Effect of Protein Hydrolysates from Limonia (L.) acidissima and Citrullus (C.) lanatus on Anthocyanin Degradation

ABSTRACT

Anthocyanins are natural plant pigments and used extensively in many food and beverage formulations due to their atrractive red, blue, or purple color. However, this color is highly sensitive to light, and therefore unstable and degraded even in the presence of ascorbic acid. This leads to the loss in the bioactivity of anthocyanin and also loss in the color of food or beverages. Hence, the present study focuses on the preservation of anthocyanin from Syzygium cumini (L.) using natural preservatives such as Limonia acidissima (L. acidissima) and Citrullus lanatus (C. lanatus) seed protein hydrolyzates at elevated temperature (35°C/5 days). Different concentrations (0.04–2%) of L. acidissima and C. lanatus protein hydrolyzates were examined for its preservative action against 5% crude extract of anthocyanin from Syzygium cumini (L.) preservation. This concentration of protein hydrolyzates followed the first order of reaction. Both concentrations of protein hydrolyzates reduced the rate of change in absorbance. However, 2% concentration of C. lanatus protein hydrolyzate was found to be very effective in providing highestthe preservation of color. Therefore, the overall study highlights the preservation effect of L. acidissima and C. lanatus protein hydrolyzate.

KEYWORDS:

• Syzygium cumini (L.)
• color stability
• peptides
• storage effect

Introduction

Anthocyanins (ACs) are colored pigments composed of phenolic compounds naturally present in fruits and vegetables. ACs are responsible for different colors like salmon-pink, orange, red, violet, and blue (Ewa, 2009). ACs are widely used in food and beverage industries as a natural and safe colorant. According to the Mermelstein (2016), there is continuous upsurge demand and market for natural food colorant rising at the rate of 10–15% annually. Additionally, these natural pigments provide numerous health benefits and biological activities like genoprotective, anticancer, cardioprotective, anti-inflammatory, antithrombotic, antihypertensive, lipid lowering, hypoglycemic, antiobesity, ocular protective, neuroprotective, and antimicrobial (Chu et al., 2010).

Therefore, the demand for natural ingredients including colorants outweighs the use of artificial and synthetic colors due to their wide acceptance and use by food and beverage industries and consumers (Katz and Williams, 2011). The sale of nonalcoholic beverages was over 1000 billion US dollars in 2014 (Statista, 2014). Many of these nonalcoholic beverages were designed to have a specific color using synthetic and natural color. However, due to increased consumerism and awareness, demand for natural colorants in non-alcoholic beverages would be beneficial, particularly ACs, as these are water-soluble and exhibit color from red to blue.

The seeds of C. lanatus fruit are a rich source of protein with minerals and also known more commonly as watermelon. L. acidissima is also called as wood apple and is native to India and Ceylon more often used in the preparation of jellies, jam, and chutney. Both the seeds are a very good source of essential amino acids, minerals, and proteins (Sonawane et al., 2016). In our previous study, protein was isolated and hydrolyzed from the seeds of C. lanatus and L. acidissima (Sonawane and Arya, 2017a, 2017b). These hydrolyzates were reported to possess very good antioxidant, antimicrobial, thermal stability and also composed of many peptides (Sonawane et al., 2018).

Healthy, natural, free from synthetic colors and preservative food products are more in demand. Therefore, natural anthocyanins isolated from fruits are gaining importance as natural colorants and functional ingredients. Additionally, these pigments have been proven as an antioxidant. However, these isolated ACs are unstable and subject to chemical degradation. Further, the degradation of ACs in aqueous solutions leads to loss of color and biological activity. Oxygen, temperature, light, enzymes, pH, and food matrix composition, e.g., carbohydrates, proteins, acids, salts, sugars, and minerals, are reported as effective parameters in the degradation of AC (Buchweitz et al., 2013a, 2013b; Cao et al., 2009; Hubbermann et al., 2006). However, this degradation of AC can be prevented by natural polymer and by forming complexes which could be further improved through molecular binding (hydrogen bonding and hydrophobic interactions) with pectin. Controlled atmospheres, encapsulation, spray drying, freeze-drying, hard-panned coating, and other novel enzymatic methods have been used effectively to enhance the stability of ACs (Cortez et al., 2017). There have been successful efforts to use polyphenolic extracts, amino acids, and polypeptides in preventing the degradation of ACs (Chung et al., 2015, 2016, 2017).

