Influence of extraction parameters and stability of betacyanins extracted from red amaranth during storage

Abstract

Natural colorants are important alternatives to synthetic colorants. They are considered harmless and positively affect biological activities owing to their antioxidant potential. The present study deals with the assessment of the extraction processes and the effects of pH (1.0, 3.0, and 5.0), extraction media (water and 50% ethanol) and storage condition (ambient and refrigeration) on betacyanin content, color values, as well as degradation kinetics of total betacyanins in red amaranth. Betacyanin content was more stable at higher than at lower pH. The degradation rate constant (K) was higher and the half-life (t_1/2) was lower at ambient temperature compared to refrigeration temperature. Betacyanin degradation was higher at ambient temperature (30 ± 2 °C) than at refrigeration temperature (4 °C). The pH, storage time, and temperature affected the stability of the color attributes. Therefore, this work suggests that water and lower temperature (4 °C) could be applied to extract more betacyanins from red amaranth and betacyanins might be used as an alternative to synthetic color.

Electronic supplementary material

The online version of this article (10.1007/s13197-018-3519-x) contains supplementary material, which is available to authorized users.

Introduction

In Bangladesh, several varieties of vegetables, such as the Indian spinach, tripatri leaves, mustard greens, amaranth leaves, spinach, goose foot and water spinach are found throughout the year. Amongst them, the red amaranth is a good source of vitamins, minerals, folic acid, protein, dietary fiber, and amino acids (Khandaker et al. 2008; Al-Mamun et al. 2016). It is also an excellent source of bioactive compounds, such as betacyanins, betaxanthins, and polyphenols that have enormous health benefits. Betalain pigments are non-toxic and may help prevent cardiovascular disorders, inflammatory response, cancer, and deteriorative diseases (Biswas et al. 2013).

Several kinds of betalains, such as amaranthin, isoamaranthin, methyl derivative of arginine betaxanthin, and betalamic acid are present in the amaranthus leaves (Biswas et al. 2013). Betalain production could vary depending on the sources, extraction process, purification, concentration, and drying conditions (Cai et al. 2005). Various studies have been performed on the extraction of betalain pigments from sources, such as red dragon fruit peels (Priatni and Pradita 2015), red dragon fruit (Ramli et al. 2014), red beetroots (deAzeredo et al. 2009), edible amaranth(Amaranthus tricolor; Biswas et al. 2013), and Basella rubra fruit (Kumar et al. 2015). Betalains have been extracted from different sources with water, methanol, and ethanol (Priatni and Pradita 2015; Kumar et al. 2015; deAzeredo et al. 2009). Ultrasonic-assisted extraction technique (Ramli et al. 2014), diffusion-extraction, reverse osmosis, and ultrafiltration (deAzeredo et al. 2009) have also been used to extract betalains from different fruit and vegetable sources.

The use of acetone and methanol is not preferred in the food industry because of their potential toxicity (Spagna et al. 2003; Patil et al. 2009); rather, ethanol is the preferred solvent. It has been suggested that pure ethanol should not be used for the extraction of pigments as some amount of water is needed to extract the hydrophilic pigment (Patil et al. 2009). Betalains are hydrophilic pigments (Priatni and Pradita 2015). The stability of betalain pigments is affected by both environmental and processing factors, such as the pH value, temperature, presence of oxygen, enzymes, metal ions, intermolecular association with other compounds (co-pigments, sugars, proteins, and degradation products), intramolecular associations, and condensation reactions (deAzeredo et al. 2009; Priatni and Pradita 2015; Kumar et al. 2015; Cai et al. 2005). Betalains are stable between pH 3.0–7.0, but the optimal pH could vary with changes in temperature, and the presence or absence of oxygen (Herbach et al. 2004). Temperature markedly influences the stability and the rate of betalain degradation. Heat and light treatments also increase the degradation rate of betalains (deAzeredo et al. 2009). Hence, the degradation of betalains that are used as food additives is the major concern for the consumers and food manufacturers. Betalain degradation has been found to follow a first–order reaction model (Casati et al. 2015). The knowledge of kinetic parameters, such as reaction rates and activation energy will be essential to predict the degradation of betalains during storage, as explained by the Arrhenius kinetic model.

