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Physicochemical properties of degraded konjac glucomannan prepared by laser assisted with hydrogen peroxide
International Journal of Biological Macromolecules 129 (2019) 78–83
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Physicochemical properties of degraded konjac glucomannan prepared by laser assisted with hydrogen peroxide Wanmei Lin 1, Yongsheng Ni 1, Lin Wang, Dengyi Liu, Chunhua Wu ⁎, Jie Pang ⁎ College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
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Article history: Received 30 July 2018 Received in revised form 2 February 2019 Accepted 5 February 2019 Available online 6 February 2019 Keywords: Konjac glucomannan Laser degradation Antioxidant activity
a b s t r a c t The exploration of methods for degrading konjac glucomannan (KGM) is of great significant and technological interest. Here, laser at the power of 10 W was employed to degrade KGM. The laser degraded konjac glucomannan (LDK) was analyzed by viscosity, Rheology, Differential scanning calorimetry (DSC), Fourier transform infrared (FT-IR) spectroscopy and laser light scatter (LLS). The viscosity of the LDK decreased from 8.38 Pa·s to 2.26 Pa·s and the average molecular weight (Mw) decreased from 7.6 × 105 Da to 5.7 × 105 Da with a polydispersity index (PDI) of 1.166. FT-IR spectra and IR images of the LDK indicated that the breakage of glucosidic bonds occurred during laser irradiation. DSC results indicated that the thermal stability of KGM has improved slightly after degradation. In addition, the determination of 1,1-diphenyl-2-picrylhrazyl (DPPH) radical scavenging activity suggested that the antioxidant activity of the LDK improved versus KGM. This stratagem provides a new pathway for efficiently degrading KGM. © 2019 Published by Elsevier B.V.
1. Introduction Konjac glucomannan (KGM), a heteropolysaccharide produced from the tubers of Amorphophallus konjac C. Koch [1], has been prevalently used in food and medical fields owing to its unique physical and chemical properties [2,3]. It is composed of β-1,4-linked D-glucose and Dmannose with a molar ratio of 1:1.6 [4], acetylated randomly at the C6 position approximately per every 19 sugar unites [5]. Apart from food and medical applications, KGM has been widely used in pharmaceutical and biological industry owing to its biocompatible, biodegradable, renewable and non-toxic nature [6–9]. But KGM has inevitable deficiencies in applications due to their large molecular weight and viscosity. For instance, it is comparatively difficult for products based on KGM to be transported during processing. In order to expand the applications of KGM, natural or synthetic materials such as sodium carboxymethylcellulose [10], zein and whey protein isolate were mixed with KGM to form the blend films [11,12]. Modifications on KGM such as carboxymethylation [13], hydrophobic grafted by amphiphilic aliphatic amines and deacetylation also have been achieved [14–16]. Although those researches have promoted the applications of KGM to some extent, degrading KGM to obtain smaller molecule polysaccharide plays a significant role in its further versatile applications. Because the degraded KGM has some particular biological functions such as anti-
⁎ Corresponding authors. E-mail addresses: [email protected] (C. Wu), [email protected] (J. Pang). 1 These authors contributed equally to this work.
https://doi.org/10.1016/j.ijbiomac.2019.02.035 0141-8130/© 2019 Published by Elsevier B.V.
oxidation [17], cellular and probiotic bacteria protection [3,18]. Thus, it is of great significance to degrade KGM [19]. Undoubtedly, the past several years have seen a considerable progress in degrading KGM such as enzymatic hydrolysis [20], acid hydrolysis and physical hydrolysis [21,22]. However, enzymatic hydrolysis not only need a strict reaction condition but also is hard to remove the residual enzymes, which needs high cost [23]. Acid hydrolysis often takes a long time to obtain the degraded products, which will affect the output and threaten the environment causing permanent damages [6,24]. Further efforts have been made to combine chemical and physical hydrolysis to degrade KGM, but inevitable shortcomings still existed. Because they need a relatively long period to obtain the qualified degraded products. For instance, Pan et al. used γ-ray irradiation at 50 kGy dose assisted with hydrogen peroxide to degrade KGM, which needs at least two days [6]. Similarly, Jin et al. combined γ-ray irradiation at 6.0 kGy dose with ethanol to prepare the degraded KGM, which takes several hours [25]. Besides, alkaline-thermal degradation method was employed to hydrolysis KGM, which is still time-consuming [2]. Thus, questions remain concerning how to develop a simple, time-saving, cost-effective and environmental-friendly method to degrade KGM. Here, we report a new strategy to degrade KGM by using laser assisted with hydrogen peroxide. In this new strategy, hydrogen peroxide was added to KGM solutions as previous researches have illustrated its excellent synergetic effect. The process of preparing the laser degraded KGM (LDK) is shown in Fig. 1. Characterizations, measurements of molecular weight (Mw) and polydispersity index (PDI) were carried out to evaluate the physical and chemical properties of the LDK. Further evaluation was achieved by the antioxidant activity test.
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Fig. 1. Schematic illustration for the preparation of LDK via laser assisted with hydrogen peroxide.
2. Materials and methods
aluminum. The heating rate was 15 °C/min with a temperature range of 25 to 600 °C.
