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Effects of κ-carrageenan on pullulan’s rheological and texture properties as well as pullulan hard capsule performances
Carbohydrate Polymers 238 (2020) 116190
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Effects of κ-carrageenan on pullulan’s rheological and texture properties as well as pullulan hard capsule performances
T
Yihui Zhanga,1, Ning Yanga,1, Yaqiong Zhangb, Jingwen Houc, Huijie Hana, Zhu Jina, Yuanyuan Shena, Shengrong Guoa,* a
School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China c Instrumental Analysis Centre, Shanghai Jiao Tong University, Shanghai, 200240, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Pullulan κ-Carrageenan Rheology Texture property
κ-Carrageenan (κ-Ca) is often used to facilitate gelling of aqueous solutions of polysaccharides. However, studies on its effects on pullulan’s rheological and texture properties and pullulan (PUL) hard capsule performances have rarely been reported. Herein, effects of κ-Ca on PUL solutions, hydrogels, films and hard capsules were investigated. It was found that the gelling temperature of 15 % (w/w) PUL solutions with 0.07 % KCl increased from 34 ℃ to 42 ℃ as the concentration of κ-Ca increased from 0.6 % to 1.2 %, and the gelling temperature rose from 25 ℃ to 37 ℃ by adding a small amount of KCl (0.07 %) for 15 % PUL solutions with 0.9 % κ-Ca. As the κ-Ca concentration increased, hardness, fracturability and adhesiveness rose for PUL gels and tensile stress increased for PUL films. PUL capsules could be easily prepared by the aid of κ-Ca, and performances of capsules could be adjusted by changing the amount of κ-Ca.
1. Introduction Hard capsules are widely applied in pharmaceutical market, and gelatin is the most commonly used material for capsule preparation (Barbosa, Al-Kauraishi, Smith, Conway, & Merchant, 2019). However, gelatin is relatively expensive, and it derived from bones and skins of animals (pigs and cattle), so gelatin capsules are not suitable for vegetarians and people of religious beliefs (Zhang, Zhao, Wang, Jiang, & Cha, 2017). Therefore, it is interesting to investigate non-gelatin or non-animal origin capsule materials. PUL is a neutral linear polysaccharide produced by Aureobasidium pullulans. PUL has the chemical structure of maltotriose repeating units interconnected by α-(1→6) glycosidic linkages, and in each maltotriose, the three glucoses are connected with each other by α-(1→4) glycosidic linkages (Singh, Saini, & Kennedy, 2008). Currently, PUL is popular in food, material, and pharmaceutical industries (Farris et al., 2012). The advantages of PUL films have been well documented. For example, Singh et al. (2008) reported that PUL films were transparent, water-soluble, with excellent mechanical properties and oxygen-barrier characteristics. Besides, Kim, Choi, Kim, and Lim (2014) reported that PUL was usually incorporated into starch to prepare films with better mechanical and physical properties than pure starch films. That makes
PUL an ideal material for edible films, as well as a biodegradable and water-soluble packaging material (Kowalczyk et al., 2019). However, the mechanical strength of pure PUL cannot meet its preparation and handling requirements when it is applied for capsules (Kristo & Biliaderis, 2007). The strength of PUL can be improved by blending it with other polymers like chitosan, carboxymethyl chitosan (Jeon, Kamil, & Shahidi, 2002) or carrageenan (Wu & Imai, 2012). κ-Ca is a natural sulfated polysaccharide extracted from marine red algae (Shen, Chang, Chen, & Dong, 2018). The chemical structure of κCa is formed by repeating units of α-(1→3)-linked galactose-4-sulphate and β-(1→4)-linked 3,6-anhydrogalactose (Li, Ni, Shao, & Mao, 2014). It is widely used in food, cosmetic and pharmaceutical industries on account of its remarkable properties such as gelling, thickening, stabilizing and texture enhancing abilities (Necas & Bartosikova, 2013). κ-Ca can easily form thermal reversible gels by the aid of gel inducing agents, usually cations, when it is cooled below gelling temperature. During the gelling process, the conformation of κ-Ca transforms from a random coil to a helix (Yuan, Du, Zhang, Jin, & Liu, 2016). It is followed by aggregation of these helices, resulting in gelation (Robal et al., 2017; Wang, Yuan, Cui, & Liu, 2018). The addition of cations can enhance the order degree of conformation and aggregation of helices to enhance the strength of κ-Ca gel (Kara, Tamerler, Bermek, & Pekcan, 2003), among
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Corresponding author. E-mail address: [email protected] (S. Guo). 1 Equal contributors. https://doi.org/10.1016/j.carbpol.2020.116190 Received 30 November 2019; Received in revised form 14 March 2020; Accepted 16 March 2020 Available online 19 March 2020 0144-8617/ © 2020 Published by Elsevier Ltd.
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which K+ is usually used (Bercea & Wolf, 2019). During the gelling process, the proportion of helices increases as the temperature decreases. So far, few studies have focused on the effects of κ-Ca on PUL’s rheological and texture properties, and the mechanism of such effects has seldom been explained. Moreover, the effects of κ-Ca in preparation of PUL capsules and their performances are useful and interesting to investigate. The aim of this study is to examine the effects of κ-Ca on PUL solutions, gels, films and capsules, and the performances of PUL hard capsules in terms of gelling temperature, hardness, fracturability, tensile strength, enlongation at break, brittleness and disintegration time.
Table 1 Compositions of five formulas (F1-F5) of PUL solutions. Formula
PUL (%, w/w a)
κ-Ca (%, w/w a)
K+ (%, w/w a)
1 2 3 4 5
15 15 15 15 15
– 0.9 0.6 0.9 1.2
– – 0.07 0.07 0.07
a The w/w refers to the dry weight of each ingredient relative to the weight of water.
distilled water with continuous stirring under heating in a water bath at 90 ℃ until the powder was totally dispersed. Then, the blended solutions were prepared by adding 0.07 % KCl and 0.3 %, 0.6 %, 0.9 % or 1.2 % κ-Ca under mild mechanical stirring, respectively. Compositions of five formulas (F1-F5) of PUL solutions are shown in Table 1. The mixtures were continuously stirred at 90 ℃ for 30 min, and then they were cooled to 45 ℃ and kept for 1 h to remove trapped air bubbles. The aforementioned solutions were poured into weighing bottles and then cooled down at 25 ℃ to form gel. Afterwards, the weighing bottles were placed in a 4 ℃ refrigerator overnight to equilibrate before testing.
2. Materials and methods 2.1. Materials Purified PUL powder was purchased from Tongliao Meihua Biochemical Science & Technology Co., Ltd (Inner Mongolia, China) with 3.20 % moisture concentration. The purity of PUL is as high as 95 % and the molecular weight of PUL was 2 × 105 Da. κ-Ca powder was commercially obtained from Zhejiang Top Biological Science & Technology Co., Ltd (Zhejiang, China), and the purity of it was as high as 99 %. Potassium chloride (KCl) was purchased from Guoyao Reagents Ltd. (Shanghai, China) and of analytical grade. The chemical structures of PUL and κ-Ca as well as the gelation mechanism of κ-Ca are shown in Fig. 1.
2.3. Casting film and hard capsule preparation About 10 g of the prepared solutions was poured onto a leveled glass plate (10 × 10 cm), followed by drying at 35 ℃ in an oven for about 8 h. Then the films were peeled off the plate and stored at 25 ± 1 ℃ and 55 ± 1% relative humidity (RH) before testing. The thickness of formed film samples was recorded using a micrometer (Guilin, China).
2.2. Solution and gel preparation PUL solutions (15 %) were prepared by dissolving PUL powder into
Fig. 1. A: Chemical structure of PUL, maltotriose repeating units are linked each other through α-(1→6) glycosidic linkages to form PUL. B: Chemical structure of κCa. C: schematic representation of the thermal reversible gelation mechanism of κ-Ca (κ-Ca network, line; potassium ion, dot). 2
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8 different points were chosen on each film as replicates. The thickness was 0.1 ± 5% cm, which was in accordance with the capsule thickness prepared. The prepared methods of hard capsules followed the description by Zhang et al. (2013) with some modifications. Stainless steel mold pins (cylindrical: cap, 7.2 ± 0.02 mm diameter; body, 7.0 ± 0.02 mm diameter) were dipped into prepared solutions and then dried in an oven at 50 ℃ for about 1 h. Capsule wall thickness measurements were taken at 8 different points on each sample with a micrometer.
