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Preparation of methyl alginate and its application in acidified milk drinks
International Journal of Biological Macromolecules 132 (2019) 651–657
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Preparation of methyl alginate and its application in acidified milk drinks Kai Xia, Peijie Zong, Xin Liu, Jingkun Zhao, Xiaodong Zhang ⁎ College of Chemical Science and Engineering, Qingdao University, Qingdao 266071, China
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Article history: Received 24 January 2019 Received in revised form 16 March 2019 Accepted 31 March 2019 Available online 01 April 2019 Keywords: Methyl alginate Acidified milk drinks Methyl esterification
a b s t r a c t A series of methyl alginate with different degree of esterification (DE) were prepared with low cost, and its application in acidified milk drinks (AMDs) was investigated. The gel strength, molecular weight, solution apparent viscosity and optical rotation value of methyl alginate all decreased with the increase of DE. Methyl alginate with DE equal or larger than 60.7% was more effective in stabilizing AMDs than high-methoxyl pectin (HMP) under the same conditions, and the higher the DE, the better its stabilizing ability. The methyl alginate had better synergistic action for stabilizing AMDs with propylene glycol alginate (PGA) than HMP. The reason for this could be attributed to that methyl alginate and PGA had the same main chain structure, and the same levorotatory optical activity. The results indicated that the methyl alginate had a potential industry application prospect for stabilizing AMDs as an alternative to HMP. © 2019 Published by Elsevier B.V.
1. Introduction Alginic acid, a cheap and easily available anionic polysaccharide, is mainly extracted from brown seaweeds such as Laminaria hyperborea, Ascophyllum nodosum and Macrocystis pyrifera [1]. The basic structure of alginic acid is composed of α-l-guluronic acid (G) and β-dmannuronic acid (M), appearing in homopolymeric blocks of consecutive G-G or M-M, or alternating G-M with 1,4-glycosidic linkages [2]. Alginic acid and its derivatives have been widely used in the food industry, pharmaceutical industry, and daily chemical industry due to its combination of renewability, biocompatibility, biodegradability and nontoxicity [3–6]. Especially, sodium alginate has the functions of detoxication, lowering blood glucose and blood fat [7]. Acidified milk drinks (AMDs) generally need the addition of stabilizers to avoid the flocculation of milk proteins and subsequent macroscopic whey separation [8,9]. Currently, the commonly used polysaccharides stabilizers in the food industry are carboxymethylcellulose (CMC), propylene glycol alginate (PGA), soybean soluble polysaccharides (SSPS), and high-methoxy pectin (HMP) [8,10–12]. It has been proved that HMP can adsorb on the surface of the casein micelles via the charged blocks of the pectin chains, and the uncharged blocks of HMP extending into solution are conducive to the stabilization of AMDs via steric repulsion [10]. As a high value functional food additive, pectin has been widely used as the gelling agent, thickener and emulsifier besides as a stabilizer [13,14]. However, its application also has been retarded by the relatively lower output and higher prices. ⁎ Corresponding author. E-mail address: [email protected] (X. Zhang).
https://doi.org/10.1016/j.ijbiomac.2019.03.243 0141-8130/© 2019 Published by Elsevier B.V.
Basically, the predominant structural feature of pectin is a chain of a(1/4)-linked D-galacturonic acid units, and some of the carboxyl groups are methyl esterified [10,15]. HMP is the pectin with more than 50% of esterified carboxyl groups [16]. It is worthwhile to note that alginic acid is also a polyuronic acid containing carboxyl groups in the C-6 position, and modifications of these carboxyl groups have obtained many new derivatives of this polysaccharide [3,17,18]. In view of the similarity of molecular structures between methyl alginate and HMP, especially that methyl alginate could be prepared at low cost through simple esterification reaction, in this study, a series of methyl alginate with different degree of esterification were prepared and its application in AMDs was investigated as the alternative of HMP.
2. Experiment 2.1. Materials Alginic acid (M/G = 48/52) was obtained from Qingdao Bright Moon Seaweed Industrial Co., Ltd., China. High-methoxyl pectin (HMP, DE: 72.0%; Mw: 38,000 g/mol), carboxymethylcellulose (CMC, DS: 0.96; Mw: 90,000 g/mol) and propylene glycol alginate (PGA, DE: 90.1%; Mw: 52,000 g/mol) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., China. Whole milk powder, purchased from Inner Mongolia Yili Industrial Co., Ltd. (China), was chosen in this study for the reason that the whole milk system was more stable than skim milk system in AMDs [19]. All other reagents used in this research were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) without further purification.
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Table 1 Formulations of polysaccharide mixtures. Formulation
A B C D E F a
Polysaccharides (g) PGA
HMP
CMC
Methyl alginatea
0.168 0.168 0.243 0.243 0 0
0.168 0 0.243 0 0.486 0
0.150 0.150 0 0 0 0
0 0.168 0 0.243 0 0.486
The DE of methyl alginate was 60.7%.
2.2. The preparation of methyl alginate Alginic acid was esterified with methanol using sulfuric acid as the catalyst. Alginic acid (50 g) and methanol (200 g) were mixed with stirring in a three-necked flask. Then 0.4 g of sulfuric acid was added into the flask. The mixture was refluxed for 3.5, 6.5, 12, 22 and 37 h, respectively. Subsequently, the mixture was cooled to room temperature, and the pH was adjusted to be 6.5–7.0 using sodium carbonate solution with the concentration of 1.5 wt%. The solid reaction product was filtered, and washed with ethanol aqueous solution (70 wt%), and finally dried at 40 °C. Thus, the methyl alginates were obtained. The degree of esterification (DE) of methyl alginate was measured using the acid-base titration method according to the determination of the degree of methyl-esterification to pectin [20]. In which, the solutions of methyl alginate before and after saponification were titrated firstly with solution of sodium hydroxide respectively, and then the DE of methyl alginate could be calculated according to the titers corresponded to the amount of carboxyl groups in methyl alginate solutions before and after saponification.
Fig. 2. Temperature dependence of the optical rotation (a) and apparent viscosity (b) for solutions of native alginate and methyl alginates on cooling.
