The interactions between anthocyanin and whey protein: A review

Abstract

Anthocyanins (ACN) are natural pigments that produce bright red, blue, and purple colors in plants and can be used to color food products. However, ACN sensitivity to different factors limits their applications in the food industry. Whey protein (WP), a functional nutritional additive, has been shown to interact with ACN and improve the color, stability, antioxidant capacity, bioavailability, and other functional properties of the ACN‐WP complex. The WP's secondary structure is expected to unfold due to heat treatment, which may increase its binding affinity with ACN. Different ACN structures will also have different binding affinity with WP and their interaction mechanism may also be different. Circular dichroism (CD) spectroscopy and Fourier transform infrared (FTIR) spectroscopy show that the WP secondary structure changes after binding with ACN. Fluorescence spectroscopy shows that the WP maximum fluorescence emission wavelength shifts, and the fluorescence intensity decreases after interaction with ACN. Moreover, thermodynamic analysis suggests that the ACN‐WP binding forces are mainly hydrophobic interactions, although there is also evidence of electrostatic interactions and hydrogen bonding between ACN and WP. In this review, we summarize the information available on ACN‐WP interactions under different conditions and discuss the impact of different ACN chemical structures and of WP conformation changes on the affinity between ACN and WP. This summary helps improve our understanding of WP protection of ACN against color degradation, thus providing new tools to improve ACN color stability and expanding the applications of ACN and WP in the food and pharmacy industries.

Full text

Received:13July2021 Revised: 30 August 2021 Accepted: 8 September 2021
DOI: 10.1111/1541-4337.12854
COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY
The interactions between anthocyanin and whey protein: A
review
Shuai Ren Rafael Jiménez-Flores Maria Monica Giusti
The Ohio State University, Department of
Food Science and Technology, Columbus,
Ohio, USA
Correspondence
Maria Monica Giusti, Department of Food
Science and Technology, The Ohio State
University,2015 Fyffe Road, Columbus,
OH 43210-1007, USA
Email: giusti.6@osu.edu
Funding information
USDA National Institute of Foodand
Agriculture, Grant/AwardNumber: Hatch
Project OHO01423
Abstract
Anthocyanins (ACN) are natural pigments that produce bright red, blue, and
purple colors in plants and can be used to color food products. However, ACN
sensitivity to different factors limits their applications in the food industry.
Whey protein (WP), a functional nutritional additive, has been shown to interact
with ACN and improve the color, stability, antioxidant capacity, bioavailability,
and other functional properties of the ACN-WP complex. The WP’s secondary
structure is expected to unfold due to heat treatment, which may increase its
binding affinity with ACN. Different ACN structures will also have different
binding affinity with WP and their interaction mechanism may also be differ-
ent. Circular dichroism (CD) spectroscopy and Fourier transform infrared (FTIR)
spectroscopy show that the WP secondary structure changes after binding with
ACN. Fluorescence spectroscopy shows that the WP maximum fluorescence
emission wavelength shifts, and the fluorescence intensity decreases after inter-
action with ACN. Moreover, thermodynamic analysis suggests that the ACN-WP
binding forces are mainly hydrophobic interactions, although there is also evi-
dence of electrostatic interactions and hydrogen bonding between ACN and WP.
In this review, we summarize the information available on ACN-WP interactions
under different conditions and discuss the impact of different ACN chemical
structures and of WP conformation changes on the affinity between ACN and
WP. This summary helps improve our understanding of WP protection of ACN
against color degradation, thus providing new tools to improve ACN color sta-
bility and expanding the applications of ACN and WP in the food and pharmacy
industries.
KEYWORDS
Anthocyanin-whey protein interaction, color stability, whey protein structure, fluorescence
technology, FTIR, preheating
1 INTRODUCTION
When consumers perceive the colors of a food product,
particularly foods they are familiar with, the brain relates
this visual information to the flavor and quality charac-
teristics of the product (Theerathammakorn et al., 2019).
Consumers often associate visual appearance with other
characteristics such as the flavor, maturity, and even
safety of a product. Therefore, food manufacturers apply
a variety of color additives to give food desirable colors to
Compr Rev Food Sci Food Saf. 2021;1–20. © 2021 Institute of Food Technologists R
1wileyonlinelibrary.com/journal/crf3

2WHEY PROTEIN - ANTHOCYANIN INTERACTIONS
attract consumers and satisfy their expectations (Coultate
& Blackburn, 2018). Increasing portions of the popula-
tion are seeking products containing naturally derived
pigments from fruit and vegetable sources because these
colorants are considered safe, natural, and part of a clean
label (de Mejia et al., 2020). In addition, many studies have
linked consumption of pigments from nature to potential
health benefits (He & Giusti, 2010; Lao et al., 2017). With
these potential benefits, there is increasing consumer
demand driving a push in the food industry to replace
synthetic colorants with colorants from natural sources.
Among natural colorants, anthocyanins (ACN) have
received increasing attention in recent years because of
their widely distributed sources, color expression, and
health benefits (Sigurdson et al., 2017; Wallace & Giusti,
2019). However, ACN are very susceptible to degradation
and color fading, which is a major issue for their appli-
cation in food industry (Li et al., 2020). Researchers have
explored various methods to improve ACN stability and
expand their applications as a healthy food colorant, such
as glycosyl acylation (Zhao et al., 2017); co-pigmentation
(Fan et al., 2019); drying procedures, such as spray drying,
freeze drying and encapsulation (Cortez et al., 2017;Tan
et al., 2018); and interaction with natural polymers such as
whey protein (Ren & Giusti, 2021a; Ren & Giusti, 2021b).
Whey proteins (WP) are widely used in food prod-
ucts because they have excellent functional and nutri-
tional properties (Kumar et al., 2018). Many studies report
the interaction between protein and phenolic compounds
(Ozdal et al., 2013). Compared with other phenolics, ACN
as a potential substitute of traditional synthetic food col-
orant attract more attention in recent years. Studies have
shown that addition of WP could improve the stability of
ACN (Corte et al., 2017;Lietal.,2020). Previous studies
demonstrated that WP could interact with ACN by forming
complexes through hydrogen bonding and hydrophobic
interactions (Zang et al., 2021;Heetal.,2018). WP are sen-
sitive to temperature because heat treatment can promote
their conformational modifications at the secondary and
tertiary structure levels, which will affect their hydropho-
bicity, functional properties, and the ability to bind other
molecules (Jiang et al., 2018). Thus, thermally modified
WP is expected to have different binding efficiency with
ACNthannativeprotein.
The objective of this paper is to review and summarize
the current knowledge of the interaction between ACN
and WP. This review describes the effect of WP on ACN
color stability, antioxidant capacity, andbioavailability; the
effect of ACN on WP structure and functional proper-
ties; the effect of WP preheating temperature on ACN-
WP interaction; and ACN-WP encapsulation technologies.
This information will help us optimize the binding condi-
tion of ACN-WP and will be helpful in understanding the
WP protection of ACN from color degradation during food
processing and storage, thus widening their applications in
the food industry.
2 ACN AS FOOD COLORANTS
2.1 Food colorants
Color plays an important role in human life. It affects every
aspect of our lives, from the clothes we wear to the food we
eat and the furnishings in our home (Yusuf et al., 2017).
Many compounds absorb parts of the light spectrum and
reflect other areas, which are perceived as colors by the
color receptors in the retina. We tend to judge the quality
attributes of food products, such as freshness, flavor, safety,
and nutritional value, from the color of food (Sigurdson
et al., 2017). Therefore, in order to provide more appeal-
ing, attractive, and pleasant products to consumers, food
colorants are widely used in food industry.
According to United States Food and Drug Administra-
tion (FDA), a food colorant is “any material, not exempted
under section 201(t) of the act, that is a dye, pigment, or
other substance made by a process of synthesis or sim-
ilar artifice, or extracted, isolated, or otherwise derived,
with or without intermediate or final change of identity,
from a vegetable, animal, mineral, or other source and that,
when added or applied to a food, drug, or cosmetic or to
the human body or any part thereof, is capable (alone or
through reaction with another substance) of imparting a
color thereto” (Code of Federal Regulations, 2016). Food
colorants may impart color to food, drinks, or non-food
applications, such as pharmaceuticals and cosmetics (de
Boer, 2013; Newsome et al., 2014).
Current FDA regulations permit two major classifi-
cations of food colorants: certified and exempt from
certification. Current permitted certified pigments are
chemically synthesized, and each batch of the colorant
produced must be evaluated for identity and sufficient
purity levels (Downham & Collins, 2000). Commonly
used synthetic colorants include FD&C Blue No. 1, Blue
No. 2, Green No. 3, Red No. 3, Red No. 40, Yellow
No. 5, Yellow No. 6, Orange B (sausage casings only),
and Citrus Red No. 2 (oranges only). Colorants exempt
from certification include a variety of compounds and
extracts derived from plant, animal, or mineral natural
sources, and fruit and vegetable juice concentrates. Col-
orants exempt from certification also include synthesized
nature-identical compounds, despite the perception that
colorants exempt from certification are all natural (Sigurd-
son et al., 2017). The most common examples of these col-
orants are carotenoids, ACN (for instance, grape skins),
flavonols, betalains, hemes, and chlorophylls.

