Current Research in Food Science 5 (2022) 2329–2337

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Current Research in Food Science

journal homepage: www.sciencedirect.com/journal/current-research-in-food-science

A review of recent strategies to improve the physical stability

of phycocyanin
Biao Yuan a, 1, Zhuxin Li a, 1, Honghong Shan a, Badamkhand Dashnyam a, Xiao Xu b,
David Julian McClements c, Bingquan Zhang e, Mingqian Tan d, Zhixiang Wang a,
Chongjiang Cao a, *
a
Department of Food Quality and Safety/ National R&D Center for Chinese Herbal Medicine Processing, College of Engineering, China Pharmaceutical University,
Nanjing, Jiangsu, 211198, China
b
School of Life Science, Shaoxing University, Shaoxing, Zhejiang, 312000, China
c
Department of Food Science, University of Massachusetts, Amherst, MA, 01003, United States
d
Academy of Food Interdisciplinary Science, School of Food Science and Technology, Dalian Polytechnic University, Dalian, Liaoning, 116034, China
e
Zhejiang Binmei Biotechnology Co. LTD, Linhai, Zhejiang, 318000, China

A R T I C L E I N F O A B S T R A C T

Keywords: There is an increasing demand for more healthy and sustainable diets, which led to an interest in replacing
Phycocyanin synthetic colors with natural plant-based ones. Phycocyanin, which is commonly extracted from Spirulina pla­
Stability tensis, has been explored as a natural blue pigment for application in the food industry. It is also used as a nu­
Encapsulation
traceutical in food, cosmetic, and pharmaceutical products because of its potentially beneficial biological
Food matrix effects
properties, such as radical scavenging, immune modulating, and lipid peroxidase activities. The biggest chal­
Bioactivity
lenges to the widespread application of phycocyanin for this purpose are its high sensitivity to chemical
degradation when exposed to heat, light, acids, high pressure, heavy metal cations, and denaturants. Conse­
quently, it is of considerable importance to improve its chemical stability, which requires a thorough knowledge
of the relationship between the structure, environment, and chemical reactivity of phycocyanin. To increase the
application of this natural pigment and nutraceutical within foods and other products, the structure, biological
activities, and factors affecting its stability are reviewed, as well as strategies that have been developed to
improve its stability. The information contained in this article is intended to stimulate further studies on the
development of effective strategies to improve phycocyanin stability and performance.

1. Introduction et al., 2021; Landim Neves, Silva and Meireles, 2021). Blue pigments
isolated from certain kinds of algae have been shown to have potential
The appearance of foods is one of the most important sensory char­ applications in foods (Fratelli et al., 2021). The utilization of this
acteristics impacting consumer acceptance (Luo et al., 2021; Martelli renewable source of natural pigments in foods may therefore improve
et al., 2014). Food colorants are therefore often added to foods to the sustainability of the modern food supply (Gephart et al., 2021;
improve their visual appeal, which may be natural or synthetic. The US Naylor et al., 2021; Tigchelaar et al., 2021; Short et al., 2021) (see
Food and Drug Administration (FDA) and the European Food Safety Table 1).
Authority (EFSA) limit the addition of many synthetic colorants in foods Researchers have reported that blue pigments isolated from Spirulina
and beverages due to their potential toxic effects on humans, which has platensis, a kind of seaweed, have a desirable color profile, good sus­
led to an interest in identifying natural colors to replace synthetic ones. tainability, and potential health benefits (Golden et al., 2021). Spirulina
Compared to red, yellow, orange, and green natural colorants, the platensis belongs to the cyanobacterium of high economic and nutritional
development of blue natural colorants are still in its infancy (Kaderides value (Belay et al., 1996; Zhou et al., 1991). The pigments of Spirulina

* Corresponding author. Department of Food Quality and Safety/ National R&D Center for Chinese Herbal Medicine Processing, China Pharmaceutical University,
Nanjing, Jiangsu, 211198, China.
E-mail address: [email protected] (C. Cao).
1
These authors contribute equally to this work.

https://doi.org/10.1016/j.crfs.2022.11.019
Received 19 March 2022; Received in revised form 10 October 2022; Accepted 19 November 2022
Available online 24 November 2022
2665-9271/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
B. Yuan et al. Current Research in Food Science 5 (2022) 2329–2337

