International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 4(5), 31-37, May (2015) Int. Res. J. Biological Sci. International Science Congress Association 31 Effect of extrusion variables (Moisture Content, Barrel Temperature and Screw Speed) on the reduction of aflatoxins in MaizeSohi Sukhman K., Sharma N.S., Bobade Hanuman, Singh Baljit and Sharma SavitaDepartment of Food Science and Technology, Punjab Agricultural University, Ludhiana, 141004, INDIA Department of Veterinary Microbiology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, 141004, INDIA Available online at: www.isca.in, www.isca.me Received 25th February 2015, revised 4th April 2015, accepted 9th May 2015 Abstract Naturally contaminated maize was extrusion processed for reduction of aflatoxins. The contaminated grains collected from different locations were screened for aflatoxins by Pressure Mini Column. The grains were quantified for aflatoxin content (AFB and AFB) by thin layer chromatography. Maize grains were extrusion processed by co-rotating twin-screw extruder using different extrusion variables; feed moisture (24, 27 and 30%), barrel temperature (140, 170 and 200°C) and screw speed (300, 400 and 500 rpm). Response surface methodology was applied to optimize the processing conditions and to assess the effect of extrusion variables on the reduction of aflatoxins. The maximum reduction of AFB1 (75%) and AFB2 (72.5%) in maize with initial concentration of 78 ppb was obtained at 24% moisture, 171 °C temperature and 369 rpm screw speed. Extrusion processed maize with final aflatoxin concentration of 19 ppb was utilized for preparation of porridge and chapatti with good consumer acceptance. Keywords: Aflatoxins, extrusion processing, maize, chapatti, porridge. Introduction Aflatoxin contamination of agricultural commodities and stored grains has long been a major problem throughout the world in tropical and sub-tropical regions, where climate and poor storage conditions are conducive for fungal growth and aflatoxin production. Aflatoxins are secondary fungal metabolites produced by toxigenic species of Aspergillus mainly Aspergillus flavus and Aspergillus parasiticus. About 18 different types of aflatoxins were recognized among which the major ones are aflatoxin B (AFB), B (AFB), G (AFG) and (AFG) distinguished by their fluorescent colour blue or green under ultraviolet light. Aflatoxin B is the most toxic form and possesses hepatotoxic, teratogenic and mutagenic properties, causing damage like hemorrhage, edema, immunosuppression and hepatic carcinoma. Naturally occurring aflatoxins and AFB are classified by the International Agency for Research on Cancer as group 1 carcinogens. Aflatoxins are common contaminants of cereal grains. Maize is the third most important crop in the world after wheat and rice. Unfortunately, aflatoxigenic fungi invade maize during its development and harvest in the field as well as during the process of transport and storage. The estimated value of maize lost to aflatoxin is $225 million per year, out of the $932 million due to all the mycotoxins in the United States. The Food and Agriculture Organization (FAO) estimates that many basic foods could be contaminated with mycotoxin producing fungi, resulting in 1000 million metric tons loss of foodstuffs each year. Based on the regulations of U.S. Food and Drug Administration (FDA), foods are not permitted to exceed the action level of 20 ppb (ng g -1 ) for total aflatoxins. The increasing number of reports pertaining to the presence of aflatoxins in food dictates the need for practical and economical detoxification procedures. Ideally, such detoxification procedures should not only reduce the concentration of toxins below regulatory limits, but also prevent production of toxic degradation products without any reduction in the nutritional value of the treated commodities. Various detoxification procedures ( physical, chemical and biological) were employed in food processing to minimize the presence of aflatoxins in food chain. Extrusion cooking has received great attention as one of the most effectiveand fastest growing food processing operation in reducing aflatoxin levels. Extrusion processing is a high temperature-short time operation in which raw food materials are thermo-mechanically cooked in a screw-barrel assembly by a combination of moisture, pressure and temperature in order to be mechanically sheared and shaped. High temperature, pressure and severe shear forces result in gelatinization of starch, denaturation of proteins, and inactivation of food enzymes and reduction of microbial counts. The present study was undertaken to study the effect of extrusion processing on reduction of AFB 1 and AFB as affected by the moisture content, barrel temperature and screw speed variables as well as to utilize the extrusion processed maize in product preparation (porridge and chapatti) and its quality assessment. Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(5), 31-37, May (2015) Int. Res. J. Biological Sci. International Science Congress Association 32 Material and Methods Screening of maize grains for aflatoxins: Raw maize grains collected from different locations (godowns, mills, grain merchants) of Punjab, India were screened for aflatoxin analysis by Pressure Mini Column. The presence of aflatoxins is observed as a compact blue fluorescent band under long wave UV light (365nm) . Experimental design: Response Surface Methodology (RSM) with Box Behnken design was adopted as a 3-factorial design with moisture content (24, 27 and 30%), barrel temperature (140, 170 and 200°C) and screw speed (300, 400 and 500 rpm) as factors affecting the reduction of AFB and AFB. The design included 17 experimental runs of three extrusion variables and for each experimental run; 500 g of maize was extruded (table-1). Extrusion processing was achieved with Clextral BC 21 co-rotating intermeshing twin screw extruder (Clextral, Firminy, France) . The extruded samples were cooled and stored in plastic bags until analyzed for aflatoxins. Table-1 Effect of extrusion conditions on product responses in maize Extrusion Variables % Reduction A: Moisture (%) B: Temperature C) C: Screw speed (rpm) AFB AFB 24 140 400 52.56 51.26 24 170 300 73.88 71.13 24 170 500 70.26 69.68 24 200 400 79.18 74.66 27 140 300 52.92 51.98 27 140 500 53.85 54.84 27 200 500 70.36 68.7 27 200 300 76.21 75.89 27 170 400 71.11 70.13 27 170 400 72.28 72.17 27 170 400 73.64 73.54 27 170 400 74.45 74.13 27 170 400 75.08 74.33 30 140 400 50.05 48.64 30 170 500 55.77 57.21 30 170 300 58.97 59.35 30 200 400 56.41 57.42 Extraction and quantification of aflatoxins: The extraction of aflatoxins was achieved by Romer’s extraction procedure. The extracted aflatoxins were trapped in chloroform and spotted on silica gel plates along with standards (AFB and AFB) for quantitation by thin layer chromatography (TLC). The developed TLC plates were examined under UV light and aflatoxin concentration was estimated by comparison of the fluorescent intensity of the spots at the Rf (retention factor) value of the toxin in the sample extracts with those of the appropriate aflatoxin standards chromatographed on the same plate10 . AFBand AFB2 concentration in maize before and after extrusion was used to calculate the percentage aflatoxin reduction for all the experimental treatments . Statistical analysis: The linear, quadratic and interactive effect of extrusion variables on the reduction of aflatoxins was represented in the form of polynomial equation: (1) Where Y is the percent aflatoxin reduction; Xi are the variables where X1 - Moisture content (A), X2 – Barrel temperature (B), 3 - Screw speed (C); is regression coefficient of intercept term; , ii and ij are linear, quadratic and interaction regression coefficients, respectively (equation-1). To evaluate the effect of moisture, temperature and screw speed on aflatoxin reduction and to determine the regression coefficients and the statistical significance for the model, analysis of variance (ANOVA) was carried out. The coefficient of determination (R) and lack of fit test was calculated to ascertain the adequacy of the model. The data obtained from the products (chapatti) prepared from extrusion processed maize was analyzed statistically using techniques of ANOVA and least significant difference (LSD) as the test for significance. AFBand AFB2 contents before and after extrusion processing were determined to calculate the aflatoxin reduction percentage for all the experimental treatments . Product preparation: The best extrusion processing conditions were selected on the basis of maximum aflatoxin reduction and quality of the extrudate. Maize processed at these optimized conditions was used for preparation of instant porridge and chapatti. These products were assessed for various quality characteristics and overall acceptability. Instant porridge Preparation: 25 g instant porridge (extruded maize) was boiled with 100 ml milk. Sugar @ 6% was added during the boiling and porridge was cooked till doneness. Porridge was served hot for sensory evaluation. Chapatti Preparation: Maize extrudates were grinded to produce flour of uniform particle size using cemotac mill (Foss, Hoganas, Sweden) having setting at No. 1. The extrusion processed maize flour was added to commercial maize flour at 10, 20 and 30 per cent level. The required quantity of water was mixed manually to obtain dough of suitable consistency. The dough was rounded and kept for half an hour at room temperature. The dough was divided into four equal parts and moulded into circular chapattis of 15.0 cm in diameter with rolling pin and board11. Traditional home baking