Endang S.Rahayu Gadjah Mada University Study on binding of AFB1 by Lactobacillus acidophilus SNP-2 Endang S. Rahayu, Hanim Z. Amanah, Rohula Utami and Tyas Utami Gadjah Mada University, Yogyakarta, Indonesia Presented in the Asian Conference of Lactic Acid Bacteria 2007, 14-17 October 2007, Shianghai, China.
28
Embed
Study on binding of AFB1 by Lactobacillus …cemycos.tp.ugm.ac.id/wp-content/uploads/2010/08/Study-on...Endang S.Rahayu Gadjah Mada University Study on binding of AFB1 by Lactobacillus
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Endang S.Rahayu Gadjah Mada University
Study on binding of AFB1 by Lactobacillus
acidophilus SNP-2
Endang S. Rahayu, Hanim Z. Amanah, Rohula Utami and Tyas UtamiGadjah Mada University, Yogyakarta, Indonesia
Presented in the Asian Conference of Lactic Acid Bacteria 2007, 14-17 October 2007, Shianghai, China.
Endang S.Rahayu Gadjah Mada University
AflatoxinsSecondary metabolites produced by Aspergillus flavus, A. parasiticus, and A. nomiusHave detrimental effects on the health
hepato-carcinogenic, mutagenic, teratogenic, and immunosuppressive effects O O OCH3
O O
O
Endang S.Rahayu Gadjah Mada University
Occurrence in food commodities
Indonesia as tropical country – aflatoxins contaminations in agricultural commodities are unavoidableMaximum level of AFB1 – 20 ppb and total aflatoxins – 35 ppb (SNI – 2007)
Endang S.Rahayu Gadjah Mada University
Peanut – Aspergillus and aflatoxins
Perbesaran 40X
Perbesaran 100X
Endang S.Rahayu Gadjah Mada University
100
80
100 100
7580
0
67
0
20
0 0
2520
0
33
0
20
40
60
80
100
120
% s
ampl
es c
onta
min
ated
by
AFB
1
Pati
Agu
s
Rem
bang
Trad
. Mar
ket
Pati
Agu
s
Rem
bang
Trad
. Mar
ket
<20 >20AFB1 contamination (ppb)
FarmerRetailer
Aflatoxin B1 contamination in peanut from several production area
Rahayu, et al, 2007
Endang S.Rahayu Gadjah Mada University
38.46 37.5
71.43
20 22.2223.08
0 2.227.5
0
22.5
0
80
66.67
14.29
32.5
23.08
14.298.89
15.38
0
10
20
30
40
50
60
70
80
90< 5 ppb> 5 < 15 ppb> 15 < 50 ppb> 50 < 80 ppb
CollectorsUnshelled peanut
FarmerUnshelled peanut Peanut Peanut
WholesalerPeanutRetailer
Percentage of peanut samples contaminated with AFB1, at distribution chain at Ciganjur, West Java region (Dharmaputra et al. 2005b)
Endang S.Rahayu Gadjah Mada University
Product # samples # samples withAFB1 < 20 ppb
# samples withAFB1 > 20 ppb
Peanut with coconut sugar cake (Ampyang)
4 4(1.5 - 2.8 ppb)
0
Fried battered peanut (peyek)
2 2(1.2 – 4.4 ppb)
0
Egg battered peanut (kacang telor)
3 30-5 – 2.9 ppb
0
Fried Peanut 3 31.0 – 2.3 ppb
0
Sweetened peanut 9 8 1 (21.2 ppb)
Peanut sauce 14 5 (0.5 – 19.5)
8 (max 100 ppb)
AFB1 contamination in Peanut Products (Rahayu et al., 2007)
Endang S.Rahayu Gadjah Mada University
Name of products
% Contaminated samples
Mean (range) % of samples with >35 ppb
Roasted peanut (47)
12.8 1.8 (0-21.0) 0
Oven peanut (3)
0 0 0
Flour coated peanut (22)
13.6 5.2 (0-62.5) 4.6
Peanut sauce (12)
91.7 41.6 (0-220.9) 25
Sweet peanut cake (enting2 gepuk) (4)
75 20.8 (0-41.7) 25
Level of aflatoxin in peanut-based products(Lilieanny et al. 2005)
Fermented milk (Megalla & Mohran 1984, Rasic et al., 1991)YogurtSourdough bread (Zinedine et al., 2002)Lb. rhamnosus GG dan Lc 705 – reduce AFB1 in the liquid media
Endang S.Rahayu Gadjah Mada University