2.1. Materials Konjac glucomannan (KGM) (purity ≥95%) was purchased from Shaotong Sanai Konjac Development Co. Ltd. (Yunnan, China). Hydrogen peroxide was purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). All chemicals are analytical grade, commercially available and are used without any further purification. 2.2. Preparation of the LDK 3 g of KGM powder was dissolved in 300 mL of distilled water, then stirred at the rate of 500 r/min for 7 h (25 °C) to obtain a 1% KGM sol. Then, all samples were divided into two groups, one group was subject to laser irradiation (JNL-1, Janus New-Materials Co., Ltd. China) at the power of 10 W for different times (0–6 min), which were coded as 0 KGM, 1 KGM, 2 KGM, 3 KGM, 4 KGM, 5 KGM and 6 KGM, respectively. Another group pretreated with 2% (V/V) hydrogen peroxide was also subjected to the same laser irradiation at the power of 10 W for different times (0–6 min), which were coded as 0+ KGM, 1+ KGM, 2+ KGM, 3+ KGM, 4+ KGM, 5+ KGM and 6+ KGM, respectively. All samples undergone dialysis treatment before vacuum drying. 2.3. The determinations of optimum irradiation time 2.3.1. Viscosity measurements After laser degradation, NDJ-55 digital viscometer was applied to measure the viscosities of all samples. Measurements were carried out at 25 °C. 2.3.2. Steady-shear measurements All samples were centrifuged at the rate of 5000 r/min for 10 min to remove bubbles. Then, using a rotational rheometer (MCR301, Anton Parr, Austria) with a 50-mm parallel plate (PP50) to collect viscosity data. The shear rate ranges from 0.1 to 150 s−1. Measurements were carried out at 25 °C. 2.4. Physicochemical properties of LDK prepared at optimum irradiation time 2.4.1. Dynamic shear analysis Dynamic oscillatory measurements were carried out by a rotational rheometer (MCR301, Anton Parr, Austria) at a frequency sweep from 0.1 to 100 Hz. G′ (storage modulus) and G″ (loss modulus) were recorded.
2.4.3. FT-IR analysis KGM and LDK were mixed with potassium bromide to make a pellet. The FT-IR spectra of KGM and LDK were recorded on a Nicolet 5700 FTIR spectrometer (Madison, USA) over a wavelength range of 4000 to 400 cm−1. 2.4.4. Infrared imaging (IR imaging) IR images of KGM and LDK were obtained using a Thermo Scientific Nicolet iN10 infrared microscope equipped with a liquid nitrogencooled MCT detector (Thermo Electron Corporation, USA). 2.5. Evaluations of LDK 2.5.1. Determinations of Mw and PDI The freeze-dried samples were dissolved in the mobile phase (NaCl) with a concentration of 0.2 mol/L and the flow velocity was 1 mL/min. The Mw and PDI were determined by laser light scatter (LLS) using a multi-angle laser photometer (DAWN-DSP, Wyatt Technology Co., USA) equipped with a He\\Ne laser (λ = 664.1 nm) in an angle range of 13° to 157.7° at 25 °C. The refractive index increment (dn/dc) measured by a double-beam-differential refractometer (DRM-1020, Otsuka Electronic Co., Japan) was 0.135 mg/mL. All samples were filtered by a sand core filter and then filtered by a 0.2 μm filter before injected. Data acquisition and analysis were achieved by using Astra software. 2.5.2. Evaluation of antioxidant activity The 1,1-diphenyl-2-picrylhrazyl (DPPH) radical scavenging assay was performed to evaluate the antioxidant activity according to the modified method of Sun et al. [26]. In brief, 9.0 mL of 70% ethanol solution of DPPH (0.1 mM) was incubated with 3.0 mL of test sample (Ai). Then 9.0 mL of 70% ethanol was incubated with 3 mL of test sample (Aj) and 9 mL of 70% ethanol solution of DPPH was incubated with 3 mL of distilled water (A0). The three reaction solutions were shaken well and incubated at 30 °C for 30 min in the dark and the optical density was measured at 517 nm. The DPPH scavenging activity was calculated according to the equation: DPPH scavenging activity ð%Þ ¼
1−
Ai −A j A0
100
ð1Þ
2.6. Statistical analysis 2.4.2. Differential scanning calorimetry (DSC) analysis DSC (200F3, Netzsch, Selb, Germany) was used to analyze the thermal stability of the samples. Samples of 3 ± 0.2 mg in a crucible were analyzed in nitrogen atmosphere with a reference sample of
All experiments were carried out in triplicate and the results were reported as mean ± SD. SPSS version 22.0 and OriginPro 9.0 were used to conduct the data analysis.
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Fig. 2. (a) Viscosities of the irradiated samples; (b) Viscosities of the irradiated samples with hydrogen peroxide pretreatment.
3. Results and discussion 3.1. Viscosity analysis One important index of macromolecular polysaccharide is viscosity. The viscosities of different samples are depicted in Fig. 2a and b. The viscosity of each sample decreased in various degrees after laser irradiation. It is noticeable that the viscosities of 2 KGM and 2+ KGM were the lowest among the corresponding group. This indicated that 2 min was the relatively feasible time for quick degradation of KGM. The viscosity loss of 2 KGM and 2+ KGM were 66.3% and 73% respectively compared with 0 KGM, which indicated that the addition of hydrogen peroxide could enhance the degradation. The result is similar to the research conducted by Hien et al. They reported that the chitosan was effectively degraded by γ-irradiation in the presence of hydrogen peroxide [24]. Similarly, Duy et al. reported the synergistic effect of γirradiation and hydrogen peroxide on the preparation of oligochitosan [27]. The common mechanism is the breakage of glycosidic bonds by rearrangement. In our research, the presence of hydrogen peroxide can enhance the formation of highly oxidative hydroxyl radicals that could react with KGM by abstracting carbon-bound hydrogen atoms. The resulting carbohydrate radicals could cause direct breakage of the glucosidic bonds by rearrangement [28]. The breakage of glycosidic bonds was illustrated by the following FT-IR spectra and IR images. Besides, the viscosities of the degraded products irradiated N2 min showed an upward trend as the irradiation time extended. During the laser irradiation, there are two main factors that influence the viscosity of the
samples. One is the degradation effect and the other is the increased concentration of solution system caused by water evaporation during laser heating. The degradation effect accounted for the decreased viscosity. The increased concentration of sample will lead to the reduction of solution mobility, which accounted for the increased viscosity. Similar explanations can be found on the research done by Vijayalakshm et al. [29]. They reported the degradation of polyethylene oxide (PEO) and polyacrylamide (PAM) via pulsed laser. And the degradation of polymers is higher in a less concentrated solution than a more concentrated solution because the mobility of polymer chains is higher in a less viscous solution. Thus, we infer that the decreased viscosities of samples irradiated for 1 and 2 min is because of the predomination of the degradation in the solutions. While water evaporation became the predomination when the irradiation time extended to N2 min. And the hydrogen peroxide also volatilized, weakening the degradation effect. In addition, the viscosities of those samples with hydrogen peroxide pretreatment are always lower than those samples irradiated by laser alone for the same degradation time. Overall, the addition of hydrogen peroxide can enhance the degradation effect and 2 min may be the best degradation time. 3.2. Steady-shear behaviors The viscosities of all samples were submitted to steady-shear flow behavior at a shear rate range of 0.1 to 150 s−1 (Fig. 3a and b). The flow curves clearly exhibit that all samples are belonged to the nonNewtonian fluids with pseudo plastic features, which is consistent
Fig. 3. (a) Steady-shear viscosities of the irradiated samples; (b) Steady-shear viscosities of the irradiated samples with hydrogen peroxide pretreatment.
