-9.
4. Pischetsrieder, M., Rinaldi, P., Gross, U., &
Severin, T. (1998). Assessment of the an- tioxidantive and prooxidalive activities of two aminoreductones formed during Mail- lard reaction: Effects on the oxidation offi- Carotene, Na-Acetylhistidine, and cis-Alkenes. J. Agric. Food Chem., 46, 2945-2950.
5. Takeuchi, H., Nakazawa, T., Okamoto, T,
Shirai, M., Kimoto, M., Nishioka, M., Con, S., Morimoti, N. and Sugiura T. (2006).
Cell elongation and cell death of Heli- cobacter pylori is modulated by the disrup- tion ofcdrA (Cell division-related gene A).
Microbiol. Immunol., 50,487-497.
Trang, V. T, Takeuchi, H., Kudo, H., Aoki, A., Katsuno, S., Shimamura, T, Sugiura, T.
Ukeda, H. (2QW). Antimicrobial activity of aminoreductone against Helicobacter py- lori J. Agric. Food Chem.. 57, 11343-
11348.
Summary
STUDY ON ANTIMICROBIAL ACTIVITIES OF AMINOREDUCTONE Maillard reaction is significant for foods because it strongly affects the quality and acceptance.
In this study, antimicrobial activities of aminoreductone (AR), a product formed during the initial stage of the Maillard reaction, were screened against food-spoilage and pathogenic bacteria and compared to the effects of common antibiotics (amikacin (AN), ciprofloxacin (CIP), meropennem (MEM) and levofloxacin (LVX)) The inhibitory effects of AR against all 7 isolates were examined by the standard disc diffusion methods, which are widely used to study the bioactivity of chemical compounds. The inhibition zone for each isolate by AR (2.5 mg) was from I5±0 mm (Bacillus cereus ATCC® 13061 ™) to 28.25±0.35 mm (Staphylococcus aureus ATCC® 25923^") in diam- eter, suggesting that all isolates exhibited sensitivity to AR. Because AR showed the advanced ef- fect of growth inhibition in combination with antibiotics, AR is a naturally formed antimicrobial agent presenting in thermally processed foods with potential heahh benefits in medical practice.
Keywords: Maillard reaction, aminoreductone. antimicrobial activities
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