WHEY PROTEIN - ANTHOCYANIN INTERACTIONS 3
FIGURE 1 Chemical structure of anthocyanins
Both synthetic dyes and color from natural are used to
compensate for color loss, enhance natural colors to make
the food more attractive and appetizing, provide color to
colorless foods, or allow consumers to identify products
on sight (Barrows et al., 2003). Food colorants should
never be used to hide defects and deceive consumers
(Code of Federal Regulations, 2016). Many studies showed
that synthetic colors have potential negative side effects,
such as allergies, hypersensitivity, behavioral problems in
children, attention-deficit/hyperactivity disorder (ADHD),
and others (Feketea & Tsabouri, 2017; Laura et al., 2019;
McCann et al., 2007; Vojdani & Vojdani, 2015). There-
fore, natural food colorants are increasingly demanded
by consumers to replace the synthetic ones (Masone &
Chanforan, 2015).
2.2 ACN
ACN are naturally water-soluble bioactive flavonoids that
are widely distributed in a variety of roots, fruits, leaves,
and flowers (Li et al., 2013). They are one of the most widely
studied natural food colorants and have been used in the
food industry to provide or improve color in food products
like jams, canned foods, dairy products, beverages, or con-
fectioneries, because of their bright and attractive and their
non-toxicity and water solubility (Montilla et al., 2011).
ACN belong to the flavonoid family, with two benzoyl
rings and a heterocyclic ring (Figure 1). They are derived
from anthocyanidins by adding sugars (Padayachee et al.,
2012). ACN structures differ in the number and position
of hydroxyl and methoxylation groups, the type and posi-
tion of sugars attached to the flavylium ring, and the num-
ber and type of aliphatic or aromatic acids connected to
the sugar groups (McGhie & Walton, 2007). According to
different substituent patterns on the different positions on
the B rings, there are six major ACN aglycones identified
in nature: pelargonidin (Pg), petunidin (Pt), peonidin (Pn),
malvidin (Mv), cyanidin (Cy), and delphinidin (Dp) (Sig-
urdson et al., 2017). The different colors of ACN, such as
blue, purple, red, and orange, are affected by the number
of hydroxyl groups and methoxyl groups (Wahyuningsih
et al., 2017). Most commonly used commercial ACN-based
colorants extracts include blackcurrants, grape skin, black
carrot, purple corn, red cabbage, and red radish (Ekici
et al., 2014).

4WHEY PROTEIN - ANTHOCYANIN INTERACTIONS
In addition to use as colorants in foods, ACN also may
impart some healthy and functional benefits to people,
such as antioxidant activities (Diaconeasa et al., 2015;
Neves et al., 2019), anti-inflammatory (Zhang et al., 2019);
anticarcinogenic properties (Wang et al., 2019; Chabir
et al., 2015), cardiovascular protection (Kimble et al., 2019;
Luís et al., 2018), antidiabetic effects (Chen et al., 2018;
Xiao & Hogger, 2015), and anti-obesity (Benn et al., 2014;
Tucakovic et al., 2018). Therefore, due to consumers’
demand for safety and health-conscious foods, the natu-
ral ACN are widely used in food and beverage products
to substitute the synthetic pigments in the food industry
(Philpott et al., 2004;Talavéraetal.,2006).
Due to ACN’s color sensitivity to pH and potential
antioxidant and antimicrobial properties, ACN are used to
develop biodegradable and edible packaging films, which
can be used to maintain the quality and extend the shelf
life of food products and also monitor the food quality
by changing color (Yong & Liu, 2020). Rawdkuen et al.
reported that ACN extracted from butterfly pea had the
highest pH sensitivity and could be used in intelligent
gelatin films to improve its antioxidant activity and act as
pH indicator (Rawdkuen et al., 2020). Fu et al. showed that
red cabbage ACN could become an excellent UV-barrier in
oxidized-chitin nanocrystals/konjac glucomannan intelli-
gent film. ACN bond with oxidized-chitin nanocrystals by
electrostatic interaction and presented good antioxidant,
antibacterial, and color change properties (Wu et al., 2020).
Clitoria ternatea ACN, heat-treated soy protein isolate, and
gellan gum could interact through electrostatic forces and
covalent bonds to generate active film. The film could con-
trol Clitoria ternatea ACN release at pH greater than 6.0
and could be potentially used to monitor seafood freshness
(Wu et al., 2021). Moghadam et al. reported that addition
of Echium amoenum ACN extract increased the thickness,
water solubility and vapor permeability, tensile strength,
and elongation of the mung bean protein film and also
inhibited E. coli and S. aureus growth (Moghadam et al.,
2021).
Despite ACN’s significant biological effects, ACN have
limited chemical stability due to its sensitivity to environ-
mental factors, such as oxygen, concentration, tempera-
ture, light, enzymes, pH, co-pigments, and food matrix
composition, such as carbohydrates, proteins, ascorbic
acids, salts, sugars, and minerals (Cao et al., 2009;He&
Giusti, 2010; Sigurdson et al., 2017; Jakobek, 2015). When
the pH is lower than 3, the flavylium cations become depro-
tonated and the ACN usually show red color. When pH
increases from 3 to 6, ACN start to lose color. When pH
is higher than 6, ACN eventually form quinoidal bases and
appear purple and blue (Sigurdson et al., 2017). The high
instability limits their commercial application as colorants
in processed foods. Therefore, it is meaningful and chal-
lenging to find an effective way to reduce the ACN loss
during food processing and storage. Several studies have
shown that the stability of ACN can be improved by bind-
ing with natural polymers to form complexes (Li et al.,
2020;Zangetal.,2021). These results may help provide
a potential method to protect ACN and thereby help to
extend their stability in food and beverage products.
3WHEY PROTEIN AS FOOD
ADDITIVE
Whey proteins (WP) are used widely as food ingredi-
ents (any substance that is added to a food to achieve
a desired effect, like to maintain or improve food safety
and freshness, nutritional value, taste, texture, and appear-
ance) or food additives (any substance was directly or indi-
rectly added to foods for specific technical and/or func-
tional purposes) currently, but traditionally were not paid
much attention, perhaps because they are by-product of
cheesemaking and were thought invaluable in the past
(Madureira et al., 2007). People began to notice the health
properties of WP in Ancient Greece; WP was used in salves
and potions to cure various illnesses in the Middle Age;
and nowadays, WP is recognized as part of a healthy diet
(Solak & Akin, 2012). WP, which makes up about 20%
of the total milk protein, contain a mixture of globular
proteins, such as β-lactoglobulin (β-LG), α-lactalbumin
(α-LA), immunoglobulins (IGs), bovine serum albumin
(BSA), lactoferrin (LF), and lactoperoxidase (LP) (Malcata
et al., 2016).
Among the various WP proportions, β-lactoglobulin (β-
LG) is widely used as a food additive due to its nutritional
and functional properties (Paul et al., 2014). Native β-LG is
the most abundant protein in WP (nearly 50–55%) and is
a highly structured globular protein with 162 amino acid
residues and a molecular weight of 18,400 Da (Rodzik et al.,
2020). β-LG exists as a dimer at neutral pH and folds into
a calyx form by antiparallel β-sheets (Havea et al., 2004).
It possesses a hydrophobic pocket located in the interior
of the calyx structure that serves as the primary binding
site for hydrophobic ligands, such as fatty acids, retinol,
β-carotene, phospholipids, vitamin D, folic acid, and
phenolic compounds (He et al., 2015; Liang & Subirade,
2012; Perez et al., 2014; Zorilla et al., 2011). Due to WP’s
special hydrophobic pocket, it attracts a lot of scholars to
study the interaction between WP, especially β-LG, and
ACN.
WP are widely used as functional ingredients in the food
industry due to their desirable functional properties, such
as foaming, emulsifying, and gelling (Hoffman & Falvo,
2004). WP contribute to foam formation by diffusing and
concentrating on the air-liquid interface, which leads to

