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anh gia s tng thich gia phng phap kim tra vi sinh vt truyn thng va phng phap o ATP quang sinh hoc trong qua trinh kim tra v sinh cua dy chuyn san xut bia
Vin Cng nghip thc phm
Trn Thi Hao
Trng i hoc Khoa hoc T nhin; Khoa Sinh hoc Chuyn ngnh: Vi sinh vt; M s: 604240 Ngi hng dn: TS. Phm Vn Thnh
Nm bo v: 2011
Abstract. Tng quan tinh hinh ng c thc phm Vit Nam, thc trng v sinh
mt s c s sn xut ch bin thc phm v vi sinh vt gy ng c thng gp
trong thc phm; cng ngh sn xut bia, nhng cng on cn thit phi kim tra
v sinh trong sn xut. Nghin cu cac phng phap kim tra v sinh trong sn
xut hin nay; gii thiu v phng phap ATP quang sinh. Kim tra qua trinh v
sinh trong dy chuyn cng ngh sn xut bia c s sn xut ca Vin Cng
nghip thc phm; cac v tr kim tra l cac v tr trong yu ca qua trinh nh cac
tec ln men v cac tec sn phm, may loc sn phm. Trinh by cac phng phap
nghin cu: phng phap kim tra vi sinh vt bng nui cy truyn thng; phng
phap xac nh sch bng ATP quang sinh. a ra kt qu v tho lun: phng
phap phn tch truyn thng; phng phap ATP quang sinh hoc. anh gia s tng
thch gia kim tra vi sinh vt bng phng phap truyn thng v o ATP quang
sinh. Kho sat ng dng phng phap to mu xac nh tng s Coliforms trn may
quang ph U-1900 ( UV-VIS spectrophotometre).
Keywords. Vi sinh vt; Sinh hoc; V sinh; Cng nghip thc phm; Ng c thc
phm
Content.
Thc phm l nhu cu thit yu hng ngy ca mi con ngi. An ton
thc phm tr thnh vn ton cu; trc nhng him hoa lun rinh rp,
e doa i sng con ngi bi cac thc phm khng an ton, i hi mi
quc gia phi c nhng quc sach m bo an ton v sinh thc phm.
2
Trn thc t, mt thc trng cn bc xc trong ngnh sn xut ch bin
thc phm nc ta l cht lng v sinh cn rt km nhiu c s sn xut.
Nhng yu cu nghim ngt v v sinh an ton thc phm trong sn xut cha
c quan tm v u t thch ang. Mt khac do li nhun m mt s t nh
cung cp s dng cac ha cht c hi ch bin, nui trng v sn xut
cac sn phm thc phm; gy nh hng khng tt ti sc khe ngi tiu
dng. Vic s dng cac thc phm khng m bo cht lng v sinh c th
gy ng c v gy ra cac bnh tiu ha cp tnh cho ngi s dng, nghim
trong hn c th dn ti t vong. V lu di, nu cac c t tch ly dn dn
ti mt ngng nht nh c th s phat sinh cac bnh nguy him, lm bin
i cu trc gen gy d tt, d dng cho cac th h tip theo.
Trc thc trng trn, ngoi vic khuyn khch cac c s sn xut thc
hin v sinh an ton thc phm Nh nc v chnh ph cn ch th v vic
tng cng cng tac bo m cht lng v sinh an ton thc phm tng
bc nng cao cht lng sn phm. Tuy nhin phng thc qun l kim tra
cht lng v sinh an ton thc phm da trn vic kim tra cht lng sn
phm cui cng khng m bo c cht lng v sinh sn phm trong khi
sn xut. iu ny ch c m bo khi c s kim soat ch ng v h
thng cac yu t cht lng v iu kin v sinh trong ton b qua trinh sn
xut, ch bin, cung ng mi c kh nng m bo an ton thc phm.
ap ng c yu cu ny khng gi khac l phi ap dng cac phng phap
kim tra nhanh mc v sinh trong cac dy chuyn sn xut, ch bin thc
phm .
Gp phn vo s kim soat v sinh an ton trong sn xut ung ni chung
v sn xut bia ni ring, chng ti tin hnh thc hin kim tra v sinh trong
dy chuyn sn xut bia, vi ti: anh gia s tng thch gia phng
phap kim tra vi sinh vt truyn thng v phng phap o ATP quang sinh
hoc trong qua trinh kim tra v sinh ca dy chuyn sn xut bia Vin cng
nghip thc phm.
I. I TNG V PHNG PHP NGHIN CU
2.1. i tng nghin cu
Kim tra qua trinh v sinh trong dy chuyn cng ngh sn xut bia
c s sn xut ca Vin Cng nghip thc phm. Cac v tr kim tra l cac v
3
tr trong yu ca qua trinh nh cac tec ln men v cac tec sn phm, may loc
sn phm.
