A New Method of Producing Resistant Starch

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Food Hydrocolloids

journal homepage:www.elsevier.com/locate/foodhyd

Synthesis and characterization of citric acid esteri ﬁ ed rice starch by reactive extrusion: A new method of producing resistant starch

Jiangping Ye

^a

, Shunjing Luo

^a

, Ao Huang

^a

, Jun Chen

^a

, Chengmei Liu

^a,∗∗

, David Julian McClements

^b,^∗

aState Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, China

bBiopolymers and Colloids Research Laboratory, Department of Food Science, University of Massachusetts Amherst, Amherst, MA, 01003, USA

A R T I C L E I N F O

Keywords:

Citric acid Rice starch Extrusion Esteriﬁcation Digestibility

A B S T R A C T

Citric acid-esterified rice starch was synthesized using a one-step reactive extrusion method. The structure, physicochemical properties, and digestibility of the esterified starch were then measured. Anin vitrodigestion study indicated that esterification significantly increased the level of resistant starch (RS) in the samples. Fourier transform infrared spectroscopy (FT-IR) confirmed esterification had occurred between the citric acid and starch, with a new peak appearing at around 1730 cm⁻¹. X-ray diffraction indicated that esterification caused a decrease in the degree of crystallinity in the starch. The swelling power and solubility of starch citrate were both lower than unmodified starch. Turbidity measurements suggested there was a slower rate of reassociation in starch citrates during storage. These results suggest that citric acid esterification promoted cross-linking within the starch that significantly reduced its digestibility. This simple method may be useful for the creation of healthier starchy foods.

1. Introduction

Starch is a major constituent of cereal grains and an important dietary source of energy for humans (BeMiller & Whistler, 2009). It is composed of anhydro-glucose units connected by α-1,4 linkages in amylose chains, which are occasionally branched by connection throughα-1,6 linkages. For nutritional purposes, starch is divided into rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) based on the rate and extent of its digestion in thein vitro Englyst assay (Englyst, Kingman, & Cummings, 1992). Rapidly digestible starch is quickly digested (< 20 min in-vitro) in the small intestine, leading to a spike in blood glucose levels. Slowly digestible starch is broken down more steadily (20–120 minin-vitro) throughout the entire small intestine, leading to a more gradual elevation of blood glucose levels. Resistant starch (RS) is not hydrolyzed (> 120 minin- vitro) during incubation in the upper gastrointestinal tract (GIT) but produces short-chain fatty acids in the large intestine through microbial fermentation, which is beneﬁcial to colon health and helps protect against colorectal cancer (Englyst & Hudson, 1996;Sajilata, Singhal, &

Kulkarni, 2006). Many commonly cooked native starches are rapidly

broken down inside the human GIT, leading to a“spike”in blood glucose levels. Consequently, there is considerable interest in the mod- iﬁcation of native starches to increase their resistance to digestion within the GIT (Xia, Li, & Gao, 2016).

The consumption of RS-rich food products reduces the energy value of a meal and leads to a slower release of glucose in the bloodstream, thereby inducing a lower insulin response (Lockyer & Nugent, 2017).

Depending on its structural and physicochemical properties, RS occurs infive subtypes:RS 1–physically protected (e.g., starch in partially ground grains);RS 2– ungelatinized starch with B-type crystallinity (e.g., potato starch, green bananas, high-amylose corn);RS 3–retro- graded starch;RS 4–chemically-modified starches due to substitution or cross-linking (e.g., esterification, etherification, or hydroxypropyla- tion); and, RS 5 – amylose-lipid complexes (Raigond, Ezekiel, &

Raigond, 2015). Various approaches are available to produce resistant starch, including enzymatic, physical, and chemical modiﬁcations, with the latter being one of the most eﬀective (Lee, Lee, & Lee, 2018).

Recently, many types of chemically modified starches have been prepared using oxidation, etherification, esterification, hydro- xypropylation, and cross-linking approaches (Masina et al., 2017).

https://doi.org/10.1016/j.foodhyd.2019.01.064

Received 20 November 2018; Received in revised form 29 January 2019; Accepted 30 January 2019

Abbreviations:RDS, rapidly digestion starch; SDS, slowly digestion starch; RS, resistant starch; FT-IR, Fourier transform infrared spectroscopy; DS, degree of substitution; RE, reaction eﬃciency; SEC, Size-exclusion chromatography; XRD, X-ray diﬀraction

∗Corresponding author.

∗∗Corresponding author.

E-mail addresses:[email protected](C. Liu),[email protected](D.J. McClements).

Available online 02 February 2019

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Esterification is one of the most widely used chemical modification methods for starch. Citric, tartaric, maleate, and polylactic acids have all been used for chemical modification of starch (Alimi & Workneh, 2018;Olivato, Müller, Carvalho, Yamashita, & Grossmann, 2014;Park et al., 2018; Sánchez-Rivera, Núñez-Santiago, Bello-Pérez, Agama- Acevedo, & Alvarez-Ramirez, 2017; Wootthikanokkhan et al., 2012;

Zuo et al., 2014). Compared with other substances, citric acid esterification is often preferred because it only involves a mild acid treatment. The use of citric acid as an esterification agent also offers other advantages, including nutritional safety, non-toxicity, and inexpen- siveness (Sánchez-Rivera et al., 2017). Also, esterified starch chains are resistant to degradation because the substituents sterically hinder digestion by enzymes (Jyothi, Moorthy, Sreekumar, & Rajasekharan, 2007;Xie, Liu, & Cui, 2006).

