*3.3. Identification of phenolic Compounds by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS) in Aloe Vera (AV)*

The identification of phenolic compounds in the gel of *Aloe vera* (AV) was performed with ultra-performance liquid chromatography (UPLC) by utilizing an Acquity UPLC H-Class (Waters Corp, Milford, MA, USA) coupled with a Waters Xevo G2-X2 Tof system with an electrospray ionization (ESI) interface. The conditions for the chromatographic analysis were as follows: the mobile phase consisted of 0.1% formic acid in water/acetonitrile at a flow rate of 0.3 mL/min and 35 ◦C. A BEH C18 Column (2.1 mm X 100 mm) was used and the chromatograms were registered through detection of the Total Ion Current. The MS conditions of analysis in negative-ion mode were as follows: drying gas (nitrogen) flow rate, 8 L/min; gas temperature, 180 ◦C; scan range, *m*/*z* 50–3000; capillary voltage, 4500 V; and nebulizer pressure, 2.5 bar [44]. A solution of 8 mg/mL of lyophilized AV was prepared, homogenized, and passed through a 0.22 µm cellulose filter prior to analysis.

### *3.4. Extraction of Polysaccharides (AP) from Aloe Vera Gel*

The extraction of *Aloe vera* gel polysaccharides (AP) followed a modification of the technique reported by Ni et al. [4]. One gram of lyophilized *Aloe vera* gel was suspended in 80 mL of a 95% water/methanol solution, homogenized with a Polytron, and centrifuged at 936× *g* (Allegra 64R; Beckman, Pasadena, CA, USA) for 10 min. The first precipitate was stored, and the supernatant was centrifuged one more time at 10,285× *g* for 15 min. The first and second precipitates were combined, re-suspended in deionized water and lyophilized. The sum of these two fractions was the polysaccharide extract (AP). The first precipitate corresponds to the cell wall fibre, and the second precipitate corresponds to micro-particles from cell organelles of *Aloe vera* [4].

#### *3.5. Indigestible Fraction and in Vitro Fermentation 3.5. Indigestible Fraction and in Vitro Fermentation*

particles from cell organelles of *Aloe vera* [4].

#### 3.5.1. Indigestible Fermentation of Aloe Vera Gel (AV) and Polysaccharides (AP)

*Molecules* **2019**, *24*, x FOR PEER REVIEW 4 of 20

The first precipitate corresponds to the cell wall fibre, and the second precipitate corresponds to micro-

Figure 5 shows the flowchart of the isolation of the indigestible fibre fractions of AV and AP The indigestible fraction (IF) of (AV) and (AP) (Figure 5) was determined following the method proposed by Saura-Calixto et al. [45] with some modifications. Briefly, 300 mg of both fractions were suspended in HCl-KCl buffer (pH 1.5). Then, 0.2 mL of a pepsin solution at a concentration of 300 mg/mL in HCl-KCl buffer (>250 units/mg, P7000; Sigma Aldrich, Toluca, Edo. Mex. Mexico) was added and incubated at 40 ◦C for 1 h with constant stirring to simulate the digestive process. 3.5.1. Indigestible Fermentation of Aloe Vera Gel (AV) and Polysaccharides (AP) Figure 5 shows the flowchart of the isolation of the indigestible fibre fractions of AV and AP The indigestible fraction (IF) of (AV) and (AP) (Figure 5) was determined following the method proposed by Saura-Calixto et al. [45] with some modifications. Briefly, 300 mg of both fractions were suspended in HCl-KCl buffer (pH 1.5). Then, 0.2 mL of a pepsin solution at a concentration of 300 mg/mL in HCl-KCl buffer (>250 units/mg, P7000; Sigma Aldrich, Toluca, Edo. Mex. Mexico) was added and incubated at 40ºC for 1 h with constant stirring to simulate the digestive process.

**Figure 5.** Indigestible fraction. The obtaining of indigestible fractions of AV and AP. AV= *Aloe vera gel, AP= Aloe* polysaccharides*;* AV-NDF = Insoluble non-digestible fraction of AV, AP-NDF = Insoluble non digestible fraction of AP, AV-SNDF = Soluble fraction of the non-digestible fibre of AV, AP-SNDF = Soluble fraction of non-digestible fibre of AP. **Figure 5.** Indigestible fraction. The obtaining of indigestible fractions of AV and AP. AV= *Aloe vera gel, AP*= *Aloe* polysaccharides; AV-NDF = Insoluble non-digestible fraction of AV, AP-NDF = Insoluble non digestible fraction of AP, AV-SNDF = Soluble fraction of the non-digestible fibre of AV, AP-SNDF = Soluble fraction of non-digestible fibre of AP.

