Pectin-non-starch nanofibers biocomposites as novel gastrointestinal-resistant prebiotics

International Journal of Biological Macromolecules 94 (2017) 131–144

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Pectin-non-starch nanofibers biocomposites as novel gastrointestinal-resistant prebiotics Alireza Chackoshian Khorasani, Seyed Abbas Shojaosadati ∗,1 Biotechnology Group, Faculty of Chemical Engineering, Tarbiat Modares University, Tehran, Iran

a r t i c l e

i n f o

Article history: Received 31 July 2016 Received in revised form 4 October 2016 Accepted 5 October 2016 Available online 6 October 2016 Chemical compounds studied in this article: Pectin (PubChem CID: 441476) Chitin (PubChem CID: 6857375) Cellulose gel (PubChem CID: 14055602) Keywords: Pectin Nanochitin Nanolignocellulose Bacterial nanocellulose Biocomposite Gastrointestinal resistant

a b s t r a c t Incorporation of nanofibers of chitin (NC), lignocellulose (NLC) and bacterial cellulose (BNC) in pectin was studied to improve prebiotic activity and gastrointestinal resistance of the pectin-nanofibers biocomposites for protection of probiotics under simulated gastrointestinal conditions. The biocomposites were prepared using various compositions of pectin and nanofibers, which were designed using D-optimal mixture method. The incorporation of the nanofibers in pectin led to a slow degradation of the pectinnanofibers biocomposites in contrast to their rapid swelling. AFM analysis indicated the homogenous distribution of interconnected nanofibers network structure in the pectin-nanofibers biocomposite. FTIR spectra demonstrated fabrication of the biocomposites based on the inter- and intra-molecular hydrogen bonding and ionic interaction of pectin-Ca2+ . XRD patterns revealed the amorphous structures of the biocomposites as compared to the crystalline structures of the nanofibers. Among the compositions, the optimal compositions were as follows: 60% pectin + 40% NC, 50% pectin + 50% NLC and 60% pectin + 40% BNC, where the prebiotic score, probiotic survival under simulated gastric and intestinal conditions were optimum. The optimal biocomposite pectin-NC exhibited the highest survival of the entrapped probiotic bacteria under simulated gastric (97.7%) and intestinal (95.8%) conditions when compared with the corresponding to free cells (76.2 and 73.4%). © 2016 Published by Elsevier B.V.

1. Introduction The administration of probiotics is both viable and sufficiently numerous in the intestinal tract which is associated with passage through the gastrointestinal conditions. It is necessary to support health-promoting claims attributed to the beneficial effects of probiotics on the human gut flora. Designing entrapment biocomposites for the protection of probiotics with the aim of providing a successful delivery is an emerging method to reduce cell death during GI passage. Majority of this technology depends on properties of the matrix ingredients. Various polymeric biocomposites fabricated using prebiotic polysaccharides cannot only protect probiotics against harsh conditions by retaining biocomposites structure before degradation, disintegration or dissolution but also increase their growth in the intestine [1–3]. Among the polymers used for encapsulation, pectin as an aqueous soluble prebiotic, has gained so much attention in the food

∗ Corresponding author at: Biotechnology Group, Faculty of Chemical Engineering, Tarbiat Modares University, P.O. Box 14155-4838, Tehran, Iran. E-mail address: shoja [email protected] (S.A. Shojaosadati). 1 http://www.modares ac ir/en/Schools/chemE/academic staff/∼SHOJA SA. http://dx.doi.org/10.1016/j.ijbiomac.2016.10.011 0141-8130/© 2016 Published by Elsevier B.V.

and pharmaceutical industries for its excellent biocompatibility, biodegradability, pH sensitivity, nontoxicity, therapeutic properties, cost-effectiveness and ability to increase in strength of the mucoadhesion on large intestinal mucosa [4,5]. The matrix made of pectin composited with biopolymers is suitable for use as a probiotic delivery vehicle since resistance of pectin-based delivery vehicles against gastric and pancreatic enzymes preserves the entrapped cells passage through the GI tract and it is selectively fermented by colonic bacteria [5,6]. N-Acetyl-glucosamine units as derivatives of glucose are linked together by ␤ (1–4) linkages to make an aminopolysaccharide called chitin with the structure being analogous to cellulose [7]. Chitin is biocompatible and non-toxic for use in a variety of human applications [8]. By passing through the GI tract, chitin did not change in weight and shape whereas some of the polysaccharides such as chitosan and starch did [9]. In nature, chitin is considered as an insoluble component of many chitin–polymer biocomposites which increases hydrogen bonds between adjacent chains of polymers leading to greater strength [7]. The increasing interest in the use of chitin as a source of nanofibers is due to the intractable structure and inherent nature of supporting cell attachment [8,10,11]. Hence, nanochitin can be used in the fabrication of nanocomposites for encapsulation in the functional foods industry.

132

A.C. Khorasani, S.A. Shojaosadati / International Journal of Biological Macromolecules 94 (2017) 131–144

Lignocellulose as an insoluble, non-digestible fiber fraction, mainly including cellulose, hemicellulose and lignin, is less considered as a prebiotic source which plays an important role in bacterial population and fermentation in the gut. Although phenolic compounds of lignocellulose can have antimicrobial effects, a low level (1.25%) of them improves the growth of probiotic bacteria and reduces Escherichia coli population in the gut [12]. In addition, lignocellulose is a bulky fiber with high water-binding capacity that can increase stomach distension and thereby enhance satiation. In vitro studies indicate that only little amounts (0–5%) of lignocellulose are fermented by the human gut [13]. The use of nanolignocellulose as a nanofiber can modify composite properties which are attributed to the high surface of nanofibers and the hydrogen bonding between lignocellulose and other ingredients of the composite [14,15]. Hence, nanofibers of lignocellulose can be used for fabrication of entrapment biocomposites due to the simplicity of the manufacturing process, low cost, light weight and properties which make them suitable for this application [16]. Cellulose as an important biopolymer and the most abundant renewable resource is used for a variety of applications due to its biocompatibility, availability, biodegradability and sustainable production potential. Intrinsic properties, such as nanoscale dimension, high surface area, unique morphology and mechanical strength contribute to expansion of the application fields of nanocellulosic materials as components in fabrication of nanocomposites [17]. Recently, a new entrapment matrix obtained by the incorporation of pectin with bacterial cellulose nanofibers was developed. This bionanocomposite showed an increase in survival rate of probiotics in the GI conditions [3]. The nanofibers show a promising potential for solving some of the problems associated with the probiotics protection in harsh conditions. Due to their nano-scale diameters, the nanofibers are advantageous in applications where resistant surface of the entrapment matrix is needed to promote the residence time of probiotic cells in the GI environment to enhance the viability [4]. The aim of this work was to fabricate new entrapment biocomposites from pectin composited with prebiotic nanofibers for probiotic delivery into the GI tract. Biocomposites of pectinnanochitin (pec-NC), pectin-nanolignocellulose (pec-NLC) and pectin-bacterial nanocellulose (pec-BNC) were developed to entrap probiotic Bacillus coagulans. These composites were evaluated as prebiotic biocomposites and examined during exposure to simulated gastric and intestinal fluids. Their optimal compositions were determined using D-optimal mixture design to obtain optimum prebiotic activity and optimum survival in gastric and intestinal conditions, simultaneously. To the best of our knowledge, these produced biocomposites addressed a new approach in the preservation of probiotics in functional foods industry, and there is still no report in the literature related to the probiotics.

