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Comparative preservation effect of water-soluble and insoluble chitosan from Tenebrio molitor waste
Accepted Manuscript Comparative preservation effect of water-soluble and insoluble chitosan from Tenebrio molitor waste
Ning Li, Xiaoli Xiong, Xia Ha, Xingyue Wei PII: DOI: Reference:
S0141-8130(19)30132-1 https://doi.org/10.1016/j.ijbiomac.2019.04.094 BIOMAC 12170
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
6 January 2019 31 March 2019 12 April 2019
Please cite this article as: N. Li, X. Xiong, X. Ha, et al., Comparative preservation effect of water-soluble and insoluble chitosan from Tenebrio molitor waste, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.04.094
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ACCEPTED MANUSCRIPT Comparative preservation effect of water-soluble and insoluble chitosan from Tenebrio molitor waste Ning Li, Xiaoli Xiong, Xia Ha, Xingyue Wei Chongqing Engineering Research Center for Processing ＆ Storage of Distinct Agricultural Products, Chongqing Technology and Business University, Chongqing 400067, China E-mail: [email protected] Edible films and coatings have been developed based on numerous natural biopolymers, which
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have been used to increase fresh-cut fruit shelf life. Here, we present the preparation, characteristics and preservation effect of water-soluble chitosan (WSC) and water-insoluble chitosan (WIC) from Tenebrio molitor waste (TMW) on fresh-cut apple slices. WIC was isolated from TMW in four steps and WSC was obtained from the WIC solution by 8 % H2O2 treatment at 40℃for 3 h. WIC and WSC were characterized by molecular weight, Fourier transform infrared spectroscopy (FTIR), morphology analysis, etc. The preservation effects of WIC and WSC for the fresh-cut apple slices were evaluated by the indexes of browning, weight loss, firmness and titratable acidity. The results showed that WSC was soluble in water and that the chemical structures of WIC and WSC were similar. However, their crystallinity, morphology and thermal properties were different. Both WSC and WIC had a good preservation effect on fresh-cut fruits. Compared with WIC, WSC might be more suitable for use in the food industry owing to its water solubility. Keywords: Tenebrio molitor waste, water-soluble chitosan (WSC), water-insoluble chitosan (WIC), preservation
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1. Introduction
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Tenebrio molitor (TM) is an edible insect that is distributed worldwide and is a convenient candidate for protein or fat isolation for the health foods and products industry. TM produces a large amount of waste during the three insect growth stages: larva, pupa and adult. The larvae molt 10–15 times and produce a large amount of exuvia during their growth. In addition, other byproducts, such as the dead beetles after oviposition and the dead larvae and shells of the pupae, are also produced. These byproducts are rarely used for other purposes and are normally considered to be wastes, which may contaminate the environment. Therefore, reasonable treatments of TM wastes (TMW) are essential. One of the important polymers, chitin, can been extracted from the bodies of these wastes. Chitin is an insoluble carbohydrate polymer, a long-chain polymer of repeating N-acetylglucosamine residues connected exclusively by linkages. This polymer is the second most abundant natural biomass resource that is derived from the exoskeletons and cuticles of insects, the shells of marine invertebrates and the cell walls of certain fungi and algae [1]. The extraction of chitin from TMW suggests it is a better source than that from other insects, due to the stable supply of raw materials and its low collection cost. Chitosan, the main derivative of chitin, is generally produced from N-deacetylation of chitin under high concentration alkaline conditions [2]. Chitosan has many applications in the environment, agriculture, cosmetics, and in food packaging (either as barrier coatings or as an antibacterial agent) industries due to its non-toxic, biodegradable, antimicrobial and anti-oxidation properties [3-5]. However, most high molecular weight chitosans are insoluble in water (namely, water-insoluble chitosan, WIC, only completely soluble under acid conditions) due to its strong intramolecular hydrogen bonding, which limits its applications. Therefore, many researchers have tried to lower the molecular weight of chitosan and improve its solubility [6]. Water-soluble chitosan (WSC, completely soluble in neutral aqueous solutions) can be 1
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obtained from WIC by enzymatic or chemical hydrolysis. WIC can be applied to pharmaceuticals, pharmaceutical ingredients, pesticides, food, etc. [6, 7]. Currently, in restaurants, school lunch programmes, food services, street and domestic houses, etc., fresh-cut fruits supply important vitamins and have become common desserts or popular snacks. However, fresh-cut fruits are susceptible to spoiling, which could directly influence or lower consumer demand. Thus, a natural edible coating to reduce decay and prolong the shelf life of fresh-cut fruits is an alternative to this problem [8]. Some research articles have reported the application of chitosan on fresh-cut fruits. Chitosan-coating treatments can effectively reduce the occurrence of enzymatic browning on the surface of fresh-cut apples during storage [9]. Chien et al.[10] reported that a chitosan coating effectively prolongs the quality attributes and extends the shelf life of sliced mango fruit. Chitosan can be used as a natural preservative due to its antimicrobial activity against a wide range of foodborne microorganisms, including Gram-negative and Gram-positive bacteria, yeasts, and moulds [11], as well as its antioxidant activity and physical protection actions, such as the formation of a barrier across the cell surface, hindering the exchange of metabolites [12,13]. The use of natural resources as a source of valuable compounds to be used in the food industry is currently a topic of growing interest. The main objective of this work was the development of a technology for the preparation of WSC and WIC from TMW. This technology can not only solve the environmental pollution problems caused by TMW but also identify an alternative natural resource for producing chitosan from their wastes. One of the potential applications of WSC is that it can be used as a preservation agent for fresh-cut fruits. Compared with other chitosan-based preservative agents, water-soluble chitosan might be more suitable for use in the food industry owing to its water solubility without the need for other additive agents and it could maintain the original flavour of foods [9].
