Investigation of Structural Features of Prunes (Prunus domestica) Insoluble Dietary Fibers

Structural characteristics of dietary fi bers are closely related to its biological functions in the human body. Previously, soluble dietary fi bers from prunes were extracted and characterized. In this work, structural analysis of insoluble dietary fi bers was conducted using monosaccharide composition, methylation, molecular weight determination and 13C-NMR data. Prunes’ non-cellulosic insoluble fi bers were found to contain, a pectic type I arabinogalactan, a fucogalactoxyloglucan and a heteroxylan. These fi ndings suggest that insoluble dietary fi bers can be composed by some pectic polysaccharides besides cellulose and hemicellulosic polymers. This paper brings important structural features of insoluble dietary fi bers from prunes that may be of biological signifi cance. Research Article Investigation of Structural Features of Prunes (Prunus domestica) Insoluble Dietary Fibers Thaisa Moro Cantu-Jungles, Marcello Iacomini and Lucimara MC Cordeiro* Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, CP 19.046, CEP 81.531-980, Curitiba, PR, Brazil Dates: Received: 12 May, 2017; Accepted: 01 June, 2017; Published: 02 June, 2017 *Corresponding author: Lucimara MC Cordeiro, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, CP 19.046, CEP 81.531980, Curitiba, PR, Brazil, Tel: +55 (41) 33611655; Fax: +55 (41) 32662042; E-mail:


Introduction
Dietary fi bers are carbohydrate polymers composed by ten or more monomeric units, which are not hydrolyzed by endogenous enzymes in the small intestine and can be partially or totally fermented in the large intestine of humans [1,2] .
Health benefi ts associated with dietary fi ber consumption result from its low caloric content, physical effects in the stomach and small intestine and fermentation in the colon. Dietary fi bers can be classifi ed as either water soluble and mostly fermentable (such as pectin) or insoluble, less fermentable, and nonviscous (such as cellulose, lignin, and some of the hemicelluloses) [3].
Soluble fi bers are generally known to increase viscosity of the stomach and small intestine content, improving satiety, reducing post-prandial glycaemia and preventing reabsorption of bile acids, thus reducing circulating blood cholesterol levels. Moreover, due to its high fermentability, soluble fi bers can positively modulate the colonic microfl ora preventing pathologies such as infectious diseases, allergy or asthma, colon cancer, obesity, liver disease, diabetes and infl ammatory bowel disease. On the other hand, insoluble fi bers are known to be poorly fermentable, but are able to increase fecal bulk and decrease transit time, increasing stool frequency [4].
However, not all soluble/insoluble fi bers behave in the same way. For example, the soluble dietary fi ber inulin was shown to increase stool frequency [5]. Likewise, insoluble fi bers, such as resistant starch, are highly fermented by the human gut microbiota [6]. This may be because carbohydrate polymers, as dietary fi bers, represent the most heterogeneous and diverse group of associated molecules found in nature. Therefore, not only water solubility, but other structural features such as monosaccharide composition, linkage types between monosaccharides, size of the polymers, branching patterns, etc., also dictates their biological activities [6,7]. Thus, the knowledge about chemical structure of food dietary fi bers is important to explore how it interacts with the human body and possibly produce health benefi ts.
Prunes, the dried fruits of plums (Prunus domestica), possess as high as 62.7% of carbohydrates and its consumption is related to laxative effects, reductions in cardiovascular risk and sugar metabolism control that may be associated with dietary fi ber constituents [8]. We have previously carried out the isolation and characterization of soluble dietary fi bers found in prunes [9] and the pectic polysaccharides homogalacturonan and rhamnogalacturonans with type I arabinogalactans side chains have been described. In this work, our objective was to further analyze the chemical structure of insoluble dietary fi bers through monosaccharide composition, linkage analysis, molecular weight determination and 13 C-NMR data and thus expand our knowledge about their structural characteristics.

