Study on Stability and Antioxidant Activity of Red Anthocyanidin Glucoside Rich Hybrid Rice, its Nutritional and Physicochemical Characteristics

Abstract

Using HPLC, it was demonstrated that the major free phenolic compound in the pericarp and seed coat of red rice was cyanidin, which belong to anthocyanidin glucoside, exist only in the episperm of red rice as content of 1.64±0.10 mg/g. Anthocyanidin glucoside play a significant role in eliminating free radicals and reacting oxygen species (ROS) to prevent cellular oxidative stress. However, the chemical stability of anthocyanidin glucoside has been one of the major drawbacks for health applications. For longer storage, our results showed that red rice anthocyanidin glucoside could be stored with 0.3 % Vitamin C or 0.5 % NaHSO₃, and be protected from light, high temperature, high pH and metal ions in environment. Antioxidant activity verified that anthocyanidin glucoside performed better than Vitamin C in ferrous ions chelating activity, scavenging activities for hydroxyl and DPPH radicals. Overall, the red rice might be used as a natural antioxidant supplement applying medical treatments in future days.

Introduction

Anthocyanidin glucoside, water-soluble natural pigments, belong to the group of flavonoids and are responsible for the red, purple and blue colours of many flowers and fruits, expected to be alternatives to synthetic colourants, and attract pollinators and seed dispersers (Zhang et al., 2014). Since ancient times, they have been utilized as medicines or tanning agents by humans, whose chemistry and synthesis studied over a long period. Studies have demonstrated that anthocyanidin glucoside exhibit various pharmacological effects including antioxidation (Zafra-stone et al., 2007), anti-inflammation (Lin et al., 2008), antineoplastic (Wang et al., 2008) and induction of leukaemia cell apoptosis (Chang et al., 2005). Various neuro-protective effects (Kang et al., 2006), immunomodulatory (Taverniti et al., 2014), the prevention of amyloid-mediated neurodysfunction (PingHsiao et al., 2011) and amelioration of dyslipidemia (Li et al., 2015) also have been reported. Moreover, as for plants themselves, anthocyanidin glucoside act as ROS cleaners to maintain normal metabolic processes in response to various abiotic adversities in plants at the cellular level (Zhou et al., 2017). Because of their potential beneficial effects on human health, considerable attention has drawn to anthocyanidin glucoside and its antioxidant properties. Many botanists devoted themselves to breed with high anthocyanidin glucoside crop cultivars, such as colorful spring wheat (Knievel et al., 2009), blueberry (Li X et al., 2017), purple sweet potato (Tensiska et al., 2017), purple endosperm rice (Zhu Q et al., 2017) and red cabbage (Tong et al., 2017).

Rice (Oryza sativa L.) is one of the most principal food crops in the world, serving as the staple food source for more than half of the world's population (Pachuau et al., 2017), which contributes 55 %–80 % to the total calories intake of people (Farooq et al., 2009). Hybrid rice that has a yield advantage of 10–20 % over conventional varieties was developed and commercially grown (Cheng et al., 2007). During previous era of advancement and technology, scientist focused on improvement of production and quality of rice but still, there is a vast range of area to improve the nutritious and quality features in rice. Hence, our main focus was to breed rice for high anthocyanidin glucoside. The most common types of rice (>85 %) are white-hulled while other types have coloured hulls, mainly purple, black, and red (Goufo and Trindade, 2014). Red rice grows taller, produces more tillers than domestic rice and shatters most of its seeds early, regarded as a major weed in many rice-growing areas of the world (Oki et al., 2002). Because the major functional components in pigments rice were anthocyanidin glucoside, traditional Chinese medicine used red rice for strengthening kidney function, treating anemia, promoting blood circulation, removing blood stasis, treating diabetes, and ameliorating sight (Deng et al., 2013). Thus, making full use of red rice has good potential prospects for nutraceutical and functional food application (Min B et al., 2011).

Organic molecules, which protect the body's cells from damage initiated by reactive oxygen species and free radicals that might otherwise exert harmful metabolic effects thus promoting health are termed as antioxidants (Goufo and Trindade, 2014). Antioxidant activity cannot be measured directly but rather by the effects of the antioxidant in controlling the extent of oxidation. There are many methods currently used for determining antioxidant activity (Mei X et al., 2017), which shown great diversity such as Ferrous ions (Fe²⁺) chelating activity (FICA) (Patel, 2013), scavenging hydroxyl, DPPH, ABTS radical (Wang et al., 2018), peroxyl radical scavenging capacity (PSC) assay (Morita et al., 2017) and oxygen radical absorbance capacity (ORAC) assay (Chen et al., 2018).

Anthocyanidin glucoside degradation, in all coloured juices and nectars, followed first-order reaction kinetics (Kirca et al., 2006). While, the change in the color of red rice during storage is attributed to the oxidative degradation of procyanidine pigments (Hayashi and Yanase, 2016). Thus, making anthocyanidin glucoside storage for a longer time has a very high application value. The objective of our work was to breed high anthocyanidin glucoside red rice, determine the anthocyanidin glucoside content of red rice and evaluate the effect of light, temperature, pH, metal ions, Vitamin C and NaHSO₃ to influence the anthocyanidin glucoside stability. Besides, in this study, anthocyanidin glucoside and antioxidant activities were determined by various assays, including FICA, scavenging activities for hydroxyl and DPPH radicals from red rice extracts.

Materials ad Methods

2.1 Plant material and Chemicals    The ruby red rice (Oryza sativa L.) germplasm was picked from Sichuan Agricultural University Rice Research Institute since 2007. After 10 years cross breeding in Hainan and Chengdu, anthocyanidin glucoside rich red hybrid rice sterile line (Z3055A) and maintainer line (Z3055B) were picked out. The hybrid red rice Z3055A/R363 was used in this study. The reagents for HPLC have following standards: Anhydrous ethanol (99 %), Cyanidin standard (98 % purity), Peonidin standard (98 % purity), Delphinidin standard (96 % purity), Malvidin standard (96 % purity), Petunidin chloride standard (96 % purity) and Pelaegonidin standard (96 % purity). Other chemical components used in this study were formic acid, methyl alcohol, ascorbic acid (Vitamin C), sodium bisulfite, DPPH (1,1-diphenyl-2-picrylhydrazyl radical) and so on. All these reagents were analytically pure and purchased from Ruijingte Chemistry Company (Chengdu, China).

