Effects of κ-carrageenan on rheological properties of dually modified sago starch: Towards finding gelatin alternative for hard capsules

Carbohydrate Polymers 132 (2015) 156–163

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Effects of ␬-carrageenan on rheological properties of dually modified sago starch: Towards finding gelatin alternative for hard capsules Mohammad-Hassan Fakharian a , Nasser Tamimi a , Hossein Abbaspour b , Abdorreza Mohammadi Nafchi a,c,∗ , A.A. Karim c a

Food Biopolymer Research Group, Food Science and Technology Department, Damghan Branch, Islamic Azad University, Damghan, Semanan, Iran Department of Biological Science, Damghan Branch, Islamic Azad University, Damghan, Semanan, Iran c Food Biopolymer Research Group, Food Technology Division, School of Industrial Technology, Universiti Sains Malaysia, Minden, 11800, Penang, Malaysia b

a r t i c l e

i n f o

Article history: Received 8 May 2015 Received in revised form 29 May 2015 Accepted 10 June 2015 Available online 19 June 2015 Keywords: Modified starch Thermoreversible gel Rheological properties Viscoelastic moduli Sol–gel transition

a b s t r a c t Composite sago starch-based system was developed and characterized with the aim to find an alternative to gelatin in the processing of pharmaceutical capsules. Dually modified (Hydrolyzed–Hydroxypropylated) sago starches were combined with ␬-carrageenan (0.25, 0.5, 0.75, and 1%). The rheological properties of the proposed composite system were measured and compared with gelatin as reference material. Results show that combination of HHSS12 (Hydrolysed–hydroxypropylated sago starch at 12 h) with 0.5% ␬-carrageenan was comparable to gelatin rheological behavior in pharmaceutical capsule processing. The solution viscosity at 50 ◦ C and sol–gel transition of the proposed composite system were comparable to those of gelatin. The viscoelastic moduli (G and G ) for the proposed system were lower than those of gelatin. These results illustrate that by manipulation of the constituents of sago starch-based composite system, a suitable alternative to gelatin can be produced with comparable properties and this could find potential application in pharmaceutical capsule industry. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Among the commercial hydrocolloids used in the food industry, gelatin has long been regarded as special and unique, as it serves multiple functions with a wide range of applications in various industries (Haug & Draget, 2009; Hazaveh, Mohammadi Nafchi, & Abbaspour, 2015; Zhang et al., 2013). Finding an ideal alternative to gelatin is very difficult. An ideal gelatin alternative should possess all, or at least some, of the important properties of gelatin, such as the “melt-in-the-mouth” property, thermal reversibility, versatility, multi-functionality, and ease of use (Karim & Bhat, 2008; Lam et al., 2013). For the production of hard capsules, the most important properties that gelatin alternatives should have are high solubility (>20%) in water, low solution viscosity and stability at 50 ◦ C, the ability to form a thermo-reversible gel with a transition temperature of 20–36 ◦ C, and good film formability (Mohammadi

∗ Corresponding author at: Food Biopolymer Research Group, Food Science and Technology Department, Damghan Branch, Islamic Azad University, Damghan, Semanan, Iran. Tel.: +98 233 5225045; fax: +98 2335225008. E-mail addresses: [email protected], [email protected] (A. Mohammadi Nafchi). http://dx.doi.org/10.1016/j.carbpol.2015.06.033 0144-8617/© 2015 Elsevier Ltd. All rights reserved.

Nafchi, Moradpour, Saeidi, & Alias, 2014). All of these requirements are based on industrial aspects of gelatin hard capsule manufacturing (Missaghi & Fassihi, 2006). Starch is an abundant natural polysaccharide that is economical and easy to use, and it may be a useful alternative to gelatin. However, starch in its native form has many drawbacks relative to gelatin for use in producing pharmaceutical capsules. For example, starch has very limited solubility in water, and at low concentrations it has high viscosity (Fouladi & Mohammadi Nafchi, 2014). In addition, the tendency to retrograde limits the use of starch in certain applications. Modification of starch can overcome some of these drawbacks (Kaur, Ariffin, Bhat, & Karim, 2012). For example, attempts to improve thermo-reversibility of starch gels with enzymatic modifications and the application of certain polysaccharides have been reported (Karim & Bhat, 2008). Sago starch is one of the important socioeconomic crops in Southeast Asia. Starch from sago is the only example of an industrial starch derived from the trunk of the sago palm (Metroxylon sagu Rottb.). Gelatinization temperature of sago starches varies from 69.5 to 70.2 ◦ C (Karim, Tie, Manan, & Zaidul, 2008; Nouri & Mohammadi Nafchi, 2014). Although modification can improve some starch properties, it may have negative effects on other functional properties. For example, hydrolysis of starch increases its solubility, but it also increases

