Optimizing the texture and retrogradation properties of Niangao (Rice Cake) made with naturally fermented rice flour

Niangao is prepared from polished round-grained or waxy rice flour and it is a popular and traditional steamed rice cake in China, but its preparation is different. On this study, Niangao was produced with naturally fermented rice flour and the effects of fermentation on physical properties and rheological characteristics were investigated. The results suggest that the water-holding capacity, texture, and color of the samples were significantly improved after natural fermentation, while amylose content was decreased. Additionally, fermentation had a marked effect on retarding the retrogradation of Niangao because fermented Niangao exhibited lower degree of retrogradation compared with the control. Control means Niangao which was produced by unfermented rice flour. Fermented Niangao was harder to be digested than the control was.


Introduction
Rice is one of the major food crops worldwide, and more than 50% of the world's population depends on rice as the primary caloric source (Zhang et al., 2010).Rice products are staple foods, especially in China, who is the world's largest rice producer and consumer.Rice products have many unique attributes, such as ease of digestion, bland taste, and hypoallergenic properties (Roman et al., 2017).Various kinds of instant foods and fresh-cooked rice products, such as cooked rice grains, rice noodles, infant rice foods, baking rice goods, and beer or wine, share the market (Wang et al., 2018).Niangao (rice cake) is a popular traditional food in China, and this food has been served at festivals and feasts for the past 2000 years.This rice cake is different from steamed sponge cake (Fagao) in China in terms of its production, mouth feel, texture, flavor, and features (such as water content, appearance, microstructural aspects, and rheological properties).During storage, the high retrogradation rate of starch leads to a shortened shelf life of Niangao.Undesirable characteristics, such as moisture loss, flavor loss, increased firmness, and poor viscosity could occur.Therefore, improving the stability of Niangao would contribute to extending its shelf-life and higher market promotion.
Many countries use natural fermentation as a common traditional process to treat grain.The taste, mouth feel, and flavor of produced rice improve after natural fermentation of whole rice grains, and safety and nutrition of products are also enhanced (Zhu, 2018).Many types of fermented food from grain, such as the Mexico's pozol (Falguni et al., 2011), Turkey's Boza (Kancabaş & Karakaya, 2013), and Ondia's chapatti (Sharma et al., 2017), are available worldwide.Fermented cassava starch has better extrusion properties, which could be beneficial to improve bread quality.Studies have shown that fermented rice food contains many functional constituents, such as amino acids, functional oligosaccharide, antioxidant activity substances (e.g., VitB12, nucleotide and aromatic compounds).Fermented rice food also contains other functional compounds which can reduce cholesterol and blood pressure.Examples of these compounds are gaba (gamma-GA-BA) and some peptides, which can inhibit the serum lipid peroxidation, reduce blood cholesterol content, and enhance the antithrombotic function in the human body.Moreover, fermented food produces some beneficial microbes, such as lactic acid bacteria, and enzymes, such as protease, amylase, and digestive enzymes (Zhou et al., 2017).
Natural fermentation mainly occurs in the starch amorphous area, in which changes in the structure and composition of amylopectin are observed.Fermented rice flour more easily forms a clear and transparent paste than the control due to starch degradation caused by microorganism fermentation (Lu et al., 2005).Fermentation has a certain degree of impact on the characteristics of rice starch particles.For instance, the particle sizes of starch are reduced and became consistent with grain sizes.This characteristic can be beneficial to form a uniform gel and make a starch grain surface acquire a kraurotic-like erosion mark.Fermentation can also change the crystallinity of starch but not its crystal form (Somsubsin et al., 2018).The gelatinisation of rice and structural characteristics of rice products are significantly related.Fermentation has significantly influence on pasting temperature, peak sticky degree, minimum viscosity, final viscosity, collapse value, and retrogradation value of rice flour.The retrogradation value of rice flour is notably reduces after fermentation, which may cause a reduction in the short-term staling ability (Yang & Tao, 2008).Starch recrystallization will also be hindered in an acidic environment after fermentation (Xiong et al., 2010).
Oncrease in amylose content and production of lactic acid contribute to the formation of a gel network and increase in gel strength.At the same time, after fermentation the gel exhibits better aging resistance properties because of the decrease in the heavy crystallization ability of amylopectin.Fermentation considerably changes the physicochemical properties of rice flour.Thus, the quality and functional properties, taste, and flavor of rice products are also obviously improved.However, the characteristics of Niangao, especially that of fermented Niangao, has not been reported yet.On China, the traditional Niangao production has become industrialized and the technology is increasingly mature.But the digestion rate of traditional Niangao is not very high, and it is in short in storage period and easy to deteriorate, that make the market popularity of traditional Niangao relatively low.On this study, the effects of natural fermentation on the structure, texture, whiteness, water-holding capacity (WHC) and retrogradation rate of Niangao were investigated to improve the properties and extend the shelf life of this food.

