The evidence thus suggests that apples have a health-promoting effect on the rat intestinal microbiota, and that this effect is mainly explained by the presence of pectin in the apples. However, there are lots of cautions to be taken when extrapolating data from animal experiments

to humans, and it should be kept in mind that rats Volasertib manufacturer metabolize the ingested apple components differently from humans. The data presented here will at a later stage be interpreted in the context of other biological changes recorded during the course of the ISAFRUIT project, which includes also human intervention studies. Methods Animals and housing Male Fischer 344 rats (5-8 weeks old) were obtained from Charles River (Sulzfeld, Germany). The animals were housed two by two in standard cages. During the

study the temperature was maintained at 22 ± 1°C and relative humidity at 55 ± 5%, air was changed 8-10 times per hour, and light was on from 9.00 to 21.00. Diets and acidified water (adjusted to pH 3.05 by citric acid to prevent growth of microorganisms) were provided ad libitum. During dosing with 1,2-dimethylhydrazine C646 cell line dihydrochloride (DMH) and 1 week thereafter, the animals were kept in flexible film isolators (Isotec 12134, Olac, Oxford, UK). Animal experiments were carried out under the supervision of the Danish National Agency for Fer-1 mouse Protection of Experimental Animals. Apple products The apples and apple products (Shampion cv. supplied by Institute for Pomology, Skierniewice, Poland)

used in this study were standardized and all GBA3 originated from the same harvest. Whole apples were cut in slices and the seeds were removed before serving to the rats. The exact contents of soluble solids and pectin in each of the products were known (Table 4). Obipektin A.G., Bischofszell, Switzerland, kindly provided the apple pectin. Table 4 Content of soluble solids and pectin in the different apple fractions Material Soluble solids (%) Unit Total pectin Water-soluble pectin Whole Fruit 12.8 g/kg 4.551 0.932 Apple purée 14.5 g/kg 4.707 2.626 Cloudy apple juice 13.0 g/l 0.379 0.379 Clear apple juice 13.5 g/l * * Pomace dried – g/kg 64.9 25.7 *Pectic substances are removed during clarification and ultrafiltration Diets and experimental design Experiment A 64 rats were randomized (by bodyweight) in four groups of sixteen animals. After one week (Week 1) of adaptation to a control diet, two groups of animals were fed the same control diet, while two other groups were fed the control diet added 10 g raw whole apple for a period of 14 weeks until euthanization. During Week 4-7, one of the control diet-fed groups and one of the apple-fed groups received by gavage 20 mg/kg bodyweight of DMH once a week (4 doses in total). Experiment B 112 rats were randomized (by bodyweight) in seven groups of sixteen animals.