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2. Materials and methods
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2.1 Materials TMW was obtained from Chongqing Bole Agri. Sci. Tech. Co., Ltd. Apples (Red Fuji) were selected based on no apparent injury with a uniform size, ripeness and colour, and purchased from a local wholesale distributor. The applied chemicals were analytical grade. 2.2 Preparation method 2.2.1 Pre-treatment of TMW TMW was dried and smashed directly. The main characteristics of the processed TMW were as follows: a particle size of 0.5 mm, a density of 126.8 kg.m-3, 1.3 % (w/w) ash, 22.5 % (w/w) crude fat and 58.7 % (w/w) nitrogenous compounds (protein and non-protein). The contents of the crude ash, the crude fat and the nitrogenous compounds in TMW were determined by a standard ash analysis method, an automated Soxhlet method and the Kjeldahl method, respectively [14-16]. 2.2.2 Extraction and purification of WIC and WSC WIC were separated from pre-treated TMW using four steps: degreasing (DG), demineralization (DM), deproteinization (DP) and deacetylation (DA). DG step: a sample of the pre-treated TMW was treated with petroleum ether (30 ℃–60 ℃) at 55 ℃for 2 h by Soxhlet with a solid-liquid ratio of 1:10 (w/v). DM step: The DG residue was immersed in 0.3 mol/L HCl solution at ambient temperatures (25 ± 2℃) for 8 h. DP step: the sample after DM treatment was dispersed in 1 % (w/w) NaOH solution for 3 h to remove organic substances such as proteins and fats. DA step: The DP residue was dispersed in a 60 % (w/w) NaOH solution at 130 ℃for 7 h (DA). The experimental solid portions in all steps were washed with deionized water until a neutral pH was achieved. Then, the sample was dried at 55 ℃for 2 h and the product was obtained (WIC). 2
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Next, 10 g of WIC was dissolved in a 0.15 mol/L HCl solution and 500 mL 0.75 mol/L H2O2 was added to the WIC solution during 100 r/min stirring at 40 ℃for 3 h. The solution was precipitated three times with ethanol, then the mixture was left to stand for 24 h and centrifuged and dried at 50 ℃(WSC). 2.3 Characteristics of WIC and WSC 2.3.1 Molecular weight and solubility testing The molecular weights of the WSC and WIC were measured by End-group analysis [17]. The solubility of the sample was tested by using the following methods: Each 1 g of sample (WSC or WIC) was dispersed in 100 mL of varying concentrations of acetic acid solutions (0 %–2 %, v/v) using a vortex. 2.3.2 FTIR spectroscopy FTIR was performed through a Nicolet Impact 400 D FTIR spectrophotometer (Shimadzu, Japan). The transparency was measured with a function of the wave number between 4000 cm−1 and 400 cm−1 with a resolution of 2 cm−1 and the number of scans was 18. 2.3.3 X-ray diffraction (XRD) analysis XRD patterns of samples were recorded by a Rigaku D/MAX 2500PC X-ray diffraction diffractometer (Rigaku, Japan) with a monochromatized X-ray beam using nickel-filtered Cu Kα radiation. The generator was operated at 40 kV and 150 mA. The diffraction intensity was recorded every 0.02 s from 5 ° to 90 ° at a rate of 4 ° /min. 2.3.4 Thermal monitoring Thermal analysis was performed on a Netzsch STA 449 C (Netzsch Instruments, Inc., Germany). Approximately 5 mg of sample was placed in an Al2O3 pan then heated to 500 ℃at a rate of 10 ℃/min, under a flow of argon at 10 mL/min. The starting temperature was 30 ℃. 2.3.5 Elemental analysis The samples were analysed with a Vario MICRO Elemental Analyzer EL III (Elementar, Germany) using 5 mg of the samples to elucidate the ratio of C, H, and N. The degree of deacetylation (DD) of the WIC or WSC was calculated from the elemental analysis by using Eq. (1):
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where DD % is the degree of deacetylation, R is the C/N value of WIC or WSC measured by elemental analysis, 6.85 corresponds to the theoretical C/N value of the chitin, and 1.71 corresponds to the difference between the theoretical C/N of chitin (6.85) and the theoretical C/N of WIC or WSC (5.14). 2.3.6 Morphology observations The morphology images of WIC and WSC were taken by SEM (Nova Nano SEM 450, FEI, USA) and a TEM (JEM-2100F, JEOL, Japan). SEM test conditions: 15 nm layers of gold particles coated on the dried powder samples, voltage 20 kV, high vacuum and room temperature. TEM test conditions: accelerating voltage 200 kV, room temperature. 2.4 Preparation of apple samples The apples were pre-washed in drinking water and then deionized water to remove any residual substances. The clean apples were cut into slices with a disinfected knife under sterile conditions. All slices without a peel used in this experiment were uniform in size, shape and weight (8 ± 0.5 g). 2.5 Preservation of fresh-cut apples One of the potential applications of WIC or WSC is that it can be used as a preservation agent for fresh-cut fruits. The preparation effect was tested by the following method: The prepared apple slices were divided into three groups randomly. The experimental groups were immersed in 10 g/L WSC or WIC solutions (WSC, deionized water solution; WIC, 1 % (v/v, aqueous acetic acid) for 2 min and the control groups were immersed in deionized water for the same time. Subsequently, the apple slices were ventilation dried on a sieve at room temperature for 3
ACCEPTED MANUSCRIPT 2 min, and then placed in the same sized plastic tray. Each parameter was measured at 0, 2, 4, 7, and 10 h. 2.5.1 Colours The colour of the cutting surfaces of all fresh-cut apples was measured with a colorimeter (NR60CP, Shenzhen 3nh Technology Co., Ltd, China). L, a and b values were recorded. 2.5.2 Weight loss Weight loss is considered as an important factor to evaluate apple quality, which is closely related to water loss. The weights of the coated fresh-cut apple slices were measured with an Analytical Balance (XPE205DR, Mettler Toledo, China). The weight loss ratio W (%) was expressed as Eq. (2) [18]:
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Where Wi (g) is the initial weight of the experimental apple slices and Wj (g) is the final weight of the experimental apple slices. 2.5.3 Firmness The firmness of fresh-cut apples is a parameter of freshness. It was determined with a texture analyser (Brookfield CT-3, USA) equipped with a P/2 probe (2 mm diameter). 2.5.4 Titratable acidity A total of 10 g of apple slices were prepared by using a direct homogenizer and a filter, then deionized water was added up to 100 mL and 10 mL of the fresh-cut apple juice was titrated by 0.1 mol/L NaOH with phenolphthalein used as the indicator. The end point of the reaction was identified as transformation of the colourlessness into a pale red (Supapvanich et al., 2011). The titratable acidity (TA, expressed as the percentage of malic acid) was calculated by using Eq. (3) [19]:
where C1 (mol/L) is the concentration of NaOH, V1 (mL) is the volume of NaOH, V2 (mL) is the total volume of
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apple juice, V3 (mL) is the titration volume of apple juice, W (g) is the total weight of the fresh-cut apple, and 0.067 is the conversion coefficient of malic acid. All of the experiments were repeated three times in parallel and the results were expressed as mean values and standard deviation. The data were analysed with Origin Pro. 8.0 Software.
3 Results and discussion
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3.1 Characteristics of WIC and WSC 3.1.1 Molecular weight and solubility testing The molecular weights of the WIC and WAC were 96.2±3.5 kDa and 9±1 kDa, respectively. Table 1 shows the solubility of both WIC and WSC in different solutions. The results showed that WSC was soluble from 0 % (deionized water) to 2 % (v/v) acetic acid concentrations, but the WIC was insoluble at acetic acid concentrations lower than 1 % (v/v). 3.1.2 FTIR spectroscopy In Fig. 1, curves a (WSC) and b (WIC) exhibited similar characteristic peaks. The -OH and -NH2 symmetric stretching vibrations were observed at 3600–3200 cm-1 and asymmetrical C-H bending vibrations of the -CH2 and -CH3 groups were observed at 1382–1385 cm-1 [20,21]. The FTIR spectra of both WSC and WIC showed characteristic bands of amide groups (1639–1655 cm-1, carbonyl stretching of amide I). A weak absorption peak was observed at 1595 cm-1 in the spectrum of WIC, assigned to the NH bending vibration of amide II. However, 4
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the peak at 1595 cm-1 did not appear in the spectrum of WSC. The shift of amide I and the disappearance of amide II indicated a difference in the molecular interaction and degree of deacetylation between WIC and WSC [4]. WSC had higher degree of deacetylation than WIC, which were also proved by the following elemental analysis. The high degree of deacetylation supported the solubility of chitosan in water. Generally, the band at 1074 cm−1 was assigned to the stretching of C-O-C in the glucose ring [22]. The spectra of WIC and WSC showed a glucose structure in the backbone at 897 cm-1 and a pyranose ring at 1255 cm-1. The weak absorption peaks of the β-(1,4)-glucoside bond were observed at 897 and 1154 cm-1 in WIC. However, the peak at 1154 cm-1 was not apparent in the spectrum of the WSC, which indicated that a number of β-(1,4) glycosidic bonds of WIC were broken and WSC was formed [21, 22]. The differences between WSC and WIC were also proven by the above molecular weight analysis. These data demonstrated that the chemical structures of the main chains of WIC and WSC were similar with appropriate shifts or disappearances of the adsorption peaks. These results were basically consistent with previous reports [20, 21].
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Fig. 1 FTIR spectra of samples. (a) WSC (b) WIC.