Materials
Waxy rice and polished round-grained rice were purchased from Tianyuan Biotechnology Company (Hanchuan, HuBei, China).Moisture (method 44-15A), protein (method 46-11A), ash (method 08-01), and lipid (method 30-10) contents of the rice flours were measured according to the methods defined by the American Association of Cereal Chemist (2000).All other chemicals used were of analytical grade, and distilled water was used in the experiment.

Preparation of Niangao
The ingredients (40 g of polished round-grained rice, 20 g of waxy rice, 180 mL of water) were mixed, sealed, and incubated at 25 °C for 2 days (natural fermentation).The fermented rice was ground into thick liquid using a ball grinder (JP-DM, China).Then, the rice slurry was quickly leached for 40 min to remove the water using a vacuum suction filter pump.The dry rice flour (moisture content of 40%) obtained from leaching was sieved through a 20 mesh screen, shaped by a mold, and then steamed for 10 min in an electric cooker preheated to 100 °C.Then, after kneading and cooled at room temperature, the Niangao samples were packaged in polyethylene bags and then cut into pieces (35×25×1.2mm 3 ).The sample made of rice soaked for only 4 h was referred to as control.A photo of Niangao is shown in Figure 1.

Analysis of amylose content
The amylose content of the rice flour starch in Niangao was determined by the method of Juliano with slight modifications (Juliano et al., 2012).The sample (100 mg) was dispersed in 1 mL of 95% ethanol and 9 mL of 1 mol/L NaOH.The flour slurry was heated for 10 min in a boiling water bath to gelatinize the starch.The starch solution was mixed with 1 mL of 1 mol/L acetic acid and 2 mL of iodine solution (0.2 g of iodine and 2.0 g of potassium iodide in 100 mL of aqueous solution).The mixture was stirred well and allowed to stand for 20 min.Absorbance was then measured at 620 nm using a WFZ UV2100 spectrophotometer (UNOCO, Shanghai).

Color measurement
The surface reflectance of the fermented Niangao and control (samples were under a plate glass with a thickness of 1.2 mm) was measured using the UltraScan XE measuring color difference meter, model RSON (Hunter Associates Laboratory, Reston, VA, USA).The instrument was calibrated against a standard white reference tile (X=93.60,Y=91.60,Z=109.70).Hunter L (lightness), a (redness), and b (yellowness) values were obtained using the setting of D65 (daylight, 65 light angle).The average value from three random locations on the surface of each sample was used for statistical analysis.The whiteness (W) was calculated as follows (equation 1):

Water-holding capacity
WHC was determined using a published method with additional modifications (Hoove & Vasanthan, 1993).Dry sample (0.5 g) was weighed precisely into a test tube (vol.7 mL), and 5 mL of distilled water added.The formation of lumps was prevented by mixing in a Vortex mixer at room temperature for 20 min and then centrifuging at 2500 rpm for 5 min (TGL-16C, Shanghai Anting Science Onstrument Co.).The supernatant and residue were separated, and the residue was weighed.The quantity of dry matter in the supernatant was determined by drying at 105 °C, until a constant weight was obtained.WHC was calculated as follows: , where G is the mass of residue in the test tube forming part of the dry matter, and G1 is the mass of dry matter in the supernatant forming another part of the dry matter.