2. Materials and methods 2.1. Materials Probiotic strain Bacillus coagulans IBRC-M 10807 and enteric strain Escherichia coli IBRC-M 10208 were obtained from Iranian Biological Research Center (IBRC). Pectin was extracted from citrus peel and dried using previously established method [18]. Bacterial nanocellulose (BNC) with the average fibril diameter of 50 nm, nanochitin (NC) with the average fibril diameter of 30 nm, and nanolignocellulose (NLC) with the average fibril diameter of 65 nm were purchased from Nano Novin Polymer Co. (Sari, Iran). Pepsin from porcine gastric mucosa (0.7 FIP-U/mg), pancreatin from porcine pancreas (350 FIP-U/g Protease, 6000 FIP-U/g Lipase, 7500 FIP-U/g Amylase), and calcium chloride were acquired

Table 1 Mixtures of pectin and nanofibers for fabrication of the entrapment biocomposites. Mixture

Pectin (X1 ) (g/g biocomposite)

Nanofiber (X2 ) (g/g biocomposite)

1 2 3 4 5

0.9 0.8 0.7 0.6 0.5

0.1 0.2 0.3 0.4 0.5

from Merck (Darmstadt, Germany). Bile salts were supplied from Sigma–Aldrich (St. Louis, MO, USA). TSB was purchased from Oxoid Ltd. (Basingstoke, Hampshire, UK). All chemicals were used as received without any further purification. 2.2. Preparation of mixtures The various compositions of pectin with NC/NLC/BNC for fabrication of entrapment biocomposites were designed using Doptimal mixture design and given in Table 1. As concentrations showed in Table 1, the ingredients were suspended in deionized water and mixed together at 60 ◦ C to produce polysaccharide mixtures with the final concentration of 1% (w/v). The mixtures were agitated at 500 rpm for 60 min. Then, they were autoclaved at 121 ◦ C for 20 min. 2.3. Entrapment of probiotic B. coagulans was grown overnight in TSB medium at 37 ◦ C. The cells were centrifuged at 6000 rpm for 10 min, then washed with sterile saline solution (0.5%, w/v) and resuspended in the same solution. The sterilized mixture prepared (Section 2.2), was mixed with the cell suspension at a ratio of 1:1 (v/v). In order to obtain the probiotic entrapped in the biocomposite, the synbiotic (probiotic cells + polysaccharide mixture) suspension was added dropwise to 5% CaCl2 solution (crosslinking agent), then kept for 1 h at 4 ◦ C to form and harden the biocomposite. The synbiotic biocomposite was centrifuged at 12,000 rpm for 10 min and then dried using freeze-dryer for 48 h. 2.4. Preparation of simulated gastric and intestinal fluids Gastric and intestinal fluids were simulated as stated previously [19]. In brief, simulated gastric fluid (SGF) was prepared by suspending pepsin in sterile saline (0.5%, w/v) to a final concentration of 3 g/L and pH was adjusted to 2.00 using 1N HCl. Simulated intestinal fluid (SIF) was prepared by suspending pancreatin USP in sterile saline to a final concentration of 1 g/L and adding bile salts (4.5%, w/v) and pH was adjusted to 8.00 using sterile 1 N NaOH. 2.5. Characterization of biocomposite 2.5.1. Scanning electron microscopy (SEM) The surface morphology of the samples was identified using the scanning electron microscopy (SEM) (XL30, Philips, Netherlands). The samples were coated with gold using the sputtering technique to improve the conductivity of the samples. The dried samples were fractured and observed at beam energy of 20.0 kV. 2.5.2. Atomic force microscopy (AFM) The surface morphology of the entrapment biocomposite was analyzed in air at ambient temperature, using the contact mode of AFM (Veeco, Autoprobe CP research, USA) with a 5 × 5 ␮m scan size at a resolution <0.1 nm. Two statistical parameters of roughness,

A.C. Khorasani, S.A. Shojaosadati / International Journal of Biological Macromolecules 94 (2017) 131–144

Rq and Ra , were calculated using the data from the images with appropriate software. 2.5.3. Fourier transform infrared spectroscopy (FTIR) FTIR spectra of the samples were obtained using a FTIR spectrophotometer (Spectrum 100, Perkin Elmer, USA). The samples were incorporated with KBr (spectroscopic grade) and pressed into a 3 mm pellet. Spectral scanning was accomplished in the range of 4000–400 cm−1 . 2.5.4. X-ray diffraction (XRD) In order to determine crystallinity of the samples, wide-angle Xray diffraction data were measured using an X-ray diffractometer (X’Pert MPD, Philips, Netherlands) equipped with Cu K␣ radiation (␭ = 0.1542 nm; 40 kV and 30 mA). Patterns were recorded by monitoring diffractions within a 2␪ angle range 10◦ – 40◦ with the scan speed at 0.02◦ /s. 2.6. Prebiotic score of biocomposite Prebiotic score (P.S.) of each mixture was evaluated by the procedure previously established [20] with the some modifications. In order to determine P.S. of the prepared mixtures, the assay was carried out by adding 1% (v/v) of an overnight culture of the bacterial strains to separate tubes containing 1 ml of TSB with 0.1% (w/v) glucose or 0.1% (w/v) polysaccharide mixture prepared (Section 2.2), then incubated at 37 ◦ C under aerobic condition. After 24 h of incubation, the sample was enumerated on TSA. Each assay was repeated three times. The prebiotic score was calculated using the following Eq. [20]: P.S. = [(bacterialog(CFU/mL)ontheprebioticat24 h) − (bacterialog(CFU/mL)ontheprebioticat0 h)]/ [(bacterialog(CFU/mL)onglucoseat24 h) − (bacterialog(CFU/mL)onglucoseat0 h)]

(1)

2.7. Survivability of probiotic under gastrointestinal conditions The survivability of the bacterial cells in the simulated gastric and intestinal fluids was determined by the procedure reported [19,21] with some modifications. Under SGF or SIF, 1.0 mL of simulated gastric or intestinal fluid was transferred into the tube containing the synbiotic biocomposite and mixed well. Then, it was incubated at 37 ◦ C for 4 h in an incubator. After incubation under SGF or SIF, the sample was centrifuged at 12,000 rpm for 15 min and decanted. The reminder was serially diluted with 0.1% sterilized peptone water for cell counts. The diluted sample was transferred onto TSA plate and incubated at 37 ◦ C for 48 h under aerobic condition. Individual colonies appeared were counted as colony forming units per gram of the synbiotic biocomposite (CFU/g) and the results were reported as Log10 values. The probiotic survival rate under gastric or intestinal condition was determined using the following equation [22]: Survivability(%) = [bacterialog(CFU/g)afterexposureto gastricorintestinalcondition]/[bacterialog(CFU/g)before exposuretogastricorintestinalcondition] × 100