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3.1.3 X-ray diffraction (XRD) WIC and WSC were characterized by XRD in order to verify the substances’ crystallinity. The XRD patterns of the isolated WIC and WSC are shown in Fig. 2. Two sharp peaks and several weak peaks were found in the XRD diffraction pattern of WIC. Two obvious sharp diffraction peaks at an angle 2θ=10.97 ° and 20.07 ° were typical fingerprints of semi-crystalline WIC and consistent with the literature data from different sources of WIC [23]. Islam et al. [24] also observed broad diffraction peaks at 2θ=10 ° and 21 ° that symbolized semi-crystalline WIC. The degree of crystallinity of WIC was 15.45 % based on the calculation of the intensity of the crystalline regions and amorphous regions (Jade 6.5, New Zealand). Two sharp peaks approximately 10° and 20° and several weak peaks from 27° to 30° were observed, which indicated that WIC was in the α-form [25]. In Fig. 2, WSC samples showed amorphous diffraction with a very broad hump at 2θ=21.15 °, which could indicate that WSC exists in a form with a noncrystalline state. A potential explanation of the crystallinity differences between WSC and WIC is the crystalline properties of WIC, due to the inter and intramolecular hydrogen bonds [26]. During the preparation of WSC, a number of β-(1,4) glycosidic bonds of WIC were broken, which could be shown by the above molecular weight and FTIR analysis and the long chains of WIC could be broken into short chains by H2O2, and WSC with a lower molecular weight that had fewer intramolecular hydrogen bonds [7]. Hence, we speculated that the WSC was amorphous due to a lack of intramolecular hydrogen bonds, which was shown in the XRD photos.
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Fig. 2 XRD patterns. (a) WSC, (b) WIC.
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3.1.4 Thermal analysis Fig. 3 demonstrates the results of the TG (thermogravimetric analysis) and DSC (differential scanning calorimetric analysis) curves for WIC and WSC. In the TG curves of the two samples, there were three characteristic temperature intervals of weight loss. The first one (30–105 ℃) with approximately 4 % (w/w) (WIC) and 6 % (w/w) (WSC) weight loss could be attributed to the evaporation of water that was physically and chemically adsorbed in the WIC and WSC [27]. The curves of the DSC showed the broad endothermic effect (maximum endothermic peaks at 89.6 ℃and 112.4 ℃for WIC and WSC, respectively). The second weight losses, occurring in the range 120–340 ℃, were 45 % (w/w) and 62 % (w/w) for WIC and WSC, respectively and were caused by depolymerisation/decomposition of polymer chains through deacetylation and cleavage of glycosidic linkages [22]. At this stage, the weight loss temperature of the WSC was lower than that of the WIC. The differences in the thermal behaviour of WIC and WSC might be ascribed to their molecular weight or polymerization degree. Commonly, the samples with a higher molecular weight and crystallinity indicated more thermal stability [28]. WSC showed a low molecular weight, and the amorphous structure was verified by End-group analysis and XRD. In contrast, WIC displayed a high molecular weight and crystallinity structure, which can be attributed to the initial decomposition temperature for WIC being 233.6 ℃, while the corresponding value for WSC was located at 195.2 ℃. The DSC curve of WIC exhibited a maximum exothermic peak, Tmax = 309.6 ℃, with an enthalpy change of −129.2 J/g. However, the DSC curve of WSC showed no obvious exothermic peak. In the last stage, the WIC decomposition temperature was higher than 340 ℃, corresponding to the thermal destruction of the pyranose ring and the decomposition of the residual carbon [22, 27].
Fig. 3 TG -DSC curves of WIC and WSC.
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3.1.5 Elemental analysis According to the WIC and WSC formula, theoretical values (w/w) of C: 44.72 %, H: 6.83 %, N: 8.69 %, C/H: 6.5455, and C/N:5.1429 were predicted. Elemental analysis exhibited the composition of WSC: C: 37.14 %, H: 5.89 %, N: 6.92 %, C/H: 6.3056, and C/N:5.3698; and the composition of WIC: C: 43.84 %, H: 7.17 %, N: 7.85 %, C/H: 6.1144, and C/N:5.5847. The DD values of WIC and WSC were 73.99 % and 86.6 % by Eq (1), respectively. 3.1.6 Morphology observations The morphology of the WSC and WIC were studied by SEM (scanning electron microscopy) and TEM (transmission electron microscopy) (Fig. 4). The surface of the WSC showed loose aggregates, like earth flaws (Fig. 4a). The surface of the WIC was fibrous and porous, with some regular flaky texture, like chrysanthemum petals (Fig. 4b). Similar surface morphology has been reported for the pupa of Vespa crabro, Archispirostreptus gigas and Zophobas morio larvae [29-31]. The WSC powder was also observed by TEM, aiming at further study of the aggregation morphology about WSC particles. The TEM image (Fig. 4c) showed that the powder of WSC was composed of loose aggregates of a fibrous network with countless like-globular perforated holes (size 20–400 nm), which was beneficial to the dissolution of WSC due to the rapid permeation of the solvent into the WSC. In addition, the XRD analysis proved that WIC was partly crystalline and that WSC was in an amorphous form. Due to the oxidation of H2O2, the lamellar WIC was cracked into small fragments, which were disordered together due to the lack of intermolecular forces and they formed an amorphous structure. The increase of water solubility and the decrease of thermal stability of WIC were due to the decreases in molecular weight and intermolecular forces.
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Fig. 4 SEM images of WSC (a) and WIC (b); TEM image of WSC (c).
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3.2 Application of WSC 3.2.1 Appearance of apple slices Apple browning is caused by an oxidation process that occurs during maturation stages. Changes in L (lightness, +L to lightness, -L to darkness), a (+a to redness, -a to greenness), b (+b to yellowness, -b to blueness) and ∆E (comprehensive evaluation index, ∆E=(∆L2+∆a2+∆b2)1/2, is a widely applied parameter for determining colour differences that can be observed by the naked eye, and all of these values are considered to be part of the browning index. As shown in Fig. 5a, “a” values increased but the increasing tendency of the control group was higher than that of the coated groups. The degree of changes in the “b” values of the WSC, WIC and control groups was slight (Fig. 5b). All of the apple slices turned brown over time accompanied by decreasing L values, but the decreasing degree of the control group was higher than that of the coated groups (Fig. 5c). In fact, the variations of lightness (∆L) between 0 h and 10 h were 8.8 and 6.3 for the control group and WSC group, respectively. During the first 4 hours, the L value of the WSC group showed more changes than the WIC group, and the opposite situation was found during the next 4 hours. The increase of redness values (a) and the decrease of L values (darkness) indicated continuous browning of the samples [32]. The ∆E values of the experimental groups were lower than that of the control group (Fig. 5d). In regards to the browning degree of the fresh-cut apple slices, the coated groups with WSC and WIC were less than that seen in the control group. In contrast, the WSC group had smaller ∆E value changes, suggesting a lower chance of the WSC group browning. In previous studies, it was also reported that chitosan had a better effect on preventing apple browning. The browning degree of apples coated with chitosan was less than 20 % at 12 h and the browning of apples coated with nothing was greater than 50 % [33].
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Fig. 5 Chromatism changes of fresh-cut apples. (a) a values; (b) b values; (c) L values; (d) ∆E values
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3.2.2 Weight loss The weight loss ratio of the uncoated (control group) and coated fresh-cut apple slices with WSC and WIC are shown in Table 2. All fresh-cut apple slices showed a gradual weight loss process, and the loss ratios of the coated groups were significantly less than that of the control group due to the formation of coating films on the surfaces of the samples. After 10 h, the uncoated fresh-cut apple slices had an average weight loss ratio of 21.89 %, whereas the coated groups were 15.00 % and 16.69 % for WIC and WSC, respectively. The water conservation effect of WIC and WSC were similar, the statistical differences among different treatments were listed in Table 2. Liu et al. [34] also demonstrated that the weight loss ratio of fresh-cut apples after 10 h coated with nothing or coated with chitosan, chitosan-ascorbic acid, or chitosan-CaCl2 were 16 %, 14 %, 13 % and 11 %, respectively. These results demonstrated that the chitosan coated apples had a good ability of water loss inhibition when compared with the control apple slices. 3.2.3 Firmness Firmness is one of the important factors for fresh-cut fruit quality. As shown in Table 2, the firmness of the fresh-cut apple slices decreased as time increased. At the beginning of the experiment, the firmness of the fresh-cut apple slices was nearly the same for both the coated and uncoated groups. After 7 h, the firmness of the coated group was higher than that of the control group due to the protective effect of WSC or WIC. We found that after 7 h WSC resulted in the highest firmness for fresh-cut apples, the statistical differences among different treatments were listed in Table 2. A physical and mechanical barrier was formed to delay the respiratory metabolism. Consequently, the water loss was through dehydration and enzymatic activities, which were related to fruit softening [35]. Liu et al [33] also demonstrated that the firmness of fresh-cut apple slices coated with chitosan was 14 % higher than those without chitosan. 9
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3.2.4 Titratable acidity As shown in Table 2, titratable acidity (TA) values slightly increased over time for all sample groups due to the accumulation of acids from D-glucose by glycolytic enzyme systems and water loss [36,37]. There were no obvious differences in TA values between coated fresh-cut apples with WSC and the control group. However, the TA values of the coated fresh-cut apples with WIC significantly increased due to the residual acetic acid on the surface of the fruits. After 4 hours, the TA values of fresh-cut apple slices coated with WIC decreased due to the gradual volatilization of acetic acid. The statistical differences among different treatments were listed in Table 2. It is widely accepted that taste is one of the most important quality parameters in determining consumer acceptability of fruits, and TA values directly affect the taste of fruit [37]. In the current study, fresh-cut apple slices coated with WIC showed some increases in acidic value that affected the taste of the fruit. Briefly, WSC was more suitable for the preservation of fresh-cut fruits.