M.-H. Fakharian et al. / Carbohydrate Polymers 132 (2015) 156–163

its tendency to retrograde (Abdorreza, Robal, Cheng, Tajul, & Karim, 2012). In such cases, the combination of different types of modification or mixing with other biopolymers might overcome problems and improve the total properties of modified starches (Fouladi & Mohammadi Nafchi, 2014). ␬-Carrageenan, which is commonly used as a gelling and stabilizing agent in food applications, is also known to have good film forming ability (Briones et al., 2004). The dependency of the ␬carrageenan sol–gel transition temperature on the potassium (K+ ) ion has been well known since the 1980s (Rochas & Rinaudo, 1980; Rochas & Rinaudo, 1982; Rochas & Rinaudo, 1984). The goal of the experiments described in this research was to combine the intrinsic properties of the dually modified sago starch and ␬-carrageenan to prepare gel that can be used for hard capsule manufacturing via dip-molding process. Hydrolyzed and hydroxypropylated sago starch easily disperses at high concentration in water, but it has no gelling properties (Aminian, Mohammadi Nafchi, Bolandi, & Alias, 2013; Fouladi & Mohammadi Nafchi, 2014). The hypothesis here was that addition of ␬-carrageenan in the K+ form as a gelling agent should overcome this problem. This combination also should allow easy control of the sol–gel transition upon cooling, which would yield a thermoreversible gel. To test this hypothesis, rheological properties, and sol–gel transitions of the composite material solutions were compared to those of gelatin (i.e., the control). 2. Materials and methods 2.1. Materials Bovine gelatin sample (bloom = 160) was purchased from Sigma Aldrich (St. Louis, MO, USA) with. Moisture content of the gelatin was 10–11% (wb). The ␬-carrageenan used was purchased from Fluka Chemika. It was a commercial sample and was mainly in the K+ form. Its characteristics were provided by the supplier. It contained 191.8 meq/100 gK+ (7.5%), 87 meq/100 g Na+ (2%), 0.6% chloride, 10 meq/100 g Ca2+ (0.4%), and 1.23 meq/100 g Mg2+ (0.03%). Its average molecular weight was 780,000 g/mol. The moisture content was 10% (wb). Acid hydrolyzed hydroxypropylated sago starch (HHSS) was prepared following the methods described Fouladi and Mohammadi Nafchi (2014). Four types of dually modified sago starch were selected for testing based on solubility and economic factors. The samples were acid hydrolyzed for 6, 12, 18, and 24 h, and then hydroxypropylated (≈0.2 molar substitution). 2.2. Preparation of solutions 2.2.1. Gelatin solutions The gelatin was allowed to swell in deionized water (18 M) for 10 min at room temperature and then placed in a water bath at 60 ◦ C for 45 min under medium agitation and finally vacuum pump used to eliminate air bubbles. The concentration was then adjusted by adding water, and the solution was kept at 50 ◦ C for 30 min. The final concentrations expressed on a dry basis (db) ranged from 20% to 30%. This range of concentrations was based on what is used in industrial applications. 2.2.2. Starch and -carrageenan solutions To prepare mixtures of starch/␬-carrageenan, the components were mixed dry and then dispersed in deionized water at room temperature under vigorous stirring. The dispersion was then placed at 90 ◦ C for 45 min to ensure complete solubilization of the constituents. The homogeneous solution was placed at 70 ◦ C for 2 h to remove residual air bubbles under vacuum, followed by storage at 55 ◦ C for 30 min. The chosen concentration of starch (A) was ∼20% (db). This concentration was chosen based on preliminary

157

dipping process experiments intended to produce a dried film with a thickness near that of pharmaceutical hard capsules (0.1 mm). The concentrations of ␬-carrageenan ranged from 1% to 0.1%, with corresponding KCl concentrations ranging from 0 to 11.70 mM. The use of ␬-carrageenan made it possible to control the temperature of the helix-coil transition by adjusting the total content of K+ (Rochas & Rinaudo, 1984). Based on the diagram provided by Rinaudo and Rochas (1981), a gelation temperature of about 25 ◦ C was chosen for ␬-carrageenan alone. Whatever the concentration of ␬-carrageenan in the range studied (0.1–1%), the total content of K+ (CT ) was kept constant by the ratio of the KCl added and the polymer concentration through the following relationship (Lafargue, Lourdin, & Doublier, 2007a): CT = CS + CP

