Effect of different drying procedures on the bioactive polysaccharide acemannan from Aloe vera (Aloe barbadensis Miller)

Carbohydrate Polymers 168 (2017) 327–336

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Effect of different drying procedures on the bioactive polysaccharide acemannan from Aloe vera (Aloe barbadensis Miller) Rafael Minjares-Fuentes a , Víctor Manuel Rodríguez-González b , Rubén Francisco González-Laredo c , Valeria Eim a , María Reyes González-Centeno a , Antoni Femenia a,∗ a

Department of Chemistry, University of the Balearic Islands, Ctra Valldemossa Km 7.5, 07122, Palma de Mallorca, Spain Facultad de Ciencias Químicas, Universidad Juárez del Estado de Durango, Av. Articulo 123 s/n Fracc Filadelfia, 35010, Gómez Palacio, Dgo, Mexico c Departamento de Ingenierías Química y Bioquímica, TecNM-Instituto Tecnológico de Durango, Blvd. Felipe Pescador 1830 Ote., 34080, Durango, Dgo, Mexico b

a r t i c l e

i n f o

Article history: Received 8 January 2017 Received in revised form 17 March 2017 Accepted 27 March 2017 Available online 29 March 2017 Keywords: Aloe vera Acemannan Drying procedures Acetylation Functional properties

a b s t r a c t The main effects of different drying procedures: spray-, industrial freeze-, refractance window- and radiant zone-drying, on acemannan, the main bioactive polysaccharide from Aloe vera gel, were investigated. All the drying procedures caused a considerable decrease in the acemannan yield (∼40%). Degradation affected not only the backbone, as indicated by the important losses of (1 → 4)-linked mannose units, but also the side-chains formed by galactose. In addition, methylation analysis suggested the deacetylation of mannose units (>60%), which was confirmed by 1 H NMR analysis. Interestingly, all these changes were reflected in the functional properties which were severely affected. Thus, water retention capacity values from processed samples decreased ∼50%, and a reduction greater than 80% was determined in swelling and fat adsorption capacity values. Therefore, these important modifications should be taken into consideration, since not only the functionality but also the physiological effects attributed to many Aloe vera-based products could also be affected. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Acemannan, the major polysaccharide found in Aloe vera gel, is mainly composed of large amounts of partially acetylated mannose units (Man >60%), followed by glucose (Glc ∼20%) and, to a minor extent, galactose (Gal <10%) (Choi & Chung, 2003; Chow, Williamson, Yates, & Goux, 2005; Femenia, Sánchez, Simal, & Rosselló, 1999; Talmadge et al., 2004). Structurally, the acemannan polysaccharide, with a molecular weight of around 40–50 kDa, could be represented by a single-chain of ␤-(1 → 4) mannose with ␤-(1 → 4) glucose inserted into the backbone; ␣-(1 → 6) galactose units may also be found as side-chains (Chokboribal et al., 2015; Chow et al., 2005; Femenia et al., 1999; Talmadge et al., 2004). The acetyl groups are the unique non-sugar functional groups present in acemannan and seem to play a key role not only in the physico-chemical properties but also in the biological activity of

∗ Corresponding author. E-mail address: [email protected] (A. Femenia). http://dx.doi.org/10.1016/j.carbpol.2017.03.087 0144-8617/© 2017 Elsevier Ltd. All rights reserved.

the Aloe vera (Campestrini, Silveira, Duarte, Koop, & Noseda, 2013; Chokboribal et al., 2015; Ni et al., 2004). Acemannan is a storage polysaccharide located within the protoplast of the parenchymatous cells of the Aloe vera gel, and not a component of the cell walls (Femenia et al., 1999). Interestingly, this polymer has been reported as the main bioactive substance present in Aloe vera gel, being responsible for most of the beneficial properties attributed to Aloe vera (Hamman, 2008; McAnalley, 1993; Reynolds, 1985; Reynolds & Dweck, 1999; t’Hart, van den Berg, Kuis, van Dijk, & Labadie, 1989), such as the reduction in blood glucose, blood pressure and the improvement of lipid profile in diabetic patients, among many others (Choudhary, Kochhar, & Sangha, 2011; Pothuraju, Sharma, Onteru, Singh, & Hussain, 2016). These beneficial effects have been attributed to the high molecular weight fractions of acemannan which are degraded by the intestinal microbiota to form oligosaccharides that inhibit intestinal glucose absorption (Boban, Nambisan, & Sudhakaran, 2006; Jain, Gupta, & Jain, 2007; Yagi et al., 2001, 2009). Furthermore, several studies have demonstrated that the acetyl groups of acemannan are mainly responsible not only for the interaction of acemannan with other biomolecules but also for enabling the transport of other bioac-