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Fig. 4. (a) Storage modulus (G′) and loss modulus (G″) as functions of angular frequency for 0 KGM, 0+ KGM, 2 KGM, 2+ KGM; (b) DSC of 0 KGM, 0+ KGM, 2 KGM, 2+ KGM.
with previous researches [30–32]. In short, all samples appeared a constant viscosity at low shear rate because the disturbance of interactions between molecules can be compensated by the new formed chain entanglements. Interestingly, the samples that harbor hydrogen peroxide keep a constant viscosity for a longer time at low shear rate. This may result from the formation of relatively solid entanglements. However, at higher shear rate, the disruption of entanglements became the predomination. Besides, the molecules were arranged in the flow direction, which leads to the reduction of apparent viscosity. There is no doubt that 2+ KGM and 2 KGM have the lowest viscosity among all samples, which is consistent with the aforementioned viscosity analysis. Therefore, 2+ KGM and 2 KGM were further evaluated in the following parts.
3.3. Dynamic shear behaviors It is of great significance to evaluate the elastic-viscosity properties of the LDK (Fig. 4a). G′ and G″ represent storage modulus and loss modulus respectively. They are dynamic rheological parameters used in characterization. As the frequency increase, both G′ and G″ of all samples increased, which is a typical feature of polysaccharide solution and the growth rate of G′ is always higher than that of G″. At low frequency, G′ was smaller than G″ but after reaching the crossover the G′ was larger than G″. This indicated that the polymer solutions had a liquid-like behavior before the crossover. But they start to entangle between polymer chains after the crossover. The high frequency
corresponding to the crossover point suggests a weaker interaction at the level of macromolecules [25]. It is noticeable that the crossover of the 2+ KGM shifted to the highest frequency. This indicated the weaker entanglement formation in this sample. 3.4. DSC analysis The thermal properties have been investigated. As shown in Fig. 4b, it is obvious that there are endothermic peaks at around 332.62 °C, 331.14 °C, 331.14 °C and 338.04 °C for 0 KGM, 0+ KGM, 2 KGM and 2 + KGM, respectively. The endothermic peak of 2+ KGM is 5.42 °C higher than 0 KGM. This indicated that the thermal stability of KGM improved after degradation. However, the reason why the gaps of the highest thermo-stability temperature between these samples were narrow was the formation of narrow molecular weights. Furthermore, the endothermic peaks of 0+ KGM and 2 KGM are lower than 0 KGM. This may due to the poor efficacy of degradation conducted by laser or hydrogen peroxide only. 3.5. FT-IR analysis FT-IR characterization was conducted for further investigation of degradation. Fig. 5a depict the FT-IR spectra of molecular vibrations of samples. FT-IR is an effective means to perform the molecular structural analysis by detecting the relative vibration and molecular rotation
Fig. 5. (a) FT-IR spectra of 0 KGM, 0+ KGM, 2 KGM, 2+ KGM; (b) IR spectra and IR images illustrate the distribution of the glycosidic bonds before and after laser irradiation.
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between atoms [33]. The intensity of the absorption peaks varies differently depending on the degradation treatment. Basically, there were no new chemical groups introduced to the KGM molecular chain after degradation. The main bands of KGM can be observed at around 4000–800 cm−1. The peaks at 3430 cm−1 were assigned to the stretching vibrations of O\\H groups and peaks at 2925 cm−1 were assigned to the stretching vibrations of C\\H groups [34,35]. The peaks at about 1728 cm−1 were assigned to the stretching vibrations of C_O groups [36]. The stretching peaks at 804 cm−1 were characteristic absorption bands of mannose [37]. Interestingly, the peak intensity at 891 cm−1 in the spectrum of 2+ KGM decrease slightly, revealing the decomposition of glycosidic bonds during laser degradation. Similar results can be found in the research done by Pan et al. [6]. This was due to the reaction between hydroxyl radicals and KGM. Hydroxyl radicals could react with KGM by abstracting carbon-bound hydrogen atoms. The resulting carbohydrate radicals could cause direct breakage of the glucosidic bonds by rearrangement [28]. In addition, the results of IR images were consentaneous with FT-IR analysis. IR images exhibit chemical changes of the KGM before and after laser degradation. We directly illustrated the distribution of glycosidic bonds at 891 cm −1 by color variation (the red color signifies high intensity while blue color represents low intensity) [38]. As shown in Fig. 5b, the red color revealed that there were abundant glycosidic bonds in KGM. However, a relatively homogeneous intensity was observed in 2+ KGM (blue color), which suggested the decreased number of glycosidic bonds. The mechanism of the degradation is based on some reactions. The primary reactions in the solutions are the formation of hydroxyl radicals ( OH) through the radiolysis of water and hydrogen peroxide as follows [39]: H 2 O2 þ ðhvÞ→H 2 ; H 2 O2 ; e− aq ; H ; H 2 O þ ðhvÞ → 2 OH
OH; H 3 Oþ
ð2Þ
Fig. 7. DPPH radical scavenging activity of 0 KGM and 2+ KGM.
