A Novel Strain of Cellulosimicrobium Funkei Can Biologically Detoxify Aflatoxin B1 in Ducklings
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bs_bs_banner A novel strain of Cellulosimicrobium funkei can biologically detoxify aflatoxin B1 in ducklings Lv-Hui Sun,1† Ni-Ya Zhang,1† Ran-Ran Sun,1 Xin to mitigate the negative effects of aflatoxicosis in Gao,1 Changqin Gu,1 Christopher Steven Krumm2 ducklings. and De-Sheng Qi1* 1Department of Animal Nutrition and Feed Science, Introduction College of Animal Science and Technology, Huazhong Aflatoxins (AF) are secondary fungal metabolites that are Agricultural University, Wuhan, Hubei 430070, China. largely produced by the fungi Aspergillus flavus and 2Department of Animal Science, Cornell University, Aspergillus parasiticus (Diaz et al., 2002). Among the Ithaca, NY 14853, USA. various dangerous AF and their metabolites, aflatoxin B1 (AFB1) is the most toxic mycotoxin, having harmful hepa- Summary totoxic, mutagenic, carcinogenic and teratogenic effects on many species of livestock. It is also classified as a Two experiments were conducted to screen microor- group one carcinogen [International Agency for Research ganisms with aflatoxin B1 (AFB1) removal potential on Cancer (IARC), 1987]. Unfortunately, AFB can easily from soils and to evaluate their ability in reducing the 1 contaminate various types of crops and is a very prevalent toxic effects of AFB1 in ducklings. In experiment 1, we contaminant of maize-based food and feed all over the screened 11 isolates that showed the AFB1 biodegra- world (Wu and Guclu, 2012; Hamid et al., 2013). The feed dation ability, and the one exhibited the highest AFB1 contaminated by AFB can pose serious problems to the removal ability (97%) was characterized and identified 1 health and productivity of livestock and can therefore as Cellulosimicrobium funkei (C. funkei). In experi- cause significantly economic losses (Rawal et al., 2010; ment 2, 80 day-old Cherry Valley ducklings were Wu and Guclu, 2012). divided into four groups with four replicates of five Several physical and chemical detoxification methods birds each and were used ina2by2factorial trial used to control AFB have been to some extent success- design, in which the main factors included adminis- 1 ful, while most of them have major disadvantages includ- tration of AFB1 versus solvent and C. funkei versus ing nutrients loss and high costs, which limited their solvent for 2 weeks. The AFB1 treatment significantly practical applications (Varga et al., 2010; Jard et al., decreased the body weight gain, feed intake and 2011). Thus, scientists have come to favor the biological impaired feed conversion ratio. AFB1 also decreased method, which is utilization of microorganisms and/or serum albumin and total protein concentration, while their enzymatic products to remove AF through microbial it increased activities of alanine aminotransferase binding and/or degradation of mycotoxins into less toxic and aspartate aminotransferase and liver damage in compounds, giving a characterization of specific, efficient the ducklings. Supplementation of C. funkei allevi- and environmentally sound detoxification (Wu et al., 2009; ated the adverse effects of AFB1 on growth perfor- Guan et al., 2011). mance, and provided protective effects on the serum Many studies have shown that AFB can be biologically biochemical indicators, and decreased hepatic injury 1 detoxified by various species of microorganisms, includ- in the ducklings. Conclusively, our results suggest ing fungi, such as Pleurotus ostreatus (Motomura et al., that the novel isolated C. funkei strain could be used 2003), Trametes versicolor (Zjalic et al., 2006), yeast such as Trichosporon mycotoxinivorans (Molnar et al., 2004) and Saccharomyces cerevisiae (Pizzolitto et al., 2013), Received 26 June, 2014; revised 13 October, 2014; accepted 22 and bacteria, such as lactic acid bacteria (Bagherzadeh October, 2014. *For correspondence. E-mail [email protected]; Kasmani et al., 2012; Nikbakht Nasrabadi et al., 2013), Tel. (+86) 27 87281793; Fax (+86) 27 87281033. †These authors con- Stenotrophomonas maltophilia (Guan et al., 2008), tributed equally to this work. doi:10.1111/1751-7915.12244 Myxococcus fulvus (Guan et al., 2010) and Rhodococcus Funding Information This project was supported by the Chinese species (Cserháti et al., 2013). Unfortunately, few of these Natural Science Foundation projects (31072058 and 31272479), Fun- microorganisms, their metabolites and/or degradation damental Research Funds for the Central Universities (2011QC050 and 2013BQ059), and Hubei Provincial Natural Science Foundation products have been utilized in animal feed due to a lack (2013CFA010). of information on the mechanisms of detoxification, the © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 2 L.