EB = (L−L0) / L0 × 100
2.4.5. Thermal analysis Differential scanning calorimetry (DSC) analysis of the films was investigated using DSC 204F1 (Netzsch, Germany). Samples of about 4 mg were sealed in aluminum pans and heated from 25 ℃ to 220 ℃ to remove the water and then the samples were cooled to 20 ℃. At last, samples were heated to 270 ℃ at a heating rate of 10 ℃ / min under nitrogen. Thermogravimetric analysis (TGA) was performed in order to measure the thermal stability and decomposition characteristics of films with a thermogravimetric analyzer (SDT-Q600, America). Samples weighing around 6−8 mg in alumina pans were heated from 30 ℃ to 600 ℃ at a rate of 10 ℃ / min−1 under nitrogen.
2.4. Characterization 2.4.1. Viscosity measurements The viscosities of PUL aqueous solutions or the solutions prepared with F1-F5 were measured using a digital rotary viscometer (NDJ-79, Shanghai, China). A rotating cylinder used for measurements is based on viscosity range of solutions (as shown in Table S1), each rotating cylinder corresponds to a certain shear rate.
2.4.6. Water content PUL hard capsules were stored at 25 ℃ and 55 % RH in desiccators for 72 h, and their weights were recorded as m0. Then they were dried at 105 ℃ in a muffle oven until the constant weights (m1) were reached. The water content was calculated using the equation as following (Chu et al., 2019):
2.4.2. Rheological measurements The rheological measurements were performed using an AR G2 rheometer (TA Instrument, USA) equipped with a plate geometry stainless steel (diameter: 40 mm, angle: 2°, gap: 500 μm). The dynamic viscoelastic tests were conducted in the linear viscoelastic region (LVR). The upper plate was covered with sealing rings to prevent evaporation of water during the test. The flow properties of the solution samples were measured at shear rate from 0.1 to 100 s−1 at 45 ℃. The flow curves of solutions were recorded and fitted by power-law model (Xiao, Tong, & Lim, 2012): τ= K·γ
n
Water content (%) = (m0−m1)/m0 × 100 %
(4)
Each test was done in triplicate. 2.4.7. Disintegration and brittleness test A disintegration apparatus (ZB-1 G, Tianjin Haiyida Technology Co. Ltd, China) was used for the disintegration test of the PUL capsules. The disintegration test was done according to Chinese pharmacopoeia (2015). Six capsules filled with talcum were tested in distilled water at 37 ± 1 ℃. The time all the six capsules tested completely disintegrate is considered as disintegration time. The brittleness of capsules was measured according to Chinese pharmacopoeia (2015). The capsules are qualified in brittleness if less than 5 of the 50 capsules tested are broken. If less than 5 of the 50 capsules tested are broken, the brittleness of capsules is considered as good.
(1)
Where τ is shear stress (Pa), K is the consistency index (Pa·s ), γ is shear rate (s−1) and n is flow behavior index. The viscoelastic properties of the solution samples were measured as following: after an equilibration at 70 ℃ for 5 min, a cooling ramp from 70 to 20 ℃ with rate of 1 ℃ /min was achieved (strain: 1%, frequency: 1 rad/s). The storage modulus G’ and loss modulus G” were recorded. The intersection of the two moduli in the curve is defined as the gelling temperature (Tgel) (Tomšič, Prossnigg, & Glatter, 2008), which indicated the transformation temperature from the solution state to gel state. n
2.4.8. Fourier transforms infrared spectroscopy (FTIR) PUL, κ-Ca and the capsules prepared by F4 were scanned using a Nicolet 6700 spectrometer (Thermo Fisher, America) with attenuated total reflectance (ATR). FTIR spectra of each sample was performed with a resolution of 4 cm−1 and a range of 600−4000 cm−1 and 32 scans.
2.4.3. Texture profile analysis Texture profile analysis (TPA) of the gel samples were carried out on a TA-XT plus texture analyzer (Stable Micro Systems, UK) under the TPA mode. The sample was compressed twice at a deformation of 50 % sample height using a P/0.5 probe. The interval between two compression phases was 5 s. The test, pre-test and post-test speed were 0.5, 2 and 3 mm/s, respectively. All measurements were done in five times.
2.4.9. X-ray diffraction (XRD) KCl powders and the capsules prepared with F4 were scanned with an X-ray diffractometer (XRD; D8 ADVANCE Da Vinci, Germany). The applied X-ray beam was 40 kV, 40 mA and the scanning range of the diffraction angle 2θ° was 3–400 with a rate of 4°/min.
2.4.4. Mechanical properties of film The tensile strength (TS, MPa) and elongation at break (EB, %) were determined using a TA-XT plus texture analyzer (Stable Micro Systems, UK) equipped with a 5 kg load cell. The specimens were of dumbbell shape (gap 70 mm, width 6 mm). The mechanical properties of film samples were measured according to ASTM standard method D882-18 (ASTM, 2018). The initial grip distance was 50 mm and the cross-head speed was 1 mm/s. Each test consisted of six replicate measurements. TS was calculated by dividing the peak stress (F, N) by the crosssectional area (A, m2). Percentage EB was calculated by dividing the increase in length at break (L) by the initial length (L0) of the film and multiplying by 100. It can also describe as the following equations (Xiao, Lim, & Tong, 2012): TS = F/A
(3)
2.4.10. Scanning electron microscopy (SEM) Both the surface and the cross-section of the capsules prepared with F4 were observed by scanning electro microscopy (SEM; Mira3/MIRA3, TESCAN, Czech Republic). Capsule specimens were immersed into liquid nitrogen for 10 min and then fractured into pieces (about 3 mm × 3 mm). A gold coating was added on the specimens with a sputter. 2.4.11. In vitro dissolution of chlorpheniramine maleate Dissolution of chlorpheniramine maleate was measured by using the Paddle method (Chinese Pharmacopoeia (Edition 2015)). A small piece of corrosion-resistant fine metal wire was used as a sinker to wind about a capsule. The dissolution test for the PUL capsules containing 2 mg chlorpheniramine maleate and 248 mg lactose was carried out in
(2) 3
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900 mL of 0.1 mol/L HCl solution, at 75 rpm and 37 ± 0.5 ℃, by using a dissolution apparatus (ZRS-8C, Tianjin University Radio Factory, China). The samples were taken out from the dissolution medium at a predetermined time and filtered through a 0.22 μm polyethersulfone membrane for analysis. The samples were analyzed using a HPLC system (Waters, Singapore). The measurement was carried out on a C18 column (250 × 4.6 mm, 5 μm) with a mobile phase of phosphate buffer - acetonitrile (80:20, v/v). The column temperature was 30 ℃, and the detective wavelength was 262 nm. 2.5. Statistical analysis Data were expressed as the mean ± standard deviation (SD). The ttest and Pearson correlation coefficients were respectively used for the significance and correlation analysis by SPSS 2015 (IBM Inc., USA) with a significance level of p < 0.05.
Fig. 3. Effects of shear rate on the viscosity of solution samples prepared with F1-F5 at 45 ℃. Viscosity was measured by an AR G2 rheometer.
F3 containing smaller amount of κ-Ca (0.6 %) look lower than those of F2, F4 and F5. Effects of shear rates on shear stress are shown in Fig. S1 and the parameters of the Power-law model for the flow curves of PUL solutions are listed in Table S2. Temperature sweep curves of solutions prepared with formula 1–5 were showed in Fig. 4. The gelling behaviors of samples were observed by measuring G’ and G’’. G’ is used to measure material resistance capability of the elastic deformation, G’’ is used to measure the size of the loss of energy in the process of viscous deformation. Fig. 4A shows G’ and G’’ values of 15 % PUL solution, and G’’ was always greater than G’, which means that the 15 % PUL solution maintained in a sol state from 70 ℃ to 20 ℃. It indicated that PUL has no gelation property at a temperature range of 70℃ to 20 ℃, which is consistent with the study of Xiao, Tong, & Tim (2012). Form Fig. 4C–E, we found that G’ increased with the increased concentration of κ-Ca (0.6, 0.9 and 1.2 %), and the values of G’ at 20 ℃ were 57.37, 453.70, 1476.00, respectively. Fig. 4 B and D show that the addition of KCl (0.07 %) also increased the G’ (from 0.02 to 15.19 at 20 ℃). From Fig. 4C–E, the Tgel of solutions prepared with F3, F4, F5 with 0.6, 0.9, and 1.2 % of κ-Ca were 34, 37 and 42 ℃, respectively. It suggested that the addition of κ-Ca could obviously increase the Tgel of PUL solutions. As the concentration of κ-Ca increased, the gel network formed by κ-Ca and KCl became denser. Thus, less decrease of temperature was required for gelling. Fig. 4 B and D show that the Tgel of solution prepared with F2 was 25 ℃, while that of F4 increased to 37 ℃ due to the addition of 0.07 % KCl. Therefore, the solution prepared with F4 can transform into gel more quickly than that of F2. The gelling promotion effects of KCl and κ-Ca ensured that the solution would not flow from the top of the molds and thus avoided the uneven thickness of capsules.
