Recrystallization kinetics of starch microspheres prepared by temperature cycling in aqueous two-phase system

Accepted Manuscript Title: Recrystallization Kinetics of Starch Microspheres Prepared by Temperature Cycling in Aqueous Two-phase System Authors: Huiping Xia, Tingting Kou, Ke Liu, Qunyu Gao, Guihong Fang PII: DOI: Reference:

S0144-8617(18)30697-0 https://doi.org/10.1016/j.carbpol.2018.06.043 CARP 13717

To appear in: Received date: Revised date: Accepted date:

30-1-2018 6-6-2018 9-6-2018

Please cite this article as: Xia H, Kou T, Liu K, Gao Q, Fang G, Recrystallization Kinetics of Starch Microspheres Prepared by Temperature Cycling in Aqueous Two-phase System, Carbohydrate Polymers (2018), https://doi.org/10.1016/j.carbpol.2018.06.043 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Recrystallization Kinetics of Starch Microspheres Prepared by Temperature Cycling in Aqueous Two-phase System Huiping Xiaa,b, Tingting Koua,b, Ke Liua,b, Qunyu Gaoa,b,*, Guihong

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Fanga,c,*

Postal address:

Carbohydrate Laboratory, School of Food Science and Engineering, South China University of

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a

Technology, Guangzhou 510640, P.R. China b

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Guangdong Province Key Laboratory for Green Processing of Natural Products and Product

Department of Nutrition and Food Hygiene，Hainan Medical University，Haikou，Hainan 571199，

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c

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Safety, Guangzhou 510640, P.R. China

author. Tel: +86-13660261703; Fax: +86-20-87113848; [email protected] (Gao),

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*Corresponding

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P.R. China

[email protected](Fang)

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Email: [email protected](Gao), [email protected](Xia), [email protected](Fang),

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[email protected](Kou), [email protected](Liu).

Highlights  The recrystallization behavior of starch microspheres (SMs) in ATPS was studied.  Temperature cycling could the recrystallization of SMs.

equation.

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 Temperature cycling could promote the stability of SMs.

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 The recrystallization kinetics of SMs were investigated through Avrami

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Abstract: The recrystallization behavior of starch microspheres (SMs) prepared by

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temperature cycling in aqueous two-phase system (ATPS) was investigated. The SMs

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were carried out under the temperature-cycled treatment at 4 °C, 30 °C or 4/30 °C for

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2 to 20 days. X-ray diffraction (XRD) results showed that the crystalline structure of

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SMs were different from that of degraded cassava starch. Compared to degraded cassava starch, the relative crystallinity of SMs under different temperature decreased,

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and the increase in relative crystallinity with the storage time was observed. All

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gelatinization temperature parameters (To, Tp and Tc) and enthalpy of gelatinization (ΔH) of SMs decreased compared with degraded cassava starch. However, these values of SMs stored at 30 °C were higher than that of SMs stored at 4 °C and

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4/30 °C. The Avrami equation was applied to analysis the recrystallization behaviors of SMs. The stability test showed that the samples stored at 30°C were more stable than that stored at 4 °C and 4/30 °C. Keywords: Starch microspheres, Aqueous two-phase system, temperature cycling, Avrami equation.

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1 Introduction Starch, as one of the large number of naturally occurring renewable natural polymers, is the major carbohydrate materials in human diet. Starch granules are composed of essentially linear amylose and highly branched amylopectin with α-Dglucopyranose, and the branched molecules form mainly crystalline regions and the linear molecules make up amorphous regions (Hoover, 2001; Kaur, Ariffin, Bhat, &

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Karim, 2012). During the gelatinization process, because of the existence of heat and water molecules, the starch granules become disordered. After cooling and storage,

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due to the molecular potential energy, the disordered high energy states gradually tend

to the ordered low energy states which is the phenomenon of starch retrogradation (Karim, Norziah, & Seow, 2000). In general, this is a return of starch from solvated,

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dispersed, amorphous state to an insoluble, aggregated or crystalline condition. Starch

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is widely used in the fields of food and non-food due to these peculiar properties

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(BeMiller & Whistler, 2009).