WHEY PROTEIN - ANTHOCYANIN INTERACTIONS 5
the reduction of the surface tension. The WP then unfold
at the interface with hydrophilic and hydrophobic groups
toward the liquid and air phase, respectively, and form
highly viscoelastic interfacial film that stabilizes the air
bubbles (Xiong et al., 2020). Similar to foaming, the role
of WP in emulsification is to provide an interfacial mem-
brane around the lipid globule to prevent creaming, coa-
lescence, flocculation, and oiling off (Kumar et al., 2018).
The gelation of WP starts with the initial unfolding of a
WP molecule followed by subsequent aggregation either
by addition of salt or decreasing the pH (Kharlamova et al.,
2018). In addition to these functional properties, WP is also
reported to have benefits in clinical practice, such as posi-
tive effects on immunomodulation, antibacterial, antiviral,
and antifungal activity among others (Vasconcelos et al.,
2018).
WP are considered one of the most valuable ingredi-
ents and contribute to a great extent in the development
of new food products. WP can be easily obtained as a
milk by-product in the cheese-making process and have
promising applications related to potential health benefits,
ranging from antibacterial effects to cognitive develop-
ment for babies to human gut health (Rocha-Mendoza
et al., 2020). They are highly bioavailable, because they
can be absorbed into the body very quickly, and they
have a high concentration of branched-chain amino acids
(BCAAs), which are highly concentrated in muscle tis-
sue (Da Silva et al., 2017; Hoffman & Falvo, 2004). The
WP bioavailability is the degree of how usable it is in the
body. High bioavailability means it is easy to digest, absorb,
and make into other proteins, so high bioavailability in
the nutritional literature directly related to absorption rate
(Sicart, 2021). For example, workout increases the tension
of our muscles and cause micro-tears in the tissues. We
take WP, which can be absorbed by our body very quickly,
and it triggers protein synthesis as soon as possible to help
speed up recovery times. Therefore, WP is widely used in
baby foods and to fuel working muscles and stimulate pro-
tein synthesis (Kimball & Jefferson, 2006). WP could also
replace other animal proteins to stabilize the meat prod-
ucts. A study showed that meatless patties containing tex-
tured WP with mushroom and vegetable flavor had similar
acceptability as commercial soya patty (Kumar et al., 2018).
Concentrations of WP exceeding 50% can create products
with functional properties that are helpful as a fat replacer
in low fat ice cream (Akbari et al., 2019). The addition of
WP to bread or bakery products not only improved the out-
put and nutritional value of the products, but also changed
the products structure to be softer and finer and increased
the fermented milk flavor (Shishkina et al., 2019). WP is
also added to fruit jams, jellies, carbonated beverages, and
fruit juices because they are soluble in low pH (Kumar
et al., 2018).
Heating is one of the most common processing to
cause the structure change of protein. During heating,
WP can undergo heat-induced denaturation, aggregation,
andgelation(Pateletal.,2015). β-LG was predominantly
present as a dimer at 25◦C in neutral pH. Heating the WP
solution at about 40◦C led to a shift of β-LG toward the
monomeric form and exposure of the free thiol group and
hydrophobic patch. Increasing the heating temperature
further led to reversible unfolding of the β-LG monomer.
Irreversible denaturation and aggregation occurred when
the heating temperature was higher than 65◦C. Then, a
thiol-disulfide exchange reaction occurred, and protein
aggregates were formed as a result (Quevedo et al., 2021).
Previous studies suggest that the exposure of WP func-
tional groups at different levels will affect the binding affin-
ity of WP with ACN (He et al., 2018). More disordered pro-
teins have stronger interactions with phenolic compounds
than globular proteins (Deaville et al., 2007). Therefore,
preheated WP might provide stronger protection for ACN
color and stability. Besides, other factors such as pH, high
pressure, ethanol, and ultrasonication, can also cause pro-
tein denaturation. The pH affected the monomer-dimer
equilibrium of β-LG and also influenced the thiol-disulfide
exchange reaction (Quevedo et al., 2021). At pH 3 and
4, WP had higher thermal stability because of increased
hydrogen bonding and decreased electrostatic interactions
(Tolkach & Kulozik, 2005). High pressure could improve
the interchange reaction between the disulfide bond and
sulfhydryl group to cause WP aggregation and denatu-
ration (Bogahawaththa et al., 2018). And WP’s surface
hydrophobicity, solubility, foaming capacity, and emulsifi-
cation stability was also increased after the high-pressure
processing (Wang et al., 2020). An ethanol concentration
of 50% caused the maximum initial extent of WP denat-
uration and WP and this denaturation is significantly
irreversible after the ethanol was removed (Nikolaidis &
Moschakis, 2018). Ultrasonic homogenization could dena-
ture WP to the maximum degree of 40%, which depending
on ultrasonic power level, treatment temperature, and the
interaction between those factors (Gregersen et al., 2019).
4 WHEY PROTEIN–ANTHOCYANIN
INTERACTION
The interactions between phenolic compounds, such as
ACN, and proteins, such as WP, are studied by many
scholars. The high abundance of hydroxyl groups on the
phenolic compounds offers a high number of sites for inter-
action with proteins (Buitimea-Cantúa et al., 2018). The
formation of their complex led to the structural, func-
tional, and nutritional changes of both protein and phe-
nolic compounds (Zhang et al., 2020; Ozdal et al., 2013).

