Effect of debranching and temperature-cycled crystallization on the physicochemical properties of kudzu (Pueraria lobata) resistant starch

International Journal of Biological Macromolecules 129 (2019) 1148–1154

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Effect of debranching and temperature-cycled crystallization on the physicochemical properties of kudzu (Pueraria lobata) resistant starch Feng Zeng a,b, Tao Li a, Hui Zhao a, Hongpei Chen a, Xiaodan Yu a,c,⁎, Bin Liu a,⁎⁎ a

College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, China School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China Department of Developmental and Behavioral Pediatrics, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Ministry of Education Shanghai Key Laboratory of Children's Environmental Health, Shanghai 200127, China

b c

a r t i c l e

i n f o

Article history: Received 16 November 2018 Received in revised form 24 December 2018 Accepted 6 January 2019 Available online 14 January 2019 Keywords: Kudzu starch Short chain amylose Slowly digestible starch Resistant starch

a b s t r a c t Resistant starch from Kudzu (Pueraria lobata) was prepared by debranching and subsequent recrystallization under isothermal and cycled temperature conditions. The granule morphology of the resistant starch was irregular in shape and size with rough surface. All the resistant starch samples exhibited B + V-type crystalline structure. The peripheral regions of native starch were better organized than that of the resistant starch samples. The solubility and swelling degree of native starch and resistant starch samples increased with the increase of temperature. The water holding capacity of native starch was much higher in comparison with resistant starch when the temperature was above 80 °C. The resistant starch showed better transmittance and freeze-thaw stability than that of native starch. These results suggest that kudzu starch is a potential resource for resistant starch preparation. © 2019 Published by Elsevier B.V.

1. Introduction Kudzu (Pueraria lobata) is a perennial leguminous vine of the genus Pueraria herb native to East Asia. It is recognized to have originated in China and is used as medicinal plant in traditional Chinese medicine [1]. Kudzu root contains some bioactive isoflavones, including daidzin, daidzein and puerarin [2,3]. These isoflavones from kudzu root showed many important physiological activities such as anti-cancer activity, and can be used to treat alcohol abuse safely and effectively. Additionally, kudzu root is also used as an edible material because starch is a main component of the fresh kudzu root. The content of starch is about 15.0–34.2% in the fresh roots [4]. Previous researches have mainly focused on functional and structural properties including kudzu starch-ascorbic acid films [5], synthesis and properties of carboxymethyl kudzu root starch [6], and crosslinking of kudzu starch [7]. Van et al. [8] compared the chemical compositions, structure and physicochemical properties of kudzu starches from different regions. Reddy et al. [9] investigated the morphology, crystallinity, pasting and thermal characteristics of starches from adzuki bean (Vigna angularis L.) and edible kudzu (Pueraria thomsonii Benth).

⁎ Correspondence to: X. Yu, College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (X. Yu), [email protected] (B. Liu).

https://doi.org/10.1016/j.ijbiomac.2019.01.028 0141-8130/© 2019 Published by Elsevier B.V.

Resistant starch (RS) is defined as the starch fraction that is poorly digested and absorbed in the small intestine but enters into the large intestine [10]. RS plays an important role in human health due to its positive physiological benefits including low glycemic response, prebiotic function on colon microflora, positive correlations with lipid metabolism and reducing the risks of colon cancer [11]. RS has five main types, i.e. physically trapped within plant cells and food matrices (RS1), native starch granules from certain plants containing ungelatinized starch (RS2), recrystallized or retrograded starch (RS3), chemically modified starch (RS4), and amylose-lipid complex (RS5) [10,12]. It is generally recognized that RS can be prepared by physical, chemical, enzymatic, and combination methods. Enzymatic method has drawn much attention as the merits of highly secure, cost-effective and suitable for commercial use. A process for improving digestion resistibility of starch was by debranching and recrystallization. Miao et al. [13] reported that gelatinized starch treated with higher enzymatic concentration and less debranching time resulted in higher amount of slowly digestible starch (SDS), while RS was formed with increased debranching time. Zhang et al. [14] treated high-amylose corn starch with high temperature-pressure and pullulanase debranching, found that storage for 24 h was favorable for RS formation. Zhang et al. [15] suggested that the combination of α‑amylase and pullulanase method is a more promising technique than high pressure to prepare RS from maize starch with greater resistance. Zhou et al. [16] prepared RS from indica by using a new method that combines α‑amylase, pullulanase

