study on binding of afb1 by lactobacillus...
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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)
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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)
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Reduction of Aflatoxins
PhysicallySegregation
Chemically : Ca(OH)2
BiologicallyLactic acid bacteriaFlavobacterium aurantiacumRhizopus oligosporus
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LAB Fermented Product – Reduction of AFB1
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
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Referensi Strains %binding
Peltonen, et al., 2000
5 Lactobacillus dan 1 BifidobacteriumLb. johnsonii LJ-1 dan Lb. paracaseiF19
5.8 - 31.3
Peltonen et al., 2001
2 strain Lactobacillus, 5 strain Bifidobacterium, dan 3 strain Lactococcus Lb rhamnosus strain Lc 1/3, Lb.amylovorus CSCC 5197 dan CSCC 5160
5.6 – 59.7
Haskard, et al., 2001
10 strain Lactobacillus, 1 Streptococcus, 1 PLactobacillus rhamnosus GG
15-78
Binding AFB1 - LAB
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Binding reversibleStrain and dose of LABTemperature pH Washing
Released by repeated aqueous washes (Haskard et al, 2001, Peltonen et al., 2001)
Conditions on cells (viable, heat killed, acid treatment, sonication)
Non-viable cells – bind more stronger and stable (Haskard, 2001)
Factors Affecting Binding
Endang S.Rahayu Gadjah Mada University
Research area of potential benefit of lactic acid bacteriaGadjah Mada University
Starter culture
• fermented food(tape, tempe, kecapferm. fish, fruits, vegetables,etc)
Probiotic• probiotic candy• probiotic fruit baseddrink, ice cream
• probiotic salad dress.• prevention diarrhea• hypercholesterolemia• lactose intolerance• binding aflatoxin
Lacticacid
bacteria
Fermented food as
probiotic agent
Bacteriocinproducer asstartercultures
Bacteriocinproducer asprobiotics
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
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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
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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)
Endang S.Rahayu Gadjah Mada University
Effect of AFB1 concentration on binding
(Condition of binding 37 oC, Cell 1010 CFU/ml)
AFB1 concentration ()
Bou
nd A
FB1
(%)
Speed of binding
Endang S.Rahayu Gadjah Mada University
(Condition 37 oC, Cells 1010 CFU/ml), AFB1 20 ppb)
Effect of pH on AFB1-LAB binding
Bou
nd A
FB1
(%)
Endang S.Rahayu Gadjah Mada University
Effect of temperature AFB1-LAB binding
(AFB1 20 ppb, Cells 1010 CFU/ml, pH 5,0)
Temperature (°C)
Bou
nd A
FB1
(%)
Endang S.Rahayu Gadjah Mada University
Effect of cell concentration on AFB1-LAB binding
(Condition : pH 5.0; 37 oC; AFB1 20 ppb)
Cell concentration (Log CFU)
Bou
nd A
FB1
(%)
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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
Endang S.Rahayu Gadjah Mada University
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