4. Conclusions
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WSC and WIC were shown to improve the quality of fresh-cut apples over time in regards to their colour, texture and titratable acidity. These findings have provided experimental evidence for the applications of WSC and WIC. Compared with WIC, WSC might be more suitable for use in the food industry owing to its water solubility. A further study on its physicochemical properties, safety and applications is in progress.
Acknowledgements
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We are grateful to Dr. Geoffrey Onaga who kindly offered the English revision. We would also thank our colleagues Dr. Wen Yun for the improvement of the language. We thank Funying Jiao, Chunyan Xie and Peiwen Jin for the data analysis. This work was supported by the project of Basic Science and Frontier Technology from Chongqing Science and Technology Commission, PRC (cstc2016jcyjA0592), the project for the Transformation of Excellent Achievements in Universities of Chongqing Education Committee (KJZH17125), and the project of Training Innovative Talents for Primary and Secondary Schools of Chongqing Education Committee (CY180801). We thank the anonymous reviewers for their careful reading and constructive comments.
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Biosci. 18 (2017)28-33. [8] M. A. Rojasgrau, A. Sobrinolopez, M.S.Tapia, O. Martinbelloso, Browning inhibition in fresh-cut 'Fuji' apple slices by natural antibrowning agents, J. Food Sci. 71(2010) S59-S65. [9] H. Qi, W. Hu, A. Jiang, M. Tian, Y. Li. Extending shelf-life of Fresh-cut ‘Fuji’ apples with chitosan-coatings, Innov. Food Sci. Emerg. 12 (2011) 62-66. [10] P. Chien, F. Sheu, F.Yang, Effects of edible chitosan coating on quality and shelf life of sliced mango fruit, J. Food Eng. 78(2007) 225-229. [11] K.Ming, X. G.Chen, X. Ke, P. Hyunjin, Antimicrobial properties of chitosan and mode of action: a state of the art review, Int. J. Food Microbiol. 144(2010) 51-63. [12] M. Friedman, V. K. Juneja, Review of antimicrobial and antioxidative activities of chitosans in food, J. Food Protect 73 (2010)1737-1761. [13] F. Joaoc, T. Frenik, S. Joséc, R.Oscars, M. Mjoao, P. Manuelae, et al., Antimicrobial effects of chitosans and chitooligosaccharides, upon staphylococcus aureus and escherichia coli, in food model systems, Food Microbio. 25 (2008)922-928. [14] AACCI, Method 08-01.01. Ash-basic method, approved methods of analysis, 11 ed. AACC International, St. Paul, MN, U.S.A, 1999. [15] J. M. Shin, Y. O. Hwang, O. J. Tu, H. B.Jo, J. H. Kim, Y. Z. Chae, et al., Comparison of different methods to quantify fat classes in bakery products, Food Chem. 136 (2013)703-709. [16] A.Jonas-Levi, J. J. I. Martinez, The high level of protein content reported in insects for food and feed is overestimated, J. Food Compos. Ana. 62 (2017)184-188. [17]T. Sun, D.Zhou, J. Xie, F. Mao, Preparation of chitosan oligomers and their antioxidant activity, Eur. Food Res. Technol. 225(2007) 451-456. [18] N. Azarakhsh, A.Osman, H. M. Ghazali, C. P. Tan, N. M. Adzahan. Lemongrass essential oil incorporated into alginate-based edible coating for shelf-life extension and quality retention of fresh-cut pineapple, Postharvest Biol. Technol. 88 (2014)1-7. [19] L.W.Wang, H.Z.Yang, D. Jiang, SB/T 10203-94: General Test Method for Fruit Juice. Ministry of Commerce of the People's Republic of China, Beijing, 1994. [20] J. Singh, P.K. Dutta, J. Dutta, A.J. Hunt, D.J. Macquarrie, J. H.Clark, Preparation and properties of highly soluble chitosan-l-glutamic acid aerogel derivative, Carbohydr. Polym. 76(2009) 188-195. [21] S. Wu, Preparation of chitooligosaccharides from clanis bilineata larvae skin and their antibacterial activity, Int. J. Biol. Macromol. 51(2012), 1147-1150. [22] H.Moussout, H. Ahlafi, M. Aazza, M. Bourakhouadar, Kinetics and mechanism of the thermal degradation of biopolymers chitin and chitosan using thermogravimetric analysis, Polym. Degrad. Stabil. 130 (2016) 1-9. [23] S. Kumari, P. Rath, A.S. H.Kumar, T.N. Tiwari, Extraction and characterization of chitin and chitosan from fishery waste by chemical method, Environ. Technol. Inno.3 (2015)77-85. [24] M.M.Islam, S. M. Masum, M.M. Rahman, M.A.Islam, A.A. Shaikh, S.K. Roy, Preparation of chitosan from shrimp shell and investigation of its properties, Int. J. Basic App. Sci. 11(2011) 77-80. [25] M. Kaya, V. Baublys, I. Satkauskiene, B. Akyuz, E. Bulut, V. Tubelyte, First chitin extraction from Plumatella repens (Bryozoa) with comparison to chitins of insect and fungal origin, Int. J. Biol. Macromol. 79 (2015) 126-132. [26] P. S. Bakshia, D.Selvakumara, K. Kadirvelu, N.S. Kumar, Comparative study on antimicrobial activity and biocompatibility of N-selective chitosan derivatives, React. Funct. Polm. 124(2018)149-155. [27] I. Corazzari, R. Nisticò, F. Turci, M.G. Faga, F. Franzoso, S. Tabasso, et al., Advanced physico-chemical characterization of chitosan by means of tga coupled on-line with ftir and gcms: thermal degradation and 11
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water adsorption capacity, Polym. Degrad. Stabil. 112(2015) 1-9. [28] Q.Yan, C. Shuai, L.Ying, H. Sun, S. Jia, J. Shi, et al., Pyrolysis of chitin biomass: TG-MS analysis and solid char residue characterization, Carbohydr. Polym. 133 (2015)163-170. [29] M. Kaya, P. Mulerčikas, I. Sargin, S. Kazlauskaitė, V. Baublys, B. Akyuz, et al., Three-dimensional chitin rings from body segments of a pet diplopod species: characterization and protein interaction studies. Mat. Sci. Eng. C 68(2016) 716-722. [30] M. Kaya, K.Sofi, I. Sargin, M. Mujtaba, Changes in physicochemical properties of chitin at developmental stages (larvae, pupa and adult) of vespa crabro (wasp), Carbohydr. Polym.145(2016) 64-70. [31] Y.S. Chu, B.T. Yee, H.T. Choon, T.R. Abdul, K. Abdan, Extraction and physicochemical characterization of chitin and chitosan from Zophobas morio larvae in varying sodium hydroxide concentration, Int. J. Biol. Macromol. 108 (2018)135-142. [32] L.R.Antoniolli, B.C. Benedetti, Effect of calcium chloride on quality of fresh-cut 'Pérola' pineapple, Pesq. Agropec. Bras. 38(2003)1105-1110. [33] X. Liu, C. Tang, W. Han, H. Xuan,, J. Ren, J. Zhang, et al., Characterization and preservation effect of polyelectrolyte multilayer coating fabricated by carboxymethyl cellulose and chitosan, Colloid. Surface. A: Physicochem, Eng. Aspects 529 (2017) 1016-1023. [34] X. Liu, J. Ren, Y. Zhu, W. Han, H. Xuan, L. Ge, The preservation effect of ascorbic acid and calcium chloride modified chitosan coating on fresh-cut apples at room temperature, Colloid. Surface. A: Physicochem. Eng. Aspects 502 (2016) 102-106. [35] S. P. Moreira, W. M. Carvalho, A.C. Alexandrino, H.C.B.Paula, M.D.C.P. Rodrigues, R.W. Figueiredo, et al., Freshness retention of minimally processed melon using different packages and multilayered edible coating containing microencapsulated essential oil, Int. J. Food Sci. Technol. 49 (2015), 2192-2203. [36] H. Liu, F. Chen, H.Yang, Y. Yao, X. Gong, Y. Xin, C. Ding, Effect of calcium treatment on nanostructure of chelate-soluble pectin and physicochemical and textural properties of apricot fruits, Food Res. Int. 42(2009)1131-1140. [37] Y. Xin, F. Chen, S. Lai, H. Yang, Influence of chitosan-based coatings on the physicochemical properties and pectin nanostructure of chinese cherry, Postharvest Biol. Technol. 133(2017) 64-71.