Table 1
The Strategy Methods of improving the stability of phycocyanin.
Methods Process conditions Results Mechanism Characteristics Disadvantages References

Addition of biopolymers
ι-Carrageenan Mixed polysaccharide The light stability of ι-Carrageenan with highly Less damage to its Although high pressure https://www.
(combined with solution and phycocyanin phycocyanin- negatively charged stretch nutrient can’t influence the sciencedirect.co
high-pressure solution, then subjected carrageenan at pH 7.0 is phycocyanin’s tertiary composition and structure of small m/science/artic
processing) the samples to high- improved and prevent structure and maintain improve the molecules le/pii/
pressure processing the attack of oxidizing secondary structure, which glycation reaction (chromophore), S0268005
treatment (450 Mpa or radicals after high- may benefit the stability of phycocyanin aggregates X20305385
600 Mpa) pressure processing phycocyanin with changes in Zhang et al.
secondary structures and (2021)
forms a more compact
protein structure
Whey protein Mixed phycocyanin The light stability of Phycocyanin-whey protein Less damage to its Phycocyanin may form a https://www.
(combined solution and whey protein phycocyanin-whey mixture with high-pressure nutrient more compact protein sciencedirect.co
high-pressure solution, then subjected improved at pH 5.0 progress may encapsulate composition and structure with the m/science/artic
treatment) the samples to high- after high pressure the chromophore inside the improve the changes in secondary le/pii/
pressure processing treatment apoprotein network and glycation reaction structures S0268005
treatment (450 Mpa or prevented the attack from X20305385
600 Mpa) oxidizing radicals Zhang et al.
(2021)
Low Whey protein was Delay the color A low concentration of whey https://www.
concentration dissolved in phycocyanin degradation of protein may help unfold sciencedirect.co
of whey solution in 100 mL tubes phycocyanin at pH 3.0 chain tetrapyrroles of m/science/artic
(0.05–0.1%) to obtain whey by light over 5 days phycocyanin through le/pii/
transformation from α-helix S0308814
to β-sheet and protected its 619317406
secondary structure from Zheng et al.
being destroyed by light (2020)
Preservatives Added preservatives into Maintaining the Increased the stability of the Adjusted the Preservative may https://www.
(citric acid, the phycocyanin solution stability of phycocyanin phycocyanin structure and sensory damage the human sciencedirect.co
sucrose and in aqueous phase at 35 disrupted the structure of characteristics of health m/science/artic
calcium ± 5 ◦ C for 45 days water and decrease protein food and extended le/pii/S1
chloride) solubility (salting out) shelf life 359511
307003455
Mishra et al.
(2008)
DSP [dithiobis To 19 ml of phycocyanin The absorbance at 614 DSP modified the amino Convenient to Exist potential risks https://www.
(succinimidyl (3.0 mg/ml) in 50 mM nm of cross-linked groups of lysine to protect handling sciencedirect.co
propionate)] phosphate buffer saline phycocyanin (Curve b) the high order structure of m/science/artic
(PBS) (pH 7.4) was added was retained at 70% of phycocyanin le/pii/S0
DSP (4.1, 8.2 and 16.4 mg) the original color 14372080300
dissolved in 1.0 ml of development in 4 M 2730
dimethyl sulfoxide urea for 8 h Fukui et al.
(DMSO) (2004)
Formaldehyde Added the formaldehyde Phycocyanin- Covalent crosslinking Convenience in Not suitable for food https://www.
into phycocyanin solution formaldehyde which between the formaldehyde ease of handling, industry sciencedirect.co
and dialyzed overnight may keep stabilize at and phycocyanin may extreme m/science/artic
100% over 120 min and prevent the dissociation of adaptability and le/pii/S1
improve the stability up phycocyanin into subunits broadest reaction 35951131931
to 1.53-folds under the specificity 7131
light Munawaroh
et al. (2020)
Oil Incorporated of EVO oil or After cooking, extra Tocopherol contained in Suitable for all Not suitable for other https://www.
sunflower oil at the virgin olive- EVO and sunflower oils was bakery products food processing sciencedirect.co
concentration of 10 g oil/ phycocyanin mixture the main responsible for the m/science/artic
100 g of dough maintained about 90% protective action against le/pii/S0023
of the originally phycocyanin degradation 643820317655
cyanobacterial biomass. Niccolai et al.
(2021)
Micelles
SDS micelles Prepared the aqueous Stabilized phycocyanin Attributed to the SDS Don’t need a SDS may confer an https://www.
solutions of phycocyanin against pH-dependent micelles entrapment of the substantial unacceptable detergent sciencedirect.co
and SDS color variation molecule in the interior and amount of taste in food products m/science/artic
stabilized by hydrophobic encapsulation le/pii/
interactions, it has the material. S0308814
capability of stabilizing a It can be used in 617311548
folded conformation at low transparent Falkeborg et al.
pH products (2018)
Microencapsulation
Electrospraying The capillary diameter The polymer showed Phycocyanin was Smaller droplet Low throughput https://www.
technique was 0.45 mm and a well thermal resistance surrounded by a barrier size with a narrow sciencedirect.co
distance between the up to 216 ◦ C (PVA) to protect against distribution; m/science/artic
capillary and the collector light, oxygen, pH, moisture, High le/pii/
was 18 cm; heat, shear or other extreme encapsulation S0960308
11% PVA and 2% PC, a conditions efficiency; 519301762
(continued on next page)

