(NCYC 125) on total aflatoxin in maize flour
| Livestock Research for Rural Development 31 (11) 2019 | LRRD Misssion | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
Aflatoxins occur as natural contaminants in cereals. Poultry feeds are mainly formulated using maize because of its high metabolizable energy. It has been suggested that fermentation can reduce levels of aflatoxins in contaminated maize. This study evaluated the effect of fermentation, under different conditions, on aflatoxin levels in contaminated maize flour. Clean (aflatoxin-free) maize was moistened in water and inoculated with Aspergillus flavus then incubated at 30°C for 60 days with frequent moistening. In the first and second experiments, maize flour was fermented with and without yeast Saccharomyces cerevisae between 5-8 days. In the third experiment, the flour to water ratio was adjusted from 1:1 to 1:1.5 and fermented for 72hours. During fermentation there was reduction in pH (6.9-5.0) as well as total aflatoxins by 52 and 53.4% when fermented either naturally or with yeast respectively. The final Aflatoxin levels reduced from 20.4ppb (unsafe for poultry) to 10.9ppb (safe) level. However, the level of aflatoxins steadily increased when fermentation proceeded after 72hours. These results show that natural fermentation (without yeast) was as effective as that with yeast since there were no significant difference in percentage of aflatoxin reduction. It is concluded that the best ratio of flour to water for fermentation was 1:1.5 and fermentation period of 72hours. Therefore, this simple method of fementation contributed to reduced aflatoxin levels in flour for poultry feed production. A prerequisite would be to carry out organoleptic tests and proximate analysis of feed material.
Key words: feed, natural fermentation, poultry
Aflatoxins (AFs) are mycotoxins produced by some species of filamentous fungi like Aspergillus flavus and Aspergillus parasiticus (Sugiharto 2019). Temperatures ranging from 26.7 to 37.8°C and 18% moisture are optimum for A. flavus to grow and produce aflatoxin (Duncan and Hagler 2008) while moisture levels in corn below 12 to 13% inhibit growth of the fungi at any temperature (Duncan and Hagler 2008). Aflatoxins are the best-known class of mycotoxins and among the most potent in terms of toxicity and carcinogenic properties (Munkvold et al 2019). The four major aflatoxins commonly isolated from foods and feeds are aflatoxins B1, B2, G1, and G2 (Obonyo and Salano 2018). Aflatoxin B1 is considered as the most potent naturally occurring carcinogen (Kew 2013; Adelekan and Nnamah 2019) categorized as class1 carcinogens by the World Health Organisation (Martinez et al 2011).
Aflatoxins occur as natural contaminants of certain agricultural commodities, particularly maize which is considered as the best substrate for fungal growth and production of toxins (Jardon-Xicotencatl et al 2015; Chauhan et al 2016). In tropical and subtropical countries, post-harvest losses due to aflatoxins tend to be high due to lack of technology, equipment and methods of reducing contamination of grain (Obonyo and Salano 2018). Additionally, there is competition of maize for human consumption and animal feed production, which leads to a tendency for poultry feeds to be formulated using moldy and substandard grains usually contaminated with mycotoxins (Thuita et al 2019). Therefore, there is growing concern that this contamination may be passed on to animal products in addition to reduced livestock performance (Thuita et al 2019).The United States Food and Drug Administration and European Commission have recommended 20ppb total aflatoxins as the range of permissible levels in poultry feed (FAO 2004). However, the levels of aflatoxins found in food (Obonyo and Salano 2018) and feed (Thuita et al 2019) commonly surpass the regulatory limits.
