Investigating the stability of encapsulated phycocyanin at acidic conditions using whey protein isolate as a wall material

Phycocyanin is a natural blue pigment derived from cyanobacteria and is usually used as a food colorant. However, phycocyanin is unstable in acidic conditions. In this research, we investigated the effect of encapsulation of phycocyanin using whey protein isolate (WPI) as the wall material to improve the stability at various pH and storage periods. Phycocyanin was extracted from dried Spirulina using a cold maceration method and subsequently encapsulated with 0.25, 0.50, 0.75, and 1.00% of WPI. Results showed that microencapsulation with WPI significantly reduced phycocyanin degradation compared to the control sample under acidic conditions ranging from pH 3.0 to 6.0. The control sample had 81.51±0.70% concentration degradation at pH 3.0. While 0.50% WPI had 50.48±1.27% degradation. Furthermore, microencapsulation exhibited a protective effect against phycocyanin degradation for 10 days of storage at 4°C in pH 4.0 and 5.0. Samples with 1.00% WPI demonstrated a lower concentration degradation each day compared to the control. WPI also demonstrated its efficacy in delaying concentration degradation at pH 4.0 and 5.0 for 8 to 10 days. Our result concluded that microencapsulation using WPI could improve the stability of phycocyanin in acidic conditions and storage periods.


Introduction
Spirulina is a unicellular, filamentous cyanobacterium that is found in seawater and freshwater.Spirulina has a spiral-shaped morphology and flagella for movement [1].Spirulina is known for having high protein content, accounting for approximately 60-70% of its dry weight.Spirulina is high in vitamins and minerals [2].Spirulina also contains pigments including chlorophyll, phycocyanin, phycoerythrin, allophycocyanin, and carotenoids.
Phycocyanin is a natural blue pigment found in Spirulina and the composition reaches 20% of the cell mass [3].Phycocyanin is commonly used in the food and beverages industry as a natural food coloring agent [4].Phycocyanin not only provides a vibrant blue color to food products but also offers a range of health benefits.Phycocyanin has been found to have several functional properties such as antioxidant [5], anti-cancer [6], and anti-inflammatory [7].
Despite its many advantages, the application of phycocyanin in the food industry has limitations, especially when exposed to acidic conditions.Phycocyanin is highly sensitive to acidic pH levels, which can lead to its degradation and loss of color.Zhang et al. [8] showed that at pH 3.0, phycocyanin tends to undergo color degradation and aggregate formation, possibly as a result of interactions occurring among phycocyanin molecules.Chaiklahan et al. [9] also observed that the concentration of IOP Publishing doi:10.1088/1755-1315/1302/1/012087 2 phycocyanin maintained around 80% at pH 6.0 and 7.0, but only around 70% at pH 5.0.This poses a challenge for the food industry, as many food products have acidic components or undergo acidification during processing.Preserving the stability of phycocyanin under acidic circumstances is essential to guarantee its efficacy as a coloring agent in food products.Phycocyanin was also found to experience degradation over time especially when stored in acidic conditions [10].The pH level of the surrounding environment plays a crucial role, as higher acidity can accelerate the degradation of the phycocyanin.
Microencapsulation is a technique that has gained attention in the food industry for its ability to protect sensitive compounds from degradation and improve their stability [11].In the case of phycocyanin, microencapsulation can be used to shield it from the detrimental effects of acidic conditions and preserve its color and bioactivity.The encapsulating matrix acts as a protective shield, preventing direct contact between phycocyanin and the acid [12].This helps to maintain the integrity of phycocyanin and prolong its stability in acidic conditions.Falkeborg et al. [13] showed that phycocyanin encapsulation with sodium dodecyl sulphate (SDS) could increase stability in acidic conditions by preventing green color conformation.Zhang et al. [8] demonstrated that the stability of phycocyanin at pH 3.0 could be enhanced by forming a complex with α-lactalbumin, β-lactoglobulin, BSA, immunoglobulins, and glycomacropeptides.
Whey protein isolate (WPI) is a byproduct of cheese that is commonly used as a microencapsulation agent due to its excellent encapsulation properties and biocompatibility.WPI forms a stable matrix around the phycocyanin molecules, protecting them from the acidic environment [14].Research conducted by Zhang et al. [8] demonstrated that the use of 10% whey protein could prevent phycocyanin aggregation under low pH (pH 3.0).However, there is no research on the effect of encapsulation using WPI on the shelf-life of phycocyanin in acidic conditions.
This research aims to explore the potential of WPI-based microencapsulation in enhancing the stability of phycocyanin in various pH and storage periods.Through this investigation, we seek to contribute to developing a novel approach that improves the utilization of phycocyanin across multiple food applications while addressing its limitations associated with pH sensitivity.The research is supported to reach the sustainable development goals of good health and well-being, in which the better stability of phycocyanin may broaden the utilization of phycocyanin for functional food and nutraceutical ingredients.