Ch tiu la chon kim tra l: tng nm men, nm mc, tng Coliform
v E.coli, tng vi sinh vt hiu kh
2.2. Trang thit b, dng c v phng php nghin cu
2.2.1. Dng c v thit b, ha cht
a. Dng c thit b
+ May o ATP HY_LITE 2
+ May quang ph UV-VIS U-1900
+ Ni kh trng
+ T m
+ T cy
+ May o pH
+ Pipet t ng, u ht
+ a petri, giy nhm, n cn, binh xt cn, a thy tinh,
b. Ha cht
+ Plate count agar ( Peptone t Carein 5,0g; cao nm men 2,5g; D+
gluco 1,0g; agar 14,0g)
Cach pha:
Ha tan 22,5 g PCA trong 1000 mL nc ct. Hp kh trng 121oC,
15pht. Sau khi kh trng pH: 7,0 0,2 25 oC.
+ Lactose broth ( Peptone 10g; lactose 10g; beef extract 6g; nc ct
1000 mL).
Cach pha:
Mi trng n: ha tan 13g lactose broth trong 1000mL nc ct.
Mi trng kp: ha tan 26g lactose broth trong 1000 mL nc ct.
Ha tan mi trng, sau em hp kh trng 121oC, 15pht. Sau khi kh
trng pH: 6,9 0,2 25 oC.
4
+ Brilliant-green lactose (bile) broth ( Peptone 10g; lactose 10g; OX
bile 20g; Brilliant green 13 mL; nc ct 1000 mL).
Cach pha:
Mi trng n: ha tan 40g trong 1000 mL nc ct.
Mi trng kp: ha tan 80g trong 1000 mL nc ct.
Ha tan mi trng, sau em hp kh trng 121oC, 15pht. Sau khi kh
trng pH: 7,2 0,2 25 oC.
Tryptone
Tryptone ( Peptone t casein ): 20g.
NaCl: 5g.
Nc ct 1000 mL.
Cach pha:
Ha tan mi trng, sau hp kh trng 121oC, 15pht. Sau khi kh trng
pH: 7,5 0.2 25 oC.
+ R2A agar (Yeast extract 0,5; proteose peptone 0,5; casein hydrolysat
0,5; glucose 0,5; starch soluble 0,5; natri pyruvat 0,3; K2HPO4 0,3; MgSO4
0,05; agar 12,0).
Cach pha:
Ha tan 15.2 g trong 1000 mL nc ct. em hp kh trng 121oC trong
vng 15 pht. Sau kh trng pH 7,2 0,2 25 oC.
+ Kovacs
+ Oxidase
+ Fluoro cult (Tryptone 5,0; NaCl 5,0; Sorbit 1,0; Tryptophan 1,0; 4-
brom-4chlor-3 indoly B-D- glactogyranoside (X-gal) 0,08; 4-
metylumbelliferyl -D-glucuronide (MGG) 0.05; 1-isopropyl -D-1-
thiogalactopyranoside 0.1 ).
Cach pha:
Mi trng n: cn 17g/1000mL nc ct
5
Mi trng kp: cn 34g/1000mL nc ct
Ha tan mi trng, sau hp kh trng 121oC, 15pht. Sau khi kh trng
pH: 6,8 0,2 25 oC.
2.2.2. Phng php nghin cu
2.2.2.1.Phng php kim tra vi sinh vt bng nui cy truyn thng
Phng phap truyn thng l phng phap ph bin nht trong cac
nghin cu vi sinh vt hoc. Trn mi trng c mi vi sinh vt s phat trin
thnh khun lc hoc s sinh kh v biu hin c ng nghim. Da trn
y c th kim tra s lng vi sinh vt trong mi n v th tch ca dch
nghin cu. Phng phap m khun lc thng c s dng kim tra s
lng nm men, nm mc.
2.2.2.1.1. Phng phap xac nh tng s Coliform v E.coli
Cy vo 5 ng ng mi trng canh thang lng nng kp vi mt
lng 10 mL mu (hoc dung dch mu pha long), trong mi ng u th
ng duham ln ngc. Tip theo, cy vo 5 ng ng mi trng canh thang
lng nng n vi mt lng 1 mL mu (hoc dung dch mu pha long),
trong mi ng u th ng duham ln ngc. Cy vo 5 ng ng mi trng
canh thang lng nng n vi mt lng 0.1 mL mu (hoc dung dch
mu pha long), trong mi ng u th ng duham ln ngc.
cac ng sau khi cho mu vo nhit 35oC trong 24 hoc 48 h v
quan sat. Vi nhng ng c biu hin c v sinh kh, chuyn tip ln lt 10
mL , 1mL, 0.1mL vo 5 ng mi trng brilliant kp v 5; 5 ng mi trng
Brilliant n, nui tip 35oC sau 48 h. sau y quan sat v oc kt qu theo
bng kt qu trong ISO 9308-phn II. Mu c coliform vi biu hin c v
sinh kh ng.