Texture and sensory property
A textural analyzer (TA-XT 2 , Stable Microsystems Ltd, Surrey, UK) was used to evaluate instrumental the sample texture.Sample (15×15×12 mm 3 ) were prepared using a stainless frame device and then placed on the platform.A cylinder-type stainless steel probe with a diameter of 50 mm was used to compress each sample to 50% of its original height with pre-test speed of 2.0 mm/s, test speed of 1.0 mm/s, and post-test speed of 1.0 mm/s.The average of at least 10 replicates was considered (Seo et al., 2007).The sensory evaluation test for Niangao was performed by trained panelists using a 9-point test.The evaluated attributes were color, off-flavor, delicate taste, sweetness, smoothness, softness, adhesiveness, and moistness for the difference test, and overall quality for the preference test.

X-Ray Diffraction (XRD)
To evaluate the recrystallization of the retrograded Niangao, the crystalline characteristics of fresh and retrograded Niangao were analyzed with a D/max-RAOOO X-ray diffractometer equipped with a copper tube operating at 35 kV and 30 mA, producing CuKα radiation of 0.154 nm wavelength.Diffractograms were achieved by scanning from 3° to 50° (2θ) at a rate of 8° /min, step size of 0.02°, divergence slit width of 1°, receiving slit width of 0.02 mm, and scatter slit width of 1°.The staling Niangao samples were stored at 4 °C for 5 days, dried at 35 °C for 24 h, and subsequently ground into powder before analysis.
All analyses were conducted in triplicate, and MDO Jade 5.0 evaluation of integrals was used to calculate the diffractograms.The crystallinity of the samples can be quantified by integrating the area under the fitting crystalline peaks.The software JADE 5.0 was used to deduct deduct the amorphous bottom, smoothing, and fitting the original peak to obtained the integrated areas.The relative crystallinity (X RC ) was indicated as X RC =(Is/Ic)×100, where Os is the integrated area of crystalline peaks in the treated samples, and Oc is the integrated area of the total strength of the diffraction peak (Primo-Martin et al., 2007).

Differential Scanning Calorimetry (DSC)
The retrogradation properties of Niangao samples were analyzed by a differential scanning calorimeter (DSC204F, NETZSCH, German) using stainless steel pans (PE 0319-0218).The equipment was calibrated with indium and tin standard, and an empty pan was used as a reference.Weighed samples (5 mg) removed from the central portion of the Niangao were hermetically sealed in aluminum to avoid moisture loss.Duplicate sample pans were prepared and each sample was heated from 20 °C to 150 °C at 5 °C/min.The temperature values obtained were the difference of the onset temperatures of transition (To) and the temperatures at the completion of transition (Tc).The peak transition temperature (Tp) was defined as the temperature at the maximum peak.The enthalpy of transition was estimated from the integrated heat flow over the temperature range of the transition, and is expressed as joule per gram of sample (J/g) (Orondi et al., 2017).

Scanning Electron Microscopy (SEM)
The structural properties of the microprofiles caused by the changes in retrogradation for Niangao were studied using a scanning electron microscope (HOTACHO X-650, NTC company, Japan).All the samples were cut into pieces with thickness of approximately 1 cm and slice area of 4 cm 2 and then dried at 35 °C for 24 h before tests.The samples were positioned on an aluminum bar using a double-sided stick tape and were plated with a thin film of gold-palladium (60:40) onto the surface.The samples were examined at an accelerating voltage of 10 kV.Omage J 1.42 q and plugin of FracLac -2.5.Release 1 d were used to analyze the scanning electron micrograph with binarization processing (Dàvila & Parés, 2007).

