Purification and characterization of aflatoxins produced by indigenous aspergilli under optimized conditions

Abstract

MOLECULAR APPROACHES FOR CHARACTERIZATION OF AFLATOXIN PRODUCING ASPERGILLUS FLAVUS ISOLATES FROM POULTRY FEED Aflatoxins are secondary toxic metabolites produced by aspergilli. Aspergillus flavus is one of the major aflatoxins producing specie. Present study was conducted to enumerate mycoflora of poultry feed and aflatoxin production potential of A. flavus. Home mixed and commercial poultry feed (n=20, each) were processed for determination of fungal load and isolation of mycoflora. Isolates were identified by culture and microscopic characters. Thin layer (TL) and high-performance liquid chromatography (HPLC) were used for screening, identification and quantification of aflatoxins produced by A. flavus respectively. A. flavus were confirmed by specie specific polymerase chain reaction (PCR). Isolation frequency of different genera, Aspergillus species and toxigenic A. flavus was calculated. The fungal count in home mixed feed was 2x102 to 1.6x104 CFU/g whereas, in commercial poultry feed from 2x101 to 6x103 CFU/g. Aspergillus was the most prevalent genus in home mixed and commercial feed followed by Mucor. Among aspergilli, the highest percentage was of flavus (95%) followed by A. niger (75%), A. fumigatus (15%) and A. terreus (5%). A total of 32.61 percent (223/685) aflatoxin producing A. flavus from commercial and 16.67 percent (23/140) from home mixed feed were detected by TLC. These aflatoxins (AFs) were identified as AFB1 and AFB2 and AFG1 by HPLC. Amplicon (500 bps) of A. flavus was observed on 2 percent agarose gel. It was concluded that poultry feed may be a source of transmission of disease producing fungi and aflatoxins to poultry birds and human beings. Key words: Aflatoxin Aspergillus flavus commercial poultry feed High performance liquid chromatography Home mixed poultry feed Polymerase chain reaction

ENZYMATIC AND AFLATOXIN PRODUCTION POTENTIAL OF ASPERGILLUS FLAVUS

Fungi especially Aspergillus species are potential candidates for production of mycotoxins and industrially important enzymes. Aspergillus flavus isolates (129) recovered from soil mixed with animal rations (n=145) had Aflatoxins (17.82%) and Enzymes (10.37%) production potential. Quantity of detected Aflatoxins varied for different isolates i.e., 3.25 to 11622.24ng, 21.34 to 194.47ng and 3.36 to 40.12ng per mL of Sabouraud‟s dextrose broth in case of AFB1, AFB2 and AFG1 as determined by High performance liquid chromatography. Optimization of non-toxigenic starch hydrolyzing A. flavus was carried out at different incubation temperatures (22, 30 and 37°C), pH (4.5, 6 and 7.5) and substrates including maize, wheat bran and rice husk (1, 3 and 5% each) for incubation period of 7 days. In optimization experiments for starch hydrolysis, most of the A. flavus (86%) produced highest enzyme (IU) at 37°C and pH 6 quantified by Dinitrosalicylic method. Maximum isolates were able to produce enzymes using rice husk followed by maize. The maximum enzyme production by A. flavus was 179.88+1.71IU using one percent of maize at pH 6 and 37°C. It was concluded that indigenous A. flavus can be used in food industry as biological source of starch hydrolyzing enzymes.