Red Amaranthus (Amaranthus Tricolor L. ) Extract as a Functional Natural Food Colour

The global shift toward regulatory clean-label food systems has accelerated the search for plant-derived colourants that offer both visual appeal and functional benefits. Betalains, a class of nitrogen-containing, water-soluble pigments, have emerged as promising alternatives to synthetic dyes due to their intense colour, antioxidant potential, and compatibility with aqueous food matrices. This technical study summarizes experimental runs conducted to evaluate the chemical composition, antioxidant strength, molecular fingerprint, and colour stability of red Amaranthus extract intended for use as a natural food colour ingredient.

Characterisation and stability assessment of red Amaranthus extract for food applications

• Antioxidant potency:

The red Amaranthus extract exhibited an ABTS•+(2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) IC₅₀ of 5.19 mg mL⁻¹ (dry basis), at 734 nm in aqueous buffer, indicating appreciable radical scavenging capacity. Total phenolic content (TPC) was measured as 235.3 mg gallic acid equivalents (GAE) per gram of dry solids, corresponding to approximately 23.5% (w/w) of total extract solids.

When the ABTS IC₅₀ was normalised to phenolic contribution, the effective in-assay antioxidant requirement corresponded to approximately 1.22 mg GAE mL⁻¹. This normalised value indicates that, although several milligrams of crude extract are required to reach 50% radical scavenging, the actual phenolic-equivalent fraction responsible for activity is relatively small, highlighting efficient antioxidant performance intrinsic to the betalain-rich matrix rather than extract mass alone.

Pigment potency of the extract was quantified as 53.3 ± 1.03 mg betanin equivalents (BE) per gram of extract (dry basis), confirming a high betalain concentration consistent with intense colour strength. In parallel, total dissolved solids (TDS) were measured at 3.68 ± 0.01 mg mL⁻¹ (temperature coefficient 1.98), indicating good solubility particularly relevant for beverage and liquid food applications.

• LC–MS/MS valiadtion of Betalain pigments

Molecular-level confirmation was achieved using LC–MS/MS analysis, providing definitive evidence of betalain dominance in the extract. A prominent base peak at m/z 194.9 was consistently observed, corresponding to a betalamic acid–derived chromophoric fragment, a well-recognized diagnostic ion for betalain pigments. This fragmentation pattern confirms the betalain-rich nature of the extract, as reported in recent literature (Araujo-León et al., 2024; Miguel, 2018).
The overall spectral profile indicates that the extract is predominantly composed of betacyanin pigments, mainly amaranthine and isoamaranthine. Classical flavonoid antioxidants were comparatively minor or not prominently detected, likely due to ion suppression effects and preferential fragmentation of nitrogen-containing betalains under positive-mode electrospray ionization (ESI⁺). This confirms that colour intensity and antioxidant behaviour are primarily governed by betalain chemistry rather than polyphenolic flavonoids.

• Chemical identity of the colour principles:

Amaranthine and isoamaranthine are betacyanins, comprising a betalamic acid core condensed with cyclo-DOPA derivatives and glycosylated moieties. Their conjugated chromophoric systems confer intense red-violet coloration, while their nitrogen-containing structures enable electron donation and radical stabilisation. Glycosylation further enhances water solubility, supporting their use in aqueous food matrices.

• Effect of pH and Temperature on Colour Stability

Colour stability studies were conducted to evaluate the practical performance of the extract under typical storage conditions relevant to food formulations. At 25 °C, the extract exhibited a ΔE* value of 4.1±0.42 , indicating a visibly perceptible colour change even under dark storage conditions. This demonstrates that light exclusion alone is insufficient to prevent betalain degradation. Over 28 days, a gradual increase in pH was observed, accompanied by a shift in hue angle (h°) toward yellow, consistent with preferential degradation of betacyanins.
Refrigerated storage at 4 °C significantly improved colour and pH stability, extending acceptable colour retention by approximately 4 days. In contrast, sub-zero storage conditions showed no statistically significant changes (p > 0.05) in colour coordinates or pH when compared with fresh extract (day 0).

• Degradation Mechanism and Stability Insight

The findings indicate that betalain degradation is primarily temperature-driven, rather than light-induced. Ambient storage conditions promote chemical instability of betalains, including decarboxylation-linked reactions, which contribute to pH elevation and selective loss of red betacyanin pigments. This results in a perceptible yellow hue shift, compromising colour quality. Refrigeration (<4 ˚C) effectively slows these degradation pathways, while sub-zero storage arrests betalain breakdown almost entirely, preserving both chromatic coordinates and pH stability comparable to freshly prepared extract.

Conclusion and Practical Implications

This integrated assessment establishes red Amaranthus extract as a betalain-rich, antioxidant-active natural food colour with strong functional potential. LC-MS/MS validation confirms the dominance of amaranthine-based pigments, whereas, antioxidant assays demonstrate efficient radical scavenging beyond mere colour contribution, and stability studies clearly identify temperature as the critical control parameter for maintaining colour integrity.
From an application standpoint, effective cold-chain management is essential, and conversion of the extract into a shelf-stable format (such as a powder) represents a scientifically sound pathway for enhancing storage stability, formulation flexibility, and commercial translation.

References:

Araújo-León, J. A., et al. (2024). Advanced mass spectrometric characterisation of betalain pigments: fragmentation pathways and analytical markers. Food Chemistry, 430, 137072.
https://doi.org/10.1016/j.foodchem.2023.137072
Miguel, M. G. (2018). Betalains in some species of the Amaranthaceae family: A review. Food Chemistry, 241, 225–232. https://doi.org/10.1016/j.foodchem.2017.08.079

Author : Debolina Kundu https://www.linkedin.com/in/debalina-k-4b617948

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