Critical Reviews in Food Science and Nutrition

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ATPS: “Aqueous two-phase system” as the “answer

to protein separation” for protein-processing food
industry

Bilal Muhammad Khan, Kit-Leong Cheong & Yang Liu

To cite this article: Bilal Muhammad Khan, Kit-Leong Cheong & Yang Liu (2019) ATPS:
“Aqueous two-phase system” as the “answer to protein separation” for protein-processing
food industry, Critical Reviews in Food Science and Nutrition, 59:19, 3165-3178, DOI:
10.1080/10408398.2018.1486283

To link to this article: https://doi.org/10.1080/10408398.2018.1486283

Published online: 16 Jul 2018.

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CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION
2019, VOL. 59, NO. 19, 3165–3178
https://doi.org/10.1080/10408398.2018.1486283

ATPS: “Aqueous two-phase system” as the “answer to protein separation”

for protein-processing food industry
Bilal Muhammad Khan, Kit-Leong Cheong, and Yang Liu
Guangdong Provincial Key Laboratory of Marine Biotechnology, STU-UNIVPM Joint Algal Research Center, Department of Biology, College of Science,
Shantou University, Shantou, Guangdong, PR China

ABSTRACT KEYWORDS
Every individual needs food for its nutritional value and flavor while the economic growth of a nation ATPS; food industry; food
depends on a thriving profit-generating industry. The food industry caters to both needs in an efficient proteins; industrial uses of
manner. Proteins can rightly be considered as the driving force behind the overwhelming success of this proteins; protein purification
industry. However, purification of proteins is not an easy undertaking due to their intricate nature while
presently employed procedures for this purpose, regrettably, are both costly, and labor- and time-
intensive in addition to being unsettling on proteins structural conformity. ATPS has accumulated a lot of
interest from the scientific community due to its mild operating conditions, high recovery yield, ease of
scaling it up, and its cost-effective and environment friendly nature. This review tries to amass some
accounts concerning the utility of ATPS for the separation and purification of proteins. Some auspicious
clues in this regard can be witnessed along with a few loopholes which need to be addressed before this
technique can truly demonstrate its potential vis-a
-vis industrial protein purification. Overall, a polymer –
salt (citrates in particular) ATPS with an added inert supplementary salt can be regarded as a better option
for purifying proteins.

1. Introduction
Without any doubt, one of the utmost imperative industrial sector
is represented by the food industry. The well-known maxim “You
cannot reason with an empty belly! It has no ears!” further reiter-
ates this eminent fact. The European food industry is worth €1017
billion and ranks 2nd on the list of large industrial enterprises (Yu
and Brooks, 2017). The demand for innovation in this industry is
being felt desperately with each passing day as consumers are
becoming more conscious about the nutrition value of their diet.
Furthermore, the amount of competition in the sector calls for
novel and feasible approaches to be adopted by the manufacturers
in order to get their share of this thriving income generating busi-
ness. Proteins can justly be regarded as one of the vital ingredients
as well as an important catalyst in a number of manufacturing and/
or storage processes undertaken in the modern food and beverage
industry. Their nutritious value aside, proteins also play a signifi-
cant role in imparting characteristic texture to the food and in its
structural assembly which enables the food to demonstrate its func-
tional property (Li-Chan and Lacroix, 2018).
The true potential of proteins in terms of monetary gains for
the industry and health benefits for consumers can only be fully
realized if their availability and purity is not in question. More-
over, their extraction and purification needs to be economically
viable and industrially proficient. The currently in vogue practi-
ces for this purpose, unfortunately, are both expensive, and
labor- and time-intensive. Downstream processing for the
purification of proteins is a tedious job due to their intricate
nature (Grilo et al., 2016; Silva and Franco, 2000). Despite being
simple and effective, separations through chromatography, how-
ever, are not only expensive but also cannot easily be scaled up
for industrial application. Extensive drops in pressure and the
need for batch-processing associated with chromatographic tech-
niques make them unsuitable for industrial scale processing of
proteins (Iqbal et al., 2016). Moreover, due to their extremely
low solubility in organic solvents, conventional liquid-liquid
extractions with organic solvents are also not regarded as the
procedure-of-choice for protein extraction (Asenjo and Andrews,
2011; Carlson, 1988). Protein’s functionality depends on its intri-
cate three dimensional structure while extreme temperature and
pH can irreversibly mutilate its structure resulting in proteins
with no function, and hence, no worth.
It is pertinent to mention that downstream-processing
accounts for over 80% of the production cost (Aguilar and
Rito-Palomares, 2010), and that retention of bioactivity in the
final product depends on the delicacy of the separation tech-
nique (Tanuja et al., 1997). Due to its mild operating conditions
and ease of scaling it up (Du et al., 2018), ATPS offers a more
viable and reliable alternative for the industrial processing of
proteins. It is for this reason that this technique has been
employed for the industrial scale protein extraction from rudi-
mentary feedstocks (Hatti-Kaul, 2001). It has gained a lot of
interest from the scientific community due to its high recovery