Fermentation has been suggested as a means of reducing occurring aflatoxin contamination in ingredients (Wacoo et al 2019) especially since it is an inexpensive method to improve the nutritional value of feed ingredients for broiler chickens (Sugiharto and Ranjitkar 2018). In recent decades, the fermentation process has been employed to produce functional feed ingredients like lactic acid bacteria (LAB), lactic acid and other organic acids that have the potential to improve broiler gastrointestinal tract, health, immune responses and production performance (Sugiharto and Ranjitkar 2018). Yeasts have great potential application in reducing the economic damage caused by toxigenic fungi and they are able to degrade toxins to less-toxic or even non-toxic substances (Pfliegler et al 2015). Saccharomyces cerevisiae which are a powerful model organism for studying the cellular workings and diseases of larger eukaryotes due to their conservation of protein amino acid sequence and function, combined with the flexibility of genetic tools and are recognized the world over for their ability to ferment sugars to ethanol and carbon dioxide (Duina et al 2014). Saccharomyces cerevisiae cell wall is composed of polysaccharides (80–90%) and that their mechanical strength is due to an inner layer formed by chains of β-D-glucans which bind toxins (Bovo et al 2015). Some strains of S. cerevisiae have been found to have high AFB1 binding capacity which could be useful for selection of starter cultures to prevent high aflatoxin contamination levels (Rahaie et al 2010; Johnston et al 2012). De Oliveira et al (2018) suggested that decontamination program based on the biological methods is the best as the physical and chemical decontamination procedures may be costly and result in nutrient loss. Therefore, the objective of this study was to determine the effect and duration of fermentation using saccharomyces cerevisiae on the level of aflatoxins in contaminated maize.
Clean (tested, aflatoxin-free using ELISA Test kit) maize kernels were moistened using distilled water and inoculated with a laboratory strain of A. flavus isolated from maize samples in Eastern Kenya. The moist maize was then incubated at 31°C for 60 days with periodic moistening of the kernels to enable uninhibited growth of fungi and aflatoxin production. Afterwards, the level of AFs in the maize was determined using the ELISA technique then confirmed using LC/MS procedure following manufacturer’s instructions (Sun et al 2015).
The maize flour samples that had been fermented and dried were used. The ELISA Test Kit was used following Manufacturer’s instructions (HELICA-Biosystem). Briefly, the aflatoxins were extracted in 70% methanol. A 20g portion from each sample was used for analysis. The extraction solvent of 100ml was added to 20g milled portion of the sample at the ratio of 1:5 of sample to extraction solvent (w/v). It was mixed by shaking in a vortex for about 2minutes. The particulate matter was allowed to settle then the extract filtered through a filter paper and then the filtrate collected to be tested for the concentration of total aflatoxins.
All the reagents were brought to room temperature before use. One dilution well was placed in a microwell holder for each standard plus each of the samples to be tested. An equal number of antibody coated microtiter wells were placed in another microwell holder. 200ml of the conjugate was dispensed into each dilution well and 100ml of each standard and sample was added to appropriate dilution well containing conjugate. It was mixed by priming pipette at least three times. A new pipette tip for each was used to transfer 100ml of contents from each dilution well to a corresponding antibody coated microtiter well and then incubated at room temperature for 15minutes.The contents from the microwells were decanted into a discard basin and washed by distilled water. The microwells were tapped on a layer of adsorbent towel to remove residual water. The required volume of substrate reagent (1ml/strip) was measured and placed in a separate container. 100ml was added to each microwell and incubated at room temperature for 5minutes. After, 100ml of stop solution was added in the same sequence as the substrate. The Absorbance optical density (OD) of each microwell was read with a Thermo Scientific™ microtiter plate reader at 450 nm. Graph pad prism7 software was used to convert the optical density (OD) data to µg/kg. The samples from ELISA were subjected to LC/MS analysis.
After inoculation, the contaminated maize was dried in an oven at 55oC then milled into flour from which 250g was obtained and introduced into a conical flask. Distilled water was added to submerge the maize sample in an air tight conical flask.
This study was carried out in three in vitro experiments.
The initial level of aflatoxins in the maize was 20.4ppb and flour to water ratio was 1:1.5 (w/v). Fermentation was carried out in two ways; through the action of native microflora (natural fermentation) and through action of yeast powder Saccharomyces cerevisiae (NCYC 125). The yeast used was from a commercial ethanol producing company in Kenya (Agro-Chemical & Food Company Ltd (ACFC). The fermentation was carried out under room temperature to mimic conditions at an ordinary farm setting while following recommendations of the ACFC. During the experimental period, pH was measured using a digital pH meter (6.7- 4.6) while the total aflatoxins were measured using the ELISA technique and confirmed using the LC/MS procedure. These measurements were taken from the start of the experiment and regular intervals: 24, 48, 72, 96 and 120hours. Each period of fermentation set-up was replicated three times with the two types of fermentation (natural vs. with yeast). The samples were dried overnight at 55oC, before the total AFs were determined.
