- Email: [email protected]
Structure and rheological characterization of konjac glucomannan octenyl succinate (KGOS)
Accepted Manuscript Structure and rheological characterization of konjac glucomannan octenyl succinate (KGOS) Geng Zhong, Fan-Bing Meng, Yun-Cheng Li, Da-Yu Liu, Xiao-Qiang Guo, Lian-Ji Zheng PII:
S0268-005X(17)30650-1
DOI:
10.1016/j.foodhyd.2017.10.015
Reference:
FOOHYD 4102
To appear in:
Food Hydrocolloids
Please cite this article as: Geng Zhong, Fan-Bing Meng, Yun-Cheng Li, Da-Yu Liu, Xiao-Qiang Guo, Lian-Ji Zheng, Structure and rheological characterization of konjac glucomannan octenyl succinate (KGOS), Food Hydrocolloids (2017), doi: 10.1016/j.foodhyd.2017.10.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical abstract
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First, in light of the special structure of KGOS, its extent of reaction cannot be
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detected by infrared spectroscopy, titration, elemental analysis or nuclear magnetic
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resonance methods, so we established the HPLC method (A), which proved to be suitable from the test results. The method also provides convenience and a basis for future research into KGOS. The existence of molecular agglomeration was verified by transmission electron microscopy and atomic force microscopy (B). Finally, the micro-properties of KGOS were analyzed by using macro-rheological methods (C). In short, this paper studied the other characteristics of the novel modified polymer polysaccharide, konjac glucomannan octenyl succinate (KGOS). We believe
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surfactant that is very valuable.
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The HPLC method was developed to determine the reaction extent of konjac glucomannan octenyl succinate (KGOS) by re-contrasting the NMR method. The molecular aggregation state of KGOS in aqueous solution was observed
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by using transmission electron microscopy and atomic force microscopy. •
Steady and dynamic shear rheology for KGOS aqueous solution under
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condition of different KGOS concentration and different ions addition were
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systematically investigated.
ACCEPTED MANUSCRIPT Structure and rheological characterization of konjac glucomannan octenyl succinate (KGOS) Geng Zhong a, Fan-Bing Meng b, *, Yun-Cheng Li b, Da-Yu Liu b, Xiao-Qiang Guo b, Lian-Ji Zheng
College of Pharmacy and Bioengineering, Chengdu University, Chengdu 610106, P. R. China c
Corresponding Author:
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Chongqing Food Industry Research Institute, Chongqing 400020, P. R. China
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College of Food Science, Southwest University, Chongqing 400716, P. R. China
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a, c
Fan-Bing Meng
College of Pharmacy and Bioengineering Chengdu University
P. R. China
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Chengdu 610106
Phone: +0086-28-84616063
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FAX: +0086-28-84616063
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Email: [email protected]
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Abstract Konjac glucomannan octenyl succinate (KGOS) has excellent emulsifying and
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thickening properties as a new polymeric surfactant. The reaction extent of KGOS
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was determined by using high-performance liquid chromatography. The molecular
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aggregation state of KGOS was observed and analyzed by transmission electron
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microscopy (TEM) and atomic force microscopy (AFM). The results showed that
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KGOS aggregated particles formed irregular spheres or ellipsoids, and the average
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particle diameter and height were approximately 30 nm and 5 nm, respectively.
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Thermal analysis indicated that the carbonization temperature of KGOS is slightly
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lower than that of konjac glucomannan (KGM). The steady shear rheology showed
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that KGOS solution fit to non-Newtonian fluid properties and the Carreau model, and
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the apparent viscosity increased with increase of KGOS concentration. The type and
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valence of the ions affected the apparent viscosity of KGOS solution. The trends of
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the dynamic mechanical spectra indicated KGOS solution could be an entanglement
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system. The ions had a similar effect on the dynamic modulus (G', G") and loss factor
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tanδ except for Fe3+ and H2PO4-.
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Keyword: Konjac glucomannan octenyl succinate (KGOS); Degree of reaction;
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Molecular group; Rheological properties
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ACCEPTED MANUSCRIPT 1. Introduction
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Konjac glucomannan (KGM) is a natural polysaccharide polymer and water-soluble
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dietary fiber derived from the tubers of Amorphophallus konjac. KGM has already
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been approved as generally regarded as safe (GRAS) in foods (Tester & Al-Ghazzewi,
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2016) and medicinal ingredients (Fang & Wu, 2004) by many countries or
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organizations. KGM has a high molecular weight range of (Mw) 105-106, which
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results in high viscosity (Li, Ji, Xia, & Li, 2012). The KGM backbone is substituted
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by 5-10% acetyl groups at the C-6 position, which have a great impact on properties
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such as water-solubility, film formation, and so on (Katsuraya, Okuyama, Hatanaka,
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Oshima, Sato, & Matsuzaki, 2003; Phillips & Williams, 2009). At the same time, the
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acetyl groups make the study of its nature difficult, including the calculation of the
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extent of its derivative reaction and elucidation of its structural properties.
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Native KGMs have been approved as having beneficial functional and nutritional
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properties, such as controlling blood cholesterol and sugar level, and promotion of
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weight loss, intestinal activity and immune function (Aoe, Kudo, & Sakurai, 2015; Bo,
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Muschin, Kanamoto, Nakashima, & Yoshida, 2013; Zhang & Yang, 2014). Moreover,
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its low cost, excellent film-forming ability, good biocompatibility, biodegradability
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and gel-forming properties support KGM’s use as a novel polymer material. Thus,
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natural KGM has promising applications in various fields, such as packing materials,
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preservative materials and controlled-release materials (Leuangsukrerk, Phupoksakul,
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Tananuwong, Borompichaichartkul, & Janjarasskul, 2014; Tester & Al-Ghazzewi,
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2016; Wang, Liu, Li, Wang, & Wang, 2015; Zhang & Yang, 2014).
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ACCEPTED MANUSCRIPT The development and use of KGM and its derivatives have received significant
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attention in recent years (Tester & Al-Ghazzewi, 2016; Zhang & Yang, 2014). Many
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new applications have been found for KGM derivatives, due to its new or distinctive
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physicochemical or functional properties. Previously, several KGM derivatives were
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prepared by degradation (Liu, Xu, Zhang, Zhou, Lyu, Zhao, & Ding, 2015),
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deacetylation (Wang, Zhan, Wu, Ye, Li, Wang, Chen, & Li, 2014), esterification
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(Zhang, Han, Yao, Pang, & Luo, 2013), oxidation (Korkiatithaweechai, Umsarika,
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Praphairaksit, & Muangsin, 2011); their properties and applications were assessed,
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including adsorption of heavy metal ions, colon-specific delivery and emulsification.
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KGM was esterified with octenyl succinic anhydride (OSA) using a microwave
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method under alkalescent conditions to yield KGOS through a method developed in
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our previous study (Meng, Zheng, Wang, Liang, & Zhong, 2014). The structure was
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characterized by means of 1H NMR spectrum, Fourier transform infrared (FT-IR)
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spectroscopy, scanning electron microscopy (SEM) and X-ray diffraction, all of
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which indicated that OSA groups had been successfully grafted onto the KGM
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molecule(Meng, et al, 2014). What’s more, further assessment indicated that KGOS
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as a new polymeric surfactant had better emulsifying and thickening properties than
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OSA modified starch (OSAS) (Meng et al., 2014), which have been used in food,
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cosmetics and pharmaceutical products as emulsifiers, stabilizers and microcapsule
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wall materials for more than half a century (Sweedman, Tizzotti, Schäfer, & Gilbert,
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2013). However, OSAS is insoluble, so as a new soluble OSA modified
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polysaccharides, KGOS might have more potential application in the above industrial
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areas. Before KGOS is applied in food and pharmaceutical industrial, more
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characteristic details should be determined, including the extent of reaction,
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aggregation properties and rheological properties, etc, to understand its nature in the
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production and application process. In the present study, an HPLC method was
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developed for detecting the extent of reaction for KGOS, because of the inaccuracy of
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the titration caused by the presence of acetyl groups and the low solubility of KGOS,
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which limit studies using 1H NMR and variable-temperature 1H NMR. In addition, the
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aggregation and rheological properties were systematically investigated. The main
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research objects in rheology are the deformation and flow of objects under an external
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force; many foods are often in liquid form during production and processing, and thus,
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rheology is widely studied in food science (Xu, Zhang, Liu, Sun, & Wang, 2015). The
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aim of this study is to provide some reference for KGOS application in food,
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cosmetics and pharmacy processing.
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2. Materials and methods
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2.1. Materials
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KGM (95.0% purity) was obtained from the Konjac Association of Chinese
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Society for Horticultural Science (Beibei District, Chongqing City, China) through
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wet-method
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2-Octen-1-ylsuccinic anhydride (99.0% purity) was purchased from Sigma Chemical
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Co. (St. Louis, MO). All chemicals were analytical grade unless otherwise stated.