3.6. Mw and PDI analysis In order to quantize the degradation effect, Mw and PDI were further investigated. Fig. 6a and b gives the information about the Mw and PDI of 0 KGM and 2+ KGM. The Mw of 2+ KGM (5.7 × 105 Da) was much lower than 0 KGM (7.6 × 105 Da). The decreased Mw of 2+ KGM indicated that the KGM has been degraded after laser irradiation. Besides, the PDI of 2+ KGM decreased from 1.468 to 1.166, which signifies the irradiation products distributed more evenly. This was due to the exact scission on glucosidic bonds during degradation.
ð3Þ
Furthermore, during irradiation, e−aq and H• can react with H2O2 as follows: e− aq þ H 2 O2 → OH þ OH −
ð4Þ
H
ð5Þ
The addition of hydrogen peroxide increased the concentration of •OH during laser irradiation. As a powerful oxidative agent, •OH could react with KGM by abstracting carbon-bound hydrogen atoms. The resulting carbohydrate radicals could cause direct breakage of the glucosidic bonds by rearrangement [28].
3.7. Antioxidant property analysis Finally, the antioxidant activity of the 2+ KGM was investigated. The antioxidant activity was gauged through DPPH radical scavenging ability (Fig. 7). DPPH is a stable lipophilic free radical which is often applied in estimating antioxidant activity of food and medicine materials [40]. The 2+ KGM displayed free radical scavenging ability of 60.2% compared with 0 KGM of 39.5%. It is commonly acceptable that the antioxidant has free radical scavenging ability is due to their hydrogen-donating ability [17]. Therefore, we infer that laser degradation can improve the antioxidant activity of KGM by promoting their hydrogen-donating ability, which is consistent with the conclusion of Jian's group [17].
Fig. 6. (a) Mw and (b) PDI of 0 KGM and 2+ KGM.
W. Lin et al. / International Journal of Biological Macromolecules 129 (2019) 78–83
4. Conclusion In summary, the degraded KGM can be obtained by laser irradiation assisted with hydrogen peroxide. The Mw decreased from 7.6 × 105 Da to 5.7 × 105 Da. And the PDI nearly approached to 1.0 revealing that the degraded products dispersed more evenly. The existence of hydrogen peroxide can increase the number of hydroxyl radicals, which could induce the breakage of glucosidic bonds in KGM molecular chains. This can be confirmed by the FT-IR spectra and IR images of the LDK. More importantly, the evaluation of DPPH radical scavenging activity demonstrated that the antioxidant activity has been improved in the LDK. Overall, this strategy can provide a simple, time-saving, cost-effective method for degrading KGM in an environmental-friendly way and expand the applications of KGM. Acknowledgement This work was financially supported by the National Natural Science Foundation of China (grant numbers 31772045, 31471704). The authors thank Yingning Yao for valuable discussion on preparation of the experiment. References [1] K. Nishinari, P.A. Williams, G.O. Phillips, Review of the physico-chemical characteristics and properties of konjac mannan, Food Hydrocoll. 6 (1992) 199–222. [2] C. Wu, Y. Li, Y. Du, L. Wang, C. Tong, Y. Hu, J. Pang, Z. Yan, Preparation and characterization of konjac glucomannan-based bionanocomposite film for active food packaging, Food Hydrocoll. 89 (2019) 682–690. [3] Y.H. Mao, A.X. Song, Z.P. Yao, J.Y. Wu, Protective effects of natural and partially degraded konjac glucomannan on Bifidobacteria against antibiotic damage, Carbohydr. Polym. 181 (2018) 368–375. [4] M. Maeda, H. Shimahara, N. Sugiyama, Detailed examination of the branched structure of konjac glucomannan, J. Agric. Chem. Soc. Jpn. 44 (1980) 245–252. [5] K. Katsurayaa, K. Okuyamab, K. Hatanakab, R. Oshimab, T. Satoc, K. Matsuzakic, Constitution of konjac glucomannan: chemical analysis and 13C NMR spectroscopy, Carbohydr. Polym. 53 (2003) 183–189. [6] T. Pan, S. Peng, Z. Xu, B. Xiong, C. Wen, M. Yao, J. Pang, Synergetic degradation of konjac glucomannan by γ-ray irradiation and hydrogen peroxide, Carbohydr. Polym. 93 (2013) 761–767. [7] J. Chen, C. Liu, Y. Chen, Y. Chen, P.R. Chang, Structural characterization and properties of starch/konjac glucomannan blend films, Carbohydr. Polym. 74 (2008) 946–952. [8] G. Conzatti, D. Faucon, M. Castel, F. Ayadi, S. Cavalie, A. Tourrette, Alginate/chitosan polyelectrolyte complexes: a comparative study of the influence of the drying step on physicochemical properties, Carbohydr. Polym. 172 (2017) 142–151. [9] Y. Hu, L. Liu, Z. Gu, W. Dan, N. Dan, X. Yu, Modification of collagen with a natural derived cross-linker, alginate dialdehyde, Carbohydr. Polym. 102 (2014) 324–332. [10] C. Xiao, Y. Lu, H. Liu, L. Zhang, Preparation and characterization of konjac glucomannan and sodium carboxymethylcellulose blend films, J. Appl. Polym. Sci. 80 (2015) 26–31. [11] K. Wang, K. Wu, M. Xiao, Y. Kuang, H. Corke, X. Ni, F. Jiang, Structural characterization and properties of konjac glucomannan and zein blend films, Int. J. Biol. Macromol. 105 (2017) 1096–1104. [12] M. Leuangsukrerk, T. Phupoksakul, K. Tananuwong, C. Borompichaichartkul, T. Janjarasskul, Properties of konjac glucomannan–whey protein isolate blend films, LWT Food Sci. Technol. 59 (2014) 94–100. [13] L. Wang, M. Xiao, S. Dai, J. Song, X. Ni, Y. Fang, H. Corke, F. Jiang, Interactions between carboxymethyl konjac glucomannan and soy protein isolate in blended films, Carbohydr. Polym. 101 (2014) 136–145. [14] J. Luan, K. Wu, C. Li, J. Liu, X. Ni, M. Xiao, Y. Xu, Y. Kuang, F. Jiang, pH-sensitive drug delivery system based on hydrophobic modified konjac glucomannan, Carbohydr. Polym. 171 (2017) 9–17.
