Abess et al 1

AUTHOR’S QUERY SHEET

Author(s): S. Abbès et al. Article title: Ability of Lactobacillus rhamnosus GAF01 to remove AFM1 in vitro and to counteract AFM1

immunotoxicity in vivoArticle no: UIMT 718810Enclosures: 1) Query sheet 2) Article proofs 3) Track changes manuscript showing language editing

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UIMT

718810

Contrib No. Given Name(s) Surname Suffix

1. Samir Abbès

2. Jalila Ben Salah-Abbès

3. Hakimeh Sharafi

4. Rania Jebali

5. Kambiz Akbari Noghabi

6. Ridha Oueslati

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1

Introduction

Aflatoxin M1 (AFM

1) is a hepato-carcinogenic metabo-

lite (IARC, 1993) found in milk of animals that have consumed feeds contaminated with aflatoxin B

1 (AFB

1)

produced by Aspergillus fungi (Park and Pohland, 1986; Moss, 1998). AFB

1 quickly appears in cows (as AFM

1

metabolite in blood after just 15 min (Moschini et al., 2006) and in milk at the first milking (Diaz et al., 2004; Masoero et al., 2007)) after ingestion of aflatoxin-con-taminated feed. We recently demonstrated that, in Beja province (Tunisia), milk and milk by-products were

contaminated with high levels of AFM1 (3–4-fold above

the permitted dose) (Abbès et al., 2012). AFM1 is such a

major concern to humans because of its frequent occur-rence in dairy products at concentrations high enough to cause adverse effects to health (IARC, 2002). As a result, guidance values for AFM

1 in milk have been established,

i.e., levels are limited to 0.05 and 0.50 µg/L in Europe and the US, respectively (Berg, 2003; Commission of the European Communities, 2006).

Effective detoxification methods would be useful in helping to remediate/mitigate the problem of AFM

1

AQ2

orIgInal arTIClE

Ability of Lactobacillus rhamnosus GAF01 to remove AFM1

in vitro and to counteract AFM1 immunotoxicity in vivo

Samir Abbès1,2, Jalila Ben Salah-Abbès1, Hakimeh Sharafi3, Rania Jebali1, Kambiz Akbari Noghabi3, and Ridha Oueslati

1Unit of Immunology, Environmental Microbiology and Cancerology, Faculty of Sciences, University of Carthage, Tunis, Tunisia, 2Animal Biotechnology Department, Higher Institute of Biotechnology of Beja, University of Jendouba, Jendouba, Tunisia, and 3National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

abstractAflatoxin M

1 (AFM

1) has been detected in many parts of the world both in raw milk and many dairy products, causing

great economic losses and human disease. Unfortunately, there are few studies dealing with AFM1 immunotoxicity/

interactions with lactic acid bacteria for potential application as a natural preventive agent. The aim of this study was to isolate (from dairy products) food-grade probiotic bacteria able to degrade/bind AFM

1 in vitro and evaluate

whether the same organism(s) could impart a protective role against AFM1-induced immunotoxicity in exposed

Balb/c mice. Bacteria (Lactobacillus plantarum MON03 and L. rhamnosus GAF01) were isolated from Tunisian artisanal butter and then tested for abilities to eliminate AFM

1 from phosphate-buffered saline (PBS) and reconstituted milk

(containing 0.05, 0.10, and 0.20 µg AFM1/ml) after 0, 6, and 24 h at 37°C. Results showed that the selected bacteria

could ‘remove’ AFM1 both in PBS and skimmed milk. The binding abilities of AFM

1 by L. plantarum MON03 and

L. rhamnosus GAF01 strains (at 108 CFU/ml) in PBS and reconstituted milk ranged, respectively, from 16.1–78.6% and 15.3–95.1%; overall, L. rhamnosus showed a better potential for removal than L. plantarum. ‘Removal’ appeared to be by simple binding; the bacteria/AFM

1 complex was stable and only a very small proportion of mycotoxin was released

back into the solution. L. rhamnosus GAF01 had the highest binding capacity and was selected for use in the in vivo study. Those results indicated that use of the organism prevented AFM

1-induced effects on total white and red blood

cells, and lymphocyte sub-types, after 15 days of host treatment. These studies clearly indicated that L. rhamnosus GAF01 was able to bind AFM

1 in vitro and—by mechanisms that might also be related to a binding effect—counteract

AFM1-induced immunotoxicity. Moreover, by itself, this bacterium was not toxic and could potentially be used as an

additive in dairy products and in biotechnology for mycotoxin detoxification.

Keywords: Aflatoxin M1, Lactobacillus strains, immunotoxicity, binding, detoxification

AQ1

Address for Correspondence: Dr Samir Abbès, Unit of Immunology, Environmental Microbiology and Cancerology, Faculty of Sciences Bizerte 7021 Zarzouna, University of Carthage, Tunis, Tunisia. Tel: 21672591906. Fax: 21672590566. E-mail: [email protected]

(Received 16 April 2012; revised 24 July 2012; accepted 02 August 2012)

Journal of Immunotoxicology, 2012; Early Online: 1–8© 2012 Informa Healthcare USA, Inc.ISSN 1547-691X print/ISSN 1547-6901 onlineDOI: 10.3109/1547691X.2012.718810

Journal of Immunotoxicology

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24July2012

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10.3109/1547691X.2012.718810

2012

Prevention against AFM1 immunotoxicity

S. Abbès et al.

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2 S. Abbès et al.

UIMT 718810 Journal of Immunotoxicology

contamination in milk/by-products. Accordingly, adequate proof of efficacy of any novel measure would also be of pivotal importance. The ability/efficacy of food additives (i.e., using mineral and bioactive compounds) purported to detoxify in vivo or in vitro have been reviewed extensively elsewhere (Abbès et al., 2010; Ben Salah-Abbès et al., 2010). The detoxification of food products contaminated with mycotoxins is restricted due to problems regarding safety issues, possible losses in nutritional quality, limited efficacy, and cost (Bata and Lasztity, 1999). This has led to the search for alternative strategies such as the use of biological agents. There has been increasing interest in one concept stating that gastrointestinal tract micro-organisms can reduce the absorption of AFM

1 in consumed foods. Consequently,

numerous investigators have shown that some dairy strains of lactic acid bacteria and bifidobacteria were able to bind effectively to AFM

1 in buffered solutions.

Thus, the aim of the present study was 2-fold: (a) to isolate lactobacillus bacteria from AFM

1-contaminated

artisanal butter and to test their ability to bind AFM1

(in a saline solution and reconstituted milk); and (b) to evaluate the ability of the isolated bacteria to mitigate/reduce any AFM

1-induced immunotoxicities in exposed

Balb/c mice.

Materials and methods

ChemicalsAFM

1 solution (0.5 ng/ml, in methanol) was obtained

from Sigma (St Louis, MO). Phosphate-buffered saline (PBS; 8 g NaCl, 1.2 g K

2HPO

4, 0.3 g KH

2PO

4) was prepared

in 900 ml distilled water, adjusting the pH to 6.5, and then diluting to a volume of 1 L. All solvents used for AFM

1 analyses were purchased from Merck (Darmstadt,

Germany) and were of high performance liquid chro-matography (HPLC) grade. In all analytical steps, water generated through a Millipore Synergy 18S Ultra-Pure Water System from Millipore (Molsheim, France) was used. AFM

1 immunoaffinity columns containing specific

monoclonal antibodies bound to a solid support material for AFM

1 clean-up were obtained from Vicam (product

code: G1007, Watertown, MA).

Sampling of artisanal butterThree samples of 1-month-old artisanal butter made from cow’s milk were collected from local producers from south-ern, central, and northern regions of Tunisia. The butter samples—designated as GAF01, MON03, and BEJ01—were intentionally collected in regions where it has been tradi-tionally manufactured in households. Collected samples were kept in sterile bags and held at 4°C; all analyses were performed within the following 24 h after collection.