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pH
WIC
WSC
Deionized water (0) 0.5 0.7 1.0 2.0
7.4 3.9 3.6 3.5 3.4
+ +
+ + + + +
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*: +, soluble; -, insoluble.
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Firmness (N)
Titratable acidity (%)
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ANOVA analysis
0
2
4
10
WSC
-
3.83±0.19
6.36±0.32
13.93±0.70
16.70±0.83
0.0321
0.0065
WIC
-
3.80±0.19
8.66±0.43
12.39±0.62
15.00±0.75
0.0386
0.0123
CK
-
5.03 ±0.25
12.33±0.62
17.55±0.88
21.90±1.09
-
-
1.37±0.07
-0.178
0.0213
1.31±0.07
0.0673
0.0007
S U
1.23±0.09
-
-
1.28±0.06
0.0061
0.0001 0.461
T P I
Ps**
WSC
1.56±0.05
1.50±0.08
1.46±0.10
1.41±0.07
WIC
1.57±0.06
1.53±0.07
1.45±0.08
1.37±0.06
CK
1.56±0.05
1.51±0.08
1.44±0.07
1.32±0.08
WSC
1.09±0.06
1.04±0.07
1.13±0.05
1.26±0.06
WIC
1.18±0.05
1.56±0.08
1.69±0.07
1.52±0.08
1.54±0.07
0.0164
CK
1.08±0.05
1.00±0.04
1.08±0.04
1.22±0.05
1.24±0.05
-
N A
M
R C
Pt***
-
*CK, control group. **Ps, P value of ANOVA between coating group and the CK group. ***Pt, P value of ANOVA of the corresponding time.
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Calculation based on uncertainty=0.05
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ANOVA of both weight loss and titratable acidity about coating varieties showed that there were statistical differences among the WSC, WIC, and CK groups; however, on firmness there was no statistical differences among the three groups. In particular, distinctions about time survived in weight loss and firmness of both WSC and WIC, and so did in titratable acidity of WSC, but there was no distinction about time in titratable acidity of WIC.
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Ning Li, Xiaoli Xiong, Xia Ha, Xingyue Wei PII: DOI: Reference:
S0141-8130(19)30132-1 https://doi.org/10.1016/j.ijbiomac.2019.04.094 BIOMAC 12170
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
6 January 2019 31 March 2019 12 April 2019
Please cite this article as: N. Li, X. Xiong, X. Ha, et al., Comparative preservation effect of water-soluble and insoluble chitosan from Tenebrio molitor waste, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.04.094
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ACCEPTED MANUSCRIPT Comparative preservation effect of water-soluble and insoluble chitosan from Tenebrio molitor waste Ning Li, Xiaoli Xiong, Xia Ha, Xingyue Wei Chongqing Engineering Research Center for Processing ＆ Storage of Distinct Agricultural Products, Chongqing Technology and Business University, Chongqing 400067, China E-mail: [email protected] Edible films and coatings have been developed based on numerous natural biopolymers, which
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have been used to increase fresh-cut fruit shelf life. Here, we present the preparation, characteristics and preservation effect of water-soluble chitosan (WSC) and water-insoluble chitosan (WIC) from Tenebrio molitor waste (TMW) on fresh-cut apple slices. WIC was isolated from TMW in four steps and WSC was obtained from the WIC solution by 8 % H2O2 treatment at 40℃for 3 h. WIC and WSC were characterized by molecular weight, Fourier transform infrared spectroscopy (FTIR), morphology analysis, etc. The preservation effects of WIC and WSC for the fresh-cut apple slices were evaluated by the indexes of browning, weight loss, firmness and titratable acidity. The results showed that WSC was soluble in water and that the chemical structures of WIC and WSC were similar. However, their crystallinity, morphology and thermal properties were different. Both WSC and WIC had a good preservation effect on fresh-cut fruits. Compared with WIC, WSC might be more suitable for use in the food industry owing to its water solubility. Keywords: Tenebrio molitor waste, water-soluble chitosan (WSC), water-insoluble chitosan (WIC), preservation
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1. Introduction
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Tenebrio molitor (TM) is an edible insect that is distributed worldwide and is a convenient candidate for protein or fat isolation for the health foods and products industry. TM produces a large amount of waste during the three insect growth stages: larva, pupa and adult. The larvae molt 10–15 times and produce a large amount of exuvia during their growth. In addition, other byproducts, such as the dead beetles after oviposition and the dead larvae and shells of the pupae, are also produced. These byproducts are rarely used for other purposes and are normally considered to be wastes, which may contaminate the environment. Therefore, reasonable treatments of TM wastes (TMW) are essential. One of the important polymers, chitin, can been extracted from the bodies of these wastes. Chitin is an insoluble carbohydrate polymer, a long-chain polymer of repeating N-acetylglucosamine residues connected exclusively by linkages. This polymer is the second most abundant natural biomass resource that is derived from the exoskeletons and cuticles of insects, the shells of marine invertebrates and the cell walls of certain fungi and algae [1]. The extraction of chitin from TMW suggests it is a better source than that from other insects, due to the stable supply of raw materials and its low collection cost. Chitosan, the main derivative of chitin, is generally produced from N-deacetylation of chitin under high concentration alkaline conditions [2]. Chitosan has many applications in the environment, agriculture, cosmetics, and in food packaging (either as barrier coatings or as an antibacterial agent) industries due to its non-toxic, biodegradable, antimicrobial and anti-oxidation properties [3-5]. However, most high molecular weight chitosans are insoluble in water (namely, water-insoluble chitosan, WIC, only completely soluble under acid conditions) due to its strong intramolecular hydrogen bonding, which limits its applications. Therefore, many researchers have tried to lower the molecular weight of chitosan and improve its solubility [6]. Water-soluble chitosan (WSC, completely soluble in neutral aqueous solutions) can be 1
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obtained from WIC by enzymatic or chemical hydrolysis. WIC can be applied to pharmaceuticals, pharmaceutical ingredients, pesticides, food, etc. [6, 7]. Currently, in restaurants, school lunch programmes, food services, street and domestic houses, etc., fresh-cut fruits supply important vitamins and have become common desserts or popular snacks. However, fresh-cut fruits are susceptible to spoiling, which could directly influence or lower consumer demand. Thus, a natural edible coating to reduce decay and prolong the shelf life of fresh-cut fruits is an alternative to this problem [8]. Some research articles have reported the application of chitosan on fresh-cut fruits. Chitosan-coating treatments can effectively reduce the occurrence of enzymatic browning on the surface of fresh-cut apples during storage [9]. Chien et al.[10] reported that a chitosan coating effectively prolongs the quality attributes and extends the shelf life of sliced mango fruit. Chitosan can be used as a natural preservative due to its antimicrobial activity against a wide range of foodborne microorganisms, including Gram-negative and Gram-positive bacteria, yeasts, and moulds [11], as well as its antioxidant activity and physical protection actions, such as the formation of a barrier across the cell surface, hindering the exchange of metabolites [12,13]. The use of natural resources as a source of valuable compounds to be used in the food industry is currently a topic of growing interest. The main objective of this work was the development of a technology for the preparation of WSC and WIC from TMW. This technology can not only solve the environmental pollution problems caused by TMW but also identify an alternative natural resource for producing chitosan from their wastes. One of the potential applications of WSC is that it can be used as a preservation agent for fresh-cut fruits. Compared with other chitosan-based preservative agents, water-soluble chitosan might be more suitable for use in the food industry owing to its water solubility without the need for other additive agents and it could maintain the original flavour of foods [9].