(2)

133

2.8. Swelling and degradation of biocomposite under gastrointestinal conditions The swelling and degradation of biocomposites in SGF and SIF were investigated by the procedure previously established [23] with the some modifications. In a typical experiment, empty tea bag was immersed in the digestive (SGF or SIF) medium for 24 h and hung to dry for 15 min. Then, the tea bag was blotted with paper towel to remove excess water and weighed (W1 ). Around 0.1 g of dried biocomposite was weighed (W2 ) and placed in the tea bag. The loaded tea bag was closed and immersed in the digestive medium, after which the loaded tea bag was taken out and blotted dry, and its weight was measured (W3 ). The swelling ratio of the biocomposite was calculated using the following equation: Swellingratio(g/g) = (W3 − W2 − W1 )/W2

(3)

As the procedure, after which the loaded tea bag was taken out and blotted dry, the biocomposite swelled was taken out the bag and completely dried. The weight of the dried biocomposite was measured (W4 ). The degradation of the biocomposite was calculated using the following equation: Degradation(%) = (W2 − W4 )/W2 × 100

(4)

2.9. Statistical analysis As described earlier, we followed D-optimal mixture design to design and optimize compositions of the biocomposites. Pectin (X1 ) and NC/NLC/BNC (X2 ) were defined as variables that influenced on the responses (Y) as follows: the difference of prebiotic score between B. coagulans and E. coli (P.S.), survivability of the entrapped probiotic under gastric and intestinal conditions. Component proportions were expressed as fractions of the mixture with a sum (X1 + X2 ) of one. The following polynomial equation of mixture cubic model was fitted to the data obtained from the experiments for the responses (Y). The polynomial equation used is: Y = ␤1 X1 + ␤2 X2 + ␤12 X1 X2 + ␤3 X1 X2 (X1 − X2 )

(5)

Where Y is the predicted response; ␤1 , ␤2 , ␤3 and ␤12 are constant coefficients for each linear and non-linear (interaction) term produced for the prediction cubic model. All experiments were carried out in triplicate and the obtained data were analyzed using Design-Expert (DX) (Version 7 trial, StatEase Inc., Minneapolis, USA) and expressed as mean standard error (SE). The statistical significance of the results was evaluated using analysis of variance (ANOVA) at the 95% confidence level. 3. Results and discussion 3.1. Characterization 3.1.1. Surface morphology of entrapment biocomposite Since SEM images taken from the nanofibers and biocomposites are similar, SEM images of NC and pec-NC biocomposites are represented. Fig. 1a and b show the fragile fracture surface of the biocomposite fabricated from only NC with average fibril diameter of 30 nm. Fig. 1c and d show pec-NC biocomposite fabricated with the optimal composition. The surface structure of pec-NC biocomposite is smooth coherent, compact and wrinkled when compared with NC biocomposite that has disrupted and patchy structures. This is attributed to the ability of the used polysaccharides to form hydrogen bonds, and ionic bonds between Ca2+ and negative functional groups of the constituents. Fig. 1e and f depict highly porous and unwrinkled surface of the pec-NC biocomposite treated under

134

A.C. Khorasani, S.A. Shojaosadati / International Journal of Biological Macromolecules 94 (2017) 131–144

Fig. 1. SEM images of (a, b) NC, (c, d) the entrapment biocomposite of pec-NC with the optimal composition and (e, f) the entrapment biocomposite after gastrointestinal digestion. Black arrows indicate B. coagulans entrapped within the biocomposite.

gastrointestinal fluids. B. coagulans entrapped within the biocomposite appeared after gastrointestinal treatment. This is due to degradation of the biocomposite by the acidic fluid and enzymatic digestion, thus removing materials which covered the probiotic cells. Since entrapment of B. coagulans within the bionanocomposite was carried out efficiently and the polysaccharides (pectin and chitin) completely covered the bacteria, the SEM image (Fig. 1f) rarely represents free bacteria. This image confirms the protecting role of the entrapment matrix which was resistant against digestion and did not release the entrapped probiotic cells. Most of the probiotics entrapped and covered by the matrix were not observed on the surface as free cells (Fig. 1e and f). 3.1.2. AFM analysis of entrapment biocomposite Since AFM images taken from the nanofibers and biocomposites are similar, AFM images of NC and pec-NC biocomposites are represented. Fig. 2a and b show topographical and phase-contrast images of the surface of pec-NC biocomposite with the optimal composition. AFM images confirm the nanoscale dimensions of the used nanofibers and display a three-dimensional arrangement of

the nanofibers. The images demonstrate that the nanofibers are intercalated into a pectin matrix. Moreover, they show a uniform morphology and a homogenous distribution of the nanofibrillar network structure, indicating a percolating NC network developed within the pec-NC biocomposite. The images present a relatively smooth surface with Ra and Rq values of 28.98 and 36.82 nm, respectively (Fig. 2a), and a continuous surface without pores and cracks and with good structural integrity. These surface properties are in agreement with the SEM images of the biocomposite. Therefore, AFM analysis indicates that the interconnected nanofibers network structure of NC, NLC and BNC is preserved and finely distributed in the entrapment biocomposite. 3.1.3. FTIR analysis of entrapment biocomposite Fig. 3 shows the FTIR spectra of the nanofibers and pectin constituting the entrapment biocomposites of pec-NC, pec-NLC and pec-BNC. Pectin, NC, NLC and BNC show characteristic spectra corresponding to their polysaccharide chemical structures. The intense

A.C. Khorasani, S.A. Shojaosadati / International Journal of Biological Macromolecules 94 (2017) 131–144

135

Fig. 2. (a) AFM topographical image and (b) phase-contrast image of the surface of entrapment biocomposite of pec-NC with the optimal composition.