Conventionally, the chemical modiﬁcation of starch is performed in stirred tank reactors (Moad, 2011). These reactors lead to uniform and gentle reaction conditions but they produce a large quantity of wastewater, gas, and residue, leading to environmental pollution. Moreover, the wet process is complex and time-consuming (Tian, Zhang, Sun, Jin,

& Wu, 2015). The use of extrusion oﬀers some solutions to these challanges. Extruders can be used to produce modiﬁed starches in a continuous reaction process with more consistent product quality.

Variations in screw design oﬀer the ability to control residence times, as well as providing opportunities for adding reagents and additives such as processing aids and stabilizers during the process (Moad, 2011).

Starch can, therefore, be conveniently esteriﬁed using this type of extrusion approach. During extrusion, citric acid is dehydrated and forms an anhydride because of the high temperatures involved. In turn, these chemical species can react with the hydroxyl (OH) groups on the starch to yield a citrate ester adduct and further heating of the reaction medium results in a cross-linking reaction (Olsson et al., 2013).

The objective of this study was to produce citrate esterified starch using a one-step reactive extrusion method. The main advantages of this method are its simplicity, high speed, use of a continuous process, relatively low cost, and limited generation of wastewater and gas. A range of complementary instrumental techniques was used to prove that the esterification reaction between starch and citric acid was successful and to characterize the properties of the esterified starch produced. Finally, the digestibility of the esterified starch was analyzed using anin vitromethod to establish its potential nutritional benefits.

The results of this study may lead to a commercially viable approach to improve the healthiness of many starch-based foods.

2. Materials and methods

2.1. Materials

Rice starch was kindly provided by Golden Agriculture Biotech Co., Ltd. (Jiangxi, China). It consisted of 22.9% amylose, 0.34% protein, and 0.20% fat (Fu, Luo, BeMiller, Liu, & Liu, 2015). Citric acid was purchased from the Nanchang Mingrui Chemical Co., Ltd. (Jiangxi, China).

Pancreatic α-amylase type VI-B from porcine pancreas (EC 3.2.1.1, A3176) and amyloglucosidase (EC 3.2.1.3, A7095) were purchased from Sigma-Aldrich (St. Louis, MO).^D-glucose assay kit was purchased from Megazyme International Ireland Ltd., (GOPOD Format K-GLUK, Wicklow, Ireland). All other chemicals and solvents were of analytical grade.

2.2. Synthesis of citrate starch composite by reactive extrusion

Citric acid (0%, 10%, 20%, 30%, 40%, of starch on a dry basis) was dissolved in 700 mL of distilled water at around 28 °C, and then the solution was slowly added to the rice starch (2000 g) with continuous agitation. The mixtures were stirred for 3 min, placed in a sealed bag, and equilibrated for 4 h. Then, reactive extrusion was performed by passing the samples through a twin-screw extruder (TSJ 30, Jiangsheng

Science and Technology Co., Ltd., Wuhan, China). The screw was 30 mm in diameter, and its length-to-diameter ratio (L/D) was 16:1.

The temperatures of the four sections of the sleeve that made up the extruder were adjusted to 80, 100, 90, and 75 °C, and the feed rate and screw speed were adjusted to 18 kg/h and 250 rpm, respectively. The extruder wasﬁrst primed using raw rice starch. When the parameters of the extruder became stable, citrate starch or control was prepared using the rest of the mixed material. The starch citrate extrudates were collected, placed in an oven, dried to constant weight at 45 °C, ground, and sieved through an 80 mesh sieve. The dry mixture was washed three times with absolute ethanol to remove the unreacted citric acid. Finally, the washed starch was air-dried at 45 °C.

2.3. Determination of the degree of substitution (DS) and reaction eﬃciency (RE)

The amount of citric acid esterified to the starch was analyzed by the method of Volkert, Lehmann, Greco, and Nejad (2010)andZuo et al. (2013)with minor modifications. Briefly, dried analyzed samples (2 g) were placed in a 250 mL conicalflask. Then 20 mL of deionized water was added with gentle shaking (to avoid adherence of the samples to the wall), and two drops of phenolphthalein were added. The solution was quickly titrated with 0.1 M of aqueous sodium hydroxide solution until the endpoint was reached,i.e.,the solution turned from colorless to pink. This change indicated that all the free citric acid had been neutralized. After that, 25 mL of 0.5 M aqueous sodium hydroxide solution was added. The stoppered conical flask was agitated and stirred for 60 min at room temperature. The excess alkali was back-titrated with a standard 0.5 M aqueous hydrochloric acid solution until the endpoint (the solution turn from pink to colorless). We then carried out a blank titration using extruded starch without citric acid. The degree of substitution (DS) was calculated as follows:

= − × × ×

A V V c

m

( 0 1) Μ 100%

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= − −

DS A

M M A

162

100 ( 1) (2)

Here,Ais the content of esteriﬁed carboxyl groups (%);Mis the molar mass of the substituent (citrate: 175 g/mol);mis the mass of the samples (mg);cis the concentration of aqueous hydrochloric acid solution (mol/L);V0is the volume of aqueous hydrochloric acid solution consumed by the blank (mL); and,V1is the volume of aqueous hydrochloric acid solution consumed by the esteriﬁed starch sample (mL).

The reaction eﬃciency (RE) was calculated as follows:

Theoretical DS = C × 162 / 175 (3)

RE = (DS / Theoretical DS) × 100% (4)

Here,Cis the mass of citric acid (g) divided by the mass of dry starch (g), and 175 is the relative molecular mass of citrate anhydride.