In vitro studiesBacteria screeningArtisanal butter samples (10 g) were homogenized in 90 ml of sterile 2% (w/v) tri-sodium citrate dihydrate

solution (pH 7.5) pre-heated to 45°C and then 10-fold serial dilutions were prepared in 0.85% (w/v) sterile saline. Using standard protocols, aerobic mesophilic bacteria were enumerated on nutrition agar (NA; Merck, GmbH, Darmstadt, Germany) and presumptive lactoba-cilli were incubated on Man-Rogosa-Sharpe broth (MRS) agar plates (Biorad, Paris, France) for 72 h at 30°C to per-mit isolation of lactic acid bacteria. All isolated colonies were cultured in MRS broth. Thereafter, to test for those organisms that might be able to bind AFM

1, the toxin was

added (at 1 mg/L) to the culture broth and incubated with shaking for 24 h. After incubation, levels of recover-able mycotoxin (i.e., non-pelleting with bacteria) were assessed via HPLC analysis of the broth supernatant. Those organisms shown to have bound/removed AFM

1

were then conclusively identified using API 50CHL kit and 16S rDNA sequencing.

AFM1 removal in PBS and reconstituted milk by isolated

bacteria and evaluation of AFM1/bacteria complex stability

Volumes of the culture broth (each containing ≈ 108 CFU/ml of given organism of interest) were centrifuged (3000 × g, 15 min) and the bacterial pellets washed with water. The pellets were then re-suspended in 5 ml PBS contami-nated with AFM

1 at 0.05, 0.10, or 0.20 µg/ml. The tubes

were then vortexed and the suspensions incubated at 37°C for 0, 4, and 24 h. An AFM

1 positive control (0.05,

0.10, and 0.20 µg/ml in PBS) in the absence of bacteria and negative controls (bacteria suspended in pure PBS) were also incubated in parallel to monitor the efficacy of bacterial AFM

1 binding.

The binding ability of AFM1 by viable lactobacilli bacte-

ria (108 CFU/ml) in reconstituted milk was also assessed in order to determine if there were any matrix effects on the efficacy of AFM

1 removal. AFM

1-free skimmed milk

powder was suspended in sterile water (at 0.2 g/ml) and portions of the reconstituted milk were then contami-nated with standard working solutions of AFM

1 at three

different concentrations, i.e., 0.05, 0.10, and 0.20 µg/ml. The Lactobacilli bacteria were then treated as described earlier, but, instead of PBS, the bacterial pellets were suspended in the contaminated skimmed milk. Each removal assay was carried out in triplicate. All designated tubes were centrifuged (15 min, 3000 × g) at the end of each incubation period; supernatants were collected, transferred to clean tubes, and then stored at 4°C until AFM

1 analysis. Unbound AFM

1 content in the super-

natants were determined by HPLC (see below). Each removal assay was performed in triplicate.

The stabilities of any bacteria-AFM1 complexes in PBS

and reconstituted milk were evaluated by determining the amount of AFM

1 remaining bound after washing.

After 6 h incubation, a supernatant sample was collected to determine binding. Bacterial pellets with bound AFM

1

were washed and re-suspended in 5 ml fresh PBS and incubated for 30 min at 37°C. The bacteria were then re-pelleted and the supernatant collected for quantifica-tion of released AFM

1. Ultimately, the bacteria with the

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Prevention against AFM1 immunotoxicity 3

© 2012 Informa Healthcare USA, Inc. UIMT 718810

highest percentages of binding and AFM1/bacteria com-

plex stability were selected for use in the in vivo studies.

Determination of AFM1

AFM1 analyses were performed using HPLC that incor-

porated an immunoaffinity column. Briefly, with each butter sample, 10 g was placed in 125 ml 75% (v/v) aceto-nitrile/water for 5 min. After blending, the samples were filtered using Whatman filter paper, and the filtrate diluted with 80 ml PBS. A sample of the filtrate (10 µl) was then passed through the Vicam AFM

1 immunoaffin-

ity column (at 5 ml/min, followed by washing with 20 ml distilled water at 5 ml/min). Bound AFM

1 was eluted

with 1.5 ml acetonitrile followed by 1.5 ml distilled water, and collected in a clean vial. These two eluted samples were mixed and analyzed using an Agilent 1100 HPLC system (Agilent Technologies, Englewood, CO) contain-ing an ACE C

18 silica (5 µm i.d., 25 × 46 mm) column pur-

chased from Advanced Chromatography Technologies (Aberdeen, Scotland); product measurements were made in-line spectrophotometrically at an OD of 435 nm. Quantification of AFM

1 was done from measures of

peak areas and extrapolating against a calibration curve prepared/analyzed in parallel using AFM

1 standards

(Sigma). Three replicate analyses were performed for each sample to determine AFM

1 content. To verify the

soundness of the assay in this protocol, recovery from bacteria-free PBS was determined for the test range of AFM

1 used; mean recovery was in the range of 89–93%.

The percentage of AFM1 bound to the bacteria was cal-

culated using the formula: 100% × (1.00 − [peak area of AFM

1 in the supernatant/peak area of AFM

1 in positive

control sample]).

In vivo studiesAnimals and treatmentForty-eight male Balb/c mice (SEXUEL, St. Doulchard, France) were used (25 ± 0.3 g, 6-week-old) here. All mice were housed (12/cage) in a pathogen-free facility main-tained at 23 ± 1°C with a 50 ± 5% relative humidity and a 12-h light/dark cycle. All mice were provided were ad libitum access to standard granulated chow (certified mycotoxin-free) and filtered drinking water. Mice were cared for under the Tunisian Code of Practice for the Care and Use of Animals for Scientific Purposes. Experimental protocols were approved in accordance with the guide-lines of the Ethical Committee of NIGEB, Tehran.

At the start of the experiment, mice were randomly allocated into four treatment groups (12/group) as fol-lows: (A) control group of distilled water, (B) AFM

1 alone

(100 µg/kg BW), (C) bacteria alone (1 mg/kg BW = 2 × 108 cfu/ml), and (D) AFM

1+Bacteria. Thereafter, each host

received the indicated treatment(s) orally (by gavage; without any need for anesthesia) daily for 2 weeks (sub-chronic study). Twenty-four hours after the final treat-ment, blood samples were collected from retro-orbital plexus for analyses of immune system-related end-points (see below).

Measures of total white and red blood cells, and lymphocyte T-cell sub-typesTotal white and red blood cells (WBC and RBC, respec-tively) were determined using a Coulter STKS blood counter (Coulter Electronics, Ltd., Luton, UK). In order to perform sub-type analyses using 2-color flow cytometry, 1 ml of heparinized blood per mouse was incubated for 20 min at room temperature together with 0.05 ml of a sus-pension containing monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) against CD4, CD8, CD54, or CD56 surface markers (Becton/Dickinson, San Jose, CA). Any erythrocytes pres-ent were lysed with 0.02 ml FACSLysing solution (Becton Dickinson) for 10 min at 4°C. The samples were then ana-lyzed on a FACScan flow cytometer (Becton Dickinson) equipped with Cellquest software. A minimum of 10,000 events/sample was analyzed. Lymphocytes (n = 2000) were measured and the percentage of positive cells in this population (gate) was assessed.

Statistical analysisData were expressed as mean ± SD of three independent experiments (in vitro study), and analyzed for statistical significance by Student’s t-test using the general linear model (n = 6 for in vivo tests). The criterion for signifi-cance was set at p < 0.05.

results

In vitro studyAFM

1 concentration in butter used for lactobacilli

bacteria isolationThe presence of AFM

1 in butter used for bacteria isolation

was detected at concentrations ranging between 54.27–112.9 ng/L. The AFM

1 levels in 12 (32%) of the positive

samples were higher than the maximum tolerance limit (i.e., 50 ng/L) accepted by Tunisia and European Union nations. The rationale for this study was to select lacto-bacilli bacteria that could tolerate a high level of AFM

1.

Isolation of micro-organisms capable of removal AFM1

Colonies (40) isolated from various samples for poten-tial AFM

1-binding activity were screened. Among them,

two strains (from butter samples MON03 and GAF01) that were able to bind the most AFM

1 were isolated and

identified using an API 50CHL kit. The organisms (i.e., Lactobacillus) most apt at binding AFM

1 in the MON03

and GAF01 were phenotyped and designated L. plan-tarum MON03 and L. rhamnosus GAF01, respectively. Moreover, 16S rDNA sequencing showed that these iso-lated organisms had 99% homology with L. plantarum and L. rhamnosus standards (data not shown).