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platensis primarily originate from the light-harvesting protein complex molecular and physicochemical origins of its instability. Therefore, this
named phycobiliprotein, which consists of phycocyanin, phycoerythrin, manuscript reviews the effects of temperature, light, pH, pressure, heavy
and allophycocyanin. Structurally, these pigments are found around the metal ions, and denaturants on phycocyanin stability. Compared to
core (allophycocyanin) of the phycobiliprotein and the periphery previous review articles, we summarize the main factors promoting
(phycoerythrin and phycocyanin) of the photosystem complex (Kan­ phycocyanin instability, explain the main reasons for this instability,
naujiya et al., 2019). The seaweed extracts are characterized by a bright and focus on potential degradation mechanisms. In addition, the
blue color because of the presence of the phycocyanin. different strategies developed to improve phycocyanin stability have
Commercially, phycocyanin is a supramolecular protein- been discussed. The knowledge presented in this article may facilitate
chromophore complex, which is usually extracted from Spirulina pla­ the utilization of phycocyanin in the food industry by increasing the
tensis, and is currently valued at around $360 per kg (Chaiklahan et al., understanding of the main factors causing its degradation.
2012; Li et al., 2021). Compared to other proteins, the phycobiliproteins
of phycocyanin possesses high fluorescence quantum yields and the 2. The structure and biological activities of the phycocyanin
apo-protein has the ability to bind other molecules (such as immuno­
globins and avidin) to form bonds, which may act as fluorescent probes Phycobilisome, the supramolecular protein complex, harvests light
that are used in fluorescence microscopy, immunoassays, and histo­ and delivers energy to the reaction centers of eukaryotic red algae and
chemistry (Eriksen, 2008; Khan et al., 2005; Romay et al., 1998; Romay prokaryotic cyanobacteria. It therefore plays an important role in
et al., 2000). Also, phycocyanin has been reported to exhibit radical photosynthesis. It is composed of a central core called allophycocyanin
scavenging, immune modulation, and lipid peroxidase activities, which and several rods that surround the core called phycocyanins (Ferraro
mean that it may also be used as a nutraceutical ingredient in foods, et al., 2020) (see Fig. 1).
cosmetics, and pharmaceuticals (Fernandez-Rojas et al., 2014; Mishra Phycobiliproteins have three main classes of pigments, each with its
et al., 2008; Patel et al., 2004). As a result of its desirable color profile own spectral features: phycocyanin (phycocyanin; blue, λmax = 617 nm);
and nutritional effects, the popularity of phycocyanin as a natural blue phycoerythrin (red, λmax = 565 nm); and allophycocyanin (bluish green,
pigment and nutraceutical ingredient is growing in food industry. λmax = 650 nm) (Ferraro et al., 2020). Of these, phycocyanin forms the
In recent years, methods to extract and improve the stability of major part of phycobiliproteins (Ma et al., 2020). The blue color of
phycocyanin have been reviewed. Jaeschke et al. mainly reviewed phycocyanin is due to a chromophore called phycocyanobilin, which
phycocyanin extraction technologies, and reported that a pulsed electric binds proteins through thioether bonds (Pez et al., 2021). As shown in
field method was particularly suitable for obtaining high yields and Fig. 2C and D, phycocyanin contains two homologous subunits: the
purities (Jaeschke et al., 2021). Fratelli et al. also summarized the α-subunit with one phycocyanobilin attached at cysteine 84 and the
technologies available to extract phycocyanin, and reported that solvent β-subunit with two phycocyanobilins attached at cysteines 84 and 155
extraction can better protect the antioxidant capacity of phycocyanin (Tang et al., 2012; (Scheer and Zhao, 2008)). The phycocyanobilins are
(Fratelli et al., 2021). Adjali et al. recently reported that the stability of open-chain tetrapyrrole chromophores with a specific arrangement of
phycocyanin can be improved by adding preservatives or by using conjugated double bonds, which are responsible for the unique radical
packaging materials (Adjali et al., 2022). These authors reviewed the scavenging and antioxidant properties of the phycocyanin, as well as its
instability of phycocyanin under different light, temperature, and pH color. Phycocyanin has been reported to prevent or reduce hepatorenal
conditions, and did not explore the underlying mechanisms of phyco­ and hematological damage because it protects the tissues via γ-gluta­
cyanin instability. Therefore, we have carried out a thorough search of myl-cysteinyl-glycine, which can scavenge reactive oxygen species and
the literature and found that the various factors affecting phycocyanin free radicals through enzymatic reactions (Belay, 2002; Falkeborg et al.,
stability have not been comprehensive reviewed, as well as the 2018; Sayed et al., 2022). In addition, phycocyanin has been reported to