2.4. Size-exclusion chromatography analysis

The molecular structures of extruded starch and esteriﬁed rice starch samples were characterized using size exclusion chromatography (SEC) as detailed elsewhere (Bai et al., 2017) with minor modiﬁcations.

Starch samples were dissolved in dimethyl sulfoxide (DMSO) with 0.5%

(w/w) LiBr at a concentration of 2 mg/mL, which was also used as the mobile phase for SEC analysis after beingfiltered through a 0.45μm hydrophilic Teflon membranefilter. Dissolved starch samples were in- jected into the SEC system equipped with a Styragel HMW 6E column (Waters Co., USA) and refractive index detector (RI). The injection volume was 100μL, theflow rate was 0.3 mL/min, and the column oven temperature 80 °C. Pullulans with different average molecular weights (Mw = 10, 60, 110, and 280 kDa) were used as standards to establish a

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calibration curve. The molecular weight-retention time (TR) equation developed from this calibration curve was: TR=−2.0874 log (MW) + 32.634 (R²= 0.999).

2.5. Determination of in vitro digestibility of starch samples

In vitrostarch digestibility of the resultant riceflour was determined at 37 °C according to a procedure described previously (Ye, Liu et al., 2018). with slight modifications. This method mimics human digestion by using amyloglucosidase and porcine pancreatic α-amylase. Pan- creatic α-amylase (0.0225 g, 10 U/mg) was suspended in 7.5 mL sodium acetate buffer (0.02 M, pH 5.5) with magnetic stirring for 30 min, and then centrifuged at 1500×g for 5 min. The supernatant was transferred into a beaker and mixed with 0.75 mL amyloglucosidase (300 U/mL) before use. The starch sample (200 mg) was added to 10 mL sodium acetate buffer (0.02 M, pH 5.5) and equilibrated at 37 °C for 30 min with shaking in a water bath, then 0.75 mL of a mixture of pancreatic α-amylase and amyloglucosidase was added. Aliquots (0.5 mL) were removed at intervals of 20 and 120 min during the digestion. Each aliquot was then mixed with absolute ethanol (4.0 mL) to inhibit the enzymes and centrifuged at 4000×g for 10 min. Subse- quently, the glucose concentration of the incubated mixture was measured using aD-glucose assay kit (GOPOD Format K-GLUK, Megazyme International Ireland Ltd., Wicklow, Ireland). The percentages of RDS, SDS, and RS in the samples were calculated using the following equa- tions:

= − ×

×

RDS G FG

(%) ( 20 TS) 0.9

100 (5)

= − ×

×

SDS G G

(%) ( 120 TS20) 0.9

100 (6)

= − +

×

RS TS RDS

(%) ( TS SDS)

100 (7)

Here, G20and G120are the amounts of glucose released within 20 and 120 min of hydrolysis, respectively; FG is the amount of free glucose in the original sample, and TS is the total starch weight of the original sample.

2.6. FT-IR spectroscopy analysis

FT-IR spectra of samples embedded in KBr pellets were obtained using an FT-IR a Nicolet 5700 (Thermo Nicolet Co., Waltham, USA) spectrometer over a wavelength range of 400–4000 cm⁻¹. Data analysis was carried out using the instrument software (OMNIC 6.2, Thermo Electron Corporation, Madison, WI, USA). Spectra were baseline-corrected at 1200 and 800 cm⁻¹by drawing a straight line and the spectra were deconvoluted. A half-band width of 26 cm⁻¹and an en- hancement factor of 2.4 with triangular apodization were employed.

Intensity measurements were performed on the deconvoluted spectra by recording the height of the absorbance bands from the baseline. The ratios of absorbance height 1047 cm⁻¹and 1022 cm⁻¹were obtained for the deconvoluted spectra (Ye, Yang, et al., 2018).

2.7. XRD analysis

Wide-angle X-ray scattering measurements of samples were performed using a Di System X-ray diﬀractometer (Bede XRD Di System, Durham, UK) equipped with a copper tube operating at 40 kV and 200 mA, producing CuK_α radiation of 0.154 nm wavelength.

Diﬀractograms were obtained by scanning from 4 to 40° (2θ) at a rate of 4°/min, step size of 0.02° at room temperature.

2.8. Swelling power and solubility analysis

A starch sample (0.4 g) was suspended in the centrifuge tube

containing 40 mL distilled water and kept in a shaking water bath at 55, 65, 75, 85, and 95 °C for 30 min. The centrifuge tube was then cooled rapidly to 25 °C. After centrifugation at 4000 rpm for 15 min, the supernatant was collected and dried at 105 °C for 4 h, and the weights of both the dried supernatant and sediment were determined. The solubility (%) was determined as the weight ratio of the dried supernatant to the dry starch. Swelling power (g/g) was determined as the weight ratio of the sediment in the tube to the dry starch.

2.9. Turbidity analysis

The method of determining turbidity (as absorbance) was a mod- iﬁed version of that ofFu and BeMiller (2017). Triplicate samples were prepared by combining 0.5 g of sample and 400 mL of distilled water in a 500-mL beaker and then covering with plastic wrap. The beakers were then immersed for 30 min in a boiling water bath with gentle mixing and cooled rapidly to about 25 °C. Initial turbidity was measured by measuring the absorbance of an aliquant at 640 nm using a UV/visible spectrophotometer equipped with a programmable cell changer (Persee, Beijing, China). The remaining sample solutions were stored in a refrigerator at 4 °C. After 1, 2, 3, 4, 5, 6, and 7 days at 4 °C, the samples were equilibrated to room temperature, vortex mixed, and then the absorbance (A640) was again determined. Distilled water was used as a blank, the A640value of which was subtracted from the A640value for the solution containing starch.

