Environmental stability and dairy-food application of Spirulina-derived phycocyanin: Color retention, microbiological quality and economic feasibility

Introduction

Consumer preference for recognisable, naturally derived ingredients has intensified interest in replacing synthetic food colours with pigments from plants, microorganisms and algae. Blue remains a technically difficult colour because relatively few natural sources provide an intense hue with acceptable food compatibility. C-phycocyanin, the principal blue phycobiliprotein of Arthrospira (commercially known as Spirulina), combines colouring capacity with reported antioxidant functionality and is therefore promising for clean-label foods. Unlike many low-molecular-weight pigments, phycocyanin is a protein-chromophore complex. Its colour is consequently sensitive to unfolding, aggregation and chromophore disruption. Temperature, pH, illumination, oxygen and food-matrix interactions can accelerate these changes. Maximum stability has commonly been reported around pH 5.5-6.0, below approximately 45 °C and in the absence of light (Chaiklahan et al., 2012; Adjali et al., 2022) [2,1] . This narrow stability window is a central barrier to wider use in thermally processed and brightly displayed foods. Model-food trials are necessary because stability in a buffered pigment solution does not necessarily predict behaviour in a complex food. Dairy matrices contain proteins, fat, lactose and minerals that may either protect the pigment or alter its optical expression. Previous work has demonstrated that food-grade phycocyanin can retain useful colour in frozen dairy systems and beverage matrices, although performance depends on formulation and storage conditions (de Amarante et al., 2020; García et al., 2021) [3,4] . The present paper addresses the third and fourth objectives of a broader pigment-development study. It evaluates the stability of extracted phycocyanin against light, pH and temperature; examines colour retention and acceptability in whipping cream and flavoured milk; monitors microbiological quality during refrigerated storage; and assesses preliminary production economics. This downstream focus avoids duplication of cultivation and extraction optimization and directly addresses feasibility as a food colourant.

Materials and Methods

Pigment and experimental design

Food-grade phycocyanin previously extracted and purified from cultivated Spirulina biomass was used. Stability was followed by absorbance at 620 nm, with higher absorbance representing greater retention of the characteristic blue pigment. Unless otherwise stated, values represent mean ± standard deviation of three determinations.

Light, pH and temperature stability

For light stability, identical pigment portions were stored in darkness, under LED daylight and under UV-A exposure. Absorbance was recorded daily for 7 days. The pH trial used pigment systems adjusted to pH 5.0, 5.5, 6.0 and 7.0 and monitored from day 0 to day 7. Thermal stability was evaluated at 30, 45, 60 and 75 °C over the same period. Retention at the final observation was calculated as final absorbance divided by initial absorbance × 100.

Preparation of model dairy foods

Whipping cream was prepared from 66.90 mL milk, 30.00 g unsalted butter, 2.50 g powdered sugar and 0.50 g vanilla flavour, with phycocyanin added according to treatment. Milk was heated to 40-45 °C, butter was melted into it, remaining ingredients were mixed, and the blend was homogenized for 2-3 min. After cooling at 4 °C for 2-4 h, it was whipped at high speed for approximately 20 min and refrigerated. Flavoured milk contained 91.80 mL milk, 8.00 g sugar, 0.10 g vanilla flavour and treatment-specific phycocyanin. Milk and sugar were heated at 70-80 °C for 5-10 min; flavour and pigment were added after cooling. Treatment T 2 contained 100 mg phycocyanin and was selected for detailed evaluation.

Sensory and colour measurements

Control and pigment treatments (0, 50, 100 and 150 mg) were assessed by a semi-trained panel of academic staff and students using a nine-point hedonic scale for appearance, colour, taste, flavour, texture and overall acceptability. The thesis did not report panel size; therefore, no unsubstantiated sample size is assigned here. Colour was measured with a Hunter 45/0-L MiniScan XE Plus after black-andwhite-tile calibration. L * , a * and b * were recorded and chroma (C * ) and hue angle (h °) were calculated. Colour of optimized products was monitored on days 0, 3, 7, 10 and 15.

Microbiological evaluation

Refrigerated products were analysed on days 0, 7, 14, 21 and 28. Serial dilutions were prepared in sterile 0.85-0.90% saline. Total plate count was determined on nutrient agar after incubation at 37 °C for 48 h. Yeasts and moulds were assessed on potato dextrose agar. Coliforms were recorded as detected or not detected. Results were expressed as CFU/g for whipping cream and CFU/mL for flavoured milk.