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tive compounds across the intestinal epithelium, enhancing their absorption in the intestine (Chokboribal et al., 2015; Reynolds & Dweck, 1999; Sharma, Mittal, & Chauhan, 2015). However, acemannan is inherently unstable and can be easily degraded by different physico-chemical factors, such as high temperature, pH changes, bacterial contamination, or enzymes, such as mannanases, present in the gel (Javed & Atta-Ur, 2014; Kim, 2006). During the manufacture of Aloe products, the Aloe vera gel is treated under different operations such as pasteurization, concentration and/or drying, dehydration being the process most used. However, the conditions applied during the drying procedure may cause irreversible modifications to the polysaccharides from Aloe vera, and in particular to acemannan, affecting its original structure, which in turn may lead to considerable changes, not only in the physico-chemical properties, but also, in the physiological and pharmacological properties attributed to the Aloe vera plant (Eshun & He, 2004; Femenia, Garcı´ıa-Pascual, Simal, & Rosselló, 2003). For instance, several authors have reported that the drying process could promote a considerable deacetylation of the acemannan polymer (Lim & Cheong, 2015; Minjares-Fuentes et al., 2016; Sriariyakul, Swasdisevi, Devahastin, & Soponronnarit, 2016), leading to a considerable reduction in its biological effect (Chokboribal et al., 2015). Thus, the main aim of this study was to evaluate the effect of different drying procedures, used on an industrial scale, on the main structural and compositional features of the acemannan polymer, the main bioactive polysaccharide present in the Aloe vera gel. In addition, functional properties related to the acemannan polymer, in particular swelling, water retention and fat adsorption capacities were also investigated.

2. Experimental 2.1. Sample Aloe vera leaves, used as a raw material, were supplied by the AMBwellness company (Mexico; http://www.amb-wellness.com/ ). Leaves between 40 and 50 cm in length and with no external damage were selected for this study. All the Aloe vera leaves came from 3-year old plants. The Aloe vera leaves were pressed to extract the gel, which was de-pectinized and filtered through a filter press as described by He, Changhong, Kojo, and Tian, (2005). The filtration process included: coarse filtration (Ø = 400–800 ␮m), moderated filtration (Ø = 100–400 ␮m), and filtration (Ø = 25–100 ␮m). The mesophyll tissue was removed by means of the coarse and moderate filters, while fibers coming from the pulp were mainly removed in the last step of the filtration procedure. Next, the filtered gel was concentrated in a quadruple-effect industrial evaporator till a moisture content of 85% on wet basis (w.b.) was achieved. A feed flow rate of ∼30,000 L/h of Aloe vera gel was introduced into the evaporator, operating in vacuum conditions with an evaporation rate of ∼4800 L/h. The concentrated Aloe vera gel was then separated into four batches for its subsequent drying.

2.2. Drying methods A different drying method was applied for each batch, in particular spray-drying (SD), industrial freeze-drying (IFD), refractance window-drying (RWD), and radiant zone-drying (RZD). In addition, fresh Aloe vera gel was lyophilized using a freeze dryer FreeZone Triad Cascade Benchtop (LABCONCO, Missouri, USA) operated at 0.010 mBar with condenser and shelf temperatures of −80 ◦ C and

−20 ◦ C, respectively. This lyophilized sample of fresh Aloe vera gel was powdered and used as a reference. Prior to analysis, all the processed Aloe vera gel samples obtained by each of the above drying procedures were packed in polyethylene bags, hermetically sealed, and stored in a desiccator with silica gel, in order to avoid absorption of moisture. 2.2.1. Spray-drying (SD) Aloe vera powder obtained by SD was produced in an industrial spray dryer (Anhydro, Massachusetts, USA) at feed flow rate of 1800 L/h. The spray dryer was operated at 150 and 75 ◦ C of inlet and outlet temperatures, respectively. 2.2.2. Industrial freeze-drying (IFD) Aloe vera powder produced by IFD was obtained in an industrial scale freeze dryer (Freeze Mobile 24, Vertis Co., Inc., New York, USA). The drying process was carried out at 25 ◦ C with an absolute pressure of 0.3 mBa. 2.2.3. Refractance window® drying (RWD) A pilot scale Refractance Window® dryer (MCD Technologies, Inc., Washington, USA) was used to obtain the Aloe vera powder. This dryer has an effective surface drying area of 1.10 m2 and length of 1.83 m in the direction of the belt movement. Drying was accomplished by spreading the homogenized concentrate of Aloe vera gel on the plastic conveyor belt that moves over the surface of circulating hot water, kept at 97 ◦ C. The thickness of the concentrate on the belt was 0.5 mm, controlled using a spreader bar. 2.2.4. Radiant zone drying (RZD) A pilot-sized Radiant Zone Dryer, model number RZD-410Z5P (Washington, USA), was used to obtain the Aloe vera powder (Savarese, 2003). The pilot system consisted of a continuous belt system with a drying area of 122 cm wide and 457.2 cm long, divided into five temperature zones. Zones 1–4 and 5 were of 76.2 cm and 152.4 cm in length, respectively. The final part of the belt was unheated allowing the product to cool prior to removal and packaging. 2.3. Scanning electron microscopy Scanning electron microscopy (SEM) was used to observe the morphology of reference and processed Aloe vera samples. Micrographs were taken with a Hitachi scanning electron microscope S-3400N (Tokyo, Japan) with an accelerating voltage of 15 kV. Samples were observed directly, without further treatment, under a pressure of 40 Pa. 2.4. Color characteristics Color of reference and processed Aloe vera samples was determined using a Konica Minolta Spectrophotometer CM − 5 (Tokyo, Japan) calibrated with black and white standards. The color parameter values of lightness (L), greenness/redness (−/+ a* ), and blueness/yellowness (−/+ b* ) were directly recorded for each sample. Chroma parameter (C), indicating color intensity, and hue angle (H◦ ) were calculated using Eqs. (1) and (2), respectively.