2.3. Characterization
Fig. 1. The FT-IR spectra of alginic acid (a) and methyl alginate (DE = 60.7%) (b).
Table 2 Physicochemical properties of the prepared methyl alginate. Properties
Molecular weight (g/mol) Gel strength (g/cm2)
Table 3 The solution apparent viscosity and surface tension of the prepared AMDs.
DE of methyl alginate (%) 0 (alginic 30.2 acid) 58,302 242.0
40.1
The FT-IR spectra were measured using a Nicolet IS 10 FT-IR spectrophotometer (Nicolet, USA) with the KBr pellet technique. Apparent viscosity of methyl alginate solutions (3.0 wt%, pH = 7) was measured using a Brookfield rheometer (DV3T, USA) at 6 r/min, and the temperature gradient was 0.5 °C/min during cooling trace from 80 °C to 25 °C. Optical rotation of methyl alginate solutions (0.25 wt%, pH = 7) was measured with an ATAGO polarimeter (POL-1/2, Japan) under the condition of temperature cooling from 80 °C to 20 °C at a rate of 0.5 °C/min. The methyl alginate and alginate fluid gels (2.5 wt%) were produced using in-situ calcium release as described by Farrés and Norton [21], CaCO3 powder was dispersed in alginate solutions, then glucono-δ-lactone was added into the alginate and CaCO3 mixtures immediately prior to sample loading, the mixtures finally stood at room temperature for 24 h, the molar ratios of Ca2+ to carboxyl and glucono-δ-lactone to Ca2+ were 0.5 and 0.6 respectively, and the gel
49.9
60.7
69.9
47,768 44,422 40,012 37,025 33,003 195.0 135.3 56.0 2 0
Properties
Apparent viscosity (mPa·s) Surface tension (mN/m)
Formulations A
B
C
D
E
F
17 63.40
19 63.49
14 63.32
16 63.37
10 63.47
12 63.53
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Chromatography (Malvern 270 max GPC, UK) as described by Ding et al. [22]. All the measurements were performed three times. 2.4. The application of methyl alginate in AMDs
Fig. 3. Effect of shear rate on the apparent viscosity of AMDs stabilized by stabilizer formulation A to F.
2.4.1. Preparation of AMDs The acidified milk drinks were prepared according to the process of Zhao et al. [23]. Distilled water (66 g) and whole milk powder (9 g) were mixed by stirring and heated to 60 °C, then kept the temperature and stirred for another 20 min to dissolve the whole milk powder completely, then cooled to room temperature. Polysaccharide mixture (0.486 g) was dissolved in 50 mL of distilled water, and then mixed with the solution of whole milk powder at room temperature. A quantitative distilled water was added into the mixture to control the total weight of system to 150 g. Then adjusted the pH to 3.8 using citrate acid aqueous solution (10 wt%), homogenized using a high-speed homogenizer (automodel 20, Primix, Japan) with the rate of 5000 rpm for 20 min at 60 °C, pasteurized at 90 °C for 30 s, and stored at 4 °C. Thus the AMDs were obtained. The formulations of polysaccharide stabilizers are shown in Table 1. Among which, formulation A was a commercial reference obtained from a local acidified milk drinks manufacture factory.
strength was measured using a Nikkansui-type gel tester (Kiya Seisakusho Ltd., Tokyo, Japan). The weight-average molecular weight of methyl alginate was measured using Gel Permeation
2.4.2. Analysis of AMDs The apparent viscosity of the AMDs was measured by a Brookfield rheometer (DV3T, USA) at 6 r/min and at room temperature. The
Fig. 4. The particle size distributions of the AMDs stabilized by stabilizer formulation A to F.
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apparent viscosity of the samples recorded as a function of shear rate was measured with shear rate ranged from 1 to 300 s−1 at room temperature. Particle size analyses were measured by a laser lightscattering-based particle sizer (Rise-2028, China). The sedimentation of AMDs was determined by the centrifugation separation method as described by Jensen et al. [10]. The surface tension of AMDs was measured by a Surface tensiometer & interface tensiometer (KINO, USA). Scanning electron microscopy (SEM) of samples was examined by scanning electron microscopy (JSM-6390LV, Japan) after freeze-drying treatment. All the analyses were carried out after the prepared samples were stored at 4 °C for 24 h, and were performed three times. 3. Results and discussion 3.1. FT-IR spectroscopy The FT-IR spectra of alginic acid and methyl alginate (DE = 60.7%) are shown in Fig. 1. In the spectrum of alginic acid (curve a), the peak at 3440 cm−1 was attributed to the stretching vibration of hydroxyl O\\H [24]. The peaks at 2922 cm−1 and 2850 cm−1 corresponded to the stretching vibration of methyl (-CH3) and methylene (-CH2) respectively. The band at 1033 cm−1 was due to C\\O stretching vibration. In the spectrum of methyl alginate (curve b), two new absorption peaks at 1733 cm−1 and 1253 cm−1 appeared, which were assigned to the C_O (methyl ester group) and the C-O-C bond stretching vibrations respectively [25]. The presence of ester group in product could verify the success of methyl esterification to alginic acid. The obtained results were in accordance with previously reported FT-IR bands of methyl alginate [26].
and sodium alginate both gradually decreased as the temperature decreased. As for this, it might be attributed to that the thermal motion of the molecular chains was enhanced at higher temperature, and the methyl alginate and sodium alginate molecules tended to be in random states.
3.4. Apparent viscosity analysis The solution apparent viscosity of sodium alginate and methyl alginate at different temperature are shown in Fig. 2b. From Fig. 2b, the solution apparent viscosity values of sodium alginate and methyl alginate all gradually increased with the decrease of the temperature. The solution apparent viscosity values of methyl alginate were lower than that of sodium alginate under the same conditions, and the solution of the methyl alginate with larger DE had lower viscosity. In addition, the changes of solution temperature had a relatively weaker impact on the solution apparent viscosity of methyl alginate with higher DE. These results might be mainly due to the decrease of the molecular weight and intermolecular hydrogen bond of methyl alginate. As mentioned in Table 2, the molecular weight of alginic acid was significantly decreased after methyl esterification, and the higher the DE of the methyl alginate, the lower the molecular weight.
