**2.1. Animals and diets**

Twenty-four male C57BL/6J mice of 12 weeks old at the beginning of the experiment were obtained from the animal facilities of CINVESTAV-Zacatenco (Mexico). The mice were housed in a temperature and humidity controlled room with a 12 h light-dark cycle. They were divided into three groups (eight mice per group) according to diet. Mice were acclimated for 7 days, having free access to a pelleted 5053 standard diet (Laboratory Rodent Diet, USA) and water. During the experimental period (6 weeks), STD mice group were fed with 5053 standard diet, whereas inulins-RNE and agavins-AAO mice groups received a diet prepared by mixing 90 g of 5053 standard diet with 10 g of Raftiline or fructans from *A. angustifolia* (AAO). All diets were made by Laboratory Rodent Diet and were available *ad libitum* to mice.

#### **2.2. Chemicals**

Agavins were extracted in our laboratory as described by López et al. [10]. Briefly, one hundred grams of milled *Agave angustifolia* stems were extracted twice with 100 ml of 80% v/v ethanol with continuous shaking for 1 h at 55 °C. The sample was filtered and the plant material re-extracted with 100 ml of water for 30 min at 55 °C. The supernatants were mixed; chloroform was used to eliminate the organic fraction. The aqueous phase was concentrated by rotary evaporation under reduced pressure. The sample was dried using a spray dryer and stored in a humidity-free container. RNE were from Beneo Orafti. The average degree polymerization for agavins is 32[11] and for RNE >23.

#### **2.3. Faeces and blood samples**

Faeces collection was performed once a week during the experimental period to evaluate the SCFAs. On day 45, mice were anaesthetized by intra-peritoneal injection of sodium pentobarbital solution. Portal vein blood samples were collected in EDTA tubes; after centrifugation for 10 minutes at 2500 r.p.m., plasma was stored at -80 °C. The concentration of serum triglycerides, cholesterol and glucose was measured using kits coupling enzymatic reaction and spectrophotometric detection of reaction end-products (BioVision).

#### **2.4. pH and SCFAs**

Segments of the caecum and proximal, medial and distal colon were immediately excised. Caecal and colonic contents of each section were collected in tubes and frozen at -80 °C. The pH measurements were made using a microelectrode (PHR-146, Lazar Research Laboratories, Inc.). Analysis of SCFAs was carried out by gas chromatography and flame ionization detection as described by Pietro Femia et al. [49] with some modifications. Briefly, 0.05 g of caecal and faecal contents were acidified with 0.05 ml of sulfuric acid and SCFAs were extracted by shaking with 0.6 ml of diethyl ether and subsequent centrifugation at 14000 r.p.m. for 30 s. One microliter of the organic phase was injected immediately into the capillary column (Nukol) of the gas chromatograph coupled to a flame ionization detector. The initial temperature was 80 °C and the final temperature was 200 °C. Nitrogen was used as carrier gas and the quantification of the samples was carried out using calibration curves for C2:0, C3:0 and C4:0 acids. A standard curve for each acid was done for their quantitation in the samples.

#### **2.5. Statistical analysis**

Results are expressed as mean values with their standard errors of the mean. Statistical differences between groups were evaluated using one-way ANOVA followed by a Tukey test using GraphPad Prism version 5 for Windows. P<0.05 was regarded as statistically significant.