2.10. Statistical analysis

All experiments were repeated three times, and the mean and standard deviation were calculated from these results. The statistical analyses were performed using SPSS (version 17.0, SPSS Inc., USA). A comparison of the means was ascertained by Duncan's test at 5% level of signiﬁcance using one-way analysis of variance (ANOVA).

3. Results and discussion

3.1. Degree of substitution and reactive eﬃciency

Citric acid undergoes dehydration when heated, forming very reactive anhydride species, which can react with starch in low moisture environments and form starch citrate (Wing, 1996). A food extruder is therefore ideal for carrying out this type of reaction because it is a one- step continuous process carried out in a reaction chamber with a high- temperature and low moisture content. We, therefore, examined the eﬃcacy of extrusion for the formation of starch citrate.

Initially, the inﬂuence of reactive extrusion on the degree of substitution (DS) of the starch citrate and its reaction eﬃciency (RE) were measured (Fig. 1). When the citric acid level increased from 10 to 40%, DS increased from 0.037 to 0.138, but there was little change in RE, with values ranging from 35.1% to 43.2%. The DS of starch citrate produced using the one-step reactive extrusion method in our study was fairly similar to that produced using high temperature/long time conditions reported by previous researchers (Kapelko-Zeberska, Zieba, Pietrzak, & Gryszkin, 2016;Mei, Zhou, Jin, Xu, & Chen, 2015;Sánchez- Rivera et al., 2017).

Additional insights into the modifications brought about by the esterification reaction were obtained by measuring the molecular weights of the samples using size-exclusion chromatography. Full molecular weight distributions, retention times (RT), and weight-average molecular weights (MW) of the starch citrate samples and control were measured (Fig. 2). The MWof extruded rice starch without citric acid added (DS = 0) was found to be 4.8 × 10⁷Da, which is similar to the values of native rice starch reported by other researchers (Shin et al., 2009;Zhong, Yokoyama, Wang, & Shoemaker, 2006). After addition of citric acid, the molecular weight significantly decreased (p < 0.05), which can be attributed to degradation of the rice starch by acid

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hydrolysis and shearing during extrusion. The MWvalues of the rice starch citrate prepared in our study were slightly lower than those reported byHung, Vien, and Lan Phi (2016), but consistent with those reported byShin et al. (2009), who found a MWof citric acid-treated rice starch of around 1400 Da. We also found that increasing the citric acid content resulted in a reduction in MW. This phenemon can be ex- plained in terms of more pronounced starch degradation at higher citric acid levels due to more acid hydrolysis of the glycosidic bonds in the starch molecules during extrusion (Olsson et al., 2013). The increased starch degradation may have facilitated the reaction of the citric acid anhydride with the hydroxyl groups on the starch chains and ac- celerated cross-linking in the starch citrate (Kapelko-Zeberska, Buksa, Szumny, Zieba, & Gryszkin, 2016).

3.2. In vitro starch digestibility

The digestive starch fractions including rapidly digested starch (RDS), slowly digested starch (SDS) and resistant starch (RS) of the control and starch citrates were presented using anin vitro digestion model to determine the impact of degree of substitution on the digestibility of starch under simulated gastrointestinal tract (GIT) conditions (Fig. 3). The percentages of RDS, SDS, and RS in the control starch were 49.7%, 15.6%, and 34.7%, respectively. When the DS of

rice starch was increased to 0.037, the RDS and RS content significantly (p < 0.05) increased and the SDS content decreased. These results indicate that a large fraction of the SDS in the extruded starch was destroyed when it was reacted with citric acid. This effect might be due to a change in the structural organization of the SDS fraction in the acid-treated starches to one that is more easily penetrated and hydrolyzed by digestive enzymes (Hung et al., 2016). The increase in RS may have been because the esterified and cross-linked structure formed by critic acid substitution was more resistant to enzyme hydrolysis than extruded starch.

When the DS values were further increased from 0.037 to 0.138, the RS content of the starch citrates signiﬁcantly (p < 0.05) increased from 37.9% to 91.0%. A positive correlation between RS and DS has also been reported in previous studies (Xia et al., 2016). This is probably because some of the hydroxyl groups on the starch molecules were esteriﬁed during the formation of starch citrate, which reduced the ability of the digestive enzymes to access the starch molecules by pre- venting access of the enzyme to the neighboring glycosidic bonds (Xie &

Liu, 2004). Previous studies have also reported that the chemical substitution of starch reduces its enzyme digestibility, probably because the bulky derivatized groups sterically hinder the formation of the enzyme-substrate complex (Xia et al., 2016). Thus, even though extrusion disrupted and degraded the structure of the rice starch, citric-acid mediated esterfication had the opposite effect, leading to a more highly substitution structure that was more resistant to attack by digestive enzymes. In addition, a greater amount of RS was observed in the samples with the higher DS values, which may be due to the formation of internal cross-links within the starch citrates (Mei et al., 2015). The mechanism for the cross-linking reaction (i.e, intermolecular di-ester formation) is the well-known Fischer-esterification between the two carboxylic acid groups of citric acid and the hydroxyl groups in starch (Olsson et al., 2013). This cross-linked strucutre is more resistant to hydrolysis by digestive enzymes, thus resulting in an increase in the RS content (Remya, Jyothi, & Sreekumar, 2018). Aflour made from high- RS starch may be useful as a functional food to replace conventional flours (maize, wheat, rice) in starchy foods, like pasta, bread, cakes, crackers, and cookies (Sánchez-Rivera et al., 2017).

