Improvement of phycocyanin stability at different temperatures using microencapsulation by whey protein isolate

Phycocyanin is a blue pigment in cyanobacteria known for its antioxidant activity and can be applied as a natural food colorant. However, one drawback to applying phycocyanin in food products is their high-temperature instability. In this research, we investigated the effect of encapsulation of phycocyanin using whey protein isolate (WPI) as the wall material to improve high-temperature stability. The study also assessed the physiochemical properties of microencapsulated phycocyanin. Phycocyanin was extracted from dry biomass Spirulina using a cold maceration method. Then, phycocyanin extract was encapsulated with prepared emulsions containing 0.25%, 0.50%, 0.75%, and 1.00% WPI as wall materials. The result showed all microencapsulated phycocyanin, regarding various concentrations of WPI, showed lower phycocyanin degradation at 60°C and 70°C at various heating times compared to control, suggesting higher stability. The control sample had 35.55±0.33% and 62.61±0.55% concentration degradation at 60°C and 70°C after 10 min heating. The microencapsulated phycocyanin with 0.50% WPI had 12.67±2.08% and 19.95±2.02% at 60°C and 70°C after 10 min heating. The encapsulation efficiency achieved 98-99% regarding various concentrations of WPI. There was no significant difference in solubility between the control and microencapsulated phycocyanin. Our result concluded that microencapsulation, using WPI as wall material, improved the high-temperature stability of phycocyanin.

Phycobiliproteins (PBPs) are pigments found in cyanobacteria, that are located on the surface of thylakoids [4].PBPs consist of three pigments, namely phycocyanin, phycoerythrin, and allophycocyanin.Phycocyanin is the main blue pigment of PBPs, representing up to 20% of the cell mass [5].Phycocyanin comprises two similar subunits, the α-subunit with one phycocyanobilin linked at cysteine 84, and the β-subunit with two phycocyanobilin attached at cysteines 84 and 155 [4].Phycocyanin is characterised by its blue colour.Phycocyanin is soluble in water and other polar solvents [6].
In the food industry, phycocyanin has been used as a natural colourant in various foods, including chewing gum, soft drinks, and candies [10].The major problem with utilising phycocyanin in food and beverage is instability in high temperatures and various pH [11].Phycocyanin tends to lose its blue colour at high temperatures due to protein degradation.A study conducted by Wu et al., [12] showed that when the temperature was increased from 45 to 75ºC, the relative concentration of phycocyanin decreased from 99.0% to 48.4%.Phycocyanin is also prone to colour degradation and forming aggregates at pH 3.0 and 4.0, most likely due to interactions between phycocyanin molecules [13].
Several studies have shown that microencapsulation is effective for maintaining phycocyanin stability.Microencapsulation is a method that uses a drying technique to encapsulate core materials in wall materials [14].Several polymers can be used as a coating or wall material for phycocyanin microencapsulation.The examples are starch (maltodextrin), gum (guar, xanthan, carrageenan, agar), fibre (pectin, cellulose, chitosan, inulin), protein (soy, whey, pea, egg white), and lipid (lecithin) [15].A study by Da Silva et al., [16] showed the use of citric acid-crosslinked maltodextrin to encapsulate phycocyanin extract with the spray drying technique.The phycocyanin microcapsules showed higher thermal stability compared to the non-encapsulated phycocyanin.Yan et al., [17] showed that the addition of chitosan and alginate could increase the preservation rate of phycocyanin to light exposure (40 days), heating (50°C), and acids (pH 1.2).
Whey protein is a by-product of cheese processing and is widely used in the food industry.Whey protein could form a protective layer surrounding the phycocyanin molecule through electrostatic or hydrophobic protein-protein interactions, thus preventing aggregation in low pH [18].A study by Zhang et al., [13] showed that whey protein was more effective in preventing aggregation in phycocyanin at pH 3.0 than pea and egg white protein.Zhang et al.,[19] also reported that whey protein isolates at a concentration of 0.05-0.1% were able to protect phycocyanin from colour degradation at pH 3.0 over 5 days in the light.However, no research proves the encapsulation efficiency of whey protein and whether it can maintain stability from high temperatures.
This research aims to investigate the effect of encapsulation of phycocyanin using whey protein isolate (WPI) as the wall material to improve high-temperature stability.The solubility, encapsulation efficiency, and antioxidant activity were also measured.The findings of this study provide evidence to overcome the limitation of phycocyanin in the food industry.

