Hình 3 .3 Kt qu xácăđ nh h ot tính kháng khu nc abacteriocin
Hình 3.4 a: s ab hăh ng; b s aăbìnhăth ng
3.7. TĨM T T
Sauăth iăgianăth căhi năđ ătƠi,ăchúngătơiăthuănh năđ cănh ngăk tăqu ăsau:
nhătínhăkh ăn ngăsinh bacteriocin
Xácă đ nhă ho tă tínhă
khángă khu nă c aă
bacteriocin
– 5/13 ch ng có kh n ngă khángă v i L.monocytogenes, ch ng có vịng kháng và ho t tính cao nh t là K3 (11,33 ± 0,33 mm, 40 AU/mL), T3P (11,33 ± 0,33 mm, 40 AU/mL)
– Có 5/13 ch ng cho kh n ngăkhángăv i S.feacalis, ch ng có vịng kháng và ho t tính cao nh t là BD68 (11,67 ± 0,33 mm, 20 AU/mL)
– Có 6/13 ch ng cho kh n ngă khángă v i S.typhi, ch ng có vịng kháng và ho t tính cao nh t là Q16 (17,00 ± 7,00 mm, 20 AU/mL)
– Có 12/13 ch ng cho kh n ngăkhángăv i S.aureus, ch ng có vịng kháng và ho t tính cao nh t là T3P (14,00 ± 1,00 mm, 320 AU/mL)
– Khơng có ch ng nào trong 13 ch ng có kh n ngă
SVTH:ăLÊăQU NHăY NăNHI 69 kháng v i E.coli
– 2 ch ng K3, T3P có kh n ngă khángă c 4 ch ng ch th S.faecalis, S.aureus, S.typhi, L.monocytogenes
Tác đ ngă c aă nhi tă đ ă vƠă pHă trênă ho tă tínhă khángăkhu n
– .Nhi tă đ : ho t tính kháng khu n nă đ nh m c 70oC, ho t tính gi m d n v i s giaăt ngănhi tăđ . L uătr nhi tăđ th p (4°C) không nh ng khơng lƠmăthayăđ i ho t tính c a chúng mà cịn có ho t tính n đ nhă vƠă caoă h nă d chă bacteriocină thôă đ i ch ngăđ nhi tăđ phòng.
o Ch ng K3: Ho t tính kháng v n gi đ c trên 50% khi m c t 70-100°Că(90.74ă±ă1.85ăđ n 53.71 ± 1.85%). Ho t tính b m t hồn tồn sau khi 121°C/ 5 phút. o Ch ng T3P: Ho t tính kháng v n gi đ c trên 50% khi m c t 70-100°Că(87.50ă±ă0.00ăđ n 58.33 ± 2.08%). Ho t tính b m t hoàn toàn sau khi 121°C/ 5 phút.
– pH: chúng có ho t tính kháng m nh trên kho ng pH r t r ng t 2 - 9, nă đ nh và m nh nh t t i kho ng pH 6 - 9.
nhădanh K tăqu ăđ nhădanhăsinhăhóaăchoăth yăK3,ăT3Păt ngă đ ngăv iăloƠiăBacillus thuringiensis (t ăl ă71,43%)
Xácă đ nhă n ngă đ ă phaă loưngă th pă nh tă c aă d chăbacteriocinăthơăcịnă căch ăv iăviăkhu năch ă th – Ch ng K3: o S.faecalis:1/2 o S.aureus: 1/8 o S.typhi:1/2 o L.monocytogenes: 1/4 – Ch ng T3P: o S.faecalis:1/2 o S.aureus:1/4 o S.typhi:1/2 o L.monocytogenes: 1/2 Th ănghi măbacteriocină nh ăch tăb oăqu năsinhă h cătrongăs a
M uă chaiă s aă th ă nghi mă cóă ch aă bacteriocină c aă ch ngăK3ăvƠăT3Păcóăth iăgianăb oăqu năs aălơuăh nă m uăchaiăs aăđ iăch ng
SVTH:ăLÊăQU NHăY NăNHI 70
PH N IV: K T LU N VÀ NGH
SVTH:ăLÊăQU NHăY NăNHI 71
4.1. K t lu n
M cătiêuăđ ătƠi:ăsƠngăl căcácăch ngăBacillus cóăkh ăn ngăsinhăbacteriocină ngă d ngătrongăb oăqu năth căph m.ăSauăth iăgianăth căhi năđ ătƠi,ăchúngătơiđưătuy nă ch năđ că2ăch ngă(K3,ăT3P)ăcóăkh ăn ngăsinhăbacteriocinăvƠăti măn ngăb oăqu nă th că ph mă (s a).ă 2ă ch ngă nƠyă cóă kh ă n ngă khángăL.monocytogenes, S.feacalis, S.typhi, S.aureus. Ho tătínhă năđ nhă ăm cănhi tăđ ă70oC, pH 6-9.ăD chăbacteiocină thơăc aă2 ch ngăcóăkh ăn ngăkéoădƠiăth iăgianăb oăqu năs aăt tăh năsoăv iăloăđ iă ch ng,ăv iăt ăl ă5%ăth iăgianăb oăqu năt ngăg pă1,63ăl năv iăch ngăK3ăvƠă1,62ăl nă v iăch ngăT3P.