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Physicochemical properties of degraded konjac glucomannan prepared by laser assisted with hydrogen peroxide Wanmei Lin 1, Yongsheng Ni 1, Lin Wang, Dengyi Liu, Chunhua Wu ⁎, Jie Pang ⁎ College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
a r t i c l e
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Article history: Received 30 July 2018 Received in revised form 2 February 2019 Accepted 5 February 2019 Available online 6 February 2019 Keywords: Konjac glucomannan Laser degradation Antioxidant activity
a b s t r a c t The exploration of methods for degrading konjac glucomannan (KGM) is of great significant and technological interest. Here, laser at the power of 10 W was employed to degrade KGM. The laser degraded konjac glucomannan (LDK) was analyzed by viscosity, Rheology, Differential scanning calorimetry (DSC), Fourier transform infrared (FT-IR) spectroscopy and laser light scatter (LLS). The viscosity of the LDK decreased from 8.38 Pa·s to 2.26 Pa·s and the average molecular weight (Mw) decreased from 7.6 × 105 Da to 5.7 × 105 Da with a polydispersity index (PDI) of 1.166. FT-IR spectra and IR images of the LDK indicated that the breakage of glucosidic bonds occurred during laser irradiation. DSC results indicated that the thermal stability of KGM has improved slightly after degradation. In addition, the determination of 1,1-diphenyl-2-picrylhrazyl (DPPH) radical scavenging activity suggested that the antioxidant activity of the LDK improved versus KGM. This stratagem provides a new pathway for efficiently degrading KGM. © 2019 Published by Elsevier B.V.
1. Introduction Konjac glucomannan (KGM), a heteropolysaccharide produced from the tubers of Amorphophallus konjac C. Koch [1], has been prevalently used in food and medical fields owing to its unique physical and chemical properties [2,3]. It is composed of β-1,4-linked D-glucose and Dmannose with a molar ratio of 1:1.6 [4], acetylated randomly at the C6 position approximately per every 19 sugar unites [5]. Apart from food and medical applications, KGM has been widely used in pharmaceutical and biological industry owing to its biocompatible, biodegradable, renewable and non-toxic nature [6–9]. But KGM has inevitable deficiencies in applications due to their large molecular weight and viscosity. For instance, it is comparatively difficult for products based on KGM to be transported during processing. In order to expand the applications of KGM, natural or synthetic materials such as sodium carboxymethylcellulose [10], zein and whey protein isolate were mixed with KGM to form the blend films [11,12]. Modifications on KGM such as carboxymethylation [13], hydrophobic grafted by amphiphilic aliphatic amines and deacetylation also have been achieved [14–16]. Although those researches have promoted the applications of KGM to some extent, degrading KGM to obtain smaller molecule polysaccharide plays a significant role in its further versatile applications. Because the degraded KGM has some particular biological functions such as anti-
⁎ Corresponding authors. E-mail addresses: [email protected] (C. Wu), [email protected] (J. Pang). 1 These authors contributed equally to this work.
https://doi.org/10.1016/j.ijbiomac.2019.02.035 0141-8130/© 2019 Published by Elsevier B.V.
oxidation [17], cellular and probiotic bacteria protection [3,18]. Thus, it is of great significance to degrade KGM [19]. Undoubtedly, the past several years have seen a considerable progress in degrading KGM such as enzymatic hydrolysis [20], acid hydrolysis and physical hydrolysis [21,22]. However, enzymatic hydrolysis not only need a strict reaction condition but also is hard to remove the residual enzymes, which needs high cost [23]. Acid hydrolysis often takes a long time to obtain the degraded products, which will affect the output and threaten the environment causing permanent damages [6,24]. Further efforts have been made to combine chemical and physical hydrolysis to degrade KGM, but inevitable shortcomings still existed. Because they need a relatively long period to obtain the qualified degraded products. For instance, Pan et al. used γ-ray irradiation at 50 kGy dose assisted with hydrogen peroxide to degrade KGM, which needs at least two days [6]. Similarly, Jin et al. combined γ-ray irradiation at 6.0 kGy dose with ethanol to prepare the degraded KGM, which takes several hours [25]. Besides, alkaline-thermal degradation method was employed to hydrolysis KGM, which is still time-consuming [2]. Thus, questions remain concerning how to develop a simple, time-saving, cost-effective and environmental-friendly method to degrade KGM. Here, we report a new strategy to degrade KGM by using laser assisted with hydrogen peroxide. In this new strategy, hydrogen peroxide was added to KGM solutions as previous researches have illustrated its excellent synergetic effect. The process of preparing the laser degraded KGM (LDK) is shown in Fig. 1. Characterizations, measurements of molecular weight (Mw) and polydispersity index (PDI) were carried out to evaluate the physical and chemical properties of the LDK. Further evaluation was achieved by the antioxidant activity test.
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Fig. 1. Schematic illustration for the preparation of LDK via laser assisted with hydrogen peroxide.
2. Materials and methods
aluminum. The heating rate was 15 °C/min with a temperature range of 25 to 600 °C.