Among the applications of starch, starch microspheres (SMs) were one of the

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most important products (Hamdi, Ponchel, & Duchêne, 1998; Lin, Pan, & Liu, 2013).

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Due to the unique advantage especially the biocompatibility and biodegradability, SMs are being run by so many researchers from different fields. Therefore, many researchers had reported the preparation of starch microspheres from different sources

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and applied them in medicine, food, chemical and other fields (Pareta & Edirisinghe, 2006; Wang, et al., 2015). In recent years, one of the classic and widely used methods

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of preparing starch-based microspheres was the emulsion technology, especially the water-in-oil (W/O) emulsification technology (Hamdi, 2008; Shi, Li, Wang, Li, & Adhikari, 2011). Generally, during the preparation, the use of organic toxic solvents

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was considered to influence the embedding of certain active substances or their safety, especially in the fields of food and pharmaceutics (Zhou, Luo, & Fu, 2014). For its safety considerations, an environmentally friendly emulsification technology based on aqueous two-phase system (ATPS) was introduced into the preparation of SMs. Since 1998, Franssen et al. (1998) have invented and applied this method to produce dextrin microsphere. Later, it was found that many water-soluble polymers exist the

immiscibility, such as dextran, ficoll, polyethylene glycol (PEG), polyvinyl alcohol, gelatin, soluble starch and so on (Cheung Shum, Varnell, & Weitz, 2012; Giuliano, 1995). Elfstrand, et al. (2006) found that the starch material and buffer were the decisive factors in the formation of SMs. Li et al. (2012) found that it is feasible to use the w/w emulsion-crosslinked methods to prepare SMs. They used octenyl succinate starch as raw materials, polyethylene glycol as the continuous phase, and

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trisodium trimetaphosphate as crosslinker to prepare SMs with a good dispersion and

good shape. In 2015, Puncha-arnon et al. (2015) studied the effect of crosslinking

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temperature and time on the microstructure and stability of starch in ATPS. In summary, the application of the ATPS to the preparation of starch microspheres has been approved by many researchers.

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To achieve SMs with round-shaped and well-defined particles, one of the

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common methods is to utilize the recrystallization (retrogradation) of starch through

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hydrogen bonding. There are many factors could affect the process of recrystallization of starch, such as the average molecular weight of starch, the distribution of chain

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length, moisture, temperature, sugars, salts and so on. Generally, the short chain of

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amylopectin needed more time to retrograde compared with amylose (Fredrisson, et al., 1998). Farhat et al. (2015) demonstrated that the rate of retrogradation depends strongly on the water content of the sample and the storage temperature. Li et al. (Li,

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Li, & Gao, 2015; Li, Zhang, Ye, & Gao, 2015) reported the different sugars and salts could accelerate or inhibit the gelatinization process of starch granules. In the industry

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production, temperature is an important factor to influence the quantity of the products. In previous studies, we found that only appropriate molecular weight of starch could form spherical SMs with sharp contours in ATPS (Xia, Li, & Gao, 2017).

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The Avrami equation was originally used to analysis the structures of metal materials and has been extensively applied to characterize the crystallization of synthetic polymers (Kim, et al., 2015; Miles, Morris, Orford, & Ring, 1985). And now, the Avrami theory has been widely applied to determining the retrogradation /recrystallization rate of starch gels and cooked rice (Shi & Gao, 2016; Yu, Ying, & Sun, 2010). We hypothesize that in ATPS, temperature might have a different effect

on the preparation of SMs. However, there are few reports about the different temperature cycling treatment on the formation of SMs in ATPS. Therefore, we aimed to explore the effect of isothermal tempering on the formation of SMs in the ATPS and to find out the relationship between recrystallization and the stability of starch microspheres by the Avrami equation in this study. 2 Materials and Methods