-H. Sun et al. a Table 1. Ability of AFB1 biodegradation by screened isolates . Cellulosimicrobium funkei T3-5 (C. funkei), which exhib- ited excellent AFB1 biodegradation ability both in vitro and b Isolate AFB1 biodegradation (%) in vivo. Our findings suggested a feasible approach for a T1-1 84.9 ± 4.0 safe and efficient method to control AFB1 levels in the ± T1-2 41.7 4.1 animal feed industry. T1-3 86.3 ± 7.3 T1-4 24.9 ± 3.8 T2-1 75.9 ± 1.8 T3-1 51.5 ± 6.9 Results T3-2 81.2 ± 4.9 T3-3 87.1 ± 4.0 Experiment 1 T3-4 20.4 ± 4.3 T3-5 94.2 ± 2.4 Screening for AFB1 biodegradation microbes. A total of ± T3-6 70.1 4.5 11 strains were isolated from three soil samples by cou- a. Values are expressed as means ± SD (n = 5). marin medium, and all of them showed various degrees of b. Isolates are screened from soil samples using coumarin as the ability to reduce concentrations of AFB1 in the liquid only carbon source. medium after 72 h incubation at 37°C (Table 1), which were calculated from the high-performance liquid chroma- efficiency and stability of detoxification under different tography (HPLC) results. The chromatograms of HPLC oxygen, pH or bile conditions, as well as their potential analysed results were shown in Fig. S2. Among the 11 side-effects needs to be further investigated. screened strains, seven showed the potential of reducing The objective of this study was to screen novel AFB1 AFB1 in the medium over 70%, and the isolate T3-5 was biodegradation microorganisms that could be applied in the most effective strain with an observed 94.2% AFB1 feed industry. Because AFB1 is a furanocoumarin deriva- reduction in the medium (Table 1). tive which has a similar chemical structure to polycyclic aromatic hydrocarbons (PAHs) (Yu et al., 2004; Guan Identification of isolate T3-5. Microscopic morphological et al., 2008), we therefore hypothesized that the microor- results showed that isolate T3-5 is a gram-positive bacte- ganism having biodegradable activity on PAHs maybe rium, which appeared as circular, yellow, a smooth also have the same effect towards AFB1. We therefore surface and an entire edge after 18 h of incubation at chose soil samples around petroleum factories, which 37°C on the Luria Bertani (LB) agar (Fig. S1). Physiologi- were contaminated with PAHs for the screening of the cal and biochemical studies showed that the T3-5 strain microorganisms that could biodegrade AFB1 (Pampanin was able to utilize most oligosaccharides including and Sydnes, 2013). Since coumarin is the basic chemical glucose, maltose, D-xylose, galactose, D-sorbitol, structure of AFB1, along with the relatively safe and inex- D-raffinose and as well as sucrose as a sole carbon pensive characterization, we used medium-containing source. The T3-5 strain could also hydrolyse cellulose, coumarin as the only carbon source to screen AFB1- gelatin and Tween 80, but not amylum (Table 2). The 16S biodegrading microorganisms by replicating the well- rDNA sequencing result of the isolate T3-5 [National established method previously conducted (Guan et al., Center for Biotechnology Information (NCBI) GenBank: 2008). Since duckling is extremely sensitive to the toxic KM032184] showed 99% deoxyribonucleic acid (DNA) effects of AFB1 (Shi et al., 2013), and thus it was used to sequence homology to that of Cellulosimicrobium funkei evaluate the detoxification effects of the isolate in this listed by NCBI (GenBank: NR_042937.1, Fig. 1). Taken study. In this study, we successfully obtained the isolate together, the morphological, physiological, biochemical Table 2. Biochemical and physiological characteristics of C. funkei T3-5. Characteristic Result Characteristic Result Characteristic Result Acid fermentation of: Phosphatidylcholine − 10% NaCl − Glucose + Tween 80 − Acid medium − Maltose + Amylum − Other test: D-Xylose + Cellulose + Motility − Galactose + Xylan − Catalase test + D-Sorbitol − Nitrate reduction + Methyl red test + D-Raffinose + Organic acid + Urease test + Sucrose + Yeast cell − V-P test − Glycerol + Growth on: Oxidase test − Hydrolysis of: 2%, 5% NaCl + Congo red tolerance + Gelatin liquefaction + 7% NaCl (+) +, Positive; −, negative; (+), weakly positive. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology C. funkei biodegradate aflatoxin B1 3 Fig. 1. Neighbour-joining phylogenetic tree based on 16S rDNA gene sequences showing the relationships among the species of the genus Cellulomonas and related specifies. Bootstrap values calculated for 1000 replications are indicated. Bar, 2 substitutions per 100 nucleotides. Accession numbers from Genbank are given in brackets. data as well as the NCBI blast results suggest that the tion of AFB1 and C. funkei or their interactions (Table 3). isolate T3-5 belonged to C. funkei, which is an aerobic and Compared with the control, the final body weight (BW), facultatively anaerobic gram-positive bacterium. The overall daily BW gain and overall daily feed intake of above strain is deposited at China Center for Type Culture ducklings were decreased (P < 0.05) 42%, 49% and 38%, Collection (CCTCC) in Wuhan University, and has a pres- along with increased (P < 0.05) 23% overall feed/gain ervation number of CCTCC NO: M 2013564.