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2.2. Preparation of KGOS
extraction
from
the
tuber
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of
the
Amorphophallus
plant.
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al., 2014): twenty grams of dry weight KGM powder and 2% of Na2CO3 (in
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proportion to KGM, w/w) were added to the reaction vessel. After homogeneous
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mixing, 20.00 g of ethanol solution (30%) was added slowly with agitation and 3%
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OSA (in proportion to KGM, w/w) was added (diluted five times with absolute
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ethanol, v/v) with slow agitation. The mixture was placed in the center of a
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microwave reactor (Shanghai Sineo Microwave Chemistry Technology Co., LTD,
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MASII) and heated at 300 W and 70 °C for 20 min, cooled to 25 °C and blended with
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40.00 mL of 30% ethanol solution for 5 min. The pH value of the solution was
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adjusted to 6.50 with HCl solution (1 N). The mixture was washed and filtered with
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30% ethanol five times to remove residual NaCl and other soluble impurities; absolute
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ethanol was used for five washes to remove residual OSA. The final solid portion was
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oven-dried at 40 °C for 24 h and passed through a 100-mesh nylon sieve.
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2.3. Determination of reaction degree
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2.3.1. The 1H NMR analysis of KGOS at different temperature
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KGOS (0.5 mg) was weighed and dissolved in D2O (1.0 mL). The solutions were
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centrifuged at 5,000 × g for 30 min to remove the insoluble material. The supernatant
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liquids were transferred to 5-mm NMR tubes. The 1H NMR spectrum was recorded in
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a 600 MHz Agilent DD2 NMR spectrometer (Agilent, USA) with varying
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temperature.
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2.3.2. HPLC analysis
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The substitution rate (SR) of KGOS was measured by the HPLC method as
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performed by an HPLC (LC-20A, Shimadzu, Japan) equipped with a Thermo BDS
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C18 column (5 µm, 250 mm × 4.6 mm, Thermo Fisher, USA) and a UV detector
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(Shimadzu) operated at 200 nm. A mixture of acetonitrile and water with 0.1% TFA
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(55:45, v/v) was used as the mobile phase. The flow rate was 1.0 mL/min.
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2.3.3. Standard curves for OS
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OSA reagent (0.2104 g) was added to a 50-mL volumetric flask and diluted to the
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desired volume (50 mL) with acetonitrile. Pipetted 0.25, 0.5, 0.75, 1 or 1.25 mL of
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OSA solution into 25-mL beakers, followed by 10 mL 1 M NaOH ethanol solution
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(30%), respectively. Samples were covered with plastic wrap and magnetically stirred
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at 400 r/min for 10 h. Then, 3 mL 4 M HCl ethanol solution (30%) was pipetted into
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beakers and the mixture was transferred into 25-mL volumetric flasks after cooling to
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room temperature and filled with acetonitrile to a total of 25 mL.
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2.3.4. Determination of free and bound OSA
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To determine the free OS content of KGOS, 0.3000 g KGOS (dry weight) was
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added to a 50-mL centrifuge tube and extracted with 5 mL of methanol with magnetic
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stirring for 1 h. The mixture was centrifuged, and 1 mL of supernatant was collected
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into a beaker and mixed with 10 mL 1 M NaOH ethanol solution (30%) and magnetic
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stirring at 400 r/min for 10 h. Next, 3 mL 4 M HCl ethanol solution (30%) was
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pipetted into a centrifuge tube and cooled to room temperature; residues were
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removed using a 0.45-µm membrane filter, and 6 mL was pipetted into a 25-mL
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volumetric flask and filled with acetonitrile to a total of 25 mL. The OS content (Wfree,
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g) was calculated using the total peak area (Afree) and the standard curve. For total OS content determination, KGOS (0.3000 g dry weight) was weighed
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into a 50-mL centrifuge tube, 10 mL 1 M NaOH ethanol solution (30%) was added,
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and the solution was magnetically stirred at 400 r/min for 10 h. Next, 3 mL 4 M HCl
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ethanol solution (30%) was pipetted into a centrifuge tube and cooled to room
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temperature. Residues were removed with a 0.45-µm membrane filter, and 6 mL was
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pipetted into a 25-mL volumetric flask and filled with acetonitrile to a total of 25 mL.
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The OS in the solution was analyzed by HPLC to yield a total peak area (Atotal) and
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corresponding weight (Wtotal, g) using a standard curve. The substitution rate (SR) was
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calculated using the following equation: SR =
− OS × 100% (1)
where is the dry weight of KGOS.
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2.4. Characterization of KGOS micelles
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Transmission electron microscopy (TEM, JEM 1200EX, JEOL) was applied to
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characterize the appearance of KGOS self-assembled micelles in aqueous solution
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(Zhu, Liu, & Du, 2013). A sample (0.3 mg/mL KGOS solution, 3 µL) was dropped
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onto the carbon-coated grid and dried under ambient conditions overnight. To stain
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the micelles, phosphotungstic acid (PTA; 0.5 w/v %) solution (10 µL) was dropped
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onto the micelles for 2 min. A filter paper was used to carefully blot the excess PTA
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solution. The grids were dried again under ambient conditions overnight. Imaging was
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performed on a JEOL JEM-2100 instrument (JEOL, Japan) at 200 kV equipped with a
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Gatan 94 Ultrascan 1k charge-coupled device camera.
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further characterize KGOS shapes. A drop of the KGOS solution (0.3 mg/mL) was
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placed on freshly cleaved mica. After 5 min of incubation at room temperature, the
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surface of the mica was gently rinsed with 5 mL of deionized water and blown with
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dry nitrogen. The sample was air-dried at room temperature and mounted on a
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microscope scanner. The shape was observed and imaged in the noncontact mode (Liu,
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Chen, Tian, Ma, Li, & Zhao, 2014).
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2.5. Thermodynamic properties of KGOS
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Thermal analyses (TG and DSC) were recorded on a TGA/DSC instrument
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(1600HT METTLER, Switzerland), and approximately 10-mg samples were heated in
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an alumina pan with a 10 K/min heating rate from 30 °C to 800 °C under N2 flow (50
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mL/min). An empty pan sealed in the same manner was used as a reference.
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2.6. Rheological measurements
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Steady shear and dynamic frequency sweeps measurements were performed using
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a Carri-Med CSL2 100 rheometer (TA Instruments, New Castle, USA) using a
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measurement system with plate-plate geometry (40 mm diameter, 500 µm gap).
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Steady shear measurements were obtained at 25 °C. The flow curves were obtained by
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registering shear stress at a shear rate that was increased from 0.01 to 500 s-1.
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Dynamic frequency sweeps measurements were performed under various
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conditions according to the references (Lin, Wu, Luo, Liu, Luo, & He, 2010; Penroj,
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Mitchell, Hill, & Ganjanagunchorn, 2005) with minor modifications: A constant
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strain (5%) determined based on strain sweep experiments at a frequency of 0.1, 1 and
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ACCEPTED MANUSCRIPT 10 Hz, which fell within the linear viscoelastic regions of all samples.
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Small-amplitude oscillatory tests were performed at 25 °C over a frequency range of
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0.1-10 Hz. The parameters obtained from the dynamic test data were storage modulus
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G', loss modulus G", loss factor tanδ (G"/G'). These were determined as the means of
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values that were within the linear viscoelastic range of each sample.
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2.6.1. Rheological properties for various concentrations of KGOS
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Powders of KGOS and KGM were dispersed in double distilled/deionized water
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with stirring. The solutions were heated to 60°C and maintained at this temperature
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for 1 h and cooled to room temperature. The concentrations of both solutions were 1%,
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1.25%, 1.5%, 1.75% and 2% (w/w).
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2.6.2. Rheological properties of KGOS solutions with various ions One percent (w/w) of KGOS was prepared as in section 2.4, and 0.02 mol of
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MgCl2, KCl, CaCl2 and FeCl3 were added to 100 g KGOS solution to study the effect
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of different cations on the rheological properties. Then, 0.02 mol of NaCl, Na2SO4,
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NaNO3 and NaH2PO4 were added to 100 g KGOS solution to study the effects of
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different anions on the rheological properties.
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2.7. Statistical analysis
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All tests were performed in triplicate. A one-way analysis of variance (ANOVA)
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and Tukey’s test were used to establish the significance of differences among the
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mean values at the 0.05 level of confidence. The statistical analyses were performed
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using SPSS version 20.0.