C = a∗2 + b∗2 H ◦ = tan−1

1⁄2

 b∗  a∗

(1) (2)

Hue angle values vary from 0◦ (pure red color), 90◦ (pure yellow color), 180◦ (pure green color) and 270◦ (pure blue color).

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Total color difference or change between dehydrated and fresh samples was also calculated using Eq. (3): E =



L0∗ − L∗

2



+ a∗0 − a∗

2



+ b∗0 − b∗

2

(3)

where L0∗ , a∗0 and b∗0 are the values of the reference sample, and L*, a* and b* the measured values corresponding to a processed sample. All parameters were measured at least in triplicate for each treatment.

2.8.2. Water retention capacity WRC was measured as the water retained by the acemannan polymer after centrifugation. Samples (0.01–0.10 g) were suspended (24 h) in phosphate buffer (5 mL) and centrifuged (18,000g; 15 min) with residual solids in the supernatant recovered by filtration (GF/C paper) and recombined with the pellet. The pellet was weighed (P1 ), and dried at 102 ◦ C overnight. After cooling the dry weight was determined (P2 ) and hence WRC was calculated using the Eq. (4) proposed by Femenia, Lefebvre, Thebaudin, Robertson, and Bourgeois (1997):

2.5. X-ray diffraction analysis WRC = X-ray diffraction of reference and processed Aloe vera samples was performed according to the method described by Ray and Aswatha (2013). X-ray analysis was carried out in a X-ray difractometer SIEMENS D5000 (Philips, Holland) equipped with Co target and Fe filter under 20 mA and 40 kV of operating power source and the scanning rate was adjusted at 5◦ /min with a scanning scope of 10–70◦ . 2.6. Alcohol insoluble residues (AIRs) AIRs from reference and processed Aloe vera samples were obtained by immersing the samples in boiling ethanol (final concentration 85% (v/v) aqueous) as described by Femenia, GarciaConesa, Simal, and Rosselló, (1998). 2.7. Isolation of acemannan polymer Isolation of acemannan was carried out as described by Rodríguez-González et al. (2011) with slight modifications. Approximately 300 mg of AIR preparations from Aloe vera, reference and dehydrated samples, were suspended in distilled water (200 mL) and stirred for 2 h at room temperature. The suspension was then centrifuged at 13,000g during 1 h at 20 ◦ C. The supernatant (containing the acemannan) was recovered and extensively dialyzed (MW cutoff 10,000–12,000). Further purification of the acemannan was carried out through gel permeation chromatography. The elution of dialyzed fractions containing acemannan was performed on a column (100 cm × 1 cm) of Sephacryl S-400-HR at a flow rate of 16 mL/h. The fractions were dissolved in 2 mL, 50 mM potassium–phosphate buffer, pH 6.5, containing 0.2 M NaCl. Fractions (2 mL) were collected and aliquots (20 ␮L) were assayed for carbohydrate by the phenol-sulphuric acid method. The appropriate fractions containing purified acemannan were combined, dialyzed, concentrated, and an aliquot was lyophilized for carbohydrate, methylation and 1 H NMR analysis. 2.8. Determination of functional properties The functional properties determined included hydration properties such as, swelling (Sw) and water retention capacity (WRC), and fat adsorption capacity (FAC). As previously described by Rodríguez-González, Femenia, Minjares-Fuentes, and GonzálezLaredo (2012), Sw and WRC of acemannan, obtained from the reference and processed samples, were measured in phosphate buffer (1 M; pH 6.3) in order to simulate pH and the buffering conditions of food products. 2.8.1. Swelling Sw was measured as bed volume after equilibration in an excess of solvent. The samples (0.01–0.10 g) were weighed into a graduated conical tube with an excess of buffer. The suspensions were stirred and after equilibration (16 h) the volumes were recorded and expressed as mL/g acemannan.