3.2. Physicochemical properties Physicochemical properties of the prepared methyl alginate with different DE were tested and shown in Table 2. It could be seen from Table 2 that both the gel strength and molecular weight of methyl alginate significantly decreased with the increase of DE. The decrease of molecular weight of methyl alginate might be owing to the fracture of alginate chains. The formation of methyl alginate gel and sodium alginate gel was mainly attributed to the chelation of calcium ions and G units from different alginate chains via the so-called “egg-box” model [27,28]. With the DE increase of methyl alginate, the G-blocks in basic structure of alginic acid were gradually destroyed, and the average length of alginate chains also had been reduced, which resulted in the reduction of gel strength. Especially, when the DE was reached up to 69.9%, the methyl alginate was lost the ability to form a gel. These results indicated that the methyl alginate with high DE values had good anticalcium precipitation property. 3.3. Optical rotation analysis According to the theory of optical rotation for organic compounds, the value of optical rotation is mainly determined by the polarizability, turns, pitch and radius of helical structure [29]. Temperature dependence of the optical rotation for solutions of sodium alginate and methyl alginate are shown in Fig. 2a. From Fig. 2a, the solution optical rotation values of sodium alginate and methyl alginate with different DE were all negative, which indicated their helical structures were left-helical. At the same temperature, the solution optical rotation of methyl alginate trended down with the increase of DE. The reasons might be that the length of alginate chains decreased, and the carboxyl groups in alginate molecules were partly substituted by the ester groups after methyl esterification, hence, the intramolecular hydrogen bonding interactions of alginate molecules were weakened, and the helical pitch of the alginate molecules increased, resulting that the values of solution optical rotation decreased [29]. The solution optical rotation of methyl alginate
Fig. 5. Sedimentation fractions of AMDs samples (a), and temperature dependence of the optical rotation for solutions (0.25 wt%, pH = 7) of methyl alginate, HMP and PGA on cooling (b).
K. Xia et al. / International Journal of Biological Macromolecules 132 (2019) 651–657
3.5. Application of methyl alginate in AMDs 3.5.1. Properties of AMDs The solution apparent viscosity and surface tension of the prepared AMDs stabilized by different polysaccharide stabilizers were measured and shown in Table 3 and Fig. 3. It could be seen from Table 3 that both the solution apparent viscosity and surface tension of the AMDs stabilized by six different polysaccharide stabilizers had no significant differences. From Fig. 3, the apparent viscosity of six AMDs samples all decreased with the increase of shear rate, which indicated that the AMDs samples exhibited non-Newtonian behavior. The AMDs samples stabilized by polysaccharide stabilizers containing methyl alginate had similar shearing resistance performance compared with the commercial stabilizer. 3.5.2. Particle size analysis The particle size of casein in AMDs could be used to analyze the stability of AMDs. The smaller the particle size of casein was, the more stable the system tended to be [30]. The particle size distribution and its statistical results of the AMDs samples stabilized by the six stabilizer formulations are shown in Fig. 4. It could be seen from Fig. 4 that the particle size distributions of the AMDs stabilized by stabilizer formulation B and D (from 0.05 μm to 2.0 μm) were both smaller than that of the samples stabilized by other four stabilizer formulations (from 0.05 μm to 2.5 μm). The statistical results of particle size showed that the D50 and Dav (the particle diameters at 50% and the average diameter
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of cumulative volume, respectively) of the AMDs stabilized by different stabilizers displayed significant difference, and the descending order of Dav was D b B b A b C b F b E. These results indicated that the particle size of AMDs stabilized by polysaccharide mixture tended to be smaller than that of single polysaccharide, and the methyl alginate could be more efficient to reduce the particle size of AMDs compared with HMP no matter it was used singly or together with PGA, or with PGA and CMC. 3.5.3. Sedimentation analysis The sedimentation fractions of the AMDs samples stabilized by stabilizer formulation A to F were measured and shown in Fig. 5a. It could be seen from Fig. 5a that the sedimentation fraction of the AMDs stabilized by stabilizer formulation D was the lowest, then in ascending order of magnitude, were the sample stabilized by stabilizer formulation B, A, C, F, and E, respectively. This order was in perfect agreement with the result of the particle size analysis (see Fig. 4). The results of sedimentation analysis also verified that the stabilizing effect of all mixture polysaccharide stabilizers were better than that of single polysaccharide stabilizers, and the stabilizing effect of the mixture polysaccharide stabilizer became better when using methyl alginate to replace HMP by the same amount. This result indicated that methyl alginate with DE of 60.7% was more effective in stabilizing AMDs than HMP under the same conditions, and methyl alginate could be used to replace HMP in commercial reference stabilizer for preparation of AMDs, although methyl alginate and HMP had the similar molecular structural formula and similar stabilizing mechanism. The difference between methyl
Fig. 6. SEM images of the AMDs samples stabilized by the stabilizer formulation A to F.
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alginate and HMP to stabilize AMDs might come from the different molecular spatial structures of two kinds of polysaccharides. As exhibited in Fig. 5b, methyl alginate and PGA had same levorotatory optical activity and similar variation of solution optical rotation on cooling, whereas, the optical activity of HMP was dextral, this might be also the reason that methyl alginate had better synergistic action than HMP with PGA for stabilizing AMDs. 3.5.4. SEM analysis The SEM images of the AMDs samples after freeze-drying treatment were evaluated and shown in Fig. 6. It could be seen that the skeleton morphology of the AMDs sample stabilized by the commercial reference stabilizer was shaped a sheet structure with large pores [23]. By contrast, the porous skeleton structure of the AMDs sample stabilized by formulation B tended to be denser, and its clusters of casein particles became smaller after using methyl alginate to replace HMP in formulation A. This was in agreement with the resulting microstructures of the AMDs stabilized by HMP and methyl alginate alone (as shown in Fig. 6E and F).