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and heat-moisture treatment, demonstrating that dual modification treatment is an efficient method for the preparation of RS. Recently, A few authors have reported the impact of temperaturecycle on the slowly digestible starch (SDS) and RS formation. Zeng et al. [17] investigated the isothermal and temperature-cycled crystallization of waxy rice starch, suggesting that the cycled temperature storage induced a greater amount of SDS and RS than the isothermal storage. Xie et al. [18] found that more imperfect crystallite was formed in the crystalline matrix under temperature-cycled retrogradation, resulting in a high yield of SDS. Shi et al. [19] suggested that the cycled temperature storage could promote the recrystallization and induce the lower digestibility of gelatinized wrinkled pea starch than the isothermal storage. It was evident that the in vitro glycemic index of SDS retrograded in temperature-cycled was lower than that in isothermal storage, suggesting that the temperature-cycled retrogradation is applicable to retard the digestion of waxy rice starch [20]. However, little attention has been devoted to synergetic debranching and temperaturecycled crystallization in RS preparation. A temperature close to glass transition temperature is good for crystal nucleation, while a slightly higher temperature near melting temperature is conducive to the growth of crystals [21,22]. When the gelatinized starch gel was stored at a cycled temperature between the nucleation temperature and propagation temperature, the recrystallization rate of starch chain could be accelerated [23]. The cycled temperature crystallization provides a stepwise nucleation and propagation, facilitating the growth of crystalline area and perfection of crystallites [22]. These processes are mainly affected by crystallization temperature, changes of temperature gradient, intervals of cycle time and crystallization time. Short chain amylose could be generated in debranching process of starch, and the cycled temperature crystallization lead to the rapid retrogradation of short chain amylose, which was beneficial to the formation of SDS or RS. Furthermore, there are few reports about RS from kudzu. The kudzu starch is reported to contain 20.8–22.9% of amylose content and 20.5 of chain length of amylopectin [8,24], which might contribute to the formation of RS. The aim of the present work was to evaluate the formation of RS and some functional characteristics by isotherm and cycled temperature crystallization at different temperatures of debranched kudzu starch. 2. Materials and methods 2.1. Materials Kudzu starch (24.4% of amylose) was bought from local market. Pullulanase (EC 3.2.1.41, 2800 ASPU/g) from Bacillus licheniformis was attained from Guangzhou Yulibao Biotechnology Co., Ltd. (Guangzhou, China). Porcine pancreas α‑amylase (EC3.2.1.1, 16 U/mg) type-B and amyloglucosidase (EC 3.2.1.3, 300 U/mL) from Aspergillus niger were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All other chemical reagents and solvents were of analytical grade. 2.2. Preparation of kudzu resistant starch Kudzu starch (15 g, dry basis) was suspended in sodium acetate buffer (0.2 M, pH 5.2) to obtain slurry. The slurry was cooked at 100 °C with continuous stirring for 30 min. After that, the temperature of starch paste was adjusted to 58 °C and debranched by pullulanase at 60 unit/g of starch for 12 h. The starch paste was sealed and heated at 100 °C for 30 min to denature the pullulanase and then cooled to room temperature (25 °C). The cooled starch paste was subjected to isothermal and temperature-cycled crystallization with different temperature cycles at time intervals of 24 h for continued four days, and the duration for each temperature was 24 h. The four days of isothermal crystallization was designed as 4/4 4/4 °C. Temperature-cycled crystallization was design as 4/−20 4/−20 °C, 4/10 4/10 °C, 4/20 4/20 °C, 4/30

