Proceedma« of the 7th Internatumal Workmg Conference on Stored-product Protection. - Volume 1

Aflatoxin b 1 binding abilities of some probiotic bacteria

Wu Xiaorong", Nagendra Shah2

Abstract

The detoxification abilities and toxm bindmg properties(ability and strength) of some probiotic bactena on aflatoxmBl were studied The detoxification abihties of the testedstrams ranged from 20 % - 50 %. About 60 % - 90 % of thebound aflatoxin B1 was washed out With water, and only10% - 40 % was strongly bound by the bacterial cells.Among the tested strains, Bifulobacteriumi infomus 1912demonstrated a sigruficantly stronger aflatoxm bmdingability than the other strains, while strain B.pseudolongum 20099 showed the lowest aflatoxin bmdmgabilities of about 10% of the added 50ng aflatoxm Bi, B.bifidum 1 showed the least aflatoxm B, bmdmg strengthduring the 180 minutes treatment.

Introduction

Aflatoxins in foods and feed attract worldwide attention asthey cause tremendous economic loses each year and healthproblems. Researchers have tried vanous methods toehmmate or reduce the aflatoxm level in foods and feeds.Recently, some research work has been carried out onantimutagenic and detoxification properties of some probioticbacteria or commercial dairy bacteria (Custy F. et al 1990;Orrhage, K 1994; Sudarshan R Nadathur 1994). Somestrams were claimed possess high aflatoxm bmdmg abihty ofup to 80 - 90 % of the added toxin or other mutagens (El-nezami, H 1998; Lankaputhra, W. E. V 1998). However,all these studies only have detected the toxms or mutagensm the supernatant part of the treated bacterial suspensions,very little work has been carried out to fmd out as to wherethe undetected toxins have gone or how strongly the toxmsare bound by the bacterial cells.The objective of this study was to mvestigate the

detoxification abilities and toxin bmdmg ability of someprobiotic bacteria of aflatoxm Bi.

IT eadung and Research Section for Microbiology, Zhengzhou GramCollege, Zhengzhou, Henan 450052, PRof Chma

2School of Life Science and Technology, Wernbee Campus, VictonaUmversity of Technology, POBox 14428, MCMC Melbourne,Victoria 8001, Australia

Materials and Methods

Reagents used

All the organic solvents used in this study were HPLCgrade (BDH Laboratory Suppher, Pole, England)Yeast extract (OXIOD); "Lab-Lemco ' Powder (umpathLtd. , Basingstoke, Hampshire, England).

Strains of the probiotic bacteria

The microbiological lab (Victoria Unrversrty oftechnology, Werribee Campus) provides four probioticstrams (B bifuium. 1900, B. pseudolongum 20099, B.tnfantis 1912, Lactobacillus easel,) , which wereonginally from The Division of Food Science andTechnology, CSIRO, Highett, Victoria, Australia. Twostrams (B bifuiuni 1 and L. casei 1) were isolated fromcommercial yoghurts (25 % frui t JUice Vaaha and Yakult )bought from Safeway. Stock cultures were kept at - 20°C in12% reconstituted skim milk (RSM) and were used formoculating workmg cultures. Working cultures were grownm 12% RSM supplemented with 1% yeast extract and0.05%L-cysteine hydrochloride (0.45 pm membrane filterstenlization) .

Preparation of probiotic bacterial cells fordetoxification study

All the bactenal strams were grown in MRS (Oxoid,Hampshire, UK) broth at 37°C for 24 hours and the cellswere harvested by centrifugation at 4000rpm for 20 min(Beckman J2-HS refrigerated centnfuge and Soroall RTIcentrifuge, Beckman Instruments inc. CA, USA). The cellpellets were washed twice with cold distilled water andlyophihsed by a Dynavac mmi-freeze drier (DynavacEngmeering Pty, Ltd. Melbourne, Australia) and stored inrefrigerator at 4°C for later use.