Determination of AFM1 binding ability by the selected bacteria

The AFM1-binding abilities of viable test strains are

summarized in Table 1. The binding abilities of AFM1 by

L. plantarum MON03 and L. rhamnosus GAF01 strains (at 108 CFU/ml) in PBS and reconstituted milk ranged

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4 S. Abbès et al.


from 16.1 (± 1.4)–78.6 (± 7.1)%, and 15.3 (± 1.5)–95.1 (± 8.3)%, depending on contamination level and incuba-tion period. L. rhamnosus GAF01 was the better binder, with ≈ 95% binding both in PBS and reconstituted milk. The differences in binding ability of L. plantarum MON03 and L. rhamnosus GAF01 at 108 CFU/ml were significant for all incubation times (0, 6, and 24 h) and with all toxin concentrations (Figure 1).

Binding was not irreversible and small amounts of AFM

1 were released back into the buffered solution,

especially for L. rhamnosus GAF01. When washing the bacteria/AFM

1 complexes, 18.5% of bound AFM

1 by

L. plantarum MON03 was released back to the solution

(Figure 2). In comparison, washing of L. rhamnosus GAF01/AFM

1 complexes resulted in 5.1% of bound AFM

1

being released back.

In vivo studyGeneral observationsThe outcome of the in vitro study indicated that L. rham-nosus GAF01 was more suitable for use in the in vivo study due to its highest capability to bind AFM

1. Moreover, L.

rhamnosus GAF01 also showed good complex stability with AFM

1.

After 2 weeks of AFM1 treatment, as expected, a

general toxicity was observed in the mice. Specifically,

Figure 1. High performance liquid chromatography (HPLC) chromatograms. (a) Untreated (positive control) aflatoxin M1 (at 0.15 µg

AFM1/ml)-phosphate buffered saline (PBS) solution; (b) AFM

1-free PBS solution; or (c) AFM

1 (0.15 µg/ml)-PBS solution after 6 h treatment

with Lactobacillus rhamnosus GAF01 at 108 CFU/ml. (See colour version of this figure online at www.informahealthcare.com/imt)

Table 1. Percentage of AFM1 removal from PBS and reconstituted milk by Lactobacillus plantarum MON03 and Lactobacillus rhamnosus GAF01 strains.

Times

PBS0.05 µg AFM

1/ml 0.10 µg AFM

1/ml 0.20 µg AFM

1/ml

0 hr 6 hr 24 hr 0 hr 6 hr 24 hr 0 hr 6 hr 24 hrL. plantarum MON03

16.1 ± 1.4 51.1 ± 4.2 71.6 ± 6.1 16.0 ± 2.4 56.3 ± 5.2 74.9 ± 8.2 15.9 ± 1.6 67.4 ± 5.8 78.6 ± 7.1

L. rhamnosus GAF01

26.2 ± 2.0 61.5 ± 5.2 84.9 ± 7.1 25.9 ± 2.5 66.6 ± 6.2 91.3 ± 7.2 26.0 ± 2.0 86.6 ± 7.1 94.7 ± 8.2

PBS only 5.1 ± 1.1 4.6 ± 0.9 4.2 ± 0.7 3.9 ± 0.8 5.0 ± 0.5 4.1 ± 0.6 4.4 ± 0.7 4.2 ± 0.1 4.2 ± 0.5Reconstituted milkL. plantarum MON03

15.3 ± 1.5 48.2 ± 4.7 72.3 ± 5.9 15.8 ± 2.2 57.1 ± 5.4 72.9 ± 8.9 15.2 ± 1.3 69.1 ± 5.3 76.9 ± 8.0

L. rhamnosus GAF01

25.1 ± 2.0 60.3 ± 5.1 85.8 ± 7.5 26.1 ± 2.7 65.3 ± 6.5 90.7 ± 7.5 25.3 ± 2.2 85.9 ± 7.4 95.1 ± 8.3

Reconstituted milk 5.3 ± 0.9 4.3 ± 0.6 4.1 ± 0.6 4.1 ± 0.7 5.2 ± 0.6 4.4 ± 0.3 4.6 ± 0.6 4.0 ± 0.3 4.4 ± 0.2Values shown are mean ± SD from three determinations per experimental scenario tested.All values shown for the bacteria-containing systems are significantly different from control at p ≤ 0.05.

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body and lymphoid organ weights were significantly decreased in the AFM

1-treated mice (data not shown).

In addition, the deaths of two mice (one on day 10 and one on day 12; ≈16% overall mortality) were noted in the AFM

1 treated group. In contrast, no overt general toxici-

ties were observed from L. rhamnosus GAF01 alone or in combination with AFM

1.

Immunological studyThe current results concerning relative levels of T-cells (and sub-types) in mice in the various treatment groups indicated that daily treatment (for 14 days) with 100 µg AFM

1/kg BW induced immune system damage. This

was reflected as significant decreases in the relative lev-els of CD4+, CD8+, CD54+, and CD56+ cells in these hosts (Figure 3). On the other hand, treatment with L. rham-nosus GAF01 did not alter blood T-cell levels. Treatment with AFM

1 + L. rhamnosus GAF01 resulted in a significant

improvement in the total counts of immune system cells toward control levels, although these values were still dif-ferent from the values of control CD4+ and CD8+ cell counts.

Total WBC and RBC levels (Figure 4) were also decreased in the AFM

1 only-treated hosts. In contrast, total WBC and

RBC counts in mice treated with L. rhamnosus GAF01 alone were comparable to the controls. The addition of L. rhamnosus GAF01 to the AFM

1 treatment regimen

resulted in a significant improvement in both total WBC and RBC counts, although again, these values still differ (albeit not significantly) from the control group levels.

Discussion

Aflatoxin M1 (AFM

1) is an immunotoxic metabolite

found in milk of animals that have consumed feeds

Figure 2. Effect of washing on bacteria:AFM1 complex. Initial

binding was determined after bacteria (108 CFU/ml) and AFM1

(0.05 µg/ml) were incubated together for 6 h at 37°C. Bacteria/AFM

1 complexes formed were subjected to centrifugation and

then washing with 5 ml PBS, before the amount of AFM1 released

was determined as outlined in the Methods. Results shown are the mean (± SD) from triplicate samples.

Figure 3. Influence of AFM1 and Lactobacillus rhamnosus GAF01

(alone or in combination) on lymphocyte sub-types. Balb/c mice received daily (for 14 days) by gavage distilled water (open bar), AFM

1 (100 µg/kg BW) (solid bar), L. rhamnosus GAF01 (1 mg/kg

BW) (shaded bold bar), or AFM1 + L. rhamnosus GAF01 (shaded

fine bar). Lymphocyte counts were then performed on blood samples collected on day 15 of the experiment (i.e., 1 day after final exposure in each regimen). Results are expressed as mean [± SE] from n = 12 mice/group. A Student’s t-test was used to compare differences between groups: aValue significantly/bnot significantly different from control at p ≤ 0.05.

Figure 4. Influence of AFM1 and Lactobacillus rhamnosus GAF01—

alone or in combination—on blood cell counts. Balb/c mice received daily (for 14 days) by gavage distilled water (open bar), AFM

1 (100 µg/kg BW) (solid bar), L. rhamnosus GAF01 (1 mg/kg

BW) (shaded bold bar), or AFM1 + L. rhamnosus GAF01 (shaded

fine bar). Erythrocyte and leukocyte counts were then performed on blood samples collected on day 15 of the experiment (i.e., 1 day after final exposure in each regimen). Results are expressed as mean [± SE] from n = 12 mice/group. A Student’s t-test was used to compare differences between groups: aValue significantly/bnot significantly different from control at p ≤ 0.05.

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6 S. Abbès et al.


contaminated with aflatoxin B1 (AFB

1). Aflatoxins are

produced by Aspergillus fungi that grow (in temperate, tropical, and subtropical climates) at a high incidence on food-stuffs/grains (Abbès et al., 2008). Since AFM

1

is a potent toxin and may pose a threat to animal and human health, practical methods to detoxify/inactivate AFM

1-contaminated milk and by-products are in great

demand. In the present study, following studies to isolate bacteria from higher contaminated Tunisian traditional butter samples that could bind with AFM

1, we evaluated

the possible protective effect of Lactobacillus rhamno-sus GAF01 against AFM

1-induced immunotoxicity in

Balb/c mice as an animal model. There is very limited literature on the occurrence of AFM

1 (levels) in butter

and milk products in Tunisia (Abbès et al., 2012). In this study, 12 out of 40 traditional butter samples tested were contaminated; all contaminated samples had a level of AFM

1 > 50 ng/L.