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2. Materials and methods
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2.1 Materials TMW was obtained from Chongqing Bole Agri. Sci. Tech. Co., Ltd. Apples (Red Fuji) were selected based on no apparent injury with a uniform size, ripeness and colour, and purchased from a local wholesale distributor. The applied chemicals were analytical grade. 2.2 Preparation method 2.2.1 Pre-treatment of TMW TMW was dried and smashed directly. The main characteristics of the processed TMW were as follows: a particle size of 0.5 mm, a density of 126.8 kg.m-3, 1.3 % (w/w) ash, 22.5 % (w/w) crude fat and 58.7 % (w/w) nitrogenous compounds (protein and non-protein). The contents of the crude ash, the crude fat and the nitrogenous compounds in TMW were determined by a standard ash analysis method, an automated Soxhlet method and the Kjeldahl method, respectively [14-16]. 2.2.2 Extraction and purification of WIC and WSC WIC were separated from pre-treated TMW using four steps: degreasing (DG), demineralization (DM), deproteinization (DP) and deacetylation (DA). DG step: a sample of the pre-treated TMW was treated with petroleum ether (30 ℃–60 ℃) at 55 ℃for 2 h by Soxhlet with a solid-liquid ratio of 1:10 (w/v). DM step: The DG residue was immersed in 0.3 mol/L HCl solution at ambient temperatures (25 ± 2℃) for 8 h. DP step: the sample after DM treatment was dispersed in 1 % (w/w) NaOH solution for 3 h to remove organic substances such as proteins and fats. DA step: The DP residue was dispersed in a 60 % (w/w) NaOH solution at 130 ℃for 7 h (DA). The experimental solid portions in all steps were washed with deionized water until a neutral pH was achieved. Then, the sample was dried at 55 ℃for 2 h and the product was obtained (WIC). 2
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DD %
6.85 R 100% (1) 1.71
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Next, 10 g of WIC was dissolved in a 0.15 mol/L HCl solution and 500 mL 0.75 mol/L H2O2 was added to the WIC solution during 100 r/min stirring at 40 ℃for 3 h. The solution was precipitated three times with ethanol, then the mixture was left to stand for 24 h and centrifuged and dried at 50 ℃(WSC). 2.3 Characteristics of WIC and WSC 2.3.1 Molecular weight and solubility testing The molecular weights of the WSC and WIC were measured by End-group analysis [17]. The solubility of the sample was tested by using the following methods: Each 1 g of sample (WSC or WIC) was dispersed in 100 mL of varying concentrations of acetic acid solutions (0 %–2 %, v/v) using a vortex. 2.3.2 FTIR spectroscopy FTIR was performed through a Nicolet Impact 400 D FTIR spectrophotometer (Shimadzu, Japan). The transparency was measured with a function of the wave number between 4000 cm−1 and 400 cm−1 with a resolution of 2 cm−1 and the number of scans was 18. 2.3.3 X-ray diffraction (XRD) analysis XRD patterns of samples were recorded by a Rigaku D/MAX 2500PC X-ray diffraction diffractometer (Rigaku, Japan) with a monochromatized X-ray beam using nickel-filtered Cu Kα radiation. The generator was operated at 40 kV and 150 mA. The diffraction intensity was recorded every 0.02 s from 5 ° to 90 ° at a rate of 4 ° /min. 2.3.4 Thermal monitoring Thermal analysis was performed on a Netzsch STA 449 C (Netzsch Instruments, Inc., Germany). Approximately 5 mg of sample was placed in an Al2O3 pan then heated to 500 ℃at a rate of 10 ℃/min, under a flow of argon at 10 mL/min. The starting temperature was 30 ℃. 2.3.5 Elemental analysis The samples were analysed with a Vario MICRO Elemental Analyzer EL III (Elementar, Germany) using 5 mg of the samples to elucidate the ratio of C, H, and N. The degree of deacetylation (DD) of the WIC or WSC was calculated from the elemental analysis by using Eq. (1):
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where DD % is the degree of deacetylation, R is the C/N value of WIC or WSC measured by elemental analysis, 6.85 corresponds to the theoretical C/N value of the chitin, and 1.71 corresponds to the difference between the theoretical C/N of chitin (6.85) and the theoretical C/N of WIC or WSC (5.14). 2.3.6 Morphology observations The morphology images of WIC and WSC were taken by SEM (Nova Nano SEM 450, FEI, USA) and a TEM (JEM-2100F, JEOL, Japan). SEM test conditions: 15 nm layers of gold particles coated on the dried powder samples, voltage 20 kV, high vacuum and room temperature. TEM test conditions: accelerating voltage 200 kV, room temperature. 2.4 Preparation of apple samples The apples were pre-washed in drinking water and then deionized water to remove any residual substances. The clean apples were cut into slices with a disinfected knife under sterile conditions. All slices without a peel used in this experiment were uniform in size, shape and weight (8 ± 0.5 g). 2.5 Preservation of fresh-cut apples One of the potential applications of WIC or WSC is that it can be used as a preservation agent for fresh-cut fruits. The preparation effect was tested by the following method: The prepared apple slices were divided into three groups randomly. The experimental groups were immersed in 10 g/L WSC or WIC solutions (WSC, deionized water solution; WIC, 1 % (v/v, aqueous acetic acid) for 2 min and the control groups were immersed in deionized water for the same time. Subsequently, the apple slices were ventilation dried on a sieve at room temperature for 3
ACCEPTED MANUSCRIPT 2 min, and then placed in the same sized plastic tray. Each parameter was measured at 0, 2, 4, 7, and 10 h. 2.5.1 Colours The colour of the cutting surfaces of all fresh-cut apples was measured with a colorimeter (NR60CP, Shenzhen 3nh Technology Co., Ltd, China). L, a and b values were recorded. 2.5.2 Weight loss Weight loss is considered as an important factor to evaluate apple quality, which is closely related to water loss. The weights of the coated fresh-cut apple slices were measured with an Analytical Balance (XPE205DR, Mettler Toledo, China). The weight loss ratio W (%) was expressed as Eq. (2) [18]:
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(2)
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0.067C1V1V2 100% V3W
(3)
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Where Wi (g) is the initial weight of the experimental apple slices and Wj (g) is the final weight of the experimental apple slices. 2.5.3 Firmness The firmness of fresh-cut apples is a parameter of freshness. It was determined with a texture analyser (Brookfield CT-3, USA) equipped with a P/2 probe (2 mm diameter). 2.5.4 Titratable acidity A total of 10 g of apple slices were prepared by using a direct homogenizer and a filter, then deionized water was added up to 100 mL and 10 mL of the fresh-cut apple juice was titrated by 0.1 mol/L NaOH with phenolphthalein used as the indicator. The end point of the reaction was identified as transformation of the colourlessness into a pale red (Supapvanich et al., 2011). The titratable acidity (TA, expressed as the percentage of malic acid) was calculated by using Eq. (3) [19]:
where C1 (mol/L) is the concentration of NaOH, V1 (mL) is the volume of NaOH, V2 (mL) is the total volume of
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apple juice, V3 (mL) is the titration volume of apple juice, W (g) is the total weight of the fresh-cut apple, and 0.067 is the conversion coefficient of malic acid. All of the experiments were repeated three times in parallel and the results were expressed as mean values and standard deviation. The data were analysed with Origin Pro. 8.0 Software.