Samples preparation
Phycocyanin was extracted from dried Spirulina platensis (California Gold Nutrition®) using a cold maceration method [15].Briefly, the dried Spirulina platensis was dissolved in distilled water for 1:100 (w/v) at 4°C overnight.The solution was centrifuged at 5000 rpm for 10 min at 10°C to collect the phycocyanin supernatant.
Whey protein (California Gold Nutrition®) solution was prepared by dissolving the whey protein in 100 mL distilled water with a final concentration of 0.25, 0.50, 0.75, and 1.00% [16].The solutions were incubated at 4°C for 24 h to complete hydration.Phycocyanin was added to the whey protein with a ratio of 1:3 (v/v).As a control, phycocyanin with no wall material was used.The solutions were homogenized for 3 min using a high-speed homogenizer (Ultra Turrax, Staufen, Germany).The mixture was dried for 12 h at 50°C.

Determination of phycocyanin stability on acidic pH
To determine pH stability, samples were dissolved in phosphate-citrate buffer at pH values of 3.0, 4.0, 5.0, and 6.0 at a concentration of 4 mg/mL.The purity and concentration were determined after samples were precipitated.For shelf-life determination, samples at pH 4 and 5 were stored at 4°C for 10 days.Every 2 days, samples were taken to analyze their concentration and purity.Data was shown as concentration degradation and relative purity.

Measurement of phycocyanin concentration and purity.
A UV-Vis spectrophotometer was used to measure the concentration and purity of phycocyanin.The concentration of phycocyanin in mg/mL Equation (1) and purity Equation ( 2) is calculated with absorbance readings at the wavelength of 280, 620, and 652 nm [17].
2.2.3.The relative purity of phycocyanin.The relative purity of phycocyanin refers to the method by Chaiklahan et al. [9], calculating the percentage of the retained phycocyanin purity (P) in the treated samples compared to the initial concentration (P0).The relative purity was calculated according to Equation (4).In pH stability analysis, P0 represented the phycocyanin content at pH 6 and P represented the phycocyanin content at pH 3.0-5.0.In shelf-life determination, P0 represented the phycocyanin content at day 0 and P represented the phycocyanin content at day 2-10.

Statistical analysis
All experiments were performed in triplicate.Data are presented as the mean±standard deviation and were analyzed using a one-way analysis of variance test (ANOVA).A post-hoc Duncan's multiple range test was used to analyze significant differences between the samples at p<0.05.