Digestion of starch in vitro
A 0.5 g portion of the sample was mixed with 25 mL of phosphate buffer (pH 6.9) and 5 mL of phosphate buffer which contained 2.6 units of α-amylase (A3176, Sigma).The flour slurry was placed in constant temperature water bath at 37 °C and oscillated.Then, 1 mL of the sample was taken out every 30 min for 3.5 h and heated in a boiling water bath and oscillated for 5 min to inactivate the enzyme.Then, the solution was cooled to room temperature.The sample (1mL) was mixed with 3 mL of 0.4 mol/L sodium acetate buffer (pH 4.75) and 20 μL of glucosidase (A3042, Sigma).Then, the mixture was oscillated under constant temperature at 55 °C for 45 min to hydrolyze starch to glucose.Glucose content was determined by DNS method.Starch content was determined by multiplying glucose content by the conversion factor (0.9), and each sample was measured in three parallel measurements.

Statistical analysis
All statistical analyses were performed using Origin software 8.0.Data were presented as means±standard deviation (SD) and calculated using one-way ANOVA of SPSS 17.0, followed by Tukey's multiple-range test.Statistical significance was defined as p< 0.05 or p< 0.01.

Composition and physical properties
The approximate compositions of the raw materials are listed in Table 1.The physical properties and color values of fermented and control samples are shown in Table 2.The apparent amylose content in the fermented Niangao was evidently lower (p<0.01)than in the control.This phenomenon was probably due to the fact that organic acid, glucoamylase and alpha-amylase were generated during fermentation and the amorphous area of the starch granule was hydrolyzed.This event apparently reduced the soluble amylose content, which was in accordance with other reports (Alonso-Gomez et al., 2016).On addition, most of the amylose fraction was quickly degraded by acid and enzymes at the start of hydrolysis (Franco et al., 2002).Niangao with lower amylose content may have chewy texture and lower retrogradation 813/817 813 rate, which would significantly delay the stalling of starch during storage at low temperature.During storage, the retrogradation of starch was partly affected by amylose chain reassociation (Auh et al., 2006).The amylopectin content of fermented Niangao was increased by 4.79% comparing with that of the unfermented Niangao.After fermentation, the recrystallization ability of the starch gel decreased during storage, resulting in good ageing resistance ability (Xiong et al., 2010).Conversely, a significant difference in WHC was found between the fermented and control (p<0.01).The WHC of the fermented sample was higher than that of the control.This characteristic may be due to the dissolution of some short-branched amylopectin during natural fermentation.This result was consistent with those studies in which the short-chain amylopectin with 5-9 glucose units was inferred to increase the swelling capacity (Jiang et al., 2013;Vandeputte et al., 2003).The change in the structure and composition of rice starch greatly influenced the quality of products after fermentation.The taste of Niangao was apparently more exquisite, and its structure was more elastic.
The lightness (L), redness (+a), and yellowness (+b) of the two kinds of Niangao differed notably (p<0.05).Overall, fermentation improved the whiteness of Niangao (P<0.01)(Table 2), which can be ascribe to starch purification and the decrease in crude lipids and ash content after fermentation (Park et al., 2012).This phenomenon may be due to the fact that rice protein and fat were degraded by microbial fermentation, and the mineral elements, which were embedded by the combination of protein, fat, and starch, were released and were more easily dissolved (Xie et al., 2017).Thus, the ash content of samples decreased, and the fermented Niangao appeared whiter and more transparent compared with the unfermented control Niangao.