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2.1 Materials

Cassava starch was supplied by Jiantai biological technology co., Ltd (Dongguan,

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China). Its starch, protein, lipid, ash and amylose contents were determined by the

methods of Mccleary, Gibson, & Mugford (1997), standard AOAC methods (1990), and iodine spectrophotometric method (Hoover & Ratnayake, 2001). These results

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were 96.3%, 0.2%, 0.2%, 0.3% and 19.1%, respectively, on a dry weight basis. The

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degraded cassava starch used in this study was produced by acid hydrolysis, which

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named as DCS. The weight-average molecular weight of hydrolyzed starch samples was 4.359 ×104 g/mol as previously described by Xia et al. (2017). Polyethylene

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glycol (PEG, 20,000 g/mol) was purchased from Uni-Chem (USA). All other reagents

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used in this work were of analytical grade. 2.2 Preparation of starch microspheres

SMs were prepared by ATPS method described by Xia et al. (2017) with some

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modifications as follows (Figure 1). Degraded cassava starch suspension (20%, by weight) was heated at 95 ºC for about 30 min until the starch granules were

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completely gelatinized to form a homogeneous aqueous solution. The solution was cooled at room temperature to 55 ºC, then was slowly poured into a beaker containing 250 mL of PEG solution (35%, w/w) under constant mechanical stirring (300 rpm).

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The agitation was maintained at 55 ºC for 15 min. After emulsification, the beaker was stored for 2, 4, 8, 14 and 20 days under three different temperature conditions: constant temperature of 4 °C, 30°C or cycles of 4°C for 1day and subsequently 30°C for 1 day. These samples were denoted 4-2d to 4-20d for storing at 4°C, 30-2d to 30-20d for 30 °C, and 4/30-2d to 4/30-20d for 4/30°C, and so on. After incubation, the precipitate was washed with deionized water and 95% (v/v) ethanol for 3 times,

respectively. And the collected SMs were dried at 45 °C in a hot-air oven for 12 h to a final moisture content of 9 ~10%, then kept in sealed containers prior to use. Starch solution

Aqueous solution of PEG Agitating

Incubating Incubation at 4 °C or 30 °C or 4/30 °C for days

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Washing drying

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Aqueous two-phase emulsion

SMs

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Figure 1. flow chat for the fabrication of SMs

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2.3 Differential scanning calorimetry (DSC)

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Gelatinization characteristics were measured and recorded using a Perkin Elmer DSC8000 (Shelton, CT, USA), equipped with a thermal analysis software (Pyris

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window, Perkin Elmer). A starch sample (dry basis, 3 mg) was weighed in a DSC pan

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and the excess water was added to obtain a starch/water ratio of 3:7. And then the well-prepared samples were kept at 25 ± 2 °C for 4 h to equilibrate the starch samples and water. The samples were scanned from 20 °C to 120 °C at the rate of 10 °C/min.

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An empty pan was used as a reference. The onset (To), peak (Tp), and conclusion (Tc) temperatures of gelatinization as well as enthalpy change of gelatinization (ΔH) were

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determined.

2.4 Analysis of recrystallization rate The recrystallization kinetics was analyzed by the Avrami equation (Baik, Kim,

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Cheon, Ha, & Kim, 1997). The basic form of the Avrami equation is: V=1-exp (-k∙ tn)

(1)

where V is the percentage of crystallization at time t, k is the crystallization rate constant (time-n), and n is the Avrami exponent and presents the forming mode of nuclei.