2.1. Materials Konjac glucomannan (KGM) (purity ≥95%) was purchased from Shaotong Sanai Konjac Development Co. Ltd. (Yunnan, China). Hydrogen peroxide was purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). All chemicals are analytical grade, commercially available and are used without any further purification. 2.2. Preparation of the LDK 3 g of KGM powder was dissolved in 300 mL of distilled water, then stirred at the rate of 500 r/min for 7 h (25 °C) to obtain a 1% KGM sol. Then, all samples were divided into two groups, one group was subject to laser irradiation (JNL-1, Janus New-Materials Co., Ltd. China) at the power of 10 W for different times (0–6 min), which were coded as 0 KGM, 1 KGM, 2 KGM, 3 KGM, 4 KGM, 5 KGM and 6 KGM, respectively. Another group pretreated with 2% (V/V) hydrogen peroxide was also subjected to the same laser irradiation at the power of 10 W for different times (0–6 min), which were coded as 0+ KGM, 1+ KGM, 2+ KGM, 3+ KGM, 4+ KGM, 5+ KGM and 6+ KGM, respectively. All samples undergone dialysis treatment before vacuum drying. 2.3. The determinations of optimum irradiation time 2.3.1. Viscosity measurements After laser degradation, NDJ-55 digital viscometer was applied to measure the viscosities of all samples. Measurements were carried out at 25 °C. 2.3.2. Steady-shear measurements All samples were centrifuged at the rate of 5000 r/min for 10 min to remove bubbles. Then, using a rotational rheometer (MCR301, Anton Parr, Austria) with a 50-mm parallel plate (PP50) to collect viscosity data. The shear rate ranges from 0.1 to 150 s−1. Measurements were carried out at 25 °C. 2.4. Physicochemical properties of LDK prepared at optimum irradiation time 2.4.1. Dynamic shear analysis Dynamic oscillatory measurements were carried out by a rotational rheometer (MCR301, Anton Parr, Austria) at a frequency sweep from 0.1 to 100 Hz. G′ (storage modulus) and G″ (loss modulus) were recorded.
2.4.3. FT-IR analysis KGM and LDK were mixed with potassium bromide to make a pellet. The FT-IR spectra of KGM and LDK were recorded on a Nicolet 5700 FTIR spectrometer (Madison, USA) over a wavelength range of 4000 to 400 cm−1. 2.4.4. Infrared imaging (IR imaging) IR images of KGM and LDK were obtained using a Thermo Scientific Nicolet iN10 infrared microscope equipped with a liquid nitrogencooled MCT detector (Thermo Electron Corporation, USA). 2.5. Evaluations of LDK 2.5.1. Determinations of Mw and PDI The freeze-dried samples were dissolved in the mobile phase (NaCl) with a concentration of 0.2 mol/L and the flow velocity was 1 mL/min. The Mw and PDI were determined by laser light scatter (LLS) using a multi-angle laser photometer (DAWN-DSP, Wyatt Technology Co., USA) equipped with a He\\Ne laser (λ = 664.1 nm) in an angle range of 13° to 157.7° at 25 °C. The refractive index increment (dn/dc) measured by a double-beam-differential refractometer (DRM-1020, Otsuka Electronic Co., Japan) was 0.135 mg/mL. All samples were filtered by a sand core filter and then filtered by a 0.2 μm filter before injected. Data acquisition and analysis were achieved by using Astra software. 2.5.2. Evaluation of antioxidant activity The 1,1-diphenyl-2-picrylhrazyl (DPPH) radical scavenging assay was performed to evaluate the antioxidant activity according to the modified method of Sun et al. [26]. In brief, 9.0 mL of 70% ethanol solution of DPPH (0.1 mM) was incubated with 3.0 mL of test sample (Ai). Then 9.0 mL of 70% ethanol was incubated with 3 mL of test sample (Aj) and 9 mL of 70% ethanol solution of DPPH was incubated with 3 mL of distilled water (A0). The three reaction solutions were shaken well and incubated at 30 °C for 30 min in the dark and the optical density was measured at 517 nm. The DPPH scavenging activity was calculated according to the equation: DPPH scavenging activity ð%Þ ¼
1−
Ai −A j A0
100
ð1Þ
2.6. Statistical analysis 2.4.2. Differential scanning calorimetry (DSC) analysis DSC (200F3, Netzsch, Selb, Germany) was used to analyze the thermal stability of the samples. Samples of 3 ± 0.2 mg in a crucible were analyzed in nitrogen atmosphere with a reference sample of
All experiments were carried out in triplicate and the results were reported as mean ± SD. SPSS version 22.0 and OriginPro 9.0 were used to conduct the data analysis.
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Fig. 2. (a) Viscosities of the irradiated samples; (b) Viscosities of the irradiated samples with hydrogen peroxide pretreatment.
3. Results and discussion 3.1. Viscosity analysis One important index of macromolecular polysaccharide is viscosity. The viscosities of different samples are depicted in Fig. 2a and b. The viscosity of each sample decreased in various degrees after laser irradiation. It is noticeable that the viscosities of 2 KGM and 2+ KGM were the lowest among the corresponding group. This indicated that 2 min was the relatively feasible time for quick degradation of KGM. The viscosity loss of 2 KGM and 2+ KGM were 66.3% and 73% respectively compared with 0 KGM, which indicated that the addition of hydrogen peroxide could enhance the degradation. The result is similar to the research conducted by Hien et al. They reported that the chitosan was effectively degraded by γ-irradiation in the presence of hydrogen peroxide [24]. Similarly, Duy et al. reported the synergistic effect of γirradiation and hydrogen peroxide on the preparation of oligochitosan [27]. The common mechanism is the breakage of glycosidic bonds by rearrangement. In our research, the presence of hydrogen peroxide can enhance the formation of highly oxidative hydroxyl radicals that could react with KGM by abstracting carbon-bound hydrogen atoms. The resulting carbohydrate radicals could cause direct breakage of the glucosidic bonds by rearrangement [28]. The breakage of glycosidic bonds was illustrated by the following FT-IR spectra and IR images. Besides, the viscosities of the degraded products irradiated N2 min showed an upward trend as the irradiation time extended. During the laser irradiation, there are two main factors that influence the viscosity of the
samples. One is the degradation effect and the other is the increased concentration of solution system caused by water evaporation during laser heating. The degradation effect accounted for the decreased viscosity. The increased concentration of sample will lead to the reduction of solution mobility, which accounted for the increased viscosity. Similar explanations can be found on the research done by Vijayalakshm et al. [29]. They reported the degradation of polyethylene oxide (PEO) and polyacrylamide (PAM) via pulsed laser. And the degradation of polymers is higher in a less concentrated solution than a more concentrated solution because the mobility of polymer chains is higher in a less viscous solution. Thus, we infer that the decreased viscosities of samples irradiated for 1 and 2 min is because of the predomination of the degradation in the solutions. While water evaporation became the predomination when the irradiation time extended to N2 min. And the hydrogen peroxide also volatilized, weakening the degradation effect. In addition, the viscosities of those samples with hydrogen peroxide pretreatment are always lower than those samples irradiated by laser alone for the same degradation time. Overall, the addition of hydrogen peroxide can enhance the degradation effect and 2 min may be the best degradation time. 3.2. Steady-shear behaviors The viscosities of all samples were submitted to steady-shear flow behavior at a shear rate range of 0.1 to 150 s−1 (Fig. 3a and b). The flow curves clearly exhibit that all samples are belonged to the nonNewtonian fluids with pseudo plastic features, which is consistent
Fig. 3. (a) Steady-shear viscosities of the irradiated samples; (b) Steady-shear viscosities of the irradiated samples with hydrogen peroxide pretreatment.