6WHEY PROTEIN - ANTHOCYANIN INTERACTIONS
With increasing demand on healthy and natural food col-
orants, ACN get more attentions by scholars. However,
the instability of ACN is a big challenge for their applica-
tions in food industry. Studies have shown the interaction
between WP and ACN could improve ACN color, stabil-
ity, and antioxidant capacity. However, the interactions of
ACN-WP are complexed, depending on the structures of
both WP and ACN. The information about ACN-WP inter-
action is very limited and therefore their interaction was
highly desirable in order to widen ACN’s application as
natural food colorants and whey protein’s application as
food additives. Therefore, the effects of ACN-WP interac-
tion on the ACN and WP physiochemical and functional
properties are summarized in this review.
Among the large number of phenolic compounds, ACN
are found in very common components of the human diet,
such as fruits and vegetables, especially in berries and
in red wine. ACN provide various colors in flowers and
fruits and have physiological functions in vegetative tis-
sues (Martín et al., 2017). The color and functional proper-
ties of ACN are, to a large extent, closely related with their
chemical structure. Therefore, ACN have been the subject
of intensive research for recent years.
WP has similar functional properties as other proteins,
but there are several potential benefits to using this par-
ticular protein. As secondary products of cheese manufac-
ture, their disposal as waste raises environmental and food
sustainability concerns (Ganju & Gogate, 2017). Repurpos-
ing this by-product would be a great benefit from an envi-
ronmental perspective. For the nutrition perspective, WP
can be better digested and absorbed in the human gastroin-
testinal tract (Ni et al., 2020). Studies showed WP could
be used as wall materials to deliver ACN into human body
and improve ACN bioavailability (Sharif et al., 2020). ACN-
loaded WP microgels can dissolve quickly in gastrointesti-
nal tract, and the formed liquid microparticles hinder the
release and degradation of ACN (Liao et al., 2021).
4.1 The effect of whey
protein–anthocyanin interaction on
anthocyanin color and stability
The interaction between WP and ACN is reported to have
effects on ACN color and stability. The WP contents sig-
nificantly (p<.05) increased the lightness L*value of
the jabuticaba ACN beverages and protected ACN from
degradation during 60 days storage at pH 4 in 5 ±2◦C
(Rocha, Viana et al., 2019). Chung and others found that
the addition of WP could prolong the color stability of
ACN in model beverages under accelerated storage con-
ditions (40◦C for 7 days) because the hydrogen bond-
ing between ACN and WP were stronger than the bind-
ing force between ACN and ascorbic acid (Chung et al.,
2015). Ren and Giusti also reported that the WP addi-
tion increased the absorbance of grape, purple corn, and
black carrot ACN and protected ACN from ascorbic acid-
mediated degradation. They also showed that ACN’ half-
life was improved by addition of WP in a dose depen-
dent manner (Ren & Giusti, 2021b). Miyagusuku-Cruzado
et al. studied the interaction of WP with different ACN
(Berberis boliviana, grape skin, purple corn, black carrot,
and red cabbage) in pH 3 model solutions. They found that
WP addition resulted in significant (p<.01) absorption
increases for all ACN,which was concentration dependent
(Miyagusuku-Cruzado et al., 2020). They also found that
WP addition resulted in significant decrease in ACN light-
ness (p<.01) and chroma (p<.01); and increase in hue
angle (p<.01), which means ACN solutions had signifi-
cantly higher light absorption and noticeably darker colors
after the addition of WP.
WP was reported to improve ACN heat stability and light
stability. The addition of WP (50 and 200 mg/L) reduced
the degradation of purple-fleshed sweet potato ACN after
heat treatment at 100◦C for 30 min and higher WP con-
centration resulted in lower ACN degradation rate (Quan
et al., 2020). Jing and Giusti reported that the milk pro-
teins can protect purple corn ACN from heat degradation
when exposed to heat at 70◦C for 120 min (Jing & Giusti,
2005). WP effectively prevented the color fading and degra-
dation of ACN in the grape skin ACN extract solution at pH
3.2 and 6.3 during the thermal treatment at 80°C for 2 h,
0.005% H2O2oxidation for 1 h, and photo illumination at
the intensity of 5000 lx for 5 days (He et al., 2016).
Antioxidant capacity is a very widely known property
of ACN. WP was found to enhance the blueberry ACN
light stability (illumination intensity 5000 x at 298 K
for 7 days) and antioxidant activity (vitamin C at 0.15
mg⋅ml−1or sucrose at 0.25 mg⋅ml−1at 25◦C in the dark
for 7 days) (Zang et al., 2021). Meng et al. found that
cyanindin-3-glucoside (C3G)-WP complexes had lower
DPPH radical scavenging activity, ABTS radical scaveng-
ing activity, reducing power, and the ability to chelat-
ing Fe2+than C3G, indicating an increased antioxidant
activity in the complexes (Meng et al., 2021). Studies
also showed that the addition of WP–dextran conjugates
significantly (p<.05) prevented C3G from degradation,
and increased the absorbance and ABTS [2,2’-azino-bis(3-
ethylbenzothiazoline-6-sulfonic acid)] radical scavenging
activity of heated bog bilberry ACN extract (Zheng et al.,
2009). Moreover, not only WP but also the Maillard-reacted
WP improved ACN color stability and antioxidant capac-
ity under heat treatment at 80◦Cfor120minatpH6(Qin
et al., 2018). It was assumed that phenolic compounds
inhibited oxidation both by binding to the proteins in
order to retard the oxidation reactions and by forming

WHEY PROTEIN - ANTHOCYANIN INTERACTIONS 7
complexes with protein molecules (Shahidi & Ambi-
gaipalan, 2015; Riedl & Hagerman, 2001). Moreover, a
study showed that the ACN-WP interaction protected the
pelargonidin-3-O-glucoside from gastrointestinal degra-
dation by promoting progressive release from the com-
plex, and the α-glucosidase enzyme inhibitory activity,
in vitro antioxidant activity, and cellular reactive oxygen
species scavenging activity of pelargonidin-3-O-glucoside
were potentially augmented after simulated gastrointesti-
nal digestion (Gowd et al., 2020). These results might be
helpful for addition of WP into ACN-rich foods and bever-
ages to improve ACN chemical stability and prolong ACN
bioactivity.
4.2 The effect of whey
protein–anthocyanin interaction on whey
protein structure
The protein structure is modified when polyphenols non-
covalently bind with protein hydrophobic groups. This
modification in protein structure subsequently led to varia-
tions in the folding of proteins (Jakobek, 2015). Thus, inter-
action between WP and ACN will change the secondary
structure of protein and further affect its physicochemical
properties. FTIR spectroscopy is commonly used to study
the secondary structure composition, structural dynamics,
conformational changes, structural stability, and aggrega-
tion of proteins (Kong & Yu, 2007). Circular dichroism
(CD) spectroscopy is also used to quantitatively analyze the
variations of the secondary structure of WP in the absence
andpresenceofACN(Gongetal.,2021). Fluorescence
quenching technology is another popular method to ana-
lyze WP structure change due to their intrinsic fluores-
cence sensitivity to structure change (Li et al., 2020).
4.3 Fourier transform infrared
spectroscopy and circular dichroism
Many studies combined FTIR and CD methods to ana-
lyze WP secondary structure change after binding with
ACN. The detailed WP structure change with different
ACN sources is summarized in Table 1.Zangetal.con-
firmed that the structure of WP changed, and the microen-
vironments of certain amino acid residues were modulated
by non-covalent binding to malvidin-3-glucoside (M3G).
There were fewer α-helix and more β-sheets formed after
WP binding with M3G (Zang et al., 2021). Gong et al.
showed that the conformational structure of WP was
altered after binding with purple potato flour ACN, where
a decrease in α-helix and β-turn contents and an increase
in β-sheet and irregular coil contents was observed (Gong
et al., 2021). Similar results were also reported by Cheng
et al., the binding between β-LG with C3G caused the alter-
ations of the secondary structures of β-LG with a decrease
in α-helix and an increase in β-sheet (Cheng et al., 2017).
However, some studies had slightly opposite results for WP
structure change. He et al. found that after the combina-
tion of native and preheated β-LG with C3G, the α-helix
and β-sheet structures in the WP were reduced and the ran-
dom coil and turn structures increased, which resulted in
more unfolded protein structures (He et al., 2018). Khal-
ifa et al. also found that the interaction between ACN
from mulberry fruits (Morus australis Poir) with WP led
toaslightriseinbothα-helix and β-turn contents, and a
decline of β-Sheet and random coil contents (Khalifa et al.,
2018). Xu et al. found that after binding with delphinidin-
3-O-glucoside, the α-helix of β-LG increased, and β-turn, β-
sheet, and random coil contents all decreased. This result
identified that some β-sheet converted to α-helix and made
the β-LG firmer (Xu et al., 2019). Those opposite results
might be due to the different ACN sources, pH, WP con-
centration, ACN-WP binding time, and buffer system.
4.4 Fluorescence quenching
spectroscopy
Tryptophan residues within a protein exhibit intrin-
sic fluorescence and are usually used to investigate
polyphenol–protein interactions and binding (Xiao et al.,
2007). The structure change of WP can be inferred from
the fluorescence change in emission peaks (Masters,
2008). Table 2shows the fluorescence spectroscopy study
for different ACNs sources with WP. The binding mecha-
nisms of ACN-WP interaction were governed by pH, other
non-protein constituents (like high lactose content), and
the ionic strength in the systems (Vuong & Hongsprabhas,
2021). A fluorometric result demonstrated that a static and
heat-stable binding between WP and ACN occurred, lead-
ing to modification in size, hydrophobicity, and secondary
structures of WP (Khalifa et al., 2018). They found that the
fluorescence intensity of the WP decreased from 2800 a.u.
to 2200 a.u. after adding mulberry ACN, indicating the
change of the WP fluorescent chromophore microenviron-
ment. The small change in chromophores further changed
the WP secondary structure and promoted ACN-WP bind-
ing (Khalifa et al., 2018). The addition of C3G quenched
the intrinsic fluorescence of the WP at 25◦C, pH 3.0 and
6.0, and 280 nm excitation in a concentration-dependent
manner, suggesting that the number of fluorophores of WP
decreased after binding with ACN (Qin et al., 2018). Salah
et al. showed that red raspberry pomace ACN extracts
quenched the fluorescence intensity of de-solvated β-LG
up to 98%, and the binding constant for ACN-WP was

