Detoxification of aflatoxin B 1 and change in microflora pattern by probiotic in vitro fermentation of broiler feed

The aim of this study was to determine the influence of spontaneous fermentation and that with the use of probiotic bacteria and yeast on aflatoxin B1 concentration and the microflora pattern during fermentation. The probiotic preparation used contained bacteria resistant to gastric juice and bile: Lactobacillus paracasei LOCK 0920, Lactobacillus brevis LOCK 0944, Lactobacillus plantarum LOCK 0945, as well as live yeasts Saccharomyces cerevisiae LOCK 0140 of high fermenting capacity. After 6-h fermentation with the probiotic, in feed mixture with a low concentration of aflatoxin B1 (1 mg/kg), the amount of aflatoxin B1 decreased by 55%. In the case of a high concentration (5 mg/kg) the decrease in aflatoxin B1 was about 39%. This tendency was sustained during the following hours of incubation (12th and 24th h). The application of probiotic bacteria and yeasts resulted in the reduction of aerobic spore forming bacteria.


INTRODUCTION
The data published by FAO in 2001 showed that 25% of agricultural products were contaminated with mycotoxins.Aflatoxins, particularly aflatoxin B 1 (AFB), are considered to be the most important of the mycotoxins due to their high toxicity and they are still of major concern to the feed industry and farmers as many raw materials which are used as components of animal feeds are prone to contamination.Poultry species differ in their susceptibility to aflatoxin B 1 , young birds are more sensitive than older ones (FAO/ WHO, 2001;Fraga et al., 2007).
The adverse effects of mycotoxins involves their mutagenic, carcinogenic (especially to kidneys and liver), teratogenic and oestrogen immunosuppressive effects.Aflatoxin B 1 is one of the strongest carcinogens and it was included by WHO and the International Agency for Research on Cancer in the list of carcinogenic substances Group I, i.e. substances with confirmed carcinogenic effect in humans (JECFA, 1998; FAO/WHO/UNEP, 1999).
Plant materials already contaminated with mycotoxins should be detoxified.It is a common practice to add different adsorbents to animal feeds to prevent the negative effects of mycotoxins (Huwig et al., 2001).Also a microbial degradation is one of the well-known strategies for the management of mycotoxins in foods and feeds.Among the different potentially decontaminating microorganisms, Saccharomyces cerevisiae and lactic acid bacteria represent unique species which are widely used in food fermentation and preservation (Baptista et al., 2004;He et al., 2010); these species are also components of probiotic preparations used in broiler production (Line et al., 1998;Patterson and Burkholder, 2003;Smulikowska et al., 2005).
The aim of this study was to determine the efficiency of a probiotic preparation composed of defined strains of lactic acid bacteria, yeasts and a yucca extract in degradation of aflatoxin B 1 in broiler feed mixture during incubation in vitro.

Material
Wheat grain was inoculated with the strain of Aspergillus flavus K30 (Collection of Pure Cultures, Institute of Food Technology of Plant Origin, Poznań University of Life Sciences) and incubated to obtain high concentration of aflatoxin B 1 in accordance with a procedure described by Xiao et al. (1996).Twenty Erlenmeyer flasks (500 ml), each containing 30 g of feed-grade wheat and 30 ml of distilled water, were autoclaved for 30 min at 120ºC and then inoculated with Aspergillus flavus K30.The culture was maintained at 30ºC in a dark room.The fermentation was terminated on day 12 of incubation, the contaminated wheat was dried for 72 h, ground and the aflatoxin B 1 (AFB) concentration was determined.The diet was formulated to meet or exceed the requirements of broilers (Table 1), adequate amount of contaminated wheat was substituted for uncontaminated one to obtain the final concentration of 1 or 5 mg of aflatoxin B 1 per kg of diet The probiotic preparation (PP) used contained (per 1 kg): 10 10 of Lactobacillus cells (L.paracasei LOCK 0920, L. brevis LOCK 0944 and L. plantarum LOCK 0945), 10 6 of yeast Saccharomyces cerevisiae LOCK 0140 cells and 50 g of Yucca schidigera extract.The strains derived from Centre of Industrial Microorganisms (LOCK), Institute of Fermentation Technology and Microbiology, Technical University of Lodz in Poland.The strains were formerly used in broiler trials (Smulikowska et al., 2005), the preparation possess full probiotic documentation and is licensed (Michałowski et al., 2004).