Economic assessment and statistical analysis

Water, cultivation medium and inoculum were costed at recorded experimental prices. Processing cost was defined as 20% of raw-material cost. Because the original cost table contained an arithmetic inconsistency, both the recorded inputs and a transparent recalculation are reported. Experimental treatment data were analysed in a completely randomized design; standard error and critical difference at the 5% level were reported where available. Values are mean ± SD (n = 3).

Results and Discussion

Effect of light

All treatments began at an absorbance of 0.93, but their trajectories separated rapidly. Dark storage retained 94.6% of initial absorbance after 7 days, compared with 66.7% under LED daylight and 26.9% under UV-A. UV-A therefore produced a 73.1% loss, whereas darkness limited loss to 5.4%. The result confirms that light protection is not merely desirable but essential for preserving this pigment. Photon-driven disruption of the protein-chromophore complex and photo-oxidation plausibly account for the accelerated decline, particularly under UV-A. Opaque or UV-barrier packaging should therefore be used during storage and retail display. Values are mean ± SD (n = 3). The most favourable condition was pH 6.0, which retained 93.5% of initial absorbance. Retention at pH 5.5 was 91.4%, compared with 88.0% at pH 7.0 and 78.0% at pH 5.0. The ranking-pH 6.0 > 5.5 > 7.0 > 5.0-agrees with the recognized stability maximum around pH 5.5-6.0 (Chaiklahan et al., 2012) [2] . Increasing acidity toward pH 5.0 probably promoted conformational destabilization and aggregation, while the modest loss at pH 7.0 may reflect a less favourable oligomeric state. The pigment is thus better suited to mildly acidic or near-neutral foods than strongly acidic beverages. [2] identified a critical stability region near 47 °C, while Martelli et al. (2014) [5] similarly described the need for stabilization at processing temperatures. Phycocyanin should therefore be added after severe heat treatment whenever possible, or protected through validated stabilizers or encapsulation. Values are mean ± SD (n = 3). Whipping cream became slightly darker (L * decreased 1.60 units) and less saturated (C * decreased 13.8%) over 15 days. Flavoured milk showed a 1.90-unit decline in L * and a 17.5% decline in C * . In both systems, negative b * values moved toward zero, indicating weakening blue intensity, while hue changed by only 1.03° in cream and 1.76° in milk. Thus, colour vividness diminished gradually without a major shift in hue identity. The cream matrix retained chroma slightly better, possibly because fat and its lower light scattering environment altered pigment protection or optical expression. Comparable successful dairy applications have been reported in ice cream and model beverages (de Amarante et al., 2020; García et al., 2021) [3,4] . T 2 (100 mg phycocyanin) provided the best overall acceptability: 8.4 in whipping cream and 8.3 in flavoured milk. It also achieved colour scores of 9.0 and 8.5, respectively. Control overall acceptability was 7.8 for cream and 7.5 for milk. Because the differences exceeded the corresponding critical differences (0.24 and 0.23), the optimized treatments were statistically preferred at the 5% level. The pigment therefore improved appearance without materially reducing taste, flavour or texture. These observations support hygienic processing, but absence of an explicitly stated detection limit and product-specific regulatory comparison prevents a definitive 28-day shelf-life claim. The PDA method was described as streak plating in the thesis; future validation should use a quantitative spread-or pour-plate protocol with replicate variability.

Effect of pH

Effect of temperature

Colour retention in dairy matrices

Microbiological quality

Preliminary economic feasibility

Conclusion

Spirulina-derived phycocyanin was most stable in darkness, at pH 5.5-6.0 and at temperatures no higher than 45 °C. UV-A and temperatures of 60-75 °C caused substantial loss of absorbance. When applied at the optimized 100-mg level, the pigment produced highly acceptable whipping cream and flavoured milk and retained its blue to bluepurple hue through 15 days, although chroma declined gradually. Microbial counts increased during 28-day refrigeration, but coliforms remained undetected. The corrected laboratory cost was approximately ₹32.09/g at the yield implied by the thesis records. Practical adoption should use light-barrier packaging, post-heat pigment addition or mild processing, refrigeration and product-specific shelf-life validation.

Limitations

The study did not report pigment purity grade, exact UV-A irradiance, LED intensity, dissolved oxygen, packaging type, sensory panel size or detection limits. Repeated-measures significance was not reported for stability datasets. The microbial yeast/mould procedure requires quantitative validation, and the economic analysis omitted commercial overheads. These omissions should be addressed before journal submission or scale-up.

Acknowledgement

AcknowledgementsThe authors acknowledge the Department of Food Microbiology and Safety and the Department of Food Chemistry and Nutrition, College of Food Technology, Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani, Maharashtra, India, for providing research facilities.

References

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