Study design and source of microorganisms
An experimental laboratory-based study was carried out to establish the efficacy of two microbial agents in reducing aflatoxin concentrations in poultry feeds. The microbial agents were obtained from previous studies that involved in vitro screening for aflatoxin reduction activity of isolates from various sources. A commercial binder, Bentonite, was included as the positive control. Bentonite, as well as the specifications for its use, were obtained from local traders in Kampala. The microbes included a bacterial strain (Bacillus spp B285) and a yeast strain (Y833); that were isolated from poultry droppings and brewer’s waste, respectively.
Preparation of the aflatoxin-reducing microbial test materials
Stock cultures of the microbial isolates, Bacillus spp (B285) and a yeast strain (Y833) were resuscitated in Trypticase Soy Broth (TSB) and Czapek Dox Broth (CDB), respectively. Purity of the isolates was confirmed by sub-culturing on Trypticase Soy Agar (TSA) and Potato dextrose agar (PDA) for the bacteria and yeast, respectively. All media were manufactured by Laboratorios Conda S.A, Spain. Working cultures were prepared by inoculation into the respective broth medium and incubated at 37 ºC, for 18 hours and 5 days, for bacteria and yeast, respectively.
From the overnight microbial broth culture, 2.0 mls were centrifuged at 3000 rpm (Zentrifugen, Germany) for 15 min at 10 ºC. The cells were washed twice with Phosphate Buffered Saline (pH 7.3) and once with double distilled water; and the cell pellet was suspended in 1.0 ml of sterile double distilled water. The microbial preparation was standardized to about 106 CFU/ml for yeast and 108 CFU/ml for bacteria by comparing with McFarland standard No.5. The viable bacterial and yeast cell concentration was confirmed by surface spread method and expressed as colony-forming units per milliliter (CFU/ml).
Compounding of the poultry feed
Freshly supplied raw materials for feed preparation were purchased from produce dealers in Kampala. The ingredients were used in compounding of about 2 kgs of broiler starter feed following an in-house formulation at the Nutrition laboratory, College of Veterinary Medicine, Animal Resources and Biosecurity (COVAB), Makerere University. The ingredients and proportions included: cotton seed cake – 300 g, silver fish meal (Rastrineobola argentea, locally known as “mukene”) – 240 g, bones – 120 g, maize – 1230 g, salt – 10 g and soya – 100 g. The ingredients were ground separately and mixed to obtain the required compounded feed. From the compounded feed, 250 g were ground further using a laboratory mill to pass No.20 and then No.14 sieve. The feed sample was mixed thoroughly for homogeneity and placed in paper bags and stored at refrigeration temperature. The compounded feed was used for the aflatoxin -reducing experiments.
Source of Aflatoxins for feed sample spiking
In-house production of aflatoxins was carried out following the methods described by [46] with modifications. An aflatoxigenic Aspergillus strain, Y20, was obtained from the National Agricultural Research Laboratory, Kawanda. Coarsely ground rice was used as the substrate. The inoculated sterile rice slurry was incubated for 7 days at 25 °C in the dark.
Aflatoxin extraction from multiple 50 g samples was made following the procedure by [47]; with modifications; and all procedures were carried out in a fume hood. For purification, the extracts were pooled and filtered through Whatman No. 1 filter paper. To 125 ml of the filtrate, 30 ml of 2% Sodium Chloride solution and 500 ml n-hexane were added, placed in a separating funnel and shaken vigorously for 5 minutes. The n-hexane layer was discarded and the lower methanolic layer was placed into another separating funnel to which 40 ml of chloroform was added; shaken and allowed to rest undisturbed for separation of layers. The chloroform layer was collected in a flask containing 5 g of Cupric carbonate, agitated and allowed to settle down. The extract was filtered through a bed of anhydrous Sodium sulphate over a filter paper; the various portions were pooled and then evaporated to dryness on a heat block. The residue was dissolved or reconstituted in 1 ml acetonitrile, to have a crude extract that was kept in a refrigerator until further analysis or use. Presence of aflatoxins in the crude extract was confirmed using Thin Layer Chromatography (TLC) plates (Sigma- Aldrich, Germany) alongside 0.04 µg/ml AFB1, 0.024 µg/ml AFB2, 0.03 µg/ml AFG1 and 0.04 µg/ml AFG2 (Romer Labs, UK) as the aflatoxin standards. The bands were visualised under UV light at 365 nm wavelength.
Further purification of the aflatoxin extract was achieved by passing it through an Aflatest Immunoaffinity column (VICAM, USA) following the manufacturer’s instructions. The column was washed with Phosphate Buffered Saline (PBS) and the toxins eluted with 100% HPLC grade methanol. Quantification of aflatoxin content was achieved using VICAM®, a fluorimetric method following the manufacturer’s instructions.
Preparation of the Aflatoxin working solution involved evaporation of the methanol by keeping the loosely closed vials at 50 °C for overnight, after which the concentration was adjusted to 1 µg/ml using PBS. The preparation was kept in an amber bottle in the refrigerator.