Extraction and purifi cation of polysaccharides
Prunes (2 kg) were blended and exhaustively extracted with water (6 L) at 100 ºC under refl ux for 2 h as previously described [9] to remove soluble dietary fi bers (SDF). The residue of hot water extraction, containing the insoluble dietary fi bers (IDF), was separated after centrifugation (8000 rpm, 15 min at 15 °C). To solubilize some of the polysaccharides present in the IDF, mainly hemicelluloses, the residue was submitted to alkaline extraction with KOH 10% (2 L each, 3x) at 100 ºC under refl ux for 2 h. Alkaline extract was then obtained by centrifugation (8000 rpm, 15 min at 15 °C), followed by neutralization with HOAc, dialysis and lyophilization, resulting in a polysaccharide fraction named herein as PK (prunes's alkaline extract) ( Figure  1). The residue remaining of this extraction contained cellulose that has not been solubilized with this treatment.
As a fi rst step of fractionation, a freeze-thaw treatment was applied in fraction PK, to give cold-water soluble (SPK) and insoluble (PPK) fractions. In this procedure, the sample was frozen and then thaw at room temperature followed by centrifugation (8000 rpm, 15 min at 15 °C).
Fraction SPK was further fractionated by Fehling's treatment. Briefl y, it was dissolved in distilled water and treated with Fehling's solutions [10] resulting, after centrifugation (8000 rpm, 15 min at 15 °C), in a xyloglucan-copper complex as the pellet (fraction PF-SPK) and a soluble fraction (SF-SPK). After neutralization with HOAc, both fractions were dialyzed against tap water and deionized with cation exchange resin. The fraction PF-SPK was later purifi ed by ultrafi ltration through a membrane with cut-off of 100 kDa (PLHK04710-Ultracel, Millipore), yielding the fractions PF-SPK-100E (eluted in 100 kDa) and PF-SPK-100R (retained in 100 kDa) ( Figure 1).
Fraction PF-SPK-100R was further purifi ed by anion exchange chromatography. It was dissolved in distilled water (50 mg/mL), centrifuged (12000 x g, 10 min at 10 ºC) and the supernatant applied to a DEAE-Sepharose Fast Flow column (3.0 cm×25 cm). The column was eluted with distilled water (F1) followed by 4.0 M NaCl solution (F2) at a fl ow rate of 1.5 mL/min. Polysaccharides in the eluted fractions were detected using phenol-sulfuric acid method [11] . The obtained fractions were concentrated and freeze-dried.
The yields were expressed as % based on the weight of dried prunes pulp that was submitted to extraction (1400 g) ( Figure 1).

Sugar composition
Polysaccharides' neutral monosaccharides composition was determined by hydrolysis with 2 M TFA (8 h/100 °C), conversion into alditol acetates using successive NaBH 4 reductions, and acetylation with Ac 2 O-pyridine (1:1, v/v, 2 mL -100 °C, 30min). A Varian gas chromatograph and mass spectrometer (Saturn 2000R), with He as carrier gas were used for analysis. For quantitative analysis, a capillary column (30 m x 0.25 mm i.d.) of DB-225 was held at 50 ºC during injection for 1 min, then programmed at 40 ºC/min to 220 ºC and held at this for 19.75 min.
The determination of uronic acid contents was conducted according to the m-hydroxybiphenyl method [12].

Determination of homogeneity and molecular weight of polysaccharides
The homogeneity of polysaccharides was evaluated by high

Methylation analysis of polysaccharide
Fraction PF-SPK-100R was O-methylated as described by Ciucanu and Kerek [13]. The per-O-methylated polysaccharide was further submitted to methanolysis in 3% HCl-MeOH (80 °C, 2 h) followed by hydrolysis with H 2 SO 4 (0.5M, 12 h) and neutralization with BaCO 3 . The material was then reduced an acetylated as described above for monosaccharides composition, except that NaBD 4 was used for reduction. The profi les and retention times of partially O-methylated alditol acetates were used for identifi cation [14].

Nuclear magnetic resonance (NMR) spectroscopy
Spectra of 13 C NMR were acquired using a Bruker spectrometer (DRX 400 MHz AVANCE III NMR -Bruker Daltonics, Germany).
Samples were dissolved in D 2 O and placed in a 5 mm inverse gradient probe, at 70 °C for analysis. Chemical shifts were expressed as  ppm and acetone CH 3 group's resonance was used as internal standard ( 30.2). The spectra were handled using the Topspin ® (Bruker) computer program.