Extraction of phycocyanin
Phycocyanin extraction was carried out by the cold maceration method with some modifications [20].Spirulina platensis dry biomass (California Gold Nutrition®) was dissolved in aquades at 1:100(w/v).The solution was incubated overnight and stored in the dark at 4°C.The solution was centrifuged at 5000 rpm for 10 min at 10°C to collect the phycocyanin solution.The obtained phycocyanin extract was referred to as crude extract.

Phycocyanin microencapsulation
The phycocyanin microencapsulation procedure was based on Pan-Uthai & Iamthan, [20] with some modifications.Emulsions were prepared by dissolving whey protein (California Gold Nutrition®) in distilled water and increasing the final amount to 100 mL.Different concentrations of whey protein were studied: 0.25, 0.50, 0.75, and 1.00%.The solutions were kept at 4°C for 24 h to complete hydration.Phycocyanin without wall material was used as a control.Phycocyanin solutions and the wall materials were mixed in a mass ratio of 1:3 (phycocyanin /wall material).The solutions were mixed with a highspeed homogeniser (Ultra Turrax, Ika Labortechnik, Staufen, Germany) at 5000 rpm for 3 min.The mixture was dried at 50°C for 12 h.Furthermore, the dried samples were powdered and stored in the dark for further analysis.

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 [21].

Determination of phycocyanin stability in different temperatures
To determine temperature stability, samples were dissolved in aquades at a concentration of 4 mg/mL.The solution was incubated in the water bath at 60 and 70°C for 10 min.The purity and concentration were measured every 2 min.Data was shown as concentration degradation and relative purity.

Concentration degradation of phycocyanin.
Phycocyanin degradation refers to Purnama et al., [22] as the percentage of degradation in phycocyanin after heating.The concentration degradation was calculated according to Equation (3) where C0 was the phycocyanin content before heating treatment and Ct was the phycocyanin content after heating treatment.
2.4.2.The relative purity of phycocyanin.The phycocyanin relative purity is referred to Chaiklahan et al., [11] as the percentage of the remaining phycocyanin purity (P) of the treated samples over the initial concentration (P0).The relative purity was calculated according to Equation (4).

Colour measurement
Colour measurement was performed by Colorimeter (NH310, 3Nh, Shenzen, China) based on CIE-Lab colour scale and distilled water was used as the control.The brightness was denoted by L*, where "0" represented black and "100" represented white.The +a* value indicated that the sample was red, whereas -a* indicated that it was green.The +b* denoted that the sample colour was yellow, whereas -b* denoted blue.

Solubility measurement
The solubility measurement was based on Pan-Utai & Iamtham, [20] with slight modification.Briefly, samples were dissolved in 10 mL of distilled water and vortexed for 3 min at room temperature.The suspension was then centrifuged for 10 min at 4200 rpm.The aliquot supernatant was transferred to a pre-weighed aluminum container and dried in an oven at 105°C until a constant mass was reached.The dry mass of soluble solids was determined, and the solubility was estimated in % by dividing it into the number of solvents.

Encapsulation efficiency
To evaluate the efficiency of phycocyanin microencapsulation, concentrations of phycocyanin and surface phycocyanin of the microcapsules were determined following the method of Pan-Uthai & Iamtham, [20].To determine phycocyanin concentration, 100 mg samples were reconstituted by adding 10 mL distilled water and continuously vibrating on a vortex mixer for 3 min.Then, the mixture was centrifuged at 4200 rpm and 10°C for 10 min.The clear supernatant was collected and filtered using Whatman paper No. 42 to measure phycocyanin concentration.Surface phycocyanin refers to the amount of phycocyanin that is presented on the surface of microcapsules after the encapsulation process.It was considered non-entrapped, meaning it had not been successfully encapsulated within the protective shell of the microcapsule but rather remained on the outer surface [23].To determine the surface phycocyanin concentration, 100 mg of samples were directly extracted with 10 mL of 95% (v/v) ethanol solution.The mixture was continuously vibrated on a vortex for 30 min, followed by centrifugation at 4200 rpm and 10°C for 10 min.After the phase separation, the clear supernatant was collected and filtered using Whatman paper No. 42, and surface phycocyanin concentration was determined by measuring its absorbance.Encapsulation efficiency was calculated by Equation (5).