Color is one of the bases for recognition and acceptability of foods and beverages. Usually, synthetic colorants are used in the food industry to give the desired color to the final product. However, these synthetic food colorants are carcinogenic and harmful to the consumer’s health. Therefore, manufacturers have recently turned to naturally derived colorants as a viable alternative. The consumers and the food manufacturers are showing growing interest in betalains because of their potent antioxidant activity (Priatni and Pradita 2015). Betalains from red amaranth could be good alternatives as natural colorants. A literature search did not reveal reports on the extraction of betalains or the stability of betalains from red amaranth leaves. Therefore, the objective of this work was to investigate the effects of the extraction parameters on the betacyanin content of red amaranth and the kinetics of their degradation.

Materials and methods

Sample collection and preparation

Fresh red amaranth (Amaranthus cruentus) was collected from the local market. The amaranth was washed with tap water to remove adhered material, dirt, and other surface impurities. Then, it was cut into small pieces manually using a stainless steel knife, and the roots were separated from the leafy parts. The freshly cut small pieces of red amaranth were kept in a dryer at 40 °C for 25 h. The dried red amaranth was ground using a blender and kept at 4 °C for further assays.

Extraction of betacyanins

In order to extract betacyanins from red amaranth, 25 g dried red amaranth powder was soaked in 1000 mL water or 50% ethanol. The pH of the solution was maintained at 1.0 ± 0.1, 3.0 ± 0.1, or 5.0 ± 0.1 using HCl and NaOH. The extraction process was performed at 50 °C for 60 min. The betacyanin extracts obtained from each of the extraction processes were filtered through a muslin cloth to remove coarse particles. Then vacuum filtration using Whatman filter paper (No. 1) was performed to remove the other dissolved minute particles. The filtered extract was used to determine the betacyanin content and to analyze the color of betacyanins. Subsequently, the filtered betacyanin extracts were stored in falcon tubes at ambient temperature (30 ± 2 °C) and refrigerated temperature (4 °C) for studying the kinetics of betacyanin degradation.

Betacyanin content

Betacyanin content was determined using the modified method given by Kumar et al. (2015). Filtered red amaranth solution (15 mL) was centrifuged at 4000 rpm for 20 min. Then, 0.5 mL of the aliquot was diluted with 2.0 mL of distilled water, and the absorbance was measured at 538 nm using a spectrophotometer (Optizen 2120 UV, Mecasys Co., South Korea). The betacyanin content was calculated based on the following equation.

$$\text{Betacyanin}\text{content}\left(\text{mg}/100\text{g}\text{of}\text{dry}\text{matter}\right)=\left(\text{A}\times \text{MW}\times \text{V}\times \text{DF}\times 100\right)/\left(€\times \text{L}\times \text{W}\right)$$

where A = absorbance; MW = molecular weight of betanin (550 g/mol); V = solution volume; DF = dilution factor; € = molar extinction coefficient (65000 L/mol cm); W = sample weight (g).

Betacyanin colour characteristics

The color measurements of betacyanin extracts were performed using the Minolta CM-2500d colorimeter (Konica Minolta Optics, Inc. Japan). Color attributes were recorded as L* (lightness), a* (redness), and b* (yellowness). The color changes (ΔE) of the specimens were evaluated using the following formula:

$$\mathrm{\Delta }\text{E}=\sqrt{{\left(\mathrm{\Delta }{\text{L}}^{\ast }\right)}^2+{\left(\mathrm{\Delta }{\text{a}}^{\ast }\right)}^2+{\left(\mathrm{\Delta }{\text{b}}^{\ast }\right)}^2}$$

where ∆L*, ∆a*, ∆b* = difference in L*, a*, and b* values between the sample color and the target color.

