3.1. Proximate Analysis of Chemical Composition of The Rice Milled Fractions
The proximate compositions of black rice flour and its different fractions are shown in Table 1
, which was calculated on a dry basis to allow comparison with data from the literature. Moisture content was found to be ~11–11.5 % for all fractions. The obtained results are within the acceptable limit (12%) recommended for long term storage, value which allows it to avoid insect infestation and microbial growth [21
]. The moisture content of some flavored rice varieties analyzed by Asaduzzaman et al. [22
] varied between 11.25% and 15.13%. Saikia et al., [23
] reported for different milled aromatic rice samples values varying from 11.6 ± 04% to 13.7 ± 0.12%, whereas Sompong et al. [24
] suggested values varying from 9.28 ± 0.06% to 13.12 ± 0.16% for the pigmented and non-pigmented aromatic rice.
The ash values were significantly different amongst the seven fractions. An increasing trend was observed with the decrease of the particle size. The first fraction had the lowest ash content of 0.92 ± 0.02 g/D.W. whereas the highest was found for F7 (4.13 ± 0.20 g/D.W.) (Table S1
). Verma and Srivastav [21
] reported different ash content for aromatic and non-aromatic Indian rice, varying from 0.35 ± 0.05% in Sarbati rice to 0.73 ± 0.05% in Khushboo rice. These results could be explained by concentration of the compounds in the bran layers of the caryopsis.
The fat concentrations varied between of 5.10 ± 0.16 g/D.W. in F4 and 5.56 ± 0.14 g/D.W. in F5 (Table S1
). Chagam et al. [25
] reported a lipid content of 3.33 ± 0.20% in the raw Chak-hao Amubi variety. Similar results for the lipid concentration were reported by Saikia et al. [23
] for the Poreiton Chakhao pigmented aromatic rice of 2.1 ± 0.08%.
The protein content also increased from 8.69 ± 0.12% in F1 to 10.87 ± 0.15% in F7. Saikia et al. [23
] reported for the milled Chakhao amubi rice protein content of 8.8%, whereas Verma and Srivastav [21
] suggested different protein content ranging from 6.87 ± 0.10% to 9.51 ± 0.25%. The increase of the protein contents as increasing the sieving is due to the concentration of the protein in the endosperm. Itani et al. [26
] reported that most of the protein fractions and fats are mainly located in the rice germ. Therefore, removing the bran and part of the rice endosperm through polishing may cause the decrease of protein concentration [27
The highest contents in fiber were obtained in F4 and F5 (3.33 ± 0.69 % and 3.56 ± 1.17%, respectively), which are an indicative of fiber’s location in the aleuronic layer of the grain. Murfidin et al. [28
] reported a lower fiber content of pigmented rice varieties ranging from 0.66 to 1.14%.
A decreasing trend for the carbohydrate content from 74.69 ± 0.06 g/D.W. to 65.16 ± 0.08 g/D.W. found in F7 was observed (Table S1
). However, it can be considered that all the flour fractions are good sources of carbohydrates. Relative higher contents of carbohydrates were reported by Verma and Srivastav [21
] in eight varieties, varying from 78.38 ± 0.12% in Swetganga rice and 82.70 ± 0.24% in Badshah Bhog rice.
Based on the obtained results, it can be concluded that sieving up to F5, the ash, fat, protein and fiber content increases, while sieving further to the seventh fraction leads to a decrease in the fiber and carbohydrates content.
3.2. Phytochemical Content
The phytochemical content of black rice fractions was previously reported by Bolea et al. [29
]. These authors reported that all the seven fractions of black rice contained a significant amount of TPC, ranging from 199.14 ± 0.097 mg GAE/100 g D.W. (in integral flour) to 248.1 ± 3.01 mg GAE/100 g D.W. (in F1) and 483 ± 0.13 mg GAE/100 g D.W. (in F4). Sieving up to F4 led to an increase in TPC being approximately two-times higher than TPC in F4, whereas up to F7 led to an increase of 1.44. Gong et al. [30
] reported significant lower TPC value for different brown rice varieties that ranged from 72.45 to 120.13 mg of GAE/100 g. Significantly higher values were reported by Zhang et al. [7
], varying between 2365 to 7367 mg of GAE/100 g D.W. among the 12 black rice varieties.