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3. Results and discussion
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3.1. Measurement of reaction degree It is difficult to precisely determine the reaction extent of OSA-modified
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polysaccharide using conventional methods such as titration; this is because these
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macromolecular compounds usually formed aggregations in aqueous solution or
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cannot dissolve in water. Tizzotti, Sweedman, Tang, Schaefer, & Gilbert (2011)
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developed a 1H NMR method to measure the reaction degree of OSA-modified starch.
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In our previous study, the 1H NMR experiment at 25°C indicated that the OSA groups
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were grafted onto the KGM via the esterification reaction and an incomplete
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deacetylation reaction in the modification (Meng, et al., 2014). However, the reaction
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degree of KGOS could not be calculated by 1H NMR because the methyl proton peak
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overlapped with some integral peaks and the solvent proton peak. To eliminate peak
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interference, the NMR temperature was gradually increased. As shown in Fig. 1, the
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integral peaks (peaks 2 and 3) still overlapped when the temperature increased to
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85°C, indicating that temperature-dependent 1H NMR could not be used to calculate
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the extent of reaction of KGOS.
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It was also difficult to use Fourier transform infrared spectroscopy (FTIR) to
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detect DS because FTIR has only been found to be reliable for high DS (≥0.3)
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(Sweedman, Tizzotti, Schäfer, & Gilbert, 2013). Elemental analysis reported by Liu,
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Li, Ma, Chen, & Zhao (2013) could also not be used to determine the DS of KGOS
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for its complex chains structure. Although convenient titration methods are widely
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used to find the DS of OSA, it is also not suitable for KGOS because an incomplete
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deacetylation reaction occurred during KGOS synthesis, resulting in inaccurate
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titration due to the presence of acetyl groups. Therefore, we tried to estimate the extent of the modification reaction by
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calculating the substitution rate (equation 1). At the same time, we chose an ethanol
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solution (30%) as a dispersant of KGOS to prevent an excessive increase in solution
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viscosity caused by KGOS swelling. As shown in Fig. 2, there were three peaks
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(identified as 1-OS acid, cis-2-OS acid and trans-2-OS acid, respectively) after OSA
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hydrolysis, giving the same result as Qiu et al. (2012). The values increased or
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decreased in the same proportion, so we chose the area of the highest peak (peak 3) to
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calculate the OS content. Standard curves for OS had the equation: Y = 4 × 10 X −
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0.005, R2=0.9996, and the SR of KGOS was 2.523±0.022%.
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3.2. Characterization of KGOS micelles
The TEM observations shown in Fig. 3A indicated that hydrophobically modified
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KGOS aggregation particles formed irregular spheres or ellipsoids, and the average
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particle diameter of KGOS was approximately 30 nm. The amplified image (Fig. 3B)
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showed that the particles were distributed individually and appeared near the bulb,
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with radial branches spread from the surface. As shown in Fig. 3C, AFM showed that
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the height for KGOS aggregate was approximately 5 nm, and a continuous hill-like
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substance was present on the mica surface. This phenomenon could further explain
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the presence of a long carbon chain spread out from the aggregate, which was caused
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by the long polymer chains and explained the multi-branch structure of KGOS. This
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appearance is different from the octenylsuccinate oat β-glucan (lower molecular
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weight of 16.8 × 104 - 7.3 × 104) reported by Liu et al. (2013) The aggregation
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clearly separated from the aqueous solution. This difference may contribute to the
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winding of long polymer chains and branches of KGOS.
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3.3. Thermodynamic properties of KGOS
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In Fig. 4, both KGM and KGOS have a smaller drop in mass at approximately
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100°C due to evaporation of water, with KGM falling by 4.95% and KGOS
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decreasing by 5.83%. In addition, there was a large decrease in mass at approximately
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300°C, which can be ascribed to the carbonization of the sample, KGM and KGOS
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decreased 77.61% and 75.17%, respectively. After 300°C, the sample tends to be
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stable. However, the DSC peak shapes of KGOS and KGM did not change
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significantly, and there was an endothermic peak for water evaporation and no
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endothermic peak for the melt. In combination with the results of Dave, Sheth,
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Mccarthy, Ratto, & Kaplan (1998), it is possible that there are many hydrogen bonds
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in the KGM and KGOS molecules, resulting in a strong interaction between KGM
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and KGOS molecular chains, which leading to the melting temperatures were much
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higher than their decomposition temperatures. However, the TG curve of KGM and
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KGOS was found to be lower (10 - 15°C) than that of KGM at the second weight loss,
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indicating that the carbonization temperature of KGOS is slightly lower than KGM.
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3.4. Rheological properties at various concentrations of KGOS
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3.4.1. Steady shear rheology
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As shown in Fig. 5, at lower shear rates, the viscosity of KGOS and KGM
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solutions was only slightly affected, the viscosity in this region is considered zero
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ACCEPTED MANUSCRIPT shear viscosity, and it depends on the polymer concentration (Fig. 5). When the shear
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rate increased to a certain threshold γ# c (intersection of the vertical line and abscissa),
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the shear viscosity of the solution substantially declined with the increasing of shear
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rate, which is known as shear thinning. This phenomenon may due to the destruction
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of the internal structure of KGOS solution. Larger aggregates could more easily
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rearrange in the shear direction (Chronakis & Alexandridis, 2001). With increasing
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concentration of KGM and KGOS, γ# c decreased (Fig. 5). The reciprocal of γ# c was
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considered to be the relaxation time of the network structure of the solution
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(Chronakis & Alexandridis, 2001). The longer relaxation time indicated that the
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strength of polymer aggregates increased and the de-aggregation time was extended. Further analysis indicated that the steady shear rheology results of KGM and
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KGOS could be fit to the Carreau model, and the nonlinear regression and resulting
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coefficients are given in Table 1. The Carreau model is as follows (Koszkul &
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Nabialek, 2004):
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where η% is the viscosity at infinite shear rate; η' is the viscosity at zero shear rate;
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γ# is the shear rate; n is flow behavior index (dimensionless); λ is a time constant.
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The zero shear viscosity η' of KGM and KGOS increased with the increase in
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concentration, and the n value decreased, which indicated an increase in the
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pseudoplasticity (n<1) of these solutions (Sudhakar, Singhal, & Kulkarni, 1996). A
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possible reason was that molecules of KGOS and KGM are irregularly bound together,
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which leads to strengthened interactions between chains. Thus, the viscosity and
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ACCEPTED MANUSCRIPT pseudoplastic characteristics of the solutions increased. However, at the same
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concentrations, the zero shear viscosity of KGOS is less than KGM. The possible
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reason was that a small amount of molecular chains of KGOS were degraded during
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preparation using a microwave method. The introduction of modified anhydride
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groups likely increased the curl degree of the intramolecular chains, resulting in the
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reduction of intermolecular crosslinking of KGOS, compared to that of KGM.
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3.4.2. Dynamic shear properties
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The mechanical spectra for various concentrations of KGM and KGOS at 25°C
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are shown in Fig. 6. The elastic modulus (G') and viscous modulus (G") of KGM and
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KGOS at the same concentration have a similar trends. At low concentration (1% to
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1.75%), with the frequency increase, the G" of KGM and KGOS were firstly higher
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and then lower than G'. According to the report of Clark and Ross-murphy (1987),
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this trend of the mechanical spectra met the characteristics of entanglement network
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systems.
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With the frequency increase, the increase range of G', which represents the
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recoverable energy when the material is subjected to deformation, was greater than for
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G" (Ma & Barbosa-Cánovas, 1995), indicated that the material will exhibit solid
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behavior. Piculell, Egermayer, & Sjöström (2003) proposed using the crossover
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modulus at the intersection of G' and G" (Gc) to characterize the number of
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cross-links and the inverse of the frequency at the intersection (τc) to characterize the
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apparent relaxation time. As shown in Fig. 6, Gc and τc increased with increasing
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concentration of KGM and KGOS, which indicated that the numbers of the
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crosslinking (as monitored by Gc) increased and the dynamics (as monitored by τc)
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slowed down (Piculell et al. 2003).
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3.5. Rheological properties of KGOS solutions with various ions
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3.5.1. Steady shear rheology
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The effects of different ions on the steady shear are shown in Fig. 7. The addition
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of various cations did not affect the fluid properties of the KGOS solution, and the
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solution fit to non-Newtonian fluid properties and the Carreau model (Table 2). K+
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had little effect on the viscosity of the solution. The viscosity increased and
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pseudoplastic behavior reinforced with addition of Mg2+ and Fe3+, especially in the
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presence of Fe3+. This could be because the increase in charge of the solution
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promoted intermolecular winding more tightly and improved the cross-linked network
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between molecules. The results showed that the type and valence of the cations may
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affect the viscosity and pseudoplasticity of KGOS solution.