The in vitro portion of our study showed the bind-ing abilities by L. plantarum MON03 and L. rhamnosus GAF01 strains (at 108 CFU/ml) toward AFM

1 in PBS.

While L. rhamnosus GAF01 was the best binder with ≈95% binding, the binding abilities of L. plantarum MON03 and L. rhamnosus GAF01 (at 108 CFU/ml) were almost immediate and significant for all incubation times (i.e., 0, 6, and 24 h) and toxin concentrations tested. Our study clearly indicates that a bacteria population of 108 CFU/ml is very suitable for binding AFM

1 in PBS and

reconstituted milk. Moreover, our study demonstrated that the matrix did not influence the AFM

1 binding

capacity of the bacteria. The amount was similar to that reported for trichothecene binding by some strains of Lactobacillus and Propionibacterium (El-Nezami et al., 1998). Similarly, Line and Brackett (1995) indicated that populations of ≥108 CFU/ml were necessary for signifi-cant removal of AFB

1. El-Nezami et al. (1998) reported

that a higher minimum (≈2 × 109 CFU/ml) was required for the effect.

Information on the binding ability of mycotoxins by dairy strains of lactic acid bacteria and bifidobacteria is available for AFB

1, but very limited for AFM

1. Our study

indicated a higher percentage binding ability against AFM

1 by the bacteria used than those published by

Pierides et al. (2000), who found that the AFM1 binding

abilities of viable Lactobacillus strains within 15–16 h ranged from 18–54%. Similarly, Peltonen et al. (2001) investigated the AFB

1 binding ability of 12 Lactobacilli,

five Bifidobacteriae, and three Lactococci strains. In their study, Lactobacillus strains bound 17–60%, Bifidobacterium strains 18–49%, and Lactococcus strains 6–41% of the AFB

1 provided.

Our studies also showed that the binding that occurred was not completely irreversible as very small amounts of AFM

1 were released back into the buffered solution,

especially for L. rhamnosus GAF01. Specifically, when L. rhamnosus GAF01/AFM

1 complexes were washed,

≈5% of the bound AFM1 was released back to the solu-

tion. This is consistent with findings for the efficient

AFB1-binding strain L. rhamnosus GG (Haskard et al.,

2001), but contrasts with results from Peltonen et al. (2001). Those authors noted that AFB

1 was not bound

strongly by Lactobacilli strains, and that 28–94% of bound AFB

1 went back into the solution. Thus, L. rhamnosus

GAF01 was more suitable for use in in vivo studies due to its demonstrably high(est) capability to bind AFM

1

and to maintain a good complex stability.The results of the blood T-cell determination studies

revealed that treatment with a daily oral dose of AFM1

induced immune system damage as evidenced by signifi-cant decreases in CD4+, CD8+, CD54+, and CD56+ levels in these hosts. Moreover, our findings indicated that the 14-day AFM

1 treatment also impacted on the numbers of

red and white blood cells. This is not all that remarkable in that the parent compound of AFM

1 (i.e., AFB

1) is immu-

notoxic (Meissonnier et al., 2008; Abbès et al., 2010). Regrettably, very few studies have been performed exam-ining the effects of AFM

1 on immune functions. Recently,

Russo et al. (2010) demonstrated the immunotoxic effects of AFM

1 on macrophage functions. Further, no work is

available regarding immunotoxicity of AFM2 even thought

both AFM1 and AFM

2 are obtained from AFB

1 and AFB

2

(via hydroxylation) and excreted into a host’s milk.The studies here showed that a co-treatment with

the isolated L. rhamnosus GAF01 enhanced blood T-cell levels (except for CD54+ cells) to those somewhat comparable to values seen with the controls. Why (cells with) this one cell marker was not affected is not clear at this time. While CD54 represents the intercellular mol-ecule of adhesion (ICAM-1) protein responsible for cell co-operation, an impact on the number of CD54+ cells appeared to also not be impacted by treatments with other Lactobacilli (i.e., L. paracasei; Jahreis et al., 2002). In contrast to the CD54+ cells, the effects noted here on the other T-cell sub-types are in keeping findings by other investigators. In rats with induced enterocolitis, the concentration of CD4+ and CD8+ cells in the intestinal lamina propria were increased to a more normal level by administration of L. plantarum (Mao, 1996). In another study, L. paracasei NCC2461 induced the development of a population of CD4+ T-cells with low proliferative capacity and that were induced to produce transform-ing growth factor (TGF)-b and interleukin (IL)-10 (von de Weid et al., 2001). In the current study, treatment with AFM

1 + L. rhamnosus GAF01 (at 1 g/kg BW) resulted in

a significant improvement in immune system cell total counts (towards control levels), although in the end they were still different than control values. Nevertheless, these findings suggested that the apparent biosorption (i.e., tight binding) of AFM

1 to the L. rhamnosus GAF01

resulted in a reduction of AFM1 bioavailability in the

gastrointestinal tract and so a mitigation of potential effects upon T-cells in these hosts. Whether there are other non-binding-related attributes from having the L. rhamnosus GAF01 in the host that impart other effects on the immune system to combat the toxicities that could be induced by AFM

1 remains to be determined.

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Many micro-organisms, including bacteria, yeasts, molds, actinomycetes, and algae, are able to remove or degrade small amounts of aflatoxin in food/feed (Styriak et al., 2001). Results of several studies suggest that bind-ing is the main mechanism of detoxification against aflatoxins (Niderkorn et al., 2006; Gratz, 2007). Strains L. rhamnosus GG and LC-705 seem to be the most effective in such detoxifications (Lahtinen et al., 2004). However, the binding mechanism itself still remains not thoroughly understood. It was suggested that carbohy-drate-rich mannoproteins or glucans might be involved in the binding, as levels of complex formation appear to be strain- and toxin-specific (Shetty and Jespersen, 2006). Raju and Devegowda (2000) attributed the bind-ing of aflatoxins by yeast cell walls to mannan oligosac-charides, while zearalenone binding was attributed to glucan components (Yiannikouris et al., 2004). Haskard et al. (2001) suggested that AFB

1 binds predominantly

to carbohydrate and protein components of viable cells. Clearly, as noted by Shetty and Jespersen (2006), system-atic studies are still needed to understand the precise binding mechanisms.

Metabolism of aflatoxins can be considered as a second mechanism for detoxification. Very few studies have addressed the potential role and importance of the gastrointestinal tract in AFB

1 metabolism. Studies

in rats identified free hydroxylated metabolites of AFB1,

i.e., AFM1 and AFL (Gratz, 2007). In the presence of pro-

biotics, an increased excretion of AFM1 was observed,

suggesting a reduced AFB1 uptake and hepatic metabo-

lism. Some of the metabolites that diffuse(d) back into the lumen were likely also bound by the probiotics and excreted in the feces. L. rhamnosus GG is known to bind AFM

1 as equally efficiently as AFB

1 (Kabak and Var,

2004). Because both AFM1 and AFM

2 are excreted into

the milk of cows fed aflatoxin-contaminated feed, this implies some measure of gastrointestinal metabolism of the mycotoxins (El-Nezami et al., 2000). The data here strongly suggest that the intestinal metabolism of AFM

1 is

an important process and that probiotic bacteria impact upon these processes.

Conclusion

The results here showed that AFM1 causes physiological

disorders (i.e., at the level of WBC overall, and T-cells specifically) in Balb/c mice. These studies also illustrated how Lactobacillus rhamnosus isolated from Tunisian artisanal butter would bind AFM

1 in vitro and decrease

its immunotoxic potential in vivo. Thus, the isolated L. rhamnosus GAF01 can be considered as a good candi-date for use as an in vivo detoxification agent during food consumption (i.e., when taken as a probiotic).

acknowledgement

The authors would like to acknowledge the Academy of Sciences for the Developing World (TWAS) and the

United Nations Educational, Scientific, and Cultural Organization (UNESCO) for the financial support granted to the first author to carry out this study as a part of TWAS-UNESCO Associateship Scheme 2010–2012 at the National Institute of Genetic Engineering and Biotechnology (NIGEB), Iran. The work was supported also by the Tunisian Ministry of Higher Education and Scientific Research (Unit of Immunology, Environmental Microbiology, and Cancerology) and the Higher Institute of Biotechnology of Beja (Animal Biotechnology Department).