Fig. 3. FTIR spectra of the nanofibers, pectin and optimal entrapment biocomposites fabricated using them.

bands at 3600–3000 cm−1 are attributed to the characteristic band for the stretching vibration of OH group. The bands at 1655–1600 and 1160–1020 cm−1 are attributed to the intramolecular hydrogen bond and C O bond, respectively. The vibrational absorption peaks of C H bond of monosaccharide are observed at 1000–850 cm−1 . The pectin composited with NC is determined by a band at 1629 cm−1 (COO− Ca2+ ) corresponding to pectin, and the bands at 3262 (NH), 1642 (amide I) and 1554 cm−1 (amide II) corresponding to NC [24–26]. The pectin composited with NLC is confirmed by the detected bands at 1600, 1506, 1426 (aromatic skeletal vibration),

1462 (C H deformations in lignin) and 1225 cm−1 (CO in lignin) corresponding to NLC [27]. 3.1.4. XRD analysis of entrapment biocomposite Fig. 4 shows the X-ray diffractograms of the nanofibers and pectin constituting the entrapment biocomposites of pec-NC, pecNLC and pec-BNC. The four peaks observed at 2␪ = 9.32◦ , 19.21◦ , 23.28◦ and 26.23◦ indicate the crystalline regions of NC and the peak at 2␪ = 19.21◦ corresponded to the region with more crystalline ordering [28,29]. NLC crystallinity is characterized by the three peaks shown at 2␪ = 16.11◦ , 22.25◦ and 29.14◦ and highlighted with the highly crys-

136

A.C. Khorasani, S.A. Shojaosadati / International Journal of Biological Macromolecules 94 (2017) 131–144

Fig. 4. X-ray diffractogram of the nanofibers, pectin and optimal entrapment biocomposites fabricated using them.

tallinity at 2␪ = 22.25◦ [30,31]. The X-ray pattern of BNC shows three crystalline peaks at 2␪ = 14.58◦ , 16.76◦ and 22.47◦ , which indicates the semi-crystalline nature of BNC. XRD spectra of the biocomposites do not show peaks corresponding to NC, NLC and BNC. This is due to the presence of pectin decreasing the crystallinity of the cellulose polysaccharides [32], and the well distribution of the nanofibers into the pectin matrix [31] since 50–60% of compositions of the biocomposites included pectin. 3.2. Prebiotic activity When grown on pectin, P.S. was 0.1 and B. coagulans fermented pectin as well as glucose as compared to E. coli. P.S. of pectin were 1.00 and 0.9 for the probiotic and enteric bacteria, respectively (Fig. 5). This could be attributed to the genes of polygalacturonase and pectin lyase which could be more expressed in B. coagulans for consumption of pectin when compared with E. coli [3,6,33]. P.S. and P.S. of pectin (1.0 and 0.1, respectively) were more than those of BNC (0.8 and 0, respectively) for B. coagulans. Among the BNC biocomposites, the highest prebiotic potential was obtained by pec-BNC (0.6 + 0.4) with P.S. of 0.07 when compared with pec-BNC (0.8 + 0.2) with P.S. of −0.09 which is the lowest (Fig. 5a). Although changes in BNC content of the biocomposites were significantly (p < 0.05) effective on the prebiotic activity, the highest P.S. (0.1) was obtained by only pectin. In agreement with the present results, microcrystalline cellulose and B. coagulans as a compartment of a synbiotic food was reported to increase in beneficial bacterial flora of the host colon [34]. Despite nanosize fibers of BNC with high surface area, insolubility of cellulose reduced the susceptibility of the BNC to the bacterial fermentation as compared to pectin as a soluble fiber, since availability of the surface area for enzymatic attack aided its fermentability [35,36]. This justified the lower P.S. of BNC as compared to P.S. of pectin. As shown in Fig. 5b, B. coagulans efficiently fermented pure NC as well as pectin. P.S. of pectin (1.0) was same as that of NC (1.0) for the probiotic, nevertheless P.S. of pectin (0.1) was more than that of NC (−0.08). Among the NC biocomposites, the highest prebiotic potential was obtained by pec-NC (0.6 + 0.4) with P.S. of 0.07 when compared with pec-NC (0.9 + 0.1) with P.S. of 0.01 as the lowest. NC was significantly (p < 0.05) effective on the pre-

biotic activity of the biocomposites but the highest P.S. (0.1) was obtained by only pectin. In the same pectin content, NC had prebiotic activity for the probiotic more than BNC since pec-NC (0.6 + 0.4) had P.S. of 1.14 but pec-BNC (0.6 + 0.4) had 0.91. In agreement with the present findings, chitin as a dietary supplement stimulated growth of the probiotic bacteria Bifidobacterium and Lactobacillus and also inhibited the growth of pathogenic bacteria Clostridium perfringens in the intestine [37]. Change in E. coli population (as a pathogenic bacteria) due to chitin supplementation was insignificant, however this study showed that E. coli population was significantly (p < 0.05) influenced by change in NC concentration of the mixtures [37]. Similar to the current study, chitin with glucan fiber supported the potential of enhanced prebiotic activity to modulate the gut microbial community [38]. As shown in Fig. 5c, P.S. and P.S. of NLC (1.62 and 0.31, respectively) were more than those of pectin (1.09 and 0.13, respectively) for B. coagulans. Among the NLC biocomposites, pec-NLC (0.5 + 0.5) with P.S. of 0.31 was better than pec-NLC (0.9 + 0.1) with P.S. of −0.02. NLC composited with pectin resulted in improvement of the prebiotic potential of the biocomposites significantly (p < 0.05). As shown in Table 2, the biocomposite of pec-NLC had the highest prebiotic activity among the biocomposites fabricated using the prebiotic nanofibers when the optimal composition of the biocomposite was as follows: 50% pectin + 50% NLC. Plant sources, which are rich in lignocellulosic materials, can be treated by using alkaline and acidic hydrolyses to produce lignocellulose. In the process of deriving lignocellulosic compounds from plant source, various prebiotic carbohydrates such as sugars and oligosaccharides can also be released. In addition, hemicellulosic polysaccharides which are constituents of lignocellulose, can act as prebiotic. These carbohydrates can be efficiently fermented by the bacteria as compared to pectin [39,40]. Moreover, the phenolic components of lignin with the side chain structure and nature of the functional groups were reported to be antimicrobial agents to inhibit pathogenic bacteria such as E. coli, Staphylococcus aureus and Pseudomonas. However, it was demonstrated that lignin increased intestinal concentrations of Lactobacilli and Bifidobacteria in broilers [12,41]. Thus, it was expected that NLC could more effectively act as a prebiotic. Both B. coagulans and E. coli fermented the prebiotic nanofibers used for fabrication of the entrapment biocomposites. Among the

Biocomposite

Response

Model

Pec-NC

P.S.

Y = 0.1X1 + 0.02X2 − 0.2X1 X2 − 0.5X1 X2 (X1 − X2 ) Y = 89.1X1 + 98.6X2 − 13.5X1 X2 (X1 − X2 ) Y = 79.5X1 + 95.9X2 + 31.7X1 X2 + 26.7X1 X2 (X1 − X2 ) Y = 0.1X1 + 0.3X2 − 0.7X1 X2 − 0.6X1 X2 (X1 − X2 ) Y = 91.2X1 + 90.6X2 + 0.6X1 X2 + 20X1 X2 (X1 − X2 ) Y = 76X1 + 85.3X2 + 9.8X1 X2 + 35.6X1 X2 (X1 − X2 ) Y = 0.1X1 − 0.03X2 − 0.3X1 X2 − 0.8X1 X2 (X1 − X2 ) Y = 92.6X1 + 99.4X2 − 4.7X1 X2 Y = 76.6X1 + 86.7X2 + 10X1 X2 + 8.1X1 X2 (X1 − X2 )

Survivability under SGF Survivability under SIF Pec-NLC

P.S.