The initial level of AFs in the maize sample was 14.3ppb and the flour to water ratio was 1:1.5 (w/v). Fermentation was either natural or with yeast but at an inclusion rate of 5% S. cerevisiae as opposed to 3% in the other two experiments. The fermentation period ran from: 24, 48, 72, 96, 120, 144, 168, and 192hours. Other fermentation conditions were similar to the first experiment.
The results of experiment one showed that fermentation for 72hours yielded the greatest reduction in total AFs. In this third experiment, the initial level of total AFs in the maize was 14.8ppb. The contaminated maize was milled and separately fermented naturally and with yeast. The ratio of flour to water was varied as shown below:
Treatment 1 = 1:1 -1g sample: 1ml tap water - no yeast
Treatment 2= 1:1 -1g sample: 1ml tap water - 3% yeast
Treatment 3= 1:1.5 -1g sample: 1.5ml tap water - no yeast
Treatment 4= 1:1.5 -1g sample: 1.5ml tap water - 3% yeast
Each treatment was replicated three times.
The data was subjected to the two way analysis of variance using the the general linear model (GLM) procedures of SAS (version 9.13) and the means were separated using Tukey’s Range Procedure.
The model was;
Yijk = μ + αi+ βj + (αβ)ij + εijk
Where: Yijk = observation associated with replication k of the factor combination ij
μ = overall mean
αi = effect of ith fermentation type (i=1,2)
βj = effect of jth duration of fermentation (j=1 ...,8)
(αβ)ij = interaction of ith fermentation type with j th duration of fermentation
εijk = random error associated with Yijk
The results indicate change in levels of aflatoxins during fermentation.
The total aflatoxins in maize flour reduced over time of fermentation. The greatest reduction from 20.4ppb to 10.9ppb in aflatoxin occurred after 72hours of fermentation Figure1. However, there was no difference between natural and yeast fermentation (3%). After 72 hours, aflatoxin concentration increased.
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| Figure 1.
The effect of fermentation time with or without Saccharomyces cerevisiae (NCYC 125) on total aflatoxin in maize flour |
After 72hours of fermentation, the total aflatoxins reduced by 60.1 and 55.2% with and without yeast respectively, then increased when the fermentation period proceeded. When maize was fermented with and without yeast, the AF levels were different (p<0.05) after 24, 96, 120, 144, and 168hours but similar after 48, 72 and 196hours of fermentation. After 72hours and 196hours of fermentation the AF levels were similar. After 72hours, the aflatoxin content is reduced irrespective of whether it is fermented with or without 5% yeast (Figure2).
 |
| Figure 2.
The effect of fermentation of maize flour with or without 5%
Saccharomyces cerevisiae (NCYC 125) on the level of total aflatoxins |
The results (Table 1) indicate that the substrate to water ratio of 1:1.5 is better than 1:1 in reducing the amount of aflatoxin.
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Table 1. Percent reduction of total aflatoxins after 72 hours of fermentation |
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Flour:water |
Fermentation type |
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With 3% yeast |
Without yeast |
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|
1:1 |
41.4±10.1 |
32.8±7.7 |
|
|
1:1.5 |
53.7±9.9 |
51.3±8.4 |
|
 |
| Figure 3. pH during 8 days of fermentation |
In all the three experiments, the pH of fermented mixtures reduced from 6.7 to 5 and 5.2 in 72hours of natural fermentation or with yeast. The pH tended to remain constant when the fermentation period increased beyond 72 hours. The changes in pH are illustrated on Figure 3. The decrease in pH was similar irrespective of whether it was fermented naturally or with yeast.
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Table 2. Percent reduction of total aflatoxins after 72 hours of fermentation in the three experiments |
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Fermentation Types |
|||
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With yeast |
Without yeast |
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|
Experiment 1 |
46.5±2.4 |
49.5±0.7 |
|
|
Experiment 2 |
60.1±6.0 |
55.2±3.7 |
|
|
Experiment 3 |
53.7±9.9 |
51.3±8.4 |
|
|
Average |
53.4±6.1 |
52.0±4.2 |
|
In the first and third experiment (Table 2), fermentation using the 3% yeast was as effective as fermentation without yeast (natural fermentation). Fermentation using 5% yeast in the second experiment reduced total aflatoxins by 60.1% compared to natural fermentation where the reduction was 55.2%. Overall, the average reduction in the three experiments was 53.4% using yeast and 52% when it is fermented naturally and were not statistically different (p>0.05).