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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RESEARCH ARTICLE

Prevention against AFM1 immunotoxicity

S. Abbès et al.

Ability of Lactobacillus rhamnosus GAF01 to remove

AFM1 in vitro and to counteract AFM1 immunotoxicity in

vivo

Samir Abbès1,2

, Jalila Ben Salah-Abbès1, Hakimeh Sharafi

3, Rania Jebali

1, Kambiz

Akbari Noghabi3, and Ridha Oueslati

1

1Unit of Immunology, Environmental Microbiology and Cancerology, Faculty of

Sciences, University of Carthage, Tunis, Tunisia, 2Animal Biotechnology Department,

Higher Institute of Biotechnology of Beja, University of Jendouba, Jendouba, Tunisia,

and 3National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

Address for Correspondence: Dr Samir Abbès, Unit of Immunology, Environmental

Microbiology and Cancerology, Faculty of Sciences Bizerte 7021 Zarzouna, University

of Carthage, Tunis, Tunisia. Tel: 21672591906. Fax: 21672590566. E-mail:

[email protected]

Aflatoxin M1 (AFM1) has been detected in many parts of the world both in raw milk and

many dairy products, causing great economic losses and human disease. Unfortunately,

there are few studies dealing with AFM1 immunotoxicity/interactions with lactic acid

bacteria for potential application as a natural preventive agent. The aim of this study was

to isolate (from dairy products) food-grade probiotic bacteria able to degrade/bind AFM1

- 2 -

in vitro and evaluate whether the same organism(s) could impart a protective role against

AFM1-induced immunotoxicity in exposed Balb/c mice. Bacteria (Lactobacillus

plantarum MON03 and L. rhamnosus GAF01) were isolated from Tunisian artisanal

butter and then tested for abilities to eliminate AFM1 from phosphate-buffered saline

(PBS) and reconstituted milk (containing 0.05, 0.10, and 0.20 µg AFM1/ml) after 0, 6,

and 24 h at 37°C. Results showed that the selected bacteria could ‘remove’ AFM1 both in

PBS and skimmed milk. The binding abilities of AFM1 by L. plantarum MON03 and L.

rhamnosus GAF01 strains (at 108 CFU/ml) in PBS and reconstituted milk ranged,

respectively, from 16.1–78.6% and 15.3–95.1%; overall, L. rhamnosus showed a better

potential for removal than L. plantarum. ‘Removal’ appeared to be by simple binding; the

bacteria/AFM1 complex was stable and only a very small proportion of mycotoxin was

released back into the solution. L. rhamnosus GAF01 had the highest binding capacity

and was selected for use in the in vivo study. Those results indicated that use of the

organism prevented AFM1-induced effects on total white and red blood cells, and

lymphocyte sub-types, after 15 days of host treatment. These studies clearly indicated

that L. rhamnosus GAF01 was able to bind AFM1 in vitro and—by mechanisms that

might also be related to a binding effect—counteract AFM1-induced immunotoxicity.

Moreover, by itself, this bacterium was not toxic and could potentially be used as an

additive in dairy products and in biotechnology for mycotoxin detoxification.

Keywords: Aflatoxin M1, Lactobacillus strains, immunotoxicity, binding, detoxification

Introduction

Aflatoxin M1 (AFM1) is a hepato-carcinogenic metabolite (IARC, 1993) found in milk of

animals that have consumed feeds contaminated with aflatoxin B1 (AFB1) produced by

- 3 -

Aspergillus fungi (Park and Pohland, 1986; Moss, 1998). AFB1 quickly appears in cows

(as AFM1 metabolite in blood after just 15 min (Moschini et al., 2006) and in milk at the

first milking (Diaz et al., 2004; Masoero et al., 2007)) after ingestion of aflatoxin-

contaminated feed. We recently demonstrated that, in Beja province (Tunisia), milk and

milk by-products were contaminated with high levels of AFM1 (3–4-fold above the

permitted dose) (Abbès et al., 2012). AFM1 is such a major concern to humans because of

its frequent occurrence in dairy products at concentrations high enough to cause adverse

effects to health (IARC, 2002). As a result, guidance values for AFM1 in milk have been

established, i.e., levels are limited to 0.05 and 0.50 µg/L in Europe and the US,

respectively (Berg, 2003; Commission of the European Communities, 2006[AU: Not in

refs. Add to list or delete here.]).

Effective detoxification methods would be useful in helping to remediate/mitigate

the problem of AFM1 contamination in milk/by-products. Accordingly, adequate proof of

efficacy of any novel measure would also be of pivotal importance. The ability/efficacy

of food additives (i.e., using mineral and bioactive compounds) purported to detoxify in

vivo or in vitro have been reviewed extensively elsewhere (Abbès et al., 2010; Ben Salah-

Abbès et al., 2010). The detoxification of food products contaminated with mycotoxins is

restricted due to problems regarding safety issues, possible losses in nutritional quality,

limited efficacy, and cost (Bata and Lasztity, 1999). This has led to the search for

alternative strategies such as the use of biological agents. There has been increasing

interest in one concept stating that gastrointestinal tract micro-organisms can reduce the

absorption of AFM1 in consumed foods. Consequently, numerous investigators have

- 4 -

shown that some dairy strains of lactic acid bacteria and bifidobacteria were able to bind

effectively to AFM1 in buffered solutions.

Thus, the aim of the present study was 2-fold: (a) to isolate lactobacillus bacteria

from AFM1-contaminated artisanal butter and to test their ability to bind AFM1 (in a

saline solution and reconstituted milk); and (b) to evaluate the ability of the isolated

bacteria to mitigate/reduce any AFM1-induced immunotoxicities in exposed Balb/c mice.

Materials and methods

Chemicals

AFM1 solution (0.5 ng/ml, in methanol) was obtained from Sigma (St Louis, MO).

Phosphate-buffered saline (PBS; 8 g NaCl, 1.2 g K2HPO4, 0.3 g KH2PO4) was prepared

in 900 ml distilled water, adjusting the pH to 6.5, and then diluting to a volume of 1 L.

All solvents used for AFM1 analyses were purchased from Merck (Darmstadt, Germany)

and were of high performance liquid chromatography (HPLC) grade. In all analytical

steps, water generated through a Millipore Synergy 18S Ultra-Pure Water System from

Millipore (Molsheim, France) was used. AFM1 immunoaffinity columns containing

specific monoclonal antibodies bound to a solid support material for AFM1 clean-up were

obtained from Vicam (product code: G1007, Watertown, MA).

Sampling of artisanal butter

Three samples of 1-month-old artisanal butter made from cow’s milk were collected from

local producers from southern, central, and northern regions of Tunisia. The butter

samples—designated as GAF01, MON03, and BEJ01—were intentionally collected in

regions where it has been traditionally manufactured in households. Collected samples

- 5 -

were kept in sterile bags and held at 4°C; all analyses were performed within the

following 24 h after collection.

In vitro studies

Bacteria screening

Artisanal butter samples (10 g) were homogenized in 90 ml of sterile 2% (w/v) tri-sodium

citrate dihydrate solution (pH 7.5) pre-heated to 45°C and then 10-fold serial dilutions

were prepared in 0.85% (w/v) sterile saline. Using standard protocols, aerobic mesophilic

bacteria were enumerated on nutrition agar (NA; Merck, GmbH, Darmstadt, Germany)

and presumptive lactobacilli were incubated on Man-Rogosa-Sharpe broth (MRS) agar

plates (Biorad, Paris, France) for 72 h at 30°C to permit isolation of lactic acid bacteria.

All isolated colonies were cultured in MRS broth. Thereafter, to test for those organisms

that might be able to bind AFM1, the toxin was added (at 1 mg/L) to the culture broth and

incubated with shaking for 24 h. After incubation, levels of recoverable mycotoxin (i.e.,

non-pelleting with bacteria) were assessed via HPLC analysis of the broth supernatant.

