**1. Introduction**

Ulcerative colitis (UC) is a chronic inflammatory disease, within intestinal bowel diseases (IBD), characterized by a tissue destruction of large intestine with relapsing phases followed by periods of remission [1]. The most common treatment strategies used include pharmacological therapy (steroidal and non-steroidal drugs) or, in the worst cases, removing portions of affected gastrointestinal tract through surgery. Although, pharmacological therapy has shown efficacies in ameliorating the severity and symptoms of IBD, typically does not lead to long periods of remission. Consequently, recent investigations have focused on the study of the impact of diet and healthy foods as potential alternative, and even the possible combination of nutraceuticals and healthy foods with pharmacological therapy in cases when the patient is not eligible for conventional therapy which could help extend remission periods and improve life quality of IBD patients [2–6].

A recent meta-analysis has associated a high intake of fruit and vegetables with a reduction of UC in European population, suggesting that the intake of their bioactive compounds (fiber, antioxidant vitamins and phytochemicals such as polyphenols, carotenoids, isoflavones) could

explain this inverse association [7]. In murine colitis models, which mimic human pathology, it has been demonstrated the preventive effect on pro-inflammatory intestinal process associated to the intake of mango [8] or pineapple [9] juices, polyphenols-rich extracts from orange [10] or its sub-products [11], apple [12] and pomegranate [13]. Polyphenols seems to be the main bioactive compounds involved in the anti-colitic action of fruits, due to its ability to inhibit some pivotal pro-inflammatory mediators as nuclear transcriptional Factor-κB (NF-κB) and specific cytokines (Tumor Necrosis Factor-α (TNF-α), and interleukin 1-β (IL-1β)) and the induction of antioxidant defence systems. Furthermore, lipophilic compounds present in vegetables and fruits, such as fat-soluble vitamins (pro-vitamin A) [14], carotenoids (β-carotene) [15] and plant sterols (PS) [16–21], have displayed important anti-inflammatory and inmmunodulatory effects. In particular, PS could be a good strategy to prevent IBD, because they show a very little absorption in the intestine (0.5–2%) and reach the colon, where could exert anti-inflammatory effects. Moreover, the European Union authorized PS addition into milk-based fruit beverages [22], among other foods, to achieve the necessary amount of 1.5–3 g PS/day (not attainable with normal diet) for the well-known cholesterol-lowering effect [23]. PS have shown promising results in animal colitis models induced by trinitrobenzene sulfonic acid (TNBS) [16,17], dextran sodium sulphate (DSS) [18–24] or high-fat diet (40–60%) [19–21]. Doses between 20–150 mg/Kg/day help to mitigate some important parameters associated to UC such as symptoms, colon shortening and presence of edema, histopathology and myeloperoxidase (MPO) activity on colon tissue.

Other bioactive compounds that have gained attention are prebiotics, which stimulate selectively beneficial intestinal bacteria, helping to maintain intestinal mucosal barrier and enhance defense against pathogenic microorganism [25]. In this sense, galactooligosacharides (GOS) have been proposed as an active ingredient to prevent or alleviate symptoms associated with IBD [26]. Human clinical trials have demonstrated that consumption of GOS contained in chocolate or banana flavored chews (3.5–7 g/day) [27] and sachets (5.5 g/day) [28,29], reduce different pro-inflammatory markers such as calprotectin in feces, plasma C-reactive protein [28] and cytokines (IL-6, IL-1β and TNF-α) [29], with a concomitant increase in levels of fecal secretory immunoglobulin A (sIgA) [28] and reduction of common ulcerative colitis symptoms [27]. However, in animal studies the effect of GOS on colitis is controversial. GOS (4 g/Kg/day) administration for 10 days increase bifidobacterial number in colon in a TNBS colitis induced murine model, but it was not linked with a reduction of pro-inflammatory markers as MPO activity and colon damage [30]. On the other hand, in mice smad-3 feed with GOS (5 g/Kg/day) for 42 days an improvement of colitis severity and increase of natural killer activity were observed after colitis induction by *Helicobacter hepaticus* [31].

Enrichment of healthy foods with bioactive compounds could be an effective strategy to help mitigate the symptoms associated with IBD and extend remission periods [3]. In this sense, milk-based fruit beverages could be a suitable nutritional matrix for this purpose due to its low-fat content and good sources of bioactive compounds such as polyphenols, carotenoids and vitamins. Moreover, previous studies of our research group have demonstrated the beneficial effect of PS enriched milk-based fruit beverages on oxidative stress prevention and intestinal epithelia integrity maintenance in Caco-2 cells [32], as well as, their systemic anti-inflammatory properties, in hypercholesterolemic post-menopausal women, through the serum increase of anti-inflammatory cytokine IL-10 with concomitant reduction of IL-1β, cytokine which play an important role on the development of IBD [33].

The present study investigates for the first time, the effect of the daily intake of a healthy food, as milk-based fruit beverages, enriched with bioactive compounds (PS and/or GOS) on clinical symptoms and inflammatory process of UC, using a pre-clinical chronic murine model induced by DSS. The aim of this preliminary study is to evaluate if PS enriched milk-based fruit beverages with/without GOS are a suitable dietetic strategy to help to mitigate the health problems associated with UC.