The loss of color in food and beverages are responsible for shelf life and quality of products (Sonawane and Arya, 2015; Sonawane et al., 2013). Therefore, a detailed and systematic study to improve the stability of ACs using natural protein hydrolyzates is required. To the best of our knowledge, there are no reports available on the preservation of AC using natural plant seed protein hydrolyzates. Hence, this study deals with the effect of different concentrations of L. acidissima and C. lanatus protein hydrolyzates on color and AC content in the Syzygium cumini (L.) during accelerated storage conditions.

Materials and Methods

The Syzygium cumini (L.) fruit was collected from the Institute of Chemical Technology campus, Matunga, Mumbai, during the summer season (April 2017); chosen fruits were fully ripened. All chemicals used in the experiment were of analytical grade.

Extraction of Anthocyanin

The solvent extraction method was carried out to extract AC from Syzygium cumini (L.) with little modification in the method reported by Sonawane and Arya (2013). The 1:10 ratio of pulp:solvent (ethanol) was used for the extraction of 18 h by using shaker at 180 RPM. The pH of the solution was maintained at 2 using a saturated solution of citric acid. The supernatant was separated at 10,000 RPM and concentrated to 70% by volume using vacuumed rotary evaporator to obtain a crude extract of AC.

Preparation of L. Acidissima and C. Lanatus Protein Hydrolyzates

Protein hydrolyzates from L. acidissima and C. lanatus defatted seed flour were prepared according to the method reported by Sonawane and Arya (2017a, 2017b).

Development of Anthocyanin and Protein Hydrolyzates Model

L. acidissima and C. lanatus protein hydrolyzates in the range of 0.04– 2% were added to 5% of crude extract of anthocyanin (prepared by using distilled water as solvent) and solutions were stored at 35 ± 2°C from 0 to 5 days in the presence of lights to understand the degradation kinetics of AC (Chung et al., 2016).

Anthocyanin and Color (Red) Intensity Determination

Anthocyanin content was determined using the pH differential method (Lee et al., 2005). Red intensity was measured at absorbance 523 nm by using a spectrophotometer (Chung et al., 2017). Before recording the red intensity of the sample, the extract was diluted in a 1:4 ratio.

Kinetic Study of Anthocyanin Degradation

The degradation kinetics of anthocyanin was evaluated by assuming first order of reactions to determine the reaction rate constant (k) and average half-life time (t_1/2) as reported by Chung et al. (2017).

Results and Discussions

Anthocyanin Content and Storage Stability

Degradation of AC in the Syzygium cumini (L.) is presented in Figure 1 for L. acidissima and C. lanatus protein hydrolyzates, respectively. Kinetic degradation of AC with the addition of L. acidissima and C. lanatus protein hydrolyzates at different doses, i.e., 0.04% to 2% is depicted in Figure 1. In both the experiments, AC in the Syzygium cumini (L.) was preserved at a concentration of 2%. The rate constant k, correlation coefficient, and half-life of the AC are presented in Table 1. Protein hydrolyzates from L. acidissima and C. lanatus were added in the range of 0.04–2% as a preservative to preserve the color of AC. The first order of reaction was confirmed by the correlation coefficient of ˃0.9. Correlation coefficient for L. acidissima and C. lanatus protein hydrolyzates was observed between 0.93 and 0.99. Hence, the concentration (0.04–2%) of L. acidissima and C. lanatus protein hydrolyzates followed the first-order reaction. Our results are in agreement with previous studies conducted for various matrices like aqueous extracts from grape juice and black carrot juice concentrates (Danişman et al., 2015; Nisha et al., 2004).

Table 1. Rate constant, R², and half-life of anthocyanin in Syzygium cumini (L.)by added L. acidissima and C. lanatus protein hydrolyzates

	L. acidissima			C. lanatus
System	Rate constant, k	R^2	t1/2 (days)	Rate constant, k	R^2	t1/2 (days)
Anthocyanin	0.246	0.99	2.81	0.246	0.99	2.81
Anthocynin +0.04%	0.223	0.99	3.07	0.174	0.95	3.98
Anthocynin +0.12%	0.213	0.97	3.25	0.181	0.93	3.82
Anthocynin +0.2%	0.216	0.96	3.21	0.141	0.94	4.91
Anthocynin +2%	0.231	0.95	3.00	0.128	0.93	5.44

Figure 1. Kinetic degradation of anthocyanin in Syzygium cumini (L.) by using (a) L. acidissima (b) C. lanatus protein hydrolyzates

The AC of Syzygium cumini (L.) showed a half-life of 2.81 days when no preservatives were added (Table 1). L. acidissima protein hydrolyzates (0.04–2%) were added in the AC extract of Syzygium cumini (L.)(Figure 1a). An increase in the stability of AC was observed with an increased concentration. However, with an increased storage period, a degradation in AC was noted. Further, the half-life of AC was increased to 3.25 days when L. acidissima protein hydrolyzate was added at a concentration of 0.12%. Further, it declined to 3 days at the concentration of 2% of L. acidissima protein hydrolyzate (Table 1). This was due to the higher rate of degradation constant which was observed when 2% of L. acidissima protein hydrolyzate was added (Table 1).