Results and Discussion
In order to extract water insoluble polysaccharides from prunes, the residue of prunes' water extraction was submitted to alkaline extraction, which resulted, after dialysis, in fraction PK. This was further fractionated through freezethaw treatment followed by centrifugation to give rise to a supernatant fraction (SPK) and a precipitated fraction (PPK) (Figure 1). The latter presented arabinose and glucose as main monosaccharides (Table 1), but NMR analysis was not possible due to its high insolubility in different solvents.
On the other hand, SPK presented arabinose and galactose as main monosaccharides ( Table 1). The 13 C-NMR spectrum (Figure 2A The anomeric signal at  107.6 was assigned to units of -L-Araf [17]. These data could indicate the presence of a type I arabinogalactan (AG-I) in fraction SPK, already reported for prunes' water extract [18]. However, besides the signals of an AG-I, SPK presented a diversity of other anomeric signals in the region between  98.0 and  104.0 (Figure 2A), as well as signifi cant content of glucose and xylose according to the monosaccharide analysis (Table 1), indicating the presence of another polysaccharide.
In order to separate the different polymers present in SPK, it was treated with Fehling's solution, resulting in a precipitated fraction (PF-SPK) and a supernatant fraction (SF-SPK) ( Figure  1). As previously observed for water extracts [18], the AG-I remained in the Fehling supernatant as could be seen in the 13 C-NMR ( Figure 2B) and monosaccharide analysis (Table 1), of fraction SF-SPK. The fraction precipitated with Fehling solution PF-SPK, had glucose and xylose as main monosaccharides (Table 1). Besides, anomeric signals of -Glcp and -Xylp could be seen at  102.3 and  99.4/98.9, respectively, in the 13 C-NMR spectrum of PF-SPK [19] ( Figure 2C). These data indicate that while the AG-I present in SPK remained soluble after Fehling treatment, the fraction containing xylose and glucose was precipitated.
Once PF-SPK had a heterogeneous profi le in HPSEC (data not shown), it was further fi ltrated with 100 kDa cutoff Milipore membrane, yelding an eluted fraction PF-SPK-100E and a retained fraction PF-SPK-100R (Figure 1). Only fraction PF-SPK-100R presented a homogeneous elution profi le when analysed by HPSEC (Figure 3). The calculated molecular weight was 66 kDa.
The monosaccharide composition of PF-SPK-100R showed glucose and xylose as main sugars, and minor amounts of arabinose, galactose and fucose (Table 1). Methylation data of PF-SPK-100R is presented on Table 2  PF-SPK-100R -3.5 12.7 34.0 11.2 38.6 a % of peak area relative to total peak areas, determined by GC-MS. b Determined using the m-hydroxybiphenyl method [12]. The presence of xylan-xyloglucan complexes has been previously identifi ed in the cell walls of olive pulp [24]. In our research group, a xyloglucan and an acid heteroxylan were also found together in alkaline extracts from starfruit (Averrhoa carambola L.) and separated through anion exchange chromatography (unpublished data). In fraction PF-SPK-100R however, due to the absence of uronic acid linked to the xylan backbone, no polymers were retained after anion exchange chromatography (Fraction F1) making its separation from the xyloglucan not possible (data not shown). Moreover, the presence of the xylan and xyloglucan was also observed in the fraction PF-SPK-100E ( Figure 5A), which demonstrated a similar 13 C-NMR spectrum as that of fraction PF-SPK-100R ( Figure 5B). Attempts were also made to separate these polymers through ultrafi ltration with 50, 30 and 10kDa membranes. However, all the retained and eluted fractions showed the presence of both polymers.
The 13 C-NMR spectrum of PF-SPK-100R ( Figure 5B) is in accordance with methylation data and presented the main signals related to the xylan and xyloglucan mixture. Anomeric signals related to (1→4) and (1,4→6)-linked -Glcp units that form the main chain of the xyloglucan are found at  102.3 and  103.2, respectively [25,26]. The signal at  98.9 was assigned to anomeric carbons of terminal and 2-O-substituted -Xylp units, while that at  99.4 and  15.8 can be assigned to C-1 and C-6 of terminal Fucp units, respectively. In addition, the signal at  104.4 can be assigned to C-1 of terminal -Galp units [25]. Signals from the heteroxylan could be seen at  101.   Some biological activities have already been attributed to type I arabinogalactans, such as immunological [28][29][30] and anti-ulcer activities [9,31]. Xyloglucan from different sources were also previously shown to display biological activities such as hypolipidemic [32], anti-tumoral [33,34], immunomodulatory [35][36][37][38] and hypoglycemic [39][40][41]. It's noteworthy, that some of these effects, such as hypolipidemic and hypoglycemic, were previously found in prunes, however, the responsible components were not fully resolved [8].

Conclusion
Prunes' non-cellulosic insoluble fi bers were found to contain, a pectic type I arabinogalactan, a fucogalactoxyloglucan and a heteroxylan, suggesting that IDF can be composed by some pectic polysaccharides besides cellulose and hemicellulosic polymers. Moreover, this paper brings important structural features of insoluble dietary fi bers from prunes that may be associated to biological functions, and provides new insights into the diversity of fruit hemicellulosic polymers.