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A similar regularity could be observed with anion addition. Cl- and NO3- did not
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affect the viscosity of the KGOS solution, but with the increasing of anionic valence,
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the viscosity of the KGOS solution increased and pseudoplasticity enhanced. SO42-
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and H2PO4- could significantly increase the viscosity of the solution. The possible
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reason was that the ions have different effect to the hydrogen-bond structure between
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water molecules, water molecules and polymer chains, which finally affect the
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polymers solubility and its solution viscosity (Yin, Zhang, Huang, & Nishinari, 2008;
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Briscoe, Luckham, & Zhu, 1996). Besides, the ligation ability of the ions to lone
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electron pairs of the hydroxyl group in KGOS structural units might also affect the
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rheological properties of the solution (Lii, Tomasik, Hung & Lai, 2002).
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3.5.2. Dynamic frequency sweeps From the results shown in Fig. 8A1, K+, Ca2+ and Mg2+ had a similar effect on the
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dynamic modulus (G', G") of KGOS solution. At frequency of 0.1-0.38 Hz, G'
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and the loss factor (Tanδ, G"/G') were greater than 1, which indicate the viscous
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properties predominate in the samples, and the solutions showed more fluid-like
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behavior; but with the frequency higher than 0.38 Hz, G'>G" and Tanδ was smaller
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than 1, which indicate the elastic properties predominate in the samples, and the
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solutions showed more solid-like behavior (Liu, Xu, & Guo, 2007; Chaisawang &
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Suphantharika, 2006). However, for the addition of Fe3+, G' always higher than G",
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and Tanδ was smaller than 1 (Fig 8A1 & A2), which indicate the elastic properties
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predominate in the sample. Compared to other ions, there was a significant reduction
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in Gc and increase in τc for Fe3+ (Fig 8 A2), which indicated an obviously reduction in
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the degree of crosslinking and a slowing down in the dynamics (Piculell, et al., 2003).
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As shown in Fig. 8B1 & B2, anions had a similar effect on the dynamic modulus (G',
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G") of the KGOS solution. Tanδ for the KGOS solutions with Cl-, NO3- were greater
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than 1 in the frequency range of 0.1-0.38 Hz, but for SO42- and H2PO4-, Tanδ was
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greater than 1 at 0.1-0.30 Hz and 0.1-0.26 Hz, respectively (Fig. 8B2). The tanδ of
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H2PO4- was lower than the other anions at low frequencies, the possible reason was
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that there were HPO42- and PO43- in H2PO4- solution, which increased the charge of the
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solution. What’s more, from the results of mechanical spectra, the KGOS solution
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contained the above ions also met the characteristics of entanglement systems.
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4. Conclusion The HPLC method was developed to determine the reaction degree of KGOS.
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Molecular agglomeration of KGOS was observed by TEM and AFM; TG and DSC
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analysis indicated the thermal properties between KGM and KGOS changed slightly.
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The study of rheological properties showed that KGOS solution fit to non-Newtonian
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fluid properties and the Carreau model. The trends of the dynamic mechanical spectra
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met the characteristics of entanglement systems. Viscosity and pseudoplasticity of
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KGOS solution could be affected by the type and valence of the ions, especially for
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the high valence cation.
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Acknowledgments
The work was supported by the National Natural Science Foundation of China
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(31601427), the Chongqing science and technology program (CSTC 2013jcyjA00028)
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and the Chengdu science and technology project (2015-NY02-00184-NC).
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Chaisawang, M., & Suphantharika, M. (2006). Pasting and rheological properties of native and anionic tapioca starches as modified by guar gum and xanthan gum. Food Hydrocolloids, 20(5), 641-649. Chronakis, I. S., & Alexandridis, P. (2001). Rheological properties of oppositely charged polyelectrolyte-surfactant mixtures: Effect of polymer molecular weight and surfactant architecture. Macromolecules, 34(14), 5005-5018. Clark, A. H., & Rossmurphy, S. B. (1987). Structural and mechanical properties of biopolymer gels. Food Polymers Gels & Colloids, 83(1), 322-338.
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Ma, L., & Barbosa-Cánovas, G. (1995). Rheological characterization of mayonnaise. Part II: Flow and viscoelastic properties at different oil and xanthan gum concentrations. Journal of Food Engineering, 25(3), 409-425. Meng, F., Zheng, L., Wang, Y., Liang, Y., & Zhong, G. (2014). Preparation and properties of konjac glucomannan octenyl succinate modified by microwave method. Food Hydrocolloids, 38, 205-210. Penroj, P., Mitchell, J., Hill, S., & Ganjanagunchorn, W. (2005). Effect of konjac glucomannan deacetylation on the properties of gels formed from mixtures of kappa carrageenan and konjac glucomannan. Carbohydrate polymers, 59(3), 367-376.
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Sudhakar, V., Singhal, R., & Kulkarni, P. (1996). Starch-galactomannan interactions: functionality and Sweedman, M. C., Tizzotti, M. J., Schäfer, C., & Gilbert, R. G. (2013). Structure and physicochemical properties of octenyl succinic anhydride modified starches: A review. Carbohydrate polymers, 92(1), 905-920.
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Wang, S., Zhan, Y., Wu, X., Ye, T., Li, Y., Wang, L., Chen, Y., & Li, B. (2014). Dissolution and rheological behavior of deacetylated konjac glucomannan in urea aqueous solution. Carbohydrate polymers, 101, 499-504.
Wang, Y., Liu, J., Li, Q., Wang, Y., & Wang, C. (2015). Two natural glucomannan polymers, from Konjac and Bletilla, as bioactive materials for pharmaceutical applications. Biotechnology Letters, 37(1), 1-8.
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Xu, J. L., Zhang, J. C., Liu, Y., Sun, H. J., & Wang, J. H. (2015). Rheological properties of a polysaccharide from floral mushrooms cultivated in Huangshan Mountain. Carbohydrate polymers, 139, 43-49.
Yin, W., Zhang, H., Huang, L., & Nishinari, K. (2008). Effects of the lyotropic series salts on the gelation of konjac glucomannan in aqueous solutions. Carbohydrate polymers, 74(1), 68-78.
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Zhang, C., Han, B., Yao, X., Pang, L., & Luo, X. (2013). Synthesis of konjac glucomannan phthalate as a new biosorbent for copper ion removal. Journal of Polymer Research, 20(1), 1-14. Zhang, C., & Yang, F. Q. (2014). Konjac glucomannan, a promising polysaccharide for OCDDS. Carbohydrate polymers, 104, 175-181.
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Zhu, Y., Liu, L., & Du, J. (2013). Probing into homopolymer self-assembly: How does hydrogen bonding influence morphology? Macromolecules, 46(1), 194-203.
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ACCEPTED MANUSCRIPT Table 1
n
R2
1%
104.696±9.047e
0.302±0.007a
0.998
1.25%
132.223±10.099d
0.263±0.008b
0.998
1.5%
301.976±13.057c
0.252±0.006b
0.998
1.75%
487.556±16.767b
0.220±0.009c
0.999
2%
854.986±24.192a
0.161±0.005d
0.999
1%
56.2715±2.966d
0.371±0.011a
0.999
1.25%
112.392±5.138d
0.264±0.009b
0.998
1.5%
209.878±7.110c
0.267±0.011b
0.998
1.75%
424.946±12.155b
0.185±0.008c
0.999
2%
803.563±16.055a
0.086±0.004d
0.999
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η0/Pa.s
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KGOS
Concentration
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Fitting parameters of Carreau equation
Assays were performed in triplicate. Mean ± standard deviation values in the same column for each
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solution followed by different superscripts are significantly different (P≤ 0.05).
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ACCEPTED MANUSCRIPT Table 2 Fitting parameters of Carreau model n
R2
Blank control
63.086±3.232cd
0.363±0.005a
0.998
KCl
59.093±3.715d
0.300±0.009c
0.999
CaCl2
59.223±5.022d
0.315±0.008c
0.998
FeCl3
107.176±4.225a
0.265±0.007d
MgCl2
73.029±1.717bc
0.269±0.006d
Na2SO4
78.426±3.276b
0.304±0.008c
NaNO3
63.315±3.105cd
0.340±0.009ab
NaH2PO4
67.709±2.035cd
0.323±0.012bc
0.999
NaCl
61.860±4.847d
0.307±0.007c
0.998
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ηo/Pa.s
0.999 0.999
0.995
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0.998
Assays were performed in triplicate. Mean ± standard deviation values in the same column for each solution followed by different superscripts are significantly different (P≤ 0.05). The blank control was
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1%KGOS solution (w/w), the concentration of the salts were 0.02 mol per 100 g KGOS solution.
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Figure Captions Fig. 1. 1H NMR spectra of KGOS at different temperatures.