The highest TFC value was found in the initial flour, of 211.14 ± 0.11 mg CE/100 g D.W., whereas sieving caused a significant decrease ranging from 84.97 ± 4.31 mg CE/100 g D.W. in F1 to 87.9 ± 6.05 mg CE/100 g D.W. in F5 and 76.82 ± 2.91 mg CE/100 g D.W. in F7 [31
]. A significant decrease was observed in F2, followed by an increase in the F3–F6. Zang et al. [7
] reported higher TFC values, ranging from 3596 ± 2020 mg QE/100 g in Heinuo 9933 variety to 85620 ± 433 mg QE/100 g in Heijing 72 variety.
Bolea and Vizireanu [31
] reported a TAC, in mg C3G mg/100 g D.W. of 9.20 ± 1.50 for the integral flour, whereas the lowest TAC value of 5.80 ± 1.20 was found in F1, followed by F2 with 5.20 ± 1.30, F3 with 3.40 ± 0.10 and F7 with 2.4 ± 0.1, respectively. Significantly higher values ranged from 1231 to 5101 mg of C3G /100 g of D.W. were reported by Zang et al. [7
The antioxidant activity was approximately 71% in whole flour, whereas sieving caused a decrease in antioxidant activity. Therefore, it can be appreciated that grinding and sieving generally lead to a decrease in the biologically active compounds, except for the total polyphenols.
3.3. Proteins Patterns of Black Rice
The rice flour with the highest protein concentration was further used to identify the main proteins fractions. It appeared that high amounts of polymers and monomers were concentrated into the stream that had the particle size lower than 90 µm (F7) after the sieving process of black rice.
The SDS-PAGE pattern of the proteins from the F7 stream obtained from black rice flour is presented in Figure 1
. Based on the solubility differences, the proteins from rice can be classified as ALB which are water soluble, GLU which are alkali soluble, GLO soluble in salt solutions and prolamins soluble in aqueous alcohol. The first vegetal storage proteins classification, provided by Osborn in 1924, depicted the main fractions as albumins, globulins, prolamins, and glutelins. The major fractions of the rice endosperm proteins are GLU (66%–78%), followed by GLO (9.6%–10.8%), ALB (3.8%–8.8%), and prolamin (2.6%–3.3%). When investigating the brown rice which was reported to be rather similar to black rice, Asano et al. [25
] showed that most of the proteins are alkali soluble ones (66.0%–67.7%), being followed by ALB and GLO (18.8%–20.8%), and finally the prolamins which are in the lowest concentration (12.5%–14.5%). Zhao et al. [32
] suggested that the rice endosperm contained 81.9% GLU and 13.2% GLO, while the rice dreg contained 84.6% of GLU. ALB and prolamin represented minor components for this particular rice. The ALB fractions were 7.7% for the rice dreg, being higher than in the rice endosperm, however, the globulin fraction in the rice dreg was lower than average.
By assessing the results of the SDS-PAGE analysis presented in Figure 1
, it can be seen that the molecular weights of ALB fractions were distributed in the range of 13–16 kDa, 20–25 kDa, and 35–50 kDa. GLU had bands around 13–25 kDa and 35 kDa regions, whereas the GLO bands were estimated to correspond to 12–17 kDa, 20–27 kDa, and 50–70 kDa. Finally, the profile obtained for the prolamin extract was characterized by the existence of two intense bands around 10 and 15 kDa (Figure 1
The molecular weights and the localization of rice proteins were reported by many researchers [2
]. Generally, 60%–80% of the total seed protein found in rice, is identified as GLU, which are classified based on their amino acid sequence similarity into 4 groups, namely GluA, GluB, GluC, and GluD [34
]. Moreover, the GLU subunits were divided into pro-glutelin that have molecular weights of 55 kDa, a large GLU subunit with a molecular weight of 34 kDa and a small subunit of glutelin, with 21 kDa [34
]. However, under reducing conditions, the SDS-PAGE analysis revealed only two bands corresponding to proteins of 20–23 kDa and 32–35 kDa, respectively.