Antioxidant activity
The antioxidant activity was determined based on ABTS •+ radical scavenging activity as described by Safari et al., [24] with slight modification.The ABTS •+ radical solution was prepared by mixing an equal volume of a 7 mmol/L ABTS •+ stock solution with a 2.45 mmol/L potassium persulfate solution in distilled water.The mixture was kept in the dark at room temperature for 16 h.This solution was diluted with methanol to an absorbance of 0.7±0.05 at 734 nm.As much as 1 mL of phycocyanin extracts was added to 2 mL of the ABTS •+ radical solution, and the absorbance of the reaction was measured in the spectrophotometer at 734 nm after incubation for 6 min.Methanol solution with ABTS •+ was used as a control.The ABTS •+ scavenging activity was evaluated using the Equation ( 6)

Statistical analysis
All experiments were performed in triplicate.Data are presented as the mean±standard deviation and were analysed using a one-way analysis of variance test (ANOVA) due to only involving one factor.Duncan's Multiple Range Test (DMRT) was used to analyse significant differences between the samples at p<0.05.DMRT is chosen due to our small sample size (5 samples).In situations where sample sizes are small, DMRT had higher statistical power and sensitivity in detecting differences between means.It can detect smaller differences with greater accuracy compared to Tukey's test, which requires a larger sample size to establish statistical significance [25].Besides, DMRT did not conduct pairwise comparisons for all groups, therefore reducing the number of comparisons that might lead to increased Type I error rates [26].

Effect of microencapsulation on the stability of phycocyanin at high temperature
Microencapsulated phycocyanin had lower concentration degradation and higher relative purity in high temperatures of 60 and 70°C compared to control, that is, the unencapsulated phycocyanin (Figure 1-4).Results also showed that the concentration degradation was decreased, and the relative purity was increased along with the increase of WPI concentrations.In a comparative study on concentration degradation and relative purity over time, significant differences were observed between the control sample and samples with 0.50% and 1.00% Whey Protein Isolate (WPI).
The control sample exhibited a higher rate of concentration degradation, reaching 13.40±0.57%after 2 minutes of heating at 70°C and escalating to 62.61±0.55%after 10 minutes.In contrast, the 0.50% WPI sample showed markedly lower degradation, starting at 2.80±0.59%and increasing to 19.95±2.02%,while the 1.00% WPI sample demonstrated the least degradation, from 2.59±0.15% to 11.38±1.20%over the same periods (Figure 1).The results suggested that encapsulation using WPI effectively delays the concentration degradation of phycocyanin, particularly in the 2 to 6-minute heating range.Regarding relative purity, the relative purity of control significantly decreased from 84.91±0.55% to 38.17±0.67%after 10 minutes of heating.Conversely, the 0.50% WPI sample maintained a higher purity level, decreasing from 95.89±0.31% to 82.21±0.78%,and the 1.00% WPI sample showed the most stability, starting at and reducing to 92.24±0.83%over the same timeframe (Figure 2).These results highlight the protective effect of WPI, as a coating material, against degradation under heat treatment.The degradation rate and relative purity at 60°C showed a similar pattern at 70°C (Figure 3).However, the concentration degradation at 60°C was 1.8 times lower than 70°C.At 60°C, the concentration degradation for the control sample was found to be 33.55±0.33%after 10 min heating.While at 70°C, the concentration degradation for the control sample was found to be 62.61±0.55%after 10 min heating.It was also found that the addition of WPI at 60°C could delay concentration degradation proven by a non-significant increase in 4-8 min heating.Similar to the concentration degradation, the relative purity at 60°C was 1.7 times lower than 70°C (Figure 4).At 60°C, the relative purity for control sample was found to be 66.56±0.61%after 10 min heating.While at 70°C, the relative purity for control sample was found to be 38.17±0.67%after 10 min heating.It was found that adding 0.75 and 1% WPI could delay the decreasing relative purity for 10 min heating.A study conducted by Chaiklahan et al. [11] showed that phycocyanin is stable at low temperatures (4°C), while its critical temperature is 47°C.Wu et al. [12] reported that an increase in temperature resulted in a higher degradation in the concentration and purity of phycocyanin.When exposed to high temperatures, the phycocyanin molecule was unfolded.Therefore, the hydrophobic groups are exposed to aggregation, resulting in degradation and loss of blue color [27].
Our results, which utilized the lower concentration of WPI at the concentration of 0.5-1.0%,were similar to Zhang et al. [13], which showed that encapsulation using 10% WPI could prevent protein aggregation compared to the untreated phycocyanin at 80°C.Another encapsulation study of capsaicin also showed that WPI-encapsulated capsaicin had a lower glass transition temperature than unencapsulated capsaicin [28].It means that WPI is proven to increase the thermal stability of bioactive compounds.WPI also showed better thermal stability for phycocyanin than other wall materials, such as carrageenan.A study conducted by Li et al. [29] showed that carrageenan-encapsulated phycocyanin had a phase separation and color degradation at 60°C.During the encapsulation, WPI molecules reorganize and form a matrix or coating around the phycocyanin due to its excellent emulsifying properties.This altered conformation could create a physical barrier that enhances the thermal stability of phycocyanin by limiting its exposure to heat [13].In contrast, carrageenan had poor colloidal stability, low net charge, and small sulfate groups per unit.Therefore, carrageenan could only bind to a few phycocyanin molecules, forming a weak electrostatic interaction [29].These result in poor protection of phycocyanin against heat.Our results are consistent with a study conducted by Zhang et al. [30] who reported that the colour of spray-dried chlorophyll was significantly affected by WPI concentration (5-15%).An increase in WPI concentration led to an increase in the brightness and a decrease in the greenness of chlorophyll.Another study also reported that encapsulation had a significant effect on the color of phycocyanin.Study by Ilter et al. [31] reported that there was an increase in the L* value of phycocyanin that was encapsulated with maltodextrin.Ilter et al. [31] also reported that there was a decrease in the L* value and an increase in the b* value of encapsulated phycocyanin with gum Arabic and sodium caseinate.It is possible that more phycocyanin was encapsulated by the wall material microcapsules (WPI), thus concealing the blue colour of phycocyanin [32].2. Results indicated that all microencapsulated powders had excellent solubility.The highest solubility was obtained from 0.5% WPI with 99.71±0.02%,while control had the lowest solubility with 99.59±0.10%.The utilization of WPI to other bioactive compounds also shows a high solubility.Zhang et al. [28] showed that WPI-encapsulated chlorophyll had a solubility of 96.57±0.14%.WPI also showed a higher solubility compared to other wall materials such as maltodextrin and gum Arabic.Ilter et al. [31] showed that phycocyanin encapsulated with maltodextrin had 79.52±2.53%solubility while with gum Arabic had 67.92±0.96%.WPI contains hydrophilic groups in their structure that will interact with water molecules through hydrogen bonding, therefore enhancing the solubility.Whereas both maltodextrin and gum Arabic contain hydrophobic segments in their structures that could limit their interaction with water molecules, therefore decreasing their overall solubility [33].WPI also has a smaller molecular weight and a more compact structure compared to maltodextrin and gum Arabic.A small molecular weight will have larger surface area-tovolume ratios.This will allow more interactions between WPI with water molecules and will increase the solubility [34].