2. 2 Extraction, fractionation and purification of red rice episperm    The fresh red rice Z3055A/R363 samples (cleaned with distilled water) were put into the drying oven (DHG-9240B, Shen Xian Co., Ltd, Shanghai, P. R. China) 80 °C for 3 hours. Then the brown rice machine (JLG-II, Da Ji Co., Ltd, Hangzhou, P. R. China) was used to separate hulls and brown rice (with episperm, embryo, aleurone layer and endosperm). Then, we used tweezers and dissecting needles to fetch the episperm (red surface). Complete rice episperm washed with deionized water and dried at 45 °C until constant weight obtained, samples meshed into powder and passed through 60 mm sieve. Extraction of episperm powder was completed by using HCl and ethanol solution 0.1 mol/L (1:15, w/v) at 60 °C for four hours inside the ultrasonic cleaners (WD-9415B, LiuYi Co., Ltd, Beijing, China). Recovered supernatants were concentrated by using the rotavapor at 45 °C (R-210, BUCHI Co., Ltd, Switzerland). Ultrasonic cleaners used to dissolve the remaining solid residues. Final extract reconstituted with deionized water on ultrasonic cleaner before storage. Then samples were stored at –20 °C for further analysis.

2. 3 Anthocyanidin glucoside contents determination    The absorbance of the solution was measured at 530, 620, and 650 nm using a Shimadzu UV-visible spectrophotometer (T6S, Puxi, Co., Ltd, Beijing, China). The anthocyanidin glucoside content calculated according to the following formula:

OD=(A₅₃₀–A₆₂₀)-0.1(OD₆₅₀)-OD₆₂₀)

One unit of anthocyanidin glucoside content was expressed as a change of 0.1 OD (unit×ne un·FW⁻¹). Each sample was replicated thrice to get stable value, all performed by An et al. (2016) with a little modification.

2. 4 Identification of anthocyanidin glucoside by High performance liquid chromatography    Anthocyanidin glucoside extracts in red rice were examined by Agilent ZORBAX SB (AGILENT LC1260, USA) High performance liquid chromatography (HPLC) analysis done according to the method of Beye et al. (2013) and Zhu Q et al. (2017) with a little modification.

2. 5 External conditions influence on stability of red rice episperm anthocyanidin glucoside

2.5.1 Light conditions    Red rice episperm extracts divided into two groups to evaluate light effect on anthocyanidin glucoside stability. One was under dark (covered) and second was under the fluorescent light placed in room temperature. The samples tested for anthocyanidin glucoside content at 0, 2, 4, 6 and 8 hr.

2.5.2 Temperature conditions    Red rice episperm extracts were covered and placed at 10, 30, 50, 70 and 90 °C temperatures under incubators. The anthocyanidin glucoside contents measured at 0, 2, 4, 6 and 8 hr in samples.

2. 6 Inherent conditions influence on stability of red rice episperm anthocyanidin glucoside

2.6.1 The pH effect    The effect of pH on the stability of red rice anthocyanidin glucoside was studied in citrate phosphate buffer solutions at five different pH i.e., 1.0, 3.0, 5.0, 7.0 and 9.0. The solution (25 mL portion) then transferred to pyrex tube and examined for 8 hr with 2 hr interval in order to determine the anthocyanidin glucoside contents.

2.6.2 Metal ions on the stability    The influence of metal ions in red rice episperm anthocyanidin glucoside stability were studied at FeCl₂, ZnCl₂, MoCl₅ and CdCl₂ salt solution (5 mg/L) and pH was adjusted to the 3 by HCl. Salt solution mixed with red rice episperm anthocyanidin glucoside (200 mg/L) to make a total of 50 mL solution, then heated at 30 °C in thermostatic water bath for 8 hr to determine the content of anthocyanidin glucoside every 2 hr.

2. 7 Vitamin C and NaHSO₃ utilization for extract anthocyanidin glucoside stability    1 mL of Vitamin C and NaHSO₃ (concentration 0 %, 0.3 %, 0.5 %, 0.7 % and 0.9 %) were mixed with 49 mL of anthocyanidin glucoside into 50 mL Pyrex tubes, heated under the constant temperature of 60 °C for 30 min, finally anthocyanidin glucoside contents were determined.

2. 8 Antioxidant Activity    Fe²⁺ chelating activity (FICA) was measured according to Patel (2013). The hydroxyl radical scavenging activity was performed by the hydroxyl radical chelating assay (Fenton reaction) Zhuang et al. (2013) and Morales (2005) to assess the antioxidant activity of the refined components from red rice episperm. The DPPH radical scavenging activity was performed by the DPPH assay according to Li et al. (2018) and Manivannan et al. (2018).

2.9 Statistical Analysis    The experiment was replicated thrice under the same conditions. Data were expressed as the mean ± standard error (SEM). One-way ANOVA was carried out with multiple comparisons using Duncan's test to compare the means of different groups at p ≤ 0.05 or 0.01. All statistical analyses were performed using the SPSS 24.0 statistical package (SPSS Inc., Chicago, IL, USA).

Results and Discussion

3. 1 Quality characteristics    After 10 years of multi-locational trail plantation and demonstrations, anthocyanidin glucoside rich red hybrid rice was featured with high quality. With 100 days' growth period, the plant height of Z3055A and Z3055B hits about 102 cm (Figure 1A). The hybrid red rice Z3055A/R363 quality features can be observed in Table 1.

Fig. 1.

Red rice Z3055A, Z3055B and grain of hybrid red rice Z3055A/R363

(A) Z3055A, sterile line; Z3055B, maintainer line. The scale bar represents 10 cm.

(B) Red hybrid brown and polished rice of Z3055A/R363.The scale bar represents 2 mm.

Table 1. Quality characteristics of hybrid red rice Z3055A/R363

Quality characteristics	Result
brown rice rate	80 %
white rice rate	71.2 %
head rice rate	60.8 %
grain length	6.1 mm
length-width ratio	2.6
Chalkiness grain rate	34 %
chalkiness degree	4.4 %
transparency	Level 2*
alkali spreading value	Grade 4.8**
gel consistency	52 mm
amylose content	27.1 %
protein content	9 %

The results were tested by rice and products quality supervision inspection and testing center of Ministry of agriculture of China.

*  Level 2 means transparency is between 0.61∼0.70.