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Fig. 2. HPLC chromatograms of hydrolyzed OSA
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Fig. 4. TG/DSC curves of KGM (A) and KGOS (B)
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Fig. 3. TEM (A & B) and AFM (C) micrographs of KGOS self-aggregation
Fig. 5. Apparent viscosity-shear rate plots for various concentrations of KGM (A) and KGOS (B)
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solutions at 25 oC
Fig. 6. Dynamic frequency sweep of KGM (A) and KGOS (B) solutions at various concentrations
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at 25 oC
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Fig. 7. Effects of various cations (A) and anions (B) on the viscosity of a 1% KGOS solution at 25
Fig. 8. Dynamic frequency sweep of KGOS solution with various cations (A1 &A2) and anions (B1&B2) at 25 oC
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S0268-005X(17)30650-1
DOI:
10.1016/j.foodhyd.2017.10.015
Reference:
FOOHYD 4102
To appear in:
Food Hydrocolloids
Please cite this article as: Geng Zhong, Fan-Bing Meng, Yun-Cheng Li, Da-Yu Liu, Xiao-Qiang Guo, Lian-Ji Zheng, Structure and rheological characterization of konjac glucomannan octenyl succinate (KGOS), Food Hydrocolloids (2017), doi: 10.1016/j.foodhyd.2017.10.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical abstract
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First, in light of the special structure of KGOS, its extent of reaction cannot be
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detected by infrared spectroscopy, titration, elemental analysis or nuclear magnetic
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resonance methods, so we established the HPLC method (A), which proved to be suitable from the test results. The method also provides convenience and a basis for future research into KGOS. The existence of molecular agglomeration was verified by transmission electron microscopy and atomic force microscopy (B). Finally, the micro-properties of KGOS were analyzed by using macro-rheological methods (C). In short, this paper studied the other characteristics of the novel modified polymer polysaccharide, konjac glucomannan octenyl succinate (KGOS). We believe
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surfactant that is very valuable.
ACCEPTED MANUSCRIPT Highlights •
The HPLC method was developed to determine the reaction extent of konjac glucomannan octenyl succinate (KGOS) by re-contrasting the NMR method. The molecular aggregation state of KGOS in aqueous solution was observed
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•
by using transmission electron microscopy and atomic force microscopy. •
Steady and dynamic shear rheology for KGOS aqueous solution under
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condition of different KGOS concentration and different ions addition were
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systematically investigated.
ACCEPTED MANUSCRIPT Structure and rheological characterization of konjac glucomannan octenyl succinate (KGOS) Geng Zhong a, Fan-Bing Meng b, *, Yun-Cheng Li b, Da-Yu Liu b, Xiao-Qiang Guo b, Lian-Ji Zheng
College of Pharmacy and Bioengineering, Chengdu University, Chengdu 610106, P. R. China c
Corresponding Author:
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*
Chongqing Food Industry Research Institute, Chongqing 400020, P. R. China
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College of Food Science, Southwest University, Chongqing 400716, P. R. China
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a, c
Fan-Bing Meng
College of Pharmacy and Bioengineering Chengdu University
P. R. China
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Chengdu 610106
Phone: +0086-28-84616063
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FAX: +0086-28-84616063
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Email: [email protected]
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Abstract Konjac glucomannan octenyl succinate (KGOS) has excellent emulsifying and
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thickening properties as a new polymeric surfactant. The reaction extent of KGOS
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was determined by using high-performance liquid chromatography. The molecular
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aggregation state of KGOS was observed and analyzed by transmission electron
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microscopy (TEM) and atomic force microscopy (AFM). The results showed that
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KGOS aggregated particles formed irregular spheres or ellipsoids, and the average
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particle diameter and height were approximately 30 nm and 5 nm, respectively.
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Thermal analysis indicated that the carbonization temperature of KGOS is slightly
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lower than that of konjac glucomannan (KGM). The steady shear rheology showed
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that KGOS solution fit to non-Newtonian fluid properties and the Carreau model, and
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the apparent viscosity increased with increase of KGOS concentration. The type and
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valence of the ions affected the apparent viscosity of KGOS solution. The trends of
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the dynamic mechanical spectra indicated KGOS solution could be an entanglement
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system. The ions had a similar effect on the dynamic modulus (G', G") and loss factor
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tanδ except for Fe3+ and H2PO4-.
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Keyword: Konjac glucomannan octenyl succinate (KGOS); Degree of reaction;
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Molecular group; Rheological properties
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Konjac glucomannan (KGM) is a natural polysaccharide polymer and water-soluble
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dietary fiber derived from the tubers of Amorphophallus konjac. KGM has already
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been approved as generally regarded as safe (GRAS) in foods (Tester & Al-Ghazzewi,
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2016) and medicinal ingredients (Fang & Wu, 2004) by many countries or
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organizations. KGM has a high molecular weight range of (Mw) 105-106, which
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results in high viscosity (Li, Ji, Xia, & Li, 2012). The KGM backbone is substituted
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by 5-10% acetyl groups at the C-6 position, which have a great impact on properties
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such as water-solubility, film formation, and so on (Katsuraya, Okuyama, Hatanaka,
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Oshima, Sato, & Matsuzaki, 2003; Phillips & Williams, 2009). At the same time, the
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acetyl groups make the study of its nature difficult, including the calculation of the
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extent of its derivative reaction and elucidation of its structural properties.
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Native KGMs have been approved as having beneficial functional and nutritional
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properties, such as controlling blood cholesterol and sugar level, and promotion of
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weight loss, intestinal activity and immune function (Aoe, Kudo, & Sakurai, 2015; Bo,
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Muschin, Kanamoto, Nakashima, & Yoshida, 2013; Zhang & Yang, 2014). Moreover,
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its low cost, excellent film-forming ability, good biocompatibility, biodegradability
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and gel-forming properties support KGM’s use as a novel polymer material. Thus,
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natural KGM has promising applications in various fields, such as packing materials,
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preservative materials and controlled-release materials (Leuangsukrerk, Phupoksakul,
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Tananuwong, Borompichaichartkul, & Janjarasskul, 2014; Tester & Al-Ghazzewi,
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2016; Wang, Liu, Li, Wang, & Wang, 2015; Zhang & Yang, 2014).
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ACCEPTED MANUSCRIPT The development and use of KGM and its derivatives have received significant
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attention in recent years (Tester & Al-Ghazzewi, 2016; Zhang & Yang, 2014). Many
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new applications have been found for KGM derivatives, due to its new or distinctive
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physicochemical or functional properties. Previously, several KGM derivatives were
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prepared by degradation (Liu, Xu, Zhang, Zhou, Lyu, Zhao, & Ding, 2015),
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deacetylation (Wang, Zhan, Wu, Ye, Li, Wang, Chen, & Li, 2014), esterification
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(Zhang, Han, Yao, Pang, & Luo, 2013), oxidation (Korkiatithaweechai, Umsarika,
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Praphairaksit, & Muangsin, 2011); their properties and applications were assessed,
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including adsorption of heavy metal ions, colon-specific delivery and emulsification.
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KGM was esterified with octenyl succinic anhydride (OSA) using a microwave
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method under alkalescent conditions to yield KGOS through a method developed in
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our previous study (Meng, Zheng, Wang, Liang, & Zhong, 2014). The structure was
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characterized by means of 1H NMR spectrum, Fourier transform infrared (FT-IR)
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spectroscopy, scanning electron microscopy (SEM) and X-ray diffraction, all of
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which indicated that OSA groups had been successfully grafted onto the KGM
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molecule(Meng, et al, 2014). What’s more, further assessment indicated that KGOS
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as a new polymeric surfactant had better emulsifying and thickening properties than
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OSA modified starch (OSAS) (Meng et al., 2014), which have been used in food,
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cosmetics and pharmaceutical products as emulsifiers, stabilizers and microcapsule
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wall materials for more than half a century (Sweedman, Tizzotti, Schäfer, & Gilbert,
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2013). However, OSAS is insoluble, so as a new soluble OSA modified
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polysaccharides, KGOS might have more potential application in the above industrial
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areas. Before KGOS is applied in food and pharmaceutical industrial, more
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characteristic details should be determined, including the extent of reaction,
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aggregation properties and rheological properties, etc, to understand its nature in the
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production and application process. In the present study, an HPLC method was
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developed for detecting the extent of reaction for KGOS, because of the inaccuracy of
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the titration caused by the presence of acetyl groups and the low solubility of KGOS,
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which limit studies using 1H NMR and variable-temperature 1H NMR. In addition, the
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aggregation and rheological properties were systematically investigated. The main
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research objects in rheology are the deformation and flow of objects under an external
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force; many foods are often in liquid form during production and processing, and thus,
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rheology is widely studied in food science (Xu, Zhang, Liu, Sun, & Wang, 2015). The
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aim of this study is to provide some reference for KGOS application in food,
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cosmetics and pharmacy processing.
