Evaluation of yeast sludge cell wall and commercial clay based toxin binders against aflatoxin B₁ on growth and serum parameters of broilers

Aflatoxins (AFs), as ubiquitous threat to poultry, are produced biologically by Aspergillus. flavus and A. parasiticus (Chen et al., 2014). Among twenty different AFs, four are most important (AFB₁, AFB₂, AFG₁ and AFG₂) with order of toxicity AFB₁ > AFG₁ > AFB₂ > AFG₂. The AFB1 is most commonly found in feedstuffs (Yang et al., 2012). AFs result in poor feed conversion, mortality, economic losses (Akkaya and Bal, 2012) and decreased total serum protein, albumin, cholesterol, glucose and uric acid (Chen et al., 2014). Clay based adsorbents and yeast glucomannan alleviate aflatoxicosis after making AFs unavailable for absorption in gastrointestinal tract (Chen et al., 2014). However, because of shortcomings of clays based toxin binders, yeast cell wall extracts (mannan contents) have been proposed as another alternate (Akkaya and Bal, 2012). This leads to formation of irreversible structure in the digestive tract between binding agent and AFs (Eraslan et al., 2006). Yeast Sludge (YS) is a waste product which is produced ater the fermentation of sugarcane molasses by Sachharomyces cerevisiae (Hashmi et al., 2006). The sonication of YS may lead to separation of cell wall which can be used as toxin binder. To best of our knowledge, no study has reported on comparative effects of YSCW against bentonites and Bent+Gluco at different levels of AFB1­. So keeping in view, the present trial was conducted to compare the YSCW against different commercial toxin binders at 100, 200 and 300 µg/Kg on broilers.

Sonication of yeast sludge
 
The YS was fractionized by sonicator in cycle mode (Branson Sonifier, Model No. 250, Serial No. BH50023, Manufacturing Company: Branson VWR Scientific USA, Input: 100, 200 / 240 VAc, 50 / 60 MHz, 2 Amp) into two layers: supernatant containing Cell Wall and residue containing Cell Soluble. The tip of sonicator horn (20 mm diameter) was located in the center of solution. Ultra-sonication of yeast cells caused the disruption of cell protein (Liu et al., 2013). The mixture was centrifuged at 1,500 g for 10 minutes (height of tube 10 cm) (Northcote and Horne, 1952) to separate supernatant and residue. The supernatant containing cell wall was then dried at 71°C.
 
Estimation of mannan oligosaccharides
 
The dried YS Cell Wall was taken for the determination of mannan oligosaccharides according to the procedure of Stock and Rice (1967). The YS Cell Wall was found to contain 2.33% mannan oligosaccharide contents.
 
Production of aflatoxins
 
AFs (B₁, B₂, G₁ & G₂) were produced by using culture of A. parasitucus (NRL 2999) described by Shotwell et al., (1966) with some modifications. The extraction of AFs was carried out by adding 30 mL chloroform / 10 g fermented rice for 30 minutes. The mixture was then centrifuged at 5,000 rpm for 10 minutes and filtered by Whatman # 1 filter paper (Oliveira et al., 2002). The filtrate was spotted on thin layer chromatography plate and eluted for the separation of AFB₁, AFG₁, AFB₂ and AFG₂. The separated AFB1 was taken in chloroform and quantified by HPLC equipped with fluorescent detector (AOAC, 1995).
 
Experimental diet and chicks
 
A total of 390 1-d-o straight-run Hubbard broiler chicks (three replicates / treatment and ten birds / replicate) were procured from local hatchery and were given ad lib starter basal diet and water under 24 hours light. The birds were kept at 95°F for brooding and temperature was reduced at the rate of 5°F per week until 70°F. On 8^th day of age, the birds were randomly categorized into different pens for various treatments (8^th to 28^th day).