To simulate small intestine digestion, pH was adjusted to 6.9, and 9 mL of tris-maleate buffer (0.1 M) was added, followed by 1 mL of α-amylase solution (3480 units/mL, A3176; Sigma Aldrich), at a concentration of 120 mg/mL in tris-maleate buffer. The mix was incubated at 37 °C for 16 h at constant stirring. Lipid digestion enzymes such as lipase or biliary juices were omitted given that the amount of fat (lower than 4%) was not significant. After the reaction time, the samples were centrifuged at 4000 *g* for 15 min, and the supernatants were saved. The precipitate was washed twice with 10 mL of distilled water and centrifuged; supernatants were collected in 80 mL beakers. The tubes containing the solid residue were placed in an oven at 60 °C for 48 h, and the residue was then weighed as the amount of insoluble non-digestible fibre in AV (AV-INDF) and AP (AP-INDF). Both insoluble non-digestible fibre fractions were stored at 4°C until the fermentation experiments. The supernatant was placed on dialysis membranes previously treated with boiling water for 20 To simulate small intestine digestion, pH was adjusted to 6.9, and 9 mL of tris-maleate buffer (0.1 M) was added, followed by 1 mL of α-amylase solution (3480 units/mL, A3176; Sigma Aldrich), at a concentration of 120 mg/mL in tris-maleate buffer. The mix was incubated at 37 ◦C for 16 h at constant stirring. Lipid digestion enzymes such as lipase or biliary juices were omitted given that the amount of fat (lower than 4%) was not significant. After the reaction time, the samples were centrifuged at 4000× *g* for 15 min, and the supernatants were saved. The precipitate was washed twice with 10 mL of distilled water and centrifuged; supernatants were collected in 80 mL beakers. The tubes containing the solid residue were placed in an oven at 60 ◦C for 48 h, and the residue was then weighed as the amount of insoluble non-digestible fibre in AV (AV-INDF) and AP (AP-INDF). Both insoluble non-digestible fibre fractions were stored at 4◦C until the fermentation experiments.

min. The supernatant was dialyzed (cellulose membrane 12000–14000 Dalton MWCO-18) at a constant The supernatant was placed on dialysis membranes previously treated with boiling water for 20 min. The supernatant was dialyzed (cellulose membrane 12000–14000 Dalton MWCO-18) at a constant water flow of 7 L/h (30 mL/15 sec) at 25 ◦C for 48 h. Finally, the content of the membranes was lyophilized and weighed to obtain the soluble fraction of the non-digestible fibre of AV (AV-SNDF) and non-digestible fibre of AP (AP-SNDF); these fractions were stored at 5 ◦C until use in the fermentation process [46].

#### 3.5.2. In Vitro Fermentation of AV-TNDF and AP-TNDF

The samples obtained in the digestion, which were the soluble fraction (SNDF) and the insoluble fraction (INDF), were combined as the total non-digestible fraction (TNDF) of AV and AP to be fermented. In vitro fermentation was assessed in the AV-TNDF and AP-TNDF fractions. Fermentation was prepared according to a modified version of Martín-Carrón and Goñi [46]. The samples were placed in a culture system under strict anaerobic conditions for 24 h. Human faeces were used as inocula, and anaerobic conditions were maintained using oxygen-free carbon dioxide.

#### Inoculum

To obtain the 10% *w*/*v* inoculum, the faeces of four volunteers were weighed and placed in beakers containing a sterile and anaerobic fermentation medium. The inocula were mixed in a Stomacher 80 Lab blender (Seward Medical, London, UK for 10 min and then filtered (1-mm mesh) before use.

#### Medium

A micromineral solution was prepared using CaCl2-2H2O, MnCl2-4H2O, CoCl2-6H2O, and FeCl3-6H2O mixed in a buffer solution ((NH4)HCO<sup>3</sup> and NaHCO3) in distilled water. A macromineral solution containing Na2HPO4, KH2PO4, and MgSO4-7H2O was made in distilled water, as was a reducing solution containing cysteine hydrochloride (C-1276, Sigma-Aldrich, Toluca, Edo. Mex., Mexico).

#### Procedure

Substrate (100 mg) of the total non-digestible fraction (AV-TNDF or AP-TNDF) was weighed in a 60 mL serum vial (Supelco; Bellefonte, PA, USA), and 8 mL of fresh medium and 2 mL of inoculum were added. Vials were sealed with rubber stoppers and placed in a water bath with constant stirring at 37 ◦C. The fermentation kinetics was monitored at 0, 4, 8 and 24 h to obtain fermented samples at each time point (AVF4, AVF8 and AVF24 or APF4, APF8 and APF24). A control for each time containing lactulose (L-7877, Sigma-Aldrich, Toluca, Edo. Mex., Mexico) and a target without substrate were included as controls.

Gas volume production and pH were measured at 0, 4, 8 and 24 h. Fermentation was stopped by adding 2.5 mL of1 M NaOH. Samples were centrifuged at 2500× *g* for 10 min, and 3 mL of supernatant was taken in duplicate for SCFA determination by gas chromatography.

#### Unfermented Fibre

The precipitate of each tube from the fermentation kinetics experiment was homogenized in 50 mL of 0.9% NaCl for 3 min. All precipitates were filtered using Dacron filters (pore size 50 µm), which were dried to a constant weight before use. Unfiltered residues were washed twice with 50 mL of 0.9% NaCl and washed twice with 5 mL of acetone to eliminate mineral and inorganic residues in addition to lipids and other hydrophobic compounds. Filter papers were dried at 60 ◦C to constant weight, assessing the unfermented fibre (UF) [47] that was calculated by the following formula:

UF = weight (filter + washed residue) − initial filter weight. (1)

Then, UF(AV-TNDF) and UF(AP-TNDF) were obtained.