Preparation of Aflatoxin spiked feeds and non-spiked feeds
Preparation of the feeds was as described by [48], with modifications. To 10 g of the compounded poultry feed, 0.8 g of Sodium chloride were added and mixed properly. A slurry was prepared by adding 10 ml PBS (pH 7.2) and then autoclaved. To 10 ml of the feed slurry, the aflatoxin working solution was added to obtain a concentration of about 40 µg/L [49], that is, 400 µL of 1 µg/ml aflatoxin working solution. To ensure proper distribution, small amounts of the feed slurry were continuously mixed with the Aflatoxin working solution and homogenised. For non-spiked feeds, instead of aflatoxin solution, 400 µL of PBS was added.
Determination of the effect of feed matrix, pH and temperature on aflatoxin reduction
A factorial study design was used to evaluate the aflatoxin reducing efficacy of the two selected microorganisms and the control (Bentonite) in aflatoxin-contaminated feed in vitro and under simulated in situ gastrointestinal tract environment (pH and temperature) of chicken according to [48]. For each treatment, the toxin reduction was tested in triplicates at pH levels of 4.5 and 6.5; and at 37 ºC, 42 ºC and at room temperature; after which the residual aflatoxin concentrations were determined.
To the Aflatoxin-spiked and non-spiked feed samples, the appropriate test reducing agents were added. For bentonite, 0.3% w/v [50] added according to the instructions given by the local agrochemical supplier. For the microbial agents, 5 mL of the standardized yeast and bacterial cell suspensions with concentration 1.0 × 106 CFU/mL and 1.0 × 108 CFU/mL, respectively, were used. In order to determine the effect of the feed matrix, PBS instead of feeds was used. The tests were carried out in triplicates and incubated at the appropriate temperatures for four (4) hours. The experimental set-up is detailed below and summarized in Table 3.
Table 3
The experimental set up for determination of aflatoxin reduction in poultry feeds, and the effect of feed matrix, pH and temperature
Reducing agent | Aflatoxin (40 µg/L) status |
Feeds | PBS* |
Spiked | Non-spiked | Spiked | Non-spiked |
5 ml 1 × 106 cfu/ml of yeast | 1 | 2 | 11 | 12 |
5 ml 1 × 108 cfu/ml of Bacillus spp | 3 | 4 | 13 | 14 |
2.5 ml 1 × 106 cfu/ml yeast + 2.5 ml 1 × 108 cfu/ml Bacillus spp | 5 | 6 | 15 | 16 |
Bentonite (Positive control) | 7 | 8 | 17 | 18 |
None (Negative control) | 9 | 10 | 19 | 20 |
PBS – Phosphate Buffered Saline. Arabic numerals 1–20 indicate the treatment groups. *The same set-up for PBS was followed for evaluating the effect of pH and temperature on Aflatoxin reduction. |
For comparison of the activity of the test materials, the treatment groups included aflatoxin spiked feeds with yeast (Group 1); non-spiked feed with yeast (Group 2); spiked feed with bacteria (Group 3); non-spiked feeds with bacteria (Group 4); spiked feeds with 50% yeast and 50% bacteria (Group 5); and non-spiked feeds with 50% yeast and 50% bacteria (Group 6). In order to determine the effect of the feed matrix, the same set up was used, however, PBS instead of feeds was used. For positive control, bentonite was added, while for the negative control, nothing was added to the spiked or non-spiked feeds.
For determination of the effect of pH on aflatoxin reduction, the pH of PBS was adjusted to 4.5 and to 6.5; one portion was spiked with aflatoxins, while the other was not. The test materials were then added following the same set-up as for the feed experiments. For the effect of temperature, PBS experimental set up as described above was employed. Two sets, one for incubation at 37 °C and the other at 42 °C were put-up. Positive and negative controls involved addition of bentonite (positive) or nothing (negative) to the spiked and non-spiked preparations (Groups 7, 8, 9, 10; and Groups 17, 18, 19 and 20).
Residual Aflatoxin concentration was determined after 4 hours of exposure. For each treatment, Aflatoxin extraction and quantification followed a procedure described by [47], using the VICAM ® fluorometric method according to the manufacturer’s instructions. Absolute values of the Aflatoxin concentrations for all the treatments were recorded.
Data Analysis
Using the absolute concentration values, percent aflatoxin reduction was calculated from the amount of unbound (residual) aflatoxins extracted after the experiments compared to negative control (without reducing agent). The formula below was employed.
![](data:image/png;base64,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)
Data were analyzed by STATA v12 and the differences between the treatments were determined using ANOVA.
For safety precautions, the cultures for aflatoxin production were autoclaved and later treated with 2% Sodium Hypochlorite before incineration. Likewise, all the plasticware and glassware were soaked overnight in Sodium Hypochlorite before disposal or washing for reuse.