Textural and sensory properties
The textural and sensory properties of Niangao are listed in Table 3. Hardness, adhesiveness, cohesiveness, and chewiness significantly differed (p<0.05).The chewiness and hardness of fermented Niangao were significantly higher than those of control (p<0.01).This phenomenon suggested that the fermented samples may be slightly firmer compared with the unfermented one.Moreover, evident increase in adhesiveness and cohesiveness were observed after fermentation (p<0.05).Adhesiveness was increasehad and depended on the combined effect of adhesive and cohesive forces, as well as the viscosity and viscoelasticity (Qi & Sun, 2010).
The trained panelists evaluated two kinds of Niangao, which had been freshly prepared (Table 3).The color, smoothness, and moistness of fermented Niangao scored higher than those of unfermented one (p<0.05).Although no obvious difference was found in softness, the overall quality of the fermented sample received a higher score.After fermentation, the reduced fat and protein contents of rice flours resulted in the release of starch grains which were originally combined.Simultaneously, the intramolecular and intermolecular in rice starch formed hydrogen bonds and an ordered network structure.Thus, fermented Niangao showed favorable chewy and flexible taste (Carmona-García et al., 2016).

X-Ray Diffraction analysis (XRD)
Final recrystallization was investigated by XRD to prove the effect of fermentation on retarding the retrogradation of Niangao.XRD patterns and corresponding crystallinity which   were observed from fermented and unfermented Niangao flour, are shown in Table 4.
Anabiosis is a straight part of amylose and amylopectin which showed a linear array, that is, from amorphous form reverting back to crystals.This parallel arrangement is a direct internal driving force of series of stalling phenomenon (Wu et al., 2010).The retrograded starch provided several peaks at approximately 2θ angles of 17°, 20°, and others (Niu et al., 2018), which were typical patterns of retrograded starch (B-type), and the pattern was clearly distinct from that of raw starch.This phenomenon was accompanied by a gradual increase in rigidity and phase separation between the polymer and the solvent (syneresis).B-type crystallinity was characterized by a well-defined peak at 17° (2θ).The formation of this peak was the result of the crystallization of melting amorphous starch, which was mainly caused by increased amylopectin fraction during storage (Thiré et al., 2003;Osella et al., 2005).The peak at 20° corresponded to the presence of crystalline V-type amylose-lipid complexes formed during processing (Romano et al., 2018).
Two major broad peaks were identified in all retrogradated Niangao samples at 2θ angles at nearby 24° and 39° (Figure 2).The intensity of the diffraction peaks of retrograded fermented Niangao was apparently lower than that of the control (Figure 2).
Given that the XRD peak of the retrograded starch was normally the dispersive broad peak (Seo et al., 2007;Niu et al., 2018), we used MDO Jade 5.0 software to smooth and fit these peaks to calculate crystallinity.Two peaks at 24° and 37° were found for the control Niangao, while two peaks at 24° and 39 o were found for fermented Niangao in each original pattern.Therefore, crystallinity was quantified by integrating the area under the fitting crystalline peaks.The relative crystallinity of fermented Niangao was markedly decreased to 72.90% compared with that of the control (100%).Both Niangao samples had strong diffraction peaks at 24°, and these peaks were characteristic diffraction peaks of B-type crystallization.The fermented Niangao had a small swelling peak at 24°, but the peak intensity of this position was much lower than that of control Niangao.These results implied that fermentation could retard the recrystallization or retrogradation behavior of rice flour in Niangao.These observations agreed with the following results on retrogradation endotherm value in DSC.