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Fig. 4. (a) Storage modulus (G′) and loss modulus (G″) as functions of angular frequency for 0 KGM, 0+ KGM, 2 KGM, 2+ KGM; (b) DSC of 0 KGM, 0+ KGM, 2 KGM, 2+ KGM.
with previous researches [30–32]. In short, all samples appeared a constant viscosity at low shear rate because the disturbance of interactions between molecules can be compensated by the new formed chain entanglements. Interestingly, the samples that harbor hydrogen peroxide keep a constant viscosity for a longer time at low shear rate. This may result from the formation of relatively solid entanglements. However, at higher shear rate, the disruption of entanglements became the predomination. Besides, the molecules were arranged in the flow direction, which leads to the reduction of apparent viscosity. There is no doubt that 2+ KGM and 2 KGM have the lowest viscosity among all samples, which is consistent with the aforementioned viscosity analysis. Therefore, 2+ KGM and 2 KGM were further evaluated in the following parts.
3.3. Dynamic shear behaviors It is of great significance to evaluate the elastic-viscosity properties of the LDK (Fig. 4a). G′ and G″ represent storage modulus and loss modulus respectively. They are dynamic rheological parameters used in characterization. As the frequency increase, both G′ and G″ of all samples increased, which is a typical feature of polysaccharide solution and the growth rate of G′ is always higher than that of G″. At low frequency, G′ was smaller than G″ but after reaching the crossover the G′ was larger than G″. This indicated that the polymer solutions had a liquid-like behavior before the crossover. But they start to entangle between polymer chains after the crossover. The high frequency
corresponding to the crossover point suggests a weaker interaction at the level of macromolecules [25]. It is noticeable that the crossover of the 2+ KGM shifted to the highest frequency. This indicated the weaker entanglement formation in this sample. 3.4. DSC analysis The thermal properties have been investigated. As shown in Fig. 4b, it is obvious that there are endothermic peaks at around 332.62 °C, 331.14 °C, 331.14 °C and 338.04 °C for 0 KGM, 0+ KGM, 2 KGM and 2 + KGM, respectively. The endothermic peak of 2+ KGM is 5.42 °C higher than 0 KGM. This indicated that the thermal stability of KGM improved after degradation. However, the reason why the gaps of the highest thermo-stability temperature between these samples were narrow was the formation of narrow molecular weights. Furthermore, the endothermic peaks of 0+ KGM and 2 KGM are lower than 0 KGM. This may due to the poor efficacy of degradation conducted by laser or hydrogen peroxide only. 3.5. FT-IR analysis FT-IR characterization was conducted for further investigation of degradation. Fig. 5a depict the FT-IR spectra of molecular vibrations of samples. FT-IR is an effective means to perform the molecular structural analysis by detecting the relative vibration and molecular rotation
Fig. 5. (a) FT-IR spectra of 0 KGM, 0+ KGM, 2 KGM, 2+ KGM; (b) IR spectra and IR images illustrate the distribution of the glycosidic bonds before and after laser irradiation.
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between atoms [33]. The intensity of the absorption peaks varies differently depending on the degradation treatment. Basically, there were no new chemical groups introduced to the KGM molecular chain after degradation. The main bands of KGM can be observed at around 4000–800 cm−1. The peaks at 3430 cm−1 were assigned to the stretching vibrations of O\\H groups and peaks at 2925 cm−1 were assigned to the stretching vibrations of C\\H groups [34,35]. The peaks at about 1728 cm−1 were assigned to the stretching vibrations of C_O groups [36]. The stretching peaks at 804 cm−1 were characteristic absorption bands of mannose [37]. Interestingly, the peak intensity at 891 cm−1 in the spectrum of 2+ KGM decrease slightly, revealing the decomposition of glycosidic bonds during laser degradation. Similar results can be found in the research done by Pan et al. [6]. This was due to the reaction between hydroxyl radicals and KGM. Hydroxyl radicals could react with KGM by abstracting carbon-bound hydrogen atoms. The resulting carbohydrate radicals could cause direct breakage of the glucosidic bonds by rearrangement [28]. In addition, the results of IR images were consentaneous with FT-IR analysis. IR images exhibit chemical changes of the KGM before and after laser degradation. We directly illustrated the distribution of glycosidic bonds at 891 cm −1 by color variation (the red color signifies high intensity while blue color represents low intensity) [38]. As shown in Fig. 5b, the red color revealed that there were abundant glycosidic bonds in KGM. However, a relatively homogeneous intensity was observed in 2+ KGM (blue color), which suggested the decreased number of glycosidic bonds. The mechanism of the degradation is based on some reactions. The primary reactions in the solutions are the formation of hydroxyl radicals ( OH) through the radiolysis of water and hydrogen peroxide as follows [39]: H 2 O2 þ ðhvÞ→H 2 ; H 2 O2 ; e− aq ; H ; H 2 O þ ðhvÞ → 2 OH
OH; H 3 Oþ
ð2Þ
Fig. 7. DPPH radical scavenging activity of 0 KGM and 2+ KGM.