The relationship between V and the enthalpy (H) from DSC is analyzed by the following equation: V= (Ht -Ho)/(H1 -Ho)

(2)

where Ho is the enthalpy at time 0, Ht is the enthalpy at time t, H1 is the maximum enthalpy. Equations (1) and (2) are combined to obtain the following relationship: ln-ln(Ht -Ho)/(H1 -Ho) =lnk+ nlnt

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(3)

The kinetic parameters (n and k) were obtained by regression analysis of

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recrystallization enthalpy from DSC. 2.5 Wide-angle X-ray diffraction patterns (XRD)

X-ray diffractometer (D8 Advance, Bruker, Germany) was used to analyze X-ray

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diffractometer. Before the measurement, starch samples were equilibrated in a

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saturated relative humidity chamber for 24 h at the 25 ± 2 °C. The operating conditions were 40 kV and 40 mA with Cu-radiation. The samples were scanned in

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the range of 4~35° (2θ). The relative crystallinity was calculated as the ratio of the

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areas of crystalline and amorphous regions of X-ray diffractograms (Nara & Komiya,

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1983).

2.6 Stability against acid environment The method used in this article to test the stability under acid environment was

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followed by Xia et al. (2017). One hundred milligram of SMs was suspended in 25 mL of 0.01 M HCl solution (pH 2.0). The suspension was incubated at 37 ± 0.5 °C

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and 500 rpm in a water bath shaker. One milliliter of sample was taken at 1, 4, 12 and 24 h and centrifuged at 3130×g for 10 min. The supernatant was analyzed for reducing sugar through the phenol-sulfuric acid method (Dubois, Gilles, Hamilton,

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Rebers, & Smith, 1955). The percentage of hydrolysis was calculated as the amount of glucose released per 100 mg of dry starch. 2.7 Stability against α-amylase One hundred milligram of starch was suspended in 25 mL of 0.02 M phosphate buffer (pH 6.9) containing 0.003 M CaCl2 and porcine pancreatic α-amylase (10 units) with vigorous mixing. The suspension was incubated at 37 ± 0.5 °C and 500 rpm in a

water bath shaker. One milliliter of sample was taken at 10, 30, 60, 90 and 120 min and centrifuged at 3130×g for 10 min. The supernatant was analyzed for reducing sugar through the phenol-sulfuric acid method (Dubois, et al., 1955) using maltose as a standard. The percentage of hydrolysis was calculated as the amount of glucose released per 100 mg of dry starch. 2.8 Statistical analysis

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Experimental data of one-way analysis of variance (ANOVA) was determined by Duncan’s multiple range tests (P < 0.05) with SPSS 17.0 Statistical Software Program

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(SPSS Incorporated, Chicago). Origin Program 8.5 (Origin Lab Company, USA) and Excel 2010 Program (Microsoft, USA) were used to analyze and report the data. Mean values from the triplicated experiments were reported.

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3 Results and discussion

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3.1. X -diffraction patterns of starch samples

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The X-diffraction patterns of SMs stored at 4, 30 °C and cycles of 4/30 °C (24 h each) for 2-20 days are shown in Figure 2, the relative crystallinity and the crystalline

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type of these starch samples are listed in Table 3. Degraded cassava starch showed a

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strong diffraction peak at 2θ =15°, 17°, 18° and 23.5°, respectively. These were indicative of A-type crystalline structure, which were very similar with native cassava starch. The results indicated that the crystalline structure of starch was little

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influenced during the hydrolysis. For all SMs samples, fabricated in ATPS, exhibited different diffraction. In the process of preparation, primary structure of starch was

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destroyed by heating, and a new diffraction was generated during recrystallization, this is the results of a recombination of starch molecules. The signature reflections at 2θ = 5.6°, 17°, 22° and 24° implied the presence of B-type pattern. The weak

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reflection at 2θ = 19.6° implied indistinct amount of V-type crystallinity, usually associated with ordered single-helical glucans or linear glucans complexed with endogenous aliphatic lipids or ethanol (Mutungi, Rost, Onyango, Jaros, & Rohm, 2009; Shi, Liang, Yan, Pan, & Liu, 2018). Therefore, all the SMs samples were ascribed to a mixture of B- and V- crystalline type. This result was different from that SMs prepared by Elstrand et al. (2006), which gave A-type diffraction patterns. The

reason may be attributed to the different source of materials and dry processing. Zeng et al. (2016) reported that different drying methods could significantly affect the crystals (formed by recrystallization). Air dried crystals exhibited a mixture of B- and V-type diffraction pattern, while freezing dried samples showed dispersive diffraction pattern tend to amorphous. Meanwhile, the crystal type could also be influenced by

(Cheetham & Tao, 1998; Shamai, Bianco-Peled, & Shimoni, 2003).