Table 1 (continued )
Methods Process conditions Results Mechanism Characteristics Disadvantages References

feed rate of 50 μLh− 1 and Without using Schmatz et al.

an electric potential of 20 high temperature (2020)
kV; or pressure
Extrusion Alginate and chitosan The microcapsules Attributed to the Simple and Involved high pressure https://www.
technique were used as coating could resistant to the hygroscopicity of chitosan, convenient; sciencedirect.co
materials; light (light for 40 days), the compact structure of Better-controlled m/science/artic
Under the high pressure, temperature (50 ◦ C), microencapsulation was microsphere sizes le/pii/
the matrix dispersion acidic (SGF pH 1.2) and formed by the interaction of S0960308
through a single or a humid (31% relative alginate and chitosan and 513000734
plurality of pathways humidity) environment prevented the entry of water Yan et al.
directly into the (2014)
continuous extraction
phase
Entrapped into Encapsulated phycocyanin The photodamage rate Phycocyanin encaged by https://link.
silica matrixes into hydrogel and constant of Si–O bands, which could springer.
controlled the gelation phycocyanin in silica is restrict the unfolding of com/article/10
time to homogeneously 25 times slower than phycobilin and protect the .1007%
dispersing it in silica the phycocyanin in linear conformation 2Fs11164-00
matrix buffer solution 9-0061-5
Li et al. (2009)
Coated with Sodium Prolonged The C-phycocyanin was High availability; https://www.
STMP/STPP trimetaphosphate/sodium antihyperalgesic effects encapsulated within Low cost sciencedirect.co
cross-linked tripolyphosphate cross- of phycocyanin in vivo amorphous chains of cross- m/science/artic
starches linked potato, banana, linked starches le/pii/
corn, cassava, and S0141813
breadfruit starches were as 020332633
wall materials for C- Lemos et al.
phycocyanin (2020)
encapsulation

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hexamer, and both subunits with molecular weight around 20 kDa have
conserved α-helix regions (Jaouen et al., 1999; Munawaroh et al., 2020).
However, when the hexamers disassemble into trimers and mono­
mers, the extinction coefficients of the phycobiliproteins decrease. The
high order structure of phycocyanin is a major factor influencing its
biological stability (Fukui et al., 2004). Also, the intensity and position
of the absorption maximum of phycocyanin have been shown to depend
on its state of aggregation (Hu et al., 2021). The aggregation and
structure of phycocyanin are influenced by temperature, pH, light,
pressure, heavy metal cations, and denaturants, which may therefore
impact its biological activity and color profile. In addition, light absor­
bance and fluorescence polarization are influenced by the structure of
the phycocyanin molecule (Moreira et al., 2019). Collectively, it can be
concluded that the color and bioactivities of the phycocyanin are
maintained through the high order structure of the phycocyanin protein.

