Genotoxicity testing of extracts from aflatoxin-contaminated peanut meal, following chemical decontamination.

One of the most important concerns in the decontamination of aflatoxin-containing feed commodities is the safety of the products for food-producing animals and for human consumption of products derived from these animals. A new method, based on the use of florisil and C18 solid phase extraction columns, was developed for the preparation of extracts from decontaminated peanut meal, which allowed testing with in vitro genotoxicity assays without interference of the residual aflatoxin B1. Recovery of degradation products in the extracts was evaluated by the use of radiolabelled [14C]-aflatoxin B1 (AFB1) added to naturally-contaminated peanut meal (3.5 mg AFB1/kg). The meal was treated by a small-scale version of an industrial decontamination process based on ammoniation. Following decontamination, more than 90% of the label could not be extracted from the meal. AFB1 accounted for about 10% of the radiolabel present in the extractable fraction, indicating a total AFB1 reduction of more than 99%. Decontamination of the meal by a number of other small- and industrial-scale ammonia-based processes resulted in similar efficiencies. Application of the extraction procedure resulted in AFB1-rich and AFB1-poor fractions, the latter containing half of the extractable decontamination products but less than 1% of the residual AFB1. Testing in the Salmonella/microsome mutagenicity assay (TA 100, with S9-mix) of the original crude extracts and AFB1-rich fractions prepared from non-treated and decontaminated meal, showed the positive results expected from the AFB1 contents as determined by HPLC analysis. Analysis and testing of the AFB1-poor fractions showed that the various decontamination processes not only resulted in a successful degradation of AFB1 but also did not produce other potent mutagenic compounds. Slight positive results obtained with these extracts were similar for the untreated and treated meals and may be due to unknown compounds originally present in the meal. Results obtained with an unscheduled DNA synthesis (UDS) and Comet assay with rat hepatocytes supported this conclusion. Positive results obtained with the micronucleus assay, using immortalized mouse hepatocytes (GKB), did not clearly reflect the mycotoxin levels and require further examination. It is concluded that the newly developed extraction procedure yields highly reproducible fractions and hence is very suitable for examining the possible formation of less potent degradation products of aflatoxins in short-term genotoxicity tests.

Absorption, distribution and excretion of aflatoxin-derived ammoniation products in lactating cows.

Peanut meal naturally contaminated with 3.5 mg/kg aflatoxin B1 (AFB1) was spiked with radiolabelled AFB1 (meal 14C-I0) and decontaminated by a small-scale copy of an industrial ammoniation process (meal 14C-I1). During the process 15% of the radioactivity was lost, whereas 90% of the remaining radiolabel could not be extracted from the meal. In the extractable part, AFB1 accounted for 10% of the radiolabel, consistent with a total AFB1 reduction of more than 99%. No degradation products were observed in the extracts. Four lactating cows were fed with a diet containing 15% of either meal 14C-I0 or 14C-I1 for 10 days. On day 9 of this treatment, respectively 23 and 67% of the radiolabel was excreted in the urine and faeces of cows fed meal 14C-I0, as compared with 2 and 101% in the case of cows fed meal 14C-I1. Milk contained respectively 1.35 (meal 14C-I0) and 0.25% (meal 14C-I1) of the radiolabel. Milk samples taken during the equilibrium stage contained respectively 5 and 0.5 ng/ml of AFB1-derived compounds. Aflatoxin M1 (AFM1) accounted for 50-80% of these compounds in the case of milk from cows fed 14C-I0, as compared with 6-20% in the case of 14C-I1. AFB1 to AFM1 carry-over rates for 14C-I0 or 14C-I1 were estimated to be respectively 0.5 and 5.9%. Only liver and kidney samples contained detectable levels of the radiolabel, being respectively 260 and 37 micrograms/kg for cows fed meal 14C-I0, and 10 and 3 micrograms/kg for those fed meal 14C-I1. In the latter case, more than 55% of the radiolabel in the liver could not be extracted, as compared with 90% in the group fed meal 14C-I1. A small part of the extractable radiolabel in the livers of cows fed meal 14C-I0 could be attributed to AFB1 and AFM1 (less than 1% of total radioactivity). In the case of the animals fed 14C-I1 there were indications for the presence of AFB1 and AFM1 (6% of total radioactivity). Decontamination of the highly contaminated (non-radiolabelled) peanut meal by two different industrial ammoniation processes, resulted in a similar reduction of the initial AFB1 levels of 3.5 mg/kg to 15 micrograms/kg. Feeding of diets containing 15% of the non-treated and two treated peanut meals to cows for a period of 10 days, resulted in AFM1 levels in milk of respectively 2.1, 0.04 and 0.07 ng/ml. AFB1 to AFM1 carry-over rates were calculated to be respectively 0.5, 2.0, and 3.6%. It is concluded that the efficient reduction of aflatoxin levels by ammoniation of contaminated peanut meal results in a strong reduction of aflatoxin-related residues in milk and meat of cows, most likely caused by a decreased bioavailability of the degradation products.