8WHEY PROTEIN - ANTHOCYANIN INTERACTIONS
TABLE 1 Effects of anthocyanins on whey protein structure under acidic environments
Circular dichroism Fourier transform infrared spectroscopy
%α-helix, %
β-sheet, % β-turn, %
random coil
λmax shift (amide
I/amide II)
(wavenumber cm–1)
Anthocyanin sources
ACN-WP
dosage WP ACN-WP
ACN-WP
dosage WP ACN-WP Reference
Malvidin-3-
glucoside(blueberry)
25 µM
ACN10 µM
WP
40.4 36.5 50 µM (1:1) 1647.83 1650.68 Zang et al., 2021
29.0 29.4
15.2 16.6 1535.22 1539.50
15.6 17.7
Purple potato 30 µM
ACN10 µM
WP
20.8 13.4 30 µM
ACN10 µM
WP
1637.27 1643.05 Gong et al., 2021
21.5 24.1
17.8 16.7 1517.70 1538.92
40.1 46
Delphinidin-3-
glucoside
60 µM
ACN10 µM
WP
19.2 20.2 60 µM
ACN10 µM
WP
– Xuetal.,2019
34.7 33.0
19.7 19.5
45.2 43.7
Cyanidin-3-glucoside 2 µMACN2µM
WP
18.1 17.8 – – Cheng et al., 2017
28.9 30.3
9.7 10.0
43.1 44.2
Cyanidin-3-glucoside – 22.8 21.4 400 µM
ACN100 WP
1650.27 1642.57 He et al., 2018
26.6 25.0
17.2 18.0 1534.73 1532.68
24.3 25.8
Chinese mulberry
fruits (Morus
australis Poir)
120 µM
ACN0.02%
WP
18.0 21.0 120 µM
ACN0.02%
WP
– Khalifa et al.,
2018
45.0 39.0
12.0 15.0
27.1 27.0
about 7.59 ×108M−1at 25◦C (Salah et al., 2020). In
addition to the change of fluorescence intensity, many
studies also observed the change in the maximum emis-
sion wavelength of WP after binding with ACN. Ren
and Giusti found that the fluorescence of WP could be
quenched by ACN up to 73%. Different ACN sources
had different maximum emission wavelength change,
with 6, 4, and 3 nm bathochromic shift for purple corn,
grape skin, and black carrot ACN extracts, respectively.
These differences could be explained by the different ACN
structures (Ren & Giusti, 2021a). Zang et al. found that
the intensity of the fluorescence signal of WP (10 µM)
clearly decreased with increasing M3G concentration
(0–50 µM) and the WP maximum fluorescence emis-
sion wavelength shifted from 328 to 331 nm. These
results were ascribable to the fluorescence quenching
of WP by M3G, which resulted in increased polarity of
the microenvironment near the Tyrosine residues in
WP (Zang et al., 2021). Similar results found that the
maximum fluorescence emission wavelength of WP
shifted from 330 to 338 nm after the addition of purple
potato ACN, which indicated that the microenvironment
around the Tryptophan and Tyrosine residues in WP
changed in polarity and became more hydrophilic due to
the interactions between WP and ACN (Gong et al., 2021).
Salah and Xu found that the fluorescence intensity spectra
of β-LG showed a bathochromic shift from 336 to 355 nm
after binding with red raspberry ACN and the quenching
efficiency was up to 97% for the ACN at the concentration
of 8 ×10−4M(Salah&Xu,2021). Arroyo-Maya et al. also

WHEY PROTEIN - ANTHOCYANIN INTERACTIONS 9
TABLE 2 Review on the fluorescence spectroscopy study and the binding forces of whey protein and anthocyanins interaction at acidic
pH
Fluorescence
intensity (a.u.)
Fluorescence
λmax shift (nm)
ACN sources WP dosage WP
ACN-
WP WP
ACN-
WP Binding force Reference
Blueberry 20 µM 4000 2000 328 331 Hydrophobic, H-bond Zang et al., 2021
Grape skin 3.6 µM630 200 372 376 Hydrophobic Ren & Giusti, 2021a
Purple corn 3.6 µM 650 190 372 378 Hydrophobic Ren & Giusti, 2021a
Black carrot 3.6 µM700 230 372 375 Hydrophobic Ren & Giusti, 2021a
Cyanidin-3-O-glucoside 20 µM 2250 250 Bathochromic
shift
Hydrophobic Meng et al., 2021
Red raspberry pomace extract 1.18 µM4400 100 336 355 Electrostatic forces Salah & Xu, 2021
Purple potato 10 µM 600 270 330 338 VdW, H-bond Gong et al., 2021
Delyphinidin-3-O-glucoside 10 µM1100 380 No shift Hydrophobic Xu et al., 2019
Cyanidin-3-O-glucoside 10 µM 1050 150 No shift H-bond, hydrophobic He et al., 2018
F. neagră grape skin 0.15 mg/ml 750 300 330 351 Electrostatic interactions Stănciuc et al., 2017
Chinese mulberry fruits 0.02%, w/v 2800 2200 – H-bond Khalifa et al., 2018
Cyanidin-3-O-glucoside 0.2 mg/ml 400 200 Hypsochromic
shift
Hydrophobic, H-bond Qin et al., 2018
Cyanidin-3-O-glucoside 2 µM 1000 460 338 334 Hydrophobic Cheng et al., 2017
Pelargonidin 1mg/ml 2800 1600 338 340 Hydrophobic Arroyo-Maya et al., 2016
Sour cherries skin 0.04 mg/ml 700 200 335 410 Hydrophobic Oancea et al., 2017
Purple carrot 54 µM5600 1900 –H-bond Chung et al., 2015
Abbreviations: H-bond, hydrogen bond; VdW, Van der Waal force.
found that there was a small bathochromic shift from 338
to 400 nm when WP bonded with pelargonidin (Arroyo-
Maya et al., 2016). However, some studies reported the
opposite direction of the maximum fluorescence emission
wavelength shift. Cheng et al. found that the maximum
fluorescence emission wavelength of β-LG exhibited a
hypsochromic shift from 338 to 334 nm with the grad-
ual addition of C3G, which clearly indicated that the
interaction between C3G and β-LG led to the increased
hydrophobicity of the microenvironment around the Tryp-
tophan residues (Cheng et al., 2017). Meng et al. detected a
huge fluorescence intensity decrease for WP (from about
2500 to 200 a.u.) after the addition of C3G at 298, 308,
and318K,butwithonlyaslighthypsochromicshiftat
the spectral maximum wavelength (Meng et al., 2021).
Xu et al. found that the interaction between delphinidin-
3-O-glucoside and β-LG was static quenching and the
intrinsic fluorescence intensity value of β-LG decreased
64.6% after addition of 60 µM delphinidin-3-O-glucoside,
but the maximum fluorescence emission wavelength
was not altered (Xu et al., 2019). These results may con-
tribute to a better understanding on the ACN-WP binding
mechanism.
4.5 Morphology studies on
anthocyanin–whey protein interactions
Morphology study is also a good way to study the size,
shape, and structure of compounds. The interaction of
ACN and WP increased the size of ACN molecules. A con-
focal laser scanning microscopy (CLSM) images showed
that WP nanoparticles distributed in a fine and homoge-
neous network and the grape berry ACN forms numer-
ous clusters, with the largest dimension at 31.36–50.64
µm. After the ACN was mixed with WP, the ACN gen-
erated into larger and more compact clusters with the
increased dimension size at 37.82–58.91 µm (Stănciuc et al.,
2017). The CLSM images from another study identified
that ACN from eggplant peel extracts aggregated inside the
WP matrix and formed small red particles with a diame-
ter of 1–2 µm (Condurache et al., 2019). The scanning elec-
tron microscopy (SEM) images revealed that spray-dried
WP-ACN powders were spherical shapes with a rough and
slightly shrunken surface (Liao et al., 2021). Their CLSM
images proved that blueberry ACN were distributed evenly
in the interiors of WP microparticles, which indicated that
ACN had been successfully encapsulated in WP powders.