3. Factors affecting the stabilities of phycocyanin

Numerous factors are considered to affect the stability of phycocy­

anin, including temperature, light, pH, pressure, heavy metal cations,
and denaturants (Berns and MacColl, 1989; Kupka and Scheer, 2008;
Sarada et al., 1999). Moreover, studies have highlighted that the water
activity, glass transition temperature, and rheology of the system can
also influence the stability of phycocyanin (Neri et al., 2011; Neri et al.,
2014). By elucidating the impact of these factors on phycocyanin sta­
Fig. 1. The industry applications of the phycocyanins.
bility, it is possible to improve its stability in food and other
applications.
inhibit the growth of cancer cells, reduce inflammation, inhibit
bleeding, prevent cataracts, and protect the heart, liver, lung, and kid­
3.1. Effect of temperature
neys (Fernandez-Rojas et al., 2014; Yu et al., 2017) (see Fig. 3).
In solution, phycocyanin forms αβ units that assemble into multi­
Temperature significantly affects the conformation of phycocyanin.
meric structures (αβ)n (n = 1–6), whose molecular weights range from
Studies have shown that a critical temperature for phycocyanin stability
around 44 to 260 kDa (Ma et al., 2020; Zhang et al., 2021). As shown in
is 47 ◦ C, as evidenced by an appreciable reduction in the half-life of
Fig. 2A and B, phycocyanin typically exists as an (αβ)3 trimer or (αβ)6
phycocyanin in solution when the temperature exceeded this value

Fig. 2. Structure of phycocyanin from Spirulina pla­

tensis. (A) Three-dimensional structure of phycocy­
anin from cyanobacterium S. platensis in form of
hexamer, image from PDB (https://www.rcsb.
org/structure/1GH0). (B) Phycocyanin exists as (αβ)
6 hexamer and both chains have the α-helix structure.
(Green, brown and pink represent α subunit. Grey,
yellow and dark brown represent β subunit). (C) (D)
Three-dimensional and secondary structure of phy­
cocyanobilin. (For interpretation of the references to
color in this figure legend, the reader is referred to the
Web version of this article.)

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Fig. 3. The biological activities, sensitive factors of the phycocyanins and the methods of protecting the phycocyanins.

(Chaiklahan et al., 2012). Similarly, in another analysis of the thermal 2014).