Li et al. [35
] used the immunoblotting analysis to quantify the seed storage GLU and prolamins, and reported a molar ratio of 1.7 for the 10 days old seeds. The purified prolamins fraction was distinguished in the SDS-PAGE as a unique band that had the molecular weight of 15 kDa.
On the other hand, the ALB fraction from the seed endosperm had the major molecular band around 20 kDa, while the band identified in the case of GLO corresponded to the molecular weights to 15, 25.5, and 200 kDa [35
]. It has been reported that, these types of GLO were also found in the soybean, represented by 7S-globulin (57 and 43 kDa) and 11S globulin (22–23 kDa and 37 kDa) [33
3.4. Kinetics of Phytochemical Thermal Degradation in Different Milled Fractions
The linear regression for the thermal degradation of TPC, TAC, TFC, and DPPH-RSA for the integral flour and F4 were confirmed by [18
]. For the other fractions, the phytochemical thermal degradation followed a first-order kinetic model and was described in terms of degradation rate k
(1/min) and activation energy (Ea
). Our results are in good agreement with previous studies that reported the use of the first order kinetic model which described the thermal degradation of phytochemical from black rice [31
By comparing the thermal degradation rate constants of all the studied compounds (Table 1
), it could be appreciated that regardless the fraction, anthocyanins degraded faster. The linear regression for the thermal degradation of TAC are shown in Figure S1
. The highest degradation rate was found in F1, as the fraction with the lowest anthocyanins content ranged from 17.43 ± 1.01 × 10−2
1/min at 60 °C to 20.42 ± 2.22 × 10−2
1/min at 100 °C. With regards to the integral flour extract, TAC degraded with the k
vales varying from 0.92 ± 0.56 × 10−2
1/min at 60°C to 1.22 ± 0.87 × 10−2
1/min at 100 °C, while in F4, the k
values ranged from 5.52 ± 1.07 × 10−2
1/min at 60 °C to 6.61 ± 0.89 × 10−2
1/min at 100 °C [18
showed the thermal degradation behavior of TPC in all studied fractions of the black rice flour. The k
values are given in Table 2
. No significant changes concerning the k
values were found for F1 in the temperature range of 60 to 90 °C. However, it can be observed that the k
values significantly decreased by increasing the sieving degree. The lowest degradation rate for TPC was found in fractions F4 as reported by Bolea et al. [18
], with k
values ranging from 0.87 ± 0.18 × 10−2
1/min at 60 °C for F4 to 0.82 ± 0.40 × 10−2
1/min for F7 (Table 1
). Increasing the temperature up to 100 °C caused an increase of the k
values, with the lowest value of 1.01 ± 0.11 × 10 −2
1/min in F4 [18
]. In the case of TPC, the lowest k
values were obtained for the fraction with the highest content of total polyphenols (F4), while the highest values of degradation constants were obtained for the fraction with the lowest content of total polyphenols (F1).
showed the thermal degradation behavior of TFC in black rice fractions. The highest degradation rate in the case of TFC was observed for F1. These values were significantly higher than those reported by Bolea et al. [18
] for TFC for F4 or for the integral flour (Table 1
). However, it can be observed that the k
values do not depend on the flavonoids content, the highest value being registered for F1, whereas the lowest for F5.
Due to the degradation of biologically active compounds, a significant decrease was recorded in the antioxidant activity, with approximately 58% in F1, 48% in F2, 65% in F3, 29% in F4, 32% in F5, and 33% and 43% in F6 and F7, respectively, after a heating process at 100 °C for 20 min. Figure S4
shows the thermal degradation behavior of DPPH RSA in fractions F1 to F7. The antioxidant activity degraded faster in F1, the estimated k
values ranging from 1.33 ± 0.11 × 10−2
1/min at 60 °C to 2.18 ± 0.32 × 10−2
1/min at 100 °C, whereas the lowest k
values were estimated by Bolea et al. [18
] in F4, ranging from 0.57 ± 0.24 × 10−2
1/min at 60 °C to 1.21 ± 0.85 × 10−2
1/min at 100 °C.