3. Results and discussion 3.1. Effects on viscosity properties of PUL solutions Fig. 2A shows that the viscosity of PUL solutions increased significantly as PUL concentration increased or temperature decreased. As the concentrations of PUL increased, the number of molecules increased per unit volume, leading to the rise of inter-molecular friction. However, when the temperature of solutions rose, the molecular kinetic energy increased, accelerating the rate of inter-molecular motion, and the hydrogen bonding connections were weakened, thus the flow resistance of solutions decreased. Fig. 2B shows the viscosity of the solutions prepared with F1-F5 at 45 ℃. The viscosity increased with increasing κ-Ca concentration. In the end, by changing the proportion of the PUL or κ -Ca, the suitable viscosity of blended solutions for capsule preparation could be reached. 3.2. Effects on rheological properties of PUL solutions As shown in Fig. 3, shear rates have great effects on the viscosity of the PUL solutions (F2 - F5) at 45 ℃. For all the PUL solutions except F1, at lower shear rates, the viscosities of the solutions decreased rapidly with shear rates; at higher shear rates, the viscosities of the solutions decreased slowly with shear rates. Such effects of shear rates on viscosity are also related to different amounts of κ-Ca and KCl in the solutions. There is no κ-Ca and KCl in F1. In comparison with F2 - F5, F1 has lower viscosities and no obvious change in viscosity with shear rates, belonging to Newtonian behavior. The viscosity of F2 is higher than that of F1 and presents shear rate dependent, implying that addition of κ-Ca changes the rheological behavior of PUL solutions. At higher shear rates, the viscosities of F2 look greater than those of F4, illustrating that addition of KCl has an effect on viscosities of the PUL solutions containing the same amount of κ-Ca (0.9 %). The viscosities of
Fig. 2. (A): Effects of concentration and temperature on viscosity of PUL aqueous solutions. (B): The Viscosity of solutions prepared with F1-F5 at 45 ℃. Viscosity was measured by a digital rotary viscometer. 4
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Fig. 4. A-E are the temperature sweep curves of solutions prepared with F1-F5 respectively during a cooling ramp from 70 to 20 ℃. G’: storage moduli. G’’: loss moduli.
bones. κ-Ca, KCl and PUL formed a system similar to a semi-interpenetrating polymer network (semi-IPN) (Dragan, 2014). As the concentration of κ-Ca and KCl increased, the gel network became denser. Besides, the addition of KCl prevented κ-Ca releasing to the surrounding aqueous phase, and reduce the ratio of κ-Ca mobile chains (Nguyen, Nicolai, Benyahia, & Chassenieux, 2014). As a result, the gel network was strengthened and thus the hardness of gel was improved. Fig. 5A also shows that fracturability increased from 115 to 245 g as the concentration of κ-Ca increased from 0.6 to 1.2 %, and we can deduce that the increase in cross-linking resulted in reduced flexibility of polymer chains (Thrimawithana, Young, Dunstan, & Alany, 2010). The adhesiveness of the samples was positively correlated with the concentration of κ-Ca, and this result was consistent with the viscosity test result of solutions. Moreover, no significant relationship was found between the
3.3. Effects on texture properties of PUL gels and mechanical properties of PUL films The TPA curves are showed in Fig. 5. Hardness, adhesiveness and fracturability of PUL gels and mechanical properties of PUL films were measured by TPA. The fracturability of F2 is not included since the software didn’t detected the peak of fracturability under the TPA mode. As shown in Fig. 5A, the hardness of PUL gels with 0.07 % KCl increased from 83 to 247 g as the concentration of κ-Ca increased from 0.6 to 1.2 %, and that of PUL gels with 0.9 % κ-Ca increased from 49 to 152 g with the addition of 0.07 % KCl. We speculate that the cross-linking happened between κ-Ca and KCl, and PUL, the linear polysaccharide, was interpenetrating in the above network and connecting with κ-Ca via H-
Fig. 5. A: Effects of κ-Ca on hardness, adhesiveness and fracurability of 15 % PUL gels; B: Effects of different concentrations of κ-Ca (0, 0.3, 0.6, 0.9 and 1.2 %) on tensile stress (TS) and enlongation at break (EB) of PUL films. Different symbols (※ △ ★) indicate significant difference (p < 0.05) from F4.
5
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springiness of the gel and the concentration of κ-Ca, and the values of springiness were all small and around 1. Capsules should have adequate mechanical strength and extensibility to meet the requirements of capsules during production, packaging, transportation and storage (Falguera, Quintero, Jiménez, Muñoz, & Ibarz, 2011). TS and EB indicate the film strength and flexibility, respectively (Tong, Xiao, & Lim, 2008). TS and EB data of PUL films with 0.07 % KCl and different concentrations of κ-Ca are summarized in Fig. 5B. The Pearson correlation coefficients between TS or EB and κ-Ca contents were analyzed, and it showed that TS and EB were both significantly (p < 0.05) correlated with the concentration of κ-Ca. TS of the films increased gradually from 40.02 to 60.74 MPa, while EB decreased from 3.4 to 1.9 % with increased κ-Ca concentrations (from 0 to 1.2 %). The pure PUL film was the most flexible. As the κ-Ca content increased, the gel network made by KCl crosslinked κ-Ca network became denser, resulting in the decrease of the flexibility and increase of the strength. What’s more, the addition of κ-Ca also increased the intermolecular hydrogen bonds between the -OSO3 group of κ-Ca backbone and hydroxyl group of PUL, thus the strength of the films was increased. In this study, the addition of κ-Ca endowed PUL films with strength that made it suitable as capsule materials.
still slowly flew down from the molds. When the molds rose from the solutions prepared with F3, F4 and F5 after dipping, it was found that the solutions quickly transferred into gels and adhered to the molds, and the wall thickness of prepared capsules were even. That’s because κ-Ca and KCl formed gel network with the addition of KCl in F3-F5, and PUL acted as a filler and a thickener in the κ-Ca network strengthen by the KCl. The wall thickness, brittleness, disintegration time and water content of capsules prepared with F2-F5 were measured by the relevant regulations of the Chinese Pharmacopoeia, and the results are displayed in Table 2. By comparing capsules prepared with F2 and F4, the addition of KCl could increase the wall thickness and disintegration time of capsules. The disintegration time of capsules increased as κ-Ca concentration increased, and that of capsules prepared with F3 and F4 met the standard of the Chinese Pharmacopoeia (less than 20 min). The water content of the capsules prepared with F2-F5 was in accordance with the provision of the Chinese Pharmacopoeia (below to 14 %). The result was in consistent with the result of first weight loss in the TGA test of the films. As a result, PUL capsules prepared with F4 (Fig. 7C) have the best properties and qualities among those formulas according to the Chinese Pharmacopoeia.
3.4. Effects on thermal properties of PUL films
3.6. FTIR analysis
The DSC curves and TGA curves of the PUL films of F1 - F5 are shown in Fig. 6. The DSC curves show no obvious peak in the range of 20–230 ℃ which indicates no phase transition of samples during this process. The curve of F1 begin to become uneven at above 260 ℃, meaning decomposition of PUL maybe occur at above 260 ℃. The other curves for F2-F5 become uneven at slight lower temperatures from 240 to 260℃. This is because that the PUL films of F2 – F5 contain small amounts of κ-Ca and/or KCl. The TGA curves present a slow downtrend at lower temperatures (< 150 ℃) with a sharp one at above 260 ℃. The slow downtrend should be attributed to the loss of adsorbed and bound water in the PUL films. The sharp downtrend might be due to decomposition of PUL. The decomposition of PUL results in rapid weight loss.
The chemical structures of PUL, κ-Ca and capsules prepared with F4 were investigated by FTIR. As shown in Fig. 8A, all samples exhibit a wide and strong absorption peak between 3200 and 3500 cm−1, which was due to the stretching vibration of hydroxyl in polysaccharides. The absorption peak between 2800 and 2900 cm−1, 900 and 1200 cm−1 were assigned to the stretching vibration of −CH2- and OeCeO, respectively. The strongest absorption peak at around 1150 cm−1 was mainly related to the stretching vibration of (1→4) glycosidic bonds (Kowalczyk et al., 2019). A week absorption peak near 1647 cm−1 was attributed to the flexural vibration of water molecules (Lasagabaster, Abad, Barral, & Ares, 2006). For κ-Ca, an obvious wide absorption peak could be seen near 1220 cm−1, which was OeSeO stretching vibration of sulfate group (Makshakova, Faizullin, & Zuev, 2020). The PUL capsules prepared with F4 showed a similar curve with PUL, where the characteristic peak of κ-Ca at 1220 cm−1 was no longer exist. That’s because the amount of κ-Ca in PUL capsule was relatively small, and the fingerprint of κ-Ca was no longer displayed.