3.3. Fourier-transform infrared spectroscopy (FT-IR)

To verify that the starch and citric acid underwent an esterification reaction, FT-IR analysis was performed on the non-esterified starch and esterified starches to observe any changes in the functional groups on the starch molecules. Previous studies have shown that the FT-IR spectra of starches are sensitive to changes in structure on a molecular Fig. 1.Effect of reactive extrusion on the degree of substitution of citric acid

and its reaction eﬃciency of rice starch at diﬀerent citric acid concentration.

Fig. 2.SEC chromatograms of control and starch citrate samples with diﬀerent DS.

Fig. 3.The percentage of RDS, SDS and RS of control and starch citrate samples with diﬀerent DS.

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level, such as starch chain conformation, crystallinity, and retrogradation (van Soest, Tournois, de Wit, & Vliegenthart, 1995). The FT- IR spectra, deconvoluted FT-IR spectra, and A1047/A1022ratio of starch citrates and their controls were determined (Fig. 4).

Zuo et al. (2013)reported that the main infrared absorption peak

positions and group assignments for starch are as follows: 3400 cm⁻¹is associated with OeH stretching and vibration of the hydrogen bond;

2930 cm⁻¹ is associated with CeH asymmetrical stretching and vibration; 1625 cm⁻¹ is associated with HeO bending vibration;

1152 cm⁻¹is associated with CeOeC asymmetrical stretching and vibration; 1080 cm⁻¹is associated withD-glucopyranose and hydroxyl- linked CeO stretching and vibration; and, 925 cm⁻¹is associated with glucosidic bond vibration. Compared with unmodiﬁed starch, a new peak was observed around 1730 cm⁻¹in all the citrate starch samples.

This band was associated with the stretching vibration of the C]O bond from the acetyl group (Gamonpilas et al., 2011;Xie et al., 2006).

The presence of this new band can, therefore, be used as an indicator of successful esteriﬁcation.

In our study, the starch citrate samples were washed with ethanol before analysis, and so any free citric acid that could have interfered with the analysis was removed. The peak at 1730 cm⁻¹observed in all starch citrates is indicative of the formation of a covalent ester bond between the citric acid and starch molecules. Moreover, the magnitude of the absorption peaks at around 3400, 2930, 1155, and 1020 cm⁻¹ became weaker in the citrate starches. These changes suggest that the hydroxyl and carboxyl groups of citric acid formed covalent bonds with the CeO groups in starch.

The deconvoluted FT-IR spectra of the control and starch citrate samples were also determined (Fig. 4B). The FT-IR absorbance band at 1047 cm⁻¹is known to be sensitive to the crystalline regions of starch whereas the band at 1022 cm⁻¹is sensitive to the amorphous regions, allowing the absorbance ratio (A1047/A1022) to be used to express the degree of molecular order in starch (van Soest et al., 1995). The absorbance ratio decreased as the DS level in the esteriﬁed starch samples increased (Fig. 4C), indicating that citrate substitution altered chain packing and generated more amorphous regions (Mei et al., 2015).

3.4. X-ray diﬀraction analysis

The X-ray diffraction patterns of dried starch citrate samples and controls were measured to provide additional information into changes in crystalline structure (Fig. 5). The extruded rice starch (DS = 0) dis- played diffraction peaks at 2θof 7.5°, 13.2°, a doublet at 17° and 18°, 19.8°, and 22.3°. It has been reported that X-ray diffraction peaks at 15°, 17°, 18°, and 23° are characteristic of the A-type starch that is common to most native rice starches (Hung et al., 2016). The peaks observed at around 13° and 20° are typical of the V-type pattern resulting from the complex of amylose with lipids (Liu et al., 2017;Ye et al., 2016). Some peaks in the X-ray diffractogram disappeared after addition of citric acid to the starch, but there were still peaks observed at 7.5°, 13.2°,

Fig. 4.FT-IR spectrum (A), deconvoluted FT-IR spectrum between 1200 cm⁻¹ and 800 cm⁻¹(B) and ratio of 1047 cm⁻¹/1022 cm⁻¹(C) of control and starch citrate samples with diﬀerent DS.

Fig. 5.XRD diﬀraction patterns of control and starch citrate samples with diﬀerent DS.

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18.1°, and 20°, which are in accordance with earlier reports for starch citrate (Shin et al., 2009;von Borries-Medrano, Jaime-Fonseca, Aguilar- Méndez, & García-Cruz, 2018).

The relative crystallinity of the starches ranged from 7.8% to 18.7%

(Fig. 5). There was a drop in the relative crystallinity of the citrate derivatives of 17%, 38%, 54%, and 58% for DS = 0.037 to 0.138, respectively, when compared to the control (DS = 0). In addition, the relative crystallinity of extruded starch (DS = 0) was 18.7%, which suggested that part of the starch was gelatinized due to the low moisture content used in this study (Liu et al., 2017). The addition of citric acid led to a decrease in relative crystallinity of the starch, which has also been reported in the previous studies (Mei et al., 2015; Xia et al., 2016).

A decrease in the intensity of the peaks associated with crystallinity with increasing DS was also observed in the present study. Previous studies have shown that citric acid solutions can penetrate into starch granules through channels and cavities, thereby disrupting the crystalline structure of the granules when they are extruded in the presence of citric acid (Mei et al., 2015). Substitution of citric acid groups on to the starch chains could restrict their molecular mobility and inhibit their ability to form crystalline regions through steric eﬀects (Xia et al., 2016). The starch citrate samples with the lowest relative crystallinity had the highest RS contents, which suggests that the decrease in digestibility may be due to the citric aicd substitution and formation of the cross-linked structure, rather than the changes in crystalline structure. Also, the tendency to decrease the digestibility of the starch may not be proportional to the degree of damaged crystal structure (Lee et al., 2018).