Those organisms shown to have bound/removed AFM1 were then conclusively identified

using API 50CHL kit and 16S rDNA sequencing.

AFM1 removal in PBS and reconstituted milk by isolated bacteria and

evaluation of AFM1/bacteria complex stability

Volumes of the culture broth (each containing ≈ 108 CFU/ml of given organism of

interest) were centrifuged (3000 × g, 15 min) and the bacterial pellets washed with water.

The pellets were then re-suspended in 5 ml PBS contaminated with AFM1 at 0.05, 0.10,

or 0.20 µg/ml. The tubes were then vortexed and the suspensions incubated at 37°C for 0,

4, and 24 h. An AFM1 positive control (0.05, 0.10, and 0.20 µg/ml in PBS) in the absence

- 6 -

of bacteria and negative controls (bacteria suspended in pure PBS) were also incubated in

parallel to monitor the efficacy of bacterial AFM1 binding.

The binding ability of AFM1 by viable lactobacilli bacteria (108

CFU/ml) in

reconstituted milk was also assessed in order to determine if there were any matrix effects

on the efficacy of AFM1 removal. AFM1-free skimmed milk powder was suspended in

sterile water (at 0.2 g/ml) and portions of the reconstituted milk were then contaminated

with standard working solutions of AFM1 at three different concentrations, i.e., 0.05,

0.10, and 0.20 µg/ml. The Lactobacilli bacteria were then treated as described earlier,

but, instead of PBS, the bacterial pellets were suspended in the contaminated skimmed

milk. Each removal assay was carried out in triplicate. All designated tubes were

centrifuged (15 min, 3000 × g) at the end of each incubation period; supernatants were

collected, transferred to clean tubes, and then stored at 4°C until AFM1 analysis.

Unbound AFM1 content in the supernatants were determined by HPLC (see below). Each

removal assay was performed in triplicate.

The stabilities of any bacteria-AFM1 complexes in PBS and reconstituted milk

were evaluated by determining the amount of AFM1 remaining bound after washing.

After 6 h incubation, a supernatant sample was collected to determine binding. Bacterial

pellets with bound AFM1 were washed and re-suspended in 5 ml fresh PBS and incubated

for 30 min at 37°C. The bacteria were then re-pelleted and the supernatant collected for

quantification of released AFM1. Ultimately, the bacteria with the highest percentages of

binding and AFM1/bacteria complex stability were selected for use in the in vivo studies.

Determination of AFM1

- 7 -

AFM1 analyses were performed using HPLC that incorporated an immunoaffinity

column. Briefly, with each butter sample, 10 g was placed in 125 ml 75% (v/v) aceto-

nitrile/water for 5 min. After blending, the samples were filtered using Whatman filter

paper, and the filtrate diluted with 80 ml PBS. A sample of the filtrate (10 µl) was then

passed through the Vicam AFM1 immunoaffinity column (at 5 ml/min, followed by

washing with 20 ml distilled water at 5 ml/min). Bound AFM1 was eluted with 1.5 ml

acetonitrile followed by 1.5 ml distilled water, and collected in a clean vial. These two

eluted samples were mixed and analyzed using an Agilent 1100 HPLC system (Agilent

Technologies, Englewood, CO) containing an ACE C18 silica (5 µm i.d., 25 × 46 mm)

column purchased from Advanced Chromatography Technologies (Aberdeen, Scotland);

product measurements were made in-line spectrophotometrically at an OD of 435 nm.

Quantification of AFM1 was done from measures of peak areas and extrapolating against

a calibration curve prepared/analyzed in parallel using AFM1 standards (Sigma). Three

replicate analyses were performed for each sample to determine AFM1 content. To verify

the soundness of the assay in this protocol, recovery from bacteria-free PBS was

determined for the test range of AFM1 used; mean recovery was in the range of 89–93%.

The percentage of AFM1 bound to the bacteria was calculated using the formula: 100% ×

(1.00 − [peak area of AFM1 in the supernatant/peak area of AFM1 in positive control

sample]).

In vivo studies

Animals and treatment

Forty-eight male Balb/c mice (SEXUEL, St. Doulchard, France) were used (25 ± 0.3 g,

6-week-old) here. All mice were housed (12/cage) in a pathogen-free facility maintained

- 8 -

at 23 ± 1°C with a 50 ± 5% relative humidity and a 12-h light/dark cycle. All mice were

provided were ad libitum access to standard granulated chow (certified mycotoxin-free)

and filtered drinking water. Mice were cared for under the Tunisian Code of Practice for

the Care and Use of Animals for Scientific Purposes. Experimental protocols were

approved in accordance with the guidelines of the Ethical Committee of NIGEB, Tehran.

At the start of the experiment, mice were randomly allocated into four treatment

groups (12/group) as follows: (A) control group of distilled water, (B) AFM1 alone (100

µg/kg BW), (C) bacteria alone (1 mg/kg BW = 2 × 108 cfu/ml), and (D) AFM1+Bacteria.

Thereafter, each host received the indicated treatment(s) orally (by gavage; without any

need for anesthesia) daily for 2 weeks (sub-chronic study). Twenty-four hours after the

final treatment, blood samples were collected from retro-orbital plexus for analyses of

immune system-related end-points (see below).

Measures of total white and red blood cells, and lymphocyte T-cell sub-

types

Total white and red blood cells (WBC and RBC, respectively) were determined using a

Coulter STKS blood counter (Coulter Electronics, Ltd., Luton, UK). In order to perform

sub-type analyses using 2-color flow cytometry, 1 ml of heparinized blood per mouse

was incubated for 20 min at room temperature together with 0.05 ml of a suspension

containing monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC) or

phycoerythrin (PE) against CD4, CD8, CD54, or CD56 surface markers

(Becton/Dickinson, San Jose, CA). Any erythrocytes present were lysed with 0.02 ml

FACSLysing solution (Becton Dickinson) for 10 min at 4°C. The samples were then

analyzed on a FACScan flow cytometer (Becton Dickinson) equipped with Cellquest

- 9 -

software. A minimum of 10,000 events/sample was analyzed. Lymphocytes (n = 2000)

were measured and the percentage of positive cells in this population (gate) was assessed.

Statistical analysis

Data were expressed as mean ± SD of three independent experiments (in vitro study), and

analyzed for statistical significance by Student’s t-test using the general linear model (n =

6 for in vivo tests). The criterion for significance was set at p < 0.05.

Results

In vitro study

AFM1 concentration in butter used for lactobacilli bacteria isolation

The presence of AFM1 in butter used for bacteria isolation was detected at concentrations

ranging between 54.27–112.9 ng/L. The AFM1 levels in 12 (32%) of the positive samples

were higher than the maximum tolerance limit (i.e., 50 ng/L) accepted by Tunisia and

European Union nations. The rationale for this study was to select lactobacilli bacteria

that could tolerate a high level of AFM1.

Isolation of micro-organisms capable of removal AFM1

Colonies (40) isolated from various samples for potential AFM1-binding activity were

screened. Among them, two strains (from butter samples MON03 and GAF01) that were

able to bind the most AFM1 were isolated and identified using an API 50CHL kit. The

organisms (i.e., Lactobacillus) most apt at binding AFM1 in the MON03 and GAF01

were phenotyped and designated L. plantarum MON03 and L. rhamnosus GAF01,

respectively. Moreover, 16S rDNA sequencing showed that these isolated organisms had

99% homology with L. plantarum and L. rhamnosus standards (data not shown).

- 10 -

Determination of AFM1 binding ability by the selected bacteria

The AFM1-binding abilities of viable test strains are summarized in Table 1. The binding

abilities of AFM1 by L. plantarum MON03 and L. rhamnosus GAF01 strains (at 108

CFU/ml) in PBS and reconstituted milk ranged from 16.1 (± 1.4)–78.6 (± 7.1)%, and 15.3

(± 1.5)–95.1 (± 8.3)%, depending on contamination level and incubation period. L.

rhamnosus GAF01 was the better binder, with ≈ 95% binding both in PBS and

reconstituted milk. The differences in binding ability of L. plantarum MON03 and L.

rhamnosus GAF01 at 108 CFU/ml were significant for all incubation times (0, 6, and 24

h) and with all toxin concentrations (Figure 1).

