Effect of Microwave Irradiation on the Physicochemical and Digestive

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Effect of microwave irradiation on the physicochemical and digestive properties of lotus seed starch Shaoxiao Zeng, Bingyan Chen, Hongliang Zeng, Zebin Guo, Xu Lu, Yi Zhang, and Baodong Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05809 • Publication Date (Web): 25 Feb 2016 Downloaded from http://pubs.acs.org on March 1, 2016

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Journal of Agricultural and Food Chemistry

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Effect of microwave irradiation on the physicochemical and digestive

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properties of lotus seed starch

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Shaoxiao Zeng *, , Bingyan Chen*, Hongliang Zeng*, Zebin Guo*, Xu Lu*,

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Yi Zhang*, , Baodong Zheng*,

†

†

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*

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Fujian, China

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†

College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002,

†

Fujian Provincial Key Laboratory of Quality Science and Processing Technology in

Special Starch, Fuzhou 350002, Fujian, China

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ACS Paragon Plus Environment


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ABSTRACT

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The objective of this study is to investigate the effect of microwave irradiation on the

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physicochemical and digestive properties of lotus seed starch. The physicochemical

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properties of lotus seed starch were characterized by light microscopy, 1H NMR,

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FT-IR spectroscopy, and HPSEC-MALLS-RI. The starch-water interaction and

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crystalline region increased due to the changed water distribution of starch granules

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and the increase of the double helix structure. The swelling power, amylose leaching,

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molecular properties and radius of gyration reduced with the increasing microwave

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power, which further impacted the sensitivity of lotus seed starch to enzymatic

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degradation. Furthermore, the resistant starch and slowly digestible starch increased

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with the increasing microwave irradiation, which further resulted in their decreasing

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hydrolysis index and glycemic index. The digestive properties of lotus seed starch

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were mainly influenced by the reduced branching degree of amylopectin and the

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strong amylose-amylose interaction.

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KEYWORDS: Lotus seed starch; microwave irradiation; physicochemical properties;

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digestive properties.

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INTRODUCTION

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Digestive property is one of the important functional characteristics for

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evaluating the nutritional quality of the starch. According to the rate and extent of

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digestion, the dietary starch has been classified into rapidly digestible starch (RDS),

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slowly digestible starch (SDS) and resistant starch (RS).1 Some reports suggested that

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the capacity to digest starch was affected by their physicochemical structures. In raw

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state, starch granules are poorly digested in vitro, perfect crystalline conformations of

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amylopectin chains and a layer of non-starch barrier material such as polysaccharides,

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protein, fatty on the surface of starch granules result in less susceptible to digestion

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enzyme.2

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Processing conditions alter the physicochemical and structural properties of the

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starch granules, further impacting the digestion. Due to the high temperatures and

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pressures applied during the process, high-pressure steaming and extrusion cooking

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allow for faster enzymatic hydrolysis by decreasing the degree of crystallinity within

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starch graunles as gelatinisation occurs.3, 4 Complete gelatinization of starch during

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boiling also improves digestibility and reduce the RS. In contrast, Kim et al found

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heat-moisture annealing reduced the digestion rate of potato starch by facilitating

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interactions between the amorphous and crystalline regions of starch.5 Simultaneously,

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lower digestibility was observed in fried and baked potato compared to other

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processing methods,6, 7 which may be partly attributed to limit the water availability

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and the formation of amylose―lipid complexes resistant to hydrolysis.

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It is well known that microwaves irradiation treatment, as a non-conventional

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forms of energy, become of important in the field of food processing, such as baking,

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cooking, thawing, blanching, dehydration, pasteurization, sterilization.8 Compared

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with the conventional heat, the unique heating is caused by the ability of polar

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molecules in food materials to absorb microwave energy and convert it into heat.

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There are several reported studies related to the interaction of microwaves with starch

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It was evident that microwave could cause the rearrangement of crystalline regions

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within the starch granules and lead to the physical and physicochemical changes,

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including water absorption ability, swelling power, paste viscosity to an extent which

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depended on starch types and treatment parameters.

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retrogradation of rice starch and lentil starch was inhibited by the microwave

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irradiation treatment.10, 11 This inhibition might affect the enzymatic sensibility of

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microwave-gelatinized gel, since a variety of ordered structures were formed to cause

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resistance to amylase due to the packing of double helices of short chains in

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predominant crystallization process.