**  Grade 4.8 means gelatinization temperature range 70∼74 °C

3. 2 Anthocyanidin glucoside contents in different plant parts    As can be seen from Figure 1B, most of the anthocyanidin glucoside observed in the episperm (outer surface layer of brown red rice), while there were no anthocyanidin glucoside in white rice (Figure 2). Results indicated that the anthocyanidin glucoside contents exist only in red rice episperm as content of 1.64 ± 0.10 mg/g. Thus, to maximize the intake of antioxidant compounds, rice should be preferentially consumed in the form of brown rice (Goufo and Trindade, 2014). The total anthocyanidin glucoside content varied greatly among diffferent black rice cultivars (79.5–473.7 mg/100 g) and red rice (7.9–34.4 mg/100 g) (Chen et al., 2012, Chen et al., 2016). Previous studies reported that anthocyanidin glucoside were enriched in apple skin, whose content can be significantly enhanced by the increased expression of the MdMYB1 and MdLOB52 genes responding to light and nitrogen environment respectively (Takos et al., 2006; Wang et al., 2017). About rice, Kawahigashi et al. (2007) reported that OsPR1.1 promoter was isolated and the promoter activity was monitored using a reporter gene OSB2, which encodes a transcription factor for anthocyanidin glucoside synthesis. Most of anthocyanidin glucoside enriched in the skin in most species, but Zhu et al. (2017) made a high-efficiency vector system for transgene stacking and used it to engineer anthocyanidin glucoside biosynthesis in the endosperm (lining part of rice) named purple endosperm rice. All of these researches provide us a new direction to search for the molecular mechanism of anthocyanidin glucoside synthesis, transportation and accumulation in natural anthocyanidin glucoside rich red rice in the future.

Fig. 2.

Anthocyanidin glucoside content of different part of rice

The location of Anthocyanidin glucoside in rice. Error bars show the SEM (n = 3). Brown rice with episperm, embryo and endosperm; polished rice contains endosperm and little embryo; red rice, Z3055A/R363; white rice, G-725 (Oryza sativa L.).

3.3 Effect of external conditions on anthocyanidin glucoside stability    Since red rice episperm color is mainly produced by anthocyanidin glucoside, it is reasonable to examine the light and thermal stability of anthocyanidin glucoside so that this information can be used in the production process. Figure 3A represented the total anthocyanidin glucoside content from the red rice episperm extracts, which contains the contents as high as 200 mg/L at the first time. In the early 2 hours, anthocyanidin glucoside exposed the light decreased a little more than darkness group, but the differences were not significant. But after 4 hours, there were remarkable differences between 2 groups. Besides, the total anthocyanidin glucoside contents decreased considerably after exposure to fluorescence light for 8 hr at room temperature. Hence, extract disclosure to light has negative correlation with anthocyanidin glucoside contents. Bononi and Tateo (2007) and Tonon et al. (2010) investigated that desiccated extract's anthocyanidin glucoside were significantly sensitive to natural light, which support current study. Furthermore, plants protect them against high irradiance is to utilise anthocyanidin glucoside as a light attenuator, due to their strong light absorption (Nichelmann and Bilger 2017; Zhu et al., 2017). Thus, anthocyanidin glucoside are sensitive to light when extracting from plants during storage (Weber et al., 2017).

Fig. 3.

External and Internal conditions influenced Anthocyanidin glucoside stability

(A) The light influenced the content of red rice episperm Anthocyanidin glucoside, error bars show the SEM (n = 3). Statistically significant differences **(P < 0.01), * (P < 0.05) and “ns” (non-significant).

(B) Temperature influenced the content of red rice episperm Anthocyanidin glucoside, error bars show the SEM (n = 3). Lowercase letters indicate the statistical significance (P ≤ 0.05).

(C) The pH influenced the content of red rice episperm Anthocyanidin glucoside, error bars show the SEM (n = 3). Lowercase letters indicate the statistical significance (P ≤ 0.05).

(D) Metal ions influenced the content of red rice episperm Anthocyanidin glucoside, error bars show the SEM (n = 3). Lowercase letters indicate the statistical significance (P ≤ 0.05).

Temperature affects the stability of anthocyanidin glucoside of red rice episperm extracts, as shown in Figure 3B. Storage temperature had a significant effect on the degradation of anthocyanidin glucoside. Total anthocyanidin glucoside contents were measured for different time intervals after incubation at various temperatures. Total extract anthocyanidin glucoside contents reduced considerably, with a corresponding elevation in temperature, after incubation at 70–90 °C for 8 h, as expected. Similar findings reported by Hellström et al. (2013) disclosed anthocyanidin glucoside degradation was faster when stored at room temperature than in the smoothie stored at 4 °C. Hence, the anthocyanidin glucoside contents degradation and disruption in antioxidant activity are prone to elevated temperature and the degradation of anthocyanidin glucoside during storage under different temperatures showed first order kinetics (Mourtzinos et al., 2008; Weber et al., 2017). Mori et al. (2007) believed that high temperature (35 °C) reduced the total anthocyanidin glucoside content to less than half compared to the control berries group (25 °C) due to the factor of methoxylation, glycosylation, and acylation leading to an increase in the thermal stability of anthocyanidin glucoside. Yamane et al. (2006) found that the expression of the main and other related anthocyanidin glucoside biosynthesis genes were more in lower temperature (20 °C). Not only on anthocyanidin glucoside, temperature also have inhibitory effects on other antioxidant compounds: the decline in phenolic acids being greater at 37 °C than at 4 °C (Zhou et al., 2004), while an 18 % loss of γ-oryzanol and 70 % loss of vitamin E was quite prominent when exposed to room temperature (25 °C) for 6 months (Pascual et al., 2013).

3. 4 pH and metal ions scavenging anthocyanidin glucoside stability    The effect of pH on the stability of red rice anthocyanidin glucoside was studied in citrate phosphate buffer solutions at five different pH i.e., 1.0, 3.0, 5.0, 7.0 and 9.0. As seen in Figure 3C, red rice episperm anthocyanidin glucoside showed two distinct stability profiles: (1) Lower pH (1.0 and 3.0), and (2) Higher pH (5.0, 7.0 and 9.0). Significant decline in anthocyanidin glucoside stability was observed at pH 5.0 and above, every 2 hours. Alkali environment has significant impact on the stability of episperm anthocyanidin glucoside, when compared with the acidic environment of pH 3.0 anthocyanidin glucoside contents were more stable at pH 9.0. Hence, internal pH 3.0 have efficient stability to store red rice episperm anthocyanidin glucoside, the current study is in accordance with Torskangerpoll (2005) who believed that alkaline solutions could have drastic effects in storage and stability of the anthocyanidin glucoside. In contrast, Babaloo and Jamei (2018) believed that highest stability of anthocyanidin glucoside was observed in pH 1 and by increasing pH from 1 to 4, absorption is decreased, which was a little different from our results. All pigments experienced dramatic colour changes when kept in the alkaline storage period, as similar findings reported by Choi et al. (2017). A previous study by Williams and Hrazdina (2010) demonstrated that maximum complex formation occurs with the 3,5-diglucoside and 3-acylglucoside-glucosides at around pH 3.1, so that it could be suitable for extracting and storing anthocyanidin glucoside from red rice episperm at pH 3.