Chuyn tip 10 mL, 1mL, 0.1 mL vo 5 ng mi trng tryptone kp
v 5; 5 ng mi trng Tryptone n, nui tip 44oC sau 24 h. Cho vo mi
ng vi giot Kovacs, nu biu hin mu hng trong ng, chng t s c mt
ca E.coli.
6
2.2.2.1.2. Tnh tng s nm men, nm mc
Chun b dch pha long v mi trng a thch. Nu tng loi mi
trng thch hp cho tng nhm vi sinh vt. Ha tan mi trng ri phn phi
vo cac lo, kh trng bng ni hp ap lc 121oC trong 15 pht. Ly 1mL
mu vi t l pha long nht nh cho vo cac hp a thch c kh
trng, mi mc pha long dng 4 a thch. Sau y rt vo mi hp a
khong 10 mL mi trng thch sau kh trng v ngui 40-50oC. y
np v xoay trn mi hp trn mt t cy, ngui n khi mt thch ng
li, ghi s mu v nng pha long ln mi ay hp petri v boc giy bao
ri em nui 35oC v oc kt qu sau 48h 3.
2.2.2.1.3. Tng vi sinh vt hiu kh
S dng mi trng R2A agar, ha tan vi lng 15,2g/1000 mL, tip
theo em hp kh trng 121oC trong 15 pht.
Ly 1 mL mu hoc mu pha long cho vo hp petri c kh
trng, rt vo khong 10 mL mi trng R2A agar kh trng, y np v
xoay trn u mi hp trn mt t cy, ngui, i khi mt thch ng li
hon ton sau y ghi tn mu v nng pha long ln ay hp petri, bao
giy ri em nui 35oC trong vng 48h 3. Khi thi gian, em ra oc
kt qu bng cach m cac khun lc trn a thch. Sau y bao cao s
khun lc trn mt n v th tch mu tng ng.
2.2.2.2. Phng php xc nh sch bng ATP quang sinh
nghin cu ng dng phng phap kim tra nhanh v sinh trong
dy chuyn sn xut bia bng o ATP quang sinh, cac bc cn phi tin
hnh nh sau:
- Nhn dng cac im kim tra: Cac im kim tra c coi l cac im kim
soat ti hn (CCP), i din cho hiu qu ca qua trinh v sinh. Kt qu thu
c s phn anh thc t hiu qu ca qua trinh v sinh.
Cac im kim tra ni chung c chia lm hai nhm:
B mt tip xc trc tip vi sn phm v nhm b mt tip xc gian tip vi
sn phm.
7
- Lp k hoch ly mu: Cn c s sn xut, xy dng k hoch ly mu,
tn sut ly mu cho ph hp tng v tr kim tra.
Thu thp d liu v xy dng gii hn thng quy: Ni dung ny gip nhn
din tinh trng v sinh hin ti ca cac v tr kim tra. Nhng d liu ny bao
gm ti cac v tr kim tra c trng thai bn v trng thai v sinh sch s.
- Xac nh cac gia tr gii hn: Sau khi c cac s liu v tinh hinh v sinh
tng v tr kim soat, kt hp vi theo di qua trinh sn xut v nhng kt qu
kim tra v sinh thng thng s tin hnh xy dng mc kim soat v sinh
bng ATP quang sinh.
- o ATP quang sinh bng thit b o HY_LITE 2.
c tnh k thut ca my:
+ Khong tuyn tnh 0-99000 RLU
+ C th lu hn 2000 kt qu o
+ Tn hiu nn < 10 RLU
+ Cac dng kim tra: HACCP, Test and store, Test only
+ u d: photomultiplier (PMT) loi cc nhy c chc nng bo v qua ti
Trong phng phap xac nh bng ATP quang sinh c hai dng dng c ly
mu c s dng
+ Que ly mu b mt:
Bi ly mu: Rt que ly mu ra khi dng c Clean-trace, bi ln b mt
kim tra vi din tch khong 10cm2
Hot ha/lc: Cn thn t que ly mu vo dng c Clean-trace, n xung v
lc hot ha mu trong vng 10 giy.
oc kt qu: t dng c Clean-trace vo b phn nhn mu ca may v i
oc kt qu khong 10 giy sau y.
+ Que ly mu nc:
Bi que ly mu: Rt que ly mu khi dng c Aqua- trace, nhng que ly
mu vo dung dch kim tra
8
Hot ha/lc: Cn thn t que ly mu vo dng c Aqua-trace, lc hot
ha mu trong vng 10 giy.
oc kt qu: t dng c Aqua-trace vo b phn nhn mu ca may v i
oc kt qu trong vng 10 giy sau y.