329

P1 − P2 P2 − k

(4)

where k = ˛ (P1 − P2 ) with ␣ = 0.028 g phosphate/mL. WRC results were expressed as g H2 O/g acemannan. 2.8.3. Fat adsorption capacity FAC was measured as the oil retained for the acemannan polymer after centrifugation. Acemannan samples (0.01–0.10 g) were mixed with sunflower oil (5 mL), left overnight at room temperature and centrifuged at 18,000g for 10 min. The excess supernatant was decanted and FAC was expressed as g oil/g acemannan. 2.9. Analysis of carbohydrate composition Carbohydrate composition was analyzed after acid hydrolysis following a modified version of the method reported by RodríguezGonzález et al. (2011). Briefly, acemannan samples were dispersed in 1 M H2 SO4 and hydrolyzed at 100 ◦ C for 2.5 h. Neutral sugars released from hydrolysis were derivatized as their corresponding alditol acetates and isothermally separated at 220 ◦ C by gas chromatography (Hewlett-Packard 5890A, Waldbronn, Germany) with a FID detector and equipped with a 30 m column DB-225 (J&W Scientific, Folsom, CA, USA) with i.d. and film thickness of 0.25 mm and 0.15 ␮m, respectively. 2.10. Methylation analysis Methylation analysis of the acemannan polymer was based on a modified sequential method using sodium hydroxide and methyl iodide (Ciucanu & Kerek, 1984). The modifications introduced to improve the overall methylation procedure were described in detail by Femenia et al. (1998). 2.11.

1H

Nuclear magnetic resonance (NMR) analysis

1 H NMR analysis of purified acemannan was carried out according to the method proposed by Bozzi, Perrin, Austin, and Arce Vera, (2007), with slight modifications. The 1 H NMR spectra at 300.13 MHz were recorded on a Bruker Avance 300 spectrometer (Massachusetts, USA), equipped with a 5 mm broadband multinuclear z-gradient (BBO) probehead. Two milligrams of purified acemannan polymer were weighted into an eppendorf tube and solubilized with 1 mL of 99.9% deuterium oxide (Sigma-Aldrich, Spain). Then, the solubilized sample was transferred to Wildman Economic 5 mm NMR tubes. Two milligrams of 99.5% nicotinamide standard (Sigma-Aldrich, Spain) were added as internal shift standard since the peaks corresponding to nicotinamide appears after 7.5 ppm, which are well-separated from other aloe-derived proton peaks (Jiao et al., 2010). Nicotinamide was used to calculate the area under the curve of the corresponding signal of acetyl groups in order to determinate the degree of acetylation of the acemannan polymer. The relative degree of acetylation of processed samples

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in relation to the fresh (reference) sample was calculated using the following Eq. (5):



Relative degree of acetylation =

AProcessed AReference



× 100

(5)

where AProcessed and AReference are the area under the curve of the signals of acetyl groups corresponding to the processed and reference Aloe vera samples, respectively. 2.12. Statistical analysis The effect of the different drying methods on acemannan polymer was statistically evaluated by one-way analysis of variance (ANOVA). Further, the Tukey-Kramer test was used as a post-hoc test with a significant level of p < 0.05. All calculations and graphics were undertaken and prepared using NCSS software version 2007 (Utah, USA) and Sigma-plot 10.0 software (California, USA), respectively. 3. Results and discussion 3.1. Physical appearance 3.1.1. SEM observations Scanning electron micrographs corresponding to the reference and processed Aloe vera samples are shown in Fig. 1. A regular and relatively rigid structure of the parenchyma cells was observed for the reference sample (Fig. 1a). Similar images have been previously reported in the literature to characterize the Aloe vera fillet tissue (Domínguez-Fernández et al., 2012; Rodríguez-González et al., 2011). On the other hand, morphological differences between the samples were observed. Specifically, SD Aloe vera sample presented spherical and/or oval shape and smooth surface particles (Fig. 1b), whereas an irregular and granular morphology with heterogeneous particle size was observed for IFD Aloe vera sample (Fig. 1c). The morphology reported for SD Aloe vera sample has also been described in the literature for other products dehydrated by spray-drying, such as mucilages, fruit juices, purees and milk (Cervantes-Martínez et al., 2014; García-Cruz, Rodríguez-Ramírez, Méndez Lagunas, & Medina-Torres, 2013; León-Martínez, MéndezLagunas, & Rodríguez-Ramírez, 2010; Santhalakshmy, Don Bosco, Francis, & Sabeena, 2015; Tan, Kha, Parks, Stathopoulos, & Roach, 2015). In the case of Aloe vera samples dehydrated either by RWD (Fig. 1d) or RZD (Fig. 1e) a smooth and flaky morphology with uniform thickness was observed. Nindo and Tang (2007) and Caparino et al. (2012) described a similar flaky morphology in carrot and mango purees dehydrated by RWD, respectively. According to these authors, the uniformity of the flake thickness is the result of a controlled feeding of concentrate using a spreader bar at the inlet section of the refractance-window dryer. 3.1.2. Color characteristics of dehydrated Aloe vera Spectral color information, used to analyze the physical appearance of fresh and dehydrated Aloe vera samples, is summarized in Table 1. It was observed that the reference and SD samples, with a L value of ∼90, were brighter than Aloe vera samples processed by IFD, RWD and RZD, which showed L values lower than 75. Similar results have also been observed in samples of strawberry puree (Abonyi et al., 2002) and mango juice (Caparino et al., 2012) dehydrated by spray-, freeze- and refractance window-drying. With regard to the Chroma (C) and Hue (H◦ ) parameters, the reference sample presented the lowest C value (∼11) and the highest H◦ value (100), whereas processed samples presented C and H◦ val-