3.5. Swelling power and solubility

The swelling power and solubility of the control and starch citrate samples were measured at different temperatures (Fig. 6). For the control, the swelling power increased as the temperature was raised, while for the starch citrates, the swelling power did not change much (Fig. 6A). Specifically, the swelling power of the extruded rice starch (DS = 0) increased from 10.4 to 16.2 (g/g) when the sample slurry was heated from 55 to 95 °C, whereas the swelling power of all the starch citrates were significantly (p < 0.05) lower than those of the control (4.8–12.1 g/g). The swelling power of the control in our study was fairly similar to that reported in a previous study, except that ours was somewhat higher at 50 and 60 °C (Hung et al., 2016). The higher swelling power in our study below 65 °C was probably because extrusion damaged the starch granules, thereby increasing the absorption of water at low temperature. In general, the swelling power decreased with increasing DS, which is consistent with earlier studies (Hung et al.,

2016;Mei et al., 2015;Remya et al., 2018).

The cross-linking of the starch by citric acid would account for the observed reduction in swelling power. Cross-linking strengthens the bonds between neighboring starch chains thereby reducing their tendency to move apart during heating. In addition, the degradation of starch due to partial acid hydrolysis of amylose and amylopectin results in a reduction of the swelling power of the treated starches (Hung et al., 2016).

The solubility of the control and starch citrate samples increased with increasing temperature, but the magnitude of this eﬀect dimin- ished with increasing DS (Fig. 6B). It has been reported that starches treated with citric or lactic acid had a higher solubility than untreated starch (Hung et al., 2016). The authors attributed this increase to the high amount of short chain amyloses produced by acid hydrolysis, which easily dissociate and diﬀuse out of the granules during swelling.

On the contrary, our results showed that the solubility of all starch citrates gradually decreased with increasing DS. A similar trend has also been observed in earlier studies (Kapelko-Zeberska, Zieba, et al., 2016;

Mei et al., 2015). It has been reported that the cross-linking of hydrolyzed short chains was the most likely cause of the reduced solubility of starch citrates in water (Kapelko-Zeberska, Buksa, et al., 2016). Thus, at a suﬃciently high degree of substitution it is diﬃcult for water to penetrate into the starch citrate. These phenomena can be also evidenced that the cross-linking existed in starch citrates, thereby inhibiting their ability to swell and dissolve.

3.6. Turbidity analysis

Previous studies have shown that turbidity measurements can be used to monitor the retrogradation of dilute starch pastes due to changes in the density distribution caused by aggregation of amylose and/or amylopectin chains (Wang, Li, Copeland, Niu, & Wang, 2015).

The increase in turbidity of starch pastes during storage has been reported to be aﬀected by factors such as granule swelling, the level of amylose and amylopectin leaching, and the amylose and amylopectin chain lengths (Jacobson, Obanni, & Bemiller, 1997). For this reason, we measured changes in the turbidity of the control and starch citrate samples (Fig. 7).

The initial turbidity of the samples decreased with increasing DS, which was attributed to a reduction in the swelling, which led to lower light scattering (Shin et al., 2007). The turbidity of the control sample increased steadily throughout storage. Presumably, the gelatinized starch underwent retrogradation, thereby leading to dense micro- domains that scattered light more strongly (Fu & BeMiller, 2017). The starch citrate with the lowest DS (0.037) exhibited a similar tendency to the control, but the magnitude of the turbidity was lower. The starch

Fig. 6.Swelling power (A) and solubility (B) of control and starch citrate samples with diﬀerent DS.

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citrates with higher DS values (0.065, 0.120, and 0.138) all exhibited similar behavior to each other, with a rise in turbidity during theﬁrst three days, followed by a leveling oﬀat longer times. According to a previous study, substitution with citrate increased the stability of cooked starch pastes towards retrogradation (Agboola, Akingbala, &

Oguntimein, 1991). These results suggest that the presence of the citric acid groups on the starch chains limited the ability of the starch molecules to come close to each other, thereby inhibiting reassociation during storage. The improved stability of starch citrate during storage could have some potential beneﬁts for certain applications in the food industry.

4. Conclusion

We have shown that rice starch citrates with DS values ranging from 0.037 to 0.138 can be synthesized using one-step reactive extrusion.

This method has advantages over conventional methods of chemically modifying starch because it can be carried out rapidly using a continuous process. As a result, the starch citrate had a higher fraction of resistant starch, making it more resistant to digestion under simulated gastrointestinal tract conditions. The starch citrates with the highest levels of amorphous structure and lowest levels of crystallinity had higher RS contents, suggesting that the decrease in digestibility was due to esteriﬁcation and cross-linking of the starch molecules, rather than to changes in their crystalline structure. Furthermore, the starch citrates with more extensive covalent cross-linking had a greater resistance to swelling, which may be important for some commercial applications.

However, further studies are required to be sure that the starch citrate will provide the desired functional attributes in food products and that it does not adversely aﬀect the sensory qualities of the foods. Moreover, ourin vitrogastrointestinal studies should be conﬁrmed usingin vivo animal or human feeding studies.