The magnitude of hyperchromic effect was strongly affected by the concentrations and species of metal ions. Among these metal ions, all of them have a strong influence to decrease the content of red rice episperm anthocyanidin glucoside in the following ascending order: Zn²⁺ > Cd²⁺ > Fe²⁺ > Mo⁵⁺ (Figure 3B). Anthocyanidin glucoside have an ability to bind metal as complexation for maintaining colour stability in various anthocyanidin glucoside-rich products (Fedenko et al., 2017). Araceli et al. (2009) believed that the blue colour was due to a complexation between anthocyanidin glucoside and some metals such as Al, Fe and Mo accounting for the B ring (with ortho-dihydroxyl group). Previous study reported that trivalent ions are more stable than divalent ions (Araceli et al., 2009; Fedenko et al., 2017). Therefore, metal free environment could be helpful in storage and stability of anthocyanidin glucoside in red rice.

3.5 Phytochemical characteristic of major anthocyanidin glucoside from red rice    The chemical compositions in the anthocyanidin glucoside were analyzed by HPLC. The results of HPLC and tentative identification are shown in Figure 4A. Six phenolic compounds were identified in the present study, which includes delphinidin, cyanidin, petunidin chloride, pelaegonidin, peonidin and malvidin. HPLC analysis revealed that the major free phenolic compound in the episperm of red rice was cyanidin. Compared to purple potato, red cabbage and color wheat (Knievel et al., 2009; Takayanagi et al., 2005; Tong et al., 2017) which contained at least two or three other compositions of their main anthocyanidin glucoside. According to the results of Saigusa et al. (2014), phenolic compounds, such as malvidin and peonidin, are the main active components of black rice, and black rice bran contained anthocyanidin glucoside 18–26 % (Laokuldilok et al., 2010). But there are significant differences in phytochemical content and antioxidant activity among the different black rice varieties (Zhang et al., 2010). Bell et al. (2014) reported that the major compositions of anthocyanidin glucoside in cherry was cyanidin-3-o-glucosiderutinoside which is similar as red rice. Cyanidin acts as antioxidant and anti-carcinogenesis properties and an important role in osteoclastic bone resorption and osteoporosis (Cheng et al., 2017), which implies the potential medical function about the red rice.

Fig. 4.

HPLC analysis, protectiveness and antioxidant capacity

(A) HPLC chromatogram (530 nm) of episperm of red rice Anthocyanidin glucoside. The peak assignments are: (1) delphinidin, (2) cyanidin, (3) petunidin chloride, (4) pelaegonidin, (5) peonidin and (6) malvidin

(B) Vitamin C additives on red rice episperm Anthocyanidin glucoside stability protection at 60 °C for 30 min. Error bars show the SEM (n = 3). Lowercase letters on the top of column indicate the statistical significance (P ≤ 0.01).

(C) NaHSO₃ additives on red rice episperm Anthocyanidin glucoside stability protection at 60 °C for 30 min. Error bars show the SEM (n = 3). Lowercase letters on the top of column indicate the statistical significance (P ≤ 0.01).

(D) FICA of the extracts, error bars show the SEM (n = 3). Statistically significant differences **(P < 0.01), * (P < 0.05)

(E) Hydroxyl radical scavenging activity of the extracts, error bars show the SEM (n = 3). Statistically significant differences **(P < 0.01)

(F) DPPH radical scavenging activity of the extracts, error bars show the SEM (n = 3). Statistically significant differences **(P < 0.01), * (P < 0.05), “ns” (non-significant).

3.6 Vitamin C and NaHSO₃ additives on anthocyanidin glucoside stability protection    Shelf life is a major consideration in developing, producing, and marketing many food products. In current study, anthocyanidin glucoside decomposed all the time. Hence, we use Vitamin C and NaHSO₃ additives to explore how they protect the stability of red rice episperm anthocyanidin glucoside. The results (Figure 4 B and C) showed that adding both Vitamin C and NaHSO₃ could increase the content of anthocyanidin glucoside after 30 min at 60 °C water baths compared to control. Besides, the contents of anthocyanidin glucoside were different with different concentration of Vitamin C and NaHSO₃. Vitamin C concentration of 0.3 % had the highest content of anthocyanidin glucoside with the preserving rate as high as 98.41 %, while, with the concentration of 0.5 %–0.9 % Vitamin C, the content of anthocyanidin glucoside declined significantly from 98.41 % to 90.04 %, anthocyanidin glucoside contents were 88.96 % without vitamin C. At the same time, NaHSO₃ had the similar influence to maintain the degradation rate of anthocyanidin glucoside, NaHSO₃ concentration of 0.5 % had the highest content of anthocyanidin glucoside with the preserving rate 96.61 % and the degradation rate of anthocyanidin glucoside without NaHSO₃ was 85.19 %. Consequently, both of Vitamin C and NaHSO₃ were effective to extend anthocyanidin glucoside shelf life compared to other internal and external factors such as light, temperature, pH and metal ions. The results of Zhang et al. (2013) were in accordance with current study that enrichment of anthocyanidin glucoside in tomatoes could significantly extend shelf life. Therefore, it is universal to use food additives in food such as Vitamin C (Shabbar et al., 2012) and NaHSO₃ as the antioxidants to retard the deterioration.

3. 7 Antioxidant activities of the extract    It is widely believed that oxidation is one of the necessary factors for developing many chronic degenerative processes, including aging (Teixeira et al., 2013). Reducing radicals power has been used as one of the antioxidant capability indicators of medicinal herbs. The methods of FICA and scavenging activities for hydroxyl and DPPH radicals are usually used to measure the antioxidant capacity of plant extracts, which can effectively indicate the changes of antioxidant capacity (Zielinska and Michalska, 2016; Feng et al., 2017). The results can be observed in Figure 4 D, E and F.