3.5.5. Effect of the DE of methyl alginate on stability of AMDs The stability of AMDs stabilized by mixture stabilizer composed of PGA and methyl alginate with different DE values (1:1 in weight) were determined and denoted as D1, D2, D3, D4 and D5 according to the corresponding DE values of methyl alginate which were 30.2%, 40.1%, 49.9%, 60.7% and 69.9%, respectively. The sedimentation fractions and particle sizes of the AMDs samples were measured and shown in Fig. 7a and b. It could be seen from Fig. 7a that the sedimentation fractions of the AMDs samples significantly decreased with the DE increase of methyl alginate, and became stable after DE of methyl alginate was greater than 60.7%. This result was in good agreement with the reports that HMP was more effective in stabilizing AMDs than low-methoxyl pectin under low pH conditions [31,32]. The reason was that methyl alginate with higher DE would have better acid resistance and anticalcium property owing to the lower proportion of carboxyl groups. The methyl alginate could be absorbed on the surface of casein particles under low pH conditions through the electrostatic interaction between its carboxyl groups with negative charge and casein particles with positive charge. When DE was higher, there would be fewer carboxyl groups existed in a smaller region of methyl alginate molecules that could interact with the casein particles, and free a more substantial portion of the methyl alginate chains that stretched in solvent for solvation, as a result, forming dispersions resistant to sedimentation [32]. From Fig. 7b, the result that the median particle diameter of AMDs tended to be smaller with the DE increase of methyl alginate, further verified that the increase of the DE of methyl alginate was beneficial to the stability of AMDs. 3.5.6. Effect of stabilizer dosage on stability of AMDs The effect of stabilizer dosage on the sedimentation fractions of the AMDs stabilized by the mixture stabilizer composed of methyl alginate (DE = 60.7%) and PGA (mass ratio of 1:1) were measured and shown in Fig. 7c. It could be seen from Fig. 7c that the sedimentation fractions of the AMDs without polysaccharide stabilizer was relatively high, indicated that the casein tended to aggregate under acidic conditions. When the dosage of stabilizer increased to 0.05 wt% of the total mass of AMDs, the sedimentation fraction of AMDs sample increased unexpectedly. The reason for this result was that the added polysaccharides were not enough to cover the casein colloid at this concentration, and the casein and polysaccharides in AMDs system could be precipitated by bridging flocculation [33]. After this point, the sedimentation fractions of the corresponding AMDs decreased constantly with the dosage increase of the polysaccharide mixture, and changed little when the dosage of stabilizer was over 0.324 wt%.
Fig. 7. The sedimentation fractions (a) and particle sizes (b) of the AMDs samples stabilized by PGA and methyl alginate with different DE. Effect of the stabilizer concentration on the sedimentation fractions of the AMDs (c).
4. Conclusions In the present work, a series of methyl alginate with different degree of esterification were prepared through a simple esterification reaction. Compared with HMP, the methyl alginate with DE equal or larger than 60.7% possessed more excellent stabilizing effect on the casein in AMDs under the same condition, and the corresponding AMDs showed
K. Xia et al. / International Journal of Biological Macromolecules 132 (2019) 651–657
good shearing resistance performance. The methyl alginate had better synergistic action for stabilizing AMDs with PGA than HMP. The present study indicated that the methyl alginate which was easily prepared with lower cost had a potential industry application prospect in AMDs as an alternative to HMP. Acknowledgements The authors gratefully acknowledge financial support from the Qingdao Municipal Science and Technology Bureau (Grant No. 17-1-1-86jch). References [1] H. Andriamanantoanina, M. Rinaudo, Characterization of the alginates from five madagascan brown algae, Carbohydr. Polym. 82 (2010) 555–560. [2] M.U. Chhatbar, R. Meena, K. Prasad, D.R. Chejara, A.K. Siddhanta, Microwaveinduced facile synthesis of water-soluble fluorogenic alginic acid derivatives, Carbohydr. Res. 346 (2011) 527–533. [3] T. Taubner, M. Marounek, A. Synytsya, Preparation and characterization of amidated derivatives of alginic acid, Int. J. Biol. Macromol. 103 (2017) 202–207. [4] N. Ganesh, C. Hanna, S.V. Nair, L.S. Nair, Enzymatically cross-linked alginichyaluronic acid composite hydrogels as cell delivery vehicles, Int. J. Biol. Macromol. 55 (2013) 289–294. [5] L. Wang, R.M. Shelton, P.R. Cooper, M. Lawson, J.T. Triffitt, J.E. Barralet, Evaluation of sodium alginate for bone marrow cell tissue engineering, Biomaterials 24 (2003) 3475–3481. [6] S.C. Angadi, L.S. Manjeshwar, T.M. Aminabhavi, Novel composite blend microbeads of sodium alginate coated with chitosan for controlled release of amoxicillin, Int. J. Biol. Macromol. 51 (2012) 45–55. [7] Y. Kimura, K. Watanabe, H. Okuda, Effects of soluble sodium alginate on cholesterol excretion and glucose tolerance in rats, J. Ethnopharmacol. 54 (1996) 47–54. [8] B. Du, J. Li, H. Zhang, L. Huang, P. Chen, J. Zhou, Influence of molecular weight and degree of substitution of carboxymethylcellulose on the stability of acidified milk drinks, Food Hydrocolloid 23 (2009) 1420–1426. [9] R.H. Tromp, C.G. de Kruif, M. van Eijk, C. Rolin, On the mechanism of stabilisation of acidified milk drinks by pectin, Food Hydrocolloid 18 (2004) 565–572. [10] S. Jensen, C. Rolin, R. Ipsen, Stabilisation of acidified skimmed milk with HM pectin, Food Hydrocolloid 24 (2010) 291–299. [11] T. Nobuhara, K. Matsumiya, Y. Nambu, A. Nakamura, N. Fujii, Y. Matsumura, Stabilization of milk protein dispersion by soybean soluble polysaccharide under acidic pH conditions, Food Hydrocolloid 34 (2014) 39–45. [12] P.J. Young, P.M. Bluestein, Stable Acidic Milk Based Beverage, US Patent 0-160-086, 2002. [13] F. Munarin, M.C. Tanzi, P. Petrini, Advances in biomedical applications of pectin gels, Int. J. Biol. Macromol. 51 (2012) 681–689. [14] S.Y. Chan, W.S. Choo, D.J. Young, X.J. Loh, Pectin as a rheology modifier: origin, structure, commercial production and rheology, Carbohydr. Polym. 161 (2017) 118–139.