Survivability under SGF Survivability under SIF Pec-BNC

P.S.

Survivability under SGF Survivability under SIF

Statistical value

Optimum value

R2

Adj. R2

Pred. R2

Adeq. Precision

p-value

0.914

0.888

0.850

13.872

< 0.0001

0.068

0.959

0.951

0.942

22.270

< 0.0001

97.7

0.968

0.958

0.945

22.202

< 0.0001

95.8

0.940

0.922

0.894

17.261

< 0.0001

0.308

0.718

0.633

0.491

7.912

0.0042

90.4

0.885

0.850

0.806

12.407

< 0.0001

85.3

0.829

0.777

0.674

9.302

0.0004

0.065

0.973

0.968

0.962

30.290

< 0.0001

97.2

0.980

0.974

0.964

30.176

< 0.0001

85.6

Optimal composition (g/ g biocomposite) Pectin

Nanofiber

0.6

0.4

0.5

0.5

0.6

0.4

A.C. Khorasani, S.A. Shojaosadati / International Journal of Biological Macromolecules 94 (2017) 131–144

Table 2 Statistical parameters, mathematical equations, optimum values and optimal compositions obtained after the ANOVA analysis of the models for responses of P.S. and survivability of the entrapped probiotic.

137

138

A.C. Khorasani, S.A. Shojaosadati / International Journal of Biological Macromolecules 94 (2017) 131–144

Fig. 5. Prebiotic scores of the biocomposites and cell densities of the probiotic and enteric bacteria grown on them fabricated using (a) pectin and BNC, (b) pectin and NC and (c) pectin and NLC.

nanofibers, NLC exhibited prebiotic potential as high as P.S of 0.31 when compared with BNC and NC which had P.S. of 0.0 and −0.08, respectively. In addition, P.S. of NLC (1.6) was higher than that of NC (1.0) and BNC (0.8) for B. coagulans (Fig. 5). Water solubility and available surface area play important roles in enhancing fermentability of the substrate. Positively charged group of amine

in NC could increase its solubility as compared to BNC. In addition, NC used with the average fibril diameter of 30 nm had surface area exposed to the bacteria larger than that of BNC with 50 nm, thereby, P.S. of NC was more than that of BNC [35,42]. Chitin has poor water solubility as compared to lignocellulose which consists of lignin, water soluble and insoluble polysaccharides. In addition, NLC fibril

A.C. Khorasani, S.A. Shojaosadati / International Journal of Biological Macromolecules 94 (2017) 131–144

139

Fig. 6. Cell density and survivability of B. coagulans entrapped by the biocomposites fabricated using pectin and BNC under (a) SGF and (b) SIF.

diameter of 65 nm creates high surface area susceptible to the bacteria. Thus, NLC was superior to NC in fermentation by the bacteria, which determined more prebiotic activity of NLC to NC based on P.S. value [12,37].

3.3. Survivability of probiotic The probiotic bacteria, which were not entrapped by the biocomposite, were more damaged under gastric and intestinal conditions as compared to the entrapped bacteria. Survivability of the free cell was approximately 76 and 73%, while reduction in the cell density was 2.1 and 2.3 log (CFU/g) under gastric and intestinal conditions, respectively. Probiotic cells (8.7 log (CFU/g)) entrapped using the pectin matrix reduced to approximately 8 and 6.7 log (CFU/g) when treated with gastric and intestinal fluids, respectively. It resulted in the survivability of 91 and 77% under gastric and intestinal treatments, respectively (Fig. 6–8 ). Similar to the results of the present study, pectin improved the viability of L. salivarius under SGI conditions containing proteases and bile salts using multiple-layer encapsulation [43,44].

The structure of the pectin matrix is dependent on pKa value of pectin and pH. Based on the esterification degree of pectin, the pKa value is between 3 and 4. In gastric condition, pectin can be protonated at pH levels lower than the pKa . This can lead to release of calcium ions and collapse of the matrix. In intestinal condition, a negatively charged pectin is formed at pH higher than the pKa . This can cause chain-chain repulsions to the network destruction [4]. Furthermore, cholate as a negative ion from bile salts incorporation with calcium ion can electrostatically form an insoluble complex in intestine, thus destructing the pectin matrix [45]. Moreover, pectin is not expected to be degraded by pepsin as a protease under the gastric fluid but it can be digested by pancreatin enzymes which contain ␣-amylase in the intestinal fluid [4,46]. Therefore, as expected, the results indicated that survivability of the probiotic entrapped by the pectin matrix was higher under the gastric condition as compared to the intestinal condition. As shown in Fig. 6a, among the BNC biocomposites, the highest gastric resistance was obtained by pec-BNC (0.5 + 0.5) with the survivability of 99.4% when compared with pec-BNC (0.9 + 0.1) with the survivability of 92.9% which is the lowest. The highest intestinal resistance was obtained by pec-BNC (0.5 + 0.5) with the surviv-

140

A.C. Khorasani, S.A. Shojaosadati / International Journal of Biological Macromolecules 94 (2017) 131–144

Fig. 7. Cell density and survivability of B. coagulans entrapped by the biocomposites fabricated using pectin and NC under (a) SGF and (b) SIF.

ability of 86.7% when compared with pec-BNC (0.9 + 0.1) with the survivability of 81.3% which is the lowest (Fig. 6b). However, optimization demonstrated that pec-BNC (0.6 + 0.4) was the optimal among the BNC biocomposites since this composition had higher prebiotic activity. The optimum P.S. and survivability under SGF and SIF are 0.065, 97.2 and 85.6%, respectively (Table 2). Consequently, BNC composited with pectin reinforced resistance of the pectin biocomposites against gastrointestinal digestion significantly (p < 0.05). In contrast to our previous study in which pec-BNC (0.2 + 0.8) optimally protected B. coagulans, this study demonstrated that the entrapment biocomposite with 40% BNC efficiently preserved the probiotic viability under GI conditions. As compared to the previous study, this study focused on reducing the BNC concentration in the biocomposite composition due to its expense [3]. Similar to the biocomposite introduced by this research, a multilayer composite produced using pectin and cationic cellulose nanofibers was studied with targeted release of probiotic in the human colon due to the low permeability and good resistance to the gastric fluid [47]. The composition of pectin with water insoluble polysaccharides such as cellulose appears especially promising to overcome the

problem of dissolution of pectin in the GI conditions. This is provided by the interactions between pectin and cellulose which are possible via their neutral sugar side chains. In contrast to the pectin matrix which is expected to shrink and collapse in the presence of GI fluids, cellulose nanofibers support pectin associated with BNC in the biocomposite to be stable against collapse. Hence, this biocomposite produced in this study, could provide a protection to probiotic cells in the GI tract while allowing enzymatic breakdown and probiotic release in the colon [48–51]. It was reported that cellulose nanofibrils could act as reinforcing agents to significantly change properties of the polymeric composites. By adding nanocellulose to pectin, the entrapment biocomposite was reinforced since polymer chains within this composite were restricted to confined domains, thus decreasing gastric and intestinal fluids permeability into the biocomposite [52]. Furthermore, microbial cellulose incorporation with hemicelluloses such as pectin is able to self-assemble into cellulose composites. This was expected for BNC acting as a cross linker in pectin network which would bring constituents closer together and lead to reduction of the pore size of the entrapment matrix. In addition, polysaccharide composition of the matrix influenced its architectural features which determined