Preparation of standard aflatoxin B 1 solutions

Standard stock solutwn of aflatoxin Bi: About Irngstandard aflatoxin B i (Sigma Chemical Co. ) was dissolved inbenzene-acetorntnle (98 + 2), transfer to a volumetric flaskof 50ml and make up to the graduation by adding benzene-acetonitrile (98 + 2), after thoroughly mixing, determinethe concentration of the standard solution by measurmg theA on a UV-Visible Spectrophotometre at 350nm and usmg

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Proceedmqs of the 7th Intematunuxl Working Conference on Stored-product Protection - Volurne 1

the followmg equation (AOAC 1995a) :tJ.gaflatoxm Bl/ml = (A x MW x 1000 x CF) IE

where CF = correction factor of the instrument and MW= 312, E= 19800 for aflatoxm BlPreparation of standard working soluium« of

aflatoxin B l: Make working standard solutions containing25ng, 50ng, 100ng and 200ng/ml and denved withtrifluoroacetic acid (Sigma Chemical Co ) by the AOACmethod (AOAC 1995b).The doses and responses trend Ime of etandard.

aflatoxin. uorkuu) solutwns: Prepare standard curve tocheck lmeanty of responses to aflatoxm Bl contents.

Sample treatment procedures for aflatoxin B 1

The lyophilized bactenal cells were suspended in distilledwater to give a cell suspension of 5mg or lOmg/mI. 1 mlofthe cell suspension was pipetted to a 1. 5ml centrifuge tube,then 50ng of standard aflatoxin B, Clng/.ul) was added toeach tube, vortexed for 20 seconds and rotated at 200rpm ona shaker at 3TC for durations of 15mm, 60mm and 180minThe levels of aflatoxm B, in three different portions wererecovered by the procedures shown below (Figure 1).

(a) The first portion was the supernatant of centrifugation(10000rpm for 5 mins ) right after incubation. Theaflatoxm remained in the supernatants were extractedtwice (2 x O. 5mO by chloroform-methanol (9 + 1) .

(b) The pellet m (a) was washed twice (2 x O. 5mO bywater, and centrifuging at 10000rpm for 5 mins. Theseparated supernatant was the second portion of atreatment The aflatoxm in this portion was alsoextracted twice (2 x 0 . Sml) by chloroform-methanol (9+ 1)

(c) The pellet of procedure (b) was then extracted twice (2x 0 . 5mO by chloroform-methanol (9 + 1) .

All the chloroform-methanol extracts of these differentportions were taken out by usmg an adjustable Eppendorfpipette, filtered through a anhydrous sodium sulphate filledmmi-column to remove water, collected the filtrate m a 5mlglass-tube and then evaporated to dryness under gentle N2stream. Then denved with 30tJ.I of tnfluoroacetic acid andmake up to 1 ml by adding water-acetonitrile (9 + 1),filtered through a O. 45.um membrane and collected thefiltrate m 1. 5ml GC VIals for HPLC use. All the treatmentswere performed m tnphcates.

Lyophilised cells

J,

Suspend in water (5-lOmg/ml)

J,

DIspense 5mg to centnfuge tubes

J,

Add 50ng ofaflatoxm HI

J,

Incubate for 15-180mm

Centrifugation ~ supernatant (a)

J, Pellet

Wash twice WIth H20 (2xO 5ml)

J,

Centrifugation ~ H20 portion (b)

J, Pellet

Add 0 5ml H20 ~ residues (c)

All the three portions (a, b and c) were extracted twice (2xO 5ml) With chloroform-methanol (9+1)

Fig.l. Procedures for sample preparation,

HPLC assay

The aflatoxm BI was detected by the AOAC official HPLCmethod with a little modification m the procedures of sample

preparation (AOAC 1995b; Azure, M. and Chong Cooper1991) The aflatoxm B, in all the samples were quantitatedby HPLC (Varian 9010) with a fluorescent detector(Vanan9070 EXCitation filter 360nm; emission filter

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Proceedings of the 7 th International Working Conference on Stored-product Protection - Volume 1