Fe²⁺, commonly found in food systems, is well known as the most powerful pro-oxidant among the various species of metal ions (Revilija et al., 2005). The purpose of ferrous ion chelating activity test was to determine the capability of testing material to bind the oxidation catalytic ferrous ion (Hsu et al., 2003). In this assay, the red rice episperm extracts and Vitamin C interfered with the formation of ferrous and ferrozine complex, suggesting that they have chelating activity and are able to capture ferrous ion before ferrozine. As we can see form the Figure 4D, both the red rice episperm extracts and Vitamin C showed relatively similar trend in high chelation activity, but Vitamin C had a lower ferrous ion chelating capability compared to the red rice episperm extracts at all concentrations. It is noteworthy that the red rice episperm extracts has a better capability in chelating iron. M. Elmastaş et al. (2006) exposed that the iron chelating capacity test indicated a gradual decrease in the absorbance of the red Fe²⁺/ferrozine complex as antioxidants compete with ferrozine in chelating ferrous ion.

Hydroxyl among all the oxygen radicals is the most reactive radical, a source of cancer, aging and several diseases. Oxidation of lipid is primarily initiated by hydroxyl radical resulting hydrogen atoms extraction from un-saturated fatty acids. Therefore, its chelation provides protection against severe damages. The scavenging of hydroxyl free radical in red rice episperm extracts was found to be higher than Vitamin C (Figure 4E). Besides, both of them with the lower concentration of 2–10 mg/L had a very high speed to enhance the scavenging rate. The concentration of 10–40 mg/L gently enhanced the scavenging rate. Slightly stable scavenging rate was observed above the concentration of 40 mg/L. Biomolecules (DNA, Proteins and Nucleic acids) are prone to reactive hydroxyl radical as it degrades them quickly (Jin and Wu, 2015; Huang et al., 2010). Reactive free radical formation (Fenton reaction, Fe²⁺+H₂O₂→Fe³⁺+OH⁻+OH·) is accelerated with the utilization of ferrous state of iron. Hydrogen and lipid peroxides oxidized by ferrous ions (Oke and Aslim, 2010). Hydrogen peroxide and hydrogen peroxide free radicals, derived from alkyl free radicals, are the major products during the propagation period (Hsu et al., 2003). Therefore, its removal leads to the increased immunity in living body against several diseases. The red rice anthocyanidin glucoside utilization could be helpful in elevating the hydroxyl scavenging activity. Hence, red rice serves as an effective source to provide immunity against several diseases.

DPPH radical scavenging activity was increased in the red rice episperm extracts and Vitamin C (Figure 4F) with the concentration between 1 to 6 mg/L and the scavenging rate was 88 % and 70 % respectively. Besides, the increasing rate was not so regular between 6–10 mg/L, while the steepness of slope becomes nearly stable after 10 mg/L and the differences between extracts and Vitamin C was not significant. DPPH is a free radical that accepts an electron or hydrogen radical to become a stable diamagnetic molecule (Omp and Tejk, 2009). The reaction between antioxidant molecules and radical, grades in decreased absorbance of DPPH, which ultimately results in scavenging activity of the radical by the donation of hydrogen (Elmastaş et al., 2006). Dang et al., (2017) reported that the fermented rice bran extracts had a strong antioxidant activity in scavenging DPPH which is accordance with our study. Kim et al., (2017) found a kind of yellow grain rice mutant which was enriched flavonoids can eliminate DPPH as well as red rice anthocyanidin glucoside. The oxygen radical absorbing capacity was ranked as follows: red > black > green > white rice (Chen XQ et al., 2012), which indicated the high potential medicine value of red rice.

Conclusions

Using heterosis, we bred anthocyanidin glucoside rich red rice, and the results revealed that anthocyanidin glucoside exist only in the red rice outer layer surface. Based on the results observed in present study and the aforementioned discussion, it is concluded that the red rice anthocyanidin glucoside have a better ability than Vitamin C in FICA, scavenging activities for hydroxyl and DPPH radicals. It can be stored with 0.3 %Vitamin C or 0.3 %–0.5 %NaHSO₃, and be protected from light, high temperature, high pH and metal ions. However, still there is a vast range of area to discover the molecular mechanisms and pathways for the synthesis of anthocyanidin glucoside and its accumulation in red rice episperm. Meanwhile, the bio-fortified red rice obtained through genetic improvement by breeding is a natural source of anthocyanidin glucoside and can be used as a natural antioxidant, which could be applied in medical treatment as supplements in the near future.

Acknowledgments    This work was supported by the International Cooperation and Exchanges Research Program of the Department of S&T of Sichuan Province (2015HH0028), Technology R&D Program of Sichuan Province (2016NZ0106).