3.5. Effects on PUL capsule performances PUL capsules were prepared with F2-F5. The viscosity of solution prepared with F1 (15 % PUL alone) was too low to prepare capsules, then the concentration of PUL in the solution was increased to 25 %. However, the prepared capsules were very soft, so they were easy to form folds on their surface and be torn when they were removed from the molds, as shown in Fig. 7A. This proves that PUL is not able to form network with consistency interaction to confer enough mechanical structure to capsules. Capsules prepared with F2 (Fig. 7B) had uneven wall thickness (80.6 ± 19.5 μm). Though the viscosity of the solution increased, it
3.7. X-ray diffraction As shown in Fig. 8B, in comparison with the X-ray diffraction patterns of neat KCl powders, no X-ray diffraction peak of KCl presents in the X-ray diffraction pattern of the capsules, indicating KCl is not in crystalline state in the capsules. The typical noncrystalline broad diffraction peak for the capsules suggests that no crystallization exists in the PUL capsule. It is well documented that PUL and κ-Ca are both of
Fig. 6. DSC curves (A) and TGA curves (B) of film samples prepared with F1-F5. 6
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Fig. 7. Photographs of capsules prepared with different formulae: A: pure PUL (25 %). B: F2 (15 % PUL and 0.9 % κ-Ca). C: F4 (15 % PUL, 0.9 % κ-Ca and 0.07 % KCl).
which were both smooth and dense without cracks or pores. It was indicated that PUL was compatible with κ-Ca.
Table 2 Functional properties of PUL capsules prepared with F1-F5. Formula
F1 F2 F3 F4 F5
Wall thickness (μm)
Brittlenessa
– 80.6 ± 19.5b 88.1 ± 7.0b 101.9 ± 5.3 123.1 ± 11.3b
– good good good good
Disintegration time (min)
Water content (%)
– 15.0 16.7 18.0 23.3
– 11.87 11.32 12.22 12.85
± ± ± ±
1.0b 0.6v 0.0 0.6b
3.9. In vitro dissolution of chlorpheniramine maleate ± ± ± ±
Chlorpheniramine maleate was selected as a model drug to investigate the dissolution performance of PUL capsules prepared with F4. As shown in Fig. S3 (Supplementary Information), it was found that no drug was released within 2 min. About 14 % of the drug was released at 5 min, while the accumulated release amount of chlorpheniramine maleate was more than 85 % at 10 min. That’s because the capsules kept integrity in the first 2 min with a swelling process and then disintegrated. Almost all of drug (≥99.25 %) was released within 15 min, which met the requirement of the Chinese Pharmacopeia.
0.35 0.89 0.55 0.50
a the capsules are qualified in brittleness if less than 5 of the 50 capsules tested are broken. If less than 5 of the 50 capsules tested are broken, the brittleness of capsules is considered as good. b indicates significant differences at p < 0.05 from F4.
amorphous structure (Kristo & Biliaderis, 2007). 4. Conclusion 3.8. Microstructure of the PUL capsules κ-Ca can initiate gelling of PUL solutions and KCl facilitates such gelling. κ-Ca has significant impact on rheological properties of PUL solutions, texture properties of PUL gels and films, and PUL hard capsule performances. κ-Ca can effectively influence the viscosity of PUL
The microstructure of the PUL capsules was observed by SEM. The surface and the cross-section of the PUL capsules prepared with F4 are shown in Fig. S2 A and B (Supplementary Information), respectively,
Fig. 8. A: FTIR spectra of the capsules prepared with PUL, F4 and κ-Ca; B: The X-ray diffraction patterns of the capsules prepared by F4 and KCl powder. 7
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Jeon, Y. J., Kamil, J. Y., & Shahidi, F. (2002). Chitosan as an edible invisible film for quality preservation of herring and Atlantic cod. Journal of Agricultural and Food Chemistry, 50(18), 5167–5178. Kara, S., Tamerler, C., Bermek, H., & Pekcan, Ö. (2003). Cation effects on sol–gel and gel–sol phase transitions of κ-Carrageenan–water system. International Journal of Biological Macromolecules, 31(4–5), 177–185. Kim, J. Y., Choi, Y. G., Kim, S. R. B., & Lim, S. T. (2014). Humidity stability of tapioca starch–pullulan composite films. Food Hydrocolloids, 41, 140–145. Kowalczyk, D., Kordowska-Wiater, M., Karaś, M., Zięba, E., Mężyńska, M., & Wiącek, A. E. (2019). Release kinetics and antimicrobial properties of the potassium sorbate-loaded edible films made from pullulan, gelatin and their blends. Food Hydrocolloids, 101, 105539. Kristo, E., & Biliaderis, C. G. (2007). Physical properties of starch nanocrystal-reinforced pullulan films. Carbohydrate Polymers, 68(1), 146–158. Lasagabaster, A., Abad, M. J., Barral, L., & Ares, A. (2006). FTIR study on the nature of water sorbed in polypropylene (PP)/ethylene alcohol vinyl (EVOH) films. European Polymer Journal, 42(11), 3121–3132. Li, L., Ni, R., Shao, Y., & Mao, S. (2014). Carrageenan and its applications in drug delivery. Carbohydrate Polymers, 103, 1–11. Makshakova, O. N., Faizullin, D. A., & Zuev, Y. F. (2020). Interplay between secondary structure and ion binding upon thermoreversible gelation of κ-carrageenan. Carbohydrate Polymers, 227, 115342. Necas, J., & Bartosikova, L. (2013). Carrageenan: A review. Veterinarni Medicina, 58(4). Nguyen, B. T., Nicolai, T., Benyahia, L., & Chassenieux, C. (2014). Synergistic effects of mixed salt on the gelation of κ-Carrageenan. Carbohydrate Polymers, 112, 10–15. Robal, M., Brenner, T., Matsukawa, S., Ogawa, H., Truus, K., Rudolph, B., & Tuvikene, R. (2017). Monocationic salts of carrageenans: Preparation and physico-chemical properties. Food Hydrocolloids, 63, 656–667. Shen, J., Chang, Y., Chen, F., & Dong, S. (2018). Expression and characterization of a κcarrageenase from marine bacterium Wenyingzhuangia aestuarii OF219: A biotechnological tool for the depolymerization of κ-carrageenan. International Journal of Biological Macromolecules, 112, 93–100. Singh, R. S., Saini, G. K., & Kennedy, J. F. (2008). Pullulan: Microbial sources, production and applications. Carbohydrate Polymers, 73(4), 515–531. Thrimawithana, T. R., Young, S., Dunstan, D. E., & Alany, R. G. (2010). Texture and rheological characterization of kappa and iota carrageenan in the presence of counter ions. Carbohydrate Polymers, 82(1), 69–77. Tomšič, M., Prossnigg, F., & Glatter, O. (2008). A thermoreversible double gel: Characterization of a methylcellulose and κ-Carrageenan mixed system in water by SAXS, DSC and rheology. Journal of Colloid and Interface Science, 322(1), 41–50. Tong, Q., Xiao, Q., & Lim, L. T. (2008). Preparation and properties of pullulan–alginate–carboxymethylcellulose blend films. Food Research International, 41(10), 1007–1014. Wang, Y., Yuan, C., Cui, B., & Liu, Y. (2018). Influence of cations on texture, compressive elastic modulus, sol-gel transition and freeze-thaw properties of kappa-carrageenan gel. Carbohydrate Polymers, 202, 530–535. Wu, P., & Imai, M. (2012). Outstanding molecular size recognition and regulation of water permeability on K-carrageenan-pullulan membrane involved in synergistic design of composite polysaccharides–structure. Procedia Engineering, 42, 1313–1325. Xiao, Q., Lim, L. T., & Tong, Q. (2012). Properties of pullulan-based blend films as affected by alginate content and relative humidity. Carbohydrate Polymers, 87(1), 227–234. Xiao, Q., Tong, Q., & Lim, L. T. (2012). Pullulan-sodium alginate based edible films: Rheological properties of film forming solutions. Carbohydrate Polymers, 87(2), 1689–1695. Yuan, C., Du, L., Zhang, G., Jin, Z., & Liu, H. (2016). Influence of cyclodextrins on texture behavior and freeze-thaw stability of kappa-carrageenan gel. Food Chemistry, 210, 600–605. Zhang, Y., Zhao, Q., Wang, H., Jiang, X., & Cha, R. (2017). Preparation of green and gelatin-free nanocrystalline cellulose capsules. Carbohydrate Polymers, 164, 358–363. Zhang, L., Wang, Y., Liu, H., Yu, L., Liu, X., Chen, L., & Zhang, N. (2013). Developing hydroxypropyl methylcellulose/hydroxypropyl starch blends for use as capsule materials. Carbohydrate Polymers, 98(1), 73–79.