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4/30 °C and 4/40 4/40 °C. The sample at 4/−20 4/−20 °C was thawed at room temperature (25 °C) after dual cycles. All of the precipitate was centrifuged, washed with distilled water to obtain a neutral pH and dried at 40 °C overnight, then ground to pass through a 100-mesh sieve. The yield of RS products is defined as the ratio (w/w) between crystallized starch (after modification) and native starch (before modification). 2.3. Scanning electron microscopy (SEM) Each sample was dispersed on double-sided stick tape placed on aluminium stubs and was covered with a gold-palladium layer (E-1010, Hitachi, Japan). The morphology of sample was observed using scanning electron microscope (TM3000, Hitachi, Japan). An accelerating voltage of 15 kV was used during micrography. 2.4. Attenuation total reflection-infrared spectroscopy The short-range structure of starch samples was analyzed by the Fourier transform infrared spectrum (FTIR) with an attenuation total reflection mode (ATR) (Tensor 27, Bruker, Germany). The spectra were scanned 32 times, with a resolution of 4 cm−1, and the spectra were corrected by the baseline to be convolved in the range of 1200–800 cm−1. Omnic 6.2 software was used to get the deconvolution spectra, the half peak width is 22 cm−1, and the resolution enhancement factor is 2.2. The absorbance amplitude of each sample was recorded at 1022 and 1047 cm−1, and the short range ordered structure of starch samples was analyzed as the ratio of 1047 cm−1/1022 cm−1 [25]. 2.5. X-ray diffraction and relative crystallinity X-ray diffraction (XRD) pattern was obtained by an X-ray diffractometer (D8 Advance, Bruker, Germany). The operating conditions were 40 kV and 40 mA with Cu Kα radiation of 1.5418 Å wavelength, and the diffractograms were recorded from 3° to 35° (2θ) at a rate of 2°/min. The relative crystallinity was analyzed by the ratio of the crystalline area to the total diffractogram area [26]. 2.6. Solubility and swelling property of KRS According to the methods of Subramanian et al. [27], starch sample (0.5 g) and 25 mL distilled water were mixed in centrifuge tube, and the mixture was subjected to water bath at 50 °C, 60 °C, 70 °C, 80 °C, and 90 °C for 30 min with oscillate occasionally. The sample was cooled to room temperature and centrifuged at 1500 ×g for 20 min. The supernatant and sediment was separated, and the weight of the supernatant is recorded after drying. The solubility (S) and swelling property (P) are calculated as follows: Sð%Þ ¼

P ð%Þ ¼

A  100% W D    100% S W 1− 100

where, A–the weight of supernatant after drying (g); W–the weight of sample (g); D–the weight of precipitate after centrifugation (g). 2.7. Water holding capacity The measurement of water holding capacity (WHC) was according to the method of Sandhu et al. [28]. Starch samples (0.5 g) and 10 mL

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distilled water was placed in centrifuge tube, the mixture was placed to water bath at 50 °C, 60 °C, 70 °C, 80 °C, and 90 °C for 30 min with shaking. After that, samples were centrifuged under 1500 ×g for 15 min. The supernatant was discarded. Water holding capacity was calculated as follows:

WHC ð%Þ ¼

m2 −m1 −m0  100% m0

Data were reported as mean values and standard deviations. One way of variance analysis was used to compare means at the 5% significance level. All the statistical analyses were conducted using the SPSS for Windows version 12.0 software (SPSS, Chicago, USA).

3. Results and discussion 3.1. Yield of KRS

where, m0—the weight of the starch (g); m1—the weight of the centrifuge tube (g); m2—the weight of starch and centrifuge tube after water removal (g). 2.8. Transparency The determination of transparency was referred to the method of Correia et al. [29]. Starch suspension (1%) was prepared and placed in a boiling water bath with stirring for 30 min, then cooled to room temperature (25 °C). The transmittance of starch paste was measured by using a 1 cm cuvette and ultraviolet spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China) at 620 nm wavelength. The transmittance of deionized water is 100%, setting as the blank control. 2.9. Freeze-thaw stability Freeze-thaw stability was measured according to the method of Hoover and Senanayake [30] with minor modifications. Starch suspension (5%, w/w) was gelatinized completely in boiling water bath. A certain amount of starch paste was put into centrifuge tube, stored at −20 °C for 24 h. The frozen paste was completely defrosted at room temperature (25 °C) and deposed to centrifugation (1500 ×g, 30 min), and the sediment was weighted. The process of freezing and thawing was repeated from 1 to 5 times. Syneresis rate was calculated as the following formula:

F ð%Þ ¼

2.11. Statistical analysis of data

w0 −w1  100% w0

where, w0—the weight of starch paste (g); w1—the weight of sediment (g). 2.10. Starch digestibility Starch digestibility was evaluated according to the method of Englyst et al. [10] with slight modifications. Starch powders (200 mg) were weighed into centrifuge tubes (50 mL) and mixed with 15 mL of sodium acetate buffer (0.2 M, pH 5.2) and glass balls. Then 10 mL mixtures of porcine pancreatic α‑amylase (290 U/mL) and amyloglucosidase (15 U/mL) were added. The starch-enzyme mixtures were incubated horizontally in a shaking water bath at 37 °C. Then 0.5 mL aliquots of hydrolyzed solution was taken out at 20 min and 120 min and mixed with 4 mL of ethanol to denature the enzymes and precipitate polymeric starch. Glucose content in the supernatant gained from centrifugation (2650 ×g, 10 min) was measured with glucose assay kit (GOPOD method). The rapidly digestible starch (RDS) was defined as the amount of glucose released in 20 min of hydrolysis. The SDS was measured as the fraction digested between 20 and 120 min of incubation. The starch not hydrolyzed within 120 min was designated as RS.

The yield of RS obtained by isothermal and temperature-cycled crystallization followed this order: 4/−20 4/−20 °C (78.8%) N 4/10 4/10 °C (75.8%) N 4/20 4/20 °C (75.2%) N 4/4 4/4 °C (72.3%) N 4/30 4/30 °C (67.13%) N 4/40 4/40 °C (64.7%). Yield of RS by 4/−20 4/ −20 °C is higher than the other treatment groups. In our previous study, temperature-cycled crystallization (4/−20 4/−20 °C) of debranched waxy rice starch yielded much higher RS (82.78%) than that of kudzu starch in this study [17]. This is probably due to the waxy rice starch generated higher amount of short chain amylose than kudzu starch in debranching process, thus more crystallites among starch molecules were formed during recrystallized period. These results indicated that the process of recrystallization can be enhanced by the cycle of temperature or multiple freezing and thawing cycles of the starch paste. Tian et al. [31] suggested that waxy rice starch subjected to temperature cycle of 4/25 °C, storage about 14 days could improve the yield of SDS which reached maximum yield of 54.5%. It is generally believed that the gelatinized amylose can form crystalline region within a few hours, while the crystallization of amylopectin should take several days or even more than a month [32,33]. Therefore, debranched kudzu starch contains a large amount of short chain amylose, which needs less time than the amylopectin to form new crystallites. In this study, the yield of RS from dual (4/10–4/20 °C) was higher than that of isothermal crystallization (4/4 4/4 °C), while yield of RS from dual (4/30–4/40 °C) was lower than isothermal crystallization. As the cycled temperature increasing from 20 °C to 40 °C, the yield of RS decreased. Cai and Shi [34] suggested that the yield of short chain amylose spherulites decreased from 88% to 50% when crystallization temperature increased from 4 °C to 50 °C. This is because that part of the short chain amylose cannot rearrange and recrystallize again at slight higher temperature storage. In addition, part of the unstable short chain amylose crystals could be dissolved when the temperature increased to 30 °C or 40 °C. Therefore, the yield of RS from 4/30 4/30 °C to 4/40 4/40 °C is lower than isothermal crystallization. The above results showed that the proper temperature cycle and crystallization temperature can promote the formation of short chain amylose crystallite and increase the yield of RS. These results are in accordance with our previous study [17].

3.2. Scanning electron micrograph of KRS The scanning electron micrograph of starch sample is shown in Fig. 1. The native kudzu starch showed sphere, hemisphere and polygon-shape granule. Our observation is in accordance with previous study [35]. The morphology of starch granule significantly changed after debranching and recrystallization. The granular structure of native kudzu starch was destroyed after gelatinization, enzymolysis and retrogradation, and the crystallites showed rough and irregular morphology with different size. Similar observations for debranched waxy rice starch and purple yam starch have been reported by Li et al. [36]. However, there was no significant difference in morphology among isothermal and temperature cycle treated samples.

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Fig. 1. Scanning electron micrograph of starch samples. (A) Native starch; (B) 4/−20 4/−20 °C; (C) 4/4 4/4 °C; (D) 4/10 4/10 °C; (E) 4/20 4/20 °C; (F) 4/30 4/30 °C; (G) 4/40 4/40 °C.