Acknowledgments

This material was partly based upon work supported by the National Natural Science Foundation of China (31772005) and National Institute of Food and Agriculture, USDA, Massachusetts Agricultural Experiment Station (MAS00491). The authors also appreciated the scholarship from China Scholarship Council (No. 201706820024).

Appendix A. Supplementary data

Supplementary data to this article can be found online athttps://

doi.org/10.1016/j.foodhyd.2019.01.064.

References

Agboola, S., Akingbala, J., & Oguntimein, G. (1991). Physicochemical and functional properties of low DS cassava starch acetates and citrates.Starch Staerke, 43(2), 62–66.

Alimi, B. A., & Workneh, T. S. (2018). Structural and physicochemical properties of heat moisture treated and citric acid modiﬁed acha and iburu starches.Food Hydrocolloids, 81, 449–455.

Bai, Y., Wu, P., Wang, K., Li, C., Li, E., & Gilbert, R. G. (2017). Eﬀects of pectin on molecular structural changes in starch during digestion.Food Hydrocolloids, 69, 10–18.

BeMiller, J., & Whistler, R. (2009).Starch chemistry and Technology(3^rded.). Amsterdam, Holland: Elsevier.

von Borries-Medrano, E., Jaime-Fonseca, M. R., Aguilar-Méndez, M. A., & García-Cruz, H.

I. (2018). Addition of galactomannans and citric acid in corn starch processed by extrusion: Retrogradation and resistant starch studies.Food Hydrocolloids, 83, 485–496.

Englyst, H. N., & Hudson, G. J. (1996). The classiﬁcation and measurement of dietary carbohydrates.Food Chemistry, 57(1), 15–21.

Englyst, H. N., Kingman, S. M., & Cummings, J. H. (1992). Classiﬁcation and mearsur- ment of nutritionally important starch fractions.European Journal of Clinical Nutrition, 46, S33–S50.

Fu, Z., & BeMiller, J. N. (2017). Eﬀect of hydrocolloids and salts on retrogradation of native and modiﬁed maize starch.Food Hydrocolloids, 69, 36–48.

Fu, Z., Luo, S.-J., BeMiller, J. N., Liu, W., & Liu, C.-M. (2015). Eﬀect of high-speed jet on ﬂow behavior, retrogradation, and molecular weight of rice starch.Carbohydrate Polymers, 133, 61–66.

Gamonpilas, C., Pongjaruvat, W., Fuongfuchat, A., Methacanon, P., Seetapan, N., &

Thamjedsada, N. (2011). Physicochemical and rheological characteristics of commercial chili sauces as thickened by modiﬁed starch or modiﬁed starch/xanthan mixture.Journal of Food Engineering, 105(2), 233–240.

Hung, P. V., Vien, N. L., & Lan Phi, N. T. (2016). Resistant starch improvement of rice starches under a combination of acid and heat-moisture treatments.Food Chemistry, 191, 67–73.

Jacobson, M. R., Obanni, M., & Bemiller, J. N. (1997). Retrogradation of starches from diﬀerent botanical sources.Cereal Chemistry, 74(5), 511–518.

Jyothi, A. N., Moorthy, S. N., Sreekumar, J. N., & Rajasekharan, K. N. (2007). Studies on the properties of citrate derivatives of cassava (Manihot esculenta Crantz) starch synthesized by microwave technique.Journal of the Science of Food and Agriculture, 87(5), 871–879.

Kapelko-Zeberska, M., Buksa, K., Szumny, A., Zieba, T., & Gryszkin, A. (2016a). Analysis of molecular structure of starch citrate obtained by a well-stablished method.LWT- Food Science and Technology, 69, 334–341.

Kapelko-Zeberska, M., Zieba, T., Pietrzak, W., & Gryszkin, A. (2016b). Eﬀect of citric acid esteriﬁcation conditions on the properties of the obtained resistant starch.

International Journal of Food Science and Technology, 51(7), 1647–1654.

Lee, S. Y., Lee, K. Y., & Lee, H. G. (2018). Eﬀect of diﬀerent pH conditions on the in vitro digestibility and physicochemical properties of citric acid-treated potato starch.

International Journal of Biological Macromolecules, 107, 1235–1241.

Liu, Y., Chen, J., Luo, S., Li, C., Ye, J., Liu, C., et al. (2017). Physicochemical and structural properties of pregelatinized starch prepared by improved extrusion cooking technology.Carbohydrate Polymers, 175, 265–272.

Lockyer, S., & Nugent, A. P. (2017). Health eﬀects of resistant starch.Nutrition Bulletin, 42(1), 10–41.

Masina, N., Choonara, Y. E., Kumar, P., du Toit, L. C., Govender, M., Indermun, S., et al.

(2017). A review of the chemical modiﬁcation techniques of starch.Carbohydrate Polymers, 157, 1226–1236.

Mei, J.-Q., Zhou, D.-N., Jin, Z.-Y., Xu, X.-M., & Chen, H.-Q. (2015). Eﬀects of citric acid esteriﬁcation on digestibility, structural and physicochemical properties of cassava starch.Food Chemistry, 187, 378–384.

Moad, G. (2011). Chemical modiﬁcation of starch by reactive extrusion.Progress in Polymer Science, 36(2), 218–237.

Olivato, J. B., Müller, C. M. O., Carvalho, G. M., Yamashita, F., & Grossmann, M. V. E.

(2014). Physical and structural characterisation of starch/polyester blends with tartaric acid.Materials Science and Engineering: C, 39, 35–39.

Olsson, E., Menzel, C., Johansson, C., Andersson, R., Koch, K., & Järnström, L. (2013). The eﬀect of pH on hydrolysis, cross-linking and barrier properties of starch barriers containing citric acid.Carbohydrate Polymers, 98(2), 1505–1513.