Effect of microencapsulation on the stability of phycocyanin at acidic conditions
The effect of microencapsulation on various pH are shown in Figure 1 and 2. Results showed that encapsulated phycocyanin had lower concentration degradation and relative purity at acidic pH compared to control.The results also showed that along with the increase in WPI concentration, the concentration degradation was decreased, and the relative purity was increased (Figure 1).In this study, pH 6.0 is used as a reference due to the highest concentration and purity in all samples.Control samples had 81.51±0.70%concentration degradation at pH 3.0.While 0.50% and 1.00% WPI had 50.48±1.27%and 47.26±1.40%concentration degradation, respectively (Figure 1A).At pH 4.0, control samples had 45.05±0.39%concentration degradation.While 0.50% and 1.00% WPI had 29.84±0.48%and 23.68±1.70%concentration degradation, respectively.At pH 5.0, control samples had 29.95±0.52%concentration degradation.While 0.50% and 1.00% WPI had 20.33±0.47%and 20.15±1.63%concentration degradation, respectively.It was also found that the addition of WPI could delay concentration degradation by a non-significant increase from pH 5.0 to pH 4.0.The relative purity had the same trend with the concentration degradation where it was higher in encapsulated phycocyanin compared to control (Figure 2).Phycocyanin showed the lowest stability at pH 3.0 and the highest stability at pH 6.0.A study conducted by Chaiklahan et al. [9] also showed that phycocyanin had the highest stability at a pH of 5.5 to 6.0.At acidic conditions, the protein will fold into a cyclic formation, causing precipitation which is shown by the discoloration of phycocyanin [9].Our result showed a similar outcome to Zhang et al. [8] who reported that the encapsulation with 10% WPI could prevent degradation and aggregation in phycocyanin at pH 3.0.The utilization of WPI as the encapsulating agent in microencapsulated phycocyanin forms a protective layer around the phycocyanin.This protective layer is formed via electrostatic or hydrophobic interactions between proteins, effectively preventing direct exposure of phycocyanin to acidic surroundings [8].Therefore, encapsulation with WPI helps to maintain the integrity of phycocyanin.
The utilization of whey protein isolates (WPI) as a coating material extends beyond phycocyanin to other bioactive compounds.Solgi et al. [19] found that WPI-encapsulated curcumin nanoparticles effectively shielded curcumin in low pH environments, simulating the intense acidity of gastric acid.Earlier studies indicated that curcumin encapsulated in this way remained highly stable at a pH of 2. This stability is likely due to the protective effect of the dense globular structure of β-lactoglobulin, which makes up 55% of WPI.The findings of our research, along with those of earlier studies, indicate that whey protein isolate (WPI) shows promise as a coating material in the encapsulation process, effectively preserving and stabilizing bioactive compounds.

Effect of microencapsulation on the stability of phycocyanin during storage
Results in Figure 3 and 4 showed the data in pH 4.0 the encapsulated phycocyanin had lower concentration degradation and higher relative purity compared to control in the same condition at the storage same time.It was found that from day 2-10, the concentration degradation in 1.00% WPI was lower compared to the control.Control samples had 44.73±5.05%concentration degradation for 2 days of storage, while 0.50 and 1.00% WPI had 43.40±2.70%and 32.63±0.73%concentration degradation, respectively (Figure 3).On day 6, control samples had 60.76±3.11%concentration degradation on day 6, while 0.50 and 1.00% WPI had 48.07±4.47%and 53.55±0.27%concentration degradation, respectively.On day 10, control samples had 84.75±0.89%concentration degradation.Samples with encapsulated 0.50 and 1.00% WPI had 76.24±2.13%and 74.79±4.47%concentration degradation, respectively.The relative purity had the same trend with the concentration degradation where it was higher in encapsulated phycocyanin compared to control (Figure 4).It was also found that the addition of 0.50% WPI could delay the degradation proven by a non-significant increase on the concentration degradation on days 8 and 10.Samples in pH 5.0 (Figure 5 and 6) had the same trend as samples in pH 4.0.The concentration degradation in 1.00% WPI was also found to be lower compared to control on days 2-10 (Figure 5).Besides, the results suggested that the addition of 1.00% WPI could delay the concentration degradation on day 8 and day 10 at pH 5.0.It is showed by a significant increase in the control sample from 67.91±1.68% to 81.96±1.45%,while the sample that encapsulated with 1.00% WPI did not have a significant increase from 55.17±5.53% to 60.46±1.48%.These findings align with Yan et al. [21], indicating that encapsulated phycocyanin using chitosan alginate, resulted in a slower degradation rate than control in an acidic environment at pH 4. Zhang et al. [20] also showed that the addition of 0.05-0.1% WPI could delay phycocyanin degradation at pH 3.0 for 5 days of light storage.This was proven by a delay in color degradation compared to the control samplei s.Whei y protei in ei ncapsulation forms a physical barriei r around thei phycocyanin molei culei s, protei cting thei m from thei acidic ei nvironmei nt.This protei ctivei layei r hei lps prei vei nt direi ct ei xposurei of phycocyanin to low pH, thei rei by minimizing dei gradation or dei naturation of thei protei in in acidic conditions [22].Bei sidei s, whei y protei in also acts as a stabilizing agei nt by ei nhancing thei structural intei grity of phycocyanin molei culei s.This stabilizing ei ffei ct rei ducei s thei suscei ptibility of phycocyanin to pH-inducei d changei s, thei rei forei increi asing its stability in acidic conditions and prolonging its shei lf-lifei [23].