3.3. ATR-FTIR spectrum

3.4. X-ray diffraction pattern and relative crystallinity

The FT-IR deconvolution spectrum of the starch samples in the ATR mode is shown in Fig. 2A. Infrared spectrum is sensitive to the changes of starch molecular level (short-range structure). In addition, the infrared spectrum can get the organization information of molecular chain structure near the surface of starch granule. Infrared spectrum can penetrate into the particle surface 2 μm depth [26]. The vibration in 1047 and 1022 cm−1 was associated with the orderly structure and disorder structure of starch, respectively. The ratio of peak height between the 1047 and 1022 cm−1 indicated the orderly structure and disordered structure [37]. It can be seen from Fig. 2A, deconvoluted infrared spectrum of RS samples were similar with the native starch. However, short-range structure of the native starch is more orderly than RS samples. Debranching and cycled temperature crystallization changed the short-range structure of the starch granule. The R(1047/1022) of dual 4/−20 °C, dual 4/20 and dual 4/30 °C were higher than that of isothermal crystallization samples, suggesting that the short-range organization of temperature-cycled samples were more orderly than isothermal sample. This indicated that temperature-cycled crystallization favored the formation of hydrogen bond and a more orderly crystal.

The X-ray diffraction (XRD) pattern of starch samples is shown in Fig. 2B, and value in the bracket is relative crystallinity (%). Native kudzu starch showed typical A-type diffraction feature with peaks at 15.3°, 17.1°, 18.2° and 23.5° (2θ). After debranching and subsequently isothermal crystallization and cycled temperature crystallization, the crystalline structure of RS samples converted from A-type to B + V complex. Previous studies have shown that the debranched and recrystallized starch exhibited B + V complex crystalline structure [16,17,38]. There was no significant difference between the isothermal and temperature-cycle crystallization samples in the XRD patterns. Park et al. [39] reported that there was no obvious difference in XRD patterns when waxy corn starch gel storage under different time and temperature cycle, but cycled temperature crystallization samples showed a higher crystallinity. Zeng et al. [17] revealed that both isothermal and temperature-cycled crystallization of debranched waxy rice starch showed similar crystalline structure. The relative crystallinity of the starch sample followed this order: native starch (38.8%) N 4/−20 4/−20 °C (37.4%) N 4/30 4/30 °C (36.6%) N 4/40 4/40 °C (36.1%) N 4/20 4/20 °C (35.3%) N 4/10 4/10 °C

Fig. 2. ATR-FTIR spectra (A) and X-ray diffraction pattern (B) of starch samples.

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Table 1 Solubility and swelling power of starch samples at different temperature. Sample

50 °C

60 °C

70 °C

80 °C

90 °C

Solubility (%) Native starch 4/−20 4/−20 °C 4/4 4/4 °C 4/10 4/10 °C 4/20 4/20 °C 4/30 4/30 °C 4/40 4/40 °C

8.55 ± 0.25a 5.89 ± 0.08b 3.13 ± 1.21c 3.49 ± 0.64bc 4.12 ± 0.28bc 4.50 ± 0.08bc 4.68 ± 1.00bc

10.47 ± 0.31a 9.48 ± 0.63ab 9.85 ± 0.69ab 7.52 ± 0.65bc 5.36 ± 0.95cd 4.91 ± 0.11cd 4.78 ± 0.91d

14.45 ± 0.73a 11.43 ± 1.54ab 12.4 ± 0.54ab 8.51 ± 0.62bc 6.44 ± 0.15c 5.04 ± 1.17c 4.81 ± 0.26c

18.57 ± 1.06a 13.13 ± 0.24ab 15.41 ± 2.00ab 11.97 ± 2.06bc 8.89 ± 0.47cd 5.32 ± 0.43d 7.30 ± 0.66cd

23.06 ± 3.35a 15.51 ± 1.44bc 17.35 ± 2.67ab 12.96 ± 1.38bcd 9.10 ± 0.31cde 5.63 ± 0.40e 7.45 ± 0.59de