ues ranging from 13.5 to 28.9 and from 74.0 to 87.1, respectively. A high H◦ value together with a low C value is often indicative of a dull color (Caparino et al., 2012). The total color change (E) was also calculated in order to quantify the potential color modification of the sample promoted by the different drying procedures in comparison with the reference sample. According to this parameter, SD Aloe vera sample exhibited the minor color change (5.8), distantly followed by Aloe vera sample dehydrated by RWD (18.6), and those processed by IFD (27.1) and RZD (30.7), which exhibited a considerable color modification. 3.2. X-ray diffraction analysis X-ray diffraction is a common technique applied to confirm the crystalline–amorphous state of dehydrated products. In general, a crystalline material shows a series of sharp peaks, while an amorphous product produces a broad background pattern. The X-ray diffraction results showed an amorphous structure for all Aloe vera samples, both fresh and processed, and no crystalline peaks were formed (data not shown). This observation is in agreement with Ray and Aswatha (2013) who observed an amorphous structure in freeze dried Aloe vera gel. Furthermore, similar X-ray patterns have also been observed in mango samples dehydrated by spray-, freeze-, drum- and refractance window-drying (Caparino et al., 2012). 3.3. Effect of the drying processes on the functional properties of acemannan The great industrial value of Aloe vera gel is mainly related to the high capacity of the Aloe vera polymers to retain water and oil (Rodríguez-González et al., 2011). However, it is known that chemical, mechanical and thermal processing may alter the physico-chemical properties of the acemannan polymer (Chokboribal et al., 2015; Lim & Cheong, 2015; Minjares-Fuentes et al., 2016). For this reason, the functional properties of acemannan from Aloe vera gel, dehydrated by the different drying procedures, were determined. As can clearly be observed in Fig. 2, swelling (Sw), water retention (WRC) and fat adsorption (FAC) capacities were significantly reduced in all cases, by the different drying processes compared with the reference sample (p < 0.05). Regarding the hydration-related properties, the reference sample exhibited a swelling value of ∼40 mL/g acemannan and a WRC of ∼7.5 g H2 O/g acemannan. In the case of the processed samples, a decrease of around 80% was observed for the Sw values of RWD and SD samples, and even a higher reduction (>90%) in the case of the RZD and IFD samples (Fig. 2a). Likewise, WRC decreased around 50% for RWD sample, while for SD, IFD and RZD samples the reduction reached about 70–75% (Fig. 2b). The decrease of hydration-related properties, and in particular of the WRC, could lead to the loss of some beneficial properties associated with Aloe vera, such as the reduction of blood glucose (Elleuch et al., 2011; Ma & Mu, 2016; Mackie, Rigby, Harvey, & Bajka, 2016; Moyano et al., 2016). Previously, Chokboribal et al. (2015) observed that fully acetylated acemannan exhibited a hydrophilic nature with good hydration properties which decreased when deacetylation occurred. Furthermore, several authors suggested that the solidification of the Aloe vera layer observed after applying a drying procedure might be the result of the deacetylation process of acemannan (Dea, Clark, & McCleary, 1986; Lim & Cheong, 2015) which could be a possible explanation for the reduction of the hydration properties of the acemannan polymer observed in this study. With regard to the FAC, the reference sample presented a value of ∼35 g oil/g acemannan whereas FAC values of the processed samples were, in all cases, lower than 5 g oil/g acemannan (Fig. 2c).

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331

Fig. 1. Microstructure of samples from (a) Aloe vera reference and processed samples obtained by (b) spray-drying, (c) industrial freeze-drying, (d) refractance window-drying and (e) radiant zone-drying.

Table 1 Color parameters (L, a*, b*), Chroma (C), Hue angle (H◦ ) and total color change (E) values of Aloe vera reference and processed samples obtained by the different drying procedures. Sample

L

a*

b*

C

H◦

E

Reference SD IFD RWD RZD

87.8 ± 1.2 90.5±0.0 70.1 ± 0.2 74.0 ± 0.6 62.5 ± 0.8

−3.2 ± 0.2 0.6 ± 0.0 7.2 ± 0.1 4.0 ± 0.1 6.9 ± 0.2

10.4 ± 0.6 13.5 ± 0.3 28.0 ± 0.1 20.5 ± 0.2 24.3 ± 0.7

10.9 ± 0.7 13.5 ± 0.4 28.9 ± 0.2 20.9 ± 0.2 25.3 ± 0.8

107.4 ± 0.1 87.1 ± 0.1 75.6 ± 0.2 78.9 ± 0.4 74.0 ± 0.3

0.0 ± 0.0 5.8 ± 0.4 27.1 ± 1.4 18.6 ± 1.7 30.6 ± 0.8

SD: Spray-Drying, IFD: Industrial Freeze-Drying, RWD: Refractance Window-Drying and RZD: Radiant Zone-Drying.