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temperature treatments, amylose content and average chain length of amylopectin

XRD results also displayed that different temperature conditions and cycling

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times had a great influence on the relative crystallinity of SMs. With the incubation

time increasing, the relative crystallinity of SMs increased. The most obvious was the sample incubated under the temperature at 30 °C for 20 days. And the relative

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crystallinity of the sample was up to 36.3%, which was almost the same or even

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higher than that of the degraded cassava starch. While the relative crystallinity of

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these samples stored at 4 °C or 4/30 °C was lower than that of the degraded starch, and the sample of 4-2d was the lowest. Elstrand et al. (2009) found that increasing the

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incubation temperature could increase the orderliness of SMs prepared in PEG system.

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This was consistent with our findings. The different systems for recrystallization of starch molecules would affect the crystalline structure. Jane et al. (2000) found that the size, amount and interaction between double helixes could also cause the

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difference in the relative crystallinity of SMs. Compared with the linear amylose, the branched amylopectin was more difficult to recrystallize. This may be the existence of

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greater steric hindrance of highly debranched amylopectin, hence less gel could be formed (Silverio, Fredriksson, Andersson, Eliasson, & Åman, 2000). In this study, in special conditions, the side chains of these amylopectin molecules from degraded

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cassava starch can been also re-linked each other to form more ordered crystals.

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Figure 2. X-ray diffraction pattern of SMs: A) SMs incubated for different days at 4 ° C; B) SMs

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incubated for different days at 4/30 ° C; C) SMs incubated for different days at 30 °C.

Relative crystallinity

DCS

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35.6 ± 0.3a

SMs-4-2d

B+V

27.4 ± 0.5g

SMs-4-4d

B+V

28.0 ± 0.2g

SMs-4-8d

B+V

30.3 ± 0.8de

SMs-4-14d

B+V

31.9 ± 0.1cd

SMs-4-20d

B+V

32.8 ± 0.2c

SMs-4/30-2d

B+V

29.8 ± 0.4f

SMs-4/30-4d

B+V

30.8 ± 0def

SMs-4/30-8d

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30.3 ± 0.3ef

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Crystalline type

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Samples

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Table 1 the crystalline type and relative crystallinity of DCS and SMs

32.1 ± 0.2cd

SMs-4/30-20d

B+V

35.5 ± 0.4a

SMs-30-2d

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31.4 ± 0.1de

SMs-30-4d

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32.1 ± 0.6cd

SMs-30-8d

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34.2 ± 0.2b

SMs-30-14d

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35.8 ± 0.5a

SMs-30-20d

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36.3 ± 0.3a

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B+V

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SMs-4/30-14d

Values in the same column with different letters in the same column are significantly different (P <

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3.2. Thermal properties of starch samples

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0.05)

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The gelatinization temperature (To, Tp and Tc), melting temperature range (Tc-To)

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and gelatinization enthalpy (H) of SMs prepared under different temperature cycling conditions are summarized in Table 2. The thermal properties and the gelatinization

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enthalpy of degraded cassava starch were 65.24 °C (To), 74.36 °C (Tp), 87.84°C (Tc) and 10.29 J/g (H), respectively. Compared with degraded cassava starch, the thermal

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parameters and the gelatinization enthalpy of SMs were reduced. This was because the SMs were prepared through recrystallization and incubation. In the process of

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gelatinization, the original crystal structure of starch was destroyed, and the hydrogen bond of the recrystallized SMs formed after incubation was less stable than the original (Karim, et al., 2000). With the increase in incubation time, the To, Tp and Tc