Fermentation
The 1 kg samples of broiler diets with different aflatoxin B 1 concentration were closed tightly in polyethylene bags and sterilized by radiation with doses ranging 25 kGy at ambient temperature.Radiation was carried out by 60 Co γ-rays at a dose rate of 0.2 Gy s -1 , Fricke dosimetry being employed.Sterilized control samples were kept tightly closed at 37 0 C and sampled after 6, 12 and 24 h for aflatoxin B 1 analysis.
The one kg samples of each unsterilized diet without supplement (spontaneous fermentation) or with addition of 1 g PP per kg (probiotic fermentation) was mixed with distilled water in a proportion of 1:1.5 (w/w) and incubated at 37ºC in an aerobic atmosphere for 24 h.Fermentation was made in triplicate.After 6, 12 and 24 h of fermentation the 10 g of each diet were sampled for microbiological and aflatoxin B 1 analysis.

Analytical procedures
The concentration of aflatoxin B 1 in wheat grain and in diets was measured with an enzyme linked immunosorbent assay (ELISA direct competitive immunoassay with horseradish peroxidase conjugate) using a commercial ELISA kits (AgraQuant Aflatoxin, Romer Labs Diagnostic, Singapore) according to the procedure described in the Agra-Quant Assay kit manual.In brief: aflatoxin B 1 was extracted from 10 g of sample with 50 ml methanol:water (70:30 v/v).Independently, 100 µl of conjugate mixture were added to each well containing standards and previously filtered samples (50 µl).After mixing, 50 µl were transferred to wells containing antibodies and incubated for 15 min.Then, the content was eliminated, and the wells were washed five times with deionized water.Any excess of water was discarded and the wells were dried.Substrate (50 µl) was then added to each well, incubated for 5 min, and the reaction was stopped by adding 50 µl stop solution.Optic density was read with an UVM 340 microplate reader (Asys, Austria) using a 450 nm filter.The aflatoxin B 1 concentration was calculated by extrapolating the optic density with the respective calibration curve (Zheng et al., 2005).

Microbiological analyses
The microbiological analyses were carried out according to the Polish Standard of PN-ISO (PN-EN ISO 4833, 2004).The following bacteria species were identified: Lactobacillus on MRS agar medium (Merck), using a doublelayer technique and anaerobic incubation at 35ºC/72 h; Clostridium on TSC agar (Merck) and anaerobic incubation at 37ºC/18-24 h; Pseudomonas on PM5 agar (BTL) and anaerobic incubation at 37ºC/24 h; the Coli group on VRBL agar (Merck) and aerobic incubation at 35ºC/24 h; total number of anaerobic bacteria on Plate Count agar (Merck) and anaerobic incubation at 35ºC/24 h, and total number of yeasts on YGC agar (Merck) and aerobic incubation at 20ºC/120 h.Anaerobic bacteria were cultured in the anaerobic atmosphere of H:N:CO 2 1:8:1 (Anaerobic Workstation Concept 400, Biotrace Int.).The specific morphology of cells was checked under a microscope (Olympus CX-41).Each determination was done in triplicate.The results are presented as colony forming units (cfu) per gram samples.

Calculations and statistical analysis
Data were collected in triplicate and subjected to three-way analysis of variance by the general linear model (GLM) procedure of Statgraphics Centurion XVI.Statistical significance was accepted at P<0.05.In case when significant difference was found, post-hoc Tuckey HSD difference test was used to compare between mean differences.For bacterial counts the analysis of variance was carried out on data submitted to logarithmic transformation.