Involvement of small intestinal motility in blood glucose response to dietary fibre in man.

Three dietary fibres with different physicochemical properties were studied in healthy humans for their effects on small intestinal motility and postprandial hyperglycaemia. Duodeno-jejunal motor activity was evaluated electromyographically for 180 min in six subjects who had ingested a test meal composed of glucose alone or glucose with 15 g of wheat bran (WB), sugar beet (SB) or ispaghula (I) fibres. Glucose and insulin concentrations were determined during the same period. Each subject received each of the four test meals randomly during a 4 d period. Addition of SB or I to the glucose meal altered duodeno-jejunal motility. Both of these fibres inhibited stationary contractile activity and increased the propagation length and velocity of propagated activity, whereas addition of WB had no effect. These results could reflect the high water-holding capacity of SB and I. Blood glycaemic response to the glucose meal was reduced by SB and I but remained unchanged with WB. Postprandial blood glucose levels were significantly correlated with the total motility index (r 0.82) and stationary activity (r 0.79). Taken together, these observations suggest that the contractile activity induced by dietary fibre in the small intestine probably plays a major role in delayed glucose absorption.

Role of viscous guar gums in lowering the glycemic response after a solid meal.

The aim of the present study was to investigate how guar gum viscosity acts on starch digestion and glucose absorption in humans. Six healthy subjects received a mixed diet composed of 60.4% carbohydrate in the form of maize glucose or pregelatinized starch, to which was added 5.6% low- or high-viscosity guar gums. Meals were ingested or instilled in the duodenum and postprandial insulin and glucose responses were monitored for 3 h. Infusion of meals containing glucose showed that the delay in the diffusion rate to the duodenal mucosa due to bolus viscosity was not significant. Infusion of meals containing starch showed that a decrease in the digestion rate of starch in the upper small intestine accounted for part of the effect of viscosity on glycemic response, whereas the main effect of guar gum was apparently to slow gastric emptying.

Sugar composition of dietary fibre and short-chain fatty acid production during in vitro fermentation by human bacteria.

The aim of the present study was to assess the relationship between the disappearance of dietary fibre sugars and the production of individual short-chain fatty acids (SCFA). The bacterial degradation of five dietary fibres whose sugars were quantified was investigated in vitro using a human faecal inoculum. Involvement of the main fibre sugars in SCFA production was evaluated by a stepwise multiple linear regression. The results show first that the nature and chiefly the associations between the fibre sugars were key variables in the fermentability. Second, the nature and the amounts of SCFA produced were closely related to the in vitro fermentation of the main sugars available: uronic acids seemed to be principally involved in the production of acetic acid whereas the production of propionic acid could be promoted by the fermentation of glucose and, to a lesser extent, by that of xylose and arabinose. Xylose tended to have a greater impact than uronic acids and glucose on the production of butyric acid. Thus, it would be possible to predict which SCFA could be specifically produced during the fermentation of a fibre, as far as the chemical composition and structure of this fibre are known.

Alterations of intestinal microflora by antibiotics. Effects on fecal excretion, transit time, and colonic motility in rats.