(1)

where,  is 0.55, CT is total ion concentration, Cs is ion concentration added by salt, and CP is ion concentration provided by the polymer. Table 1 represents the details of concentration calculations. 2.3. Characterizations The rheological measurements were performed using an AR1000 rheometer (TA Instruments Inc., New Cattle, DE, USA) equipped with cone and plate geometry stainless steel (diameter: 40 mm, angle: 2◦ ; truncation: 54 ␮m). During measurements, samples were covered with paraffin oil to avoid evaporation. Standard procedures were established to characterize the rheological behavior of the samples under near industrial processing conditions to simulate the different stages of the development of hard capsules. The linear viscoelastic region (LVR) was determined prior to dynamic viscoelastic studies. This is the first step in measuring the properties of samples at a temperature of 50 ◦ C (temperature at which stock solutions of gelatin are stored). Then, the behavior of gels during cooling and heating were measured in order to determine the key parameters of interest in this study, namely the kinetics of gel formation, the gelling and melting temperature. 2.3.1. Flow properties The samples were loaded on the rheometer plate and heated to 60 ◦ C. The flow curves were performed at 50 ◦ C in linear mode (from 0 to 100 s−1 and back from 100 to 0 s−1 ) and logarithmic mode (100 to 0.01 s−1 with stabilization for 1% min between each point). 2.3.2. Viscoelastic properties The samples were loaded on the rheometer plate and heated to 60 ◦ C. Following the pretested protocols viscoelastic properties of samples were evaluated: Sol characterization • A mechanical spectrum was achieved at 50 ◦ C in the linear area of 100 to 0.1 rad/s with strains of 0.1% in logarithmic mode. Sol–gel transition • A cooling ramp from 50 ◦ C to 20 ◦ C with rates of 1 ◦ C/min was achieved (strain amplitude of 0.1%, frequency measurement 1 rad/s). • A heating ramp of 20 ◦ C to 50 ◦ C with rates of 1 ◦ C/min was then performed (strain amplitude of 0.1%, frequency measurement 1 rad/s). Gel characterization • A mechanical spectrum was achieved at 20 ◦ C in the linear area of 100 to 0.1 rad/s to strains of 0.1%. • Finally, for gel cure evaluations, after storage at 60 ◦ C for 1 h, the variation of the modulus G’ and G” over time at 20 ◦ C after rapid cooling from 60 ◦ C (30 ◦ C/min) was recorded.

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Table 1 Compositions of the starch-␬-carrageenan solution. Description

Starch alone HHSS ␬-carrageenan alone ␬C 1 ␬C 0.75 ␬C 0.5 ␬C 0.25 ␬C 0.1 Mixtures HHSS ␬C 1 HHSS ␬C 0.75 HHSS ␬C 0.5 HHSS ␬C 0.25 HHSS ␬C 0.1

Starch concentrations (%)

␬-carrageenan concentration CP (%)

20

0

0 0 0 0 0 25–30 25–30 25–30 25–30 25–30

␬-carrageenan concentration CP × 103 (eq/l) 0

KCL concentration Cs × 103 (eq/l)

0

1 0.75 0.5 0.25 0.1

23.58 17.69 11.79 5.90 2.36

0 3.24 6.49 9.73 11.70

1 0.75 0.5 0.25 0.1

23.58 17.69 11.79 5.90 2.36

0 3.24 6.49 9.73 11.70

CT constant = 1.3 × 10−2 eq/l, ␬C: ␬-carrageenan. The content of K+ ␬-carrageenan was 2.36 × 10−3 eq/g (M0 = 424 g/mol). For concentration of 1% ␬-carrageenan without salt added, the total ionic concentration is CT = (2.36 × 10−3 eq/g) × (10 g/l) × 0.55 = 1.3 × 10−2 eq/l. HHSS: hydrolyzed–hydroxypropylated sago starch.