A.C. Khorasani, S.A. Shojaosadati / International Journal of Biological Macromolecules 94 (2017) 131–144

141

Fig. 8. Cell density and survivability of B. coagulans entrapped by the biocomposites fabricated using pectin and NLC under (a) SGF and (b) SIF.

the different diffusivities of the matrix [53,54]. Therefore, 40% bacterial nanocellulose could promote the construction of the pec-BNC to limit permeability of the GI fluids through the entrapment biocomposite. As shown in Fig. 7a, among the NC biocomposites, the highest gastric resistance was obtained by pec-NC (0.5 + 0.5) with the survivability of 98.9% when compared with pec-NC (0.8 + 0.2) with the survivability of 89.8% which is the lowest. The highest intestinal resistance was obtained by pec-NC (0.5 + 0.5) with the survivability of 95.8% when compared with pec-NC (0.9 + 0.1) with the survivability of 91.8% which is the lowest (Fig. 7b). However, optimization demonstrated that pec-NC (0.6 + 0.4) was the optimal among the NC biocomposites since this composition had the higher prebiotic activity. The optimum P.S. and survivability under SGF and SIF are 0.068, 97.7 and 95.8%, respectively (Table 2). Consequently, NC composited with pectin resulted in improvement of the gastric and intestinal resistance of the pectin biocomposites significantly (p < 0.05). Similar to the present research, a nanocomposite scaffold composed of pectin, chitin and nano CaCO3 was developed for tissue engineering and drug delivery [7].

One possible explanation for the higher survivability obtained by the pec-NC biocomposite as compared to the pec-BNC and pecNLC biocomposites in the GI fluids is that chitin could stabilize pectin in the pec-NC biocomposite using ionic interactions which could be formed between amino groups of chitin and carboxyl groups of pectin, improving the sustainability of the biocomposite in the aqueous conditions of GI tract. The electrostatic bond is due to the positive and negative charges which are attributed to the NH2 groups on a chitin polymer and COOH groups on a pectin polymer, respectively. In contrast to chitin being a stabilizer for pectin to maintain its solid status, calcium ion-crosslinked pectin might not be so sustainable in the GI fluids and can be more easily released from the biocomposite, resulting in decrease of the survivability [55]. Fiber–fiber interactions between NC in the biocomposite such as van der Waals attraction, electrostatic repulsion and hydrogen bonding led to adhesion of NC fibers, and thus creating a compact surface for the matrix when they were dried of water. This occurred due to pore shrinkage during the freeze-drying because of the high surface tension of water [8,56]. Therefore, pecNC biocomposite, due to its compact surface, hindered the uptake of the GI fluids into the biocomposite. Less survivability obtained

142

A.C. Khorasani, S.A. Shojaosadati / International Journal of Biological Macromolecules 94 (2017) 131–144

Fig. 9. Swelling ratio (−) and degradation (- - -) of the optimal biocomposites in (a) SGF and (b) SIF.

by the pure NC as compared to the pec-NC biocomposite was due to the ability of pancreatin to efficiently degrade chitin [57]. This result reflected the fact that the composition of pectin and NC was a potent entrapment biocomposite resistant to the intestinal fluid. It is worth noting that bile salts could be adsorbed at the interface of nanochitin network, leading to impeding of the adsorption and penetration of the digestion enzymes of the intestinal fluid into the pec-NC biocomposite and thereby increasing survivability of the probiotic entrapped within the matrix [58]. This confirmed why the survivability obtained by pec-NC (95.8%) under the intestinal condition was more than that by pec-BNC (85.6%) and pec-NLC (85.3%). As shown in Fig. 8a, among the NLC biocomposites, the highest gastric resistance was obtained by pec-NLC (0.9 + 0.1) with the survivability of 93.8% when compared with pec-NLC (0.7 + 0.3) with the survivability of 89.5% which is the lowest. The highest intestinal resistance was obtained by pec-NLC (0.5 + 0.5) with the survivability of 85.3% when compared with pec-NLC (0.8 + 0.2) with the survivability of 80.5% which is the lowest (Fig. 8b). However, optimization demonstrated that pec-NLC (0.5 + 0.5) was the optimal among the NLC biocomposites since this composition resulted in

the highest prebiotic activity and survivability under SIF. The optimum P.S. and survivability under SGF and SIF were 0.308, 90.4 and 85.3%, respectively (Table 2). Consequently, NLC composited with pectin made the pectin biocomposites stronger against gastric and intestinal fluids, significantly (p < 0.05). Lignin can fix the other polysaccharides in the matrix construction due to its branched and complex three-dimensional networks [59]. Distribution of lignocellulose nanofibers in the pectin-based composite can improve resistance properties of the composite against dissolution of the network under the GI conditions due to hydrogen bonding and van der Waals attraction between pectin and lignocellulose, particularly, the presence of the CO group of hemicellulose in the lignocellulose nanofibers which facilitate better bonding between the lignocellulose nanofibers and the biocomposite [14,60]. The digestive enzymes rarely degrade lignin because of its polyphenolic structure [59]. On the other hand, lignocellulose digestion requires cellulases that efficiently hydrolyze glycosidic bonds of the cellulose fibrils, and a wide range of other glycoside hydrolases that break down the network of hemicelluloses. The required enzymes are not in the human GI tract, thereby composite material fabricated using lignocellulose cannot be digested [61,62].

A.C. Khorasani, S.A. Shojaosadati / International Journal of Biological Macromolecules 94 (2017) 131–144

The prebiotic nanofibers used for fabrication of the biocomposites were efficiently successful in reinforcing the biocomposites against both gastric and intestinal conditions. Under the gastric condition, BNC and NC optimally increased the survivability with addition of 60% pectin to the nanofibers as compared to NLC that increased with the addition of 50% pectin. The pec-NC biocomposite containing 40% NC provided the highest protection with the survivability of 95.8% as compared to the others similarly protecting the probiotic under the intestinal condition. Consequently, from the obtained data, the survivability under the gastric condition was more than that under the intestinal condition. This was attributed to the mechanism of pH-sensitive swelling of the biocomposite involved in the deprotonation of carboxyl groups of pectin at high pH. At pH 2 (SGF), carboxyl groups in pectin was protonated. Due to the dominant effect of the protonated carboxyl groups in pectin, the pec-nanofibers deswelled. On the other hand, if pH further increases to 7.4, the carboxyl groups on the biocomposites become progressively ionized. At pH 8 (SIF), carboxyl groups in pectin was deprotonated, increasing negatively charged carboxylate groups in the pectin-based biocomposites and thus, the swelling increased due to a large swelling force created by the electrostatic repulsion between the groups. Hence, the biocomposites under the intestinal condition had the highest swelling ratio, leading to the fast release of the probiotic and exposure to the enzymatic digestion [63]. Besides, the nanofibers used in this study, increased the resistance of the biocomposites to destruction by the dissolution in the GI tract due to the insolubility in the aqueous medium. On the other hand, the biocomposites with high pectin contents had a more open structure, which permitted the release of the probiotic [64]. Moreover, high surface area of the nanofibers allowed the probiotic cells to adhere to the surface of the biocomposite.

