Black rice (Oryza sativa L.) was purchased from Heilongjiang Xinhe Ecological Agriculture Development Co., Ltd (China). Caffeic acid (assay > 98%), DPPH and ABTS were supplied by Shanghai Yuanye Biotechnology Co., Ltd (China). Lipase Novozyme 435 was obtained from the Zhejiang Novocata Biotechnology Co., Ltd (China). 4A zeolite was purchased from the Langfang Nake Biotechnology Co., Ltd (China). AB - 8 macroporous adsorption resin was procured from Tianjin Haiguang Chemical Co., Ltd (China). Other solvents and materials were analytical grade.
2.2 Preparation of the anthocyanin extracts from black rice
Anthocyanin extracts from black rice were obtained basing on the modified method reported by Hao et al (Hao et al., 2015). The black rice flour was extracted twice with ethanol/water/1% hydrochloric acid (50:50:0.5, v/v/v) by solid - liquid ratio (1:10) at 30 oC for 2 h. All extracts were centrifuged at 4000 r/min for 10 min and the supernatants were collected. The collected filtrates were subjected to a vacuum evaporator to remove the remaining ethanol. And then the concentrated extracts were loaded on an AB - 8 macroporous adsorption resin to remove major impurities according to the following manner: 10 g AB - 8 resin was added into 50 mL crude anthocyanin extracts with stirring at room temperature for 24 h. The anthocyanin extracts contained with AB - 8 resin were filtered and subsequently recovered with 70% ethanol containing 1% hydrochloric acid. The ethanol eluent was evaporated under vacuum to yield the anthocyanin extracts. Finally, the anthocyanin extracts were freeze-dried in a freeze dryer at - 50 oC for 24 h to obtain anthocyanin powders.
2.3 Acylation of anthocyanin extracts
Anthocyanins from black rice were acylated according to the modified method described by Yan et al. (2016) (Yan et al. 2016). The appropriate masses of anthocyanins and caffeic acid basing on different weight ratio were added into a 250 mL three-neck flask. The black rice anthocyanins and caffeic acid were thoroughly mixed at the weight ratio of 1:0, 2:1, 1:1 and 1:2, respectively. And then 5 mL distilled water, 60 mL tert-butyl alcohol, 15 g 4A zeolite, and lipase Novozyme 435 (20 wt% black rice anthocyanins) were added in the reactant mixture and stirred at room temperature for 24 h. After the reaction finished, the organic reagents were evaporated and freeze-dried in a freeze dryer at - 50 oC for 24 h. The acylated anthocyanins prepared at the weight ratio of 1:0, 2:1, 1:1 and 1:2 labeled as AN/CA 1:0, AN/CA 2:1, AN/CA 1:1 and AN/CA 1:2, respectively. Fig. 1 (a) represents the synthesis routine of acylated anthocyanins from black rice see.
2.4 UV analysis
The UV analysis of the anthocyanins from black rice were investigated by Puruisi UV - 18 model instrument (l = 200 - 400 nm). The measurements were repeated intriplicates. In the text, the results of UV ranges will be given in terms of wave length (nm).
2.5 FTIR analysis
The chemical structure of the anthocyanins from black rice were measured by Perkin - Elmer Spectrum 100 FT - IR spectrometer with the prepared monomers at 4 cm−1 resolution after 32 scans by casting a thin film with KBr.
2.6 Thermal properties
The thermal weight loss value of acylated anthocyanins during heat was investigated by thermal gravimetric analysis (TGA), which was carried out by TA Instruments Q50 TGA from 25 to 400 oC at a heating rate of 20 oC/min, in a nitrogen atmosphere.
2.7 Assays for antioxidant
The antioxidant capacity was determined by three assays, including DPPH radical scavenging assay, ABTS radical scavenging assay, and ferric reducing antioxidant power (FRAP) assay, respectively. DPPH and ABTS scavenging assay were performed according to the method reported by Sahreen et al. with slight modification (Sahreen et al., 2010). The absorbance was taken at 517 nm and 734 nm by UV-visible spectrophotometer for DPPH and ABTS radical scavenging activity, respectively. And the scavenging rate was calculated by Eq. (1):
where A is the absorbance of samples, A0 is the absorbance of standard solution prepared under the same conditions but without sample added.
The FRAP of acylated anthocyanins concentration (AAC) was measured basing on the method reported by Thaipong et al. with slight modification (Thaipong et al., 2006). The FRAP stock solution was prepared from sodium acetate buffer (0.3 mol/L, pH 3.6), FeCl3 solution (0.02 mol/L), and TPTZ solution (0.01 mol/L with 0.04 mol/L HCl) with the volume ratio of 10:1:1, respectively. 0.02 mL sample was mixed with 3.98 mL FRAP stock solution and stirred 30 min at room temperature. The absorbance of the samples was investigated at 593 nm. The standard curve was linear between 0 and 25 mM Trolox. Results are expressed in mM TE/mg AAC.
2.8 Stability studies
2.8.1 pH stability
The pH stability of acylated anthocyanins was investigated at pH 3.0, 4.0, 5.0, 6.0 and 7.0, respectively. 75 mg anthocyanin extracts were mixed with 75 mL citrate phosphate buffer at 25 oC and was divided into the test tube. And then evaluated their absorbance using UV spectrometer at 520 nm. Further, the anthocyanin contents were calculated per hour based on the percentage of the absorbance at each pH value until 5 hours.
2.8.2 Thermal stability
The effect of temperature on acylated anthocyanins from black rice was evaluated basing on the methodology reported by Swer with some modification (Swer et al., 2019). 75 mg acylated anthocyanins were dissolved in 75 mL citrate phosphate buffer. The mixed solution was divided into test tube with stopper and were subjected to heating in water bath at 50 oC, 60 oC, 70 oC, 80 oC and 90 oC, respectively. And then, the mixture was read immediately at 520 nm in UV spectrophotometer after cooled to room temperature, in which the same solvent was used as blank. The anthocyanin contents were tested per hour based on the percentage of the absorbance before heating until heated for 5 hours.
2.8.3 Light stability
75 mg acylated anthocyanins were mixed with 75 mL citrate phosphate buffer and divided into two parts. One part was put into an amber colored bottle whereas the other one was put into a transparent bottle and exposed continuously to ultraviolet light (UV intensity ≥ 90 uw/cm2). Changes in color intensity were determined by UV-visible spectrophotometer by measuring the Abs at 520 nm after 0, 1, 2, 3, 4 and 5 h of each treatment.
2.8.4 Theoretical section
The degradation kinetics of the acylation of anthocyanins from black rice is basing on Eq. (2).
where, C is the anthocyanin content at a specific time, C0 is the initial value of anthocyanin content, t is time (in hours), and k is the rate constant (per unit time).
The k values could be obtained through the slope of the straight line basing on Eq. (2).
The relationship between k and its halftime (t1/2) is used by Eq. (3).
The temperature dependence on the rate constant is typically represented through the Arrhenius equation:
The Arrhenius model was used to describe the dependence of degradation rate constants on temperature：
where, E0 is the activation energy (kJ/mol), R is the universal gas constant, k0 is the initial value of the rate constant for degradation (1/min), and T is the absolute temperature (K). The E0 can be calculated from the slope of ln(k) vs. 1/T plot.
2.9 Statistical analysis
All the experiments were performed in triplicates with duplicate samples. Data was expressed in terms of average values and evaluated by one-way analysis of variance (ANOVA) using SPSS 22.0 (SPSS Inc., USA) and Origin 8.5 (Origin Institute Inc, USA). And p values of less than 0.05 were considered as statistically significant.