RESULTS
After 6 h keeping at 37 o C in diets that had been previously subject to radiation sterilization the amount of AFB was reduced to 0.89 mg/kg (by 10%) in the sample containing initially 1 mg AFB /kg , and to 4.57 mg/kg (by 8.5%) in the sample with the higher dose of the toxin.The concentration of AFB in irradiated feed did not change during further 24 h (Figure 1).The concentration of AFB decreased after 6 h of incubation at 37 o C in non supplemented diet by 16-20% and did not decrease substantially during further 12 and 24 h of incubation (Figure 1, Table 2).However, in broiler diets with initial concentration of 1 mg AFB/kg supplemented with probiotic after 6 h of incubation at 37 o C the concentration of AFB was reduced to 0.45 mg/kg (by 55%) and in diet with 5 mg AFB/kg to and 3.03 mg/kg (by 39%), respectively.Aflatoxin B 1 concentration was reduced further during prolonged incubation, and after 24 h incubation it decreased by 73% in the sample containing initially 1 mg AFB /kg and by 53% in the sample with a higher dose of AFB (the interaction AxT and PxT significant; Table 2).The differences were statistically significant (P<0.05) in comparison with the irradiated sample and sample without probiotic (Figure 1).
Main effects of experimental treatments on bacteria and yeasts number was shown in Table 2. Initial dietary aflatoxin B 1 did not affect bacteria and yeasts counts.Lactobacillus, total bacteria and yeasts counts increased (P<0.01)during incubation in diets without and with probiotic preparation.However, the increase in bacterial counts was higher by 2 orders of magnitude, in yeasts counts by 4 orders of magnitude in the diets supplemented with the probiotic preparation (interactions probiotic x time were highly significant).
The inhibition of viable aerobic spore forming bacteria increased in unsupplemented diets and decreased in the diets supplemented with probiotic (interactions probiotic x time were highly significant).Significant interaction (P<0.05) was found also between initial dietary aflatoxin B 1 level and time of incubation for anaerobic spore forming bacteria, their numbers decreased in the diet containing 1 mg aflatoxin B 1 after 6 h, than slightly increased, while in the diet containing 5 mg aflatoxin B 1 it increased with time of incubation (Table 2).
In both diets initially the bacteria from the Coli group as well as Pseudomonas and Clostridium were present at average amounts 2.0, 4.1 and 1.0 log CFU/g, respectively, but they were not found after 6 or 12 h of incubation.The effect was connected neither with aflatoxin B 1 concentration nor with the presence of probiotic.