stability, it was reported that the antioxidant potential of phycocyanin
decreased more than 50% after 21 and 35 days of exposition at the 3.4. Effect of high-pressure processing
temperature of 40 and 50 ◦ C, respectively. A decrease in blue hue has
also been reported when the phycocyanin is heated (Colla et al., 2017). Recently, high-pressure processing has been applied to pasteurize
Further, researchers found that the phycocyanin lost color when heated beverages at reduced temperatures. This technique needs high hydraulic
to 60 ◦ C for 30 min (Rahman, Sarian, van Wijk, Martinez-Garcia and van pressures (400–600 MPa) and can extend the shelf life and improve the
der Maarel, 2017; Zhang et al., 2021). This phenomenon may be due to safety of foods (Li et al., 2021; Xia et al., 2020). However, high-pressure
the conformational changes of the apoprotein disturbing the coupling processing can promote phycocyanin denaturation and aggregation,
and energy transfer pathways between the embedded chromophores (Li thereby altering its color (Zheng et al., 2020).
et al., 2021). Heating caused the phycocyanin to partially unfold,
thereby exposing hydrophobic groups that were normally located in the 3.5. Effect of heavy metal cations
interior of the molecular. Furthermore, phycocyanin subunits aggregate
during heat treatment, which can also cause degradation of phycocyanin The existence of excess cysteinyl residues and anionic amino acid
and reduce the blue color. Other researchers reported that the aggre­ residues in phycocyanin means it can coordinate with metal cations
gation and degradation of phycocyanin subunits during heating could be (Han et al., 2018). When heavy metal cations bind to phycocyanin, the
modeled using the Weibull model (Faieta et al., 2022). fluorescence tends to be quenched due to a decrease in α-helix and β-turn
structures and increase in random coil and β-sheet structures (Bhayani
3.2. Effect of light et al., 2016). Other researchers have reported that ion the type and
concentration of metal influenced the light absorption of phycocyanin
Phycocyanin is unstable in the presence of light with the degradation (Chi et al., 2020). These effects have been attributed to the binding of
rate depending on the intensity and wavelength of light used. Re­ cationic ions to anionic groups on the protein surfaces, thereby altering
searchers have reported that the antioxidant potential of phycocyanin the structure of phycocyanin. Other researchers reported that addition
decreased under fluorescent (20 W, 0.16 A) and ultraviolet (15 W, λ = of Cu2+ and Zn2+ ions reduced the color of phycocyanin solutions
265 nm) light (Colla et al., 2017). When exposed to light a cyclohelical significantly, which was attributed to the ability of the metal ions to
conformation was adopted, it caused nonfluorescent, deactivation, and inhibit electron transfer and photosynthetic oxygen (Memije-Lazaro
photo-degradation due to intramolecular energy transfer or radical re­ et al., 2018). Heavy metal ions reduced the color of phycocyanin solu­
actions (Li et al., 2009). Other researchers found that adding epi­ tions may attribute to the formation of supramolecular fibrous networks
gallocatechin gallate can stabilize the phycocyanin when exposed to (Han et al., 2018). Taken together, these studies indicated that metal
light (Yang et al., 2022). ions disrupted the structure and aggregation of phycocyanin, thereby
altering its color and biological activity.
3.3. Effect of pH
3.6. Effect of denaturants
Phycocyanin is found to be unstable at the pH values between 4.0
and 4.8, which is fairly close to its isoelectric point (pH 3.4) (Li et al., Phycocyanin may also lose its color and biological activities when
2021). Some researchers reported that phycocyanin was insoluble under exposed to certain kinds of protein denaturants. For instance, the ability
acidic conditions (pH 3.0) but highly soluble under neutral conditions of urea to decrease these activities was attributed to its ability to bind to
(pH 7.0) (Antelo et al., 2008). The net charge of phycocyanin at its the surface of the phycocyanin, thereby disrupting the molecular in­
isoelectric point is zero, resulting in a reduced electrostatic repulsion teractions and destabilizing the native state, and the color and biological
between the molecules, thereby promoting the aggregation and pre­ activities of the protein molecules were reduced (Mishra et al., 2008).
cipitation. Research have reported that phycocyanin exists in a
monomer-trimer system at pH 4.5 to 6.0 (Mishra et al., 2008). However, 4. Strategies to improve the stability of phycocyanin
it shifted towards the monomer when the system is diluted. Phycocyanin
has also been reported to be unstable to aggregation at low pH values The chemical stability of phycocyanin depends on its molecular
and high ionic strengths due to a reduction in the electrostatic repulsion characteristics, as well as solution and environmental conditions. Some
between the protein molecules (Hattori et al., 1965; Newsome et al., of the methods used to protect phycocyanin are unsuitable for