To estimate the temperature dependences of the k
values on temperature, the constants obtained from Equation (1) were fitted to an Arrhenius equation. The activation energy values are given in Table 1
. It can be observed, that the Ea
values for the TPC thermal degradation increased from 7.05 kJ/mol for F1 to 12.58 kJ/mol for F2, decreased for F3 and F4 up to 3.51 kJ/mol and subsequently increased for F5 to F7 up to 18.77 kJ/mol. It can be stated that the k
values were less dependent on the temperature in F4, therefore TPCs are thermostable in this fraction and less thermostable in the other.
In the case of TFC, an increase from 1.99 kJ/mol in F1 to 10.78 kJ/mol in F2 can be observed in Table 1
. The highest temperature dependence and therefore the lowest thermal stability was found for F5, with an Ea
value of 21.93 kJ/mol. Flavonoids were found to be more thermostable in the F3 and less stable in F5.
For TAC, the highest temperature dependence and therefore the lowest thermal stability was found for F7, with an Ea value of 13.79 kJ/mol, while the highest thermostability was found for F1, with the lowest Ea value of 4.31 kJ/mol.
For the antioxidant activity, the Ea
values were found to decrease up to F3 (9.39 kJ/mol) from 16.61 kJ/mol in F1 to 8.27 kJ/mol in F2. The F4 presented the highest value and therefore the lowest thermal stability [18
], with an Ea
value of 19.93 kJ/mol (Table 1
). When comparing to integral flour, it seems that the antioxidant activity is more heat stable, since the lowest Ea
value was estimated of 7.45 kJ/mol.
Overall, it can be appreciated that the thermal stability of the biologically active compounds in the different black rice flour fractions depends on the degree of grinding and sieving, the anthocyanins being the compounds that degrade at the highest rate, significantly affecting the antioxidant activity.
3.5. The Effect of Temperature on the Black Rice Protein Fractions
The use of proteins as a natural biopolymer is reasonably increasing in the matter of their application in several fields such as food industry, packaging, and environmental protection. In particular, rice proteins have very interesting properties such as having good nutritional, hypoallergenic, and healthful properties for human consumption [36
]. However, although rice proteins have high nutritional value and are hypoallergenic and healthful for human consumption, few studies concerning their structural and conformational properties are reported in literature [36
]. As a consequence, there is limited data in literature regarding the thermal denaturation of rice proteins fractions from the perspectives of their use as food ingredients, such as in gels, puddings, ice creams, and baby formulas. For example, Ju et al. [33
] studied the denaturation and hydrophobic properties of rice flour proteins and concluded that heat denaturation of globulin and glutelin resulted in significant increases in surface hydrophobicity. Rice is routinely subjected to various heat treatments during processing such as steaming, drying, tempering, and roasting. These thermal treatments often lead to substantial denaturation or to the unfolding of the proteins native structure [19
]. In our study, we investigated the heat-induced changes of the three rice fractions by fluorescence spectroscopy techniques, which enabled us evaluate in detail the folding–unfolding events of the targeted fractions.
3.5.1. Intrinsic Fluorescence
The fluorescence spectroscopy measurements were performed to observe the folding, unfolding events or the conformational changes that affect the microenvironment of tryptophan (Trp) and tyrosine (Tyr) residues found in the ALB, GLO, and GLU fractions extracted from the black rice flour with particles smaller than 90 µm. The excitation wavelength of 292 nm was used to monitor the changes in the vicinity of Trp residues, 280 nm for both Trp and Tyr, and 274 nm for Tyr.