DISCUSSION
Broiler feed used in the current study was heavily contaminated with aflatoxin B 1 , much above the level 0.02 mg/kg permitted for poultry (Directive 2002/32/ EC).Probiotic preparations are aimed to populate the intestinal tract with bacteria (Patterson and Burkholder, 2003) or yeasts (Line et al., 1998), which influence the environment of the gut and favour the establishment of beneficial rather than detrimental species.The current study shows that during in vitro fermentation, despite the high or very high aflatoxin B 1 (AFB) level in broiler feed, the lactic acid bacteria (LAB) and yeasts multiplyied considerably in feed with addition of probiotic in comparison to feed without the probiotic.The influence of applied probiotic on the Clostridium growth was inconclusive, but in our earlier study the probiotic added to the feed reduced significantly Clostridium number in excreta of 3-week-old chickens (Smulikowska et al., 2005).
In the present study the concentration of aflatoxin B 1 in a feed mixture decreased considerably due to probiotic activity.From practical point of view the most important is the time of 6 h of fermentation, as the passage rate of feed in broilers ranges between 4-8 h.During first 6 h of fermentation in the feed with a low AFB concentration the amount of aflatoxin B 1 decreased by 55%, in the feed with the high AFB concentration (5 mg/kg) the reduction in aflatoxin B 1 equalled about 39%.The prolonged time of incubation (12 and 24 h) resulted in further decrease of AFB concentration.After 24 h of fermentation the amount of aflatoxin B 1 was reduced by 73% in samples with lower and by 53% in samples with a higher aflatoxin B 1 concentration.
Biological detoxification of mycotoxins in food, raw products, mixed protein feeds and also in human and animal organisms is a novel and very promising process.Among the organisms that have been used for mycotoxin elimination, are: Acinetobacter calcoaceticus bacteria (Kusumaningtyas et al., 2006), Aspergillus (Wiseman and Marth, 1981), Alternaria, Botrytis, Cladosporium, Phaffia, Penicillum and Rhizopus moulds (Niderkorn et al., 2006), lactic acid bacteria of the Lactobacillus strains (El-Nezami et al., 1998) and Saccharomyces yeasts (Kusumaningtyas et al., 2006).Shetty and Jespersen (2006) examining the ability to bind aflatoxin B 1 by different strains of lactic acid bacteria and yeast obtained the best results with strains of Saccharomyces cerevisiae, Candida krusei, Lactobacillus plantarum and Lactobacillus fermentum, which eliminated 60% or more of the aflatoxin B 1 from saline.Peltonen et al. (2001) shown that a fairly high efficiency (50%) of binding of aflatoxin B 1 from a buffered saline solution is displayed by strains of Lactobacillus rhamnosus and Lactobacillus amylovorus, while Lactococcus lactis spp.cremoris, Bifidobacterium animalis and Bifidobacterium lactis were slightly less efficient (40-50%).Experiments on decontaminating chicken feed containing aflatoxin B 1 by using Saccharomyces cerevisiae, Rhizopus oligosporus or both species were conducted by Kusumaningtyas et al. (2006).Both separate and combined use of these organisms yields similar results in reducing the concentration of aflatoxin B 1 being most evident (over 60% reduction) after 5 days of action.
Nevertheless, special interest has been paid to lactic acid bacteria due to their favourable influence on animal and human organisms and the widespread use in the production of fermented food and probiotic preparations.These bacteria inhibit both the growth of moulds and their production of mycotoxins (Shetty and Jespersen, 2006).Probiotics also can improve the feed utilization by means of specific hydrolytic enzymes production that decompose carbohydrates as well as can increase the activity of the host's enzymes, such as β-galactosidases, saccharase and maltase (Ŝtyriak et al., 2001).
In order to investigate the mechanisms which account for the removal of mycotoxins by LAB, the effects of viable and heat inactivated bacteria were compared in a number of studies (El-Nezami et al., 1998;Fink-Gremmels, 1999).Additionally, the bacteria were treated with enzymes (such as pronase E and lipase) or periodate, which cause alterations to the structure of the cell walls (Haskard et al., 2001;Lahtinen et al., 2001).On the basis of the results obtained in these experiments, it was postulated that the removal of aflatoxin B 1 and zearalenone is due to the non-covalent binding of the toxins to the carbohydrate moieties of the cell walls.However, since a decrease in their toxic effects was also observed in the case of cytosolic preparations of LAB, it was hypothesized that other mechanisms (e.g., interactions with short chain fatty acids) might also play a role (Allameh et al., 2005).
Contamination of broiler feeds with AFB can occur occasionally irrespective of the grain quality monitoring procedures (Fraga et al., 2007).The results of the present study indicate that the probiotic preparation containing LAB bacteria, Saccharomyces cerevisiae yeasts and Yucca shidigera extract aimed at beneficial modulation of gut microbiota, can also effectively decrease the level of AFB during 6 h fermentation of feed mixture.The in vivo experiment on broilers is needed to confirm a usefulness of the preparation in protection of broilers against the occasional AFB contamination.

Table 1 .
Composition of the feed mixture for broiler chickens, g/kg -1

Table 2 .
Main effects of supplementation with probiotic preparation, dietary aflatoxin B 1 level and time of incubation on bacterial and yeasts counts, log CFU/g diet and on aflatoxin B 1 concentration in feed, mg/kg