Effectiveness of Density Segregation and Sodium Carbonate Treatment-on the Detoxification of Fusarium-Contaminated Corn Fed to Growing Pigs''

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1 J Sci Food Agric 1995,68, Effectiveness of Density Segregation and Sodium Carbonate Treatment-on the Detoxification of Fusarium-Contaminated Corn Fed to Growing Pigs'' Roland G RotterJ Barbara A Ratter,$ Brian K Thompson, Dan B Prelusky and H Locksley Trenholm Centre for Food and Animal Research, Research Branch, Agriculture and Agri-Food Canada, K W Neatby Building, Ottawa, Ontario, Canada, K1A OC6 (Received 10 July 1994; revised version received 10 January 1995; accepted 22 February 1995) Abstract: The effectiveness of using rinsing, density segregation or 0.1 M sodium carbonate treatments of Fusarium-contaminated corn containing deoxynivalenol (DON) and zearalenone were tested both chemically and by feeding the material to growing pigs. Uninfected and Fusarium graminearum-inoculated corn (containing 40.3 mg DON and 0.94 mg zearalenone kg-' corn) were subjected to various treatments: no treatment (control), rinsed, rinsed and all floating material removed, soaked in 0.1 M sodium carbonate for 24 h or soaked in 0.1 M sodium carbonate for 24 h after removal of the floating material. All corn samples were analysed for DON and zearalenone. Rinsing the contaminated corn reduced the concentrations of both mycotoxins by 44% while soaking in 0.1 M sodium carbonate reduced the toxin concentrations an additional 35%. In both cases, removal of the floating material decreased concentrations another 8 and 4%, respectively. In the feeding study, 10-week-old barrows were fed diets containing 17.5% of the treated corn samples over a 21 day period. Daily feed consumption and weight gains were significantly improved in pigs fed the rinsedminus-floating corn and the sodium carbonate-soaked corn with and without floating material compared to the untreated contaminated corn. The chemical and biological data demonstrated that rinsing combined with the removal of floating material or soaking in 0.1 M sodium carbonate with or without removal of floating material were effective in reducing the toxicity of the contaminated corn. Due to a higher energy requirement needed to dry the corn soaked in sodium carbonate, the combination of rinsing and removal of the floating material is recommended as a more general and practical method of detoxification of contaminated grain. Key words: detoxification, Fusarium, corn, density segregation, sodium carbonate, swine. * CFAR Contribution No $ Present address : Toxicological Evaluation Division, Bureau of Chemical Safety, Food Directorate, Health Protection Branch, Health Canada, Ottawa, Ontario, Canada, KIA OL2. 8 To whom correspondence should be addressed J Sci Food Agric /95/%09.00 Crown Copyright (Canada). Printed in Great Britain

2 332 R G Rotter et a1 INTRODUCTION Deoxynivalenol (DON, vomitoxin) and zearalenone (ZEN) are naturally occurring secondary fungal metabolites called mycotoxins that contribute to feed and food contamination. Produced by Fusarium fungi, these two toxins commonly occur together. Nutritional and toxicologic studies have shown that swine are sensitive to the adverse effects of both DON and ZEN, as reflected in poor growth and a variety of reproductive problems, respectively (Trenholm et a1 1984, 1988; Friend et a1 1990; Rotter et a1 1994). Reducing the toxic effects of mycotoxins is of major interest to the agricultural industry. A number of approaches have been taken to detoxify mycotoxins, but only a few have had practical application. Ideally, a method should reduce the amounts of the toxins to safe levels, while not creating new toxic compounds nor reducing the nutritional value of the treated commodity (Young 1985). In addition, the method should be simple, inexpensive and, if possible, employ readily available technology. A considerable research effort has been directed towards decontamination techniques for aflatoxin. Although a variety of chemical approaches has been tested (Huff 1980; Huff and Hagler 1982; Clavero et a1 1993; Tabata et a1 1994), to date only ammoniation has actually been successful on a practical basis (Park et a1 1988). Detoxification of grain contaminated with DON and ZEN has been recently reviewed (Hagler 1991 ; Scott 1991 ; Charmley and Prelusky 1994). Physical methods of decontamination include: sieving and dehulling (Trenholm et a1 1991), density segregation of contaminated from noncontaminated kernels (Huff and Hagler 1985; Babadoost et a1 1987; Hagler 1991) and food-processing practices such as milling (Scott et a1 1984), cleaning and washing (Scott et a1 1983) and baking (Abbas et a1 1988). Chemical treatments for decontamination of DON-contaminated grain on a small experimental scale include application of ozone, chlorine and ammonia (Young 1986), sodium bisulphite (Young et a1 1987) and sodium carbonate (Trenholm et a1 1992). Detoxification of ZEN was also