The experimental diet (Table 1) for each treatment was as follow: 1. Basal diet (NC), 2. NC + 100 µg AFB₁/kg (PC1), 3. NC + 200 µg AFB₁/kg (PC2), 4. NC + 300 µg AFB₁/kg (PC3), 5. PC1 + YS Cell wall (YSCW1), 6. PC1 + Bent (Bent1), 7. PC1 + Bentonite + Glucomannan (Bent + Gluc1), 8. PC2 + YS cell wall (YSCW2), 9. PC2 + Bentonite (Bent2), 10. PC2 + Bentonite + Glucomannan (Bent + Gluco2), 11. PC3 + YS Cell Wall (YSCW3), 12. PC3 + Bentonite (YSCS3), 13. PC3 + Bentonite + Glucomannan (Bent + Gluco3). All YSCW, Bentonite and Bentonite + Glucomannan were added at 1%, 1% and 0.5% of total feed, respectively. Iso-caloric and iso-nitrogenous feed was formulated according to NRC (1994) recommendations. The birds were offered with starter diet until 21^st day of age and changed to grower diet. Weekly feed intake (FI) and weight gain (WG) were recorded. The protocol for the current study was approved by Ethical Review Committee of the University of Veterinary and Animal Sciences, Lahore, Pakistan (Approval No: DR/142 dated 06-04-2016). On slaughtering, liver and gizzard weight of birds were recorded.


 
Biochemical parameters
 
After slaughtering, the serum was stored at -20^oC until analyses (Yildirim et al., 2011). The total serum protein was determined by Biuret method (Breuer & Breuer Diagnostics, Germany) (Henry et al., 1974). Commercial Randox Kits (Rec, 1972) were used to determine serum albumin (Human Diagnostics, Germany). Serum cholesterol (mono reagent with LCF, CHOD-PAP method), aspartate aminotransferase (ASAT) (GOT IFCC mod) and alanine aminotransferase (GPT IFCC mod) were analyzed by Clinical Chemistry analyzer (Company: Elitech Group, Microlab 300 Voltage: 220, 50/60 Hz) using commercial kits according to the company recommendations (Human Diagnostics, Germany).
 
Experimental design
 
The data were statistically analyzed using completely randomized design by 2-way ANOVA under factorial arrangement using GLM procedure of SAS 9.1 software. The model included the main effects of AFs levels and toxin binders (Steel et al., 1997). Duncan Multiple Range test was used to compare the significant difference of means of treatments at 0.05 level of probability (Duncan, 1955).

Production parameters
 
The AFs intoxication significantly affected FI (p>0.05) as compared to NC and negatively affected WG and FCR at 100, 200 and 300 µg AFB1/kg. The NC showed comparatively better FI and WG than other dietary treatments, thus indicating negative effects of AFs which matches with the findings of Santin et al., (2006). However, YSCW, Bent and Bent + Gluc showed non-significant effects at different AFB₁ levels on FI but were unable to show non-significant effect as NC.

Among positive controls, PC2 and PC3 showed 37.42% and 36.38% decrease in WG leading to 1.41 and 1.35 times increase in FI as compared to NC, respectively (Table 2). The presence of glucomannan in YSCW and Bent + Gluc may be responsible for better WG (87.9, 83.45 and 75.48% for YSCW and 83.37, 82.83 and 78.35 for Bent + Gluc vs 66.26, 62.6 and 63.6% for PC than NC) at 100, 200 and 300 µg AFB₁/kg, respectively. This caused to affect FCR i.e. 1.02, 1.12 and 1.12 times for YSCW and 1.11, 1.11 and 1.15 times for Bent + Gluc vs 1.37, 1.41 and 1.35 times for PC than NC at 100, 200 and 300 µg AFB₁/kg. The interaction of toxin binders with AFs levels explains their effectiveness on the growth of birds. The current findings agree with Mogadam and Azizpour (2011) who found higher FI (7.4%), WG (24%) and better FCR (13.7%) with 0.1% yeast glucomannan compared with 1.5% Na-bentonite and NC birds. Neeff et al., (2013) noted birds fed AFB1 alone had 27% reduction in WG, whereas birds fed both AFB1 and HSCAS caused 18% reduction in WG. This suggests partially effectiveness of HSCAS in alleviating the decrease in WG by AFB1 and matches with non-significant findings of YSCW and Ben + Gluc on WG at different AFs levels.



Modified glucomannan trap the toxins (Girish and Devegowda, 2006) in small intestine and impart beneficial health impacts. Insufficient AFs concentration / tolerance from 100 to 300 µg AFB₁/kg may be responsible for non-significant results for FI, WG and FCR on positive control (PC1 to PC3) (Table 2). The dietary treatments, toxin binders and AFs levels showed significant effect (p<0.01) on FCR whereas, non-significant difference among YSCW and other toxin binders showed their effectiveness.
 