The effects of intragastric antibiotics in rats were examined on fecal microflora and excretion and through transit time and cecocolonic myoelectric activity. A solution of nonabsorbable antibiotics infused into the stomach for 20 days had a dramatic effect on the quantity, composition, and bacterial content of rat feces. Both the dry weight and the water content of feces were increased. The amount of short-chain fatty acids in the feces was dramatically lowered. However, neither total nor cecocolonic transit time of solids was affected. The cyclic organization of cecocolonic myoelectric activity was altered by antibiotic treatment, and the motility index, ie, the quantity of myoelectric activity recorded on the colon, progressively increased. An infusion of short-chain fatty acids modified this motor pattern but did not restore activity to a level comparable to that of control animals. In conclusion, intragastric antibiotics dramatically reduced intestinal microflora and increased fecal excretion of dry matter and water but did not affect the transit time of solid gut contents, although they did influence cecocolonic motility.

Alpha-amylase (EC 3.2.1.1) susceptibility rather than viscosity or gastric emptying rate controls plasma responses to starch in healthy humans.

The relationship between starch alpha-amylase (EC 3.2.1.1) susceptibility, plasma responses and gastric emptying rates has been investigated in humans. Nine randomly chosen healthy subjects were given three carbohydrate test meals (25 g starch or equivalent glucose units): two maize starch pastes with (a) 240 (S24) or (b) 500 (S50) g amylose/kg, and a glucose solution (GS). At 30 min, in vitro starch alpha-amylolysis was 48 (SD 4)% for S24 and 35 (SD 4)% for S50. Test meals differed in viscosity (mPa x s: S24, 54,000; S50, 190; GS, 4). Carbohydrates were labelled with 99mTechnetium and isotope gastric emptying was measured by external gamma counting. Carbohydrate isotopic gastric emptying patterns were exponential. Half gastric emptying time (min) was significantly (P less than 0.05) shorter for S50 (19(SD 2] than for GS (26(SD 2] or S24 (29(SD 2]. No correlation was found between half gastric emptying time and plasma response values. Values for peak insulin (pmol/l) above fasting were significantly (P less than 0.05) different: GS, 306 (SD 11); S24, 227 (SD 11); S50, 187 (SD 11). It is concluded that alpha-amylase susceptibility of the test carbohydrates is a determining factor in the insulin response of healthy subjects, while viscosity of the test meals and gastric emptying rate have no effect.

[Aflatoxin inactivation after ammonia treatment. In vitro studies on detoxified peanut meals]. / Inactivation des aflatoxines par traitement a l'ammoniac. Etudes in vitro de tourteaux d'arachide détoxiqués.

The maximum allowable tolerance of aflatoxins in animal feeds is becoming lower and lower, and it is obvious that the fairly high level of aflatoxin B1 found in almost all peanut meals in recent years restricts this protein source for use in the diets of most animal species. Among the different chemical methods for aflatoxin inactivation, treatment by gaseous ammonia under a pressure of 2 to 3 bars, appears a very attractive solution because it may be achieved by a fairly easy and rapid procedure. This treatment markedly reduces--up to 95 p. 100--the aflatoxins content of the meal. An increase in the nitrogen content, mainly in the non protein form, is observed. Ammoniation has no adverse effect on in vitro pepsin digestibility and even improves the sensitivity of the meal towards proteases. It slightly reduces protein deamination in the artificial rumen and nitrogen solubility in a buffer solution at pH 7,5; these effects seem to be favourable for the utilization of the treated meals by rumiinants. The amino acid compostion of the meal is not significantly changed, particularly with regard to total and available lysine. However, cystine undergoes partial destruction; but this loss could be counterbalanced by a supply of synthetic methionine.

["Milk aflatoxin" and transfer of toxicity]. / "Milk aflatoxine" et toxicité de relais

Diet including 20 p. 100 of lyophilised milk produced by Goat who consumes peanut meals containing 1,530, 79 and 54 microng/kg aflatoxins with 1,136, 64 and 54 microng/kg aflatoxin B1, is given to duckling during 23 days. There is no influence of this diet on growth, feed efficiency and liver or kidney histology. In contrast, direct consummation of Peanut meal, with the highest level of aflatoxins, produces 18 p. 100 mortality and characteristic injuries of aflatoxiocsis.