440nm) installed with Varian Star Work Station.0.5cm/min chart speed; the mobile solvents were water-acetonitrile-methanol(66 + 17 + 17); flow rate 1. OmI/min.50p.1of TFA-derived sample was injected automatically bythe autosampler (Model Varian 9100, Varian AnalyticalInstruments, CA, USA).LC column and guard-column: 15cm x 4. 6mm id

ALLSPHEREODS-2,5p.m LC column and 7 .5cm x 4.6mmALL-GUARDCARTRIDGES(Alltech Associates Pty. Ltd.,Australia) .Calculation of Aflatoxin B1 contents: Calculate the

aflatoxin B1 contents of individual sample as follows. Usethe responses of standard containing 2 - 4ng of aflatoxin B1for calculation (AOAC1995b).

Aflatoxin B1(ng/rnl) = (P /P') x ex 1000/50Where P and P' = peak areas for sample and standard

respectively, per 50p.1injection; C = aflatoxin B1 contentin standard solution (about 2ng).

Statistical analysis

All the results were compared the significant level ofdifferences by ANOVAin Unistat and Microsoft Excel 97.

Results and Discussion

Results of responses to aflatoxin B1 doses

Figure 2 shows the responses of injections were linearlyrelated to the doses of aflatoxin B1 in the whole tested range(from O.5ng to 4. Ong). Thus, the amount of aflatoxin B 1amount in all the samples analysed can be calculated by theformula listed above.

9.E+06

8.E+06

7.E+06

6.E +06

5.E+06 - ._-------~--------.j

4.E+08 ! --3E+06 t I

2.E+06t 7/ jl.E+06 r "",.,. "~ ----O.E+OO ~"" ---~---~-------- "-

o

Fig. 2. Standard curve for the responses (peak areas) toaflatoxin B1 doses (ng),

The different detoxification abilities among strainstested

The toxin amount recovered from the supernatant part(a) indicates the detoxification ability of the treated strain.The more toxin recovered (high percentage of recovery),

the less ability of detoxification the strain demonstrated.The detoxification abilities of the treated strains are shownin Figures 3.

60 HIO

d~to.3tifi:t;ati(ln tsme {min}

Fig.3. Detoxification abillities of various probiotic bacterialstrains.

The influence of treatment periods

The influence of treatment periods on the detoxificationeffects is reflected in Figure 4, 5 and 6. Figure 4 shows thetime influences on the recoveries from the supernatantportion (a). The results indicate the detoxification effect isnot affected by the treatment time in most strains, only thestrain B. pseudolongurn 20099 demonstrated a highly timedependent detoxification ability. The longer the treatedtime, the more toxin it bound. The strain L. casei had thelowest detoxification ability during the longest treatmenttime of 180 minutes. The toxin recovered from the water-washing portion (Figure 5) was also not significantlyinfluenced by the treatment time. The strain B. bifidurn 1demonstrated a significant (P < 0.05) lower recovery attreatment time of 60 minutes than those of 15 and 180minutes treatment. Whereas, the toxin-binding ability ofthe strain B. infantis 1912 decreased with the increase oftreatment time (P<0.05).

'L--- -- B bifidum 1900 -

I

---B. infantis 1912--ill- B. bifidum 1

100~-------------------'

1\· :: -- "-~--~~~~~

40

~!2:._" ~

15 60 180detoxification time (min)

-- B. pseudo/ongum20099--L. casei--L. case; 1

Fig.4 . Influence of treatment time on the aflatoxin B 1

recovery from supernatant.

