Authored by Bin Zhao*
Abstract
The effects of nitrogen application at different levels (0, 45, 135 and 225 kg/ha in 2015 and 0, 90, 180, 270 kg/ha in 2017) on the physicochemical characteristics of Fagopyrum tataricum Gaertn starch were studied by field experiments. Starch from Fagopyrum tataricum Gaertn with application of nitrogen showed lower amylose content, particle size, and retrogradation, and higher structure complexity, pasting temperature, gelatinization enthalpy, relative crystallinity, light transmittance, solubility of the starch. Nitrogen application did not change the ‘A’-type crystalline pattern of Fagopyrum tataricum Gaertn starch. This study indicated that nitrogen level and years and the interactions among nitrogen fertilizer levels and years significantly affected physicochemical properties of Fagopyrum tataricum Gaertn starch. The integrated results also provide some information into the management of the fertilization conditions to obtain starches with special properties for applications in food or non-food industries.Keywords:Fagopyrum tataricum gaertn; Nitrogen; Starch properties
Introduction
Fagopyrum tataricum Gaertn (Tartary buckwheat) is an annual dicotyledonous pseudo cereal of the Polygonate family [1]. It can grow under low input conditions and be adapted to marginal lands with harsh environments [2], and it is cultivated mainly in southern China, northern India, Bhutan, and Nepal [4]. Fagopyrum tataricum Gaertn is one of the main minor grain crops and important medicinal crops rich in flavonoids over the world. Fagopyrum tataricum Gaertn has a great efficacy to reduce hypoglycemia hypolipidemic, even can be used to support the prevention and treatment of diabetes, high blood fat, hypertension and other diseases because of inutrient— rich and it’s rich in flavonoid. Nitrogen is an essential nutrient for Fagopyrum tataricum Gaertn due to its effects on the yield and quality of Fagopyrum tataricum Gaertn. Starch physicochemical characteristics represent the important parameters of Fagopyrum tataricum Gaertn quality.Recently, Fagopyrum tataricum Gaertn is catching more attentions because it contains high nutritional content (e.g., antioxidants, protein, dietary fiber, and resistant starch) and disease-preventative roles [3]. Fagopyrum tataricum Gaertn can be used for the production of many products, such as noodles, breads, cakes, teas and alcoholic drink [6]. The starch is the major component of Fagopyrum tataricum Gaertn seed and its content is high and around 70% [4]. Fagopyrum tataricum Gaertn starch had special physicochemical properties and is thereby capable of being exploited as a potential source of retrograded starch [5]. And it is also responsible for the textural properties of Fagopyrum tataricum Gaertn products in the absence of gluten protein [6]. Compared with wheat and corn starch, Fagopyrum tataricum Gaertn starch has a higher amylose content (20%-28%), a better water-binding capacity, more stable pasting properties, and lower percentage of retrogradation [7]. Fagopyrum tataricum Gaertn starch consists of amylopectin and amylose. The amylose content is one of most important traits to impact the quality of Fagopyrum tataricum Gaertn.
The structure and physicochemical properties of Fagopyrum tataricum Gaertn starch are affected by genetic background, soil conditions, climatic conditions and agricultural treatments during Fagopyrum tataricum Gaertn growth and development [8]. Nitrogen is an important and non-negligible environmental factor affecting crop quality. Many studies suggested that nitrogen fertilizer is not contributing to crop quality improvement, but many research results showed that appropriate nitrogen can maintain and improve crop quality [9,10]. Previous studies suggested that nitrogen fertilizer can significantly lower the peak viscosity, hot paste viscosity, cool paste viscosity, breakdown viscosity, amylose content and improve the physicochemical properties of rice starch [11,12], and affect the biosynthesis and quality of barley starch [13]. However, the effects of nitrogen fertilizer on starch were mainly studied on maize [14], wheat [15] and barley [13], rice [16]. To the best of our knowledge, there are few reports on how nitrogen application affects the physicochemical properties of Fagopyrum tataricum Gaertn starch. Therefore, it is essential to compare the physicochemical characteristics of Fagopyrum tataricum Gaertn starch of varied levels of nitrogen application and to evaluate the most optimum amount of nitrogen fertilizer applied to achieve high quality for its potential use in the food and non-food industries.