These results are consistent with those of Femenia et al. (2003), who observed that the FAC of Aloe vera was reduced during convective drying as air-drying temperature increased. This behavior was justified since structural and compositional changes in the main

Aloe vera polysaccharides, and particularly those affecting acemannan, caused by thermal processes such as drying, seem to reduce the ability to capture oil (Femenia et al., 2003; Rodríguez-González et al., 2011). It is worth pointing out that the efficiency of Aloe vera

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(a)

250

Yield (mg Acemannan / g dehydrated sample)

50

Swelling

( mL / g acemannan )

40

30

20

10

200

150

100

50

0 REFERENCE

SD

IFD

RWD

RZD

Sample

(b)

REFERENCE

10

( g water / g acemannan )

Water Retention Capacity

6

RWD

RZD

bioactive polysaccharide was isolated from fresh and processed samples and subjected to carbohydrate, methylation and 1 H NMR analysis.

4

0 REFERENCE

SD

IFD

RWD

RZD

Sample 40

30 ( g oil / g acemannan )

IFD

Fig. 3. Acemannan yield from Aloe vera reference and processed samples obtained by the different drying procedures: spray drying (SD), industrial freeze drying (IFD), refractance window drying (RWD) and radiant zone drying (RZD).

2

Fat Adsorption Capacity

SD

Sample

8

(c)

0

20

3.4.1. Yield As can be observed in Fig. 3, the acemannan yield was significantly reduced by the different drying procedures in comparison with the reference sample (p < 0.05); although, no significant differences were observed among the different processed samples (p > 0.05). These results are in agreement with Femenia et al. (2003) who observed a considerable decrease in the acemannan yield as the process temperature increased. Thus, these authors reported that the acemannan yield decreased from ∼11% up to ∼27% when air-drying temperature increased from 30 to 90 ◦ C. Further, Sriariyakul et al. (2016) observed a considerable reduction of the acemannan content, higher than 40%, when Aloe vera puree was dehydrated using hot air in combination with far infrared radiation and a high voltage electric field. In contrast, Rodríguez-González et al. (2011) observed that the acemannan yield increased during pasteurization of Aloe vera gel, probably as a result of the generation of new hydrogen bonds between acemannan chains and different mannose-containing oligosaccharides.

10

0 REFERENCE

SD

IFD

RWD

RZD

Sample Fig. 2. Functional properties determined for acemannan polysaccharide obtained from Aloe vera gel reference and processed samples obtained by the different drying procedures. (a) Swelling (Sw), (b) water retention capacity (WRC) and (c) fat adsorption capacity (FAC).

polysaccharides in binding organic molecules might play an important role in its reported capacity to reduce levels of cholesterol, carcinogens and other toxic compounds (Rodríguez-González et al., 2011). 3.4. Effect of the drying process on acemannan In order to evaluate the main effects of SD, IFD, RWD and RZD procedures on the composition and structure of acemannan, this

3.4.2. Carbohydrate composition The results of the carbohydrate analysis of isolated acemannan, from both reference and processed Aloe vera samples, are summarized in Table 2. As can be seen, mannose was the predominant sugar, followed by glucose and also to a minor extent by galactose. Mannose from the reference sample accounted for approximately 75% of the total sugars, the remaining 25% being formed mainly of glucose (∼22%) and galactose (∼3%). The content of mannose was similar to the values reported by Chang, Chen, and Feng, (2011) and Rodríguez-González et al. (2011), but relatively higher than the percentage reported by Chokboribal et al. (2015). Regarding the processed samples, slight but significant differences were observed in the carbohydrate profile of the acemannan, depending on the drying procedure applied. Thus, in the case of the RWD sample, acemannan exhibited similar mannose content to that of the reference sample (p > 0.05). However, a small but significant loss of mannose units (∼4–5%) was detected for samples dehydrated by SD, IFD and RZD (p < 0.05), but no significant changes between SD, IFD and RZD samples (p > 0.05). These results are in agreement with Femenia et al. (2003) who observed that a con-

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333

Table 2 Carbohydrate composition (mol%) of acemannan polysaccharide isolated from Aloe vera reference and processed samples obtained by the different drying procedures.

Man Gal Glc

Reference

SD

IFD

RWD

RZD

75.4 ± 2.2 2.9 ± 0.5 21.7 ± 0.8

70.0 ± 1.1 0.6 ± 0.1 29.4 ± 0.8

70.5 ± 1.7 0.3 ± 0.1 29.2 ± 2.7

74.3 ± 2.2 0.9 ± 0.2 24.9 ± 2.1

71.1 ± 1.1 1.9 ± 0.5 27.0 ± 1.9

SD: Spray-Drying, IFD: Industrial Freeze-Drying, RWD: Refractance Window-Drying and RZD: Radiant Zone-Drying. Man: mannose, Gal: galactose, and Glc: glucose.