Kinetics of degradation and stability of betacyanins

Stability and degradation kinetics of the extracted betacyanins from red amaranth were examined after 4, 8, and 12 days of storage at 4 °C and 30 ± 2 °C. The degradation of betacyanins was calculated using the standard equation for a first-order reaction as follows:

$$lnC=ln{C}_o-k$$ 1

where C is the concentration at time t; C_o the concentration at time zero; k is the first-order rate constant (day⁻¹); and t the storage time (day).

The half-life (t_1/2) of betacyanins was expressed as the following equation:

$${\text{t}}_{1/2}=\frac{0.693}{\text{k}}$$ 2

Temperature dependence of reaction rates was determined using the Arrhenius equation, given below:

$$k=k_o.exp\left(-\frac{E_a}{\mathit{RT}}\right)$$ 3

Taking ln of both sides of Eq. (3), we get the following equation:

$$ln\left(\text{k}\right)=-\frac{{\text{E}}_{\text{a}}}{\text{R}}\left(\frac{1}{\text{T}}\right)+ln\left({\text{k}}_{\text{o}}\right)$$ 4

where, k is the reaction rate constant (h⁻¹), k_o is the pre-exponential constant (h⁻¹), E_a is the activation energy (kJ/mol), R is the universal gas constant (8.314 kJ/mol k) and T is the absolute temperature (^oK).

Activation energy was calculated from the graph using Eq. (4), by plotting ln k versus 1/T and the slope = $-\frac{{\text{E}}_{\text{a}}}{\text{R}}$.

Color retention

$$\%R=\frac{A_{\mathit{tx}}}{A_{\mathit{to}}}\times 100$$

where %R = percentage of color retention; At_x = concentration of betacyanins at a specified storage time, At_o = concentration of betacyanins at time zero, or the initial time of the experiment.

Statistical analyses

Each experiment was performed in triplicates. Data were analyzed using the statistical software R, version 3.2.2. A multifactorial analysis of variance was carried out. Individual effects of the factors were calculated at a particular point of time during the study. Differences were considered to be significant at P ≤ 0.05.

Results and discussion

Effects of solvent and pH on betacyanin content

Figure 1 shows the effects of solvent and pH on the betacyanin content extracted from red amaranth under various storage conditions. The amount of extracted betacyanins ranges from 15.79 to 159.09 mg/100 g in water and 14.32 to 114.76 mg/100 g in 50% ethanol at pH 1.0, 3.0, and 5.0. These results were higher than those observed for edible portions of amaranth seed (0.07–0.96 mg/100 g), amaranth stalks (0.56–1.54 mg/100 g), amaranth leaves (16.90–20.93 mg/100 g), amaranth flowers (0.95–6.02 mg/100 g), and amaranth sprouts (2.69 mg/100 g), reported by Li et al. (2015). The results of this study were consistent with the findings of Ramli et al. (2014) who found that the betacyanin content ranged from 71.34 to 82.79 mg/100 g for red pitaya flesh and 17.64 to 18.67 mg/100 g for red pitaya peel. These variations might be due to different extraction methods and origin of samples. In this study, water extracted samples had higher betacyanin contents than ethanol extract samples. The lower amount of betacyanin content in ethanol extract samples might be related to the nucleophilic attack by ethanol on the aldimine bond. Usually, ethanol has a high electron density in the presence of oxygen atom (Wybraniec and Mizrahi 2005; Wybraniec 2005). Castellar et al. (2006) also found higher betacyanin content in water than ethanol for Opuntia stricta fruits.

Fig. 1.

Effects of solvent and pH on betacyanins content (mg/100 g of dry matter) under various storage conditions at a 4 °C, b 30 °C. ^{a, b}Means followed by different superscript alphabets are significantly different (P ≤ 0.05) among different pH within the same solvent. ^A–CMeans followed by different superscript alphabets are significantly different (P ≤ 0.05) among storage duration within the same solvent