Considering the adverse effects of aflatoxin (AF) in poultry production, mitigation measures are desirable. Several strategies have been developed and tested to reduce aflatoxin levels in animal feed, but their adoption is low due to either high costs or technical difficulties (Udomkun et al 2017). In the current study, the effect of fermentation on AF levels in contaminated maize was evaluated. The method chosen was to mimic what is easily implemented among subsistence farmers. This is because it is more practical to ferment contaminated ingredients rather than the complete poultry feed. The use of yeast in fermentation of animal feed ingredients is a possible means of reducing widely occurring aflatoxin contamination (Hayo 2018, Wacoo et al 2019).
The findings of the current study show that spontaneous fermentation (through the action of indigenous microflora) and fermentation using yeast reduced levels of total aflatoxins by 52.1 and 53.5% respectively. The levels of aflatoxin before fermentation was considered unsafe (20.4 and 14.3ppb) but these reduced to safe (10.9 and 5.7ppb) levels for poultry feed formulation (FAO 2004). The highest reduction of total aflatoxin occurred within 72hours of fermentation which corroborates other studies. For example, Adeleken and Nnamah (2019) reported reduction in aflatoxin content in maize from initial concentration of 58.00 to 3.1ppb in the same steeping period. Similar findings have also been reported by other workers (Assohoun et al 2013; Okeke et al 2015; Poloni et al 2017).
It has been postulated that S. cerevisiae binds toxic metabolites of filamentous fungi to the cell wall thereby significantly reducing AFB1 concentrations (Gonçalves et al 2015; Chlebicz and Śliżewska 2019). Similarly, spontaneous natural fermentation without yeast has shown reduction in levels of total aflatoxins. This is thought to be due to lactic acid bacteria that removes toxins through non covalent binding of mutagens by fractions of the cell wall skeleton of the lactic acid bacteria (Zhang and Ohta, 1991). However, another alternative mechanism of aflatoxin B1 removal has been reported in which lactic acid bacteria fermentation opens up the aflatoxin B1 lactone ring resulting in its complete detoxification (Nout 1994). The current study design could not explain the mode of action of removal of aflatoxins neither could it account for observed difference in aflatoxin reduction in the two methods chosen.
The findings of the current study show that during fermentation using both methods, the ratio of flour to water (1:.1 and 1:.1.5) had effect (p<0.05) on final aflatoxin levels (Table 1). The highest reduction in total aflatoxin levels was observed in fermentation with the ratio of 1:1.5 (1g sample: 1.5ml tap water). This corroborates the work of Biernasiak et al (2006) who used the same ratio to detoxify mycotoxins by probiotic preparation for broiler chickens. Similarly, there was a drop in pH after fermentation as compared to the pH of non-fermented samples (Figure 3). This is due to metabolic processes releasing organic acids, acetic acid, ethanol and a few other minor products (Műller 2008). Due to detectable amounts of lactic and acetic acids after fermentation, lactic acid bacteria dominate the culture system and result in lower pH, an important characteristic for the product safety (Poloni et al 2017). The low pH of fermented feeds acidifies the upper digestive tract and thereby improves the barrier function of the gizzard against pathogens (Sugiharto and Ranjitkar 2018). Additionally, it has been reported that the low pH in fermented feeds increases the resistance of poultry diets to fungal contamination (Londero et al 2014). However, such studies deserve attention as a follow up to the current one.
The authors would like to thank the Center of Excellence in Sustainable Agriculture and Agribusiness Management (CESAAM), Egerton University and National Research Fund of Kenya Multidisciplinary Project [NRF: 2018] for financially supporting this study. We would also like to thank Mr. Micah Lagat of the Mycotoxin Research Laboratory, Egerton University for the technical expertise rendered in this study.
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Received 27 July 2019; Accepted 3 October 2019; Published 2 November 2019