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of SMs also increased, which indicated that incubation time could not be neglected during the formation. The melting temperature range (Tc - To) gives an indication of the quality and heterogeneity of the recrystallized amylopectin. Thus, a wide melting range might imply a large amount of crystals of varying stability, whereas a narrow range could suggest crystals of a more homogeneous quality and similar stability (Fredriksson, et al., 1998). In ATPS, the gelatinization properties and enthalpy of SMs

incubated at 4 °C were the lowest compared with that of SMs incubated at 30 °C or 4/30 °C. The results demonstrated that 4 °C was not conducive to the growth of nuclei, only a small amount of starch crystals formed in this system. Many literatures indicated that temperature conditions had a significant effect on the nucleation and growth of crystals (Miao, Jiang, & Zhang, 2009; Silverio, et al., 2000; Zeng, et al., 2014). However, the SMs incubated at 30 °C showed a higher gelatinization

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properties and enthalpy, which indicated that this condition was more favorable for

the formation and growth of nuclei. This result is not the same as the result of other

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researchers (Shi, et al., 2016; Zeng, et al., 2014). They reported temperature-cycled induced the formation of crystal. The difference between our results and previous

studies possibly was due to the differences in botanical variety. In addition, the

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system for recrystallization was also different, one was ATPS, the other was water

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solution. It meant that the system for recrystallization was significantly affect the properties of the crystals.

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For SMs, the  H value was significantly lower than that of DCS, it meant that

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less amount of double helical or crystalline order formed during the recrystallization

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than those existed in DCS. The  H value of SMs stored at 30 °C or 4 °C increased almost to 3.15 J/g from 2 days to 20 days. However, these values of the cycled conditions exhibited little change.  H reflects the number of double helix and

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crystalline order, a parameter that is influenced by amylose content, amylopectin chain-length distribution and amylose-lipid complexes (Gunaratne & Hoover, 2002).

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The crystallites under the constant temperature melted at a higher  H than that of the cycled conditions. The results were consistent with the report of Elfstrand, et al. (2009). They thought that the ordering of starch molecules increased during

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incubation at the higher temperature. Park et al. (2009) also suggested that the temperature-cycled waxy maize starch sample melted at a lower  H than those formed under constant temperature storage at 4 °C. These results were also in agreement with the increased relative crystallinity of SMs (seen in Section 3.1). Table 2. Thermal characteristics of DCS and SMs.

To (°C)

Tp (°C)

Tc (°C)

Tc- To (°C)

H (J/g)