solutions, the Tgel, hardness, fracturability and adhesiveness of PUL gel, and the mechanical strength of PUL film. κ-Ca can prolong the disintegration time of PUL capsules. These results are interesting and useful to develop PUL capsules using κ-Ca and KCl as additives. CRediT authorship contribution statement Yihui Zhang: Conceptualization, Investigation, Writing - original draft, Formal analysis. Ning Yang: Data curation, Writing - review & editing, Formal analysis. Yaqiong Zhang: Resources. Jingwen Hou: Resources. Huijie Han: Validation. Zhu Jin: Writing - review & editing. Yuanyuan Shen: Supervision. Shengrong Guo: Conceptualization, Project administration, Supervision, Resources. Declaration of Competing Interest None. Acknowledgement We acknowledge financial supports from National Natural Science Foundation of China (grant numbers 81773647), Medicine-Engineering Joint Foundation at Shanghai Jiao Tong University (grant numbers YG2019QNB36). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2020.116190. References ASTM D882-18 (2018). Standard test method for tensile properties of thin plastic sheeting. Philadelphia: American Society for Testing and Materials. Barbosa, J. A., Al-Kauraishi, M. M., Smith, A. M., Conway, B. R., & Merchant, H. A. (2019). Achieving gastroresistance without coating: Formulation of capsule shells from enteric polymers. European Journal of Pharmaceutics and Biopharmaceutics, 144, 174–179. Bercea, M., & Wolf, B. A. (2019). Associative behaviour of κ-carrageenan in aqueous solutions and its modification by different monovalent salts as reflected by viscometric parameters. International Journal of Biological Macromolecules, 140, 661–667. Chu, Y., Xu, T., Gao, C., Liu, X., Zhang, N., Feng, X., ... Tang, X. (2019). Evaluations of physicochemical and biological properties of pullulan-based films incorporated with cinnamon essential oil and Tween 80. International Journal of Biological Macromolecules, 122, 388–394. Dragan, E. S. (2014). Design and applications of interpenetrating polymer network hydrogels. A review. Chemical Engineering Journal, 243, 572–590. Falguera, V., Quintero, J. P., Jiménez, A., Muñoz, J. A., & Ibarz, A. (2011). Edible films and coatings: Structures, active functions and trends in their use. Trends in Food Science & Technology, 22(6), 292–303. Farris, S., Introzzi, L., Fuentes-Alventosa, J. M., Santo, N., Rocca, R., & Piergiovanni, L. (2012). Self-assembled pullulan–silica oxygen barrier hybrid coatings for food packaging applications. Journal of Agricultural and Food Chemistry, 60(3), 782–790.
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Effects of κ-carrageenan on pullulan’s rheological and texture properties as well as pullulan hard capsule performances
T
Yihui Zhanga,1, Ning Yanga,1, Yaqiong Zhangb, Jingwen Houc, Huijie Hana, Zhu Jina, Yuanyuan Shena, Shengrong Guoa,* a
School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China c Instrumental Analysis Centre, Shanghai Jiao Tong University, Shanghai, 200240, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Pullulan κ-Carrageenan Rheology Texture property
κ-Carrageenan (κ-Ca) is often used to facilitate gelling of aqueous solutions of polysaccharides. However, studies on its effects on pullulan’s rheological and texture properties and pullulan (PUL) hard capsule performances have rarely been reported. Herein, effects of κ-Ca on PUL solutions, hydrogels, films and hard capsules were investigated. It was found that the gelling temperature of 15 % (w/w) PUL solutions with 0.07 % KCl increased from 34 ℃ to 42 ℃ as the concentration of κ-Ca increased from 0.6 % to 1.2 %, and the gelling temperature rose from 25 ℃ to 37 ℃ by adding a small amount of KCl (0.07 %) for 15 % PUL solutions with 0.9 % κ-Ca. As the κ-Ca concentration increased, hardness, fracturability and adhesiveness rose for PUL gels and tensile stress increased for PUL films. PUL capsules could be easily prepared by the aid of κ-Ca, and performances of capsules could be adjusted by changing the amount of κ-Ca.
1. Introduction Hard capsules are widely applied in pharmaceutical market, and gelatin is the most commonly used material for capsule preparation (Barbosa, Al-Kauraishi, Smith, Conway, & Merchant, 2019). However, gelatin is relatively expensive, and it derived from bones and skins of animals (pigs and cattle), so gelatin capsules are not suitable for vegetarians and people of religious beliefs (Zhang, Zhao, Wang, Jiang, & Cha, 2017). Therefore, it is interesting to investigate non-gelatin or non-animal origin capsule materials. PUL is a neutral linear polysaccharide produced by Aureobasidium pullulans. PUL has the chemical structure of maltotriose repeating units interconnected by α-(1→6) glycosidic linkages, and in each maltotriose, the three glucoses are connected with each other by α-(1→4) glycosidic linkages (Singh, Saini, & Kennedy, 2008). Currently, PUL is popular in food, material, and pharmaceutical industries (Farris et al., 2012). The advantages of PUL films have been well documented. For example, Singh et al. (2008) reported that PUL films were transparent, water-soluble, with excellent mechanical properties and oxygen-barrier characteristics. Besides, Kim, Choi, Kim, and Lim (2014) reported that PUL was usually incorporated into starch to prepare films with better mechanical and physical properties than pure starch films. That makes
PUL an ideal material for edible films, as well as a biodegradable and water-soluble packaging material (Kowalczyk et al., 2019). However, the mechanical strength of pure PUL cannot meet its preparation and handling requirements when it is applied for capsules (Kristo & Biliaderis, 2007). The strength of PUL can be improved by blending it with other polymers like chitosan, carboxymethyl chitosan (Jeon, Kamil, & Shahidi, 2002) or carrageenan (Wu & Imai, 2012). κ-Ca is a natural sulfated polysaccharide extracted from marine red algae (Shen, Chang, Chen, & Dong, 2018). The chemical structure of κCa is formed by repeating units of α-(1→3)-linked galactose-4-sulphate and β-(1→4)-linked 3,6-anhydrogalactose (Li, Ni, Shao, & Mao, 2014). It is widely used in food, cosmetic and pharmaceutical industries on account of its remarkable properties such as gelling, thickening, stabilizing and texture enhancing abilities (Necas & Bartosikova, 2013). κ-Ca can easily form thermal reversible gels by the aid of gel inducing agents, usually cations, when it is cooled below gelling temperature. During the gelling process, the conformation of κ-Ca transforms from a random coil to a helix (Yuan, Du, Zhang, Jin, & Liu, 2016). It is followed by aggregation of these helices, resulting in gelation (Robal et al., 2017; Wang, Yuan, Cui, & Liu, 2018). The addition of cations can enhance the order degree of conformation and aggregation of helices to enhance the strength of κ-Ca gel (Kara, Tamerler, Bermek, & Pekcan, 2003), among
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Corresponding author. E-mail address: [email protected] (S. Guo). 1 Equal contributors. https://doi.org/10.1016/j.carbpol.2020.116190 Received 30 November 2019; Received in revised form 14 March 2020; Accepted 16 March 2020 Available online 19 March 2020 0144-8617/ © 2020 Published by Elsevier Ltd.
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which K+ is usually used (Bercea & Wolf, 2019). During the gelling process, the proportion of helices increases as the temperature decreases. So far, few studies have focused on the effects of κ-Ca on PUL’s rheological and texture properties, and the mechanism of such effects has seldom been explained. Moreover, the effects of κ-Ca in preparation of PUL capsules and their performances are useful and interesting to investigate. The aim of this study is to examine the effects of κ-Ca on PUL solutions, gels, films and capsules, and the performances of PUL hard capsules in terms of gelling temperature, hardness, fracturability, tensile strength, enlongation at break, brittleness and disintegration time.
Table 1 Compositions of five formulas (F1-F5) of PUL solutions. Formula
PUL (%, w/w a)
κ-Ca (%, w/w a)
K+ (%, w/w a)
1 2 3 4 5
15 15 15 15 15
– 0.9 0.6 0.9 1.2
– – 0.07 0.07 0.07
a The w/w refers to the dry weight of each ingredient relative to the weight of water.
distilled water with continuous stirring under heating in a water bath at 90 ℃ until the powder was totally dispersed. Then, the blended solutions were prepared by adding 0.07 % KCl and 0.3 %, 0.6 %, 0.9 % or 1.2 % κ-Ca under mild mechanical stirring, respectively. Compositions of five formulas (F1-F5) of PUL solutions are shown in Table 1. The mixtures were continuously stirred at 90 ℃ for 30 min, and then they were cooled to 45 ℃ and kept for 1 h to remove trapped air bubbles. The aforementioned solutions were poured into weighing bottles and then cooled down at 25 ℃ to form gel. Afterwards, the weighing bottles were placed in a 4 ℃ refrigerator overnight to equilibrate before testing.