Binding was not irreversible and small amounts of AFM1 were released back into

the buffered solution, especially for L. rhamnosus GAF01. When washing the

bacteria/AFM1 complexes, 18.5% of bound AFM1 by L. plantarum MON03 was released

back to the solution (Figure 2). In comparison, washing of L. rhamnosus GAF01/AFM1

complexes resulted in 5.1% of bound AFM1 being released back.

In vivo study

General observations

The outcome of the in vitro study indicated that L. rhamnosus GAF01 was more suitable

for use in the in vivo study due to its highest capability to bind AFM1. Moreover, L.

rhamnosus GAF01 also showed good complex stability with AFM1.

After 2 weeks of AFM1 treatment, as expected, a general toxicity was observed in

the mice. Specifically, body and lymphoid organ weights were significantly decreased in

the AFM1-treated mice (data not shown). In addition, the deaths of two mice (one on day

10 and one on day 12; ≈ 16% overall mortality) were noted in the AFM1 treated group. In

- 11 -

contrast, no overt general toxicities were observed from L. rhamnosus GAF01 alone or in

combination with AFM1.

Immunological study

The current results concerning relative levels of T-cells (and sub-types) in mice in the

various treatment groups indicated that daily treatment (for 14 days) with 100 µg

AFM1/kg BW induced immune system damage. This was reflected as significant

decreases in the relative levels of CD4+, CD8

+, CD54

+, and CD56

+ cells in these hosts

(Figure 3). On the other hand, treatment with L. rhamnosus GAF01 did not alter blood T-

cell levels. Treatment with AFM1 + L. rhamnosus GAF01 resulted in a significant

improvement in the total counts of immune system cells toward control levels, although

these values were still different from the values of control CD4+ and CD8

+ cell counts.

Total WBC and RBC levels (Figure 4) were also decreased in the AFM1 only-

treated hosts. In contrast, total WBC and RBC counts in mice treated with L. rhamnosus

GAF01 alone were comparable to the controls. The addition of L. rhamnosus GAF01 to

the AFM1 treatment regimen resulted in a significant improvement in both total WBC

and RBC counts, although again, these values still differ (albeit not significantly) from

the control group levels.

Discussion

Aflatoxin M1 (AFM1) is an immunotoxic metabolite found in milk of animals that have

consumed feeds contaminated with aflatoxin B1 (AFB1). Aflatoxins are produced by

Aspergillus fungi that grow (in temperate, tropical, and subtropical climates) at a high

incidence on food-stuffs/grains (Abbès et al., 2008). Since AFM1 is a potent toxin and

may pose a threat to animal and human health, practical methods to detoxify/inactivate

- 12 -

AFM1-contaminated milk and by-products are in great demand. In the present study,

following studies to isolate bacteria from higher contaminated Tunisian traditional butter

samples that could bind with AFM1, we evaluated the possible protective effect of

Lactobacillus rhamnosus GAF01 against AFM1-induced immunotoxicity in Balb/c mice

as an animal model. There is very limited literature on the occurrence of AFM1 (levels) in

butter and milk products in Tunisia (Abbès et al., 2012). In this study, 12 out of 40

traditional butter samples tested were contaminated; all contaminated samples had a level

of AFM1 > 50 ng/L.

The in vitro portion of our study showed the binding abilities by L. plantarum

MON03 and L. rhamnosus GAF01 strains (at 108 CFU/ml) toward AFM1 in PBS. While

L. rhamnosus GAF01 was the best binder with ≈ 95% binding, the binding abilities of L.

plantarum MON03 and L. rhamnosus GAF01 (at 108 CFU/ml) were almost immediate

and significant for all incubation times (i.e., 0, 6, and 24 h) and toxin concentrations

tested. Our study clearly indicates that a bacteria population of 108 CFU/ml is very

suitable for binding AFM1 in PBS and reconstituted milk. Moreover, our study

demonstrated that the matrix did not influence the AFM1 binding capacity of the bacteria.

The amount was similar to that reported for trichothecene binding by some strains of

Lactobacillus and Propionibacterium (El-Nezami et al., 1998). Similarly, Line and

Brackett (1995) indicated that populations of ≥ 108 CFU/ml were necessary for

significant removal of AFB1. El-Nezami et al. (1998) reported that a higher minimum (≈

2 × 109

CFU/ml) was required for the effect.

Information on the binding ability of mycotoxins by dairy strains of lactic acid

bacteria and bifidobacteria is available for AFB1, but very limited for AFM1. Our study

- 13 -

indicated a higher percentage binding ability against AFM1 by the bacteria used than

those published by Pierides et al. (2000), who found that the AFM1 binding abilities of

viable Lactobacillus strains within 15–16 h ranged from 18–54%. Similarly, Peltonen et

al. (2001) investigated the AFB1 binding ability of 12 Lactobacilli, five Bifidobacteriae,

and three Lactococci strains. In their study, Lactobacillus strains bound 17–60%,

Bifidobacterium strains 18–49%, and Lactococcus strains 6–41% of the AFB1 provided.

Our studies also showed that the binding that occurred was not completely

irreversible as very small amounts of AFM1 were released back into the buffered

solution, especially for L. rhamnosus GAF01. Specifically, when L. rhamnosus

GAF01/AFM1 complexes were washed, ≈ 5% of the bound AFM1 was released back to

the solution. This is consistent with findings for the efficient AFB1-binding strain L.

rhamnosus GG (Haskard et al., 2001), but contrasts with results from Peltonen et al.

(2001). Those authors noted that AFB1 was not bound strongly by Lactobacilli strains,

and that 28–94% of bound AFB1 went back into the solution. Thus, L. rhamnosus GAF01

was more suitable for use in in vivo studies due to its demonstrably high(est) capability to

bind AFM1 and to maintain a good complex stability.

The results of the blood T-cell determination studies revealed that treatment with

a daily oral dose of AFM1 induced immune system damage as evidenced by significant

decreases in CD4+, CD8

+, CD54

+, and CD56

+ levels in these hosts. Moreover, our

findings indicated that the 14-day AFM1 treatment also impacted on the numbers of red

and white blood cells. This is not all that remarkable in that the parent compound of

AFM1 (i.e., AFB1) is immunotoxic (Meissonnier et al., 2008; Abbès et al., 2010).

Regrettably, very few studies have been performed examining the effects of AFM1 on

- 14 -

immune functions. Recently, Russo et al. (2010) demonstrated the immunotoxic effects

of AFM1 on macrophage functions. Further, no work is available regarding

immunotoxicity of AFM2 even thought both AFM1 and AFM2 are obtained from AFB1

and AFB2 (via hydroxylation) and excreted into a host’s milk.

The studies here showed that a co-treatment with the isolated L. rhamnosus

GAF01 enhanced blood T-cell levels (except for CD54+

cells) to those somewhat

comparable to values seen with the controls. Why (cells with) this one cell marker was

not affected is not clear at this time. While CD54 represents the intercellular molecule of

adhesion (ICAM-1) protein responsible for cell co-operation, an impact on the number of

CD54+ cells appeared to also not be impacted by treatments with other Lactobacilli (i.e.,

L. paracasei; Jahreis et al., 2002). In contrast to the CD54+ cells, the effects noted here

on the other T-cell sub-types are in keeping findings by other investigators. In rats with

induced enterocolitis, the concentration of CD4+ and CD8

+ cells in the intestinal lamina

propria were increased to a more normal level by administration of L. plantarum (Mao,

1996[AU: Mao et al. in refs.]). In another study, L. paracasei NCC2461 induced the

development of a population of CD4+ T-cells with low proliferative capacity and that

were induced to produce transforming growth factor (TGF)-β and interleukin (IL)-10

(von de Weid et al., 2001[AU: von der Weid in refs.]). In the current study, treatment

with AFM1 + L. rhamnosus GAF01 (at 1 g/kg BW) resulted in a significant improvement

in immune system cell total counts (towards control levels), although in the end they

were still different than control values. Nevertheless, these findings suggested that the

apparent biosorption (i.e., tight binding) of AFM1 to the L. rhamnosus GAF01 resulted in

a reduction of AFM1 bioavailability in the gastrointestinal tract and so a mitigation of

- 15 -

potential effects upon T-cells in these hosts. Whether there are other non-binding-related

attributes from having the L. rhamnosus GAF01 in the host that impart other effects on

the immune system to combat the toxicities that could be induced by AFM1 remains to be

determined.