3.6. Mw and PDI analysis In order to quantize the degradation effect, Mw and PDI were further investigated. Fig. 6a and b gives the information about the Mw and PDI of 0 KGM and 2+ KGM. The Mw of 2+ KGM (5.7 × 105 Da) was much lower than 0 KGM (7.6 × 105 Da). The decreased Mw of 2+ KGM indicated that the KGM has been degraded after laser irradiation. Besides, the PDI of 2+ KGM decreased from 1.468 to 1.166, which signifies the irradiation products distributed more evenly. This was due to the exact scission on glucosidic bonds during degradation.
ð3Þ
Furthermore, during irradiation, e−aq and H• can react with H2O2 as follows: e− aq þ H 2 O2 → OH þ OH −
ð4Þ
H
ð5Þ
The addition of hydrogen peroxide increased the concentration of •OH during laser irradiation. As a powerful oxidative agent, •OH could react with KGM by abstracting carbon-bound hydrogen atoms. The resulting carbohydrate radicals could cause direct breakage of the glucosidic bonds by rearrangement [28].
3.7. Antioxidant property analysis Finally, the antioxidant activity of the 2+ KGM was investigated. The antioxidant activity was gauged through DPPH radical scavenging ability (Fig. 7). DPPH is a stable lipophilic free radical which is often applied in estimating antioxidant activity of food and medicine materials [40]. The 2+ KGM displayed free radical scavenging ability of 60.2% compared with 0 KGM of 39.5%. It is commonly acceptable that the antioxidant has free radical scavenging ability is due to their hydrogen-donating ability [17]. Therefore, we infer that laser degradation can improve the antioxidant activity of KGM by promoting their hydrogen-donating ability, which is consistent with the conclusion of Jian's group [17].
Fig. 6. (a) Mw and (b) PDI of 0 KGM and 2+ KGM.
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4. Conclusion In summary, the degraded KGM can be obtained by laser irradiation assisted with hydrogen peroxide. The Mw decreased from 7.6 × 105 Da to 5.7 × 105 Da. And the PDI nearly approached to 1.0 revealing that the degraded products dispersed more evenly. The existence of hydrogen peroxide can increase the number of hydroxyl radicals, which could induce the breakage of glucosidic bonds in KGM molecular chains. This can be confirmed by the FT-IR spectra and IR images of the LDK. More importantly, the evaluation of DPPH radical scavenging activity demonstrated that the antioxidant activity has been improved in the LDK. Overall, this strategy can provide a simple, time-saving, cost-effective method for degrading KGM in an environmental-friendly way and expand the applications of KGM. Acknowledgement This work was financially supported by the National Natural Science Foundation of China (grant numbers 31772045, 31471704). The authors thank Yingning Yao for valuable discussion on preparation of the experiment. References [1] K. Nishinari, P.A. Williams, G.O. Phillips, Review of the physico-chemical characteristics and properties of konjac mannan, Food Hydrocoll. 6 (1992) 199–222. [2] C. Wu, Y. Li, Y. Du, L. Wang, C. Tong, Y. Hu, J. Pang, Z. Yan, Preparation and characterization of konjac glucomannan-based bionanocomposite film for active food packaging, Food Hydrocoll. 89 (2019) 682–690. [3] Y.H. Mao, A.X. Song, Z.P. Yao, J.Y. Wu, Protective effects of natural and partially degraded konjac glucomannan on Bifidobacteria against antibiotic damage, Carbohydr. Polym. 181 (2018) 368–375. [4] M. Maeda, H. Shimahara, N. Sugiyama, Detailed examination of the branched structure of konjac glucomannan, J. Agric. Chem. Soc. Jpn. 44 (1980) 245–252. [5] K. Katsurayaa, K. Okuyamab, K. Hatanakab, R. Oshimab, T. Satoc, K. Matsuzakic, Constitution of konjac glucomannan: chemical analysis and 13C NMR spectroscopy, Carbohydr. Polym. 53 (2003) 183–189. [6] T. Pan, S. Peng, Z. Xu, B. Xiong, C. Wen, M. Yao, J. Pang, Synergetic degradation of konjac glucomannan by γ-ray irradiation and hydrogen peroxide, Carbohydr. Polym. 93 (2013) 761–767. [7] J. Chen, C. Liu, Y. Chen, Y. Chen, P.R. Chang, Structural characterization and properties of starch/konjac glucomannan blend films, Carbohydr. Polym. 74 (2008) 946–952. [8] G. Conzatti, D. Faucon, M. Castel, F. Ayadi, S. Cavalie, A. Tourrette, Alginate/chitosan polyelectrolyte complexes: a comparative study of the influence of the drying step on physicochemical properties, Carbohydr. Polym. 172 (2017) 142–151. [9] Y. Hu, L. Liu, Z. Gu, W. Dan, N. Dan, X. Yu, Modification of collagen with a natural derived cross-linker, alginate dialdehyde, Carbohydr. Polym. 102 (2014) 324–332. [10] C. Xiao, Y. Lu, H. Liu, L. Zhang, Preparation and characterization of konjac glucomannan and sodium carboxymethylcellulose blend films, J. Appl. Polym. Sci. 80 (2015) 26–31. [11] K. Wang, K. Wu, M. Xiao, Y. Kuang, H. Corke, X. Ni, F. Jiang, Structural characterization and properties of konjac glucomannan and zein blend films, Int. J. Biol. Macromol. 105 (2017) 1096–1104. [12] M. Leuangsukrerk, T. Phupoksakul, K. Tananuwong, C. Borompichaichartkul, T. Janjarasskul, Properties of konjac glucomannan–whey protein isolate blend films, LWT Food Sci. Technol. 59 (2014) 94–100. [13] L. Wang, M. Xiao, S. Dai, J. Song, X. Ni, Y. Fang, H. Corke, F. Jiang, Interactions between carboxymethyl konjac glucomannan and soy protein isolate in blended films, Carbohydr. Polym. 101 (2014) 136–145. [14] J. Luan, K. Wu, C. Li, J. Liu, X. Ni, M. Xiao, Y. Xu, Y. Kuang, F. Jiang, pH-sensitive drug delivery system based on hydrophobic modified konjac glucomannan, Carbohydr. Polym. 171 (2017) 9–17.
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