Material and Methods
Plant materials and experimental design
Seeds of the variety No. 2019 of Fagopyrum tataricum Gaertn were collected in the test farm (109.7E, 38.3N, altitude 1080 m), Yulin, Shaanxi Province, China. The soil type is sandy loam. The crop previous to Fagopyrum tataricum Gaertn was nursery. Soil had 0.15% total nitrogen, 1.82 mg/kg available phosphorus, and 21.65mg/kg available potassium. These seed samples were grown under the same conditions. The meteorological data was offered by agricultural meteorological information service platform.The experiment was arranged in a randomized block design. The nitrogen level included 0, 45, 135 and 225 kg/ha in 2015 and 0, 90, 180, 270 kg/ha in 2017 (non, low, medium and high nitrogen level). All the groups were treated with potassium (K) 37.5 kg/ha and phosphorus (P) 37.5 kg/ha. Seeds were sown on July 10 and the harvest time was October 7. Nitrogen fertilizer was applied in the form of base fertilizer and then no longer topdressing during the growth of Fagopyrum tataricum Gaertn. Other practices were in conformity with local recommendations.
Starch isolation
Five hundred grams of the Fagopyrum tataricum Gaertn seeds of the different treatments were separately taken, ground with universal high speed smashing machines (FW100, Taisite LTD, Tianjin, China) and sieved with 200 mesh (< 75 μm) sieve to obtain Fagopyrum tataricum Gaertn flour. Fagopyrum tataricum Gaertn flour with 80% ethanol with the solid-liquid ratio of 1:20 was treated at 50 °C for 30 min by ultrasonic treatment at the power of 500 W to remove the flavonoids. Then add distilled water at the solid-liquid ratio of 1:10 (g/mL), and soak in a water bath at 30 °C for 18 h to remove protein. Let it stand for a few minutes and then pour off the upper suspension and place it in an oven at 40 °C. After removing the fat with petroleum ether, the excess petroleum ether was washed away with 80% ethanol and washed repeatedly with water. Centrifuged at 4000 r/min for 10 min and the supernatant was removed. The upper gray matter was scraped until only white material and the lower white material was poured in a Petri dish or beaker. Finally, the Fagopyrum tataricum Gaertn starch was dried in an oven at 40 °C for 24 h and sieved with 100 mesh (< 150 μm) sieve. The starch was sealed in self-sealing plastic bags and stored at 4 °C for use.Amylose content
The amylose contents of the starches were measured and calculated according to the method of Yang and Zhang et al. [17]. The absorbance of the solutions of Fagopyrum tataricum Gaertn starches at different nitrogen levels was measured at 434 and 604 nm using the Blue Star B spectrophotometer (Saith Chemical Co., Ltd, Wuhan, China).Scanning electron microscopy (SEM) observation
The starch samples were affixed to the loading platform of the surface processor using a double-sided adhesive tape, and then put into the processor. The surface morphology of starch granules was observed and then photographed using scanning electron microscopy (JSM-6360LV, Jeol, Japan). The magnification was 2000×.Granule size analysis
The granule size was carried out using a laser diffraction particle size analyzer (Mastersizer 2000E, Malvern, England). The starch was in tap water and stirred. The instrument could measure starch samples between 0.1 and 1000 μm.Bivariate flow cytometric analysis
Starch granules were stained according to the method of Zhang, Feng et al. [18]. A bio fluorescent microscope (Imager M2, Carl Zeiss, Germany) was utilized to examine the starch staining efficiency. However, the starch suspension reagent was ddH2O. The Fagopyrum tataricum Gaertn starch granule suspensions were measured using BD FACSAria™ III, BD, America and analyzed using BD Bioscience [18].Pasting properties
Pasting properties of starch samples were determined using the Rapid Viscosity Analyzer (RVA4500, Parten, Stockholm, Sweden). Three grams of each sample with 14.0% moisture were directly weighed into aluminum RVA canisters, after which, 25.0 mL distilled water was added to achieve a total weight of 28.0 g. The parameters were set: The slurry was held at 50 °C for 1 min, then heated from 50 °C to 95 °C for 3.7 min, and held at 95 °C for 2.5 min. Finally, the sample was allowed to cool to 50 °C for 3.8 min and held at 50 °C for 2 min [19].Thermal properties