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It was also reported that

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Lotus seed (Nelumbo nucifera Gaertn) is an edible herb commonly used in

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Ayurveda and Traditional Chinese Medicine. Studies have reported that lotus seed has

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antioxidant,12 immunoregulatory,13 anti-inflammatory14 and antiviral properties.15

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Starch, the main component of lotus seed (60% dry weight), consists of 40%

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amylose,16 which is relate to the rapid aging property. Studies have mostly focused on

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the physicochemical properties, structure and prebiotic effects of lotus seed starch.17

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However, few studies have attempted to investigate the digestion of lotus seed starch

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by microwave-gelatinized. Meanwhile, there were no reports to deal with the relation

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between its physicochemical and digestive properties.

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Therefore, in this study, the objective of this study was to investigate the effect of

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microwave irradiation on the physicochemical and digestive properties of lotus seed

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starch. The effect of microwave irradiation on the physicochemical properties of lotus

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seed starch was investigated using light microscopy, 1H NMR, FT-IR spectroscopy,

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HPSEC-MALLS-RI, respectively. In addition, the digestive properties were assessed

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in vitro.

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MATERIALS AND METHODS

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Materials. Frozen fresh lotus seeds were obtained from Fujian Green Acres

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Industrial Investment and Development Co., Ltd. (Fuzhou, Fujian, China). Amylose

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(Sigma-A0512), Porcine pancreatic a-amylase (EC3.2.1.1, 16 U/mg) was purchased

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from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), Amyloglucosidase (EC

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3.2.1.3; 1000,00 U/ml) from Aspergillus niger was purchased from Aladdin Industrial

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Co. (Shanghai, China). Other chemicals and solvents were of analytical grade.

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Preparation of Lotus Seed Native Starch. The preparation of lotus seed

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native starch was used the previous method reported by Guo, et al.18 Lotus seed starch

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contained, on average, 10.23% (d.b.) moisture, 0.42% (d.b.)ash, 0.43% (d.b.) protein

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and 0.35% (d.b.) lipid.

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Microwave Gelatinization of Lotus Seed Native Starch. For micorwave

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gelatinized treatment (MTLS). Native lotus seed starch slurries (30% dry basis) was

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heated in a microwave station(XH-300B，Beijing Xianghu Instrument Co., Ltd. China)

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at different power levels (2.4, 4.0, 6.4, or 8.0 W/g) until fully gelatinized. With regard

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to water bath gelatinized treatment (WTLS), native lotus seed starch sample was

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placed in hot water bath until fully gelatinized. The temperature of the starch sample

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was detected by resistance sensor.

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Swelling power and Leaching of Lotus Seed Amylose. 30% starch

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slurry was heated to 50 - 90°C in microwave power（8.0W/g）and in water bath.,

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respectively. Swelling power (SP) was reported as the ratio of the sediment weight to

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the dry weight of starch.19 The leaching amylose in the supernatant were measured

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using the iodine blue value and amylose (Sigma-A0512) is used as a standard product.

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Light Microscopy. The bright-field light images of the starch paste was

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viewed under 400× magnification in multi-function optical microscope (CX21FS1,

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Olympus, Japan). The starch samples were stained with 0.2% iodine solution.

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Nuclear Magnetic Resonance (NMR). A low field pulsed NMI 20-Analyst

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(Shanghai Niumag Corporation, China) with 18.4 MHz was used in the experiment.

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Approximately 1g of sample treated by microwave was placed in a 15 mm glass tube

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and inserted in the NMR probe. Carr–Purcell–Meiboom–Gill (CPMG) sequences

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were employed to measure spin-spin relaxation time T2, Typical pulse parameters

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were as follows: the echo time (TE) was 200µs, the echo number was set to 1000, the

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waiting time (TW) was 2s, seight scans were acquired for each measurement.

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Fourier Transform Infrared Spectroscopy (FTIR). Starch sample and

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potassium bromide were dried to constant weight at 105 °C. FT-IR analyses of the

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samples were carried out with fourier transform infrared spectrometer (VERTEX70,

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Bruker Co., Ltd, USA) following the method of Zhang et al.20 The spectra were

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acquired at wavelength from 400 to 4000 cm-1 with 4 cm-1 resolution.

