study on binding of afb1 by lactobacillus...

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.


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


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)


Peanut – Aspergillus and aflatoxins

Perbesaran 40X

Perbesaran 100X


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


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)


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)


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)


Reduction of Aflatoxins

PhysicallySegregation

Chemically : Ca(OH)2

BiologicallyLactic acid bacteriaFlavobacterium aurantiacumRhizopus oligosporus


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


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


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


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


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)


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.


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


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


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)


RESULTS


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)


Effect of AFB1 concentration on binding

(Condition of binding 37 oC, Cell 1010 CFU/ml)

AFB1 concentration ()

Bou

nd A

FB1

(%)

Speed of binding


(Condition 37 oC, Cells 1010 CFU/ml), AFB1 20 ppb)

Effect of pH on AFB1-LAB binding

Bou

nd A

FB1

(%)


Effect of temperature AFB1-LAB binding

(AFB1 20 ppb, Cells 1010 CFU/ml, pH 5,0)

Temperature (°C)

Bou

nd A

FB1

(%)


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

(%)


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

(%)


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


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

study on binding of afb1 by lactobacillus...

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