included in several studies. Formaldehyde (Bennett et a1 1980) and calcium hydroxidemonomethylamine (Ca(OH)2-CH,NH2) (Bauer et a1 1987) both reduced ZEN levels by 85%. In most cases, a single method of decontamination has been tested, but a combination of physical/chemical treatments might prove to be more effective. Although many physical and chemical detoxification procedures have been utilised with Fusarium-contaminated grain, very few have been tested by feeding the treated material to animals. Young et a1 (1987) showed that autoclaving contaminated corn in the presence of sodium bisulphite reduced DON concentration with no adverse performance effects on feeding the treated corn to growing pigs. Bauer et a1 (1987) confirmed reduction of ZEN by testing the material with female pigs. The present study was undertaken to test the effectiveness of a density segregation method in combination with sodium carbonate treatment, by feeding the material to growing pigs. MATERIALS AND METHODS Treatment of corn samples Each of five treatments was applied to approximately 500 kg of clean corn, and corn experimentally inoculated with Fusarium graminearum, both of which were grown at the Centre for Food and Animal Research (CFAR) Greenbelt Farm in The treatments were as follows: (i) no treatment oven-dried for 24 h at 50 C in aluminium roasting pans (Dried); (ii) rinse (defined as a complete immersion of material) in tap water for approximately 5 min, all the water drained off and all material oven-dried for 24 h (H20 Rinse); (iii) rinse in tap water for approximately 5 min, removal of all floating material (F), draining the water and oven-drying the remaining material for 24 h (H20 Rinse-F); (iv) the material was soaked in 0.1 M sodium carbonate ph 11.3 for 24 h and handled subsequently as described under (ii) (Sod Carb Soak); and (v) the material was placed in 0.1 M sodium carbonate, floating material was removed and the remaining material was soaked for 24 h and handled subsequently as described under (iii) (Sod Carb Soak-F). Prior to oven-drying, corn samples subjected to treatments (iv) or (v) were rinsed once with tap water to remove residual sodium carbonate. In treatments (ii)-(v), inclusive, the proportion of corn to immersion solution was 1 : 2 (w/w). After drying to an average 93.5% DM, the corn samples were ground using a Sprout-Waldron model 105-A grinder (Muncy, AP) to a particle size of approximately 0.74 mm. Diet formulation All pigs were fed a common basal diet formulated to meet National Research Council requirements (Rotter et a1 1992, see Table 1, footnote) that included either the control or contaminated corn samples at 175 g kg-' of diet. This level was used to ensure that the contaminated diet (dried only corn) contained approximately 7 mg DON kg- '. The original pre-treated contaminated corn contained mg DON kg-' (see Table 1). Experimental design and procedures Pure-bred Yorkshire barrows of approximately 10 weeks of age (14.3 kg mean body weight) were obtained

3 DetoxiJication of Fusarium-contaminated corn 333 from a barrier-maintained, minimal disease swine facility. The pigs were group-housed for 7 days before being allocated to individual pens for an additional 7 days adaptation period. Throughout this 14 day period, all animals were fed a common diet (Ottawa Pig Grower). Feed and water were provided ad libitum at all times. The experiment was conducted in two separate, but identically set-up, trials arranged in randomised complete block designs using a total of three pigs per treatment (in each trial). One mix of each diet was used in both trials. The pigs were weighed on days 0, 2, 4, 7, 14 and 21 of the experimental period. The mean starting and finishing body weights were 18.2 and 31.0 kg, respectively. All pigs were sacrificed at the end of the experimental period, and their stomachs removed for examination and grading for colour, thickness and inflammation (Rotter et a1 1992). Analyses The chemical composition of all diets was determined according to standard AOAC (1980) procedures (crude protein, method number 7.015; dry matter, method number 7.007; ether extract, method number 7.055; and ash, method number 7.009). All corn samples and complete diets were also analysed for DON (Trenholm et a1 1985) and zearalenone (Trenholm et a1 1981) using the modified procedures described by Rotter et al (1992). Blood samples (EDTA-tubes) were collected from cranial vena cava at the end of the experiment and used to measure haematocrit, platelets and total leukocyte count (QBC@ V Hematology Analyzer, Becton Dickinson, Franklin Lakes, NJ, USA). The experiment was set up using a randomised complete block design. All performance data (average daily feed consumption (DFC), average daily weight gain (DWG), and feed efficiency (FE)) and stomach scores were analysed using the analysis of variance (Agriculture Canada software number S022-RPS) according to principles outlined by Snedecor and Cochran (1980). RESULTS AND DISCUSSION Mycotoxin