From the data, it can be stated that betacyanin content was the highest at pH 5.0 for both solvents, and the degree of extraction was higher with water than that with 50% ethanol throughout the storage period. Our results are in accordance with the observations of Azeredo (2009) and Castellar et al. (2003) that betacyanins are stable at pH 5.0–6.0. Below pH 3.0, betanin, a betacyanin pigment, is degraded by C₁₅-isomerisation or C₁₇-decarboxylation due to change of spectral band position (Azeredo 2009). More degradation was observed in ethanolic solution because of the single and double decarboxylation forms (Wybraniec and Mizrahi 2005; Wybraniec 2005). The betacyanin contents of all the extracts decreased during the storage period. The degradation of betacyanins was faster at ambient conditions than at refrigerated conditions. This phenomenon is similar to the earlier observation that the degradation rate of betacyanins was faster at higher temperatures as compared to lower temperatures (Azeredo2009). Heat, temperature, light, and acidity could enhance betanin isomerization, decarboxylation, or cleavage (Azeredo 2009; Czyzowska et al. 2006; Fernández-López and Almela 2001). In our study, the color changed from light pink to yellowish brown at ambient temperature. Biswas et al. (2013) suggested that the brown color is due to the betalainic dye present in amaranth. A good correlation was found between betacyanin content and storage day (R² = 0.82, 0.72, and 0.87 for pH 1.0, 3.0, and 5.0, respectively for extraction with water and R² = 0.63, 0.63, and 0.73 for pH 1.0, 3.0, and 5.0, respectively for extraction with ethanol at refrigeration temperature; whereas R² = 0.72, 0.87, and 0.93 for pH 1.0, 3.0, and 5.0, respectively for extraction with water and R² = 0.63, 0.73, and 0.80 for pH 1.0, 3.0, and 5.0, respectively for extraction with ethanol at room temperature). There were no significant differences at different pH values on the 4th, 8th, and 12th day in ethanol solution for both the storage conditions.

Color attributes of betacyanin extracts from red amaranth

Color attributes of betacyanin extracts from red amaranth under various storage conditions are shown in Fig. 2I–IV. L*, a*, and b* values of the water extract were lower under weak acidic conditions (pH 5.0) than under strong acidic conditions (pH 1.0) during storage. This is mainly because more betacyanins were found at higher pH, compared to lower pH. On the other hand, betacyanins extracted at pH 3.0 had higher L* and b* values than betacyanins extracted at pH 1.0 and 5.0 for the ethanol extracts. However, a* values were dependent on pH and storage temperature. This might be related to the interaction between ethanol–water and ethanol-betacyanins. The L* and b* values increased for all samples during storage at both storage conditions. The b* values were higher at room temperature than at 4 °C. On the contrary, a* values were dependent on the extraction parameters and did not give a specific pattern. The changes in L* and b* values correlated with the yellow color. The yellow color was more prominent at room temperature than at freezing temperature. The degradation of betacyanins may be responsible for a* values. However, our results showed that betacyanins and a* values have a higher correlation at pH 3.0 and 5.0 than at pH 1, for both types of extraction samples. We found that R² = 0.67 and 0.58 for pH 3.0 and 5.0, respectively for extraction with water, whereas R² = 0.28 and 0.057 for pH 3.0 and 5.0, respectively for extraction with ethanol at refrigeration temperature. On the other hand, we also found that R² = 0.69 and 0.79 for pH 3.0 and 5.0, respectively, for extraction with water, whereas R² = 0.69 and 0.70 for pH 3.0 and 5.0, respectively, for extraction with ethanol at room temperature. Color values of extracted betacyanins could be described clearly by ΔE values. The ΔE values were higher at 12 days of storage as compared to 4 days of storage for all samples. L*, a*, and ΔE values were not significantly affected at pH 3.0 and 5.0 in ethanol extract throughout the storage time. The changes in color throughout the storage period are due to degradation of betacyanins and the generation of other isoforms, such as betaxanthin, methyl derivative of arginine betaxanthin, and betalamic acid. Narkprasom et al. (2012) showed that the formation of isoforms of betacyanins is dependent on pH, heat, and temperature and the alcohol concentration. In our study, L* and b* values were much lower, whereas a* values were higher compared to the powder of different parts of the Amaranthus species (Li et al. 2015). The deviation may probably be due to variation in cultivar, environment, and the processing of samples.