143

GI tract, thereby, preventing network structure from collapsing to increase the probiotic survival. 4. Conclusions The present study introduced three types of entrapment biocomposites developed based on the prebiotic nanofibers of chitin, lignocellulose and bacterial cellulose incorporated with pectin, as gastrointestinal-resistant prebiotic materials for protection of probiotics passing through the GI tract. D-optimal mixture design method was adequate to determine the optimal compositions of the biocomposites for obtaining optimum prebiotic activity and probiotic survival under gastrointestinal conditions, simultaneously. All the optimal biocomposites exhibited completely amorphous structures and non-porous compact surfaces which resulted from inter- and intra-molecular hydrogen bonding, ionic interactions of pectin-Ca2+ and high surface area accessed by the nanofibers. It is noteworthy that such structure played an important role in high survival of the entrapped probiotics when compared with the free probiotics under harsh conditions. Interestingly, the nanofibers incorporation with pectin led to a rapid swelling of the pec-nanofibers biocomposites in contrast to their slow degradation. The nanofibers of chitin with optimum ratio of 40% improved prebiotic activity, gastric and intestinal resistance properties of the pec-NC biocomposite more efficiently as compared to the other nanofibers. Therefore, the proposed biocomposites have a great potential for application in the functional foods industry as a new approach for not only protected delivery of probiotics into the GI tract but also, increase in shelf life of probiotics in foods during storage. References

3.4. Biocomposite swelling and degradation in simulated gastrointestinal fluids Fig. 9 shows that the swelling ratios of the biocomposites were between 1.1 and 1.5. This demonstrated that the type of digestive medium did not affect the swelling. The swelling of the biocomposites in SGF and SIF could be explained by the diffusion of Ca2+ ions from the matrix network into the swelling medium. Calcium ions were the crosslinkers of the pectin-based matrix network and their loss could result in an increase in the water permeation [65]. Therefore, the swelling of the biocomposites occurred through water diffusion and it was influenced by neither acidic/basic environment nor enzyme degradation. Although the swelling ratios of the biocomposites were similar, their degradation percentage was significantly different (Fig. 9). In addition, swelling of the biocomposites was rapid but their degradation occurred slowly. It should be noted that after swelling, all the biocomposites kept their original state and were not degraded until the latest hour (Fig. 9). The pectin matrix without the nanofibers showed degradation of approximately 50 and 55% in SGF and SIF, respectively. In contrast, degradation of the pec-NC biocomposite was 15 and 10% in SGF and SIF, respectively (Fig. 9). These results demonstrated that the nanofibers could efficiently reinforce pectinbased biocomposites against digestive media. This was due to the lack of suitable enzymes in gastrointestinal fluids for the degradation of the non-starch nanofibers used in the pec-nanofibers biocomposites [61,62]. Degradation of the biocomposites occurred at the latest hour, whereas their swelling occurred in the first hour. This indicated structure resistance of the biocomposites to the destruction by swelling. This structure prevented release of the probiotic cells into the digestive media. Therefore, the presence of the nanofibers in the construction of the biocomposites caused the uptake of the nutrient dissolved in the aqueous media in the

[1] A.K. Anal, H. Singh, Trends Food Sci. Technol. 18 (2007) 240–251. [2] M.T. Cook, G. Tzortzis, D. Charalampopoulos, V.V. Khutoryanskiy, J. Control. Rel. 162 (2012) 56–67. [3] A.C. Khorasani, S.A. Shojaosadati, Int. J. Biol. Macromol. 83 (2016) 9–18. [4] S. Alborzi, L.-T. Lim, Y. Kakuda, LWT-Food Sci. Technol. 59 (2014) 383–388. [5] T.W. Wong, G. Colombo, F. Sonvico, AAPS PharmSciTech 12 (2011) 201–214. [6] F. Nazzaro, F. Fratianni, P. Orlando, R. Coppola, Pharmaceuticals 5 (2012) 481–492. [7] P.S. Kumar, C. Ramya, R. Jayakumar, V.-K. Lakshmanan, Colloids Surf. B: Biointerfaces 106 (2013) 109–116. [8] P. Ratanajiajaroen, M. Ohshima, J. Microencapsul. 29 (2012) 549–558. [9] Y. Okamoto, M. Nose, K. Miyatake, J. Sekine, R. Oura, Y. Shigemasa, S. Minami, Carbohydr. Polym. 44 (2001) 211–215. [10] A.M. Salaberria, J. Labidi, S.C. Fernandes, Eur. Polym. J. 68 (2015) 503–515. [11] Y. Zhang, J. Jiang, L. Liu, K. Zheng, S. Yu, Y. Fan, Nanoscale Res. Lett. 10 (2015) 1–11. [12] M. Bogusławska-Tryk, R. Szymeczko, A. Piotrowska, K. Burlikowska, K. ´ zewska, ˙ Pak. Vet. J. 35 (2015) 212–216. Sli [13] C.S. da Silva, J.J. van den Borne, W.J. Gerrits, B. Kemp, J.E. Bolhuis, Physiol. Behav. 107 (2012) 218–230. [14] A. Sutka, J. Gravitis, S. Kukle, A. Sutka, M. Timusk, Soft Mater. 13 (2015) 18–23. [15] D. Aydemir, A. Kiziltas, D.J. Gardner, Y. Han, G. Gunduz, Int. J. Polym. Anal. Charact. 20 (2015) 231–239. [16] P. Alvarez, C. Blanco, R. Santamaría, M. Granda, Compos. Part A: Appl. Sci. Manuf. 36 (2005) 649–657. [17] D. Koutsianitis, C. Mitani, K. Giagli, D. Tsalagkas, K. Halász, O. Kolonics, C. Gallis, L. Csóka, Ultrason. Sonochem. 23 (2015) 148–155. [18] C.D. May, Carbohydr. Polym. 12 (1990) 79–99. [19] G. Blaiotta, B. La Gatta, M. Di Capua, A. Di Luccia, R. Coppola, M. Aponte, Food Microbiol. 36 (2013) 161–169. [20] J. Huebner, R.L. Wehling, R.W. Hutkins, Int. Dairy J. 17 (2007) 770–775. [21] T. Faye, A. Tamburello, G. Vegarud, S. Skeie, J. Dairy Sci. 95 (2012) 558–566. [22] R. Bedani, A.D.S. Vieira, E.A. Rossi, S.M.I. Saad, LWT-Food Sci. Technol. 55 (2014) 436–443. [23] M. Xu, M.-J. Dumont, J. Food Eng. 165 (2015) 52–59. [24] K.G. Nair, A. Dufresne, Biomacromolecules (Washington) 4 (2003) 666–674. [25] I. Rumengan, E. Suryanto, R. Modaso, S. Wullur, T. Tallei, D. Limbong, Int. J. Fish. Aquat. Sci. 3 (2014) 12–18. [26] A. Fellah, P. Anjukandi, M.R. Waterland, M.A. Williams, Carbohydr. Polym. 78 (2009) 847–853. [27] P. Zhang, S.-J. Dong, H.-H. Ma, B.-X. Zhang, Y.-F. Wang, X.-M. Hu, Ind. Crops Products 76 (2015) 688–696.