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Proceedings of the 7th International Working Conference on Stored-product Protection - Volume 1

~~~~-----==========::;1- B. bifidum 1900:::=~:il~J8~~912

20'/o.~======-~==========:;;:;;;:;18

16 ~=3~~~;:~~'~~~~~~""E==114 .1_ lII-. - -I(

~~:~_~'~_~_--=Z:::=---- -=-- __ ----I8 - ~_-...____ ---6 -1------------- ---- ...=--------------14 ~------~.-- ----------------2o +-------..,..---~------___l

15 60 180detoxification time (min)

_ ..._~-------_. - -_._-------------'

Fig.5. Influence of treatment time on aflatoxin B1 recovery

from water washing portion.

The toxin recovered from the residue cells indicates thetoxin-binding strength of the bacterial cells, the more toxinrecovered from this portion, the stronger binding ability thebacterial cells possess. The toxin binding strength of strainB. bifidum 1 and L. casei seemed greatly affected by thetreatment time (P < 0.05), with the strongest toxin-binding ability during the treatment time of 60 minutes. Thetoxin binding ability of strain B. pseudolongum 20099increased with the treatment time lasted.

'~B-bij-id-u';'--19-00- ----- B. pseudu/ongum2li099 ll!1 --b--B. infantis 1912 ~L caset I

! I --If-- B. bifidum 1 --.- L casei 1 i I

11 O%r----------===================~I i

8

6

4

2

! 015 60 . 180

detoxification time (min)

Fig. 6 . The toxin binding strength of the tested probioticbacterial strains.

The differences of toxin binding abilities amongstrains

The toxin amount recovered from the water washing partplus that from bacterial cell residues indicates the toxinbinding abilities of the tested strains. The higher recoverypercentage of these two parts, the more toxin the bacterialcells bind. The toxin binding ability and the influence oftime on it are clearly revealed in Figure 7.Among the strains tested, B. pseudolongum 20099 had a

significantly (P <0.05) lower toxin-binding ability (9.17-12.94%) than all the other strains, which was quiteinconsistent with the detoxification ability it demonstrated.

More studies need to find out where the undetected aflatoxinB1 has gone or become of. B. infantis 1912 showedsignificantly higher toxin-binding ability than the otherstrains. The toxin binding ability of all the strains was nottime dependent.

B. biftdllm /900 B. B. inJmlis /9/2 L case! B. b!fiJlIm / 1. casei /J,sef/t!fJI01Igfml

201199

Fig. 7. Toxin binding ability of the tested probiotic bacterialstrains.

Toxin binding strength

The ratio of the recovery percentage from the cell residueportion (a) to that from cell residues plus that from thewater-washing portion (b + c) reveals the toxin bindingstrength of the bacterial cells, which is shown in Figure 8.

30 .-----------------------,o 15mins 080mins 0180mins!

20 l·~···J, +·····································"",ILlJ.;···················ct

15

10

BlliO_I900 II.ps~ lI.i_.19I221»!19

Fig. 8 . The toxin binding strength of the tested probioticbacterial strains.

The strain B. infantis 1912 demonstrated a significantlyhigher toxin-binding ability (P < 0.05) than the otherstrains in the 15 minutes treatment; and strain B. bifidum1 boundmuch less (P <0 .05) toxin than the other strains inthe 180 minutes treatment. There was no significantdifference of toxin binding strength between all the strainsduring the 60 minutes treatment.The toxin binding strength was also not affected by the

detoxification time for all the treated strains except B.bifidum 1, the toxin binding strength of this strain wasgreatly affected by the treated time. The strongest binding(about 37% was bound) happened in the treatment of 60

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Proceedma« 0/ the 7th International Work'tng Conference on Stored-product Protection. - Volume 1

minutes, and the least of 8.5 % for the treatment of 180mmutes All the results indicated that the toxin bindingstrength of a strain was not proportional to the duration oftreatment.