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Preparation of methyl alginate and its application in acidified milk drinks Kai Xia, Peijie Zong, Xin Liu, Jingkun Zhao, Xiaodong Zhang ⁎ College of Chemical Science and Engineering, Qingdao University, Qingdao 266071, China
a r t i c l e
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Article history: Received 24 January 2019 Received in revised form 16 March 2019 Accepted 31 March 2019 Available online 01 April 2019 Keywords: Methyl alginate Acidified milk drinks Methyl esterification
a b s t r a c t A series of methyl alginate with different degree of esterification (DE) were prepared with low cost, and its application in acidified milk drinks (AMDs) was investigated. The gel strength, molecular weight, solution apparent viscosity and optical rotation value of methyl alginate all decreased with the increase of DE. Methyl alginate with DE equal or larger than 60.7% was more effective in stabilizing AMDs than high-methoxyl pectin (HMP) under the same conditions, and the higher the DE, the better its stabilizing ability. The methyl alginate had better synergistic action for stabilizing AMDs with propylene glycol alginate (PGA) than HMP. The reason for this could be attributed to that methyl alginate and PGA had the same main chain structure, and the same levorotatory optical activity. The results indicated that the methyl alginate had a potential industry application prospect for stabilizing AMDs as an alternative to HMP. © 2019 Published by Elsevier B.V.
1. Introduction Alginic acid, a cheap and easily available anionic polysaccharide, is mainly extracted from brown seaweeds such as Laminaria hyperborea, Ascophyllum nodosum and Macrocystis pyrifera [1]. The basic structure of alginic acid is composed of α-l-guluronic acid (G) and β-dmannuronic acid (M), appearing in homopolymeric blocks of consecutive G-G or M-M, or alternating G-M with 1,4-glycosidic linkages [2]. Alginic acid and its derivatives have been widely used in the food industry, pharmaceutical industry, and daily chemical industry due to its combination of renewability, biocompatibility, biodegradability and nontoxicity [3–6]. Especially, sodium alginate has the functions of detoxication, lowering blood glucose and blood fat [7]. Acidified milk drinks (AMDs) generally need the addition of stabilizers to avoid the flocculation of milk proteins and subsequent macroscopic whey separation [8,9]. Currently, the commonly used polysaccharides stabilizers in the food industry are carboxymethylcellulose (CMC), propylene glycol alginate (PGA), soybean soluble polysaccharides (SSPS), and high-methoxy pectin (HMP) [8,10–12]. It has been proved that HMP can adsorb on the surface of the casein micelles via the charged blocks of the pectin chains, and the uncharged blocks of HMP extending into solution are conducive to the stabilization of AMDs via steric repulsion [10]. As a high value functional food additive, pectin has been widely used as the gelling agent, thickener and emulsifier besides as a stabilizer [13,14]. However, its application also has been retarded by the relatively lower output and higher prices. ⁎ Corresponding author. E-mail address: [email protected] (X. Zhang).
https://doi.org/10.1016/j.ijbiomac.2019.03.243 0141-8130/© 2019 Published by Elsevier B.V.
Basically, the predominant structural feature of pectin is a chain of a(1/4)-linked D-galacturonic acid units, and some of the carboxyl groups are methyl esterified [10,15]. HMP is the pectin with more than 50% of esterified carboxyl groups [16]. It is worthwhile to note that alginic acid is also a polyuronic acid containing carboxyl groups in the C-6 position, and modifications of these carboxyl groups have obtained many new derivatives of this polysaccharide [3,17,18]. In view of the similarity of molecular structures between methyl alginate and HMP, especially that methyl alginate could be prepared at low cost through simple esterification reaction, in this study, a series of methyl alginate with different degree of esterification were prepared and its application in AMDs was investigated as the alternative of HMP.
2. Experiment 2.1. Materials Alginic acid (M/G = 48/52) was obtained from Qingdao Bright Moon Seaweed Industrial Co., Ltd., China. High-methoxyl pectin (HMP, DE: 72.0%; Mw: 38,000 g/mol), carboxymethylcellulose (CMC, DS: 0.96; Mw: 90,000 g/mol) and propylene glycol alginate (PGA, DE: 90.1%; Mw: 52,000 g/mol) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., China. Whole milk powder, purchased from Inner Mongolia Yili Industrial Co., Ltd. (China), was chosen in this study for the reason that the whole milk system was more stable than skim milk system in AMDs [19]. All other reagents used in this research were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) without further purification.
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Table 1 Formulations of polysaccharide mixtures. Formulation
A B C D E F a
Polysaccharides (g) PGA
HMP
CMC
Methyl alginatea
0.168 0.168 0.243 0.243 0 0
0.168 0 0.243 0 0.486 0
0.150 0.150 0 0 0 0
0 0.168 0 0.243 0 0.486
The DE of methyl alginate was 60.7%.
2.2. The preparation of methyl alginate Alginic acid was esterified with methanol using sulfuric acid as the catalyst. Alginic acid (50 g) and methanol (200 g) were mixed with stirring in a three-necked flask. Then 0.4 g of sulfuric acid was added into the flask. The mixture was refluxed for 3.5, 6.5, 12, 22 and 37 h, respectively. Subsequently, the mixture was cooled to room temperature, and the pH was adjusted to be 6.5–7.0 using sodium carbonate solution with the concentration of 1.5 wt%. The solid reaction product was filtered, and washed with ethanol aqueous solution (70 wt%), and finally dried at 40 °C. Thus, the methyl alginates were obtained. The degree of esterification (DE) of methyl alginate was measured using the acid-base titration method according to the determination of the degree of methyl-esterification to pectin [20]. In which, the solutions of methyl alginate before and after saponification were titrated firstly with solution of sodium hydroxide respectively, and then the DE of methyl alginate could be calculated according to the titers corresponded to the amount of carboxyl groups in methyl alginate solutions before and after saponification.
Fig. 2. Temperature dependence of the optical rotation (a) and apparent viscosity (b) for solutions of native alginate and methyl alginates on cooling.