Thermal analysis
The enthalpy values of retrograded starch reflect the melting of the crystallites, which were formed by association between adjacent double helices during gel storage (Santiago-Ramos et al., 2018).This endotherm peak was due to the melting of retrograded amylopectin (Gunaratne et al., 2011) rather than amylose.The degree of starch retrogradation was measured using the enthalpy values (H) of the endothermic peak from 40 °C to 60 °C in the DSC thermograms.This peak was characteristic of the melting of retrogradated amylopectin.Table 5 lists the retrogradation transition temperature (To, Tp, and Tc) and H of samples after 5 days of storage at 4 °C and 16 days of storage at -18 °C.Fermented Niangao had lower H compared with the control at 4 °C and -18 °C.The results revealed that fermentation effectively inhibited starch retrogradation in Niangao.For both kinds of Niangao, the increase in the values of H at 4°C storage condition was greater than that those at -18 °C storage.Compared with those at -18 °C storage, the retrogradation of Niangao at 4 °C storage was more rapid.This phenomenon may be due to the fact that the protein and lipid contents of rice flour markedly decreased after fermentation such that starch, which was originally combined with protein or lipid, was released during fermentation, thereby purifing the rice starch (Lu et al., 2003).Compared with the known hydrophilicity of wheat gluten, lipids and rice proteins are hydrophobic (Peng et al., 2017).These modifications made rice starch acquire higher WHC to prevent starch molecular recrystallization during storage.Means (three replicate) ± Standard Deviation (n=3).X RC, which is the relative crystallinity, was calculated as follows: X RC =(Is/Ic)×100, where Is is the integrated area of fitting crystalline peaks (24° and 39°) in the treated samples, and Ic is the integrated area of fitting crystalline peaks (24° and 37°) in the control.Relative crystallinity of the control is 100.

Morphological properties
The scanning electron micrographs of the texture profile of the fermented and control Niangao samples at magnifications of 300×100 μm and 500×100 μm are illustrated in Figure 3 and listed in Table 6.The texture of fermented and control Niangao exhibited significant variations in shape.Although these surfaces appeared to have a honeycomb-like structure, which may be due to the high loss of water during storage, the fermented sample apparently had smoother and more intact structure than unfermented samples (Figure 3).Therefore, the microstructure could be used as the evaluation index to distinguish fresh from retrograded Niangao.
Fractal dimension reflects the complexity of a system.Fractal dimension becomes higher when a system is more complex.This characteristic shows the complexity of the curve, and the trends of filling with the scope of the plane (Xie et al., 2017).For Niangao, the higher fractal dimension was, the more incomplete the internal structure was, and the finer particles were.The pore equivalent diameter is inversely proportional to the fractal dimension.The smaller the pore equivalent diameter is, the smaller the inner structure particle is and the whole plane tended to be filled.Table 6 shows that the unfermented Niangao had higher fractal dimension and lower average pore equivalent diameter compared with the fermented Niangao.The structure of the control was evidently more compact, indicating the higher crystallinity of the structure and higher retrogradation.

Digestion rate of starch
The digestion rates of starch in fermented and control Niangao are shown in Figure 4.The digestion rate of starch in fermented Niangao was faster than that of control Niangao with time.The hydrolysis reached balance after 3 hours.This phenomenon illustrated that fermented Niangao was more digestible than control Niangao and the nutrients of the former were more easily absorbed by people.

Conclusions
Natural fermentation of rice, when used to produce Niangao, would cause marked changes in the amylose content and side chain length distribution of amylopectin in rice starch.These characteristics could improve the processing, texture profiles, and functional properties of Niangao, and ultimately the quality of Niangao products.Meanwhile, the WHC, moisture content, reducing sugar content, and color were also improved.On addition, fermentation enhanced the extensibility of Niangao, resulting in finer, smoother, and more delicious taste.Finally, fermentation inhibited the retrogradation of Niangao and enhanced the nutritional value of this food.Understanding the mechanism to prevent the retrogradation of Niangao at the molecular level would be useful in further kinetic studies.

Figure 4 .
Figure 4. Digestion rate of fermented and control Niangao.

Table 1 .
Proximate composition of raw materials.

Table 2 .
The physical properties of fermented and control Niangao.

Table 3 .
Textural and sensory properties of fermented and control Niangao.

Table 4 .
Relative crystallinity (X RC ) in retrograded samples as determined using x-ray diffraction.

Table 6 .
The pore equivalent diameter and fractal dimension of the SEM of Niangao samples.