Swelling power (%) Native starch 4/−20 4/−20 °C 4/4 4/4 °C 4/10 4/10 °C 4/20 4/20 °C 4/30 4/30 °C 4/40 4/40 °C

3.95 ± 2.21a 2.75 ± 0.87a 5.15 ± 2.60a 3.31 ± 0.81a 3.18 ± 0.29a 2.37 ± 1.64a 2.88 ± 0.95a

6.17 ± 1.16ab 4.16 ± 0.06ab 6.43 ± 1.73a 3.94 ± 0.23ab 3.61 ± 2.15ab 4.49 ± 0.71ab 3.23 ± 0.31b

6.83 ± 1.79ab 4.31 ± 0.52ab 6.88 ± 1.41a 4.75 ± 0.83ab 4.02 ± 0.22b 4.56 ± 0.93ab 3.74 ± 0.13b

7.32 ± 3.20a 4.54 ± 0.58c 7.21 ± 0.72ab 4.90 ± 0.45c 5.13 ± 0.30c 5.47 ± 0.19abc 5.28 ± 0.52bc

9.17 ± 2.86a 5.26 ± 1.45b 8.26 ± 0.04ab 5.41 ± 1.05b 5.72 ± 0.88ab 6.42 ± 0.49ab 6.40 ± 1.27ab

Results are mean ± SD. Means with different letters in a column are significantly different (P b 0.05). Values are the means of triplicates.

(34.9%) N 4/4 4/4 °C (34.2%). Van et al. [8] reported that the Vietnamese kudzu starch also showed A-type crystal structure with relative crystallinity of 38.6%. The RS at 4/−20 4/−20 °C showed higher crystallinity than the other RS. This might be due to low temperatures (−20 °C) which can promote the water molecules separated from the starch paste form ice crystals, and shorten the distance between the amylose molecule, which made it form hydrogen bonds easily and formed an orderly structure. The higher crystallization temperature (30 °C and 40 °C) showed higher relative crystallinity, which might be attributed to that higher temperature storage similar to annealing treatment, making the starch double helix molecular chain arrange orderly.

Solubility of RS samples was lower than that of native kudzu starch, which might be attributed to the compact crystalline structure of RS samples as shown in the SEM graphs (Fig. 1). Swelling power of RS was much lower in comparison with native kudzu starch at relatively higher temperature (80–90 °C). This indicated that the solubility and swelling power of RS was less sensitive result from debranching and crystallization process. Therefore, desired starch properties could be achieved as soon as the appropriate recrystallized condition was applied.

3.6. Water holding capacity 3.5. Solubility and swelling power Solubility and swelling power reflects the extent of interaction between starch and water. The solubility of starch is the dissolution percentage of starch granule when heated to a certain temperature, reflecting the ability of amylose molecule escaping from the starch granule. Swelling power is the mass fraction of per gram dried starch absorbs water at a certain temperature, indicating the characteristics of amylose in starch granule [40]. Both of solubility and swelling power are mainly depend on the size, molecular structure and amylose content of starch granule. The solubility and swelling power of native kudzu starch and RS at different temperatures are shown in Table 1. It can be seen that the solubility and swelling power of the starch samples are increased with the increased temperature. These results are in accordance with RS from purple yam prepared by autoclaving and multi-enzyme hydrolysis [36]. This is due to the chemical bond inside the starch granule became loose as the temperature increased, resulted in the swell and collapse of starch granule. The crystalline structure of starch was damaged, and the number of exposed hydrogen bonds in the crystalline region increased, which made free water molecule easier to infiltrate into starch molecule inside. Therefore, the solubility and swelling power of starch increased gradually.

Water holding capacity (WHC) is the ability of starch to overcome gravity to retain water, which is related to starch granule morphology, molecule structure and the content of amylose. The water holding capacity of starch samples at different temperatures is shown in Table 2. The WHC of the native kudzu starch and RS samples were all increased with increased temperature. When the temperature was below 80 °C, the water holding capacity of RS is greater than that of native starch (expect for 4/4 4/4 °C and 4/10 4/10 °C). This might be resulted from the increased amount of short chain amylose during the debranching process, leading to an increase of the hydrophilic hydroxyl groups on the outside of the glucose unit. A tremendous increase in WHC was observed in native starch when temperature was above 80 °C. The dramatic increase in WHC has been associated with the destruction of starch granule structure, which facilitates the swelling of starch granules [41]. Native kudzu starch contains tight packed double helix structure, which restricts water enter into the inner part of the starch granules at low temperature. Starch molecule chain in the crystalline region expose and combined with water when heated at a higher temperature. Starch granule absorbing water at certain temperature could lead to starch gelatinization and the increasing of water holding capacity.