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application in food products because they involve chemicals that are not 4.1.3. Addition of edible preservatives
safe for consumption. Therefore, alternative approaches are being Preservatives are used to ensure that foods remain safe and unspoiled
developed to protect phycocyanin in foods, such as controlling food throughout their shelf lives. Dithiothreitol and sodium azide have been
matrix composition and encapsulation. shown to protect phycocyanin from degradation, however, these are
toxic substances that are not suitable for food applications (Mishra et al.,
4.1. Addition of biopolymers 2008). Thus, it is important to identify other kinds of food-grade pre­
servatives that can protect the color and biological activities of
In the food industry, it is common practice to add biopolymers, such phycocyanin.
as polysaccharides and proteins, to phycocyanin formulations to inhibit Citric acid has been shown to protect phycocyanin from degradation
its aggregation and improve its stability. in aqueous solutions when stored at 35 ± 5 ◦ C for 45 days (Fabre et al.,
2022; Mishra et al., 2008). The apoprotein part of the phycocyanin can
4.1.1. Addition of polysaccharides be stabilized due to strong molecular interactions with the citrate anion,
The stability of food proteins can be enhanced by physically com­ as this ion disrupted the water structure in phycocyanin solutions (Resch
plexing or chemically conjugating them to polysaccharides or other et al., 2005). Excessive use of preservatives may have negative effects on
carbohydrates (Faieta et al., 2020). Protein glycation is a common human health and so they should only be used in relatively small
modification method used to optimize the properties of food proteins, amounts.
which involves forming covalent crosslinks between the carbonyl groups
of reducing sugars and amino groups of proteins. For example, glycation 4.1.4. Addition of crosslinkers
of phycocyanin has been reported to improve its functionality (Liu et al., Glutaraldehyde and formaldehyde are the most commonly used
2020; Zheng et al., 2020). crosslinkers for proteins because of their low price and strong reactivity
Anionic food polysaccharides have been used to protect the biolog­ (Hernandez-Munoz et al., 2005). Dithiobis (succinimidyl propionate)
ical activities and color of phycocyanin because they can stabilize the and formaldehyde have been used to prevent the deactivation of
protein structure and inhibit protein aggregation. The negatively charge phycocyanin by crosslinking the protein. The dithiobis (succinimidyl
groups on the polysaccharides form electrostatic links to the positively propionate) is convenient and easy to handle, with extreme adaptability
charged groups on the proteins, thereby improving the stability and and the broadest reaction specificity. Previous research has found that
functionality of the phycocyanin (Barbiroli et al., 2017). As an example, dithiobis (succinimidyl propionate) could modify the amino groups of
forming ι-carrageenan-phycocyanin complexes was shown to improve lysine and protect the high order structure of the phycocyanin. The
the colloidal and color stabilities of the protein (Zhang et al., 2021). absorbance at 614 nm of formaldehyde-crosslinked phycocyanin was
Moreover, the light stability of the phycocyanin and ι-carrageenan retained at 70% of the original value when the proteins were incubated
complexes at pH 7.0 was enhanced after a high-pressure treatment. in a urea solution (4 M) for 8 h (Fukui et al., 2004). Similarly,
Similarly, researchers reported that the blue color of phycocyanin phycocyanin-formaldehyde complexes were reported to maintain 100%
remained stable after heat treatment at 90 ◦ C at pH 2.5 and 3.0 after stability over 120 min when exposed to light (Munawaroh et al., 2020).
adding λ-carrageenan (Buecker, Grossmann, Loeffler, Leeb and Weiss, However, some of these crosslinking agents are toxic and unsuitable for
2022b). The same research team showed that carrageenan-phycocyanin food processing. Consequently, it is important to identify food-grade
complexes were more stable to color fading at pH 2.5 than 4.5 (Buecker, crosslinking agents that can be used to protect the color and activity
Grossmann, Loeffler, Leeb and Weiss, 2022a). Presumably, the addition of phycocyanin, such as some enzymes.
of ι-carrageenan altered the molecular structure of phycocyanin in a way
that made it more resistant to degradation. 4.1.5. Addition of edible oil
Also, sugars have been used as stabilizing agents for phycocyanin in Studies have shown that the stability of phycocyanin in some food
both high and low moisture environments. For instance, researchers products can be improved by incorporating oils. For instance, adding
reported that the half-life of phycocyanin increased from 19 min to sunflower oil or extra virgin olive oil (10 g oil/100 g bakery dough)
around 30–44 min at pH 7 after heating at 60 ◦ C for 15 min when protected phycocyanin from thermal degradation during cooking (Nic­
20–40% glucose or sucrose was added (Faieta et al., 2020). Interestingly, colai et al., 2021). These researchers reported that the tocopherol con­
the viscosity, water activity, and glass transition temperature of sugars tained in the oils was responsible for protecting the phycocyanin from
have been reported to influence the stability of phycocyanin. Besides degradation.
that, coating the polysaccharides on the surface of phycocyanin can
prevent the protein aggregation of phycocyanin. 4.2. Encapsulation strategy