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2. Materials and methods
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2.1. Materials
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KGM (95.0% purity) was obtained from the Konjac Association of Chinese
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Society for Horticultural Science (Beibei District, Chongqing City, China) through
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wet-method
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2-Octen-1-ylsuccinic anhydride (99.0% purity) was purchased from Sigma Chemical
21
Co. (St. Louis, MO). All chemicals were analytical grade unless otherwise stated.
22
2.2. Preparation of KGOS
extraction
from
the
tuber
5
of
the
Amorphophallus
plant.
ACCEPTED MANUSCRIPT KGOS was prepared using a previously described microwave method (Meng, et
2
al., 2014): twenty grams of dry weight KGM powder and 2% of Na2CO3 (in
3
proportion to KGM, w/w) were added to the reaction vessel. After homogeneous
4
mixing, 20.00 g of ethanol solution (30%) was added slowly with agitation and 3%
5
OSA (in proportion to KGM, w/w) was added (diluted five times with absolute
6
ethanol, v/v) with slow agitation. The mixture was placed in the center of a
7
microwave reactor (Shanghai Sineo Microwave Chemistry Technology Co., LTD,
8
MASII) and heated at 300 W and 70 °C for 20 min, cooled to 25 °C and blended with
9
40.00 mL of 30% ethanol solution for 5 min. The pH value of the solution was
10
adjusted to 6.50 with HCl solution (1 N). The mixture was washed and filtered with
11
30% ethanol five times to remove residual NaCl and other soluble impurities; absolute
12
ethanol was used for five washes to remove residual OSA. The final solid portion was
13
oven-dried at 40 °C for 24 h and passed through a 100-mesh nylon sieve.
14
2.3. Determination of reaction degree
15
2.3.1. The 1H NMR analysis of KGOS at different temperature
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KGOS (0.5 mg) was weighed and dissolved in D2O (1.0 mL). The solutions were
17
centrifuged at 5,000 × g for 30 min to remove the insoluble material. The supernatant
18
liquids were transferred to 5-mm NMR tubes. The 1H NMR spectrum was recorded in
19
a 600 MHz Agilent DD2 NMR spectrometer (Agilent, USA) with varying
20
temperature.
21
2.3.2. HPLC analysis
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The substitution rate (SR) of KGOS was measured by the HPLC method as
6
ACCEPTED MANUSCRIPT described by Qiu, Bai, & Shi (2012) with some modification. HPLC analysis was
2
performed by an HPLC (LC-20A, Shimadzu, Japan) equipped with a Thermo BDS
3
C18 column (5 µm, 250 mm × 4.6 mm, Thermo Fisher, USA) and a UV detector
4
(Shimadzu) operated at 200 nm. A mixture of acetonitrile and water with 0.1% TFA
5
(55:45, v/v) was used as the mobile phase. The flow rate was 1.0 mL/min.
6
2.3.3. Standard curves for OS
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OSA reagent (0.2104 g) was added to a 50-mL volumetric flask and diluted to the
8
desired volume (50 mL) with acetonitrile. Pipetted 0.25, 0.5, 0.75, 1 or 1.25 mL of
9
OSA solution into 25-mL beakers, followed by 10 mL 1 M NaOH ethanol solution
10
(30%), respectively. Samples were covered with plastic wrap and magnetically stirred
11
at 400 r/min for 10 h. Then, 3 mL 4 M HCl ethanol solution (30%) was pipetted into
12
beakers and the mixture was transferred into 25-mL volumetric flasks after cooling to
13
room temperature and filled with acetonitrile to a total of 25 mL.
14
2.3.4. Determination of free and bound OSA
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To determine the free OS content of KGOS, 0.3000 g KGOS (dry weight) was
16
added to a 50-mL centrifuge tube and extracted with 5 mL of methanol with magnetic
17
stirring for 1 h. The mixture was centrifuged, and 1 mL of supernatant was collected
18
into a beaker and mixed with 10 mL 1 M NaOH ethanol solution (30%) and magnetic
19
stirring at 400 r/min for 10 h. Next, 3 mL 4 M HCl ethanol solution (30%) was
20
pipetted into a centrifuge tube and cooled to room temperature; residues were
21
removed using a 0.45-µm membrane filter, and 6 mL was pipetted into a 25-mL
22
volumetric flask and filled with acetonitrile to a total of 25 mL. The OS content (Wfree,
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g) was calculated using the total peak area (Afree) and the standard curve. For total OS content determination, KGOS (0.3000 g dry weight) was weighed
3
into a 50-mL centrifuge tube, 10 mL 1 M NaOH ethanol solution (30%) was added,
4
and the solution was magnetically stirred at 400 r/min for 10 h. Next, 3 mL 4 M HCl
5
ethanol solution (30%) was pipetted into a centrifuge tube and cooled to room
6
temperature. Residues were removed with a 0.45-µm membrane filter, and 6 mL was
7
pipetted into a 25-mL volumetric flask and filled with acetonitrile to a total of 25 mL.
8
The OS in the solution was analyzed by HPLC to yield a total peak area (Atotal) and
9
corresponding weight (Wtotal, g) using a standard curve. The substitution rate (SR) was
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calculated using the following equation: SR =
− OS × 100% (1)
where is the dry weight of KGOS.
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2.4. Characterization of KGOS micelles
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Transmission electron microscopy (TEM, JEM 1200EX, JEOL) was applied to
14
characterize the appearance of KGOS self-assembled micelles in aqueous solution
15
(Zhu, Liu, & Du, 2013). A sample (0.3 mg/mL KGOS solution, 3 µL) was dropped
16
onto the carbon-coated grid and dried under ambient conditions overnight. To stain
17
the micelles, phosphotungstic acid (PTA; 0.5 w/v %) solution (10 µL) was dropped
18
onto the micelles for 2 min. A filter paper was used to carefully blot the excess PTA
19
solution. The grids were dried again under ambient conditions overnight. Imaging was
20
performed on a JEOL JEM-2100 instrument (JEOL, Japan) at 200 kV equipped with a
21
Gatan 94 Ultrascan 1k charge-coupled device camera.
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ACCEPTED MANUSCRIPT Atomic force microscopy (AFM, Dimension Icon, Bruker, Germany) was used to
2
further characterize KGOS shapes. A drop of the KGOS solution (0.3 mg/mL) was
3
placed on freshly cleaved mica. After 5 min of incubation at room temperature, the
4
surface of the mica was gently rinsed with 5 mL of deionized water and blown with
5
dry nitrogen. The sample was air-dried at room temperature and mounted on a
6
microscope scanner. The shape was observed and imaged in the noncontact mode (Liu,
7
Chen, Tian, Ma, Li, & Zhao, 2014).
8
2.5. Thermodynamic properties of KGOS
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Thermal analyses (TG and DSC) were recorded on a TGA/DSC instrument
10
(1600HT METTLER, Switzerland), and approximately 10-mg samples were heated in
11
an alumina pan with a 10 K/min heating rate from 30 °C to 800 °C under N2 flow (50
12
mL/min). An empty pan sealed in the same manner was used as a reference.
13
2.6. Rheological measurements
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Steady shear and dynamic frequency sweeps measurements were performed using
15
a Carri-Med CSL2 100 rheometer (TA Instruments, New Castle, USA) using a
16
measurement system with plate-plate geometry (40 mm diameter, 500 µm gap).
17
Steady shear measurements were obtained at 25 °C. The flow curves were obtained by
18
registering shear stress at a shear rate that was increased from 0.01 to 500 s-1.
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Dynamic frequency sweeps measurements were performed under various
20
conditions according to the references (Lin, Wu, Luo, Liu, Luo, & He, 2010; Penroj,
21
Mitchell, Hill, & Ganjanagunchorn, 2005) with minor modifications: A constant
22
strain (5%) determined based on strain sweep experiments at a frequency of 0.1, 1 and
9
ACCEPTED MANUSCRIPT 10 Hz, which fell within the linear viscoelastic regions of all samples.
2
Small-amplitude oscillatory tests were performed at 25 °C over a frequency range of
3
0.1-10 Hz. The parameters obtained from the dynamic test data were storage modulus
4
G', loss modulus G", loss factor tanδ (G"/G'). These were determined as the means of
5
values that were within the linear viscoelastic range of each sample.
6
2.6.1. Rheological properties for various concentrations of KGOS
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Powders of KGOS and KGM were dispersed in double distilled/deionized water
8
with stirring. The solutions were heated to 60°C and maintained at this temperature
9
for 1 h and cooled to room temperature. The concentrations of both solutions were 1%,
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1.25%, 1.5%, 1.75% and 2% (w/w).