3. Results 3.1. Gelatin characterization in capsule processing industry The rheological behaviors of gelatin were characterized in conditions similar to those used in the process of obtaining capsules in industry. 3.1.1. Flow properties The flow properties were determined at 50 ◦ C for gelatin concentrations of 20 to 30% (g/g of water). In this range of concentrations, Newtonian behavior was observed which is in agreement with literature reports (Leuenberger, 1991; Wulansari, Mitchell, Blanshard, & Paterson, 1998). The viscosities at different concentrations are shown in Table 2. 3.1.2. Sol characterization The mechanical spectrum of gelatin at 50 ◦ C was investigated. The modulus G” is greater than G’ over the entire frequency range considered. Moreover, a strong dependence of modulus with frequency was observed: G ␣ ␻1 and G ␣ ␻1.8 . This was observed for all gelatin concentrations studied and reflects the behavior the macromolecular solution of gelatin at this temperature (Lam et al., 2013). 3.1.3. Sol–gel transition Variations of G and G during cooling between 50 ◦ C and 25 ◦ C are shown in Fig. 1(a) at a concentration of 25% gelatin. At 50 ◦ C, G was greater than G and the value of the elastic modulus was low (G ≈ 5 × 10−2 Pa and G ≈ 1 Pa). Between 50 ◦ C and 37 ◦ C, the modulus varied slightly; the modulus increased rapidly between Table 2 Gelling temperature, melting temperature, and solution viscosity (at 50 ◦ C) of gelatin at different concentration. Concentration (%)

Viscosity (Pa s)*

20 21 23 25 27 30

0.24 0.25 0.57 0.69 0.85 1.15

± ± ± ± ± ±

0.02e 0.02e 0.05d 0.04c 0.02b 0.02a

TGEL

TM

30 30 31 32 32 33

33 33 35 36 37 38

* Values are means ± SD. Means with different letter are significantly different at 5% level of probability. Gelation temperatures, TGEL and melting temperature TM  (G = G ) during cooling from 50 to 25 ◦ C and heating from 25 ◦ C to 50 ◦ C. The rate of heating or cooling was 1 ◦ C/min. Frequency: 1 rad/s. Strain amplitude: 1%.

37 ◦ C and 25 ◦ C. At a temperature of 32 ◦ C, the moduli G and G intersected. The point of intersection was taken as the gelation temperature (TGEL ), marking the transition from one state (solution) to another (gel) state (Doublier & Cuvelier, 2006). After the transition temperature for gelatin in these experiments, the modulus reached values of about 6 × 103 Pa for G and 5 × 102 Pa for G at 25 ◦ C. Variations of G and G during heating of the gel between 25 ◦ C and 50 ◦ C are shown in Fig. 1(b). At 25 ◦ C, the value of the modulus was significantly greater than that observed at the end of the ramp cooling. This reflects the evolution of the gel over time when stored at 25 ◦ C for about 30 min. At this temperature, G ∼ 4 × 104 Pa and G ∼ 2 × 103 Pa. During the heating, storage modulus and loss modulus cross at 37 ◦ C, thus defining the melting temperature (TM ) corresponding to the transition from gel to sol. The modulus then decrease gradually until 50 ◦ C; the values at this temperature were similar to those obtained before the cooling ramp. This demonstrates the thermo-reversible nature of gelatin gels. The temperatures of gelling and melting for different gelatin concentrations studied are given in Table 2. There was a slight increase in TGEL from 30 ◦ C to 33 ◦ C when the concentration was increased from 20% to 30%. The melting temperature TM was greater by several degrees (3 ◦ C to 5 ◦ C) compared to TGEL and varies in a comparable manner. 3.1.4. Gel characterization The mechanical spectrum of gelatin at 20 ◦ C is shown in Fig. 1(c). The viscoelastic behavior was dominated by the elastic component G . Over the entire frequency range, G was much higher than G (with G > 10G ). G was constant over a wide frequency range (G ≈ 3 × 104 Pa). The weak dependency of G on frequency and much higher value of G than G indicate that the system behaves as a gel (Clark & Ross-Murphy, 1987). The same behavior was observed for all gelatin concentrations studied. The slight increase of G at low frequency was due to the fact that the gel evolved over time. The highest frequency used was 102 rad/s. The time required to obtain each point of the spectrum varied with the inverse of the frequency. Fig. 1(d) illustrates this evolution of gelatin gel over time after rapid cooling from 60 ◦ C to 20 ◦ C (∼30 ◦ C/min). Since in this temperature range G is much higher than G , gel formation was almost instantaneous. G increased rapidly over time and in the first 20 min of measurement reached about 3 × 104 Pa then increased more slowly until a near constant value was attained. After 6 h G was about 4 × 104 Pa. This aspect of the gelation kinetics of gelatin has been widely described (Ross-Murphy, 1992; te Nijenhuis, 1997), and research has demonstrated that gelatin gels never reach equilibrium. It has been shown

