Cell Culture

Balb/c 3T3 clone A31 and Caco-2 were purchased from ATCC, Manassas, VA, USA. Balb/c 3T3 clone A31 cells were maintained in DMEM with 10% Calf Bovine Serum (CBS; ATCC, Manassas, VA, USA) and 1% penicillin-streptomycin (10,000 unit/mL) at 37 ◦C in a 5% CO2 atmosphere. Caco-2 cells were cultured in DMEM with FBS 10% ( *w*/*w*), non-essential amino acids (1% *w*/*w*), L-glutamine (1% *w*/*w*) and penicillin–streptomycin (1% *w*/*w*). The cells were grown in a humidified atmosphere (5% CO2) at 37 ◦C until 80% confluence and sub-cultured twice a week [27].

### Neutral Red Uptake (NRU)

The *in vitro* 3T3 NRU test was performed as described by the ISO 10993-5:2009 "Biological evaluation of medical devices—Part 5: Tests for *in vitro* cytotoxicity". Briefly, 2.5 × 10<sup>4</sup> 3T3 cells/well were incubated in 96-well plates and cultured for 24 h at 37 ◦C and 5% CO2 in humidified hair with increases doses of BTG and PLOMW (0.39, 0.78, 1.56, 3.12, 6.25, 12.5 and 25 mg mL−1) overnight in DMEM. Cell viability was measured by neutral red uptake (NRU) assay [28]. The NRU assay provided an incubation (3-h) with neutral red (50 μg/mL in DMEM) followed by an extraction with acetic acid, ethanol and water (1:50:49 *v*/*v*/*v*). The absorbance was measured at 540 nm in a microplate reader Epoch (BioTek, Winooski, VT, USA). A percentage of viability was calculated as follows:

$$\% \text{Viability} = \frac{(\text{Absorbance}\_{540\text{nm}} \text{test material}) - (\text{Absorbance}\_{540\text{nm}} \text{blank})}{(\text{Absorbance}\_{540\text{nm}} \text{control}) - (\text{Absorbance}\_{540\text{nm}} \text{blank})} \tag{4}$$

Cell Viability Assay

MTT staining assay was used to evaluate cell viability as described by Mosmann [29]. Briefly, 1 × 10<sup>4</sup> Caco-2 cells/well were incubated in 96-well plates and cultured for 24 h at 37 ◦C and 5% CO2 in humidified hair with increases doses of BTG and PLOMW (3.12, 6.25, 12.5 and 25 mg mL−1); then, fresh MTT was added to each well and after 2 h of incubation 1 mL of DMSO was used to solubilize the formazan products. The optical density (OD) was measured at 570 nm in a microplate reader Epoch (BioTek). A percentage of viability was calculated as follows:

$$\% \text{Viability} = \frac{(\text{OD} \text{5} \text{0nm} \text{test material}) - (\text{OD} \text{5} \text{0nm} \text{blank})}{(\text{OD}\_{570\text{nm}} \text{control}) - (\text{OD}\_{570\text{nm}} \text{blank})} \tag{5}$$

Con A/o-Pd Assay

Caco-2 cells were trypsinized, washed in saline solution and divided into three groups: (1) Control (2.5 × 10<sup>5</sup> cells in 5 mL of saline solution); (2) BGT (2.5 × 10<sup>5</sup> cells in 5 mL of BGT 12.5 mg mL−1); (3) PLOMW (2.5 × 10<sup>5</sup> cells in 5 mL of PLMW 12.5 mg mL−1). Each group was incubated at 30 ◦C for 15 min, under gentle shaking. Then, the cells were washed twice with 5 mL Phosphate Buffered Saline (PBS) and centrifugated (2000 rpm) for 5 min. Subsequently, the cells were transferred to a clean tube and given a final wash. After an additional centrifugation (2000 rpm) for 5 min, the supernatant was removed. The pellet was stirred with a vortex mixer and washed twice with 12 mL of PBS and centrifugated (2000 rpm) for 5 min. Furthermore, the cells were transferred to a clean tube and washed prior to the addition of the next reagen<sup>t</sup> (5 mL of PBS containing 1.0 mM calcium chloride and 10 mg L−<sup>1</sup> biotinylated concanavalin A from Canavalia ensiformis (Con-A)). The mixture was incubated at 30 ◦C for 30 min under gentle shaking, centrifuged (2000 rpm) for 5 min. The cells were washed twice with PBS and transferred to a clean tube. 5 mL of PBS containing 5 mg/L streptavidin peroxidase was added and each tube was incubated at 30 ◦C for 60 min. Finally, the cells were washed and transferred to a clean tube and resuspended with 1 mL of o-phenylenediamine dihydrochloride (o-pd) solution (containing 0.4 mg o-pd and 0.4 μL 30% H2O2 in 1.0 mL 0.05 M citrate phosphate buffer). The oxidation of o-pd was stopped after 2 min with 1.0 mL of 1.0 M H2SO4 and the optical density measured at 490 nm (spectrophotometer Epoch, BioTek) [30].

### *2.6. Preparation of Puddings*

Puddings were prepared using starch and PLOMW as gelling agent. Pears were purchased in a local supermarket and after washing, they were peeled. In order to prepare the puree, 50 mL of water was added to the fruit pulp (650 g) divided into smaller pieces. The pulp was then heated and stirred at low speed for 15 min, setting the temperature at 100 ◦C. Subsequently, after inactivation of the fruit enzymes, the heating was stopped and the pulp was ground for 1 min at high speed. The formulation of the pudding was determined after evaluating the consistency, according to the different quantity of gelling agent. Specifically, the pudding (PPLOMW) was prepared by mixing 100 mL of milk, 10 g of starch, 50 g of pear puree and 0.5 g of PLOMW. The starch was solubilized at room temperature in 75.0 mL of milk. The pear puree solubilized in the remaining volume of milk was then added under stirring at 80 ◦C for 5 min. The tare gum was gradually added without interrupting stirring and heating, to favor its hydration. The pudding was packaged in 60 g glass jars, immediately sealed with a metal lid. The pudding samples were stored at 4 ◦C in the refrigerator until the analysis carried out when the containers were opened (day 0), after seven, fourteen and twenty-eight days from the preparation. A control pudding (BCTG) was also prepared as control under the same conditions as described above, but using commercial tara gum (CTG) instead of PLOMW.

### *2.7. Characterization of Pudding*