2.3. Characterization
Fig. 1. The FT-IR spectra of alginic acid (a) and methyl alginate (DE = 60.7%) (b).
Table 2 Physicochemical properties of the prepared methyl alginate. Properties
Molecular weight (g/mol) Gel strength (g/cm2)
Table 3 The solution apparent viscosity and surface tension of the prepared AMDs.
DE of methyl alginate (%) 0 (alginic 30.2 acid) 58,302 242.0
40.1
The FT-IR spectra were measured using a Nicolet IS 10 FT-IR spectrophotometer (Nicolet, USA) with the KBr pellet technique. Apparent viscosity of methyl alginate solutions (3.0 wt%, pH = 7) was measured using a Brookfield rheometer (DV3T, USA) at 6 r/min, and the temperature gradient was 0.5 °C/min during cooling trace from 80 °C to 25 °C. Optical rotation of methyl alginate solutions (0.25 wt%, pH = 7) was measured with an ATAGO polarimeter (POL-1/2, Japan) under the condition of temperature cooling from 80 °C to 20 °C at a rate of 0.5 °C/min. The methyl alginate and alginate fluid gels (2.5 wt%) were produced using in-situ calcium release as described by Farrés and Norton [21], CaCO3 powder was dispersed in alginate solutions, then glucono-δ-lactone was added into the alginate and CaCO3 mixtures immediately prior to sample loading, the mixtures finally stood at room temperature for 24 h, the molar ratios of Ca2+ to carboxyl and glucono-δ-lactone to Ca2+ were 0.5 and 0.6 respectively, and the gel
49.9
60.7
69.9
47,768 44,422 40,012 37,025 33,003 195.0 135.3 56.0 2 0
Properties
Apparent viscosity (mPa·s) Surface tension (mN/m)
Formulations A
B
C
D
E
F
17 63.40
19 63.49
14 63.32
16 63.37
10 63.47
12 63.53
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Chromatography (Malvern 270 max GPC, UK) as described by Ding et al. [22]. All the measurements were performed three times. 2.4. The application of methyl alginate in AMDs
Fig. 3. Effect of shear rate on the apparent viscosity of AMDs stabilized by stabilizer formulation A to F.
2.4.1. Preparation of AMDs The acidified milk drinks were prepared according to the process of Zhao et al. [23]. Distilled water (66 g) and whole milk powder (9 g) were mixed by stirring and heated to 60 °C, then kept the temperature and stirred for another 20 min to dissolve the whole milk powder completely, then cooled to room temperature. Polysaccharide mixture (0.486 g) was dissolved in 50 mL of distilled water, and then mixed with the solution of whole milk powder at room temperature. A quantitative distilled water was added into the mixture to control the total weight of system to 150 g. Then adjusted the pH to 3.8 using citrate acid aqueous solution (10 wt%), homogenized using a high-speed homogenizer (automodel 20, Primix, Japan) with the rate of 5000 rpm for 20 min at 60 °C, pasteurized at 90 °C for 30 s, and stored at 4 °C. Thus the AMDs were obtained. The formulations of polysaccharide stabilizers are shown in Table 1. Among which, formulation A was a commercial reference obtained from a local acidified milk drinks manufacture factory.
strength was measured using a Nikkansui-type gel tester (Kiya Seisakusho Ltd., Tokyo, Japan). The weight-average molecular weight of methyl alginate was measured using Gel Permeation
2.4.2. Analysis of AMDs The apparent viscosity of the AMDs was measured by a Brookfield rheometer (DV3T, USA) at 6 r/min and at room temperature. The
Fig. 4. The particle size distributions of the AMDs stabilized by stabilizer formulation A to F.
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apparent viscosity of the samples recorded as a function of shear rate was measured with shear rate ranged from 1 to 300 s−1 at room temperature. Particle size analyses were measured by a laser lightscattering-based particle sizer (Rise-2028, China). The sedimentation of AMDs was determined by the centrifugation separation method as described by Jensen et al. [10]. The surface tension of AMDs was measured by a Surface tensiometer & interface tensiometer (KINO, USA). Scanning electron microscopy (SEM) of samples was examined by scanning electron microscopy (JSM-6390LV, Japan) after freeze-drying treatment. All the analyses were carried out after the prepared samples were stored at 4 °C for 24 h, and were performed three times. 3. Results and discussion 3.1. FT-IR spectroscopy The FT-IR spectra of alginic acid and methyl alginate (DE = 60.7%) are shown in Fig. 1. In the spectrum of alginic acid (curve a), the peak at 3440 cm−1 was attributed to the stretching vibration of hydroxyl O\\H [24]. The peaks at 2922 cm−1 and 2850 cm−1 corresponded to the stretching vibration of methyl (-CH3) and methylene (-CH2) respectively. The band at 1033 cm−1 was due to C\\O stretching vibration. In the spectrum of methyl alginate (curve b), two new absorption peaks at 1733 cm−1 and 1253 cm−1 appeared, which were assigned to the C_O (methyl ester group) and the C-O-C bond stretching vibrations respectively [25]. The presence of ester group in product could verify the success of methyl esterification to alginic acid. The obtained results were in accordance with previously reported FT-IR bands of methyl alginate [26].
and sodium alginate both gradually decreased as the temperature decreased. As for this, it might be attributed to that the thermal motion of the molecular chains was enhanced at higher temperature, and the methyl alginate and sodium alginate molecules tended to be in random states.
3.4. Apparent viscosity analysis The solution apparent viscosity of sodium alginate and methyl alginate at different temperature are shown in Fig. 2b. From Fig. 2b, the solution apparent viscosity values of sodium alginate and methyl alginate all gradually increased with the decrease of the temperature. The solution apparent viscosity values of methyl alginate were lower than that of sodium alginate under the same conditions, and the solution of the methyl alginate with larger DE had lower viscosity. In addition, the changes of solution temperature had a relatively weaker impact on the solution apparent viscosity of methyl alginate with higher DE. These results might be mainly due to the decrease of the molecular weight and intermolecular hydrogen bond of methyl alginate. As mentioned in Table 2, the molecular weight of alginic acid was significantly decreased after methyl esterification, and the higher the DE of the methyl alginate, the lower the molecular weight.