References

•  An,  J.P.,  Li,  H.H.,  Song,  L.Q.,  Su,  L.,  Liu,  X.,  You,  C.X.,  Wang,  X.F., and  Hao,  Y.J. (2016) The molecular cloning and functional characterization of MdMYC2, a bHLH transcription factor in apple. Plant Physiol. Biochem., 108, 24-31.
•  Araceli,  C.,  Madelourdes,  P. H.,  Maelena,  P.,  Joséa,  R., and  Carlosandrés,  G. (2009). Chemical studies of anthocyanins: a review. Food Chem., 113, 859-871.
•  Babaloo,  F. and  Jamei,  R. (2018). Anthocyanin pigment stability of Cornus mas-Macrocarpa under treatment with pH and some organic acids. Food Sci. Nutr., 6, 168-173.
•  Bell,  P. G.,  Gaze,  D. C.,  Davison,  G. W.,  George,  T. W.,  Scotter,  M. J., and  Howatson,  G. (2014). Montmorency tart cherry (Prunus cerasus L.) concentrate lowers uric acid, independent of plasma cyanidin-3-o-glucosiderutinoside. J. Funct. Foods, 11, 82-90.
•  Beye,  C.,  Tounkara,  L. S.,  Destain,  J.,  Zgoulli,  S.,  Ndoye,  A. S., and  Thonart,  P. (2013). Study of the sorption behavior of hibiscus sabdariffa, anthocyanins on a macroporous resin. J. Food Process Eng., 36, 579-590.
•  Bononi,  M. and  Tateo,  F. (2009). Stabilization of cranberry anthocyanins in nutraceutical capsules. Int. J. Food Sci. Nutr., 58, 142-149.
•  Chang,  Y. C.,  Huang,  H. P.,  Hsu,  J. D.,  Yang,  S. F., and  Wang,  C. J. (2005). Hibiscus anthocyanins rich extract-induced apoptotic cell death in human promyelocytic leukemia cells. Toxicol. Appl. Pharmacol., 205, 201-212.
•  Chen,  C.,  Wang,  L.,  Wang,  R.,  Luo,  X.,  Li,  Y., and  Li,  J., et al. (2018). Phenolic contents, cellular antioxidant activity and antiproliferative capacity of different varieties of oats. Food Chem., 239, 260-267.
•  Chen,  M. H.,  McClung,  A. M., and  Bergman,  C. J. (2016). Bran data of total flavonoid and total phenolic contents, oxygen radical absorbance capacity, and profiles of proanthocyanidins and whole grain physical traits of 32 red and purple rice varieties. Data in Brief, 8, 6-13.
•  Chen,  X. Q.,  Nagao,  N.,  Itani,  T., and  Irifune,  K. (2012). Anti-oxidative analysis, and identification and quantification of anthocyanin pigments in different coloured rice. Food Chem., 135, 2783-2788.
•  Cheng,  J.,  Lin,  Z.,  Qian,  L.,  Tickner,  J.,  Zhen,  T., and  Li,  X., et al. (2018). Cyanidin chloride inhibits ovariectomy-induced osteoporosis by suppressing RANKL-mediated osteoclastogenesis and associated signaling pathways. J. Cell. Physiol., 233, 2502-2512.
•  Cheng,  S. H.,  Zhuang,  J. Y.,  Fan,  Y. Y.,  Du,  J. H., and  Cao,  L. Y. (2007). Progress in research and development on hybrid rice: a super-domesticate in China. Ann. bot., 100, 959-966.
•  Choi,  I.,  Lee,  J. Y.,  Lacroix,  M., and  Han,  J. (2017). Intelligent pH indicator film composed of agar/potato starch and anthocyanin extracts from purple sweet potato. Food Chem., 218, 122-128.
•  Dang,  L. A. R.,  Rashid,  N. Y. A.,  Jamaluddin,  A.,  Sharifudin,  S. A.,  Kahar,  A. A., and  Long,  K. (2017). Cosmeceutical potentials and bioactive compounds of rice bran fermented with single and mix culture of aspergillus oryzae, and rhizopus oryzae. J. Saudi Soc. Agric. Sci., 16, 127-134.
•  Deng,  G. F.,  Xu,  X. R.,  Zhang,  Y.,  Li,  D.,  Gan,  R. Y., and  Li,  H. B. (2013). Phenolic compounds and bioactivities of pigmented rice. Crit. Rev. Food Sci. Nutr., 53, 296-306.
•  Farooq,  M.,  Basra,  S. M. A.,  Wahid,  A.,  Khaliq,  A., and  Kobayashi,  N. (2009). Rice seed invigoration: a review. Org. Farm. Pest Contr. Remed. Soil Pollut., 1, 137-175.
•  Fedenko,  V. S.,  Shemet,  S. A., and  Landi,  M. (2017). UV-vis spectroscopy and colorimetric models for detecting anthocyanin-metal complexes in plants: An overview of in vitro and in vivo techniques. J. Plant Physiol., 212, 13-28.
•  Feng,  C.Y.,  Wang,  W.W., and  Ye,  J.F. et al. (2017). Polyphenol profile and antioxidant activity of the fruit and leaf of Vaccinium glaucoal-bum from the Tibetan Himalayas. Food Chem., 219, 490-495.
•  Filipiak,  K.,  Hidalgo,  M.,  Silvan,  J. M.,  Fabre,  B.,  Carbajo,  R. J., and  Pinedalucena,  A., et al. (2014). Dietary gallic acid and anthocyanins cytotoxicity on human fibrosarcoma ht1080 cells. a study on the mode of action. Food Funct., 5, 381-389.
•  Goufo  P. and  Trindade  H. (2014). Rice antioxidants: phenolic acids, flavonoids, anthocyanins, proanthocyanidins, tocopherols, tocotrienols, γ-oryzanol, and phytic acid. Food Sci. Nutr., 2, 75-104.
•  Hayashi,  S. and  Yanase,  E. (2016). A study on the color deepening in red rice during storage. Food Chem., 199, 457-462.
•  Hellström,  J.,  Mattila,  P., and  Karjalainen,  R. (2013). Stability of anthocyanins in berry juices stored at different temperatures. J. Food Compos. Anal., 31, 12-19.
•  Hsu,  C. L.,  Chen,  W.,  Weng,  Y. M., and  Tseng,  C. Y. (2003). Chemical composition, physical properties, and antioxidant activities of yam flours as affected by different drying methods. Food Chem., 83, 85-92.
•  Huang,  C. Y.,  Pu,  H.,  Zhang,  Z. Y., and  Song,  J. (2010). Evaluation of antioxidant and antitumour activities of lemon essential oil. J. Med. Plant Res., 4, 1910-1915.
•  Jin,  T. and  Wu,  Y. X. (2015). Antioxidant activities of protein hydrolysates from little hairtail (trichiurus haumela) of east china sea. J. Food Agric. Environ., 7, 354-360.