Fig. 2.

I Effects of solvent and pH on hunter color L values of betacyanins extracts under various storage conditions at a 4 °C, b 30 °C. II Effects of solvent and pH on hunter color a values of betacyanins extracts under various storage conditions at a 4 °C, b 30 °C. III Effects of solvent and pH on hunter color b values of betacyanins extracts under various storage conditions at a 4 °C, b 30 °C. IV Effects of solvent and pH on color changes (ΔE) of betacyanins extracts under various storage conditions at a 4 °C, b 30 °C. ^{a, b}Means followed by different superscript alphabets are significantly different (P ≤ 0.05) among different pH within the same solvent. ^A–CMeans followed by different superscript alphabets are significantly different (P ≤ 0.05) among storage duration within the same solvent

Assessment of kinetic parameters of betacyanin extracts from red amaranth

Color retention of betacyanin extracts from red amaranth is shown in Table 1. Color retention highly correlated with the content of betacyanins for all samples. The color retention was higher at pH 3.0 and 5.0 in water as compared to pH 1.0 at both storage conditions. However, color retention was not observed in 50% of the ethanol extracts at ambient conditions. The pH-dependent changes in betacyanin pigment structure may result in this phenomenon. The color retention was poor at ambient storage condition (30 ± 2 °C) than at refrigerated storage condition. This could be due to higher betacyanin content during storage at refrigeration temperature and their degradation with increasing time. The effect of temperature on the stability of betacyanins may be responsible for the same. This phenomenon is relevant with an earlier report that red dragon fruit showed higher color stability at 4 °C than at 25 °C (Woo et al. 2011). The pH and storage time did not show significant effects at refrigeration temperature in any of the samples.

Table 1.

Color retention percentage (% R) as kinetic parameters for betacyanins degradation from red amaranth under various conditions

Span of storage	Storage temperature	Ambient temperature (30 ± 2 °C)
	Extracting solvent	Water			50% ethanol
	Solvent pH	1	3	5	1	3	5
4th Day		A46.58 ± 0.17b	A56.36 ± 0.42a	A49.43 ± 2.6b	A25.2 ± 9.83a	A37.60 ± 15.59a	A30.57 ± 15.39a
8th Day		B20.55 ± 10.97a	B35.10 ± 7.63a	A39.23 ± 11.20a	A24.05 ± 9.17a	AB18.91 ± 7.56a	A15.81 ± 6.87a
12th Day		B15.30 ± 5.02a	C21.89 ± 8.40a	B23.86 ± 4.3a	A23.72 ± 10.21a	B14.57 ± 4.78a	A12.32 ± 4.77a

Span of storage	Storage temperature	Stored temperature (4 °C)
	Extracting solvent	Water			50% ethanol
	Solvent pH	1	3	5	1	3	5
4th Day		A77.65 ± 37.67a	A84.68 ± 14.02a	A82.86 ± 10.51ab	A76.55 ± 25.67a	A102.87 ± 33.41a	A81.62 ± 35.55a
8th Day		A78.35 ± 33.28a	A80.08 ± 13.99a	A82.33 ± 7.85a	A95.36 ± 8.63a	A103.91 ± 31.16a	A81.83 ± 32.95a
12th Day		A76.35 ± 33.27a	A78.71 ± 10.56a	A79.88 ± 7.75a	A74.67 ± 23.71a	A102.25 ± 32.83a	A80.92 ± 34.05a

Mean ± SD

^{a, b}Means followed by different superscript alphabets in each row are significantly different (P ≤ 0.05) among different pH within the same solvent

^A–CMeans followed by different superscript alphabets in each column are significantly different (P ≤ 0.05) among storage durations

