**3. Results**

#### *3.1. Evaluation of Clinical Symptoms of Induced Chronic Colitis in Mice*

Mice exposed to DSS in drinking water (2% *w*/*v*) showed marked clinical symptoms (DAI values) during all experiment, being slightly lower in the first DSS cycle (7 days) (see Figure 2a). Administration of MfB significantly reduced DAI values between 24–67% compared to DSS group, with the exception of the first DSS cycle. MfB-G beverage attenuated DSS derived symptoms during the first cycles (7 and 21 days), reducing DAI values between 40–54% with respect to the DSS group. However, from the third DSS cycle (35 days) onwards, an increase of DAI (1.6 fold compared to the DSS group) was observed. With the aim to evaluate the effect of beverages on specific illness derived symptoms, Figure 2b,c show the evolution of mice weight, stool consistency and presence of blood in feces, respectively. In general, MfB and MfB-G administration did not have any differences in terms of weight loss compared to DSS group during the study, with the exception of MfB-G beverage, where a statistical (*p* < 0.05) higher weight loss at 10 day (20% vs. DSS group) was observed. Both beverages statistically (*p* < 0.05) reduced the presence of blood in feces (4 to 6-fold) during the acute phase of the illness (7 days) with respect to the DSS group (Figure 2c). However, in subsequent DSS cycles, this protective effect was not observed with both beverages and sometimes the incidence of blood in faces increased in the MfB-G mice group (third and fifth cycle). With respect to stool consistency, MfB prevented completely the appearance of diarrhea during the first two DSS cycles (7 and 21 days) and reduced up to 50% their incidence during the third DSS cycle, compared to DSS group, (see Figure 2c). However, after fourth (49 days) DSS cycle MfB did not show differences in the stool consistency in comparison to DSS group, although a slight beneficial effect was observed at the end of the study. On the other hand, MfB-G partially prevented the loss of stool consistency during the second DSS cycle, but this loss rapidly increased in the subsequent DSS cycles after which stool consistency remained constant for the rest of the study. The loss of stool consistency observed in the MfB-G group during the third cycle coincides with the

increase of blood presence in feces. This suggests that the higher acute diarrhea observed is related to colonic epithelium erosion.

**Figure 2.** Effect of plant sterol enriched milk-based fruit beverages without (MfB) or with galactooligosacharides (MfB-G) on clinical symptoms in DSS-induced chronic colitis model. (**a**) Disease activity index (DAI) score after DSS (dextran sulphate sodium) administration in each cycle; (**b**) body weight per mouse during all the experiments; (**c**) stool consistency loss (lines) and presence of blood in feces (bars) at the end of each DSS administration cycle. Bars/markers show the mean ± standard deviation (*n* = 8). Different lowercase letters (a–c) indicate statistically significant differences (*p* < 0.05) among mice groups (DSS, DSS + MfB and DSS + MfB-G) within the same DSS cycle. (\*) Denotes statistically significant differences (*p* < 0.05), compared to the DSS group, at the same day.

#### *3.2. Colon Length Shortening and Presence of Edema*

Figure 3a shows that mice exposed to DSS (6.10 ± 0.28 cm), DSS + MfB (7.20 ± 0.35 cm) and DSS + MfB-G (6.33 + 0.35 cm), suffered a significant shortening (*p* < 0.05) of colon length in comparison to the control (8.53 ± 0.29 cm). DSS + MfB mice showed longer colon (18%) than DSS mice, significantly preventing (*p* < 0.05) the colon shortening induced by DSS. Mice that received DSS showed higher presence of edema in colonic tissue than the control group (39–50 vs. 28 mg/cm, respectively) (see Figure 3b). Administration of beverages did not show any significant (*p* > 0.05) protective effect with respect to edema development. Mice that received MfB and MfB-G showed very similar colon length and tissue edema compared to control, which is according with the absence of clinical symptoms observed on these mice groups.

**Figure 3.** Evaluation of colon length (**a**) and presence of edema (**b**) after administration of plant sterol enriched milk-based fruit beverages without (MfB) or with galactooligosacharides (MfB-G) for 56 days on dextran sulphate sodium (DSS)- chronic colitis induction. Bars show the mean ± standard deviation (*n* = 8). Different lowercase letters (a–c) indicate statistically significant differences (*p* < 0.05) among samples.

**(b)** 

## *3.3. Histopathological Analysis*

As is shown in Figure 4A, control, MfB and MfB-G mice groups (Figure 4a,c,e, respectively) had a morphologically normal colon without signs, or very low levels of leucocyte infiltration. In contrast, DSS group (Figure 4b) suffered a marked inflammation characterized by a loss of epithelial architecture, reduction of number of globet cells and crypts, as well as a strong increase of immune cell infiltration compared to control (Figure 4a), what it was reflected in a high histological score (9/10 points). Treatment with DSS + MfB (Figure 4d) or DSS + MfB-G (Figure 4f) mice showed similar histopathology

alteration compared to DSS group (b), with immune cell infiltration and distortion of crypts, although the epithelial architecture was more preserved resulting in a significant (*p* < 0.05) lower histological score (34 and 10%, respectively) values. However, DSS + MfB-G (Figure 4f) mice showed a higher histological score (25%) and damage in the epithelial architecture, with distortion of crypts and high presence of immune cell in comparison to the DSS + MfB group (Figure 4d).

A

**Figure 4.** The representative photographs of hematoxylin and eosin staining (magnification ×200) (**A**) and the corresponding score (**B**) of colon tissues obtained after administration of plant sterol enriched milk-based fruit beverages without (MfB) or with galactooligosacharides (MfB-G) for 56 days on dextran sulphate sodium (DSS)- chronic colitis induction. Data are expressed as the means ± standard deviation of six mice in each group. Representative images of staining of colon tissues from each (*n* = 3) *p* < 0.05 vs. normal control. Different lowercase letters (a–c) indicate statistically significant differences (*p* < 0.05) among samples.

#### *3.4. Presence of MPO in Colonic Tissue*

Similar to that observed in the histopathological analysis, mice receiving DSS showed high levels of immune cells infiltration, raising from 2 to 3.5-fold MPO levels with respect to control (see Figure 5). Daily administration of beverages (DSS + MfB or DSS + MfB-G groups) failed to attenuate the increase of MPO levels induced by DSS. MfB and MfB-G mice groups had very similar MPO values with respect to the control, suggesting that both beverages apparently do not trigger an inflammatory response in the colon in the absence of colitis.

**Figure 5.** Presence of myeloperoxidase (MPO) levels in colonic tissue after administration of plant sterol enriched milk-based fruit beverages without (MfB) or with galactooligosacharides (MfB-G) for 56 days on dextran sulphate sodium (DSS)- chronic colitis induction. Bars show the mean ± standard deviation (*n* = 8). Different lowercase letters (a–c) indicate statistically significant differences (*p* < 0.05) among samples.