2.2.2.3. ng dng my o quang UV-VIS U-1900
c tnh k thut
H quang: Ratio beam
Bc sng: 190-1100nm
rng di quang ph: 4 nm
Stray light: trong 0.5 % (220 nm NaI; 340 nm NaNO2)
chnh xac bc sng: 0.5 nm
tai lp bc sng: 0.3 nm
Dng o: Photometry, Wavelength scan, Time scan, Multiple, Wavelength
measurement
Photometric range: Abs (-3.000 ti 3.000)
% T (0 ti 300%T)
Nng (0.000 ti 9999)
Photometric Accuracy : 0.002 Abs ( 0 ti 0.5 Abs)
0.004 Abs (0.5 ti 1.0 Abs)
% 0.3 T
Photometric repeatability : 0.001 Abs ( 0 ti 0.5 Abs)
0.002 Abs (0.5 ti 1.0 Abs)
% 0.15 T
Tc scan bc sng: 10, 100, 200, 400, 800, 1200, 2400, 3600 nm/pht
Baseline stability ( tri ): 0.0004 Abs/h (500nm, 2 ting sau khi bt may)
Baseline flatness: 0.002 Abs (200 ti 950 nm)
Baseline level: 0.00015 Abs (500nm)
9
Ngun sang: WI v n D2
Detector: Silicon photodiode
Tin hnh: Cac mu mi trng phat quang c chun b. S dng
cuvet 20 mm cho php o. Chon di qut bc sng t 190 nm - 1100 nm.
II. KT QU V THO LUN
Chng ti thc hin kim tra v sinh mt s im trong yu trong dy
chuyn sn xut bia nh:
+ Cac tec ln men
+ May loc sn phm
+ Cac tec sn phm
3.1. Phng php phn tch truyn thng
3.1.1. Kim tra cc tec ln men
Tec ln men 1
Bng 2: Kt qu kim tra v sinh tec ln men 1
Ch tiu phn tch Ra ln 1 Ra ln 2 Ra ln 3 Ra ln 4
Tng vi sinh vt hiu kh 5.4 x106 5.8 x106 5.2 x104 1.5 x103
Tng nm men, nm mc 4.2 x 103 3.7 x10
3 3.2 x10
3 1.0 x 10
2
Tng E.coli (MPN) 0 0 0 0
Tng Coliform (MPN) 4 2,0 2,0
10
Ch tiu phn tch Ra ln 1 Ra ln 2 Ra ln 3 Ra ln 4
Tng vi sinh vt hiu kh 4.2 x106 3.7 x106 2.1 x104 1.0 x103
Tng nm men, nm mc 3.6 x103 3.7x10
3 1.9 x10
3 1.0 x10
2
Tng E.coli (MPN) 0 0 0 0
Tng Coliform (MPN) 2,0 4,0 2,0 2,0
Tec ln men 3
Bng 4: Kt qu kim tra v sinh tec ln men 3
Ch tiu phn tch Ra ln 1 Ra ln 2 Ra ln 3 Ra ln 4
Tng vi sinh vt hiu kh 5.7 x106 5.5 x10
6 4.7 x10
4 1.8 x10
3
Tng nm men, nm mc 3.4 x103 3.3x10
3 2.6 x10
3 0.5 x 10
2
Tng E.coli (MPN) 0 0 0 0
Tng Coliform (MPN) 4,0 2,0
11
Bng 6: Kt qu kim tra v sinh may loc sn phm t 2
Ch tiu phn tch Ra ln 1 Ra ln 2 Ra ln 3 Ra ln 4
Tng vi sinh vt hiu kh 6.1 x103 6.0 x10
3 2.0 x10
2 5 x10
Tng nm men, nm mc 4.7 x102 4.8 x10
2 3.0 x10
2 12
Tng E.coli (MPN) 0 0 0 0
Tng Coliform (MPN) 4,0
12
Bng 9: Kt qu kim tra v sinh ca tec sn phm 2
Ch tiu phn tch Ra ln 1 Ra ln 2 Ra ln 3 Ra ln 4
Tng vi sinh vt hiu kh 6.0 x103 6.1 x10
3 8.0 x10
2 12
Tng nm men, nm mc 5.7 x102 5.4 x10
2 6.2 x10 14
Tng E.coli (MPN) 0 0 0 0
Tng Coliform (MPN) 2,0
13
4 V sinh ln 4 295 297 285
Khi v sinh cac tec ln men ln ra th 1 v th 2 nc cn bn,
cac tec o c u khong 6000 RLU. T ln 3 tr i mc v sinh
kha hn, cac tec o c khong 500 RLU. Kt thc qua trinh kim tra (ln
4) mu o c t mc di 300 RLU c 3 tec
3.2.2. Kim tra my lc sn phm
Mu ly may loc sn phm c tin hnh sau khi x thao vi loc, x
b cn ln u tin trn cac bn loc.