DCS

65.24 ± 0.52a

74.36 ± 0.48a

87.84 ± 0.80a

19.10 ± 0.54bc

10.29 ± 0.12a

4-2d

49.60 ± 0.35g

57.35 ± 0.31j

65.62 ± 0.22j

16.02± 0.29h

5.81± 0.38gf

4-4d

50.62 ± 0.18g

57.43 ± 0.15j

67.92 ± 0.64hi

17.3± 0.32g

6.59 ± 0.46ef

4-8d

50.97 ± 0.26fg

58.74 ± 0.42ghi

69.82 ± 0.48e

18.85± 0.38bc

7.63 ± 0.65d

4-14d

51.58 ± 0.21ef

59.85 ± 0.33ef

70.93 ± 0.53cd

19.35± 0.35b

8.52 ± 0.24bc

4-20d

49.80 ± 0.85g

60.28 ± 0.23def

70.2 ± 0.46de

20.40 ± 1.03a

8.62 ± 0.32b

4/30-2d

49.46 ± 0.52g

57.67 ± 0.24j

67.44 ± 0.71i

17.98 ± 0.49efg

6.13± 0.59fg

4/30-4d

49.49 ± 0.29g

57.96 ± 0.66ij

67.89 ± 0.44hi

18.40 ± 0.38def

7.14 ±0.53de

4/30-8d

50.20 ± 0.45gh

58.68 ± 0.37hi

68.91 ± 0.28fg

18.71 ± 0.42def

7.25 ± 0.26de

4/30-14d

50.85 ± 0.36fg

58.92 ± 0.91gh

69.71 ± 0.37ef

18.86 ± 0.36cd

7.43± 0.34d

4/30-20d

51.6 ± 0.21ef

59.59 ± 0.52fg

70.89 ± 0.13cd

19.29 ± 0.18bc

7.72 ± 0.42cd

30-2d

51.97 ± 0.82de

60.07 ± 0.30ef

67.84 ± 0.34hi

15.87 ± 0.29h

5.70 ± 0.68g

30-4d

52.35 ± 0.25cde

60.67± 0.94de

68.92 ± 0.63fg

16.57 ± 0.47h

7.52 ± 0.29d

30-8d

52.72 ± 0.41cd

61.14 ± 0.53cd

68.59 ± 0.48gh

17.83 ± 0.22gf

8.46 ± 0.45bc

30-14d

53.00 ± 0.63c

61.88 ± 0.49bc

71.18 ± 0.24c

18.46 ± 0.67ef

8.60 ± 0.24b

62.49 ± 0.36b

72.41 ± 0.42b

18.48 ± 0.15ef

8.85 ± 0.81b

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53.93 ± 0.27b

30-20d

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Samples

To, Tp, Tc indicate the temperature of the onset, peak, conclusion of gelatinization, respectively.

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ΔH indicates enthalpy of gelatinization. Values in the same column with different letters in the same column are significantly different (P < 0.05)

3.3 The retrogradation kinetics of starch samples The plot of enthalpy of SMs against incubation time are shown in Figure 3 using Eq. (3), and the retrogradation kinetic parameters of starch samples are listed in Table

3. As can be seen from the best fit line presented by Figure 2 and the R2 values listed in Table 3, the Avrami model fit the experimental data satisfactorily. Usually, the crystallization rate constant (k) and the Avrami exponent (n) are significant parameters in this model. The crystallization rate constant (k) reflects crystals growth and the speed of crystallization, and the Avrami exponent (n) is associated with the means of crystal nucleation (Kim, Ciacco, & D'Appolonia, 1976).

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The crystallization rate constant of SMs obtained under cycled temperature 4/30 °C was 1.256, which was higher than that of starch samples under 4 °C (k =

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0.690, R2=0.9822) and 30 °C (k = 0.764, R2=0.9454). The results presented that the recrystallization rate of crystal at 4/30 °C was much faster than that of isothermal condition. When the Avrami exponent n≤1, the nucleation mode is instantaneous

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nucleation, corresponding to the one-dimensional crystal growth modes. In the system,

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the value n of SMs obtained by recrystallization under different temperatures was less

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than 1. It was not the same as the recrystallization in water. Obviously, the n-values could be affected by the starch sources and the recrystallization system. Shi et al.

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(2016) found that the value of n under the temperature at 30 °C was greater than 1,

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and the nucleation of the crystals continued to nucleation and the nuclei required for the crystals formed during the entire storage period. The results indicated that the nucleation mode of the crystal was a single nucleation or an instant nucleation. It

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meant that the nuclei required for crystallization were mainly concentrated in the initial stage of storage, and the nuclei formed in later stages were smaller (Mciver,

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Axford, Colwell, & Elton, 1968). Therefore, SMs incubated under different temperature in ATPS had different crystallization rate, while the nucleation mode of

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the crystal was instantaneous nucleation.

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Figure 3. Plot of enthalpy of retrograded starch against storage time: (A) sample stored at 4 °C, (B) sample stored at 4/30 °C, (C) sample stored at 30 °C.

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Remark:  (t) represents ln-ln(Ht -Ho)/(H1 -Ho) in equation (3) in the section 2.4. Table 3. The retrogradation kinetics parameters of starch samples

k(d-n)

n

R2

4 °C

0.690 ± 0.079b

0.4798 ±0.0373ab

0.9822

4/30 °C

1.256 ± 0.141a

0.3619 ± 0.0664b

0.9082

30 °C

0.764 ± 0.149b

0.5035 ± 0.0688a

0.9454

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Storage condition

Remark: the d in the units of k represents day.