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Mw of Starch Polymers. Lotus seed starch and each gelatinized starch sample

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solution were prepared using the previous method reported by Zeng et al.21 An aliquot

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of the filtered sample (1 ml) was subjected to HPSEC-MALLS-RI (Dawn HELEOS-П,

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Wyatt, USA) coupled to columns (P8514-806, Showa Denko, Tokyo, Japan), MALLS

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(632.8 nm, DAWN DSP, Wyatt Technology, Santa Barbara, CA), and an RI detector

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(Optilab DSP,Wyatt Technology, Santa Barbara, CA) connected in series. The mobile

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phase consisted of DMSO with 50 mmol/L LiBr, the flow velocity was 0.3 ml/min,

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and the dn/dc value was set at 0.074.22

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In-vitro Starch Digestibility. The in vitro starch digestibility was measured

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using the method reported by Zeng et al.23 Glucose concentration (GC) was

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determined in triplicate by the 3, 5-dinitrosalicylic acid (DNS) method, which was

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measured by absorbance at a wavelength of 540 nm. Starch hydrolysis content (%)

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was calculated by the following equation (Eq 1):

0 .9 × G C × V × 100 % SW × SC

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Dt =

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where Dt is the digested starch (g/100 g dry starch) at time t, GC is glucose

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concentration (mg/mL), V is the volume of digestive juice (mL), SC is the starch

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content (%), and SW is the sample weight (mg), 0.9 is the molar mass conversion from

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glucose to starch.

(1)

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The rapidly digestible starch (RDS) content was measured as the amount of

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glucose released in 20 min of incubation. The slowly digestible starch (SDS) fraction 7



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was defined as the fraction digested between 20 and 180 min of hydrolysis. The starch

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not hydrolyzed within 180 min was designated resistant starch (RS) content.

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Glycemic Index and Hydrolysis Kinetic. The kinetics of the in vitro starch

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digestion were evaluated to determine the in vitro digestion rate of lotus seed starch

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under different processing conditions.24 The results were fitted by a modified

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first-order kinetic model (Eq.(2)); Eq.(3) was used to calculate the area under the

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digestogram (AUC) between times t1 and t2; Hydrolysis index (HI) was calculated

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from ratio between AUC of the sample (0-180 min) and AUC of white bread,

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glycemic index (GI) was calculated from the hydrolysis index formula (Eq.(4))25

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C=

Dt − D0 = [1 − exp(−kt)] D∞ − 0

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K0 =

dDt = D∞ − 0 ⋅ K dx

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D∞ = D 0 + D∞ − 0

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t2 D∞ − 0   AUC exp =  D∞ ⋅ t + ⋅ exp(− kt ) K  t1

(3)

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GI = 39.71 + 0.549 HI

(4)

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where Dt is the digested starch (g/100 g dry starch) at time t, D0 is the digested

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starch (g/100 g dry starch) at time t= 0, D∞ is the digested starch (g/100 g dry starch)

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at time t= ∞, C is the ratio of digested starch at digestion time t, K is the the digestion

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rate constant, The value of K can be obtained from the slope of a linear-least-squares

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fit of a plot of ln (1

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starch at time t= 0.

−

(2)

C) against t. K0 is the first-order hydrolysis kinetic constant of

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Statistical Analyses. Triplicate measurements were performed. Graphs were

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constructed using OriginPro 8.1. Data were analyzed and significant differences were

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determined by DPS 9.05 (Science Press, Beijing, China). P ≤ 0.05 was considered to

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be statistically significant.

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RESULTS AND DISCUSSION

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Swelling Power (SP) and Amylose Leaching (AML). When starch

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granules are heated in excess water, they undergo a process called gelatinization.

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During gelatinization, the molecular order within granules was collapsed, the starch

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granules swelled up >10× their initial volume. Different starch types have different

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thermal transitions and swelling patterns. Starch swelling power (SP) is dependent on

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the content of conjugated lipids in starch and on the amylopectin structure.26 The SP

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curves of gelatinized lotus seed starch at different temperatures are shown in Figure

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1(A). As the temperature elevating from 50 °C to 90 °C, the SP value increased

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gradually before 70 °C and rising sharply between 70 °C to 80 °C due to starch

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gelatinization, and had different SPmax values. In the water bath, the SP of WTLS

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reached a maximum value (10.87) at 80 °C, the reduction in SP was due to the

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irreversible rupture of granules at 90 °C. Microwave irradiation played a prominent

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role in reducing lotus seed starch swelling during gelatinization. The SP of MTLS was

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decreased with the increasing microwave power. SPmax values of MTLS were 7.42,

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6.97 and 6.73, respectively at 2.4-6.4W/g. At 8.0 W/g, the SPmax value of MTLS was

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only 5.83, which was 46.36% lower compared to WTLS. These reported that a

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limitation of the SP of granules induced during microwave irradiation treatments. This

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phenomenon may be attributed to restructure occurred inside of granules, Upon

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irradiation, the amylose and amylopectin chains in starch molecules produced

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partially degraded dextrins which had higher proportion of short chains. This is bad

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for granules swelling. These results were agree with those of T. Palav et al.