Conclusion
Thei study novei lty is markei d by thei ei mploymei nt of highei r concei ntrations of whei y protei in isolatei (WPI) as thei wall matei rial at concei ntrations of 0.5% and 1.00%.Additionally, wei ei xplorei d thei phycocyanin stability of thei compound across a pH rangei of 3.00 -6.00 and ei xtei ndei d its shei lf lifei to up to 10 days.Our rei sults concludei d that ei ncapsulation with WPI could maintain stability at acidic pH (pH 3.0-6.0)by dei crei asing phycocyanin dei gradation and increi asing rei lativei purity.Rei sults also showei d that microei ncapsulation could maintain thei stability of phycocyanin for tei n days of storagei at pH 4.0 and 5.0.Thei data dei monstratei d a consistei nt trei nd in ei ach day of storagei whei rei 1.00% WPI had lowei r concei ntration dei gradation than control.Bei sidei s, it also suggei stei d that adding 0.50 and 1.00% of WPI could dei lay thei concei ntration dei gradation at pH 4.0 and 5.0 on days 8-10.Futurei rei sei arch could ei xaminei thei fei asibility of using phycocyanin microei ncapsulation in acidic foods and ei xplorei altei rnativei ei ncapsulation mei thods, likei frei ei zei -drying.

Figure 1 .
Figure 1.Effect of phycocyanin microencapsulation on pH stability expressed in concentrations degradation.Data with small superscript letters indicate a significant difference between pH.Data with capital letters indicate a significant difference between samples within the same pH (p<0.05).

Figure 2 .
Figure 2. Effect of phycocyanin microencapsulation on pH stability expressed in relative purity.Data with small superscript letters indicate a significant difference between pH.Data with capital letters indicate a significant difference between samples within the same pH (p<0.05).

Figure 3 .
Figure 3.Effect of phycocyanin microencapsulation on phycocyanin stability during storage at pH 4.0 expressed in concentrations degradation.Data with small superscript letters indicate a significant difference between days.Data with capital letters indicate a significant difference between samples within the same days (p<0.05).

Figure 4 .
Figure 4. Effect of phycocyanin microencapsulation on phycocyanin stability during storage at pH 4.0 expressed in relative purity.Data with small superscript letters indicate a significant difference between days.Data with capital letters indicate a significant difference between samples within the same days (p<0.05).

Figure 5 .
Figure 5.Effect of phycocyanin microencapsulation on phycocyanin stability during storage at pH 5.0 expressed in concentrations degradation.Data with small superscript letters indicate a significant difference between days.Data with capital letters indicate a significant difference between samples within the same days (p<0.05).

Figure 6 .
Figure 6.Effect of phycocyanin microencapsulation on phycocyanin stability during storage at pH 5.0 expressed in relative purity.Data with small superscript letters indicate a significant difference between days.Data with capital letters indicate a significant difference between samples within the same days (p<0.05).