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Fig. 1. Storage and loss moduli G , G for a 25% gelatin sample during a cooling ramp (a) and heating ramp (b). Temperature was ramped from 50 ◦ C to 20 ◦ C at 1 ◦ C/min. Frequency: 1 rad/s. Strain amplitude: 1%. (c) Mechanical spectrum of 25% gelatin gel. (d) Changes in modulus G and G as a function of time for a 25% gelatin gel at frequency: 1 rad/s. G : filled symbols, G : empty symbols. The temperature was 20 ◦ C. Strain amplitude: 1%.

that G’ increases over time due to increased junction zones in the network and the stiffening of the chains that connect them. These phenomena are attributed to greater degrees of helix formation (te Nijenhuis, 1997). It should also be noted that the stability of the gels depends on their thermal history (te Nijenhuis, 1997). Increased storage time during cooling and/or a higher temperature storage led to greater stability of helices and consequently an increase in melting temperature of the gel. Results shows that the modulus G varies with the concentration to the power 2.1 (G ∝ C2.1 ). However, it is accepted that G varies linearly with the square of the concentration over a wide concentration range (Ferry, 1948; te Nijenhuis, 1997). Previously it was observed that G did not equilibrate and continually increased over time. For this reason, the relationship between G and concentration varies from that observed other studies (Clark & Ross-Murphy, 1987; Lafargue, Pontoire, Buléon, Doublier, & Lourdin, 2007b).

3.2. Characterization of dually modified starch/-carrageenan blends 3.2.1. Flow properties The flow properties of starch/␬-carrageenan mixture were determined at 50 ◦ C. The total concentration of starch/␬carrageenan was kept as 25%. Fig. 2(a) represents the flow curves of the HHSS12-␬C0.5 mixture. The flow curves of dually modified starch HHSS12 at 20%, 23%, and 25% and ␬-carrageenan at 0.5% (␬C0.5) are also presented. The mixture HHSS12-␬C0.5 (total concentration 20%) behaved similarly to starch alone except that shear thinning was more pronounced. The apparent viscosity of the mixture was much higher than that of the constituents alone. The viscosity of the mixture was also higher than that of 25% starch. These results indicate a strong synergy between starch

Fig. 2. (a) Flow curves of the mixture HHSS12-␬C0.5 (), 20%HHSS12 and 0.5% ␬carrageenan, ␬C0, 5 (×), and starch dispersions HHSS12 20% (䊐), 23% (), and 25% ( ). (b) Flow curve of the HHSS12-␬C0.5. Shear rate up 0 to 100 s−1 empty symbols, and down 100 to 0 s−1 filled symbols. The temperature was 50 ◦ C.

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3.2.2. Viscoelastic properties Fig. 4(a) shows the mechanical spectrum at 50 ◦ C of the mixture of HHSS12-␬C0.5, 0.5% ␬-carrageenan alone (␬C0.5), and 25% HHSS12 alone. The ␬C0.5 shows a behavior typical of a macromolecular solution with G ␣ ␻2 and G ␣ ␻1 . The point of intersection of the modulus is 100 rad/s. At this temperature, ␬-carrageenan chains are in disordered conformation. This behavior was observed for all concentrations studied of ␬-carrageenan. The mechanical spectrum of the mixture HHSS12-␬C0.5 is quite similar to that of starch alone. For both, G depended on frequency (G ␣ ␻0.92 ) and G was greater than G at high frequencies (10–100 rad/s). Also a typical behavior that reported for starch with G’ which tends to plateau at low frequencies. The point of intersection of the modulus was 0.25 rad/s. The mechanical spectrum of mixture HHSS12-␬C0.5 actually resembled that of a 30% solution of starch. This behavior reflects the synergy between the major constituents of the mixture previously observed by viscometric measurements (Lafargue et al., 2007a).

Fig. 3. (a) Flow curves of mixtures of 25% starch HHSS12 with ␬-carrageenan at different concentrations. (b) Flow curves for 0.5% ␬-carrageenan and mixtures of 25% dually modified sago starches/␬C0.5. Measurements were taken at 50 ◦ C.