3 Results and discussion
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3.1 Characteristics of WIC and WSC 3.1.1 Molecular weight and solubility testing The molecular weights of the WIC and WAC were 96.2±3.5 kDa and 9±1 kDa, respectively. Table 1 shows the solubility of both WIC and WSC in different solutions. The results showed that WSC was soluble from 0 % (deionized water) to 2 % (v/v) acetic acid concentrations, but the WIC was insoluble at acetic acid concentrations lower than 1 % (v/v). 3.1.2 FTIR spectroscopy In Fig. 1, curves a (WSC) and b (WIC) exhibited similar characteristic peaks. The -OH and -NH2 symmetric stretching vibrations were observed at 3600–3200 cm-1 and asymmetrical C-H bending vibrations of the -CH2 and -CH3 groups were observed at 1382–1385 cm-1 [20,21]. The FTIR spectra of both WSC and WIC showed characteristic bands of amide groups (1639–1655 cm-1, carbonyl stretching of amide I). A weak absorption peak was observed at 1595 cm-1 in the spectrum of WIC, assigned to the NH bending vibration of amide II. However, 4
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the peak at 1595 cm-1 did not appear in the spectrum of WSC. The shift of amide I and the disappearance of amide II indicated a difference in the molecular interaction and degree of deacetylation between WIC and WSC [4]. WSC had higher degree of deacetylation than WIC, which were also proved by the following elemental analysis. The high degree of deacetylation supported the solubility of chitosan in water. Generally, the band at 1074 cm−1 was assigned to the stretching of C-O-C in the glucose ring [22]. The spectra of WIC and WSC showed a glucose structure in the backbone at 897 cm-1 and a pyranose ring at 1255 cm-1. The weak absorption peaks of the β-(1,4)-glucoside bond were observed at 897 and 1154 cm-1 in WIC. However, the peak at 1154 cm-1 was not apparent in the spectrum of the WSC, which indicated that a number of β-(1,4) glycosidic bonds of WIC were broken and WSC was formed [21, 22]. The differences between WSC and WIC were also proven by the above molecular weight analysis. These data demonstrated that the chemical structures of the main chains of WIC and WSC were similar with appropriate shifts or disappearances of the adsorption peaks. These results were basically consistent with previous reports [20, 21].
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Fig. 1 FTIR spectra of samples. (a) WSC (b) WIC.
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3.1.3 X-ray diffraction (XRD) WIC and WSC were characterized by XRD in order to verify the substances’ crystallinity. The XRD patterns of the isolated WIC and WSC are shown in Fig. 2. Two sharp peaks and several weak peaks were found in the XRD diffraction pattern of WIC. Two obvious sharp diffraction peaks at an angle 2θ=10.97 ° and 20.07 ° were typical fingerprints of semi-crystalline WIC and consistent with the literature data from different sources of WIC [23]. Islam et al. [24] also observed broad diffraction peaks at 2θ=10 ° and 21 ° that symbolized semi-crystalline WIC. The degree of crystallinity of WIC was 15.45 % based on the calculation of the intensity of the crystalline regions and amorphous regions (Jade 6.5, New Zealand). Two sharp peaks approximately 10° and 20° and several weak peaks from 27° to 30° were observed, which indicated that WIC was in the α-form [25]. In Fig. 2, WSC samples showed amorphous diffraction with a very broad hump at 2θ=21.15 °, which could indicate that WSC exists in a form with a noncrystalline state. A potential explanation of the crystallinity differences between WSC and WIC is the crystalline properties of WIC, due to the inter and intramolecular hydrogen bonds [26]. During the preparation of WSC, a number of β-(1,4) glycosidic bonds of WIC were broken, which could be shown by the above molecular weight and FTIR analysis and the long chains of WIC could be broken into short chains by H2O2, and WSC with a lower molecular weight that had fewer intramolecular hydrogen bonds [7]. Hence, we speculated that the WSC was amorphous due to a lack of intramolecular hydrogen bonds, which was shown in the XRD photos.
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Fig. 2 XRD patterns. (a) WSC, (b) WIC.
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3.1.4 Thermal analysis Fig. 3 demonstrates the results of the TG (thermogravimetric analysis) and DSC (differential scanning calorimetric analysis) curves for WIC and WSC. In the TG curves of the two samples, there were three characteristic temperature intervals of weight loss. The first one (30–105 ℃) with approximately 4 % (w/w) (WIC) and 6 % (w/w) (WSC) weight loss could be attributed to the evaporation of water that was physically and chemically adsorbed in the WIC and WSC [27]. The curves of the DSC showed the broad endothermic effect (maximum endothermic peaks at 89.6 ℃and 112.4 ℃for WIC and WSC, respectively). The second weight losses, occurring in the range 120–340 ℃, were 45 % (w/w) and 62 % (w/w) for WIC and WSC, respectively and were caused by depolymerisation/decomposition of polymer chains through deacetylation and cleavage of glycosidic linkages [22]. At this stage, the weight loss temperature of the WSC was lower than that of the WIC. The differences in the thermal behaviour of WIC and WSC might be ascribed to their molecular weight or polymerization degree. Commonly, the samples with a higher molecular weight and crystallinity indicated more thermal stability [28]. WSC showed a low molecular weight, and the amorphous structure was verified by End-group analysis and XRD. In contrast, WIC displayed a high molecular weight and crystallinity structure, which can be attributed to the initial decomposition temperature for WIC being 233.6 ℃, while the corresponding value for WSC was located at 195.2 ℃. The DSC curve of WIC exhibited a maximum exothermic peak, Tmax = 309.6 ℃, with an enthalpy change of −129.2 J/g. However, the DSC curve of WSC showed no obvious exothermic peak. In the last stage, the WIC decomposition temperature was higher than 340 ℃, corresponding to the thermal destruction of the pyranose ring and the decomposition of the residual carbon [22, 27].
Fig. 3 TG -DSC curves of WIC and WSC.
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3.1.5 Elemental analysis According to the WIC and WSC formula, theoretical values (w/w) of C: 44.72 %, H: 6.83 %, N: 8.69 %, C/H: 6.5455, and C/N:5.1429 were predicted. Elemental analysis exhibited the composition of WSC: C: 37.14 %, H: 5.89 %, N: 6.92 %, C/H: 6.3056, and C/N:5.3698; and the composition of WIC: C: 43.84 %, H: 7.17 %, N: 7.85 %, C/H: 6.1144, and C/N:5.5847. The DD values of WIC and WSC were 73.99 % and 86.6 % by Eq (1), respectively. 3.1.6 Morphology observations The morphology of the WSC and WIC were studied by SEM (scanning electron microscopy) and TEM (transmission electron microscopy) (Fig. 4). The surface of the WSC showed loose aggregates, like earth flaws (Fig. 4a). The surface of the WIC was fibrous and porous, with some regular flaky texture, like chrysanthemum petals (Fig. 4b). Similar surface morphology has been reported for the pupa of Vespa crabro, Archispirostreptus gigas and Zophobas morio larvae [29-31]. The WSC powder was also observed by TEM, aiming at further study of the aggregation morphology about WSC particles. The TEM image (Fig. 4c) showed that the powder of WSC was composed of loose aggregates of a fibrous network with countless like-globular perforated holes (size 20–400 nm), which was beneficial to the dissolution of WSC due to the rapid permeation of the solvent into the WSC. In addition, the XRD analysis proved that WIC was partly crystalline and that WSC was in an amorphous form. Due to the oxidation of H2O2, the lamellar WIC was cracked into small fragments, which were disordered together due to the lack of intermolecular forces and they formed an amorphous structure. The increase of water solubility and the decrease of thermal stability of WIC were due to the decreases in molecular weight and intermolecular forces.