Table 2 Water-holding capacity of starch samples at different temperatures. Samples

50 °C

60 °C

70 °C

80 °C

90 °C

Native starch 4/−20 4/−20 °C 4/4 4/4 °C 4/10 4/10 °C 4/20 4/20 °C 4/30 4/30 °C 4/40 4/40 °C

77.86 ± 4.24d 165.13 ± 9.77c 199.89 ± 2.16ab 196.53 ± 1.75b 215.12 ± 3.27a 200.30 ± 0.87ab 200.59 ± 1.68ab

103.86 ± 2.98b 211.00 ± 0.54a 219.56 ± 2.17a 212.42 ± 4.19a 231.69 ± 2.36a 230.75 ± 13.31a 222.19 ± 10.73a

278.14 ± 1.16d 318.43 ± 2.67b 274.81 ± 4.12d 256.21 ± 0.58e 327.76 ± 3.61ab 299.77 ± 0.40c 329.88 ± 1.99a

840.25 ± 2.76a 379.27 ± 10.82c 366.60 ± 7.66c 434.11 ± 8.56b 377.74 ± 1.05c 458.47 ± 1.34b 445.00 ± 9.39b

1469.09 ± 4.48a 395.92 ± 6.05e 426.32 ± 0.46de 485.66 ± 14.84bc 414.11 ± 16.50de 499.06 ± 11.62b 447.73 ± 5.80cd

Results are mean ± SD. Means with different letters in a column are significantly different (P b 0.05). Values are the means of triplicates.

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Table 3 Freeze-thaw stability of starch samples. Samples

1st (%)

2nd (%)

3rd (%)

4th (%)

5th (%)

Transparency

Native starch 4/−20 4/−20 °C 4/4 4/4 °C 4/10 4/10 °C 4/20 4/20 °C 4/30 4/30 °C 4/40 4/40 °C

12.00 ± 1.12ab 12.23 ± 2.99ab 14.11 ± 4.06ab 20.30 ± 4.36a 10.53 ± 3.84ab 12.82 ± 1.94ab 6.59 ± 2.50b

26.94 ± 4.61ab 19.83 ± 0.47bc 20.27 ± 0.02abc 32.64 ± 5.96a 13.87 ± 2.02c 15.39 ± 1.69bc 7.89 ± 2.81c

34.72 ± 2.62a 32.19 ± 4.67a 27.68 ± 3.95ab 40.96 ± 1.52a 29.97 ± 4.36ab 29.85 ± 8.25ab 14.33 ± 0.53b

51.54 ± 5.89a 39.28 ± 2.38ab 36.04 ± 1.16abc 42.75 ± 7.44ab 32.45 ± 2.11bc 43.01 ± 6.43ab 18.31 ± 3.48c

52.56 ± 4.49a 41.79 ± 3.10ab 36.54 ± 3.28abc 43.26 ± 5.30ab 33.46 ± 3.53bc 46.01 ± 7.85ab 20.82 ± 1.48c

62.82 ± 1.62d 66.87 ± 2.78cd 67.07 ± 3.17cd 68.15 ± 0.95bcd 70.14 ± 2.44abc 74.16 ± 2.46ab 75.71 ± 1.47a

Results are mean ± SD. Means with different letters in a column are significantly different (P b 0.05). Values are the means of triplicates.