Table 3 Methylation analysis (mol%) of isolated acemannan polysaccharide from Aloe vera reference and processed samples obtained by the different drying procedures. Reference

SD

IFD

RWD

RZD

Man Terminal 1,4 1,6 1,3,4 1,4,6 1,3,4,6

0.2 70.3 1.3 1.6 1.3 0.2

0.4 66.7 0.9 0.4 0.1 0.1

0.5 65.9 0.9 0.9 0.1 0.1

0.4 69.4 1.1 0.6 0.2 0.1

0.5 67.4 0.8 0.5 0.1 0.2

Gal Terminal 1,6 1,3,4 1,3,6 1,4,6

1.3 0.3 0.4 0.4 0.3

0.1 n.d. 0.2 0.1 0.1

0.1 n.d. 0.1 0.1 0.1

0.2 n.d. 0.2 0.2 0.1

0.4 n.d. 0.3 0.1 0.1

Glc 1,4 1,3,4 1,4,6 Glucitol

17.0 0.5 4.2 0.3

23.7 0.2 6.1 0.2

24.5 0.1 5.9 0.2

21.7 0.2 4.5 0.2

22.2 0.3 6.4 0.2

MW (kDa)

49

26

24

26

23

n.d. = no detected. SD: Spray-Drying, IFD: Industrial Freeze-Drying, RWD: Refractance Window-Drying and RZD: Radiant Zone-Drying. Man: mannose, Gal: galactose, and Glc: glucose. MW: molecular weight.

vective drying procedure reduced the content of mannose units, suggesting the degradation of the acemannan polymer. Moreover, a considerable decrease in galactose was observed in all processed samples, reaching up to ∼90% in the case of the IFD sample, indicating a considerable degradation of the galactose side-chains, which are characteristic of the acemannan polymer (Chokboribal et al., 2015; Manna & McAnalley, 1993). Overall, these results suggest that the acemannan polymer was not only affected by the high temperature applied, but also by other factors such as drying time and the heat transfer mechanism (convection, conduction and radiation). Thus, the SD process occurs when small liquid droplets come into immediate contact with a stream of air at very high temperature, the residence time in the spray drier being in the order of seconds (Filkova & Mujumdar, 1995; Lee, 1983; Toledo, 2007). On the other hand, in the RWD and RZD procedures, the thermal energy is transferred to the wet product by radiation and conduction, the drying time being, generally, in the order of minutes (Chakraborty, Savarese, Harbertson, Harbertson, & Ringer, 2009; Nindo & Tang, 2007). 3.4.3. Methylation analysis In order to gain more insight into these observations, glycosidic linkage analysis was performed on the isolated acemannan polymer from the different samples. The results of the methylated polymers obtained from the different drying procedures are shown in Table 3. Relative sugar mole ratios obtained from alditol acetates were consistent with those from methylated alditol acetates. The presence of only a few types of methylated ethers from each sugar and the virtual absence of unmethylated monomers in the

hydrolysates of the methylated acemannan fractions indicated a complete methylation. Methylation analysis revealed important structural differences among the acemannan polymers obtained from the different drying procedures. Despite the fact that (1,4)-linked mannosyl residue was predominant in all samples, the residue was degraded by the different drying methods applied. Further, significant differences were found in the average molecular weight (MW), the degree of acetylation and also in the abundance of side chains. The effect of the different drying procedures on the molecular weight (MW) of acemannan polymer was determined from the ratio (1,4)-, (1,3,4)-, (1,4,6)- and (1,3,4,6)-linked residues to terminally linked mannosyl units (Femenia et al., 1999). Thus, a MW of 49 kDa was estimated for acemannan from the fresh sample whereas the average MW of acemannan polymer from processed samples varied from 23 to 26 kDa. Previously, Medina-Torres et al. (2016) observed that high temperature of processing together with the shear-forces present in the spray-drying chamber promoted a significant reduction of the MW of polysaccharides from Aloe vera mucilage. In fact, it has been observed that shear-forces might cause the scission of branching, leading to a MW reduction of branched molecules (Cave, Seabrook, Gidley, & Gilbert, 2009). Further, a significant decrease of (1,3,4)-linked mannosyl residues was detected in the acemannan polymer, being around 40% in the case of the IFD sample, and up to more than 60% for the SD and RZD Aloe vera samples. These losses were accompanied by a considerable decrease in (1,4,6)-linked mannosyl units. These residues may correspond to the acetylation of the polysaccharide backbone since acetyl groups have been detected at C3 or C6 of mannose units of the active component acemannan (Campestrini et al., 2013; Chokboribal et al., 2015; McAnalley, 1993). Several authors have suggested that the deacetylation process could be implicated in the MW reduction of different acetylated polymers (Chen, Li, & Li, 2011; Du, Li, Chen, & Li, 2012; Jian, Siu, & Wu, 2015). This could be one of the main causes of the significant reduction observed in the functional properties related to the acemannan polymer. In addition, lower recoveries of galactosyl residues were detected in all dehydrated samples. These observations together with the loss of (1,3,4,6)-linked mannosyl units suggested a lower degree of branching, in particular of galactose side-chains since galactose units attached to C6 of acetylated mannose residues have been detected in Aloe acemannan (Chokboribal et al., 2015; Manna & McAnalley, 1993). The observed deacetylation process together with the lower degree of branching would support the modification of the topology of acemannan, which may affect its biological properties. In fact, the distribution of acetyl groups and galactosyl units along the main chain can have a significant effect on the interactive properties of mannans (Dea et al., 1986). 3.4.4. 1 H NMR analysis NMR technique has shown itself to be an essential tool for assessing the identity and the quality of Aloe vera gel preparations (Bozzi et al., 2007; Chokboribal et al., 2015; Diehl & Teichmuller, 1998). Fig. 4 shows the 1 H NMR spectra of acemannan polymer