2. Materials and methods 2.1. Materials Purified PUL powder was purchased from Tongliao Meihua Biochemical Science & Technology Co., Ltd (Inner Mongolia, China) with 3.20 % moisture concentration. The purity of PUL is as high as 95 % and the molecular weight of PUL was 2 × 105 Da. κ-Ca powder was commercially obtained from Zhejiang Top Biological Science & Technology Co., Ltd (Zhejiang, China), and the purity of it was as high as 99 %. Potassium chloride (KCl) was purchased from Guoyao Reagents Ltd. (Shanghai, China) and of analytical grade. The chemical structures of PUL and κ-Ca as well as the gelation mechanism of κ-Ca are shown in Fig. 1.
2.3. Casting film and hard capsule preparation About 10 g of the prepared solutions was poured onto a leveled glass plate (10 × 10 cm), followed by drying at 35 ℃ in an oven for about 8 h. Then the films were peeled off the plate and stored at 25 ± 1 ℃ and 55 ± 1% relative humidity (RH) before testing. The thickness of formed film samples was recorded using a micrometer (Guilin, China).
2.2. Solution and gel preparation PUL solutions (15 %) were prepared by dissolving PUL powder into
Fig. 1. A: Chemical structure of PUL, maltotriose repeating units are linked each other through α-(1→6) glycosidic linkages to form PUL. B: Chemical structure of κCa. C: schematic representation of the thermal reversible gelation mechanism of κ-Ca (κ-Ca network, line; potassium ion, dot). 2
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8 different points were chosen on each film as replicates. The thickness was 0.1 ± 5% cm, which was in accordance with the capsule thickness prepared. The prepared methods of hard capsules followed the description by Zhang et al. (2013) with some modifications. Stainless steel mold pins (cylindrical: cap, 7.2 ± 0.02 mm diameter; body, 7.0 ± 0.02 mm diameter) were dipped into prepared solutions and then dried in an oven at 50 ℃ for about 1 h. Capsule wall thickness measurements were taken at 8 different points on each sample with a micrometer.
EB = (L−L0) / L0 × 100
2.4.5. Thermal analysis Differential scanning calorimetry (DSC) analysis of the films was investigated using DSC 204F1 (Netzsch, Germany). Samples of about 4 mg were sealed in aluminum pans and heated from 25 ℃ to 220 ℃ to remove the water and then the samples were cooled to 20 ℃. At last, samples were heated to 270 ℃ at a heating rate of 10 ℃ / min under nitrogen. Thermogravimetric analysis (TGA) was performed in order to measure the thermal stability and decomposition characteristics of films with a thermogravimetric analyzer (SDT-Q600, America). Samples weighing around 6−8 mg in alumina pans were heated from 30 ℃ to 600 ℃ at a rate of 10 ℃ / min−1 under nitrogen.
2.4. Characterization 2.4.1. Viscosity measurements The viscosities of PUL aqueous solutions or the solutions prepared with F1-F5 were measured using a digital rotary viscometer (NDJ-79, Shanghai, China). A rotating cylinder used for measurements is based on viscosity range of solutions (as shown in Table S1), each rotating cylinder corresponds to a certain shear rate.
2.4.6. Water content PUL hard capsules were stored at 25 ℃ and 55 % RH in desiccators for 72 h, and their weights were recorded as m0. Then they were dried at 105 ℃ in a muffle oven until the constant weights (m1) were reached. The water content was calculated using the equation as following (Chu et al., 2019):
2.4.2. Rheological measurements The rheological measurements were performed using an AR G2 rheometer (TA Instrument, USA) equipped with a plate geometry stainless steel (diameter: 40 mm, angle: 2°, gap: 500 μm). The dynamic viscoelastic tests were conducted in the linear viscoelastic region (LVR). The upper plate was covered with sealing rings to prevent evaporation of water during the test. The flow properties of the solution samples were measured at shear rate from 0.1 to 100 s−1 at 45 ℃. The flow curves of solutions were recorded and fitted by power-law model (Xiao, Tong, & Lim, 2012): τ= K·γ
n
Water content (%) = (m0−m1)/m0 × 100 %
(4)
Each test was done in triplicate. 2.4.7. Disintegration and brittleness test A disintegration apparatus (ZB-1 G, Tianjin Haiyida Technology Co. Ltd, China) was used for the disintegration test of the PUL capsules. The disintegration test was done according to Chinese pharmacopoeia (2015). Six capsules filled with talcum were tested in distilled water at 37 ± 1 ℃. The time all the six capsules tested completely disintegrate is considered as disintegration time. The brittleness of capsules was measured according to Chinese pharmacopoeia (2015). The capsules are qualified in brittleness if less than 5 of the 50 capsules tested are broken. If less than 5 of the 50 capsules tested are broken, the brittleness of capsules is considered as good.
(1)
Where τ is shear stress (Pa), K is the consistency index (Pa·s ), γ is shear rate (s−1) and n is flow behavior index. The viscoelastic properties of the solution samples were measured as following: after an equilibration at 70 ℃ for 5 min, a cooling ramp from 70 to 20 ℃ with rate of 1 ℃ /min was achieved (strain: 1%, frequency: 1 rad/s). The storage modulus G’ and loss modulus G” were recorded. The intersection of the two moduli in the curve is defined as the gelling temperature (Tgel) (Tomšič, Prossnigg, & Glatter, 2008), which indicated the transformation temperature from the solution state to gel state. n
2.4.8. Fourier transforms infrared spectroscopy (FTIR) PUL, κ-Ca and the capsules prepared by F4 were scanned using a Nicolet 6700 spectrometer (Thermo Fisher, America) with attenuated total reflectance (ATR). FTIR spectra of each sample was performed with a resolution of 4 cm−1 and a range of 600−4000 cm−1 and 32 scans.
2.4.3. Texture profile analysis Texture profile analysis (TPA) of the gel samples were carried out on a TA-XT plus texture analyzer (Stable Micro Systems, UK) under the TPA mode. The sample was compressed twice at a deformation of 50 % sample height using a P/0.5 probe. The interval between two compression phases was 5 s. The test, pre-test and post-test speed were 0.5, 2 and 3 mm/s, respectively. All measurements were done in five times.
2.4.9. X-ray diffraction (XRD) KCl powders and the capsules prepared with F4 were scanned with an X-ray diffractometer (XRD; D8 ADVANCE Da Vinci, Germany). The applied X-ray beam was 40 kV, 40 mA and the scanning range of the diffraction angle 2θ° was 3–400 with a rate of 4°/min.
2.4.4. Mechanical properties of film The tensile strength (TS, MPa) and elongation at break (EB, %) were determined using a TA-XT plus texture analyzer (Stable Micro Systems, UK) equipped with a 5 kg load cell. The specimens were of dumbbell shape (gap 70 mm, width 6 mm). The mechanical properties of film samples were measured according to ASTM standard method D882-18 (ASTM, 2018). The initial grip distance was 50 mm and the cross-head speed was 1 mm/s. Each test consisted of six replicate measurements. TS was calculated by dividing the peak stress (F, N) by the crosssectional area (A, m2). Percentage EB was calculated by dividing the increase in length at break (L) by the initial length (L0) of the film and multiplying by 100. It can also describe as the following equations (Xiao, Lim, & Tong, 2012): TS = F/A
(3)
2.4.10. Scanning electron microscopy (SEM) Both the surface and the cross-section of the capsules prepared with F4 were observed by scanning electro microscopy (SEM; Mira3/MIRA3, TESCAN, Czech Republic). Capsule specimens were immersed into liquid nitrogen for 10 min and then fractured into pieces (about 3 mm × 3 mm). A gold coating was added on the specimens with a sputter. 2.4.11. In vitro dissolution of chlorpheniramine maleate Dissolution of chlorpheniramine maleate was measured by using the Paddle method (Chinese Pharmacopoeia (Edition 2015)). A small piece of corrosion-resistant fine metal wire was used as a sinker to wind about a capsule. The dissolution test for the PUL capsules containing 2 mg chlorpheniramine maleate and 248 mg lactose was carried out in
(2) 3
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900 mL of 0.1 mol/L HCl solution, at 75 rpm and 37 ± 0.5 ℃, by using a dissolution apparatus (ZRS-8C, Tianjin University Radio Factory, China). The samples were taken out from the dissolution medium at a predetermined time and filtered through a 0.22 μm polyethersulfone membrane for analysis. The samples were analyzed using a HPLC system (Waters, Singapore). The measurement was carried out on a C18 column (250 × 4.6 mm, 5 μm) with a mobile phase of phosphate buffer - acetonitrile (80:20, v/v). The column temperature was 30 ℃, and the detective wavelength was 262 nm. 2.5. Statistical analysis Data were expressed as the mean ± standard deviation (SD). The ttest and Pearson correlation coefficients were respectively used for the significance and correlation analysis by SPSS 2015 (IBM Inc., USA) with a significance level of p < 0.05.