procedure was followed to bake chapattis on iron plate (Tawa). Chapattis were cooled and comparative evaluation was done using following criteria: Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(5), 31-37, May (2015) Int. Res. J. Biological Sci. International Science Congress Association 33 Characteristic Score grade Dough handlingNon-sticky, Sticky, Slightly sticky, Full sticky Puffing of chapatti Full, Partial, Nil Quality assessment: Water Absorption Index (WAI): The method outlined by Anderson12 was used to determine water absorption index of porridge. WAI measures the volume occupied by the granule or starch polymer after swelling in excess of water. The ground extrudates were suspended in distilled water at room temperature (34 °C ) for 30 min, gently stirred during this period, and then centrifuged at 3000g for 15 min. The supernatant liquid was poured carefully into tared evaporating dish. The remaining gel was weighed and water absorption index was calculated as the grams of gel obtained per gram of solid. Water absorption index (g g-1) = Weight of sediment (g) Weight of dry solids (g) Water Solubility Index (WSI): Water solubility index of porridge determines the amount of free polysaccharides or polysaccharides released from the granule on addition of excess water. The WSI was the weight of dry solids in the supernatant from the water absorption index test described above12expressed as percentage of the original weight of the sample. Water solubility index (%) = Weight of dissolved solid in supernatant (g) × 100 Weight of dry solids (g) Bulk Density: Bulk density of porridge was calculated by measuring the weight of known volume of sample. Samples were poured into a graduated cylinder, gently tapped ten times and filled to 500 ml. Results were expressed as g ml -1 . Bulk Density (g ml-1) = Weight of extrudates (g) Volume of extrudates (ml) Texture: Textural quality of porridge and chapatti was examined by using a TA-XT2i Texture Analyser. For porridge, the compression probe (50 mm diameter, aluminium cylinder) was applied to measure the force required to break the sample which indicates hardness. Hardness of porridge was expressed in Newton (N) keeping 1.0 mm s -1 pre-test speed, 2.0 mm s -1 test speed, 10.0 mm s -1 post-test speed and 5 mm distance13as testing conditions. For chapatti, the force required to cut chapatti was evaluated by using texture analyser14. Strips measuring 4 cm × 2cm were cut from each chapatti. One strip at a time was placed on the center of the sample holder and the blade was allowed to cut the chapatti strip. The force (N) required to cut chapatti strip into two pieces was recorded. The speed was maintained at 1.70 mm s -1 . Viscosity: Viscosity of porridge (cP) was measured using Brookfield viscometer at room temperature with spindle No. 2 at a speed of 60 rpm. Colour Analysis: Colour analysis of the prepared products (porridge and chapatti) was done by using Hunter Lab colorimeter. Readings were displayed as L* (100 for white; 0 for black), a* (+, red; -, green) and b* (+, yellow; -, blue) colour parameters according to the CIELAB system of colour measurement. Sensory Evaluation: Sensory evaluation by panel of judges was conducted for sensory attributes (color, texture, taste, flavor and overall acceptability) for the prepared products using 9- point hedonic scale15. Results and DiscussionThe aflatoxin content in the maize samples ranged between 78 and 140 ppb. Keeping in view the aflatoxins limit of 20 ppb for foods and feed ingredients set by FDA, maize grains with aflatoxin concentration of 78 ppb was selected for extrusion processing. The effect of extrusion processing variables (moisture content, barrel temperature and screw speed) on percent reduction in AFB and AFB in maize was evaluated and the data obtained on aflatoxin reduction from the experiment is summarized in table-1. The experimental data fit the polynomial equation well. This is shown by R value of 0.98 each for AFB and AFB. The models showed non-significant lack of fit with value of 0.45 and 0.59 (more than 0.05) for AFB and AFB, respectively (table-2). The quadratic model obtained from regression analysis for reduction in AFB and AFB was developed in following equation: Table-2 ANOVA and model statistic for product responses in maize Model % Reduction AFB 1 AFB 2 F value 67.51 61.51 Mean 65.70 65.00 SD 1.64 1.63 C V 2.50 2.50 R 2 0.98 0.98 Adjusted R 2 0.97 0.97 Predicted R 2 0.91 0.92 Adequate precision 25.33 22.84 -value for lack of fit 0.45 0.59 SD- Standard deviation, CV- Coefficient of variation, R- Coefficient of determination AFB1 reduction = 73.31 6.83A +9.10B 1.47C 5.07AB +0.10AC 1.69BC 6.19A 7.57B 2.40C2 (2) AFB2 reduction = 72.86 5.51A +8.74B 0.99C 3.66AB 0.17AC 2.51BC 6.69A 8.18B 1.83C (3) As found in the regression equations 2 and 3, temperature had a