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Fig. 4. 1 H NMR spectra of acemannan purified polysaccharide from Aloe vera reference and processed samples obtained by the different drying procedures spray drying (SD), industrial freeze drying (IFD), refractance window drying (RWD) and radiant zone drying (RZD).

corresponding to the reference and processed Aloe vera samples. The signals corresponding to malic acid and acemannan polymer, two main natural components found in Aloe vera gel, are clearly present in the NMR spectrum of all samples. In the 1 H NMR spectrum, the acetyl groups of acemannan generate a characteristic signal (2.00–2.26 ppm), which can be considered as the fingerprint of Aloe vera (Campestrini et al., 2013; Diehl, 2008; Diehl & Teichmuller, 1998). As can be observed in Fig. 4, the corresponding signal of acemannan polymer from all processed samples was very low in comparison with the reference sample. Similar results were observed by Bozzi et al. (2007) when the quality of different commercial Aloe vera powders was evaluated. In addition, a quantitative analysis was performed to determine the degree of acetylation of acemannan. The results showed that the relative degree of acetylation of acemannan was reduced around 40% in the case of the IFD sample. Meanwhile, this reduction was of 70, 52 and 60% in SD, RWD and RZD samples, respectively. Interestingly, these results correlate well with the losses of (1,3,4)-linked mannosyl residues observed by methylation analysis, confirming the deacetylation of acemannan promoted by the different drying procedures evaluated. The deacetylation of the acemannan polymer promoted by different drying processes has also been observed by other authors using FTIR analysis (Lim & Cheong, 2015; MinjaresFuentes et al., 2016; Sriariyakul et al., 2016).

4. Conclusions The effects of different drying methods, in particular, SD, IFD, RWD and RZD, on the main bioactive polymer from Aloe vera, known as acemannan, were evaluated. The results indicated that the acemannan polymer was severely affected by the different drying methods. Overall, the different drying procedures reduced the content of acemannan by around 40%. Regarding the carbohydrate composition, small but significant losses of mannose, the main sugar present in acemannan, were caused by all the drying processes evaluated. Moreover, galactose, the main sugar forming

the side chains of acemannan, was severely degraded up to trace amounts in the case of samples processed by SD, IFD and RWD. In fact, methylation analysis revealed that all drying procedures promoted considerable losses of (1,3,4)-, (1,4,6)- and (1,3,4,6)mannosyl residues, suggesting the deacetylation of mannose units, and also, suggesting the reduction of the degree of branching of acemannan. These losses led to a considerable reduction of the MW of acemannan polymer, from 49 in the fresh Aloe vera gel to around 23–26 kDa in the processed samples. In addition, 1 H NMR technique was a useful tool in estimating the degree of acetylation of acemannan of processed samples, since the reduction of the signal intensity corresponding to acetyl groups was concomitant with the loss of (1,3,4)-mannosyl residues observed after methylation analysis. Furthermore, the significant reduction of the functional properties of acemannan, in particular Sw, WRC and FAC, could be the result of structural and compositional modifications, such as deacetylation, reduction of the MW and loss of side chains, caused by the different drying processes. Overall, the physico-chemical alterations of the main bioactive acemannan polymer from Aloe vera, observed in all the drying procedures evaluated, may well have important implications for the beneficial activities attributed to the Aloe vera plant. Therefore, further studies of the biological significance of these modifications are needed. Acknowledgements The authors would like to acknowledge the financial support (AGL2012–34627 and RTA2015-00060-C04-00) of the Spanish Government (MICINN) and the European Regional Development Fund (FEDER); and the Government of the Balearic Islands for the research fellowship (FPI/1477/2012) of the “Conselleria d’Educació, Cultura i Universitats” and the European Social Fund (FSE). References Abonyi, B. I., Feng, H., Tang, J., Edwards, C. G., Chew, B. P., Mattinson, D. S., et al. (2002). Quality retention in strawberry and carrot purees dried with refractance windowTM system. Journal of Food Science, 67, 1051–1056.

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