(a)
(b)
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Fig. 4 SEM images of WSC (a) and WIC (b); TEM image of WSC (c).
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3.2 Application of WSC 3.2.1 Appearance of apple slices Apple browning is caused by an oxidation process that occurs during maturation stages. Changes in L (lightness, +L to lightness, -L to darkness), a (+a to redness, -a to greenness), b (+b to yellowness, -b to blueness) and ∆E (comprehensive evaluation index, ∆E=(∆L2+∆a2+∆b2)1/2, is a widely applied parameter for determining colour differences that can be observed by the naked eye, and all of these values are considered to be part of the browning index. As shown in Fig. 5a, “a” values increased but the increasing tendency of the control group was higher than that of the coated groups. The degree of changes in the “b” values of the WSC, WIC and control groups was slight (Fig. 5b). All of the apple slices turned brown over time accompanied by decreasing L values, but the decreasing degree of the control group was higher than that of the coated groups (Fig. 5c). In fact, the variations of lightness (∆L) between 0 h and 10 h were 8.8 and 6.3 for the control group and WSC group, respectively. During the first 4 hours, the L value of the WSC group showed more changes than the WIC group, and the opposite situation was found during the next 4 hours. The increase of redness values (a) and the decrease of L values (darkness) indicated continuous browning of the samples [32]. The ∆E values of the experimental groups were lower than that of the control group (Fig. 5d). In regards to the browning degree of the fresh-cut apple slices, the coated groups with WSC and WIC were less than that seen in the control group. In contrast, the WSC group had smaller ∆E value changes, suggesting a lower chance of the WSC group browning. In previous studies, it was also reported that chitosan had a better effect on preventing apple browning. The browning degree of apples coated with chitosan was less than 20 % at 12 h and the browning of apples coated with nothing was greater than 50 % [33].
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Fig. 5 Chromatism changes of fresh-cut apples. (a) a values; (b) b values; (c) L values; (d) ∆E values
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3.2.2 Weight loss The weight loss ratio of the uncoated (control group) and coated fresh-cut apple slices with WSC and WIC are shown in Table 2. All fresh-cut apple slices showed a gradual weight loss process, and the loss ratios of the coated groups were significantly less than that of the control group due to the formation of coating films on the surfaces of the samples. After 10 h, the uncoated fresh-cut apple slices had an average weight loss ratio of 21.89 %, whereas the coated groups were 15.00 % and 16.69 % for WIC and WSC, respectively. The water conservation effect of WIC and WSC were similar, the statistical differences among different treatments were listed in Table 2. Liu et al. [34] also demonstrated that the weight loss ratio of fresh-cut apples after 10 h coated with nothing or coated with chitosan, chitosan-ascorbic acid, or chitosan-CaCl2 were 16 %, 14 %, 13 % and 11 %, respectively. These results demonstrated that the chitosan coated apples had a good ability of water loss inhibition when compared with the control apple slices. 3.2.3 Firmness Firmness is one of the important factors for fresh-cut fruit quality. As shown in Table 2, the firmness of the fresh-cut apple slices decreased as time increased. At the beginning of the experiment, the firmness of the fresh-cut apple slices was nearly the same for both the coated and uncoated groups. After 7 h, the firmness of the coated group was higher than that of the control group due to the protective effect of WSC or WIC. We found that after 7 h WSC resulted in the highest firmness for fresh-cut apples, the statistical differences among different treatments were listed in Table 2. A physical and mechanical barrier was formed to delay the respiratory metabolism. Consequently, the water loss was through dehydration and enzymatic activities, which were related to fruit softening [35]. Liu et al [33] also demonstrated that the firmness of fresh-cut apple slices coated with chitosan was 14 % higher than those without chitosan. 9
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3.2.4 Titratable acidity As shown in Table 2, titratable acidity (TA) values slightly increased over time for all sample groups due to the accumulation of acids from D-glucose by glycolytic enzyme systems and water loss [36,37]. There were no obvious differences in TA values between coated fresh-cut apples with WSC and the control group. However, the TA values of the coated fresh-cut apples with WIC significantly increased due to the residual acetic acid on the surface of the fruits. After 4 hours, the TA values of fresh-cut apple slices coated with WIC decreased due to the gradual volatilization of acetic acid. The statistical differences among different treatments were listed in Table 2. It is widely accepted that taste is one of the most important quality parameters in determining consumer acceptability of fruits, and TA values directly affect the taste of fruit [37]. In the current study, fresh-cut apple slices coated with WIC showed some increases in acidic value that affected the taste of the fruit. Briefly, WSC was more suitable for the preservation of fresh-cut fruits.
4. Conclusions
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WSC and WIC were shown to improve the quality of fresh-cut apples over time in regards to their colour, texture and titratable acidity. These findings have provided experimental evidence for the applications of WSC and WIC. Compared with WIC, WSC might be more suitable for use in the food industry owing to its water solubility. A further study on its physicochemical properties, safety and applications is in progress.
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We are grateful to Dr. Geoffrey Onaga who kindly offered the English revision. We would also thank our colleagues Dr. Wen Yun for the improvement of the language. We thank Funying Jiao, Chunyan Xie and Peiwen Jin for the data analysis. This work was supported by the project of Basic Science and Frontier Technology from Chongqing Science and Technology Commission, PRC (cstc2016jcyjA0592), the project for the Transformation of Excellent Achievements in Universities of Chongqing Education Committee (KJZH17125), and the project of Training Innovative Talents for Primary and Secondary Schools of Chongqing Education Committee (CY180801). We thank the anonymous reviewers for their careful reading and constructive comments.
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ACCEPTED MANUSCRIPT Table 1 Solubility of WIC and WSC in varying concentrations of acetic acid* Concentration (%)
pH
WIC
WSC
Deionized water (0) 0.5 0.7 1.0 2.0
7.4 3.9 3.6 3.5 3.4
+ +
+ + + + +
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*: +, soluble; -, insoluble.
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Firmness (N)
Titratable acidity (%)
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ANOVA analysis
0
2
4
10
WSC
-
3.83±0.19
6.36±0.32
13.93±0.70
16.70±0.83
0.0321
0.0065
WIC
-
3.80±0.19
8.66±0.43
12.39±0.62
15.00±0.75
0.0386
0.0123
CK
-
5.03 ±0.25
12.33±0.62
17.55±0.88
21.90±1.09
-
-
1.37±0.07
-0.178
0.0213
1.31±0.07
0.0673
0.0007
S U
1.23±0.09
-
-
1.28±0.06
0.0061
0.0001 0.461
T P I
Ps**
WSC
1.56±0.05
1.50±0.08
1.46±0.10
1.41±0.07
WIC
1.57±0.06
1.53±0.07
1.45±0.08
1.37±0.06
CK
1.56±0.05
1.51±0.08
1.44±0.07
1.32±0.08
WSC
1.09±0.06
1.04±0.07
1.13±0.05
1.26±0.06
WIC
1.18±0.05
1.56±0.08
1.69±0.07
1.52±0.08
1.54±0.07
0.0164
CK
1.08±0.05
1.00±0.04
1.08±0.04
1.22±0.05
1.24±0.05
-
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Pt***
-
*CK, control group. **Ps, P value of ANOVA between coating group and the CK group. ***Pt, P value of ANOVA of the corresponding time.
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Calculation based on uncertainty=0.05
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ANOVA of both weight loss and titratable acidity about coating varieties showed that there were statistical differences among the WSC, WIC, and CK groups; however, on firmness there was no statistical differences among the three groups. In particular, distinctions about time survived in weight loss and firmness of both WSC and WIC, and so did in titratable acidity of WSC, but there was no distinction about time in titratable acidity of WIC.
E C
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