and ␬-carrageenan at 50 ◦ C. Non-thixotropic and non-Newtonian behavior of combinations of ␬-carrageenan and dually modified sago starches are shown in Fig. 2(b). 3.2.1.1. (a) Influence of -carrageenan content in starch/-car blends. Fig. 3(a) presents the flow curves of mixtures of 25% starch (HHSS12) with concentrations of ␬-carrageenan ranging from 0.25% to 1% compared with 25% HHSS12 alone. Increasing the concentration of ␬-carrageenan in the mixture caused a significant increase in apparent viscosity with pronounced increases in shear thinning behavior. The shape of the flow curve of the mixture ␬C0.25-HHSS12 (0.25% ␬-carrageenan) was similar to starch alone, with a slight synergistic effect. At lower concentration of ␬-carrageenan (␬-C = 0.1%), the flow curves of the mixtures were comparable with that of starch alone, reflecting the limited influence of ␬-carrageenan at this concentration (not shown in the figures). 3.2.1.2. (b) Influence of starch with different extents of hydrolysis in starch/-car blends. Fig. 3(b) represents the flow curves at 50 ◦ C of mixtures of starches hydrolyzed for various times (and 20% hydroxypropylated) and ␬C0.5. In all cases, the apparent viscosity of mixtures is much greater than that of starches alone (shows before), confirming the synergistic effect for all starches. Shearthinning behavior was more pronounced when starches were less hydrolyzed, meaning that the molecular weights of the starches were higher (Abdorreza et al., 2012; Fouladi & Mohammadi Nafchi, 2014).

3.2.3. Sol–gel transitions Viscoelastic modulus G and G between 20 ◦ C and 50 ◦ C for ␬carrageenan at a concentration of 0.5% and for the HHSS12-␬C0.5 mixture with total concentration of 25% are shown in Fig. 4. The heating and cooling rates were 1 ◦ C/min. CT was kept constant at 1.3 × 10−2 eq/l by adjustment of the K+ ion concentration. During the cooling at temperatures above 27 ◦ C, the modulus G” was constant for ␬C0.5 (Fig. 4(b)). In this temperature range, the ␬carrageenan behaves as a macromolecular solution. From 27 ◦ C, G” rapidly increased and finally reached 10 Pa at 20 ◦ C. If, as in the gelatin system, the gelation temperature of the system was defined as G = G , then TGEL of ␬C0.5 is 25 ◦ C. Without adjusting K+ ion concentration, previous studies showed that the gelation temperature was around 20 ◦ C (Lafargue et al., 2007b). For the ␬0.5-HHSS12 mixture, G was slightly higher than G , reflecting the dispersal behavior previously described on the basis of the mechanical spectrum. From about 32 ◦ C, both modulus increased and intersected at a temperature of 28 ◦ C. At 20 ◦ C, G was approximately 500 Pa and G was about 60 Pa. At the beginning of heating ramp (Fig. 4(c)), G’ and G” for ␬C0.5 and for the HHSS12␬0.5 mixture were significantly higher than values obtained at the end of cooling. G and G were approximately 50 Pa and 15 Pa for ␬C0.5 and 600 Pa and 80 Pa for the HHSS12-␬C0.5 mixture. This difference reflects the evolution of the gel over time at 20 ◦ C (approximately 30 min). The TM was 30 ◦ C for ␬C0.5 and 38 ◦ C for the mixture. The thermo-reversibility of ␬-carrageenan gel-based mixtures were previously observed (Tomsic, Prossnigg, & Glatter, 2008). Transition temperatures for ␬-carrageenans and mixtures determined by rheological measurements are presented in Table 3. Table 3 Gelling temperatures (TGEL ), melting temperatures (TM ), Storage, and loss moduli of ␬-carrageenan alone and the mixture HHSS12-␬-carrageenan determined from cooling and heating ramps at 1 ◦ C/min and 1 rad/s. Sol–gel transition temperatures (G = G ) Compositions ␬-carrageenan ␬C1 ␬C0.75 ␬C0.5 ␬C0.25 Mixtures HHSS12-␬C1 HHSS12-␬C0.75 HHSS12-␬C0.50 HHSS12-␬C0.25

TGEL (◦ C)

TM (◦ C)

G (Pa)

G (Pa)

26 34 47.0 26 33 13.2 25 30 3.3 Cannot be detected by rheometer –

–

34 31 28 26.5

110 88.3 28.7 2.0

CT was constant at 1.3 × 10−2 eq/l.

50 47.5 38 37

26800 1135 803 20.3

9.9 4.3 1.5

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161

Fig. 4. (a) Mechanical spectrum of ␬C0.5 (solid lines 䊏, 䊐), HHSS12 (solid lines 䊉, ), and the mixture ␬C0.5-HHSS12 (䊏, 䊐). Concentration of HHSS12 alone was 25% and in combination total concentration was 25%. Measurement temperature: 50 ◦ C. (b) Variation of viscoelastic modulus G and G as a function of temperature for ␬C0.5 and for the mixture of ␬C0.5 and HHSS12 in cooling and heating ramp and (d) cooling ramp for 25% HHSS24 alone and in combination with ␬-carrageenan Heating/cooling rate: 1 ◦ C/min. Frequency: 1 rad/s. Strain amplitude: 1%. G : filled symbols, G : empty symbols.