3.7. Freeze-thaw stability and transparence The retrogradation property of starch can be evaluated by freezethaw stability. The starch molecular chain can close to each other in the freeze-thaw process, causing water push out. The extent of syneresis rate reflects the stability of starch paste in the freeze-thaw process. The higher syneresis rate means the worse freeze-thaw stability. The freezethaw stability of the starch samples is shown in Table 3. It could be seen that native kudzu starch and RS showed the lowest syneresis rate in the first cycle. Syneresis rate gradually increased from second to the fifth cycle. The increased amplitude of syneresis rate tended to be stable after the fifth freeze-thaw cycle. Srichuwong et al. [42] also showed that as the times of cycle increased, the water segregation rate of starch paste increased gradually. After four times of freeze-thaw cycles, the syneresis rate of RS samples is lower than that of native starch, suggesting that the freeze-thaw stability is improved after debranchingrecrystallized treatment, which might be suitable for application in frozen food. The transparency of RS sample was higher than that of native starch. The RS samples at 4/30 4/30 °C and 4/40 4/40 °C showed higher transparency in comparison with native starch (P b 0.05). These results suggested that debranching and crystallization process could improve the transparency of kudzu starch.

3.8. Digestion characteristics The percentage of RDS, SDS and RS of native kudzu starch and treated starch samples is shown in Table 4. The 4/10 4/10 °C sample showed the highest content of SDS than the other samples. The contents of RS fragment followed this order: 4/−20 4/−20 °C (43.93%) N 4/4 4/4 °C (43.50%) N 4/20 4/20 °C (42.58%) N 4/30 4/30 °C (39.89%) N 4/40 4/40 °C (38.55%) N 4/10 4/10 °C (34.50%) N native starch (24.15%). The RS content of cycled temperature crystallization samples is higher than that of the native starch. After different cycled temperature crystallization, treated samples showed lower sensitivity to enzymatic digestion. RS was formed by pullulanase debranching and recrystallization, which belonged to RS3. Previous studies have shown that the sensitivity of starch to digestive enzymes can be decreased by cycled temperature crystallization [17–20]. It is possible that the compact organization of molecules in the crystallites was responsible for its low sensitivity to digestive enzyme, as the interaction among short chain amylose molecule

Table 4 The level of RDS, SDS and RS of starch samples. Samples

RDS (%)

SDS (%)

RS (%)

Native starch 4/−20 4/−20 °C 4/4 4/4 °C 4/10 4/10 °C 4/20 4/20 °C 4/30 4/30 °C 4/40 4/40 °C

41.25 ± 0.64ab 25.06 ± 1.73d 32.69 ± 1.35c 24.61 ± 1.09d 46.19 ± 2.62a 41.69 ± 1.29ab 36.75 ± 1.87bc

34.60 ± 0.22b 31.01 ± 0.27b 23.82 ± 0.35cd 40.89 ± 1.42a 11.22 ± 1.77e 18.42 ± 1.68d 24.70 ± 2.88c

24.15 ± 0.41e 43.93 ± 1.47a 43.50 ± 1.71ab 34.50 ± 0.33d 42.58 ± 0.85ab 39.89 ± 0.39bc 38.55 ± 1.01c

Results are mean ± SD. Means with different letters in a column are significantly different (P b 0.05). Values are the means of triplicates.

was enhanced. In addition, the SEM results suggested that compact pack and smooth surface of the short chain amylose crystallite might hinder the enzyme adhere to the matrix. According to the XRD results, the debranched and recrystallized starch exhibited B + V-type which were complex crystalline structure. The recrystallized starch was defined as RS3 which generally showed B-type XRD, and the amyloselipid complex which showed V-type XRD was classified as RS5. A complex crystalline structure of short chain amylose crystallite is the structure basis of the kudzu RS. It is necessary to research weather the B-type crystallites or the V-type crystallites play the key role in anti-digestion property during digestion process. Pullulanase debranching and cycled temperature recrystallization is an effective method to decrease the sensitivity of starch toward digestive enzyme.

4. Conclusions Debranching and subsequent cycled temperature crystallization (4/ −20 4/−20 °C) obtain higher yield of RS. The solubility and swelling power of native kudzu starch and cycled temperature recrystallized starch were increased with increased temperature. The syneresis rate of native starch and cycled temperature treated starch increased with the increased times of freezing and thawing. The results of syneresis rate suggested that freeze-thaw stability of RS samples is better than native starch. The content of SDS was the highest in 4/−20 4/−20 °C, and RS content of cycled temperature crystallization sample was higher than that of native starch. The crystalline type of RS exhibited B + V complex. The above properties of RS from kudzu suggest that kudzu starch is a potential resource for RS preparation.

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