Emission spectra obtained after the selective excitation of Trp (Figure S5a
) indicated that the maximum fluorescence intensity was registered at 358 nm for ALB fraction, 384 nm for GLO, and 360 nm for GLU. According to Shin et al. [40
], the high mobility of amino acids that are exposed to a polar environment, is indicated by the red shifts, while the burial in the non-polar environment generates a blue shift in λmax
. Depending on the environmental properties of the protein, Lakowicz [41
] showed that the Trp residues can emit from 308 to 352 nm. It has been reported that Trp residues are buried in a non-polar environment if the maximum fluorescence emission (λmax
) is lower than 330 nm. If the λmax
is higher than 330 nm, the Trp is assigned to a polar environment, which in most cases implies a solvent exposure [42
]. The significant higher value obtained for the GLO fractions is an indicative of the higher exposure of Trp residues to a polar microenvironment.
When heating the ALB fraction, small blue-shifts around 1–2 nm in the λmax at 50 °C and 60 °C were observed, indicating the partial folding of the polypeptide chains, whereas at higher temperatures, a 2 nm red-shift was found, as an unfolding indicative. For the GLO fraction, heating in the temperature range of 50 to 60 °C caused significant 5 nm blue-shifts in the λmax, followed by 2 nm red-shifts at higher temperatures. In the case of GLU, no significant heat induced changes were observed in the temperature range of 50–70 °C. Heating at temperatures up to 100 °C caused 2 nm red-shifts in λmax, indicating the unfolding of polypeptide chains.
When excited at 280 nm, the λmax
corresponding to the maximum fluorescence intensity for the investigated protein fractions were 355 nm for ALB, 357 nm for GLO and 359 nm for GLU (Figure S5b
). These values indicated a higher exposure of Trp and Tyr residues to the solvent in the GLU fractions. The unfolding of polypeptide chains was observed for ALB at temperatures higher than 70 °C. GLO unfolded by heating up to 70 °C and folded at higher temperatures, whereas in the GLU fractions, the Tyr and Trp residues seemed to be more exposed at temperatures higher than 80 °C.
When excited at the wavelength of 274 nm, the protein fractions displayed the fluorescence intensity maximum at 355 nm for ALB, at 356 nm for GLO, while in the case of GLU the maximum fluorescence intensity was recorded at 359 nm (Figure S5c
). Heating up to 60 °C caused several folding events and unfolding events at temperatures ranging from 70 to 100 °C for the ALB fractions. For the GLO, up to 60 °C, the unfolding of the polypeptides chains was observed, followed by a folding process up to 100 °C, whereas the GLU unfolds at temperatures higher than 60 °C. The red-shifts suggested that the unfolding of the protein structure strengthened the intermolecular β-sheet hydrogen bonds [43
Based on the λmax
values, it could be appreciated that in the black rice flour fractions, the Trp and Tyr residues are exposed to the solvent, whereas the heat treatment caused the folding at the lower temperatures range and the unfolding at higher temperatures. Heat-induced denaturation and the unfolding of rice protein structures could release more hydrophilic groups [43
] at pH 7.0, possibly resulting in the increase of surface hydrophobicity. Our results are in good agreement with those reported by Zhao et al. [44
], who suggested that the heat treatments caused the increase of β-turns at the expense of β-sheets and random coils of rice endosperm proteins. Consequently, the partial unfolding process, which is dominant, revealed diverse protein structural changes that are induced by processing and consistent with the protein surface hydrophobicity increase. Ellepola et al. [19
] suggested that rice GLO possesses a relatively high thermal stability (with a denaturation temperature of 70.9 ± 0.04 °C for the milled rice flour, 97.6 ± 0.27 °C for crude rice GLO, and 98.5 ± 0.39 °C for purified GLO), suggesting that the protein could retain its functionality in heat-processed rice products.
3.5.2. Synchronous Fluorescence Spectra
The fluorescence synchronous spectra are another method to investigate the amino acid residues microenvironment by modifying the highest wavelength value (λmax
) obtained at emission, which corresponds to the polarity change around the hydrophobic groups of the molecule [45
]. In the tested temperature range, the synchronous spectra at Δλ of 15 nm indicated the presence of a red shift of 3 nm for the ALB fraction (from 286 nm at 25 °C to 289 nm at 100 °C), and small blue shifts of 1 nm and 2 nm for the GLO and GLU fractions (from 282 at 25 °C to 281 nm at 100 °C and from 295 nm to 293 nm, respectively) (Figure 2
). The red-shifts for the ALB fractions suggested the exposure of the Tyr residues to a more polar microenvironment, whereas the blue shifts in Δλ for GLO and GLU are an indicative of the heat-induced burial of Tyr residues to a more non-polar microenvironment.)