analysis of the corn samples showed that each of the three components of the decontamination procedures (rinsing, removal of floating kernels and soaking) was effective in reducing DON and ZEN concentrations to varying degrees (Table 1). Rinsing the contaminated corn in water substantially reduced (44-50%) concentrations of both toxins, while removal of floating kernels reduced the DON concentration by an additional 4-8%. The substantial reduction in toxin concentration suggests the presence of surface contamination and loss of fine particles during the removal of corn from the water. The floating material removed from the H,O Rinse-F and Sod Carb Soak-F samples contained (mg kg-'): DON, and 273.0, respectively, and ZEN 7-63 and 7.51, respectively. Soaking the corn in 0.1 M sodium carbonate further reduced the DON concentration by 35% as compared to the material only rinsed in water, while removal of the floating kernels again reduced the contamination by an additional 4%. The differences in DON and ZEN concentrations between H,O Rinse and Sod Carb Soak treatments were probably due to soaking the material in sodium carbonate which allowed additional time for the toxins to leach from the grain (Trenholm et a1 1992). Some decomposition of the toxins was also possible. Trenholm et a1 (1992) showed that soaking of contaminated corn in 0.1 M sodium carbonate for a longer (72 h) period decreased DON concentration by 95% and ZEN by 87%. They also reported that the eficiency of DON and ZEN removal was increased from 65 to TABLE 1 Concentrations of DON and ZEN in contaminated corn and in naturally contaminated diets" Treatment Contaminated corn Control diet (C) Contaminated diet (NC) DON (mg kg-') ZEN (mg kg-') DON (mg kg-') ZEN (mg kg-') DON (mg kg-') ZEN (mg kg-') Dried only H,O Rinse H,O Rinse-F ND 0.30 ND 3.28 ND Sod Carb Soak ND 242 ND Sod Carb Soak-F ND 1.63 ND All values are presented on a dry matter basis. All diets contain 175 g of treated clean or contaminated corn per kg. The basal diet consisted (per kg) of 514 g corn, 250 g barley, 170 g soya bean meal, 13 g limestone, 18 g dicalcium phosphate, 5 g iodised salt, 20 g fat, 5 g mineral mix, 5 g vitamin mix (Rotter et a1 1992). On average diets contain 177 g protein, 892 g dry matter, 51.6 g ash and 50 g fat per kg. Clean corn contained 0.03 mg kg- ' of DON and ZEN, respectively. F, floaters.

4 3 34 R G Rotter et al 72% and from 61 to 87%, respectively, by using a 1 M sodium carbonate wash followed by two washes with water as compared to three washes with water only (each wash being for 30 min). Long soaking, however, reduces the commercial application of this procedure because of additional washing and drying costs. In contrast, a short rinsing procedure can quickly lower the mycotoxin concentration to an acceptable level, and at the same time reduce the drying time. Babadoost et a1 (1987) tested density segregation using water and a sodium chloride solution (300 g litre-') on grain sorghum naturally contaminated with DON and ZEN. Floatation in the sodium chloride solution reduced the DON and ZEN concentrations by 64 and 84%, respectively. Huff and Hagler (1985) showed that removal of DON-contamined corn kernels with water and then with sucrose (300 g litre-') (d - 1.1) reduced the DON concentration by 53% and 77%, respectively. Although chemical analysis of treated samples of contaminated feedstuffs may show significant reductions in toxin concentrations, the species to be fed the treated feedstuffs are the true indicators of successful detoxification. Rotter et al (1990) reported a 50% reduction in detectable ochratoxin A in barley ensiled for 28 days, but no reduction in toxicity was observed when fed to growing chicks. In the present study, the DON and ZEN concentrations were sufficiently reduced by detoxification/dilution that the corn could be used in a study with growing pigs. The actual dietary concentrations of DON and ZEN measured are shown in Table 1. The DON and ZEN concentrations seen in the control diets were derived solely from standard 'clean' constituents of the diet. Analysis of the performance of the current data showed that there were diet x treatment interactions (P < 0.01) for DFC and DWG throughout the experiment, but there was no effect on FE (Table 2). These interactions reflect the differences in response pattern between control and contaminated diets, that is, the response to the different processing treatments with the contaminated diet. Pigs fed the control corn diets had similar DFC and DWG, regardless of the treatment used. In contrast, rinsing the material in water, removal of the floating material and/or soaking the contaminated corn in sodium carbonate solution resulted in improved DFC and DWG compared to the dried-only contaminated corn. Better improvements were seen when floater-removed contaminated diets were fed compared to their respective treatments where floaters were not removed. Over the 21 day study, DWG results were almost double