10 WHEY PROTEIN - ANTHOCYANIN INTERACTIONS
Their CLSM also showed the dynamic release of ACN
during digestion. ACN were protected by the liquid WP
microparticles with a core-shell structure. After 30 min in
gastric digestion, the liquid WP microparticles started to
crack, and some ACN diffused from the interior to the sur-
face (Liao et al., 2021). SEM micrographs showed differ-
ent microstructures for spray-dried and freeze-dried WP-
ACN complex (Zhang et al., 2020). Spray-dried samples
had smooth spherical shape with some surfaces exhibit-
ing wrinkles and dimples, which might be due to the rapid
water loss during the spray-drying process (Wilkowska
et al., 2017). However, freeze-dried samples showed a wrin-
kled spongy shape and their surfaces distributed with
multi-cavities, which could be explained by the rapid sub-
limation of frozen water from the alginate matrix (Smrdel
et al., 2008).
4.6 Anthocyanin–whey protein binding
sites
According to the studies, the ACN-WP binding sites can be
calculated using the equation:
log [(𝐹0−𝐹
)∕𝐹]= log 𝐾s+𝑛 log[𝑄](1)
where F0and Frepresent the fluorescence intensities of
proteins alone and in the presence of a given concentra-
tion of ACN (quencher), respectively; Qis the concentra-
tion of free ACN; Ksis the binding constant; and nrep-
resents the binding site. Based on this equation, Oancea
et al. found that the binding site value was lower than 1
for β-LG at each preheating temperature, which suggests a
weak binding between sour cherry (Prunus cerasus L) skin
ACN and β-LG. This weak binding might be explained by
other compounds presented in the ACN extracts that might
compete with ACN at the protein binding sites (Oancea
et al., 2017). Chung et al. reported slightly small nvalues
around 0.5 to 0.7, which also suggested a weak interaction
between the purple carrot extract ACN and WP (Chung
et al., 2015). Similar results also were described by Stănciuc
et al., who reported that the nvalues were around 0.3–0.75
for grape extract ACN with different preheated WP and the
n increased as the temperature increased (Stănciuc et al.,
2017). Salah and Xu found the nvalue for red raspberry
ACN and β-LG interaction was approximately 0.9 (Salah
&Xu,2021). Cheng et al. showed that the nvalue for C3G
and β-LG binding was approximately 1 (Cheng et al., 2017).
He et al. got a slightly higher nvalue for C3G and β-LG
interaction at approximately 1.2 (He et al., 2018). Xu et al.
also reported a nvalue about 1.2–1.5 for delphinidin-3-O-
glucoside and β-LG (Xu et al., 2019). Meng et al. showed
the binding sites between C3G and WP were about 1.4–
1.5 and it increased with increasing measurement temper-
ature (Meng et al., 2021). Increasing pH from acid to alkali
pH significantly decreased the numbers of binding sites
of WPI because of the alteration of protein conformation
and surface charges and the ACN ionized forms (Vuong &
Hongsprabhas, 2021). However, some studies did not agree
with the equation for the n calculation (Lissi & Abuin, 2011;
Grossweiner, 2000). They believed the equation should
be:
log [(𝐹0−𝐹
)∕𝐹]= log 𝐾s+𝑚 log[𝑄](2)
where mrepresents the kinetic reaction order (molecu-
larity in Q). mmeasures the number of Qmolecules that
interact simultaneously with each site and does not express
the number of independent and equivalent binding sites
(Chipman et al., 1967). Therefore, according to this restric-
tive definition of m, the value of m is always very close to
one.
4.7 Whey protein–anthocyanin binding
force
There are mainly four types of non-covalent binding forces
involved in the interaction between biomacromolecules
and small molecules: hydrophobic interaction, hydrogen
bonding, electrostatic interaction, and Van der Waals
force (Tang et al., 2014). Thermodynamic analysis was
used to determine binding force between ACN and WP.
Table 2summarizes the binding forces for different ACN
sources with native WP at acidic pH and Figure 2showed
the hydrophobic interaction mechanism of cyanidin-3-
glusocide and β-LG (Docking experiments were conducted
using mCule docking software https://mcule.com/apps/
1-click-docking/). The positive values of enthalpy ΔH and
entropy ΔS revealed that hydrophobic interactions dom-
inated between M3G and WP, and the negative Gibbs
free energy ΔG revealed that this interaction was spon-
taneous (Zang et al., 2021). Ren and Giusti explored the
interaction between WP and purple corn, grape skin,
and black carrot ACN extracts. They found that all of
the three ACN extracts bond WP through hydrophobic
forces (Ren & Giusti, 2021a). Xu et al. studied the inter-
action between delphinidin-3-O-glucoside and β-LG and
their interaction was mainly hydrophobic forces (Xu et al.,
2019). He et al. found both native and preheated β-LG
bound C3G mainly via hydrophobic forces and hydrogen
bonds (He et al., 2018). Chevalier et al. found that the
blueberry puree including ACN could interact with WP
through hydrogen bonding and hydrophobic interactions
(Chevalier et al., 2019). Arroyo-Maya indicated that the
bindingofpelargonidintoβ-LG was spontaneous and
hydrophobic interactions played an important role in this