The thermal characteristics of the test starches were determined by a differential scanning calorimeter (DSC) (DSC2000, TA instrument, United States). The DSC analyzer was calibrated using indium. A total of 3.0 mg of the dried starch sample was mixed with two times volume of water sealed in an aluminum pan at room temperature in 2 hours. The sample pans were heated to 110 °C at a rate of 10 °C/min, while one empty aluminum pan was used as the control. The starch gelatinization parameters that the DSC curve involved were onset temperature (To), completion temperature (Tc), peak temperature (Tp) and phase transition enthalpy ΔH [20].X-ray diffraction analysis
XRD patterns of samples were obtained on an X-ray diffractometer (D8 ADVANCE A25; Bruker, Germany). The starch samples were scanned at 4°-60° (2θ) and a rate of 8 °/min at a target voltage of 40 kV and a current of 40 mA [5]. Relative crystallinity (%) was calculated by using software (MDI Jade 6).Retrogradation
One percent starch pastes of the samples were prepared and placed into 20 mL graduated test tubes with stopper, 20 mL per tube, and stored at 25 °C for 24 h. During this storage period, the supernatant volumes of the tubes were counted every hour. Accordingly, the retrogradation curve of the supernatant volume percentages with time was drawn [21].Light transmittance
The light transmittance was measured according to Chao et al. [21] with the Blue Star B spectrophotometer (Lab tech Ltd, Beijing, China) using distill water as their control.Solubility
The solubility of the starch samples was determined by using the method described by Chao et al. and Liu et al. [22,23]. Each 200 mg starch sample was transferred to a dry centrifugal tube, weighed, and mixed with 10 mL distilled water. Each tube was incubated in a shaking water bath at 60, 70, 80, and 90 °C for 30 min, cooled to room temperature, and centrifuged at 2000 r/min for 20 min. The supernatant was carefully decanted, and the tube with the remaining contents was weighed. The residue obtained after drying the supernatant represents the amount of starch dissolved in water. The solubility of the samples was calculated on a dry-weight basis (DB) through the following equation:Solubility = the weight of dried supernatant weight of starch
Data processing
All the tests and measurements were repeated trice and the data thus obtained were statistically analyzed by SPSS 19.0 (SPSS Inc., Chicago, IL, USA). All data represent the mean ± standard deviation. Difference significances among the means were tested by the analysis of variance and the least significant difference (LSD) at p < 0.05.Results and Discussion
The temperature variation of fagopyrum tataricum Gaertn during grain-filling stage
Grain-filling stage is the key period of dry matter and starch accumulation and the temperature is an important parameter that affects the crop growth and development [24]. Fagopyrum tataricum Gaertn began to produce more grains at about 40 days after seeding emergence and there were an end of filling stage at around 80 days for most of the Fagopyrum tataricum Gaertn grains [25]. Therefore, the date from 40 to 80 days after emergence was selected in order to investigate the effect of temperature change on amylose accumulation and starch characteristics. Compared with the maximum temperature in 2017 from 40 to 57 days, the higher maximum temperature and minimum temperature in 2015 could be seen in in the Figure 1A and the similar trend can also be observed at mean temperature (Figure 1C). As shown in the Figure.1B, there were a lower temperature difference from 40 to 50 days and a higher temperature difference from 50 to 80 days in 2017. Zhang et al. reported that the content of starch and amylose and the activities of key enzymes involved in starch synthetic metabolism in developing rice grains were influenced by different temperatures during grain filling stage [26]. And some studies indicated that heat stress during grain filling generally decreases starch contents and enlarges starch granules [27]. High temperature during early grain development play a more important role in affecting the starch pasting properties, crystalline structure, and granule size of cereal starches than that during late grain development [28].
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