•  Kang,  T. H.,  Hur,  J. Y.,  Kim,  H. B.,  Ryu,  J. H., and  Kim,  S. Y. (2006). Neuroprotective effects of the cyanidin-3-o-beta-d-glucopyranoside isolated from mulberry fruit against cerebral ischemia. Neurosci. Lett., 391, 122-126.
•  Kawahigashi,  H.,  Hirose,  S.,  Iwai,  T.,  Ohashi,  Y.,  Sakamoto,  W.,  Maekawa,  M., and  Ohkawa,  Y. (2007). Chemically induced expression of rice OSB2 under the control of the OsPR1. 1 promoter confers increased anthocyanin accumulation in transgenic rice. J. Agric. Food Chem., 55, 1241-1247.
•  Kim,  B.,  Woo,  S.,  Kim,  M. J.,  Kwon,  S. W.,  Lee,  J., and  Sung,  S. H., et al. (2018). Identification and quantification of flavonoids in yellow grain mutant of rice (oryza sativa l.). Food Chem., 241, 154-162.
•  Kirca,  A.,  Ozkan,  M., and  Cemeroglu,  B. (2006). Stability of black carrot anthocyanins in various fruit juices and nectars. Food Chem., 97, 598-605.
•  Knievel,  D. C.,  Esm,  A. A.,  Rabalski,  I.,  Nakamura,  T., and  Hucl,  P. (2009). Grain color development and the inheritance of high anthocyanins blue aleurone and purple pericarp in spring wheat (triticum aestivum l.). J. Cereal Sci., 50, 113-120.
•  Laokuldilok,  T.,  Shoemaker,  C. F.,  Jongkaewwattana,  S., and  Tulyathan,  V. (2010). Antioxidants and antioxidant activity of several pigmented rice brans. J. Agric. Food Chem., 59, 193-199.
•  Lejr,  E.,  Gealy,  D. R.,  Gbur,  E. E.,  Talbert,  R. E., and  Mcclelland,  M. R. (2005). Rice and red rice interference. ii. rice response to population densities of three red rice (Oryza Sativa) ecotypes. Weed Sci., 53, 683-689.
•  Li,  X.,  Liu,  H.,  Lv,  L.,  Yan,  H., and  Yuan,  Y. (2018). Antioxidant activity of blueberry anthocyanins extracts and their protective effects against acrylamide-induced toxicity in HepG2 cells. Int. J. Food Sci. Technol., 53, 147-155.
•  Lin,  J. Y.,  Li,  C. Y., and  Hwang,  I. F. (2008). Characterisation of the pigment components in red cabbage (Brassica oleracea L. var.) juice and their anti-inflammatory effects on LPS-stimulated murine splenocytes. Food Chem., 109, 771-781.
•  Elmastaş,  M.,  Gülçin,  İ.,  Işildak,  Ö.,  Küfrevioğlu,  Ö. İ.,  İbaoğlu,  K., and  Aboul-Enein,  H. Y. (2006). Radical scavenging activity and antioxidant capacity of bay leaf extracts. J. Iranian Chem. Soc., 3, 258-266.
•  Manivannan,  A.,  Bhardwaj,  R.,  Padmanabhan,  S.,  Suneja,  P.,  Hebbar,  K. B., and  Kanade,  S. R. (2018). Biochemical and nutritional characterization of coconut (cocos nucifera L.) haustorium. Food Chem., 238, 153-159.
•  Mei,  X.,  Yi,  C., and  Huang,  G. (2017). Preparation methods and antioxidant activities of polysaccharides and their derivatives. Mini Rev. Med. Chem., 17, 863-868.
•  Min,  B.,  McClung,  A. M., and  Chen,  M. H. (2011). Phytochemicals and antioxidant capacities in rice brans of different color. J.Food Sci., 76, C117-C126.
•  Morales,  F. J. (2005). Assessing the non-specific hydroxyl radical scavenging properties of melanoidins in a fenton-type reaction system. Anal. Chim. Acta., 534, 171-176.
•  Mori,  K.,  Goto-Yamamoto,  N.,  Kitayama,  M., and  Hashizume,  K. (2007). Loss of anthocyanins in red-wine grape under high temperature. J. Exp. Bot., 58, 1935-1945.
•  Morita,  M.,  Naito,  Y.,  Yoshikawa,  T., and  Niki,  E. (2017). Antioxidant capacity of blueberry extracts: peroxyl radical scavenging and inhibition of plasma lipid oxidation induced by multiple oxidants. J. Berry Res., 7, 1-9.
•  Mourtzinos,  I.,  Makris,  D. P.,  Yannakopoulou,  K.,  Kalogeropoulos,  N.,  Michali,  I., and  Karathanos,  V. T. (2008). Thermal stability of anthocyanins extract of hibiscus sabdariffa l. in the presence of beta-cyclodextrin. J. Agric. Food Chem., 56, 10303-10310.
•  Nichelmann,  L. and  Bilger,  W. (2017). Quantification of light screening by anthocyanins in leaves of berberis thunbergii. Planta, 246, 1069-1082.
•  Oke,  F. and  Aslim,  B. (2010). Biological potentials and cytotoxicity of various extracts from endemic Origanum minutiflorum O. Schwarz & PH Davis. Food Chem. Toxicol., 48, 1728-1733.
•  Oki,  T.,  Masuda,  M.,  Kobayashi,  M.,  Nishiba,  Y.,  Furuta,  S., and  Suda,  I., et al. (2002). Polymeric procyanidins as radical-scavenging components in red-hulled rice. J. Agric. Food Chem., 50, 7524-7529.
•  Omp,  S. and  Tejk,  B. (2009). DPPH antioxidant assay revisited. Food Chem., 113, 1202-1205.
•  Pachuau,  L.,  Dutta,  R. S.,  Roy,  P. K.,  Kalita,  P., and  Lalhlenmawia,  H. (2017). Physicochemical and disintegrant properties of glutinous rice starch of mizoram, india. Int. J. Biol. Macromol., 95, 1298-1304.
•  Pascual,  C. D. S. C. I.,  Massaretto,  I. L.,  Kawassaki,  F.,  Barros,  R. M. C.,  Noldin,  J. A., and  Marquez,  U. M. L. (2013). Effects of parboiling, storage and cooking on the levels of tocopherols, tocotrienols and γ-oryzanol in brown rice (Oryza sativa, L.). Food Res. Int., 50, 676-681.
•  Patel,  R. M. (2013). Ferrous ion chelating activity (FICA)-a comparative antioxidant activity evaluation of extracts of eleven naturally growing plants of gujarat, india. Int. J. Sci. Res., 2, 426-428
•  Shih,  P.H.,  Wu,  C.H.,  Yeh,  C.T., and  Yen,  G.C. (2011). Protective effects of anthocyanins against amyloid β-peptide-induced damage in neuro-2a cells. J. Agric. Food Chem., 59, 1683-1689.
•  Revilija,  M.,  Turid,  R., and  Ivar,  S. (2005). Characterisation of Fe²⁺ as pro-oxidant in oxidation of phospholipids. Antimicrob. Agents Ch., 44, 2903-2904.