WHEY PROTEIN - ANTHOCYANIN INTERACTIONS 11
FIGURE 2 In silico molecular docking of β-Lactoglobulin with cyanindin-3-glucoside
process (Arroyo-Maya, 2016). Gong et al. found that purple
potato ACN bound WP through hydrogen bonds and van
der Waals forces (Gong et al., 2021). Chung et al. found a
relatively high negative value of the enthalpy change, sug-
gesting that the interaction between highly denatured WPI
and ACN was mainly caused by hydrogen bonding (Chung
et al., 2015). Khalifa et al. found that ACN-WP complex
was mainly molded by hydrophobic effects of WP surface
and subsequently was stabilized by hydrogen bonding and
van der Waals forces (Khalifa et al., 2018). Salah and Xu
reported a negative enthalpy and a positive entropy, which
implied that interaction between red raspberry ACN and
β-LG was formed through electrostatic forces (Salah & Xu,
2021).
4.8 Docking study for
anthocyanin–whey protein interaction
Docking is a molecular modeling technique that is used
to predict how a protein interacts with small molecules
(Roy et al., 2015). The docking results could provide
more detailed evidence about the ACN-WP binding site.
Previous studies have shown that the internal cavity of
the β-barrel, the hydrophobic surface pocket in the groove
between the α-helix and the β-barrel, and the outer surface
near Tryptophan 19-Arginine 124 were the three possible
binding sites for β-LG (Roufik et al., 2006). Pan et al.
showed that hydrogen bond and hydrophobic interaction
were the main binding forces to maintain the stability of
the C3G and cow milk protein complexes and β-CA is the
best C3G carrier protein among the four (α-LA, β-LG, αs1-
CA, and β-CA) cow milk proteins (Pan et al., 2021). Zang
et al. showed that there were eight amino acids involved in
the M3G-WPI hydrophobic binding. Among these amino
acids, Pro 38, Asn 90, and Asn 88 formed C─H bonds with
M3G on the hydroxyl groups at positions 5 of the A ring, 3″
of the galactosyl group of M3G, and 3″ and 4″ of the galac-
tosyl group of M3G. At the same time, the ACN stability
was improved because WP wrapped the active binding
sites on the ACN’s A and B rings (Zang et al., 2021). Gong
et al. studied the binding between WP with petunidin-3-
glucoside (Pt3G) and peonidin-3-glucoside (Pn3G). They
found the binding sites were different for Pt3G and Pn3G.
Pt3G was surrounded by 12 amino acids residues of WP
and two hydrogen bonds were formed between Asn109,
Asn90, and Pt3G. Pn3G was surrounded by 13 amino acid
residues of WP with five hydrogen bonds formed. They
also demonstrated that WP could improve ACN stability
because the active groups in the WP could interact with the
hydroxyl groups of the A, B, and C rings of Pt3G and Pn3G
through hydrogen bonds (Gong et al., 2021;Langetal.,
2019; Khalifa et al., 2018). Aprodu et al. reported that β-LG
generated hydrogen bonding with both the aglycon and

12 WHEY PROTEIN - ANTHOCYANIN INTERACTIONS
the sugar moiety of Pn3G (Aprodu et al., 2019). A docking
study for the interaction of C3G with β-LG showed that
there was a strong multi-centered hydrophobic interac-
tion between C3G with the nearby amino acid residues.
Besides, Glu18, Asn 109, and Ser 116 residues formed
three hydrogen bonds with C3G (Cheng et al., 2017).
Xu et al. reported eight amino acid residuals involved
in the binding between delphinidin-3-O-glucoside and
β-LG and four hydrogen bonds were presented in the
ACN-β-LG complex (Xu et al., 2019). All of these results
provide evidence that the ACN-WP complex are stabilized
mainly through both hydrogen bonding and hydrophobic
interaction.
4.9 The effect of whey protein–phenolic
interaction on whey protein functional
properties
The interaction with phenolic compounds could improve
the bioactivities and functional properties of WP. The WP
emulsion stability and antioxidant capacity were reported
to be improved by the addition of ACN. The addition of
black rice ACN into WP-stabilized emulsions improved the
emulsions’ stability in a dose-dependent manner during a
storage study at 35◦C for 5 days because ACN could inhibit
droplet aggregation. Furthermore, the black rice ACN also
improved the antioxidant capacity of the emulsions by
inhibiting both lipid oxidation and WP oxidation (Yi et al.,
2020). Viljanen and others found raspberry and blackberry
juices inhibited protein oxidation in WP emulsions. The
antioxidant effect and turbidity increased with increasing
concentration of berries and WP concentration (Viljanen
et al., 2005). Further, WP-phenolic particles are able to pro-
duce stable foams as well as maintain anti-inflammatory
activity of polyphenols entrapped within such particles
(Schneider, 2016). Another study from Viljanen et al. found
that ACN could inhibit formation of carbonyl compounds
once associated with the proteins, which further inhibited
WP oxidation (Viljanen et al., 2005). The ACN-WP inter-
action was also able to change the sensory properties of
WP. The addition of ACN-absorbed WP microgels changed
the red-green value (a*) of the reduced-fat cheddar cheese
and improved the overall sensory evaluation scores of the
final product in the color and mechanical aspects com-
pared with the product with only WP microgels without
ACN (Wen et al., 2020).
Similar with ACN, other phenolic compounds also inter-
acted with WP to change WP physicochemical proper-
ties. Studies showed that phenolic-modified WP exhibits
an enhanced emulsifying capacity (Berton-Carabin et al.,
2013;Wangetal.,2014). The surface hydrophobicity of WP
declined by nearly 50% when combined with 240 µmol/g
(−)-epigallocatechin-3-gallate (EGCG) at pH 7.0 (Cao &
Xiong, 2017). Alkaline-modulated covalent binding of
EGCG to WP could enhance its foaming and improve its
emulsifying properties (Jia et al., 2016).
4.10 The effect of preheating
temperature on whey protein–anthocyanin
interaction
Temperature is one of the most important factors that
affects protein–phenolic interactions. Heat treatment pro-
motes denaturation and aggregation of WP, which results
in its secondary and tertiary structure changes (Busti et al.,
2005). Since the hydrophobicity interaction between pro-
tein and ACN is affected by the structure of protein, it may
be helpful for their interaction if the protein is thermally
induced by heat. A previous study showed that preheated
WP had a better protective effect on the thermal, oxidation,
and photo stability of grape skin ANC extracts than native
WP. The optimal effects occurred when WP was preheated
at 50◦C for 15 min, which decreased the thermal, oxidative,
and photo degradation of grape skin ACN at 71.59%, 32.22%,
and 56.92% at pH 3.2 and 54.91%, 22.89%, and 46.68% at
pH 6.3, respectively (He et al., 2016). The ACN-WP bind-
ing affinity and the structural conformations of WP are
reported to be affected by different WP preheating temper-
atures. Ren and Giusti found that the interaction between
purple corn, grape skin, and black carrot ACN extracts with
preheated WP always had a higher binding affinity than
with native WP, but the binding affinity decreased gradu-
ally with increasing WP preheating temperature from 40 to
80◦C (Ren & Giusti, 2021a). He et al. found that when the
WP preheating temperature increased, the C3G-WP bind-
ing affinity increased and when the WP preheating tem-
perature reached 55–85°C, the WP conformation changed
at a markedly increased rate. They also showed that the
highest binding affinity was observed when WP was pre-
heated at 85°C for 30 min at pH 6.3 with a binding con-
stant of 14.10 (±0.33) ×105M−1. On the other hand, they
found that the binding affinity of ACN-β-LG decreased
at pH 3.6 when WP preheating temperature increased,
which might be due to protein aggregation at high temper-
ature (He et al., 2018). Oancea et al. found that the bind-
ing affinity between β-LG and sour cherries skins ACN
increased with the increasing β-LG preheating tempera-
ture from 25 to 80◦C, but decreased when the tempera-
ture increased from 80 to 100◦C (Oancea et al., 2017). Stăn-
ciuc et al. found that addition of different preheated WP to
grape skin ACN extract resulted in different maximum flu-
orescence emission wavelength shift, with bathochromic
shifts of 21 nm at 25◦C, 22.5 nm at 70◦C, and 20 nm at
90◦C, respectively, and the allure of spectra after heating