Liver and gizzard weight
 
The dietary treatments, toxin binders, AFs and Toxin binders × AFs showed non-significant effect for relative gizzard (Table 2). AFs affect mainly liver (Quezada et al., 2000). The dietary treatments, toxin binders and AFs showed significant effect (p<0.01) for relative liver weight which agrees with Yunus et al., (2011) who found increase in relative liver weight by AFs contamination. Chen et al., (2014) found significant interaction between AFB1 and HSCAS. Non-significant effect was observed on relative liver weight of NC birds in comparison to birds given toxin binders and AFB1 at 100 µg/kg, indicating detoxifying efficacy of toxin binders. Another possible explanation could be low AFB1 levels in this study. The toxin binders in combination with 200 and 300 µg AFB1/kg showed significant increase in relative liver weight as compared to toxin binders with 100 µg AFB1/kg (Table 2). Chen et al., (2014) found glucomannan (p<0.05) and HSCAS (p = 0.02) to decrease liver weight than control. The AFs may inhibit hepatic protein synthesis and lipid metabolism which expose liver to accumulate lipids.
 
Serum and liver biochemistry
 
Low AFs levels showed non-significant effect on performance but decreased total serum protein, albumin, cholesterol and uric acid (Table 3) which matches with the findings of Yang et al., (2012). AFs administration was found to increase ALAT (7.8±3.03 U/l) as compared to control (5.4±1.14 U/l). However, the devastating effect on ALAT was restored significantly by bentonites (Eraslan et al., 2006). Moderate to severe liver intoxication affects the liver functions tests that include increase in ASAT. Consequently, there are hepatocytes degeneration and leakage of enzymes (Tessari et al., 2010). The dietary treatments showed significant effects (p<0.01) for ASAT. Moreover, the AFs levels and toxin binders showed significant effect (p<0.01) for ASAT (Table 3). Yang et al., (2012) found significant increase in serum ASAT and ALAT by feeding 75% and 100% contaminated corn. The NC showed non-significant effect with YSCW and Bent + Gluc at 100 and 200 µg AFB₁/kg intervention comparatively at 300 µg AFB₁/kg for ASAT. Eraslan et al., (2006) found bentonites to affect non-significantly on total proteins, albumin and ASAT whereas Chen et al., (2014) found significant effect (p<0.05) of HSCAS on serum albumin and ASAT for birds fed 1 and 2 mg AFB₁/kg, respectively. The dietary treatments, AFs levels and toxin binders affected non-significantly to ALAT (p = 0.17, p = 0.43, p = 0.74) and significantly to albumin (p<0.05) and uric acid (p<0.01, p<0.01, p<0.01 and p = 0.17).



The 0.5 and 1 mg AFB1/kg negatively affected serum protein concentrations (Chen et al., 2014). This may be due to reduced serum total protein or inhibition of amino acid transport and mRNA transcription which resulted in inhibition of DNA and protein synthesis (Yang et al., 2012). The supplementation of sodium bentonites improved total protein (3±0.52 vs 2.19±0.06 g/dL), serum albumin (1.42±0.26 vs 1.15±0.06 g/dL) and cholesterol (88.66±6.65 vs 114.8±13.7 mg/dL) as compared to AFs contaminated feed (Mogadam and Azizpour, 2011). Compared with birds fed control diet, feeding AFB₁ to broilers resulted in significant reduction in serum albumin and total protein (Kececi et al., 1998). Sodium bentonites and AFs decreased serum cholesterol (88.66±6.65 vs 114.8±13.7 mg/dL) significantly as compared to NC (Mogadam and Azizpour, 2011). Quezada et al., (2000) found interaction of AFB1 with HSCAS for serum albumin in birds fed 1 mg AFB₁/kg (p<0.05). Decreased total serum protein and cholesterol were not ameliorated by zeolites whereas decreased serum cholesterol in broilers were improved by adding 5 g/kg bentonites alone with AFs (Monson et al., 2015). It can be concluded that Yeast Sludge Cell Wall can be great asset as toxin binder to detoxify the toxic effects of AFs in comparison with bentonites and glucomannan products.

This research was funded by Higher Education Commission, Pakistan. We are grateful to Higher Education Commission, Pakistan for funding this research.