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reported the lack of starch granule swelling was induced by microwave gelatinaztion

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and result in the absence of a continuous network of amylose chains.

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27

who

Amylose leaching (AML) is the other important feature in gelatinization. When

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starch granules are gradually heated in excessive water, starch granules swell and the

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amylose molecules leach out into the supernatant, result in a network surrounding the

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swollen granules, which inhibits further swelling of starch granules.28-30 Studies on

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AML provide information on the extent of interaction between starch chains in the

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amorphous and crystalline domains of the native granule. The extent of AML in both

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WTLS and MTLS is shown in Figure 1(B). When the temperature was below 60 °C,

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AML of WTLS and MTLS had no obvious discrepancy. Whereas the extent of AML

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on WTLS was higher than that of MTLS following heating up to 70, 80 and 90 °C. In

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the end of gelatinization, the amount of AML was decreased with the increasing

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microwave power. The AML of 8.0 W/g was 68.58 mg/g, which was 48.35% lower

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than that of WTLS (132.82 mg/g). When the temprtature was above 70 °C, the crystal

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structure of starch was disrupted, and the water molecules formed hydrogen bonds

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with hydroxyl groups in amylose or amylopectin, a large number of AML was

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founded in WTLS. According to the previous report, the decrease in AML on MTLS

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was mainly attributed to :(1) additional interaction between amylose-amylose (AM–

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AM) or amylose-amylopectin (AM–AMP) chains; (2) lipid complexed amylose

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chains.31 In this study, the decrease in AML was mainly due to the former, since few

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lipids (0.35%) was associated with lotus seed starch granules, thermal energy

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imparted to amylose chains was higher during microwave gelatinization, resulting in

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facilitating AM - AM interaction in the starch granule and the less amount of AML.

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Light Microscopy of Starch Granules. Amylose and amylopectin leaching

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can be assessed with diluted iodine. The suspension of MTLS and WTLS was

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confirmed by microscope investigation. At 50 °C (Figures 2A and 2a), all of the

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granules were slightly swollen, and a small amount of AML from the starch granules,

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with no significant differences between MTLS and WTLS. At 60 °C (Figures 2B and

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2b), starch granules began to swell and conglomerate. The change of WTLS was more

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pronounced. At 70 °C (Figures 2C and 2c), starch granules were significantly swollen

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and granules morphology was different in both treatments. In the case of WTLS,

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extensive AML was observed, which present blue in the image. Unlike WTLS, the

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granule surface of MTLS was collapsed and wrinkled, which suggested the mobility

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of the starch protons may occurred in the beginning of gelatination. When the

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temperature was up to 80 °C and 90 °C. starch granules swelled dramatically and the

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solution became a homogenous blue mixture, and only small remnants of starch

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granules could be observed (Figures 2D and 2d). On the other hand, the AML of

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MTLS was reduced and microscopic images exhibited amaranth color (Figures 2E

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and 2e). These results were consistent with the results from AML, further indicating

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that microwave irradiation could restrain the AML in gelatinization.

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The State of Water in Gelatinized Starch Measured by NMR. To further

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study the mobility and distribution of water in gelatinized starch, Starch–water

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interactions have mainly been studied using 1H NMR, and the present study utilized

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the CPMG pulse sequence to measure the transverse relaxation time (T2). T2 is the

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time required for an excited spin-spin proton to reach dynamic equilibrium after

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energy exchange with adjacent protons. It reflects the difference in the degrees of

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freedom of water. A higher degree of freedom is associated with longer transverse

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relaxation time, while a lower degree of freedom corresponds to a shorter transverse

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relaxation time.32 It was observed that there were three different water distributions in

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MTLS and WTLS. The first one was the minor peaks occurring between 1 and 10 ms,

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probably representing bound water (T21) of gels. The components with a relaxation

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time 10 ms

Effect of Microwave Irradiation on the Physicochemical and Digestive

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