#### **2. Materials and Methods**

#### *2.1. PS Milk-Based Fruit Beverage Formulation*

Two Ps-enriched (1 g/100 mL beverage) milk-based fruit beverages were used: MfB and MfB-G, without or with addition of GOS (1.8 g/100 mL beverage), respectively. Both beverages contained skimmed milk with the addition of milk fat and whey protein concentrate enriched with milk fat globule membrane (MFGM, Lacprodan ® MFGM-10 from Arla Foods Ingredients, Sønderhøj, Denmark), mandarin juice from concentrate (45%) (Source of β-cryptoxanthin 205 μg/100 mL beverage) and banana puree (4%). PS were added during beverage formulation as microencapsulated free microcrystalline PS (β-sitosterol 80%; sitostanol 12%; campesterol; 7%; campestanol 1% and stigmasterol 0.7%) from tall oil in a powder form (Lipohytol ® 146 ME Dispersible from Lipofoods, Barcelona, Spain). GOS syrup (Vivinal ® GOS from Friesland Campina Ingredients, Amersfoort, Netherland) containing approximately 59% GOS, 21% lactose, 19% glucose and 1% galactose based on dry matter. In order to guarantee microbiological stability, beverages were subjected to pasteurization (90 ◦C for 30 s) and filled aseptically in 250-mL tetra bricks.

#### *2.2. Animals and Treatment*

Female C57BL/6 mice, aged 6–8 weeks with weights ranging between 18 to 20 g, provided by Harlan Interfauna Iberica (Barcelona, Spain) were used in this study. Animals were acclimatized for 7 days under a 12-h light/ dark cycle at 22 ◦C/60% humidity and fed with a standard laboratory rodent diet and water *ad libitum* before experiments. All experiments were approved by the Institutional Ethics Committee of the University of Valencia, Spain (2017/VSC/PEA/00143).

Chronic colitis induction was performed by repeated administration of a 2% ( *w*/*v*) of DSS (in drinking water) for 7-day cycles according to Marín et al. [34] The DSS phases were interrupted by 7-days cycles of access to drinking water without DSS. MfB and MfB-G were orally administered everyday by gavage (0.15 mL) to achieve 35 and 95 mg/Kg body weight for PS and GOS, respectively (see Figure 1). Animals were randomly assigned into six intervention groups with 8 mice/group (*n* = 48): control (mice received water during all experiment), DSS (access to drinking water containing 2% DSS), DSS + MfB (drinking water containing 2% DSS + 0.15 mL of MfB), DSS + MfB-G (drinking water containing 2% DSS + 0.15 mL of MfB-G), MfB (received only 0.15 mL of MfB) and MfB-G (received only 0.15 mL of MfB-G).

**Figure 1.** Experimental design of the chronic colitis induction by dextran sulphate sodium (DSS) (2%, *w*/*v*) and plant sterol enriched milk-based fruit beverages without (MfB) or with galactooligosacharides (MfB-G) administration in the procedures.

Water intake was monitored three times a week to guarantee equitative DSS intake among different colitis mice groups and the type of beverages did not affect drink behavior. No statistically significant differences (*p* > 0.05) in water intake was found among different mice group (ml/mouse/day); control (4.3 ± 0.5), DSS (4.2 ± 0.8), DSS + MfB (4.5 ± 0.7), DSS+MfB-G (4.8 ± 1.1), MfB (4.6 ± 0.4) and MfB-G (4.5 ± 0.5).

#### *2.3. Disease Activity Index (DAI)*

The disease activity index (DAI) was determined according to the parameters outlined in Table 1. Animal body weights, stool consistency and visible blood in feces were recorded three times a week during all experiments. Stool consistency and blood in feces parameters were evaluated checking fresh feces contained in each animal group cage and scored following the method described by Marín et al. [34]. Also, blood presence around mouse perianal area was taken as indicator of blood in feces. DAI was calculated by combining scores (WS: weight loss; SC: stool consistency; BF: bloody feces) following the next formula DAI: (WS + SC + BF)/3 according to the methodology described by Marín et al. [34].

**Table 1.** Scoring system to calculate the disease activity index (DAI \*).


\* DAI was calculated by combining scores (WS: weight loss; SC: stool consistency; BF: bloody feces).

#### *2.4. Colon Shortening and Presence of Edema*

Animals were sacrificed at day 56 by cervical dislocation and their colons (ileo-cecal junction to anal verge) were removed. Fecal residues were washed with cold phosphate buffered saline (PBS) and the length and weight were measured. Presence of edema in colonic tissue was evaluated working out the weight/length (mg/cm) ratio. Then, colons were cut in four portions of approximately 2 cm and frozen immediately at −80 ◦C until use.

## *2.5. Histological Analysis*

Portions of approximately 2 cm from the colon of every group of mice were fixed in freshly prepared 4% formaldehyde in PBS buffer (pH 7.2), embedded in paraffin, and sectioned at 4 μm. Sections were stained with hematoxylin and eosin for histopathological analysis and examined by light microscopy [35]. The slides were analysed and scored as previously described Cooper et al. [36] according to the criteria listed in Table 2. Scores were calculated by adding the score for the three parameters, giving a maximum score of 10.




**Table 2.** *Cont.*

Histological score is calculated by adding the score of three parameters up to a total score of 10 points. DSS: dextran sulphate sodium;

#### *2.6. Myeloperoxidase (MPO) Assay*

In the model of experimental colitis, neutrophil infiltration into the colon was assessed indirectly by measuring myeloperoxidase (MPO) following the method elaborated by Bradley et al. [37]. Colon samples were weighed and blended in homogenizer with phosphate buffer (0.22 M) containing 0.5% of hexadecyl-trimethyl-ammonium bromide. Samples were centrifuged at 12000 g/4 ◦C/20 min and supernatants were placed on 96-well plates. Estimation of MPO content in colonic tissues was evaluated spectrophotometrically (450 nm) by measuring the 3,3-,5,5--tetramethyl-benzidine (MPO substrate) oxidation in presence of H2O2 [34].