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3.4. Stability of SMs against acid and α-amylase environment The stability of SMs obtained under different temperature cycling conditions

against acid environment, presented by the rate of hydrolysis in different time at pH 2.0, is shown in figure 3. The acid environment is to explore the degradation of SMs simulated the pH in the stomach. This method could indirectly represent the release rate of SMs in gastric juice (Puncha-Arnon, et al., 2015). As shown in Figure 3, the

hydrolysis rate of SMs prepared under different temperature decreased with the increase of incubation days. It meant that the stability of SMs increased. The maximum hydrolysis rate of the sample was only 5.94 % (sample 4/30-2d hydrolysis for 24 h), which implied that the structure of recrystallized microspheres could be relative stable at pH 2.0. It was because that the amylopectin or amylose was recrystallized to form crystals, which have some resistance to be hydrolyzed. The

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most stable sample was 30-20d after 2 h acid hydrolysis, the hydrolysis rate was only 0.44%.

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Figure 4 shows the hydrolysis rate of SMs under α-amylase. The regularity is

basically similar with the hydrolysis under the acid environment. The stability of the samples was affected greatly by the incubation temperature. The lowest hydrolysis

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rate of SMs was the sample prepared at 30 °C, but the degree of hydrolysis was much

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higher than that under the acid environment. This indicated the efficient catalytic

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activity of the enzyme. These results were consistent with the results of crystallinity of SMs. The reason may be that during the recrystallization, amorphous regions of

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SMs turned to more ordered or crystalline regions. With higher the crystallinity and

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larger the crystalline regions, the ability of resist to be hydrolyzed could be stronger. Shi et al. (2016) have reported that temperature cycled was favor for recrystallization and the hydrolysis rate was the lowest. However, our results had shown that 30 °C

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was more conducive to the recrystallization of SMs. The n-values of starch samples stored at 30 °C were different between water solution and ATPS. It meant that the

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model of crystal nucleation formation was different. Therefore, the properties of starch samples could be influenced. These will provide some basis for the application

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of SMs.

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Figure 4. Acid hydrolysis rate of SMs samples stored under different temperature at pH 2.0 in different incubation time. (A) SMs stored at 4 °C, (B) SMs stored at 4/30 °C, (C) SMs stored at 30°C

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Figure 5. The hydrolysis rate of SMs samples stored under different temperature against α-amylase in different incubation time. (A) SMs stored at 4 °C, (B) SMs stored at 4/30 °C, (C) SMs stored at 30°C.

4 Conclusions The retrogradation behaviors of SMs prepared in ATPS depended on the different temperature storage were investigated using a series of methods. The different temperature had significant impacts on the crystalline structure, thermal properties

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and stability against acid and α-amylase. The crystalline structure of SMs was transferred from A-type to a mixture of B- and V-type. With the storage time

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increasing, the relative crystallinity, gelatinization temperature (To, Tp and Tc) and H

of SMs were increased. These values of SMs stored at 30 °C were higher than that of

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samples stored at 4°C or 4/30°C. The Avrami equation analysis revealed that the

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recrystallization rate of crystal at 4/30°C condition was much faster than that of

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isothermal condition. The nucleation mode of all SMs was instantaneous nucleation.

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The stability test revealed that SMs were stable under acid environment. However, the

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stability against α-amylase varied depending on incubation days and temperature, the samples stored at 30°C were more stable than that stored at 4 °C and 4/30 °C. The

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samples stored at 30 °C were the most stable among these recrystallized SMs.

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Therefore, the stored temperature at 30 °C was more suitable to prepare SMs in ATPS. These results were different from water solution, in which lower temperature was beneficial to recrystallize. These results suggested that the storage temperature could

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be a feasible way to modulate the properties of SMs.

Acknowledge The authors gratefully acknowledge financial support from the State Key Program of National Natural Science of China (Grant No. 31230057) and Natural Science Program of Hainan Province (817142).

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