•  Saigusa,  N.,  Yamamoto,  K.,  Tsutsui,  M., and  Teramoto,  Y. (2014). Microbial production of novel pigments from black rice anthocyanins. Food Sci. Technol. Res., 20, 1013-1016.
•  Abbas,  S.,  Wei,  C. D.,  Hayat,  K., and  Xiaoming,  Z. (2012). Ascorbic acid: microencapsulation techniques and trends—a review. Food Rev. Int., 28, 343-374.
•  Takos,  A.M.,  Jaffé,  F.W.,  Jacob,  S.R.,  Bogs,  J.,  Robinson,  S.P., and  Walker,  A.R. (2006) Light-induced expression of aMYBgene regulates anthocyanins biosynthesis in red apples. Plant Physiol., 142, 1216-1232.
•  Taverniti,  V.,  Fracassetti,  D.,  Del,  B. C.,  Lanti,  C.,  Minuzzo,  M., and  Klimis-Zacas,  D., et al. (2014). Immunomodulatory effect of a wild blueberry anthocyanins -rich extract in human caco-2 intestinal cells. J. Agric. Food Chem., 62, 8346-8351.
•  Teixeira  J,  Silva  T,  Andrade  PB, and  Borges  F. (2013). Alzheimer's disease and antioxidant therapy: how long how far? Curr. Med. Chem., 20, 2939-2952.
•  Tensiska,  T.,  Marta,  H.,  Cahyana,  Y., and  Amirah,  N. S. (2017). Application of Encapsulated Anthocyanins Pigments from Purple Sweet Potato (Ipomoea Batatas L.) in Jelly Drink. KnE Life Sci. 2, 482-493.
•  Tonon,  R. V.,  Brabet,  C., and  Hubinger,  M. D. (2010). Anthocyanin stability and anti-oxidant activity of spray-dried açai (Euterpe oleracea Mart.) juice produced with different carrier agents. Food Res. Int., 43, 907-914.
•  Torskangerpoll,  K. and  Andersen,  Ø. M. (2005). Colour stability of anthocyanins in aqueous solutions at various ph values. Food Chem., 89, 427-440.
•  Wang,  H.,  Dong,  Y., and  Xiu,  Z. L. (2008). Microwave-assisted aqueous two-phase extraction of piceid, resveratrol and emodin from polygonum cuspidatum by ethanol/ammonium sulphate systems. Biotechnol. Lett., 30, 2079-2084.
•  Wang,  L. S. and  Stoner,  G. D. (2008). Anthocyanins and their role in cancer prevention. Cancer Lett., 269, 281-290.
•  Wang,  L.,  Gulati,  P.,  Santra,  D.,  Rose,  D., and  Zhang,  Y. (2018). Nanoparticles prepared by proso millet protein as novel curcumin delivery system. Food Chem., 240, 1039-1046.
•  Wang,  Y.,  Xue,  Y., and  Li,  J. (2005). Towards molecular breeding and improvement of rice in china. Trends Plant Sci, 10, 610-614.
•  Wang,  Y.,  Wang,  N.,  Xu,  H.,  Jiang,  S.,  Fang,  H.,  Zhang,  T., ... and  Chen,  X. (2017). Nitrogen Affects Anthocyanin Biosynthesis by Regulating MdLOB52 Downstream of MdARF19 in Callus Cultures of Red-Fleshed Apple (Malus sieversii f. niedzwetzkyana). J. Plant Growth Regul., 1-11.
•  Weber,  F.,  Boch,  K., and  Schieber,  A. (2017). Influence of copigmentation on the stability of spray dried anthocyanins from blackberry. LWT - Food Sci. Technol., 75, 72-77.
•  Williams,  M. and  Hrazdina,  G. (2010). Anthocyanidin glucoside as food colorants: effect of ph on the formation of Anthocyanidin-rutin complexes. J. Food Sci., 44, 66-68.
•  Yamane,  T.,  Jeong,  S. T.,  Goto-Yamamoto,  N.,  Koshita,  Y., and  Kobayashi,  S. (2006). Effects of temperature on anthocyanin biosynthesis in grape berry skins. Am. J. Enol. Vitic., 57, 54-59.
•  Zafra-Stone,  S.,  Yasmin,  T.,  Bagchi,  M.,  Chatterjee,  A.,  Vinson,  J. A., and  Bagchi,  D. (2007). Berry anthocyanins as novel antioxidants in human health and disease prevention. Mol. Nutr. Food Res., 51, 675-683.
•  Zhang,  M. W.,  Zhang,  R. F.,  Zhang,  F. X., and  Liu,  R. H. (2010). Phenolic profiles and antioxidant activity of black rice bran of different commercially available varieties. J. Agric. Food Chem., 58, 7580-7587.
•  Zhang,  Y.,  Butelli,  E., and  Martin,  C. (2014). Engineering anthocyanins biosynthesis in plants. Curr. Opin. Plant Biol., 19, 81-90.
•  Zhang,  Y.,  Butelli,  E.,  Stefano,  R. D.,  Schoonbeek,  H.,  Magusin,  A., and  Pagliarani,  C., et al.> (2013). Anthocyanins double the shelf life of tomatoes by delaying overripening and reducing susceptibility to gray mold. Curr. Biol., 23, 1094-1100.
•  Zhou,  L. J.,  Li,  Y. Y.,  Zhang,  R. F.,  Zhang,  C. L.,  Xie,  X. B.,  Zhao,  C., and  Hao,  Y. J. (2017). The small ubiquitin-like modifier E3 ligase MdSIZ1 promotes anthocyanin accumulation by sumoylating MdMYB1 under low-temperature conditions in apple. Plant Cell Environ. 40, 2068-2080.
•  Zhou,  Z.,  Robards,  K.,  Helliwell,  S., and  Blanchard,  C. (2004). The distribution of phenolic acids in rice. Food Chem., 87, 401-406.
•  Zhu,  H.,  Zhang,  T. J.,  Zheng,  J.,  Huang,  X. D.,  Yu,  Z. C., and  Peng,  C. L., et al. (2017). Anthocyanins function as a light attenuator to compensate for insufficient photoprotection mediated by nonphotochemical quenching in young leaves of acmena acuminatissima, in winter. Photosynthetica, 1-10.
•  Zhu,  Q.,  Yu,  S.,  Zeng,  D.,  Liu,  H.,  Wang,  H., and  Yang,  Z., et al. (2017). Development of “purple endosperm rice” by engineering anthocyanins biosynthesis in the endosperm with a high-efficiency transgene stacking system. Mol. Plant, 10, 918-929.
•  Zhuang,  H.,  Tang,  N., and  Yuan,  Y. (2013). Purification and identification of antioxidant peptides from corn gluten meal. J. Funct. Foods, 5, 1810-1821.
•  Zielinska,  M. and  Michalska,  A. (2016). Microwave-assisted drying of blueberry (Vaccinium corymbosum L.) fruits: drying kinetics, polyphenols, anthocyanins, antioxidant capacity, colour and texture. Food Chem., 212, 671-680.