11
2.6.2. Rheological properties of KGOS solutions with various ions One percent (w/w) of KGOS was prepared as in section 2.4, and 0.02 mol of
13
MgCl2, KCl, CaCl2 and FeCl3 were added to 100 g KGOS solution to study the effect
14
of different cations on the rheological properties. Then, 0.02 mol of NaCl, Na2SO4,
15
NaNO3 and NaH2PO4 were added to 100 g KGOS solution to study the effects of
16
different anions on the rheological properties.
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2.7. Statistical analysis
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All tests were performed in triplicate. A one-way analysis of variance (ANOVA)
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and Tukey’s test were used to establish the significance of differences among the
20
mean values at the 0.05 level of confidence. The statistical analyses were performed
21
using SPSS version 20.0.
22
3. Results and discussion
10
ACCEPTED MANUSCRIPT 1
3.1. Measurement of reaction degree It is difficult to precisely determine the reaction extent of OSA-modified
3
polysaccharide using conventional methods such as titration; this is because these
4
macromolecular compounds usually formed aggregations in aqueous solution or
5
cannot dissolve in water. Tizzotti, Sweedman, Tang, Schaefer, & Gilbert (2011)
6
developed a 1H NMR method to measure the reaction degree of OSA-modified starch.
7
In our previous study, the 1H NMR experiment at 25°C indicated that the OSA groups
8
were grafted onto the KGM via the esterification reaction and an incomplete
9
deacetylation reaction in the modification (Meng, et al., 2014). However, the reaction
10
degree of KGOS could not be calculated by 1H NMR because the methyl proton peak
11
overlapped with some integral peaks and the solvent proton peak. To eliminate peak
12
interference, the NMR temperature was gradually increased. As shown in Fig. 1, the
13
integral peaks (peaks 2 and 3) still overlapped when the temperature increased to
14
85°C, indicating that temperature-dependent 1H NMR could not be used to calculate
15
the extent of reaction of KGOS.
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It was also difficult to use Fourier transform infrared spectroscopy (FTIR) to
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detect DS because FTIR has only been found to be reliable for high DS (≥0.3)
18
(Sweedman, Tizzotti, Schäfer, & Gilbert, 2013). Elemental analysis reported by Liu,
19
Li, Ma, Chen, & Zhao (2013) could also not be used to determine the DS of KGOS
20
for its complex chains structure. Although convenient titration methods are widely
21
used to find the DS of OSA, it is also not suitable for KGOS because an incomplete
22
deacetylation reaction occurred during KGOS synthesis, resulting in inaccurate
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titration due to the presence of acetyl groups. Therefore, we tried to estimate the extent of the modification reaction by
3
calculating the substitution rate (equation 1). At the same time, we chose an ethanol
4
solution (30%) as a dispersant of KGOS to prevent an excessive increase in solution
5
viscosity caused by KGOS swelling. As shown in Fig. 2, there were three peaks
6
(identified as 1-OS acid, cis-2-OS acid and trans-2-OS acid, respectively) after OSA
7
hydrolysis, giving the same result as Qiu et al. (2012). The values increased or
8
decreased in the same proportion, so we chose the area of the highest peak (peak 3) to
9
calculate the OS content. Standard curves for OS had the equation: Y = 4 × 10 X −
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0.005, R2=0.9996, and the SR of KGOS was 2.523±0.022%.
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3.2. Characterization of KGOS micelles
The TEM observations shown in Fig. 3A indicated that hydrophobically modified
13
KGOS aggregation particles formed irregular spheres or ellipsoids, and the average
14
particle diameter of KGOS was approximately 30 nm. The amplified image (Fig. 3B)
15
showed that the particles were distributed individually and appeared near the bulb,
16
with radial branches spread from the surface. As shown in Fig. 3C, AFM showed that
17
the height for KGOS aggregate was approximately 5 nm, and a continuous hill-like
18
substance was present on the mica surface. This phenomenon could further explain
19
the presence of a long carbon chain spread out from the aggregate, which was caused
20
by the long polymer chains and explained the multi-branch structure of KGOS. This
21
appearance is different from the octenylsuccinate oat β-glucan (lower molecular
22
weight of 16.8 × 104 - 7.3 × 104) reported by Liu et al. (2013) The aggregation
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ACCEPTED MANUSCRIPT particles of octenylsuccinate oat β-glucan presented regular spheres, and the surface
2
clearly separated from the aqueous solution. This difference may contribute to the
3
winding of long polymer chains and branches of KGOS.
4
3.3. Thermodynamic properties of KGOS
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In Fig. 4, both KGM and KGOS have a smaller drop in mass at approximately
6
100°C due to evaporation of water, with KGM falling by 4.95% and KGOS
7
decreasing by 5.83%. In addition, there was a large decrease in mass at approximately
8
300°C, which can be ascribed to the carbonization of the sample, KGM and KGOS
9
decreased 77.61% and 75.17%, respectively. After 300°C, the sample tends to be
10
stable. However, the DSC peak shapes of KGOS and KGM did not change
11
significantly, and there was an endothermic peak for water evaporation and no
12
endothermic peak for the melt. In combination with the results of Dave, Sheth,
13
Mccarthy, Ratto, & Kaplan (1998), it is possible that there are many hydrogen bonds
14
in the KGM and KGOS molecules, resulting in a strong interaction between KGM
15
and KGOS molecular chains, which leading to the melting temperatures were much
16
higher than their decomposition temperatures. However, the TG curve of KGM and
17
KGOS was found to be lower (10 - 15°C) than that of KGM at the second weight loss,
18
indicating that the carbonization temperature of KGOS is slightly lower than KGM.
19
3.4. Rheological properties at various concentrations of KGOS
20
3.4.1. Steady shear rheology
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As shown in Fig. 5, at lower shear rates, the viscosity of KGOS and KGM
22
solutions was only slightly affected, the viscosity in this region is considered zero
13
ACCEPTED MANUSCRIPT shear viscosity, and it depends on the polymer concentration (Fig. 5). When the shear
2
rate increased to a certain threshold γ# c (intersection of the vertical line and abscissa),
3
the shear viscosity of the solution substantially declined with the increasing of shear
4
rate, which is known as shear thinning. This phenomenon may due to the destruction
5
of the internal structure of KGOS solution. Larger aggregates could more easily
6
rearrange in the shear direction (Chronakis & Alexandridis, 2001). With increasing
7
concentration of KGM and KGOS, γ# c decreased (Fig. 5). The reciprocal of γ# c was
8
considered to be the relaxation time of the network structure of the solution
9
(Chronakis & Alexandridis, 2001). The longer relaxation time indicated that the
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strength of polymer aggregates increased and the de-aggregation time was extended. Further analysis indicated that the steady shear rheology results of KGM and
12
KGOS could be fit to the Carreau model, and the nonlinear regression and resulting
13
coefficients are given in Table 1. The Carreau model is as follows (Koszkul &
14
Nabialek, 2004):
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η = η% + (η' − η% )[1 + (λγ# )* ](, -)/* (2)
where η% is the viscosity at infinite shear rate; η' is the viscosity at zero shear rate;
16
γ# is the shear rate; n is flow behavior index (dimensionless); λ is a time constant.
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The zero shear viscosity η' of KGM and KGOS increased with the increase in
18
concentration, and the n value decreased, which indicated an increase in the
19
pseudoplasticity (n<1) of these solutions (Sudhakar, Singhal, & Kulkarni, 1996). A
20
possible reason was that molecules of KGOS and KGM are irregularly bound together,
21
which leads to strengthened interactions between chains. Thus, the viscosity and
14
ACCEPTED MANUSCRIPT pseudoplastic characteristics of the solutions increased. However, at the same
2
concentrations, the zero shear viscosity of KGOS is less than KGM. The possible
3
reason was that a small amount of molecular chains of KGOS were degraded during
4
preparation using a microwave method. The introduction of modified anhydride
5
groups likely increased the curl degree of the intramolecular chains, resulting in the
6
reduction of intermolecular crosslinking of KGOS, compared to that of KGM.
7
3.4.2. Dynamic shear properties
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The mechanical spectra for various concentrations of KGM and KGOS at 25°C
9
are shown in Fig. 6. The elastic modulus (G') and viscous modulus (G") of KGM and
10
KGOS at the same concentration have a similar trends. At low concentration (1% to
11
1.75%), with the frequency increase, the G" of KGM and KGOS were firstly higher
12
and then lower than G'. According to the report of Clark and Ross-murphy (1987),
13
this trend of the mechanical spectra met the characteristics of entanglement network
14
systems.