WHEY PROTEIN - ANTHOCYANIN INTERACTIONS 13
at 90◦C suggested a more open protein structure (Stăn-
ciuc et al., 2017). Similar results were also reported by
Oancea et al., who showed that the addition of the same
amount of sour cherries skin ACN to different preheated
β-LG led to a different maximum fluorescence emission
wavelength shift. When the β-LG preheating temperature
reached the range of 50 to 90◦C, there were significant
red-shifts between 78 and 76 nm, while only 75 nm were
observed for native β-LG (Oancea et al., 2017). Ren and
Giusti showed that maximum fluorescence intensity Fmax
of ACN-WP complexes increased significantly (p<.05) as
the WP preheating temperature increased from 40 to 60◦C,
but no significant change was observed when temperature
increased from 60 to 80◦C (Ren & Giusti, 2021a). These
results could be explained because the WP disulfide-linked
aggregation decreased the exposure of Tryptophan as tem-
perature increased from 40 to 60◦C(Palazoloetal.,2000),
thus decreasing the ACN quenching effect. However, when
WP preheating temperature continued increasing, the WP
structure became more unfolded and the breaking of the
disulfide bond and the exposure of all the previously buried
Tryptophan increased ACN quenching (Stănciuc et al.,
2012). Chung et al. found that the addition of highly dena-
tured WP (95◦C for 30 min) significantly inhibited color
fading and enhanced the stability of the purple carrot ACN
more than native WP during accelerated storage at 40◦C
for 7 days, with about 24% higher relative absorbance and
30% less color loss than native WP. This result could be
explained because the unfolding of the protein may have
increased the interactions with the ACN, possibly due to
greater exposure of the hydrophobic groups (Chung et al.,
2015). These results should help facilitate the widespread
application of WP and ACN in thermal processing in the
food industry.
4.11 Anthocyanin–whey protein
encapsulation and digestion
Encapsulation is a technique used to entrap active ingre-
dients within wall materials to form microcapsules. It is
a viable approach for stabilizing active food ingredients,
such as ACN (Norkaew et al., 2019). The encapsulation
technique via spray drying is an effective way to preserve
phenolic compounds from thermal degradation (Mueller
et al., 2018). Many studies have reported that WP could
be successfully used as wall materials for the encapsula-
tion of ACN extracts and juices (Sharif et al., 2020; Rocha,
de Barros et al., 2019; Bilek et al., 2017; Tao et al., 2017).
The aglycon and glucosyl moiety of the encapsulated ACN
molecules could interact with WP through hydrophobic
interactions and hydrogen bonds (Aprodu et al., 2019).
Their results showed that the encapsulated ACN-WP
powders had elevated phytochemical contents, significant
antioxidant activity, and good thermal stability. Constantin
et al. found that there was an interaction between grape
skin ACN with amine groups of WP during freeze-drying
encapsulation. The ACN-WP complex displayed high sta-
bility in the dark at a temperature of 4◦C up to 3 months
and showed slower ACN release in the simulated gas-
tric fluid (Constantin et al., 2020). Wang et al. reported
that ACN-loaded chitosan hydrochloride/carboxymethyl
chitosan-WP nanocomplexes improved ACN stability. The
encapsulated ACN had an increased half-life from 290.61
to 415.97 min after heating at 90◦C for 300 min compared
with nonencapsulated ACN. The nanocomplexes main-
tained higher DPPH and hydroxyl free radical scaveng-
ing activities (Wang et al., 2021). Addition of red raspberry
pomace ACN to β-LG increased the antioxidant activity of
β-LG-nanoparticles. The ACN-WP nanoparticles showed
higher stability in the mouth (pH 6.8), simulated gas-
tric (pH 2), and simulated intestinal (pH 6.9) environ-
ments, and higher heat stability than that of unencap-
sulated ACN (Salah et al., 2020). ACN from sour cherry
peel-encapsulated WP were found to decrease the release
from 45% to 98.00% after 120 min of reaction in the gas-
trointestinal tract, which identified that the absorption
rate of ACN was improved and ACN could protect WP to
release effectively into the intestine (Oancea et al., 2018).
Ge et al. reported that the ACNs-loaded chitosan/β-LG
nanocomplexes had less ACN release than the ACN solu-
tions in the simulated gastric and intestinal fluid (Ge et al.,
2019). The microcapsules of the WP and ACN-rich extract
from Thai black rice released high amounts of ACN and
total phenolic compounds under in vitro intestinal diges-
tion and remarkably increased the antioxidant activity of
the intestinal fluids (Norkaew et al., 2019). Stănciuc et al.
found that the encapsulation of grape skin ACN with WP
decreased ACN antioxidant activity and improved the ACN
thermal stability (Stănciuc et al., 2017). All of these stud-
ies proved that the stability and bioavailability of ACN is
significantly improved by nanocomplexes encapsulation,
especially when the WP was involved as wall materials.
Therefore, extending ACN stability by interaction with WP
could be used as a valuable method for the further applica-
tion of ACNs in food and pharmacy.
ACN-WP encapsulation also affects WP digestion. Liao
et al. studied the in-vitro digestion behaviors of milk
proteins with blueberry ACN. The FTIR spectra showed
that the amide A peaks of WP-ACN microparticles had
significant shift for ACN-WP microparticles than WP only
during the intestinal digestion. And their fluorescence
study showed that there were more obvious red shifts for
the λmax of WP-ACN complexes than WP only (Liao et al.,
2021). These identified that WP changed less during diges-
tion after binding with ACN, which might be explained

14 WHEY PROTEIN - ANTHOCYANIN INTERACTIONS
that ACN inhibited digestive enzymes and the WP became
less susceptible to such enzymes (Zhao et al., 2020).
Khalifa and others found that mulberry ACN could inter-
act with WP both noncovalently and covalently to form
ACN-WP adducts. Mulberry ACN could unfold the WP
structure through fluctuating their α-helix and β-sheet,
which resulted in an increased chance for the digestive
enzymes to access the peptide bonds and consequentially
improved the WP’s digestibility (Khalifa et al., 2021). Yu
et al. found that grape seed polyphenol extract improved
the digestion of whey protein by trypsin (Yu et al.,
2016).
5CONCLUSION AND PERSPECTIVE
The applications of ACN as natural food colorants have
become more popular in recent decades. Although the sen-
sitivity of ACN to environmental conditions has limited
their application in the food industry, the color and sta-
bility of ACN could be improved by interaction with WP
to form complexes. Characterization of the ACN-WP com-
plexation by CD spectroscopy reveals that the contents of
α-helix, β-sheet, β-turn, and random coil change due to
their interaction. The FTIR spectra show a change in the
amide I and amide II peaks when ACN-WP complexes are
formed. The WP intensity decreases significantly in the
fluorescence spectra after binding with ACN, which indi-
cates the strongly binding affinity between ACN and WP.
Thermodynamic analysis of the ACN-WP binding suggests
that ACN react with WP through hydrophobic, hydrogen
bonding, electrostatic, and Van der Waal force. In addi-
tion, the color, antioxidation, digestion and absorption,
and some functional properties of the ACN-WP complex
are also improved.
Although the interaction between ACN and WP has
been extensively studied, there are still many aspects that
need to be further elucidated. First, the binding affinity
between ACN and WP depends on ACN chemistry struc-
ture. Different ACN may have different binding forces
with WP and need to be explored case by case. Second,
preheated WP will cause its secondary structure change,
which will further affect its interaction with ACN. The dif-
ferent preheating treatments need to be studied in order
to acquire the optimal preheating temperature. Third, the
optimum dosage of WP to be used in combination with
ACN still needs to be determined as it may affect the sen-
sory properties and rheological properties of the ACN-WP
complex, very important properties for food and bever-
age products. Once these challenges are addressed, WP
addition may become a powerful and feasible strategy to
increase ACN color expression and stability in food matri-
ces and together, impart functional benefits in addition to
color.
ACKNOWLEDGMENTS
This work was supported in part by the USDA National
Institute of Food and Agriculture, Hatch Project
OHO01423, Accession Number 1014136.
AUTHOR CONTRIBUTIONS
Shuai Ren: conceptualization, data curation, formal
analysis, methodology, project administration, resources,
software, validation, visualization, writing-original draft,
writing-review & editing. Rafael Jiménez-Flores: for-
mal analysis, resources, writing-review & editing. Maria
Monica Giusti: conceptualization, data curation, formal
analysis, funding acquisition, investigation, methodology,
project administration, resources, supervision, validation,
visualization, writing-original draft, writing-review & edit-
ing.
CONFLICT OF INTEREST
The authors declare no conflicts of interest.
ORCID
Shuai Ren https://orcid.org/0000-0003-4219-1469
Maria Monica Giusti https://orcid.org/0000-0002-2348-
3530
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How to cite this article: Ren, S., Jiménez-Flores,
R., & Giusti, M. M. (2021). The interactions between
anthocyanin and whey protein: A review. Compr
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