Park, E. Y., Ma, J.-G., Kim, J., Lee, D. H., Kim, S. Y., Kwon, D.-J., et al. (2018). Eﬀect of dual modiﬁcation of HMT and crosslinking on physicochemical properties and digestibility of waxy maize starch.Food Hydrocolloids, 75, 33–40.

Raigond, P., Ezekiel, R., & Raigond, B. (2015). Resistant starch in food: A review.Journal of the Science of Food and Agriculture, 95(10), 1968–1978.

Remya, R., Jyothi, A. N., & Sreekumar, J. (2018). Eﬀect of chemical modiﬁcation with citric acid on the physicochemical properties and resistant starch formation in different starches.Carbohydrate Polymers, 202, 29–38.

Sajilata, M. G., Singhal, R. S., & Kulkarni, P. R. (2006). Resistant starch - a review.

Comprehensive Reviews in Food Science and Food Safety, 5(1), 1–17.

Sánchez‐Rivera, M. M., Núñez‐Santiago, M. d. C., Bello‐Pérez, L. A., Agama‐Acevedo, E.,

& Alvarez‐Ramirez, J. (2017). Citric acid esteriﬁcation of unripe plantainﬂour:

Physicochemical properties and starch digestibility.Starch Staerke, 69(9–10).

Shin, S. I., Lee, C. J., Kim, M. J., Choi, S. J., Choi, H. J., Kim, Y., et al. (2009). Structural characteristics of low-glycemic response rice starch produced by citric acid treatment.Carbohydrate Polymers, 78(3), 588–595.

Fig. 7.Turbidity of control and starch citrate samples with diﬀerent DS during 4 °C storage.

(8)

Shin, S. I., Lee, C. J., Kim, D.-I., Lee, H. A., Cheong, J.-J., Chung, K. M., et al. (2007).

Formation, characterization, and glucose response in mice to rice starch with low digestibility produced by citric acid treatment.Journal of Cereal Science, 45(1), 24–33.

van Soest, J. J., Tournois, H., de Wit, D., & Vliegenthart, J. F. (1995). Short-range structure in (partially) crystalline potato starch determined with attenuated total reﬂectance Fourier-transform IR spectroscopy.Carbohydrate Research, 279, 201–214.

Tian, Y., Zhang, X., Sun, B., Jin, Z., & Wu, S. (2015). Starch sodium dodecenyl succinate prepared by one-step extrusion and its properties.Carbohydrate Polymers, 133, 90–93.

Volkert, B., Lehmann, A., Greco, T., & Nejad, M. H. (2010). A comparison of diﬀerent synthesis routes for starch acetates and the resulting mechanical properties.

Carbohydrate Polymers, 79(3), 571–577.

Wang, S., Li, C., Copeland, L., Niu, Q., & Wang, S. (2015). Starch retrogradation: A comprehensive review.Comprehensive Reviews in Food Science and Food Safety, 14(5), 568–585.

Wing, R. E. (1996). Starch citrate: Preparation and ion exchange properties.Starch Staerke, 48(7‐8), 275–279.

Wootthikanokkhan, J., Wongta, N., Sombatsompop, N., Kositchaiyong, A., Wong-On, J., Ayutthaya, S. I. N., et al. (2012). Eﬀect of blending conditions on mechanical, thermal, and rheological properties of plasticized poly(lactic acid)/maleated ther- moplastic starch blends.Journal of Applied Polymer Science, 124(2), 1012–1019.

Xia, H. P., Li, Y. Y., & Gao, Q. Y. (2016). Preparation and properties of RS4 citrate sweet potato starch by heat-moisture treatment.Food Hydrocolloids, 55, 172–178.

Xie, X. J., & Liu, Q. (2004). Development and physicochemical characterization of new resistant citrate starch from diﬀerent corn starches.Starch Staerke, 56(8), 364–370.

Xie, X., Liu, Q., & Cui, S. W. (2006). Studies on the granular structure of resistant starches (type 4) from normal, high amylose and waxy corn starch citrates.Food Research International, 39(3), 332–341.

Ye, J., Hu, X., Zhang, F., Fang, C., Liu, C., & Luo, S. (2016). Freeze-thaw stability of rice starch modiﬁed by improved extrusion cooking technology.Carbohydrate Polymers, 151, 113–118.

Ye, J., Liu, C., Luo, S., Hu, X., & McClements, D. J. (2018a). Modiﬁcation of the digestibility of extruded rice starch by enzyme treatment (β-amylolysis): An in vitro study.

Food Research International, 111, 590–596.

Ye, J., Yang, R., Liu, C., Luo, S., Chen, J., Hu, X., et al. (2018b). Improvement in freeze- thaw stability of rice starch gel by inulin and its mechanism.Food Chemistry, 268, 324–333.

Zhong, F., Yokoyama, W., Wang, Q., & Shoemaker, C. F. (2006). Rice starch, amylopectin, and amylose: Molecular weight and solubility in dimethyl sulfoxide-based solvents.

Journal of Agricultural and Food Chemistry, 54(6), 2320–2326.

Zuo, Y., Gu, J., Yang, L., Qiao, Z., Tan, H., & Zhang, Y. (2013). Synthesis and characterization of maleic anhydride esteriﬁed corn starch by the dry method.

Zuo, Y., Gu, J., Yang, L., Qiao, Z., Tan, H., & Zhang, Y. (2014). Preparation and characterization of dry method esteriﬁed starch/polylactic acid composite materials.