3.2. Physicochemical properties Physicochemical properties of the prepared methyl alginate with different DE were tested and shown in Table 2. It could be seen from Table 2 that both the gel strength and molecular weight of methyl alginate significantly decreased with the increase of DE. The decrease of molecular weight of methyl alginate might be owing to the fracture of alginate chains. The formation of methyl alginate gel and sodium alginate gel was mainly attributed to the chelation of calcium ions and G units from different alginate chains via the so-called “egg-box” model [27,28]. With the DE increase of methyl alginate, the G-blocks in basic structure of alginic acid were gradually destroyed, and the average length of alginate chains also had been reduced, which resulted in the reduction of gel strength. Especially, when the DE was reached up to 69.9%, the methyl alginate was lost the ability to form a gel. These results indicated that the methyl alginate with high DE values had good anticalcium precipitation property. 3.3. Optical rotation analysis According to the theory of optical rotation for organic compounds, the value of optical rotation is mainly determined by the polarizability, turns, pitch and radius of helical structure [29]. Temperature dependence of the optical rotation for solutions of sodium alginate and methyl alginate are shown in Fig. 2a. From Fig. 2a, the solution optical rotation values of sodium alginate and methyl alginate with different DE were all negative, which indicated their helical structures were left-helical. At the same temperature, the solution optical rotation of methyl alginate trended down with the increase of DE. The reasons might be that the length of alginate chains decreased, and the carboxyl groups in alginate molecules were partly substituted by the ester groups after methyl esterification, hence, the intramolecular hydrogen bonding interactions of alginate molecules were weakened, and the helical pitch of the alginate molecules increased, resulting that the values of solution optical rotation decreased [29]. The solution optical rotation of methyl alginate
Fig. 5. Sedimentation fractions of AMDs samples (a), and temperature dependence of the optical rotation for solutions (0.25 wt%, pH = 7) of methyl alginate, HMP and PGA on cooling (b).
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3.5. Application of methyl alginate in AMDs 3.5.1. Properties of AMDs The solution apparent viscosity and surface tension of the prepared AMDs stabilized by different polysaccharide stabilizers were measured and shown in Table 3 and Fig. 3. It could be seen from Table 3 that both the solution apparent viscosity and surface tension of the AMDs stabilized by six different polysaccharide stabilizers had no significant differences. From Fig. 3, the apparent viscosity of six AMDs samples all decreased with the increase of shear rate, which indicated that the AMDs samples exhibited non-Newtonian behavior. The AMDs samples stabilized by polysaccharide stabilizers containing methyl alginate had similar shearing resistance performance compared with the commercial stabilizer. 3.5.2. Particle size analysis The particle size of casein in AMDs could be used to analyze the stability of AMDs. The smaller the particle size of casein was, the more stable the system tended to be [30]. The particle size distribution and its statistical results of the AMDs samples stabilized by the six stabilizer formulations are shown in Fig. 4. It could be seen from Fig. 4 that the particle size distributions of the AMDs stabilized by stabilizer formulation B and D (from 0.05 μm to 2.0 μm) were both smaller than that of the samples stabilized by other four stabilizer formulations (from 0.05 μm to 2.5 μm). The statistical results of particle size showed that the D50 and Dav (the particle diameters at 50% and the average diameter
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of cumulative volume, respectively) of the AMDs stabilized by different stabilizers displayed significant difference, and the descending order of Dav was D b B b A b C b F b E. These results indicated that the particle size of AMDs stabilized by polysaccharide mixture tended to be smaller than that of single polysaccharide, and the methyl alginate could be more efficient to reduce the particle size of AMDs compared with HMP no matter it was used singly or together with PGA, or with PGA and CMC. 3.5.3. Sedimentation analysis The sedimentation fractions of the AMDs samples stabilized by stabilizer formulation A to F were measured and shown in Fig. 5a. It could be seen from Fig. 5a that the sedimentation fraction of the AMDs stabilized by stabilizer formulation D was the lowest, then in ascending order of magnitude, were the sample stabilized by stabilizer formulation B, A, C, F, and E, respectively. This order was in perfect agreement with the result of the particle size analysis (see Fig. 4). The results of sedimentation analysis also verified that the stabilizing effect of all mixture polysaccharide stabilizers were better than that of single polysaccharide stabilizers, and the stabilizing effect of the mixture polysaccharide stabilizer became better when using methyl alginate to replace HMP by the same amount. This result indicated that methyl alginate with DE of 60.7% was more effective in stabilizing AMDs than HMP under the same conditions, and methyl alginate could be used to replace HMP in commercial reference stabilizer for preparation of AMDs, although methyl alginate and HMP had the similar molecular structural formula and similar stabilizing mechanism. The difference between methyl
Fig. 6. SEM images of the AMDs samples stabilized by the stabilizer formulation A to F.
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alginate and HMP to stabilize AMDs might come from the different molecular spatial structures of two kinds of polysaccharides. As exhibited in Fig. 5b, methyl alginate and PGA had same levorotatory optical activity and similar variation of solution optical rotation on cooling, whereas, the optical activity of HMP was dextral, this might be also the reason that methyl alginate had better synergistic action than HMP with PGA for stabilizing AMDs. 3.5.4. SEM analysis The SEM images of the AMDs samples after freeze-drying treatment were evaluated and shown in Fig. 6. It could be seen that the skeleton morphology of the AMDs sample stabilized by the commercial reference stabilizer was shaped a sheet structure with large pores [23]. By contrast, the porous skeleton structure of the AMDs sample stabilized by formulation B tended to be denser, and its clusters of casein particles became smaller after using methyl alginate to replace HMP in formulation A. This was in agreement with the resulting microstructures of the AMDs stabilized by HMP and methyl alginate alone (as shown in Fig. 6E and F).