4.1.2. Addition of proteins Encapsulation technologies can be used to protect sensitive bioactive
Numerous researchers have investigated the effects of proteins on the molecules by packaging them within particles with micrometer- or
stability of phycocyanin. For example, Zhang et al. reported that nanometer-scale dimensions (Tie et al., 2022; Wang et al., 2022; Yu
phycocyanin-whey protein complexes formed using high pressure (450 et al., 2022).
MPa) treatment can encapsulate the chromophore and decrease the
damage caused by oxidizing radicals (Zhang et al., 2021). Other re­ 4.2.1. Formation of micelles
searchers found that the whey protein blocked the aggregation of Encapsulation in sodium dodecyl sulfate (SDS) surfactant micelles
phycocyanin and stabilized it at pH 3.0 (Zhang et al., 2020). Also, it has has been shown to protect phycocyanin from degradation (Falkeborg
been reported that adding a low concentration (around 0.05–0.1%) of et al., 2018), which was mainly attributed to the ability of the micelles to
whey protein could delay the photo-degradation of phycocyanin at pH stabilize the blue circular helical regions of the phycocyanobilin via
3.0 (Zhang et al., 2021). This effect may be due to that whey proteins hydrophobic interactions. Moreover, the SDS-micelles coat the phyco­
protected the phycocyanin structure from being disrupted (Newsome cyanin and prevent it from aggregating. Other researchers reported that
et al., 2014). In general, these studies showed that certain kinds of anthocyanins could also be stabilized through their interactions with
proteins can prevent conformational changes, aggregation, and discol­ SDS (Liu et al., 2014), which in this case was attributed to electrostatic
oration of phycocyanin, which may be beneficial for some food attraction between cationic protein groups and anionic charged
applications. SDS-micelles. Even though SDS can protect phycocyanin in acidic en­
vironments, this anionic surfactant is unsuitable for food applications.
Therefore, it is still necessary to identify food-grade surfactants to

2334
B. Yuan et al. Current Research in Food Science 5 (2022) 2329–2337

prepare micelles for this purpose. Supervision. David Julian McClements: Conceptualization, Supervi­
sion. Bingquan Zhang: Conceptualization, Supervision. Mingqian Tan:
4.2.2. Formation of microencapsulation Supervision, Writing – review & editing, Funding acquisition. Zhixiang
Microencapsulation involves converting a fluid form of a material Wang: Supervision, Writing – review & editing, Funding acquisition.
into a powdered form, which can protect them (Li et al., 2019). There Chongjiang Cao: Supervision, Writing – review & editing, Funding
has been some research on the protection of phycocyanins using acquisition.
microencapsulation (Pan et al., 2019). Methods such as extrusion,
electro-spraying, spray drying, freeze drying, and fluid bed coating have
Declaration of competing interest
been used for this purpose (Yan et al., 2014).
Electro-spraying produces ultrafine particles by applying an electric
The authors declare that they have no known competing financial
potential to a polymer solution under ambient temperature and pres­
interests or personal relationships that could have appeared to influence
sure. When polyvinyl alcohol was used for the encapsulation of phyco­
the work reported in this paper.
cyanin, the diameter of the particles produced by electro-spraying was
395 ± 71 nm and the encapsulation efficiency was 75.1 ± 0.2%
Acknowledgment
(Schmatz et al., 2020). The encapsulated phycocyanin exhibited good
thermal resistance up to 216 ◦ C.
This work was supported by the National Key Research and Devel­
The extrusion method has been used to microencapsulate phycocy­
opment Program of China (2018YFD0901106), the National Natural
anin in a biopolymer matrix consisting of chitosan and alginate (Yan
Science Foundation of China (No. 31901699), Scientific Research Start-
et al., 2014). The diameters of the microcapsules produced using this
up Fund of Shaoxing University (No. 20195015), the Fundamental
method were about 1.03 mm. Encapsulation was shown to improve the
Research Funds for the Central Universities (No. 2632021ZD01), China
resistance of the phycocyanin to light exposure (40 days), heating
Postdoctoral Science Foundation (No. 2020T130138ZX), Jiangsu Plan­
(50 ◦ C), acids (simulated gastric fluids, pH 1.2) and relative humidity
ned Projects for Postdoctoral Research Funds.
(31% RH). This phenomenon was related to the dense structure of the
phycocyanin/alginate/chitosan microcapsules. In situ mineralization of
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