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With the frequency increase, the increase range of G', which represents the
16
recoverable energy when the material is subjected to deformation, was greater than for
17
G" (Ma & Barbosa-Cánovas, 1995), indicated that the material will exhibit solid
18
behavior. Piculell, Egermayer, & Sjöström (2003) proposed using the crossover
19
modulus at the intersection of G' and G" (Gc) to characterize the number of
20
cross-links and the inverse of the frequency at the intersection (τc) to characterize the
21
apparent relaxation time. As shown in Fig. 6, Gc and τc increased with increasing
22
concentration of KGM and KGOS, which indicated that the numbers of the
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2
crosslinking (as monitored by Gc) increased and the dynamics (as monitored by τc)
3
slowed down (Piculell et al. 2003).
4
3.5. Rheological properties of KGOS solutions with various ions
5
3.5.1. Steady shear rheology
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The effects of different ions on the steady shear are shown in Fig. 7. The addition
7
of various cations did not affect the fluid properties of the KGOS solution, and the
8
solution fit to non-Newtonian fluid properties and the Carreau model (Table 2). K+
9
had little effect on the viscosity of the solution. The viscosity increased and
10
pseudoplastic behavior reinforced with addition of Mg2+ and Fe3+, especially in the
11
presence of Fe3+. This could be because the increase in charge of the solution
12
promoted intermolecular winding more tightly and improved the cross-linked network
13
between molecules. The results showed that the type and valence of the cations may
14
affect the viscosity and pseudoplasticity of KGOS solution.
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A similar regularity could be observed with anion addition. Cl- and NO3- did not
16
affect the viscosity of the KGOS solution, but with the increasing of anionic valence,
17
the viscosity of the KGOS solution increased and pseudoplasticity enhanced. SO42-
18
and H2PO4- could significantly increase the viscosity of the solution. The possible
19
reason was that the ions have different effect to the hydrogen-bond structure between
20
water molecules, water molecules and polymer chains, which finally affect the
21
polymers solubility and its solution viscosity (Yin, Zhang, Huang, & Nishinari, 2008;
22
Briscoe, Luckham, & Zhu, 1996). Besides, the ligation ability of the ions to lone
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electron pairs of the hydroxyl group in KGOS structural units might also affect the
2
rheological properties of the solution (Lii, Tomasik, Hung & Lai, 2002).
3
3.5.2. Dynamic frequency sweeps From the results shown in Fig. 8A1, K+, Ca2+ and Mg2+ had a similar effect on the
5
dynamic modulus (G', G") of KGOS solution. At frequency of 0.1-0.38 Hz, G'
6
and the loss factor (Tanδ, G"/G') were greater than 1, which indicate the viscous
7
properties predominate in the samples, and the solutions showed more fluid-like
8
behavior; but with the frequency higher than 0.38 Hz, G'>G" and Tanδ was smaller
9
than 1, which indicate the elastic properties predominate in the samples, and the
10
solutions showed more solid-like behavior (Liu, Xu, & Guo, 2007; Chaisawang &
11
Suphantharika, 2006). However, for the addition of Fe3+, G' always higher than G",
12
and Tanδ was smaller than 1 (Fig 8A1 & A2), which indicate the elastic properties
13
predominate in the sample. Compared to other ions, there was a significant reduction
14
in Gc and increase in τc for Fe3+ (Fig 8 A2), which indicated an obviously reduction in
15
the degree of crosslinking and a slowing down in the dynamics (Piculell, et al., 2003).
16
As shown in Fig. 8B1 & B2, anions had a similar effect on the dynamic modulus (G',
17
G") of the KGOS solution. Tanδ for the KGOS solutions with Cl-, NO3- were greater
18
than 1 in the frequency range of 0.1-0.38 Hz, but for SO42- and H2PO4-, Tanδ was
19
greater than 1 at 0.1-0.30 Hz and 0.1-0.26 Hz, respectively (Fig. 8B2). The tanδ of
20
H2PO4- was lower than the other anions at low frequencies, the possible reason was
21
that there were HPO42- and PO43- in H2PO4- solution, which increased the charge of the
22
solution. What’s more, from the results of mechanical spectra, the KGOS solution
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contained the above ions also met the characteristics of entanglement systems.
2
4. Conclusion The HPLC method was developed to determine the reaction degree of KGOS.
4
Molecular agglomeration of KGOS was observed by TEM and AFM; TG and DSC
5
analysis indicated the thermal properties between KGM and KGOS changed slightly.
6
The study of rheological properties showed that KGOS solution fit to non-Newtonian
7
fluid properties and the Carreau model. The trends of the dynamic mechanical spectra
8
met the characteristics of entanglement systems. Viscosity and pseudoplasticity of
9
KGOS solution could be affected by the type and valence of the ions, especially for
10
the high valence cation.
11
Acknowledgments
The work was supported by the National Natural Science Foundation of China
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(31601427), the Chongqing science and technology program (CSTC 2013jcyjA00028)
14
and the Chengdu science and technology project (2015-NY02-00184-NC).
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Korkiatithaweechai, S., Umsarika, P., Praphairaksit, N., & Muangsin, N. (2011). Controlled release of diclofenac from matrix polymer of chitosan and oxidized konjac glucomannan. Marine Drugs, 9(9), 1649-1663.
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Liu, H., Xu, X. M., & Guo, S. D. (2007). Rheological, texture and sensory properties of low-fat mayonnaise with different fat mimetics. LWT-Food Science and Technology, 40(6), 946-954. Liu, J., Chen, F., Tian, W., Ma, Y., Li, J., & Zhao, G. (2014). Optimization and characterization of curcumin loaded in octenylsuccinate oat β-glucan micelles with an emphasis on degree of substitution and molecular weight. Journal of Agricultural and Food Chemistry, 62(30),
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ACCEPTED MANUSCRIPT Phillips, G. O., & Williams, P. A. (2009). Handbook of hydrocolloids. (2nd ed.). Florida: CRC Press. Piculell, L., Egermayer, M., & Sjöström, J. (2003). Rheology of mixed solutions of an associating polymer with a surfactant. Why are different surfactants different? Langmuir, 19(9), 3643-3649. Qiu, D., Bai, Y., & Shi, Y.-C. (2012). Identification of isomers and determination of octenylsuccinate in modified starch by HPLC and mass spectrometry. Food Chemistry, 135(2), 665-671. rheological aspects. Food chemistry, 55(3), 259-264.
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konjac (Amorphophallus konjac) glucomannan. Journal of the Science of Food and Tizzotti, M. J., Sweedman, M. C., Tang, D., Schaefer, C., & Gilbert, R. G. (2011). New 1H NMR procedure for the characterization of native and modified food-grade starches. Journal of
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ACCEPTED MANUSCRIPT Table 1
n
R2
1%
104.696±9.047e
0.302±0.007a
0.998
1.25%
132.223±10.099d
0.263±0.008b
0.998
1.5%
301.976±13.057c
0.252±0.006b
0.998
1.75%
487.556±16.767b
0.220±0.009c
0.999
2%
854.986±24.192a
0.161±0.005d
0.999
1%
56.2715±2.966d
0.371±0.011a
0.999
1.25%
112.392±5.138d
0.264±0.009b
0.998
1.5%
209.878±7.110c
0.267±0.011b
0.998
1.75%
424.946±12.155b
0.185±0.008c
0.999
2%
803.563±16.055a
0.086±0.004d
0.999
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η0/Pa.s
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KGOS
Concentration
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KGM
Fitting parameters of Carreau equation
Assays were performed in triplicate. Mean ± standard deviation values in the same column for each
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R2
Blank control
63.086±3.232cd
0.363±0.005a
0.998
KCl
59.093±3.715d
0.300±0.009c
0.999
CaCl2
59.223±5.022d
0.315±0.008c
0.998
FeCl3
107.176±4.225a
0.265±0.007d
MgCl2
73.029±1.717bc
0.269±0.006d
Na2SO4
78.426±3.276b
0.304±0.008c
NaNO3
63.315±3.105cd
0.340±0.009ab
NaH2PO4
67.709±2.035cd
0.323±0.012bc
0.999
NaCl
61.860±4.847d
0.307±0.007c
0.998
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ηo/Pa.s
0.999 0.999
0.995
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0.998
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Figure Captions Fig. 1. 1H NMR spectra of KGOS at different temperatures.
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Fig. 2. HPLC chromatograms of hydrolyzed OSA
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Fig. 4. TG/DSC curves of KGM (A) and KGOS (B)
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Fig. 3. TEM (A & B) and AFM (C) micrographs of KGOS self-aggregation
Fig. 5. Apparent viscosity-shear rate plots for various concentrations of KGM (A) and KGOS (B)
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Fig. 6. Dynamic frequency sweep of KGM (A) and KGOS (B) solutions at various concentrations
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Fig. 7. Effects of various cations (A) and anions (B) on the viscosity of a 1% KGOS solution at 25
Fig. 8. Dynamic frequency sweep of KGOS solution with various cations (A1 &A2) and anions (B1&B2) at 25 oC
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