Fig. 3. Effects of shear rate on the viscosity of solution samples prepared with F1-F5 at 45 ℃. Viscosity was measured by an AR G2 rheometer.
F3 containing smaller amount of κ-Ca (0.6 %) look lower than those of F2, F4 and F5. Effects of shear rates on shear stress are shown in Fig. S1 and the parameters of the Power-law model for the flow curves of PUL solutions are listed in Table S2. Temperature sweep curves of solutions prepared with formula 1–5 were showed in Fig. 4. The gelling behaviors of samples were observed by measuring G’ and G’’. G’ is used to measure material resistance capability of the elastic deformation, G’’ is used to measure the size of the loss of energy in the process of viscous deformation. Fig. 4A shows G’ and G’’ values of 15 % PUL solution, and G’’ was always greater than G’, which means that the 15 % PUL solution maintained in a sol state from 70 ℃ to 20 ℃. It indicated that PUL has no gelation property at a temperature range of 70℃ to 20 ℃, which is consistent with the study of Xiao, Tong, & Tim (2012). Form Fig. 4C–E, we found that G’ increased with the increased concentration of κ-Ca (0.6, 0.9 and 1.2 %), and the values of G’ at 20 ℃ were 57.37, 453.70, 1476.00, respectively. Fig. 4 B and D show that the addition of KCl (0.07 %) also increased the G’ (from 0.02 to 15.19 at 20 ℃). From Fig. 4C–E, the Tgel of solutions prepared with F3, F4, F5 with 0.6, 0.9, and 1.2 % of κ-Ca were 34, 37 and 42 ℃, respectively. It suggested that the addition of κ-Ca could obviously increase the Tgel of PUL solutions. As the concentration of κ-Ca increased, the gel network formed by κ-Ca and KCl became denser. Thus, less decrease of temperature was required for gelling. Fig. 4 B and D show that the Tgel of solution prepared with F2 was 25 ℃, while that of F4 increased to 37 ℃ due to the addition of 0.07 % KCl. Therefore, the solution prepared with F4 can transform into gel more quickly than that of F2. The gelling promotion effects of KCl and κ-Ca ensured that the solution would not flow from the top of the molds and thus avoided the uneven thickness of capsules.
3. Results and discussion 3.1. Effects on viscosity properties of PUL solutions Fig. 2A shows that the viscosity of PUL solutions increased significantly as PUL concentration increased or temperature decreased. As the concentrations of PUL increased, the number of molecules increased per unit volume, leading to the rise of inter-molecular friction. However, when the temperature of solutions rose, the molecular kinetic energy increased, accelerating the rate of inter-molecular motion, and the hydrogen bonding connections were weakened, thus the flow resistance of solutions decreased. Fig. 2B shows the viscosity of the solutions prepared with F1-F5 at 45 ℃. The viscosity increased with increasing κ-Ca concentration. In the end, by changing the proportion of the PUL or κ -Ca, the suitable viscosity of blended solutions for capsule preparation could be reached. 3.2. Effects on rheological properties of PUL solutions As shown in Fig. 3, shear rates have great effects on the viscosity of the PUL solutions (F2 - F5) at 45 ℃. For all the PUL solutions except F1, at lower shear rates, the viscosities of the solutions decreased rapidly with shear rates; at higher shear rates, the viscosities of the solutions decreased slowly with shear rates. Such effects of shear rates on viscosity are also related to different amounts of κ-Ca and KCl in the solutions. There is no κ-Ca and KCl in F1. In comparison with F2 - F5, F1 has lower viscosities and no obvious change in viscosity with shear rates, belonging to Newtonian behavior. The viscosity of F2 is higher than that of F1 and presents shear rate dependent, implying that addition of κ-Ca changes the rheological behavior of PUL solutions. At higher shear rates, the viscosities of F2 look greater than those of F4, illustrating that addition of KCl has an effect on viscosities of the PUL solutions containing the same amount of κ-Ca (0.9 %). The viscosities of
Fig. 2. (A): Effects of concentration and temperature on viscosity of PUL aqueous solutions. (B): The Viscosity of solutions prepared with F1-F5 at 45 ℃. Viscosity was measured by a digital rotary viscometer. 4
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Fig. 4. A-E are the temperature sweep curves of solutions prepared with F1-F5 respectively during a cooling ramp from 70 to 20 ℃. G’: storage moduli. G’’: loss moduli.
bones. κ-Ca, KCl and PUL formed a system similar to a semi-interpenetrating polymer network (semi-IPN) (Dragan, 2014). As the concentration of κ-Ca and KCl increased, the gel network became denser. Besides, the addition of KCl prevented κ-Ca releasing to the surrounding aqueous phase, and reduce the ratio of κ-Ca mobile chains (Nguyen, Nicolai, Benyahia, & Chassenieux, 2014). As a result, the gel network was strengthened and thus the hardness of gel was improved. Fig. 5A also shows that fracturability increased from 115 to 245 g as the concentration of κ-Ca increased from 0.6 to 1.2 %, and we can deduce that the increase in cross-linking resulted in reduced flexibility of polymer chains (Thrimawithana, Young, Dunstan, & Alany, 2010). The adhesiveness of the samples was positively correlated with the concentration of κ-Ca, and this result was consistent with the viscosity test result of solutions. Moreover, no significant relationship was found between the
3.3. Effects on texture properties of PUL gels and mechanical properties of PUL films The TPA curves are showed in Fig. 5. Hardness, adhesiveness and fracturability of PUL gels and mechanical properties of PUL films were measured by TPA. The fracturability of F2 is not included since the software didn’t detected the peak of fracturability under the TPA mode. As shown in Fig. 5A, the hardness of PUL gels with 0.07 % KCl increased from 83 to 247 g as the concentration of κ-Ca increased from 0.6 to 1.2 %, and that of PUL gels with 0.9 % κ-Ca increased from 49 to 152 g with the addition of 0.07 % KCl. We speculate that the cross-linking happened between κ-Ca and KCl, and PUL, the linear polysaccharide, was interpenetrating in the above network and connecting with κ-Ca via H-
Fig. 5. A: Effects of κ-Ca on hardness, adhesiveness and fracurability of 15 % PUL gels; B: Effects of different concentrations of κ-Ca (0, 0.3, 0.6, 0.9 and 1.2 %) on tensile stress (TS) and enlongation at break (EB) of PUL films. Different symbols (※ △ ★) indicate significant difference (p < 0.05) from F4.
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springiness of the gel and the concentration of κ-Ca, and the values of springiness were all small and around 1. Capsules should have adequate mechanical strength and extensibility to meet the requirements of capsules during production, packaging, transportation and storage (Falguera, Quintero, Jiménez, Muñoz, & Ibarz, 2011). TS and EB indicate the film strength and flexibility, respectively (Tong, Xiao, & Lim, 2008). TS and EB data of PUL films with 0.07 % KCl and different concentrations of κ-Ca are summarized in Fig. 5B. The Pearson correlation coefficients between TS or EB and κ-Ca contents were analyzed, and it showed that TS and EB were both significantly (p < 0.05) correlated with the concentration of κ-Ca. TS of the films increased gradually from 40.02 to 60.74 MPa, while EB decreased from 3.4 to 1.9 % with increased κ-Ca concentrations (from 0 to 1.2 %). The pure PUL film was the most flexible. As the κ-Ca content increased, the gel network made by KCl crosslinked κ-Ca network became denser, resulting in the decrease of the flexibility and increase of the strength. What’s more, the addition of κ-Ca also increased the intermolecular hydrogen bonds between the -OSO3 group of κ-Ca backbone and hydroxyl group of PUL, thus the strength of the films was increased. In this study, the addition of κ-Ca endowed PUL films with strength that made it suitable as capsule materials.
still slowly flew down from the molds. When the molds rose from the solutions prepared with F3, F4 and F5 after dipping, it was found that the solutions quickly transferred into gels and adhered to the molds, and the wall thickness of prepared capsules were even. That’s because κ-Ca and KCl formed gel network with the addition of KCl in F3-F5, and PUL acted as a filler and a thickener in the κ-Ca network strengthen by the KCl. The wall thickness, brittleness, disintegration time and water content of capsules prepared with F2-F5 were measured by the relevant regulations of the Chinese Pharmacopoeia, and the results are displayed in Table 2. By comparing capsules prepared with F2 and F4, the addition of KCl could increase the wall thickness and disintegration time of capsules. The disintegration time of capsules increased as κ-Ca concentration increased, and that of capsules prepared with F3 and F4 met the standard of the Chinese Pharmacopoeia (less than 20 min). The water content of the capsules prepared with F2-F5 was in accordance with the provision of the Chinese Pharmacopoeia (below to 14 %). The result was in consistent with the result of first weight loss in the TGA test of the films. As a result, PUL capsules prepared with F4 (Fig. 7C) have the best properties and qualities among those formulas according to the Chinese Pharmacopoeia.