4.2. ngh :
ăhoƠnăthi năh năchoăcơngătrìnhănghiênăc uvƠăđ ngăd ngăbacteriocin c aă cácăch ngăviăkhu năth nghi m,ăchúngătơiăcóănh ngăđ ăngh ănh măc ngăc ăthêmă k tăqu ămƠăđ ătƠiăđ tăđ căc ngănh ăh ngăphátătri n:
Nghiên c u t iă uăhóaămơiătr ngălênămenăđ s n xu t bacteriocin b ng quy ho ch th c nghi m
Tách chi t và tinh s ch bacteriocin Th nghi m tính an tồn c a bacteriocin
Th nghi m l i hi u qu c a bacterion trong b o qu n s a và m t s th c ph m khác
SVTH:ăLÊăQU NHăY NăNHI 72
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SVTH:ăLÊăQU NHăY NăNHI 82
PH L C
PH ăL CăMỌIăTR NG ậ HịAăCH T
Môiătr ngă ThƠnhăph n
Nutrient Broth Pepton
Caoăn mămen NaCl N căc t 10 gam 5 gam 5 gam 1000mL MHB (pH ~ 7,4 ± 0,2)
Infusion from meat Casein acid hydrolysate Starch N căc t 300 gam 7,5 gam 1,5 gam 1000 mL
MHA (Mueller Hinton Agar) Mueller Hinton Broth
Agar N c c t
21,5 gam 20 gam 1000 mL
Môiătr ngălênămenăcácălo iă đ ngă( emăh păkh ătrùngă ă 110oC/15 ậ 20 phút).pH : 7.4 NB ng Phenol red N c c t 8 gam 10 gam 0,01 gam 1000 mL HịAăCH T
H2O2 5% (B oă qu nă trongă chaiămƠuă ă4oC)
H2O2 H2O
5 mL 100 mL
Thu cănhu măCrystal violet Dungăd chă1
Crystal violet C n96o Dungăd chă2 ammonium oxalate N c c t 2gam 20 mL 0.8gam 80 mL
Thu cănhu mălugol I2
KI2 N c c t
1 gam 2gam 300 mL
Thu cănhu măSafraninăO Safranină Oă (dungă d chă 2%ă trongăc nă96o) Ethanol N căc t 0.25gam 10 mL 90 mL
SVTH:ăLÊăQU NHăY NăNHI 83
PH ăL CăTH NGăKÊ
2.1. K t qu x lý th ng kê ANOVA m t y u t xácă đ nh kh n ngă sinhăbacteriocin c a các ch ng Bacillus d a vào
đ ng kính vịng vơ khu n ( = mm) b ng ph n m m Microsoft Excel.
2.1.1. Listeria monocytogenes
Anova: Single Factor
SUMMARY
Groups Count Sum Average Variance
Row 1 3 21 7 0 Row 2 3 0 0 0 Row 3 3 21 7 0 Row 4 3 0 0 0 Row 5 3 34 11.33333 0.333333 Row 6 3 0 0 0 Row 7 3 34 11.33333 0.333333 Row 8 3 0 0 0 Row 9 3 26 8.666667 0.333333 Row 10 3 0 0 0 Row 11 3 0 0 0 Row 12 3 0 0 0 Row 13 3 0 0 0 ANOVA Source of
Variation SS df MS F P-value F crit
Between Groups 815.7436 12 67.97863 883.7222
9.49E-
31 2.147926 Within Groups 2 26 0.076923
2.1.2. Streptococcus feacalis
SVTH:ăLÊăQU NHăY NăNHI 84 SUMMARY
Groups Count Sum Average Variance
Row 1 3 0 0 0 Row 2 3 0 0 0 Row 3 3 0 0 0 Row 4 3 0 0 0 Row 5 3 21 7 0 Row 6 3 0 0 0 Row 7 3 21 7 0 Row 8 3 0 0 0 Row 9 3 0 0 0 Row 10 3 25 8.333333 0.333333 Row 11 3 35 11.66667 0.333333 Row 12 3 0 0 0 Row 13 3 32 10.66667 0.333333 ANOVA Source of
Variation SS df MS F P-value F crit
Between Groups 791.5897 12 65.96581 857.5556 1.4E-30 2.147926 Within Groups 2 26 0.076923
Total 793.5897 38
2.1.3. Salmonella typhi
Anova: Single Factor SUMMARY
Groups Count Sum Average Variance
Row 1 3 0 0 0 Row 2 3 51 17 7 Row 3 3 0 0 0 Row 4 3 50 16.66667 6.333333 Row 5 3 37 12.33333 0.333333 Row 6 3 48 16 1 Row 7 3 26 8.666667 2.333333 Row 8 3 43 14.33333 16.33333 Row 9 3 0 0 0
SVTH:ăLÊăQU NHăY NăNHI 85 Row 10 3 0 0 0 Row 11 3 0 0 0 Row 12 3 0 0 0 Row 13 3 0 0 0 ANOVA Source of
Variation SS df MS F P-value F crit
Between Groups 2099.026 12 174.9188 68.21833 1.65E- 16 2.147926 Within Groups 66.66667 26 2.564103 Total 2165.692 38 2.1.4. Staphylococcus aureus