that seen in pigs fed the contaminated dried-only diet, and only slightly lower than control-fed animals. Soaking contaminated corn for 24 h also resulted in a higher DWG. The inverse linear relationship between DFC and DWG and dietary DON indicated that the feed intake and weight gain were associated with the concentration of DON in the diet. If other toxins or dietary factors were involved, the detoxification treatment (floatation, soaking) affected all factors in a manner similar to DON. Previous studies by Young et al (1987) have shown that DON levels in infected corn can be reduced by autoclaving the material with sodium bisulphite. A 7 day feeding trial conducted subsequently showed that TABLE 2 Mean DFC, DWG and FE of swine fed control (C) and naturally contaminated (NC) diets Treatment DFC (kd DWG (kd FE Days 0-7 Days 0-21 Days 0-7 Days 0-21 Days 0-7 Days 0-21 C NC C NC C NC C NC C NC C NC Dried only H2O H,O Rinse-F Sod Carb Soak Sod Carb Soak-F SEM Factor df Analysis of variance: mean squares Diet (D) *** 2.101*** 1.426*** 0,492*** 0.667*** 0.006' Treatment (T) ** 0.179** 0.111** 0.028* 0.117" DxT *** 0.212**, 0.118*** 0.065*** Error P = 0.068; P =

5 Detoxijication of Fusarium-contaminated corn 335 pig's feed intake and body weight gain improved compared with pigs fed untreated inoculated corn and were similar to controls. Similarly, Bauer et a1 (1987) confirmed detoxification of T-2 toxin, diacetoxyscripenol and zearalenone with calcium hydroxide monomethylamine using biological assays such as skin irritation and chick embryo tests and feeding trials with female pigs. Of the blood parameters measured, only the haematocrit (P < 0.002) and platelet counts (P < 0.01) were significantly different for the type of basal diet. The average haematocrit counts were and 38.39% (SEM = 0.36), while the platelet counts were and x lo9 litre-' (SEM = 12.8) for the control and contaminated diet-fed pigs, respectively. The only differences in stomach grading parameters in the current study were noted in the appearance of the fundic region (P < 0.06). The fundic appearance was scored using a scale 1 (smooth) to 7 (deeply corrugated) with the midrange value being 3.5 (wrinkled). The difference in fundic region resulted primarily from presence (4.1) or absence (2-7) of floaters and was observed for both clean and contaminated diets. As indicated previously the removed floaters were heavily contaminated with DON and ZEN. Previously, Rotter et al(1992) reported much healthier appearances of stomachs of pigs fed DON-contaminated diets compared to control, as evidenced by reduced erosion of and increased folding of the esophageal region. There are several possible factors responsible for the difference between the current and the previous study. First, the corn and barley used in the current study were ground through a 0.48 cm screen, whereas Rotter et a1 (1992) used a 0.32 cm screen. Patience and Thacker (1989) suggested that using a fine grind of cereals in the diet is responsible for a high degree of ulceration in pigs. Secondly, the better esophageal appearance might be a result of a reduced feed intake, while differences in fundic appearance result from ingestion of contaminated diet. Rotter et a1 (1994) observed previously that when a pair-fed treatment was included in the experimental protocol to enable examining the effects of differences in feed intake, both diets (contaminated and pair-fed) showed a better stomach appearance with differences in the fundic region (contaminated diet = 4.0; pair-fed = 2-6). The present study showed that a quick rinse procedure of contaminated corn combined with removal of floating material can substantially reduce the DON and ZEN concentration. The method is simple enough to be used at the farm level. Although absolute decontamination is never possible, toxin concentrations were reduced to levels low enough to consider using the material in diets for growing pigs. This approach would reduce the overall drying cost of the material. Although soaking the contaminated corn in sodium carbonate solution resulted in a further decrease in concentration (increased leaching and/or degradation of toxin), this approach would reduce the usefulness of grain for storage. This procedure, however, could be used for other types of feeding systems (liquid feeding), processing such as wet milling of grain or for mycotoxincontaminated grain used in ethanol fermentation. The effectiveness of all treatments was confirmed in the animal study. ACKNOWLEDGEMENTS This paper is dedicated in memory of R M Warner whose ideas contributed to this research. The authors thank S M Croteau, S Clarkin, L Robinson, Dr K Hartin, N Breedyk and S Nelson for their valuable technical assistance. The authors acknowledge financial assistance from the Ontario Ministry of Agriculture and Food, Agricultural Research Institute of Ontario and Ontario Pork Producers Marketing Board (ARIO Project no OP1001). 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