3.5.5. Effect of the DE of methyl alginate on stability of AMDs The stability of AMDs stabilized by mixture stabilizer composed of PGA and methyl alginate with different DE values (1:1 in weight) were determined and denoted as D1, D2, D3, D4 and D5 according to the corresponding DE values of methyl alginate which were 30.2%, 40.1%, 49.9%, 60.7% and 69.9%, respectively. The sedimentation fractions and particle sizes of the AMDs samples were measured and shown in Fig. 7a and b. It could be seen from Fig. 7a that the sedimentation fractions of the AMDs samples significantly decreased with the DE increase of methyl alginate, and became stable after DE of methyl alginate was greater than 60.7%. This result was in good agreement with the reports that HMP was more effective in stabilizing AMDs than low-methoxyl pectin under low pH conditions [31,32]. The reason was that methyl alginate with higher DE would have better acid resistance and anticalcium property owing to the lower proportion of carboxyl groups. The methyl alginate could be absorbed on the surface of casein particles under low pH conditions through the electrostatic interaction between its carboxyl groups with negative charge and casein particles with positive charge. When DE was higher, there would be fewer carboxyl groups existed in a smaller region of methyl alginate molecules that could interact with the casein particles, and free a more substantial portion of the methyl alginate chains that stretched in solvent for solvation, as a result, forming dispersions resistant to sedimentation [32]. From Fig. 7b, the result that the median particle diameter of AMDs tended to be smaller with the DE increase of methyl alginate, further verified that the increase of the DE of methyl alginate was beneficial to the stability of AMDs. 3.5.6. Effect of stabilizer dosage on stability of AMDs The effect of stabilizer dosage on the sedimentation fractions of the AMDs stabilized by the mixture stabilizer composed of methyl alginate (DE = 60.7%) and PGA (mass ratio of 1:1) were measured and shown in Fig. 7c. It could be seen from Fig. 7c that the sedimentation fractions of the AMDs without polysaccharide stabilizer was relatively high, indicated that the casein tended to aggregate under acidic conditions. When the dosage of stabilizer increased to 0.05 wt% of the total mass of AMDs, the sedimentation fraction of AMDs sample increased unexpectedly. The reason for this result was that the added polysaccharides were not enough to cover the casein colloid at this concentration, and the casein and polysaccharides in AMDs system could be precipitated by bridging flocculation [33]. After this point, the sedimentation fractions of the corresponding AMDs decreased constantly with the dosage increase of the polysaccharide mixture, and changed little when the dosage of stabilizer was over 0.324 wt%.
Fig. 7. The sedimentation fractions (a) and particle sizes (b) of the AMDs samples stabilized by PGA and methyl alginate with different DE. Effect of the stabilizer concentration on the sedimentation fractions of the AMDs (c).
4. Conclusions In the present work, a series of methyl alginate with different degree of esterification were prepared through a simple esterification reaction. Compared with HMP, the methyl alginate with DE equal or larger than 60.7% possessed more excellent stabilizing effect on the casein in AMDs under the same condition, and the corresponding AMDs showed
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good shearing resistance performance. The methyl alginate had better synergistic action for stabilizing AMDs with PGA than HMP. The present study indicated that the methyl alginate which was easily prepared with lower cost had a potential industry application prospect in AMDs as an alternative to HMP. Acknowledgements The authors gratefully acknowledge financial support from the Qingdao Municipal Science and Technology Bureau (Grant No. 17-1-1-86jch). References [1] H. Andriamanantoanina, M. Rinaudo, Characterization of the alginates from five madagascan brown algae, Carbohydr. Polym. 82 (2010) 555–560. [2] M.U. Chhatbar, R. Meena, K. Prasad, D.R. Chejara, A.K. Siddhanta, Microwaveinduced facile synthesis of water-soluble fluorogenic alginic acid derivatives, Carbohydr. Res. 346 (2011) 527–533. [3] T. Taubner, M. Marounek, A. Synytsya, Preparation and characterization of amidated derivatives of alginic acid, Int. J. Biol. Macromol. 103 (2017) 202–207. [4] N. Ganesh, C. Hanna, S.V. Nair, L.S. Nair, Enzymatically cross-linked alginichyaluronic acid composite hydrogels as cell delivery vehicles, Int. J. Biol. Macromol. 55 (2013) 289–294. [5] L. Wang, R.M. Shelton, P.R. Cooper, M. Lawson, J.T. Triffitt, J.E. Barralet, Evaluation of sodium alginate for bone marrow cell tissue engineering, Biomaterials 24 (2003) 3475–3481. [6] S.C. Angadi, L.S. Manjeshwar, T.M. Aminabhavi, Novel composite blend microbeads of sodium alginate coated with chitosan for controlled release of amoxicillin, Int. J. Biol. Macromol. 51 (2012) 45–55. [7] Y. Kimura, K. Watanabe, H. Okuda, Effects of soluble sodium alginate on cholesterol excretion and glucose tolerance in rats, J. Ethnopharmacol. 54 (1996) 47–54. [8] B. Du, J. Li, H. Zhang, L. Huang, P. Chen, J. Zhou, Influence of molecular weight and degree of substitution of carboxymethylcellulose on the stability of acidified milk drinks, Food Hydrocolloid 23 (2009) 1420–1426. [9] R.H. Tromp, C.G. de Kruif, M. van Eijk, C. Rolin, On the mechanism of stabilisation of acidified milk drinks by pectin, Food Hydrocolloid 18 (2004) 565–572. [10] S. Jensen, C. Rolin, R. Ipsen, Stabilisation of acidified skimmed milk with HM pectin, Food Hydrocolloid 24 (2010) 291–299. [11] T. Nobuhara, K. Matsumiya, Y. Nambu, A. Nakamura, N. Fujii, Y. Matsumura, Stabilization of milk protein dispersion by soybean soluble polysaccharide under acidic pH conditions, Food Hydrocolloid 34 (2014) 39–45. [12] P.J. Young, P.M. Bluestein, Stable Acidic Milk Based Beverage, US Patent 0-160-086, 2002. [13] F. Munarin, M.C. Tanzi, P. Petrini, Advances in biomedical applications of pectin gels, Int. J. Biol. Macromol. 51 (2012) 681–689. [14] S.Y. Chan, W.S. Choo, D.J. Young, X.J. Loh, Pectin as a rheology modifier: origin, structure, commercial production and rheology, Carbohydr. Polym. 161 (2017) 118–139.
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