Influence of bacterial cell amount on thedetoxification results

The influences of different bacterial cell amounts (5mgand 10 mg) on the detoxification effect are shown in Table1.The data m table 1 indicates that the detoxification effects

are independent to the bactenal cell amount in the 15minutes treatments, but the detoxification effects, toxinbinding ability and toxm binding strength all are significantlydifferent m the 180 minutes treatments. The more bactenalcells used, the better detoxification effects achieved, themore and stronger the toxin was bound. ThIS is inaccordance With the results reported by Elnezami H. et al insome way, but the detoxification effects are not exactlyproportional to the bacterial cell amounts used, so thedetoxification process of probiotic bacteria can not be simplyexplained as a physical affinity phenomenon.

Table 1. Influences of bacterial cell amounts on the detoxification efficiency.

15 minutes 60 minutes 180 mmutesStram Celf(mg)

a * b t c" c/(b+c)* a b+c c/Cb-r c) a b+c c/(b+ c)

B. mfantis 1912 10 59.70 ± 21.88 ± 39.11 ± 50.90 ± 32.34± 44.67± 61. 70± 27.91± 34.72±4.98 1.64 3. 81 4.21 1.99 2.85 1.01 2.61 1.17

5 59.24± 19.34± 35.38 ± 64.01 ± 22.22± 31.99 ± 69.76± 19.56± 22.45 ±5.37 0.44 3.50 4.53 2.85 8.66 6.03 1.55 0.92

L. casei 10 61.65 ± 14.43± 32.33± 57.88 ± 22.73± 22.44 ± 64.28± 26.19± 28.06 ±6.18 0.12 0.80 4.56 0.66 4.24 2.74 1.41 0.83

5 59.92± 15.98± 20.11 ± 57.45 ± 19.36± 25.47± 77.20 ± 15.27± 21.87 ±4.37 2.81 3.40 1.38 1.34 2.08 2.36 0.83 3.50

* a, band c represent the three different recovery portions III Figurre 1 respectivelly. Ail the figures III the table are mean values ± standarddeviations

Conclusions

Strains of probiotic bactena showed diverse levels ofdetoxification properties to aflatoxin B l :Generally, there were no significant differences of

detoxification effects among most of the tested strains andbetween different durations of treatment. Only the stramB. pseudolongum 20099 demonstrated a more effectivedetoxification ability of 50% of the added aflatoxin BI thanthe other strains during the 180 mmutes treatment, and italso showed the lowest toxin binding strength among all thestrains tested. The strain B tn/antis 1912 had both goodtoxin binding ability and strength.The detoxification properties of most tested probity

bacteria strains were not time dependent but were cell-amount dependent.

References

AOACOfficialMethods Analysis (1995a), 16th edition. 49.2.02 and 49.3.06AOACOfficialMethods AnalYSIS(l995b), 16th edition. 49.2.17.Azure, M. and Chong Cooper. 1991. Determmation of

aflatoxms in foods using HPLC and a commercial ELISAsystem. Journal of Food Protection, 54, 4, 291 - 294.Custy F. Fernandes and Khem M. Shahani. 1990.Anticarcmogemc and immunological properties of dietarylactobacilli. Journal of Food Protection, 53, 8, 704-710.El- nezanu, H. , Kankaanpaa P. , Salminen S. , Ahokas J.1998. Ability of dairy strains of lactic acid bacteria to binda common food carcinogen, aflatoxin B-1 Food& ChemicalToxicology, 36, 4, 321 - 326,EI- nezarru, H. , Kankaanpaa P. , Salminen S. , Ahokas J.1998. Physicochemical alterations enhance the ability ofdairy strains of lactic acid bacteria to remove aflatoxin fromcontaminated media Journal of Food Protection, 61, 4,466-468.Lankaputhra, W. E. V. , N. P. Shah. 1998. Antimutagenicproperties of probiotic bactena and of orgamc acids.Mutation Research, 397, 169 - 182.Orrhage, K., E. Sillerstrom, J. A. Gustafsson, C E.Nord, J. Rafter. 1994. Binding of mutagenic heterocyclicammes by mtestmal and lactic acid bacteria. MutationResearch-Molecular Mechamsm of Mutagenesis, 311,239 - 248.Sudarshan R Nadathur , Steven J. Gould and Alan T.Bakalinsky. 1994. Antimutagenicity of fermented milk. J.Dairy SCIence, 77, 11, 3287 - 3295.

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