Effect of microencapsulation on the encapsulation efficiency of phycocyanin.
Encapsulation efficiency is a determining factor in evaluating the performance of encapsulation [35].The high encapsulation efficiency value ensures that bioactive substances are highly bioavailable.The phycocyanin microencapsulation efficiency is shown in Table 3.The encapsulation efficiency of phycocyanin exceeded 98% in all treatments, which indicates that WPI could be a suitable wall material for phycocyanin encapsulation processing.The sample with 0.50% WPI had the highest encapsulation efficiency at 99.31±0.39%,while 0.75% WPI had the lowest efficiency at 98.59±0.42%.Our results are similar to Zhang et al. [30] who showed that WPI had a high encapsulation efficiency in chlorophyll.The encapsulation efficiency of chlorophyll increased along with the increasing in WPI concentrations, with the highest efficiency of 99.76% in 15% WPI.Compared to other wall materials, WPI possessed a higher encapsulation efficiency.A study of phycocyanin encapsulation using 1.5-2.5% sodium alginate showed that the encapsulation efficiency ranged from 53-71% with 1.5% having the lowest efficiency and 2.5% having the highest efficiency [36].The high efficiency of WPI could be attributed to its globular structure which has a compact, three-dimensional, and folded conformation that allows WPI to form a stable encapsulating matrix [37].This structure aided in entrapping the bioactive compounds effectively.On the other hand, alginate encapsulation forms a gel-like matrix rather than a specific globular shape.This process might result in the formation of larger pores or less uniform encapsulation structures, potentially leading to lower encapsulation efficiency compared to the tighter, more uniform encapsulation provided by WPI [36].