Furthermore, the synchronous spectra at Δλ of 60 nm showed small red shifts for all the tested fractions when rising the temperature from 25 °C to 100°C (from 282 nm to 283 nm, from 278 nm to 280 nm, and from 282 nm to 284 nm for ALB, GLO, and GLU, respectively), therefore indicating the exposure of the Trp residues to a more polar microenvironment (Figure 3
Wherefore, it can be appreciated that the thermal treatment induced the exposure of Tyr and Trp residues in the ALB fractions, whereas for the GLO and GLU fractions, the Tyr residues were buried in a more non-polar microenvironment, while Trp residues became exposed.
3.5.3. Quenching Experiments
In order to check the accessibility of fluorescent residues of black rice proteins fractions to different quenchers, experiments with acrylamide and KI were performed. Acrylamide and KI are external quenchers (charged and non-charged) used to analyze the solvent accessibility and the polarity of the microenvironment close to the Trp residues. The selection of the two quenchers was based on the different accessibility of the quenchers, as acrylamide quenches the exposed and partially exposed Trp residues, while KI quenches only the fluorescence of the exposed Trp located at or near to the surface of the molecules. As expected, when quenching with acrylamide, the KSV
were higher than those obtained with KI, regardless of the applied heating conditions (Table 2
), thus indicating that the KSV
value for the terminal Trp residues are higher than those for the buried ones.
From Table 2
and Table 3
it can be observed a significantly higher Trp residues accessibility for acrylamide and KI in GLO, followed by GLU and ALB fractions. Heating caused important conformational changes, as indicated by the sequential decrease of KSV
values at 50 °C for GLO and GLU and an increase for the ALB fractions. The highest values for the acrylamide quenching constants were measured after a thermal treatment at 60°C for ALB (15.11 ± 3.32 mol−1
L), at 100 °C for GLO (19.63 ± 2.70 mol−1
L) while for the GLU fraction the highest value was recorded at 100 °C (14.15 ± 1.67 mol−1
L) (Table 2
). In the case of ALB fractions, heating at temperatures higher than 60 °C had no significant effect on the quenching constants values. However, the accessibility of acrylamide to Trp was higher when heating comparing to 25 °C. Therefore, it can be appreciated that the conformational transition of the polypeptide chains decreases the distance between the Trp residues and the quenching agent. The increased KSV
values at high temperatures indicated several structural rearrangements as well as the partial exposure of Trp residues within the protein molecules.
A different thermal behavior was observed for GLO and GLU (Table 2
), with a decrease at 50 °C, followed by an increase at 60 °C and 70 °C for GLU. In the temperature range of 70–80 °C, the KSV
values decreased, whereas at higher temperature increased again. For all the rice protein fractions, the KSV
values variations indicated the sequential character of the structural and conformational changes being thermally induced.
For the KI quenching experiments, the maximum KSV
value for ALB was calculated at 100 °C (6.47 ± 0.53 mol−1
L) and the minimum value was recorded at 80 °C (4.70 ± 0.57 mol−1
L). For the GLO fraction, the maximum KSV
was found at 70 °C (6.65 ± 1.80 mol−1
L) and the minimum at 80 °C (4.53 ± 1.50 mol−1
L), while the highest value for the GLU fraction was recorded at 90 °C (4.38 ± 1.15 mol−1
L) and the lowest one at 70 °C (1.37± 0.42 mol−1
L) (Table 3
). It seems that Trp residues are more exposed to the solvent at pH 7.0 and 25 °C in the GLO and less exposed in the GLU fractions. For the ALB and GLU fractions, heating caused an increase in regards to the accessibility of Trp with KI, which could suggest an unfolding of the molecule, with the exception of 70 °C. Within the tested temperature range, local heat-induced conformational changes that bury the Trp residues from the molecule surface occur in the GLO fractions.