Many micro-organisms, including bacteria, yeasts, molds, actinomycetes, and

algae, are able to remove or degrade small amounts of aflatoxin in food/feed (Styriak et

al., 2001). Results of several studies suggest that binding is the main mechanism of

detoxification against aflatoxins (Niderkorn et al., 2006; Gratz, 2007). Strains L.

rhamnosus GG and LC-705 seem to be the most effective in such detoxifications

(Lahtinen et al., 2004). However, the binding mechanism itself still remains not

thoroughly understood. It was suggested that carbohydrate-rich mannoproteins or glucans

might be involved in the binding, as levels of complex formation appear to be strain- and

toxin-specific (Shetty and Jespersen, 2006). Raju and Devegowda (2000) attributed the

binding of aflatoxins by yeast cell walls to mannan oligosaccharides, while zearalenone

binding was attributed to glucan components (Yiannikouris et al., 2004). Haskard et al.

(2001) suggested that AFB1 binds predominantly to carbohydrate and protein components

of viable cells. Clearly, as noted by Shetty and Jespersen (2006), systematic studies are

still needed to understand the precise binding mechanisms.

Metabolism of aflatoxins can be considered as a second mechanism for

detoxification. Very few studies have addressed the potential role and importance of the

gastrointestinal tract in AFB1 metabolism. Studies in rats identified free hydroxylated

metabolites of AFB1, i.e., AFM1 and AFL (Gratz, 2007). In the presence of probiotics, an

increased excretion of AFM1 was observed, suggesting a reduced AFB1 uptake and

- 16 -

hepatic metabolism. Some of the metabolites that diffuse(d) back into the lumen were

likely also bound by the probiotics and excreted in the feces. L. rhamnosus GG is known

to bind AFM1 as equally efficiently as AFB1 (Kabak and Var, 2004). Because both AFM1

and AFM2 are excreted into the milk of cows fed aflatoxin-contaminated feed, this

implies some measure of gastrointestinal metabolism of the mycotoxins (El-Nezami et

al., 2000). The data here strongly suggest that the intestinal metabolism of AFM1 is an

important process and that probiotic bacteria impact upon these processes.

Conclusion

The results here showed that AFM1 causes physiological disorders (i.e., at the level of

WBC overall, and T-cells specifically) in Balb/c mice. These studies also illustrated how

Lactobacillus rhamnosus isolated from Tunisian artisanal butter would bind AFM1 in

vitro and decrease its immunotoxic potential in vivo. Thus, the isolated L. rhamnosus

GAF01 can be considered as a good candidate for use as an in vivo detoxification agent

during food consumption (i.e., when taken as a probiotic).

Acknowledgement

The authors would like to acknowledge the Academy of Sciences for the Developing

World (TWAS) and the United Nations Educational, Scientific, and Cultural

Organization (UNESCO) for the financial support granted to the first author to carry out

this study as a part of TWAS-UNESCO Associateship Scheme 2010–2012 at the

National Institute of Genetic Engineering and Biotechnology (NIGEB), Iran. The work

was supported also by the Tunisian Ministry of Higher Education and Scientific Research

(Unit of Immunology, Environmental Microbiology, and Cancerology) and the Higher

Institute of Biotechnology of Beja (Animal Biotechnology Department).

- 17 -

Declaration of Interest: The authors report no conflicts of interest.

The authors alone are responsible for the content and writing of the

paper.

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Table 1. Percentage of AFM1 removal from PBS and reconstituted milk by Lactobacillus

plantarum MON03 and Lactobacillus rhamnosus GAF01 strains.

PBS

0.05 µg AFM1/ml 0.10 µg AFM1/ml 0.20 µg AFM1/ml

Times 0 hr 6 hr 24 hr 0 hr 6 hr 24 hr 0 hr 6 hr 24 hr

L. plantarum

MON03

16.1 ± 1.4 51.1 ± 4.2 71.6 ± 6.1 16.0 ±

2.4

56.3 ±

5.2

74.9 ±

8.2

15.9 ±

1.6

67.4 ±

5.8

78.6 ±

7.1

L. rhamnosus

GAF01

26.2 ± 2.0 61.5 ± 5.2 84.9 ± 7.1 25.9 ±

2.5

66.6 ±

6.2

91.3 ±

7.2

26.0 ±

2.0

86.6 ±

7.1

94.7 ±

8.2

PBS only 5.1 ± 1.1 4.6 ± 0.9 4.2 ± 0.7 3.9 ± 0.8 5.0 ±

0.5

4.1 ±

0.6

4.4 ± 0.7 4.2 ±

0.1

4.2 ±

0.5

Reconstituted

milk

L. plantarum

MON03

15.3 ± 1.5 48.2 ± 4.7 72.3 ± 5.9 15.8 ±

2.2

57.1 ±

5.4

72.9 ±

8.9

15.2 ±

1.3

69.1 ±

5.3

76.9 ±

8.0

L. rhamnosus

GAF01

25.1 ± 2.0 60.3 ± 5.1 85.8 ± 7.5 26.1 ±

2.7

65.3 ±

6.5

90.7 ±

7.5

25.3 ±

2.2

85.9 ±

7.4

95.1 ±

8.3

Reconstituted

milk

5.3 ± 0.9 4.3 ± 0.6 4.1 ± 0.6 4.1 ± 0.7 5.2 ±

0.6

4.4 ±

0.3

4.6 ± 0.6 4.0 ±

0.3

4.4 ±

0.2

Values shown are mean ± SD from three determinations per experimental scenario tested.

All values shown for the bacteria-containing systems are significantly different from control at p ≤ 0.05.

Figure 1. High performance liquid chromatography (HPLC) chromatograms. (a)

Untreated (positive control) aflatoxin M1 (at 0.15 µg AFM1/ml)-phosphate buffered

saline (PBS) solution; (b) AFM1-free PBS solution; or (c) AFM1 (0.15 µg/ml)-PBS

solution after 6 h treatment with Lactobacillus rhamnosus GAF01 at 108 CFU/ml.

Figure 2. Effect of washing on bacteria:AFM1 complex. Initial binding was determined

after bacteria (108 CFU/ml) and AFM1 (0.05 µg/ml) were incubated together for 6 h at

37°C. Bacteria/AFM1 complexes formed were subjected to centrifugation and then

washing with 5 ml PBS, before the amount of AFM1 released was determined as outlined

in the Methods. Results shown are the mean (± SD) from triplicate samples.

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Figure 3. Influence of AFM1 and Lactobacillus rhamnosus GAF01 (alone or in

combination) on lymphocyte sub-types. Balb/c mice received daily (for 14 days) by

gavage distilled water (open bar), AFM1 (100 µg/kg BW) (solid bar), L. rhamnosus

GAF01 (1 mg/kg BW) (shaded bold bar), or AFM1 + L. rhamnosus GAF01 (shaded fine

bar). Lymphocyte counts were then performed on blood samples collected on day 15 of

the experiment (i.e., 1 day after final exposure in each regimen). Results are expressed as

mean [± SE] from n = 12 mice/group. A Student’s t-test was used to compare differences

between groups: a Value significantly/

bnot significantly different from control at p ≤ 0.05.

Figure 4. Influence of AFM1 and Lactobacillus rhamnosus GAF01—alone or in

combination—on blood cell counts. Balb/c mice received daily (for 14 days) by gavage

distilled water (open bar), AFM1 (100 µg/kg BW) (solid bar), L. rhamnosus GAF01 (1

mg/kg BW) (shaded bold bar), or AFM1 + L. rhamnosus GAF01 (shaded fine bar).

Erythrocyte and leukocyte counts were then performed on blood samples collected on

day 15 of the experiment (i.e., 1 day after final exposure in each regimen). Results are

expressed as mean [± SE] from n = 12 mice/group. A Student’s t-test was used to

compare differences between groups: a

Value significantly/b not significantly different

from control at p ≤ 0.05.

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