Bng 12: Kt qu kim tra bng phng phap ATP quang sinh
may loc sn phm
S th t Tn mu t 1 (RLU) t 2 (RLU) t 3 (RLU)
1 V sinh ln 1 790 795 787
2 V sinh ln 2 792 620 690
3 V sinh ln 3 254 265 235
4 V sinh ln 4 150 120 137
hai ln v sinh u, may loc sn phm vn cha c sch, sau ln
th 3 v sinh kt qu RLU c ba t u nh hn 300 RLU. may loc sn
phm ch sau 3 ln c th t yu cu v sinh.
3.2.3. Kim tra cc tec sn phm
Bng 13: Kt qu kim tra bng phng phap ATP quang sinh
cac tec sn phm
S th t Tn mu Tec 1 (RLU) Tec 2 (RLU) Tec 3 (RLU)
1 V sinh ln 1 870 734 674
2 V sinh ln 2 723 627 580
3 V sinh ln 3 225 243 235
14
4 V sinh ln 4 131 127 130
Sau ln 1 v ln 2, qua trinh v sinh vn khng thay i ang k. n
ln 3, tt c cac tec u t kt qu di 300 RLU. Cng nh may loc sn
phm, tec sn phm cng t yu cu sau 3 ln v sinh.
3.3. nh gi s tng thch gia kim tra vi sinh vt bng phng php
truyn thng v o ATP quang sinh
3.3.1. Mu bit trc nng
so sanh s tng thch, chng ti chun b cac mu th bit trc
nng . Mu c chun b t nm men Saccharomyces carlbergensis dng
trong sn xut bia, vi nng ban u l 3.5 x 106 t bo/mL Cac mu c
chun b vi nhiu mc nng khac nhau vi h s pha long l 10-1, 10-2,
10-3
, 10-4
kt qu c trinh by bng di y.
Bng 14: Kt qu phn tch mu bit trc nng bng c
phng phap truyn thng v phng phap ATP quang sinh.
Tn mu Tng vi sinh vt
(CFU) o ATP (RLU)
Mu ban u 3.5 x 106 2302
Mu 10-1 3.5 x 105 747
Mu 10-2 3.5 x 104 243
Mu 10-3 3.5 x 103 158
Mu 10-4 3.5 x 102 132
Qua bng kt qu ta thy kt qu o c t phng phap ATP quang
sinh v tng s t bo nm men m c c s tng thch, s n v RLU
tng dn khi s lng t bo tng dn. giai on pha long gia nng
mu 10-4 v mu 10-3, kt qu thay i khng nhiu, nhng c s nhy vot
gia mu 10-1 v 10-2 v c bit l t mu ban u so vi mu pha long
10 ln ( Mu 10-1).
15
3.3.2. Mu trn dy truyn sn xut bia
a. Kim tra tec ln men
Tec 1
Bng 15: Kt qu phn tch tec ln men 1 bng phng phap truyn
thng v phng phap ATP quang sinh
Tn mu Tng vi sinh vt
hiu kh (CFU) o ATP (RLU)
Ra ln 1 5.8 x 106 6608
Ra ln 2 5.4 x106 5460
Ra ln 3 5.2 x 104 520
Ra ln 4 1.5 x 103 295
Tec 2
Bng 16: Kt qu phn tch tec ln men 2 bng phng phap
truyn thng v phng phap ATP quang sinh
Tn mu Tng vi sinh vt
hiu kh (CFU) o ATP (RLU)
Ra ln 1 4.2 x 106 5500
Ra ln 2 3.7 x106 4673
Ra ln 3 2.1 x 104 510
Ra ln 4 1.0 x 103 297
Tec 3
Bng 17: Kt qu phn tch tec ln men 3 bng phng phap
truyn thng v phng phap ATP quang sinh
Tn mu Tng vi sinh vt
hiu kh (CFU) o ATP (RLU)
16
Ra ln 1 5.7 x 106 6740
Ra ln 2 5.5 x106 6580
Ra ln 3 4.7 x 104 495
Ra ln 4 1.8 x 103 285
Nhn xt:
Qua trinh v sinh, ra ln 1 v ln 2 cha c s thay i nhiu th hin
c phng phap xac nh truyn thng v phng phap ATP quang sinh. T
ln ra th 3, cac tec ln men mi thy biu hin r rt s thay i v sinh,
v n ln ra cui (ln 4) cng thi s m bo v sinh mi c hon tt.