A similar trend is depicted in Figure 1b for the preservation of AC in Syzygium cumini (L.) when different concentrations (0.04–2%) of C. lanatus protein hydrolyzates were added. The half-life of AC was increased with increased concentration of C. lanatus protein hydrolyzates, that is, 2.81 to 5.44 days. From Table 1, it is evident that protein hydrolyzates from C. lanatus play an effective role in the preservation of AC in Syzygium cumini (L.) as compared to that of L. acidissima protein hydrolyzate. This might be due to the lower degradation rate constant that was observed in the case of C. lanatus protein hydrolyzates as compared to L. acidissima protein hydrolyzate (Table 1). The composition of peptides present in protein hydrolyzates and source of protein may affect and contribute to the preservation of AC in Syzygium cumini (L.)(shown in supplementary Table, Sonawane et al., 2018). Similar results were observed by Chung et al. (2017) who reported an increase in the half-life (from 3.57 to 6.22 days) of purple carrot ACs, when added with 0.1% amino acids to that the AC and ascorbic acid model (Chung et al., 2017).

Color Stability and Color Fading of Anthocyanin

Color stability of AC was determined by measuring red intensity at 523 nm and is depicted in Figure 2. In both the cases of L. acidissima and C. lanatus protein hydrolyzates, color stability was observed at 2%. From Figure 2, it is clear that both the concentrations of protein hydrolyzates stabilized the degradation of color. Different concentration of L. acidissima and C. lanatus protein hydrolyzates reduced the rate of change in absorbance with respect to storage days. The absorbance of AC in Syzygium cumini (L.) showed a reduction from 1.2 to 0.394 during the accelerated storage of 0 to 5 days. A reduction in the absorbance from 1.2 to 0.772 was noted when AC was added with L. acidissima at 2% concentration, whereas C. lanatus addition resulted in a less reduction, i.e., from 1.2 to 0.820. Therefore, C. lanatus protein hydrolyzates were observed to be more effective in stabilizing the color as compared to that of L. acidissima protein hydrolyzates. This might be due to the less degradation rate constant of anthocyanin in C. lanatus protein hydrolyzates to that of L. acidissima protein hydrolyzates (Table 1). Flavylium cations are responsible to exhibit red color at pH 2 and preserved during storage as the concentration of protein hydrolyzate was increased (Trkyilmaz and Khan, 2012).

Figure 2. Color stability of anthocyanin in Syzygium cumini (L.) by using (a) L acidissima (b) C. lanatus protein hydrolyzates

The degradation of the color of Syzygium cumini (L.) with a combination of different concentrations of 0.04–2% of L. acidissima and C. lanatus protein hydrolyzate strongly correlated with the AC (Table 2). This was due to the fact that the degradation in color is highly correlated with the degradation of AC (Cavalcanti et al., 2011; Yu et al., 2013).

Table 2. Correlation between the color and AC of Syzygium cumini (L.) added with L. acidissima and C. lanatus protein hydrolyzates

System	L. acidissima (R^2)	C. lanatus (R^2)
AC	0.993	0.993
AC+ 0.04	0.980	0.974
AC+ 0.12	0.955	0.959
AC+0.2	0.953	0.958
AC + 2	0.938	0.965

Conclusions

The degradation of anthocyanin in the Syzygium cumini (L.) added with natural plant protein hydrolyzates from L. acidissima and C. lanatus followed the first order of reactions. C. lanatus was observed to be very effective in the preservation color as compared to that of L. acidissima protein hydrolyzate. The most significant effect was observed with the addition of 2% protein hydrolyzate. The mechanism behind the preservation of natural color like anthocyanin using peptides or protein hydrolyzates from L. acidissima and C. lanatus or other plant seed source needs to be further explored through detailed studies.

Acknowledgments

Authors are thankful to UGC-SAP for providing financial support during the conduct of this research.

Disclosure Statement

Authors do not have any conflict of interests.

Supplementary Material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by University Grant Commission, New Delhi, Government of India; UGC SAP.