#### 3.5.3. Quantification of SCFAs in AV-TNDF and AP-TNDF

SCFA quantification was carried out in AV-TNDF, AP-TNDF and lactulose fermented according the methodology reported by Saura-Calixto et al., [45] and adapted from Zhao et al., [48]. Briefly, 500 µL of supernatant of each fermentation time sample was mixed with 400 mL of 2-methylvaleric acid as an internal standard (10987-8; Sigma, Toluca, Edo. Mex., Mexico) and 100 mL of HClO<sup>4</sup> to maintain a constant pH in the samples. The mixture was centrifuged at 10,000 at 4 ◦C for 15 min, and the supernatant was placed in gas chromatography vials. A Clarus 500/580 GC gas chromatographer (Perkin-Elmer, Inc. Shelton, CO., USA) was used with a TG-WAXMS-A GC column (Thermo Fisher Scientific, CDMX, Mexico) and a flame ionization detector with an injector temperature of 270 ◦C and

a detector temperature of 300 ◦C. Glacial acetic acid, propionic acid, butyric acid, and 4-methylvaleric acid (Sigma, 320099, 94425, 19215 and 10987-8) were used as standards to obtain calibration curves and assess retention times. The oven was heated to 95 ◦C at 2 min and increased at 120 ◦C/min to 240 ◦C; the carrier flow was 1 mL/min, and the carrier gas flow was 20 mL/min.

#### 3.5.4. Quantification of Phenolic Compounds

Total phenolic compounds were quantified using the Folin–Ciocalteu method [49] in the AV, AP, AV-TNDF, AP-TNDF, AVF4, AVF8, AVF24, APF4, APF8 and APF24 samples. The samples were homogenized in 0.5 mL methanol, added Folin-Ciocalteu reagent, and saturated sodium carbonate solution and water. After 60 min, the absorbance was measured at 760 nm. A calibration curve was performed with aloin, because it is the most representative phenolic compound in *Aloe vera* [50]. The concentration of total phenolic compounds was expressed as milligram equivalents of aloin/100 g sample.

### 3.5.5. Identification of Phenolic Compounds by HPTLC

HPTLC was accomplished according to a modified version of the methodology published by Paillat, et al., [51]. At the beginning volumes of standard solutions of ferulic acid, *p*-hydroxybenzaldehyde (PHB), *p*-hydroxybenzoic acid (APHB), *p*-cresol, *p*-creosol, and vanillic acid (722820, 54590, 54630, 61030, 41340, and 68654, Sigma-Aldrich, Química S.A. de C.V.) were applied at a concentration of 3 mg/mL. After that, were applied 15 µL of AV, AV-TNDF, AP, AP-TNDF and the kinetic fermentation samples (AVF4, AVF8, AVF24 and APF4, APF8, APF24) at a concentration of 12.5 mg/mL. Samples were placed on TLC silica gel 60 F254 plates (E. Merck, Darmstadt, Germany) using an ATS 4 TLC sampler (CAMAG, Muttenz, Switzerland) at a constant application rate of 120 nL s−<sup>1</sup> and developed in a CAMAG automated developing chamber ADC2 (47% moisture) saturated and preconditioned for 5 min to a 50-mm distance with an *n*-hexane: chloroform:methanol:acetic acid solvent system (5:36:4:0.5 *v*/*v*/*v*/*v*). Plates were scanned at 254 nm and 366 nm in a CAMAG TLC III scanner (slit size 4 mm <sup>×</sup> 0.3 mm) at a scanning speed of 10 mm s−<sup>1</sup> and a data step resolution of 50 <sup>µ</sup>m.

#### *3.6. Antioxidant Activity*

### 3.6.1. Reducing Power and Hydroxyl Radical Scavenging Activity

The reducing power of AV, AV-TNDF, AP, and AP-TNDF was assessed by the Oyaizu method [52]. Various concentrations of methanolic extracts (12.5, 6.25, 3.12, 1.56, and 0.78 mg/mL) were mixed with 2.5 mL 200 mmol/L sodium phosphate buffer (pH 6.6) and 2.5 mL 1% potassium ferricyanide. The mixture was incubated at 50 ◦C for 20 min. After 2.5 mL of 10% trichloroacetic acid (*w*/*v*) were added, the mixture was centrifuged at 650 rpm for 10 min. The upper layer (5 mL) was mixed with 5 mL deionised water and 1 mL 0.1% of ferric chloride; the absorbance was measured at 700 nm, a higher absorbance indicating a higher reducing power. The assays were carried out in triplicate and the results were expressed as mean values ± standard deviations. Finally, the reducing power in the kinetic fermentation samples (AVF4, AVF8, AVF24, APF4, APF8 and APF24) was determined but only with a concentration of 12.5 mg/mL. This was the concentration that gave the best results in the previous samples and was chosen for this second part of the work.