Nu dng phng phng kim tra nhanh bng ATP quang sinh thi tec ln
men hon thnh qua trinh v sinh khi ch s RLU nh hn 300.
b. Kim tra may loc sn phm
t 1
Bng 18: Kt qu phn tch may loc sn phm t 1 bng phng
phap truyn thng v phng phap ATP quang sinh
Tn mu Tng vi sinh vt
hiu kh (CFU) o ATP (RLU)
Ra ln 1 6.2 x 103 790
Ra ln 2 5.9 x103 792
Ra ln 3 3.0 x 102 254
Ra ln 4 0.5 x 102 150
t 2
Bng 19: Kt qu phn tch may loc sn phm t 2 bng phng
phap truyn thng v phng phap ATP quang sinh
Tn mu Tng vi sinh vt o ATP (RLU)
17
hiu kh (CFU)
Ra ln 1 6.1 x 103 795
Ra ln 2 6.0 x103 620
Ra ln 3 2.0 x 102 265
Ra ln 4 0.4 x 102 120
t 3
Bng 20: Kt qu phn tch may loc sn phm t 3 bng phng
phap truyn thng v phng phap ATP quang sinh
Tn mu Tng vi sinh vt
hiu kh (CFU) o ATP (RLU)
Ra ln 1 5.9 x 103 787
Ra ln 2 5.7 x103 690
Ra ln 3 1.0 x 102 235
Ra ln 4 2.0 x 10 117
Nhn xt: may loc sn phm t sau ln ra th ba s m bo v sinh
c hon tt, c ba t ch s RLU o c u nh hn 300.
a. Kim tra cac tec sn phm
b. Tec 1
Bng 21: Kt qu phn tch tec sn phm 1 bng phng phap
truyn thng v phng phap ATP quang sinh
Tn mu Tng vi sinh vt
hiu kh (CFU) o ATP (RLU)
Ra ln 1 6.3 x 103 870
18
Ra ln 2 6.2 x103 723
Ra ln 3 10 x 102 225
Ra ln 4 2.0 x 10 131
Tec 2
Bng 22: Kt qu phn tch tec sn phm 2 bng phng phap
truyn thng v phng phap ATP quang sinh
Tn mu Tng vi sinh vt hiu
kh (CFU) o ATP (RLU)
Ra ln 1 6.0 x 103 734
Ra ln 2 6.1 x103 627
Ra ln 3 8.0 x 102 243
Ra ln 4 1.2 x 10 127
Tec 3
Bng 23: Kt qu phn tch tec sn phm 3 bng phng phap
truyn thng v phng phap ATP quang sinh
Tn mu Tng vi sinh vt
hiu kh (CFU) o ATP (RLU)
Ra ln 1 5.4 x 103 674
Ra ln 2 4.8 x103 580
Ra ln 3 9.0 x 102 235
Ra ln 4 1.7 x 10 130
Nhn xt: cac tec ln men sau ln ra v sinh th 3, cac tec t yu cu
v v sinh, cac ch s RLU o c c 3 tec sau ln ra th 3 u nh hn
300.
19
3.4. Kho st ng dng phng php to mu xc nh tng s Coliform
trn my quang ph U-1900 ( UV-VIS spectrophotometre)
S dng nc thi sau v sinh ca xng sn xut kim tra
Coliforms. T ngun nc thi xac nh c Coliforms tng s (k hiu
M) em pha long ra cac nng khac nhau M x 5 -1; M x 10 -1; M x 10 -2
c cac mu c cha Coliforms khac nhau nui cy, sau em o trn
quang ph UV-VIS.
Chng ti tin hnh scan bc sng ba mc nng ( M x 5 -1; M
x 10 -1
; M x 10 -2
). t bc sng 190nm -1100nm, tc 100nm/pht. Kt qu
thu c nh sau:
Nng M x 10-2mL
Bng 24: Kt qu scan mu c nng pha long 10-2 bc sng
190 1100nm
Peak Valley
Th t WL Abs WL Abs
1 967 0.467 1078 0.265
2 655 0.930 908 0.322
3 614 0.957 644 0.897
4 284 3.587 559 0.830
5 243 3.776 277 3.551
Nng M x 10-1
Bng 25: Kt qu scan mu c nng pha long 10-1 bc sng
190 1100nm
Peak Valley
Th t WL Abs WL Abs
1 972 0.409 1074 0.220
20
2 292 3.646 907 0.258
3 242 3.753 273 3.591
Nng M x 5-1
Bng 26: Kt qu scan mu c nng pha long 5-1 bc sng
190 1100nm
Peak Valley
Th t WL Abs WL Abs
1 970 0.447 910 0.306
2 655 0.951 644 0.914
3 614 0.979 558 0.840
4 292 3.637 276 3.587
5 241 3.793
c 3 mc nng , hp th t cao nht bc sng 241nm 243nm.