Effect of microencapsulation on antioxidant activity
Table 4 shows that the antioxidant activity was decreased along with the increase in WPI concentration.This was indicated by an increase in IC50.The highest antioxidant activity was shown in control samples with IC50 of 126.31±3.96ppm while the lowest was 1% concentration had IC50 of 506.70±15.80ppm.Our results are in accordance with Pan-Utai & Iamtham, [20] where phycocyanin had the lowest IC50 for about 760±100 ppm and encapsulated phycocyanin using maltodextrin-gum arabic had the highest IC50 or about 1350±110 ppm.Hadiyanto et al. [36] also reported that alginate-encapsulated phycocyanin had higher IC50 values compared the unencapsulated which are 197.67±9.33 ppm for the encapsulated and 280.29±0.75ppm for the unencapsulated phycocyanin.Those studies prove that a decrease in antioxidant activity in encapsulated phycocyanin does not only occur in WPI as a coating material but also in other wall materials.During encapsulation, phycocyanin is enclosed within the WPI matrix, which might limit their interaction with the surrounding environment and potentially reduce their availability for antioxidant reactions.The WPI matrix might also act as a barrier, hindering the phycocyanin from accessing their target sites or interacting efficiently with oxidizing agents, thus reducing the overall antioxidant activity of phycocyanin [38].

Conclusion
In this study, we have demonstrated the use of WPI to improve the stability of phycocyanin in high temperatures.Our results showed that encapsulation with WPI showed the ability to maintain stability at different temperatures (60 and 70°C) by decreasing phycocyanin degradation and increasing relative purity.WPI could form a protection film surrounding the phycocyanin, thus protecting phycocyanin from high temperatures.Microencapsulation increases the L* (lightness), a* (redness), and b* (blueness) value resulting in a decrease in phycocyanin blue color compared to control.Encapsulation also showed no significant effect on phycocyanin solubility and high encapsulation efficiency exceeding 95%.Microencapsulated phycocyanin showed lower antioxidant activity compared to the control samples due to the limited number of phycocyanin that react with ABTS reagent.It was also found that increasing WPI concentrations resulted in lower antioxidant activity.

Figure 1 .
Figure 1.Effect of microencapsulation on thermal stability expressed in concentration degradation at 70°C.Data with small superscript letters indicates a significant difference between heating times.Data with capital superscript letters indicates a significant difference between samples in the same heating time (p<0.05).

Figure 2 .
Figure 2. Effect of microencapsulation on thermal stability expressed in relative purity at 70°C.Data with small superscript letters indicates a significant difference between heating times.Data with capital superscript letters indicates a significant difference between samples in the same heating time (p<0.05).

Figure 3 .
Figure 3.Effect of microencapsulation on thermal stability expressed in concentration degradation at 60°C.Data with small superscript letters indicates a significant difference between heating time.Data with capital superscript letters indicates a significant difference between samples in the same heating times (p<0.05).

Figure 4 .
Figure 4. Effect of microencapsulation on thermal stability expressed in relative purity at 60°C.Datawith small superscript letters indicates a significant difference between heating times.Data with capital superscript letters indicates a significant difference between samples in the same heating time.

3. 2 . 1 .
Effect of microencapsulation on the color of phycocyanin.The colour of microencapsulated and the control phycocyanin are shown in Table 1.The control sample had lower L*, a*, and b* value compared to encapsulated.The control sample had 38.65±1.26for L* value, -1.21±0.86 for a* value, and -8.31±0.09for b* value, while 1% WPI had 48.28±0.63 for L* value, 1.85±0.75for a* value, and 0.34±0.43 for b* value.As the WPI ratio increased, the value of L* increased from 46.62±0.44 to 48.28±0.63, the value of a* increased from -0.17±1.28 to 1.85±0.75,and the value of b* increased from -2.57±1.23 to 0.34±0.43.Increasing in L* value means increasing in lightness intensity.Increasing in a* value means increasing in redness intensity.A positive a* value indicates red, while a negative indicates green.Increasing in b* value means decreasing blueness intensity.A positive b* value indicates yellow, while a negative b* value indicates blue.Therefore, WPI concentration significantly affected the colour of the microencapsulated phycocyanin.

Table 1 .
Effect of microencapsulation on the colour of phycocyanin.
Effect of microencapsulation on the solubility of phycocyanin.The effect of microencapsulation on phycocyanin solubility is shown in Table

Table 2 .
Effect of microencapsulation on solubility of phycocyanin.

Table 3 .
Effect of WPI concentration on encapsulation efficiency.

Table 4 .
Effect of microencapsulation on antioxidants activity.Different letters within the same column indicate a significant difference (p < 0.05)