3.4. Effects on thermal properties of PUL films
3.6. FTIR analysis
The DSC curves and TGA curves of the PUL films of F1 - F5 are shown in Fig. 6. The DSC curves show no obvious peak in the range of 20–230 ℃ which indicates no phase transition of samples during this process. The curve of F1 begin to become uneven at above 260 ℃, meaning decomposition of PUL maybe occur at above 260 ℃. The other curves for F2-F5 become uneven at slight lower temperatures from 240 to 260℃. This is because that the PUL films of F2 – F5 contain small amounts of κ-Ca and/or KCl. The TGA curves present a slow downtrend at lower temperatures (< 150 ℃) with a sharp one at above 260 ℃. The slow downtrend should be attributed to the loss of adsorbed and bound water in the PUL films. The sharp downtrend might be due to decomposition of PUL. The decomposition of PUL results in rapid weight loss.
The chemical structures of PUL, κ-Ca and capsules prepared with F4 were investigated by FTIR. As shown in Fig. 8A, all samples exhibit a wide and strong absorption peak between 3200 and 3500 cm−1, which was due to the stretching vibration of hydroxyl in polysaccharides. The absorption peak between 2800 and 2900 cm−1, 900 and 1200 cm−1 were assigned to the stretching vibration of −CH2- and OeCeO, respectively. The strongest absorption peak at around 1150 cm−1 was mainly related to the stretching vibration of (1→4) glycosidic bonds (Kowalczyk et al., 2019). A week absorption peak near 1647 cm−1 was attributed to the flexural vibration of water molecules (Lasagabaster, Abad, Barral, & Ares, 2006). For κ-Ca, an obvious wide absorption peak could be seen near 1220 cm−1, which was OeSeO stretching vibration of sulfate group (Makshakova, Faizullin, & Zuev, 2020). The PUL capsules prepared with F4 showed a similar curve with PUL, where the characteristic peak of κ-Ca at 1220 cm−1 was no longer exist. That’s because the amount of κ-Ca in PUL capsule was relatively small, and the fingerprint of κ-Ca was no longer displayed.
3.5. Effects on PUL capsule performances PUL capsules were prepared with F2-F5. The viscosity of solution prepared with F1 (15 % PUL alone) was too low to prepare capsules, then the concentration of PUL in the solution was increased to 25 %. However, the prepared capsules were very soft, so they were easy to form folds on their surface and be torn when they were removed from the molds, as shown in Fig. 7A. This proves that PUL is not able to form network with consistency interaction to confer enough mechanical structure to capsules. Capsules prepared with F2 (Fig. 7B) had uneven wall thickness (80.6 ± 19.5 μm). Though the viscosity of the solution increased, it
3.7. X-ray diffraction As shown in Fig. 8B, in comparison with the X-ray diffraction patterns of neat KCl powders, no X-ray diffraction peak of KCl presents in the X-ray diffraction pattern of the capsules, indicating KCl is not in crystalline state in the capsules. The typical noncrystalline broad diffraction peak for the capsules suggests that no crystallization exists in the PUL capsule. It is well documented that PUL and κ-Ca are both of
Fig. 6. DSC curves (A) and TGA curves (B) of film samples prepared with F1-F5. 6
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Fig. 7. Photographs of capsules prepared with different formulae: A: pure PUL (25 %). B: F2 (15 % PUL and 0.9 % κ-Ca). C: F4 (15 % PUL, 0.9 % κ-Ca and 0.07 % KCl).
which were both smooth and dense without cracks or pores. It was indicated that PUL was compatible with κ-Ca.
Table 2 Functional properties of PUL capsules prepared with F1-F5. Formula
F1 F2 F3 F4 F5
Wall thickness (μm)
Brittlenessa
– 80.6 ± 19.5b 88.1 ± 7.0b 101.9 ± 5.3 123.1 ± 11.3b
– good good good good
Disintegration time (min)
Water content (%)
– 15.0 16.7 18.0 23.3
– 11.87 11.32 12.22 12.85
± ± ± ±
1.0b 0.6v 0.0 0.6b
3.9. In vitro dissolution of chlorpheniramine maleate ± ± ± ±
Chlorpheniramine maleate was selected as a model drug to investigate the dissolution performance of PUL capsules prepared with F4. As shown in Fig. S3 (Supplementary Information), it was found that no drug was released within 2 min. About 14 % of the drug was released at 5 min, while the accumulated release amount of chlorpheniramine maleate was more than 85 % at 10 min. That’s because the capsules kept integrity in the first 2 min with a swelling process and then disintegrated. Almost all of drug (≥99.25 %) was released within 15 min, which met the requirement of the Chinese Pharmacopeia.
0.35 0.89 0.55 0.50
a the capsules are qualified in brittleness if less than 5 of the 50 capsules tested are broken. If less than 5 of the 50 capsules tested are broken, the brittleness of capsules is considered as good. b indicates significant differences at p < 0.05 from F4.
amorphous structure (Kristo & Biliaderis, 2007). 4. Conclusion 3.8. Microstructure of the PUL capsules κ-Ca can initiate gelling of PUL solutions and KCl facilitates such gelling. κ-Ca has significant impact on rheological properties of PUL solutions, texture properties of PUL gels and films, and PUL hard capsule performances. κ-Ca can effectively influence the viscosity of PUL
The microstructure of the PUL capsules was observed by SEM. The surface and the cross-section of the PUL capsules prepared with F4 are shown in Fig. S2 A and B (Supplementary Information), respectively,
Fig. 8. A: FTIR spectra of the capsules prepared with PUL, F4 and κ-Ca; B: The X-ray diffraction patterns of the capsules prepared by F4 and KCl powder. 7
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solutions, the Tgel, hardness, fracturability and adhesiveness of PUL gel, and the mechanical strength of PUL film. κ-Ca can prolong the disintegration time of PUL capsules. These results are interesting and useful to develop PUL capsules using κ-Ca and KCl as additives. CRediT authorship contribution statement Yihui Zhang: Conceptualization, Investigation, Writing - original draft, Formal analysis. Ning Yang: Data curation, Writing - review & editing, Formal analysis. Yaqiong Zhang: Resources. Jingwen Hou: Resources. Huijie Han: Validation. Zhu Jin: Writing - review & editing. Yuanyuan Shen: Supervision. Shengrong Guo: Conceptualization, Project administration, Supervision, Resources. Declaration of Competing Interest None. Acknowledgement We acknowledge financial supports from National Natural Science Foundation of China (grant numbers 81773647), Medicine-Engineering Joint Foundation at Shanghai Jiao Tong University (grant numbers YG2019QNB36). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2020.116190. References ASTM D882-18 (2018). Standard test method for tensile properties of thin plastic sheeting. Philadelphia: American Society for Testing and Materials. Barbosa, J. A., Al-Kauraishi, M. M., Smith, A. M., Conway, B. R., & Merchant, H. A. (2019). Achieving gastroresistance without coating: Formulation of capsule shells from enteric polymers. European Journal of Pharmaceutics and Biopharmaceutics, 144, 174–179. Bercea, M., & Wolf, B. A. (2019). Associative behaviour of κ-carrageenan in aqueous solutions and its modification by different monovalent salts as reflected by viscometric parameters. International Journal of Biological Macromolecules, 140, 661–667. Chu, Y., Xu, T., Gao, C., Liu, X., Zhang, N., Feng, X., ... Tang, X. (2019). Evaluations of physicochemical and biological properties of pullulan-based films incorporated with cinnamon essential oil and Tween 80. International Journal of Biological Macromolecules, 122, 388–394. Dragan, E. S. (2014). Design and applications of interpenetrating polymer network hydrogels. A review. Chemical Engineering Journal, 243, 572–590. Falguera, V., Quintero, J. P., Jiménez, A., Muñoz, J. A., & Ibarz, A. (2011). Edible films and coatings: Structures, active functions and trends in their use. Trends in Food Science & Technology, 22(6), 292–303. Farris, S., Introzzi, L., Fuentes-Alventosa, J. M., Santo, N., Rocca, R., & Piergiovanni, L. (2012). Self-assembled pullulan–silica oxygen barrier hybrid coatings for food packaging applications. Journal of Agricultural and Food Chemistry, 60(3), 782–790.
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