Tuy nhin dung dch to mu c mu xanh lc, l vng nhin thy nm trong
di ph t 400 nm- 800nm. Do vy chng ti tin hnh kho vng bc
sng t 400 nm 800 nm. Kt qu thu c nh sau:
Nng ban u M
Bng 27: Kt qu scan mu M bc sng 400 800nm
Peak Valley
Th t WL Abs WL Abs
1 613.5 0.928 569.0 0.825
Nng M x 5 -1
21
Bng 28: Kt qu scan mu M x 5-1 bc sng 400 800nm
Peak Valley
Th t WL Abs WL Abs
1 615 0.992 568 0.886
Nng M x 10 -1
Bng 29: Kt qu scan mu M x 10-1 bc sng 400 800nm
Peak Valley
Th t WL Abs WL Abs
1 615 0.909 588.5 0.775
Nng M x 10 -2
Bng 30: Kt qu scan mu M x 10-2 bc sng 400 800nm
Peak Valley
Th t WL Abs WL Abs
1 656 1.301 640.5 1.272
2 617 1.301 565.5 1.089
Nng M x 10 -3
Bng 31: Kt qu scan mu M x 10-3 bc sng 400 800nm
Peak Valley
Th t WL Abs WL Abs
1 657 0.972 636.5 0.960
2 617.5 0.985 561 0.823
22
c 5 mc nng , scan t bc sng 400nm 800nm, chng ti thu c
hp th cao nht bc sng 617 nm. Chng ti tin hnh o mu nc
thi ly b cha nc thi sau khi v sinh bc sng 617 nm, thu c kt
qu nh sau:
Bng 32: Kt qu phn tch mu ly b cha nc thi sau khi v
sinh bng phng phap truyn thng v o bng may UV-VIS 1900
ca Hitachi.
Tn mu ABS MPN
(coliforms)
M 1.634 13 x 105
M x 10-1
1.442 80 x 104
M x 10-2
1.266 50 x 103
M x 10-3
1.223 40 x 102
M x 10-4
0.852 23 x 10
M x 10-5
0.074 40
Nhn xt:
Nhin vo bng kt qu trn chng ta thy c s tng thch gia hp
thu o c trn may quang ph UV-VIS 1900 v phng phap xac nh
coliform truyn thng (Phng phap nhiu ng xac nh coliform: ISO 9308-
2). S khac nhau v hp th ABS gia cac mc nng khac nhau rt r.
Qua , cho thy ban u c th dng thit b UV VIS 1900 ca Hitachi vo
xac nh s c mt ca coliform.
III. KT LUN V KIN NGH
T kt qu nghin cu c ti cho chng ta mt s kt lun sau:
1. Phng phap nui cy vi sinh vt truyn thng v phng phap o ATP
quang sinh hoc c s tng thch nht nh.
23
2. Phng phap ATP quang sinh c nhiu u im, d thc hin v cho kt
qu nhanh, tng i chnh xac cho cac php th nhanh v v sinh an
ton thc phm.
T kt qu nghin cu cho thy, khi o bng phng phap ATP quang
sinh c kt qu nh hn 300 RLU, thi mt s khu vc v sinh trong dy
truyn sn xut bia t yu cu v v sinh.
Phng phap ATP quang sinh c nhng u im nh sau:
+ L phng phap o nhanh, n gin, d thc hin, d mang i hin
trng.
+ T kt qu o c bng ATP quang sinh khng ch phat hin c s
lng vi sinh vt sng m cn kim tra c hm lng tn d cac cht
dinh dng lm mi trng cho vi sinh vt phat trin.
+ Nh vy gip cac nh may, c s sn xut v ch bin thc phm khc
phc c cac s c v sinh tc thi.
Tuy nhin phng phap ny cng c mt s hn ch:
+ Phng phap ch kim tra c tng s vi sinh vt m cha c phn loi
c tng loi vi sinh vt.
+ Gia thnh ca mi que th ly mu cng tng i t v mi que ly
mu ch dng c mt ln duy nht, nu thao tac ly mu khng ng s
kha tn km khi ly i, ly li nhiu ln.
3. trong nghin cu ny, chng ti tin hnh th ng dng ca may quang
ph UV-VIS 1900 Hitachi v thy c s tng thch kha cht ch gia
hp th v tng s Coliform trong mu. Bc u c th ng dng may o
UV-VIS 1900 Hitachi vo vic xac nh mc c mt ca coliform mt
cach tng i.
Kin ngh:
1. Phng phap o ATP quang sinh cn l phng phap mi, c th em
ra ng dng rng ri v c hiu qu hn, chng ta cn phi u t thi
gian nghin cu v tham kho nhiu hn na.