144

A.C. Khorasani, S.A. Shojaosadati / International Journal of Biological Macromolecules 94 (2017) 131–144

[28] A.M. Salaberria, S.C. Fernandes, R.H. Diaz, J. Labidi, Carbohydr. Polym. 116 (2015) 286–291. [29] S. Ifuku, M. Nogi, K. Abe, M. Yoshioka, M. Morimoto, H. Saimoto, H. Yano, Biomacromolecules 10 (2009) 1584–1588. [30] S. Iwamoto, T. Endo, ACS Macro Lett. 4 (2014) 80–83. [31] C.G. Zhou, Q. Gao, S. Wang, Y.S. Gong, K.S. Xia, B. Han, M. Li, Y. Ling, React. Funct. Polym. 100 (2016) 97–106. [32] J. Gu, J.M. Catchmark, Carbohydr. Polym. 88 (2012) 547–557. [33] L.V. Thomassen, D.M. Larsen, J.D. Mikkelsen, A.S. Meyer, Enzyme Microbial Technol. 49 (2011) 289–297. [34] J.W. Gagné, J.J. Wakshlag, K.W. Simpson, S.E. Dowd, S. Latchman, D.A. Brown, K. Brown, K.S. Swanson, G.C. Fahey, BMC Vet. Res. 9 (2013) 1. [35] G.R. Gibson, Clin. Nutr. Suppl. 1 (2004) 25–31. [36] V. Kumar, A.K. Sinha, H.P. Makkar, G. de Boeck, K. Becker, Crit. Rev. Food Sci. Nutr. 52 (2012) 899–935. [37] S.K. Halder, A. Adak, C. Maity, A. Jana, A. Das, T. Paul, K. Ghosh, P.K.D. Mohapatra, B.R. Pati, K.C. Mondal, Indian J. Exp. Biol. 51 (2013) 924–934. [38] A.M. Neyrinck, S. Possemiers, W. Verstraete, F. De Backer, P.D. Cani, N.M. Delzenne, J. Nutr. Biochem. 23 (2012) 51–59. [39] B.A. Acosta-Estrada, J.A. Gutiérrez-Uribe, S.O. Serna-Saldívar, Food Chem. 152 (2014) 46–55. [40] E. Conde, P. Gullón, A. Moure, H. Domínguez, J.C. Parajó, Food Bioproducts Process. 87 (2009) 208–214. [41] B. Baurhoo, C. Ruiz-Feria, X. Zhao, Anim. Feed Sci. Technol. 144 (2008) 175–184. [42] D. Narayanan, R. Jayakumar, K. Chennazhi, Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol. 6 (2014) 574–598. [43] Y. Zhang, J. Lin, Q. Zhong, Food Hydrocoll. 52 (2016) 804–810. [44] Y. Zhang, J. Lin, Q. Zhong, Food Res. Int. 71 (2015) 9–15. [45] W. Krasaekoopt, B. Bhandari, H. Deeth, Int. Dairy J. 14 (2004) 737–743. [46] T. Chelpanova, F. Vitiazev, N. Mikhaleva, E. Efimtseva, Rossiiskii fiziologicheskii zhurnal imeni IM Sechenova/Rossiiskaia akademiia nauk 98 (2012) 734–743.

[47] S.L. Mølgaard, M. Henriksson, M. Cárdenas, A.J. Svagan, Carbohydr. Polym. 114 (2014) 179–182. [48] B.A. McKenna, P.M. Kopittke, J.B. Wehr, F. Blamey, N.W. Menzies, Physiol. Plant. 138 (2010) 205–214. [49] W. He, Q. Du, D.-Y. Cao, B. Xiang, L.-F. Fan, Int. J. Pharm. 348 (2008) 35–45. [50] G. Agoda-Tandjawa, S. Durand, C. Gaillard, C. Garnier, J. Doublier, Carbohydr. Polym. 90 (2012) 1081–1091. [51] A.W. Zykwinska, M.-C.J. Ralet, C.D. Garnier, J.-F.J. Thibault, Plant Physiol. 139 (2005) 397–407. [52] W.J. Orts, J. Shey, S.H. Imam, G.M. Glenn, M.E. Guttman, J.-F. Revol, J. Polym. Environ. 13 (2005) 301–306. [53] P. Lopez-Sanchez, E. Schuster, D. Wang, M.J. Gidley, A. Strom, Soft Matter 11 (2015) 4002–4010. [54] J. Gu, J.M. Catchmark, Cellulose 21 (2014) 275–289. [55] P.-H. Chen, T.-Y. Kuo, J.-Y. Kuo, Y.-P. Tseng, D.-M. Wang, J.-Y. Lai, H.-J. Hsieh, Carbohydr. Polym. 82 (2010) 1236–1242. [56] J. Wu, J.C. Meredith, ACS Macro Lett. 3 (2014) 185–190. [57] C.-R. Shen, C.-L. Liu, H.-P. Lee, J.-K. Chen, Molecules 18 (2013) 2978–2987. [58] M.V. Tzoumaki, T. Moschakis, E. Scholten, C.G. Biliaderis, Food Funct. 4 (2013) 121–129. [59] T. Gidenne, Livest. Prod. Sci. 81 (2003) 105–117. [60] X. Li, Y. Meng, S. Wang, A.V. Rajulu, S. Tjong, J. Polym. Sci. Part B: Polym. Phys. 42 (2004) 666–675. [61] A. Brune, Nat. Rev. Microbiol. 12 (2014) 168–180. [62] J. Ni, G. Tokuda, Biotechnol. Adv. 31 (2013) 838–850. [63] C.-Y. Yu, B.-C. Yin, W. Zhang, S.-X. Cheng, X.-Z. Zhang, R.-X. Zhuo, Colloids Surf. B: Biointerfaces 68 (2009) 245–249. [64] M. Hiorth, I. Tho, S.A. Sande, Eur. J. Pharm. Biopharm. 56 (2003) 175–181. [65] G. Pasparakis, N. Bouropoulos, Int. J. Pharm. 323 (2006) 34–42.