The aim of this study was primarily to compare the temperatures of gelling of ␬-carrageenan-starch mixtures to those of gelatin. The transition temperature (TGEL @ G = G ) was obtained by temperature ramps at a fixed frequency for mixtures and for ␬-carrageenan alone. Unfortunately in low concentrations of ␬-carrageenan the transition could not be detected by rheometers. Fig. 4(d) shows the influence of the concentration of ␬carrageenan on the gelation of the mixture with 25% HHSS24. ␬-Carrageenan concentrations of 0.0%, 0.25%, 0.5%, 0.75%, and 1% were used. At temperatures above TGEL , G was greater than G , and both modulus increased with the concentration of ␬carrageenan. G exceeded G over the entire temperature range; this phenomenon was also observed at a very low concentration of ␬-carrageenan (0.1%, data not shown). The criterion G = G can be applied in this case. The modulus at 20 ◦ C increased dramatically with the addition of ␬-carrageenan, with G equal to 2 × 103 Pa at 1% ␬-carrageenan. The addition of 0.25% ␬-carrageenan allowed the system to gel with G approximately 101 Pa. This phenomenon was also observed upon a heating ramp. Increased concentrations of ␬carrageenan resulted in increased melting temperatures of the gels (data not shown).

3.2.4. Viscoelastic properties of gels at 20 ◦ C The mechanical spectra of the mixture HHSS12-␬C0.5 and spectra of constituents ␬C0.5 and 25% HHSS12 are investigated (results not shown). The behavior of HHSS12, the behavior at 20 ◦ C was very similar to that observed at 50 ◦ C. This is consistent with the fact that the sample was not gelled under these conditions. The viscoelastic

behavior of the mixture HHSS12-␬C0.5 was characteristic of a gel with G = 800 Pa and G = 30 Pa (at 0.1 rad/s). Results show that for different mixtures, gel behavior was observed even at low concentrations of ␬-carrageenan. G at 0.1 rad/s was 20 Pa for HHSS12-␬C0.25, 800 Pa for HHSS12-␬C0.5, and about 2700 Pa for HHSS12-␬C1. ␬-Carrageenan alone does not form a gel under these conditions. At low concentrations of ␬-carrageenan (HHSS12-␬C0.25mixture), the dependency of modulus at higher frequencies and the fact that G was similar to G indicate that that this mixture formed a weak gel. The values of G and G determined from the mechanical spectrum at 20 ◦ C and 0.1 rad/s for mixtures HHSS12-␬C compared with ␬-carrageenan alone are shown in Table 3. As the dually modified sago starch does not form gels under any conditions tested, the gelation of the mixture with ␬-carrageenan was attributed to the presence of ␬-carrageenan, which in small amounts is able to form a continuous network. The mixtures gelled at 20 ◦ C. Under these conditions ␬-carrageenan alone does not form a gel (≤0.25%) either. The transition temperatures of the mixtures differed from that of ␬-carrageenan alone by 10 ◦ C during cooling and about 20 ◦ C during heating. The strengthening of the gel and increased transition temperatures are likely due to an increase in the total ionic concentration (CT ) of the system (CT = CS + CP ). At a constant salt concentration, the behavior of composite gels can be attributed to the effects of ␬-carrageenan, which causes an artificial increase in CT . This could be due to a phenomenon of phase separation with the formation of a zone enriched in ␬-carrageenan and thus an increase of CP in this area. The phenomenon of incompatibility between the polysaccharides when