C nhng nghin cu su hn v ng dng ca may quang ph UV-VIS vo
vic xac nh tng s vi sinh vt ni chung cng nh tng s coliforms. Cng
24
c thm mi tng quan gia hp th v nng vi sinh vt, t c th
cho ra mt kt qu tng ng gia n v hp th ABS v n v ca vi
sinh vt tng s cng nh tng s coliforms
References :
Ting Vit
1. Kiu Hu nh (2010), Gio trnh vi sinh vt hc thc phm, Nh xut bn
giao dc Vit Nam.
2. Nguyn Ln Dng, Nguyn ng c, ng Hng Min (1976), Mt s
phng php nghin cu vi sinh vt hc, Nh xut bn khoa hoc k
thut, tp3.
3. Nguyn Ln Dng (1971), Kim tra s lng v phn lp cc nhm vi sinh
vt, Nh xut bn i hoc tng hp, tp 1
4. Nguyn Vn t (1974), Phn tch lng thc thc phm, Nh xut bn
khoa hoc k thut.
5. Nguyn Quang Ho (1980), Thc hnh vi sinh vt hc, Nh xut bn giao
dc.
6. Lu Lm, Nguyn Yn, Trn Quang (1969), V sinh thc phm, V k
thut.
7. Hong Tch Mch, H Huy Khi (1977), V sinh dinh dng v v sinh
thc phm, Nh xut bn Y hoc.
8. V Vn Ng (1987), K thut xt nghim vi sinh vt hc, Nh xut bn Y
hoc.
9. o Th Nguyn (1995), Vi sinh vt y hc, Nh xut bn Y hoc.
10. Lng c Phm (2000), Vi sinh vt hoc v an ton v sinh thc phm,
Nh xut bn Nng nghip.
11. Phm Vn S, Bi Th Nh Thun, Nguyn Phng Tin (1975), V sinh
thc phm, Nh xut bn Y hoc.
25
12. Phm Vn Thnh (2002), Nghin cu ng dng phng php phn tch
nhanh kim tra v sinh an ton thc phm. Bao cao ti nm
2002. B Cng nghip
13. Vn Tinh (1983), Kim tra cht lng sn phm nng nghip, Nh xut
bn Thnh ph H Ch Minh.
14. Bi Nh Thun, Nguyn Phng Tin, Bi Minh c (1991), Kim nghim
cht lng v thanh tra v sinh an ton thc phm, Nh xut bn Y
hoc.
15. ng dng cc phng php phn tch cng c trong kim sot mi trng
v thc phm (1999), Hi tho khoa hoc H ni.
16. ng dng cc phng php phn tch cng c hin i trong mi trng
v an ton lng thc thc phm (1994), Hi tho khoa hoc H ni.
17. Phm Thu Yn (2001), Kim tra qu trnh v sinh trong sn xut thc
phm, nh gi s tng thch gia phng php kim tra truyn
thng v phng php o ATP quang sinh hc, kha lun tt nghip.
Ting Anh
18. Adam M.R anh Moss M.O (1995), Food Microbiology, The Royal
Society Of Chemistry.
19. Claude Moreau (1974), Mousissures toxique dans Ladimentation
edition, Pari.
20. Harry H (1962), Practical food Microbiology and Technology Wesport,
Connecticut.
21. R.D. Gonzalez. L.M. Tamagnini, P.D. Olmos, G.B. de Sousa (2003),
Evalution of a chromogenic medium for total Coliformss and
Escherichia coli determination in ready-to-eat foods, Food
Microbiology, 20, pp. 601-604.
22. Denis Byamukama, Frank Kansiime, Robert L. Mach, and Andreas H.
Farnleitner (200), Determination of Escherichia coli Cotamination
with Chromocult Coliforms Agar Showed a High Level of
Discrimination Efficiency for Differing Fecal Pollution Levels in
26
Tropical Waters of Kampala, Uganda , Applied and Environmental
Microbiology, pp. 864-868
23. K. Geissler, M. Manafi, I. Amoros and J.L. Alonso (2000), Quantitative
detarmination of total Coliforms and Escherichia coli in marine waters
with chromogenic and flourogenic media, Joural of Applied
Microbiology, 88, pp. 280-285.
24. ICMSF (2005), Microorganisms in food, Second Edition, kluwer
Academic, Plenum Publishers
25. Jay, J.M. (2000), Modern food microbiology, Sixth edition, Aspen
Pushlisher
26. Manning, S.D., (2004), Escherichia coli infections, Chelsea House
Publishers.
27. Standard Method the Examination of Water and Wastewater 21th
edition,
2005, 9215B.
28. Standard Method the Examination of Water and Wastewater 21th
edition,
2005, 9215D
29. ISO 9308-2: First edition 1990-10-01
Recommended