Referensi Strains %binding
Peltonen, et al., 2000
5 Lactobacillus dan 1 BifidobacteriumLb. johnsonii LJ-1 dan Lb. paracaseiF19
Foodbiopreservatives● Bacteriocin ● Biomass of LAB
Endang S.Rahayu Gadjah Mada University
Lactobacillus acidophilus SNP-2 (infant origin)
Resistant to low pH (pH 2.0) & bile salt (10%)Has antagonistic effect againts Shigella, Salmonella choleraecius, Vibrio parahaemolyticus, E. coli, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereusAble to assimilate cholesterol serum (3.60%) & deconjugate bile salt (1.97%)Resistant to spray drying & freeze dryingGrow well in low cost medium (coconut water & green bean malt extract)
Endang S.Rahayu Gadjah Mada University
Aims of this research
to study factors that influence the binding of AFB1 by viable cells of Lactobacillus acidophilusSNP-2, such as pH, concentration of AFB1, time, temperature and concentration of cells; to study the binding of AFB1 by the non-viable cells after the physico-chemical treatments of cells such as acidity, heat, and sonication.
Endang S.Rahayu Gadjah Mada University
Materials and Methods
Lactic acid bacteria strains from several sourcesBiomass of LAB were produced using MRS, incubation for 24h, 37°C – followed by centrifugation to obtain biomass pelletBinding was carried out in phosphate buffer saline (PBS)Incubation, 24h, 37°C
Endang S.Rahayu Gadjah Mada University
Variation
Concentration of toxins20, 40, 60, 80, and 100 ppb
Concentration of cells108, 109 and 1010 CFU/ml
Temperature : 4, 28, and 37 °CpH : 3.0; 4.0; 5,0; 6,0; and 7,3Washing
Endang S.Rahayu Gadjah Mada University
Physico-chemical treatments – dead cells
Heat (121 °C, 15 min)Acid (soaking in 2M HCl, for 1 h)Sonication (60 min by ultrasonic oscillator, 4 °C)
Endang S.Rahayu Gadjah Mada University
RESULTS
Endang S.Rahayu Gadjah Mada University
Genus - spesies No strain % AFB1 bound
Lactobacillus acidophilus SNP-2 61.85
Lactobacillus bulgaricus FNCC-0041 37.56
Streptococcus thermophilus FNCC-0040 31.09
Lactobacillus plantarum FNCC-0265 28.52
Lactobacillus sp. Dad-13 26.12
Lactobacillus plantarum-pentosus
FNCC-0249 9.33
Lactobacillus sp. RS-12 7.21
AFB1 binding by lactic acid bacteria (pH 7.3; 37oC)
Stability AFB1-Lactobacillus acidophilus SNP-2 complexes after 5x washing
25.17
56.8561.52
20.04
3.81
42.20 45.61
7.77
010203040506070
Hidup Panas Asam Sonikasi
% p
engi
kata
n AF
B1
Aw al P1 P2 P3 P4 P5
Viable Heat Killed Acid Sonication
P0
Bou
nd A
FB1
(%)
Endang S.Rahayu Gadjah Mada University
Binding of AFB1 by Lactobacillus acidophilus SNP-2
L. acidophilus SNP2 Binding AFB1 (%)
% relative to viable cells
Viable cell 25,17 100
Heat killed cells 56,85 225,9
Dead cells (acid) 61,52 244,5
Cell fraction (sonication)
20,04 79,6
AFB1: 0.25 µg; cells 1010 CFU/ml; pH 7,3; temperature 37ºC, 24 h
Endang S.Rahayu Gadjah Mada University
ConclusionsProbiotic, Lactobacillus acidophilus SNP-2 is the most potential LAB for AFB1 binding Concentration of AFB1 did not give any effect on the percentage of binding, however it affect the speed of binding The higher the toxin concentration the faster of binding The lower pH the more bound AFB1The higher the temperature the bigger its ability to bind AFB1The higher concentration of the cells the more bound of AFB1Heat-killed probiotic – stronger and stable binding