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one of the components is gelling agent can cause dramatic changes in properties of gels, including a strengthening modulus and a decrease of critical gelation (Lorenzo, Zaritzky, & Califano, 2015; Zasypkin, Braudo, & Tolstoguzov, 1997), as was observed for blends in this study. The gelation temperatures of mixtures were similar regardless of the concentration of ␬-carrageenan and the molecular weight of starch (due to different hydrolysis time). Under the same concentrations, the gelation temperature (TGEL ) of the mixtures was comparable to that of gelatin, about 28–35 ◦ C. However, for the mixtures evaluated, the modulus G was lower (≈03 Pa) than the G of gelatin (≈104 Pa). 4. Discussion In this study of starch-carrageenan films obtained by casting, the main objective was to find a suitable replacement for gelatin in the manufacture of pharmaceutical hard capsules. Dually modified sago starch, which forms films that are readily soluble in water at high concentrations (up to 35%), was combined with ␬-carrageenan which forms gels during cooling. The gelation temperature of ␬carrageenan was controlled by adjusting the total system ionic concentration (CT ) using K+ . In this study, the total ionic concentration of the system was maintained at 1.3 × 10−2 eq/l; under these conditions, the gelation temperature of ␬-carrageenan alone is 25 ◦ C. 4.1. Comparison with gelatin The main goal of the research described in this research was to finding replacements for gelatin for hard pharmaceutical capsules. In this section the thermo-reversible material prepared in the current research is compared to gelatin in solution, sol–gel transition, and gel. The behavior of mixtures at 50 ◦ C was significantly different from that of gelatin solutions. The behavior of gelatin at this temperature is Newtonian with viscosities of approximately 0.24 to 0.69 Pa s at concentrations ranging 20 to 25%. Dually modified sago starches alone had almost Newtonian behavior. The dually modified sago starch must be used at a concentration of around 20% to prepare films of required thickness (0.1 mm) and, in combination with ␬-carrageenan, higher concentrations produce a viscous solution that leads to high film thickness in dipping. Viscosity is an important parameter for materials used in the manufacturing of hard capsules as viscosity determines the thickness of material on the molding fingers during the dipping step. The graph in Fig. 5 shows shear rates equivalent to those used industrially to control the viscosity in the dipping bath. The apparent viscosities determined at 1 s−1 of different formulations of 20% starch/␬-carrageenan blends are compared with those of gelatin at concentrations of 25–30%. Except for mixtures of HHSS6, apparent viscosities of starches evaluated were comparable to those used for the manufacture of capsules containing gelatin. However, the use of starches of high molecular weight requires the addition of small amounts of ␬-carrageenan to limit the apparent viscosity (concentration of ␬-carrageenan lower than 0.5%). For the starches of lower molar masses (HHSS18, HHSS24), ␬-carrageenan was used at concentrations of up to 0.75% while maintaining an apparent viscosity appropriate for industrial processes. Jellification is essential during the dipping step to set the material in contact with molding fingers. The gelation temperature and gel strength are two parameters that must be considered. In the same range of concentrations, temperatures of gelation of evaluated starch mixtures were comparable to those of gelatin, about 35 ◦ C, irrespective of the concentration of ␬-carrageenan and molecular weight of starch. This behavior is explained by the fact that the total ionic strength of the formulations was kept constant

Fig. 5. Viscosities of 20% starches studied compared to those of gelatin (shaded area) at concentrations of 25–30%. Measurement temperature was 50 ◦ C. Apparent viscosity of mixtures was determined at 1 s−1 .

by adjusting the amount of KCl. Despite its very low content in the mixture, the ␬-carrageenan governed the gelation process and starch only played a reinforcing role. The mechanical spectrum of 20% HHSS12 in the presence of 1% and 0.25% ␬-carrageenan are compared with a 25% gelatin gel. In the presence of 0.25% ␬carrageenan, gels obtained had lower G (≈102 Pa) than those of gelatin. For 1% ␬-carrageenan blend, the modulus G was on the order of 2 × 103 Pa, significantly lower than G of the gelatin which reached about 104 Pa. In order to obtain gel properties as close as possible to those of gelatin, it is necessary to use a formulation containing a high ␬-carrageenan content. The use of ␬-carrageenan at high concentrations results in a significant increase of apparent viscosity at 50 ◦ C. As a compromise, a relatively low apparent viscosity and sufficiently high rigidity results when low molar weight starches are used. In the range of concentrations studied (20–25% for starch and 0.25–1% for ␬-carrageenan at constant total ionic concentration), dramatic increases in apparent viscosity and stronger gels were observed. These effects were attributed to the thermodynamic incompatibility between the components that induces a phase separation. The solubilized starch molecules and ␬-carrageenan are mutually exclusive in the environment and cause significant differences in the rheological behavior of mixtures in comparison with the constituents alone. The composite gels had temperatures of gelation similar to that of gelatin. 5. Conclusion This study aimed at gaining a better understanding on the properties of blends of dually modified sago starch and ␬-carrageenan for the manufacturing of pharmaceutical capsules of plant origin. Based on the reference behavior of gelatin under conditions similar to those used industrially for the manufacture of capsules, rheological properties of starch and ␬-carrageenan mixtures with different degrees of modification were analyzed. The data generated is relevant for understanding the solution characterization, sol–gel transition, and gel characterization. Both viscosity in solution and stiffness in gels could be adjusted using high levels of ␬-carrageenan and was relatively independent of the molecular weight of the starch. The experimental results obtained have opened up some opportunities. The determination of rheological properties of starch and ␬-carrageenan mixtures has revealed

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