Table 2 shows the kinetic parameters, such as reaction rate constants (k), half-life (t_1/2), and activation energy (E_a) of betacyanins from red amaranth. Reaction rate constants (k) and half-life (t_1/2) of betacyanins were calculated from Fig. 3a, b. Low temperature (4 °C) resulted in a longer half-life because of a lower reaction rate constant compared to higher temperature (30 °C) for all conditions. Higher temperature may accelerate the reaction rate and subsequently decrease the half-life (Wang and Xu 2007; Tsai et al. 2010). This might be due to higher betacyanin content at lower temperature than at higher temperature. However, different pH and solvent extractions did not follow the same trend for the reaction rate and half-life. The reaction rate could be increased or decreased depending on various factors, such as moisture, pH, soluble solid, and solvent. Kirca and Cemeroglu (2003) found that the reaction rates accelerated with increased concentration of the reaction species as the number of molecules increased. Cai (2002) and Chandran et al. (2014) also mentioned that the stability of betacyanins could be influenced by temperature and air. In this study, the rate constant and half-life were high, consistent with the findings of Cai (2002)who reported rate constants ranging from 0.192 to 0.817 day⁻¹ and half-lives ranging from 0.031 to 36.74 days for Amaranthus cruentus at different temperatures and conditions. The activation energy was calculated using the Arrhenius equation which is shown in Fig. 4 I (a–c), II (d–f) (supplementary information). The activation energy of water extraction sample was 68.76–119.75 kJ/mol; whereas it was 22.97–125.34 kJ/mol for ethanol extraction samples. Priatni and Pradita (2015) found that the activation energy of betacyanins extracted from red dragon fruit peels was 14.37 kJ/mol. According to Vaillant et al. (2005)and Ruiz-Gutiérrez et al. (2015), the activation energy of betacyanins in pitaya juices and red cactus pear were 71.12–112.13 kJ/mol and 1.5888 kJ/mol, respectively. Higher E_a values are associated with the dependence of betacyanin degradation on higher temperature. The difference in activation energy values could be due to the difference in soluble solid contents (Kara and Erçelebi 2013) and treatment of samples resulting in compositional change (Kirca et al. 2007).

Table 2.

The degradation rate constant (k), half-life (t_1/2), and activation energy (E_a) of extracted betacyanins content from red amaranth under various conditions

pH	Temperature (°C)	Water				50% ethanol
		k (day−1)	t1/2 (day)	R2	Ea (kJ/mol)	k (day−1)	t1/2 (day)	R2	Ea (kJ/mol)
1	4	0.0016	433.12	0.33	119.75	0.0031	223.54	0.66	22.97
	30	0.1391	4.98	0.93		0.0073	94.93	0.89
3	4	0.0091	76.15	0.02	68.76	0.0011	630.00	0.55	125.34
	30	0.1182	5.86	1.00		0.1178	5.88	0.93
5	4	0.0045	154.00	0.88	80.72	0.0080	86.62	0.13	71.13
	30	0.0913	7.59	0.95		0.1135	6.100	0.93

Fig. 3.

a Degradation kinetics of betacyanin from red amaranth at different pH (1.0, 3.0, 5.0) using water. b Degradation kinetics of betacyanin from red amaranth at different pH (1.0, 3.0, 5.0) using ethanol

Conclusion

In this work, betacyanins were efficiently extracted from red amaranth and the effects of various parameters (pH, extraction media, and storage conditions) on betacyanin content, color properties and stability were also investigated. Extraction at pH 3.0 and 5.0 resulted in higher betacyanin content and better thermal stability at refrigeration storage condition than at ambient atmospheric conditions. Low pH resulted in higher L*, a*, and b* values than high pH in water extraction samples at both the temperature conditions. However, the color retentions were higher at pH 3.0 and 5.0 in water extraction samples as compared to pH 1.0 at both storage conditions. Color retention highly correlated with the content of betacyanins for all samples. The change in color (ΔE) or color loss gradually increased during storage and was highest in 50% ethanol at ambient temperature. On the other hand, degradation rate constant (K) was lower, and half-life (t_1/2) was higher at refrigeration temperature as compared to ambient temperature. Therefore, it can be concluded that water, pH 5.0, and refrigeration temperature could be the suitable parameters for extraction of betacyanins from red amaranth that would be used as natural colorants in the food industry to avoid the carcinogenic effects of the synthetic colorants.

Electronic supplementary material

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