CONTACT Yang Liu, PhD liuyanglft@stu.edu.cn; Bilal Muhammad Khan PhDkhan@stu.edu.cn, ranezai@yahoo.com Guangdong Provincial Key Laboratory of
Marine Biotechnology, STU-UNIVPM Joint Algal Research Center, Department of Biology, College of Science, Shantou University, Shantou, Guangdong 515063, PR China.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/bfsn.
© 2018 Taylor & Francis Group, LLC
3166 B. M. KHAN ET AL.
Table 1. A list of important food industry proteins which are selected for this review.
Protein Importance
Thaumatin low-calorie protein sweetener and taste-modifier; 2000–
3000 times sweeter than sucrose
Miraculin taste-modifier
Bovine serum Emulsifier
albumin (BSA)
Lysozyme Preservative
Trypsin enhances the function (emulsification, solubility,
foaming and gelling) and nutrition of dietary
proteins; enhances digestibility of proteins; yields
bioactive peptides; diminishes allergens
concentration in foodstuffs; nutritive-supplement
Invertase breaks down sucrose into fructose and glucose, known
as “invert sugar” or “inverted sugar syrup,” a vital
ingredient in candy manufacturing and commercial
baking; utilized in the manufacture of synthetic
honey, jams, liquid-sugar, and non-crystallizing
creams
a-Amylase used for the commercial starch hydrolysis; utilized in the
manufacture of baked foodstuffs, brewed beverages,
gastrointestinal aids, cakes, juices and starch syrups
b-glucanase b-glucan hydrolysis; beer formation; piglets’ and
hatchlings’ feed manufacture
yield, ease of scaling up, and its cost-effective and environment
friendly nature (Iqbal et al., 2016). It can be setup for various
types of analyses particularly for the separation and purification
of bio-molecules (Asenjo and Andrews, 2011; Grilo et al., 2016;
Hatti-Kaul, 2000; van Berlo et al., 1998). Furthermore, in com-
parison to customary liquid-liquid extraction, ATPS is innocu-
ous, noninflammable and is not prone to emulsification
(Asenjo and Andrews, 2012).
The composition of ATPS (polymers and/or salt used) deter-
mines the partitioning behavior of proteins between the two
phases (Asenjo and Andrews, 2011). The upper less-polar hydro-
phobic phase (generally PEG) will normally be rich in proteins
while for the separation of proteins from each other, some altera-
tions in the molecular weight of the polymer and/or the ionic
strength of the salt will be needed (Iqbal et al., 2016). Several types
of ATPSs have been employed for protein processing, such as
polymer – polymer [PEG – dextran (Cascone et al., 1991;
Diamond and Hsu, 1990); PEG – maltodextrin (Alves et al.,
2000)], PEG – salt (Andrews and Asenjo, 2010; Balasubramaniam
et al., 2003; Cascone et al., 1991; de Belval et al., 1998;
Franco et al., 1996; Hachem et al., 1996; Merchuk et al., 1998),
non-ionic surfactant (Liu et al., 1998), ionic surfactants (Xiao et
al., 2000), polymer – polymer (PEG – dextran) – salt (Fan and
Figure 1. Schematic diagram of composition and operation of an ATPS.
Glatz, 1999; Zaslavsky, 1994), and “Smart polymers” [single poly-
mer] (Johansson et al., 1999).
This review is centered on the importance of certain pro-
teins in the food and beverage industry (Table 1) as well as the
superiority of ATPS, in reference to protein purification, over
conventional techniques. This process can be rightly termed as
the “answer to protein separation” for the food and beverage
industry due to its potential for further maximizing the gains
from this booming manufacturing sector.
2. Aqueous two-phase system (ATPS)
2.1. Definition
An aqueous two-phase system (ATPS) is a combination of either
two immiscible polymers or a polymer and an inorganic salt dis-
solved in water above their critical concentrations. Since the two
solutions thus formed are immiscible, they will form two distinct
phases wherein the upper phase will be composed of one of the two
polymers while the lower phase will have the other polymer or the
inorganic salt depending on the composition of the system
(Figure 1). An ATPS is different from conventional aqueous-
organic extraction systems in that it contains a much more amount
of water (80-99% by weight), and retains exceptionally less interfa-
cial tension of 10–5N.m¡1 (Liu et al., 2011).
2.2. History
Martinus Willem Beijerinck, in 1896, mixed starch and gelatin
aqueous solutions and thus inadvertently
discovered ATPS
(Mazzola et al., 2008). It was Per-Ake Albertsson, however,
who really introduced it as an analytical tool in the 1950s (Liu
et al., 2016). It has, subsequently, found use in a range of sepa-
ration procedures (Albertsson, 1986; Grilo et al., 2016; van
Berlo et al., 1998). A range of substances can be dissolved in
water to form an ATPS (Hatti-Kaul, 2000) but the most utilized
and worked out of ATPS systems consist of either two polymers
or a polymer and an inorganic salt (van Berlo et al., 1998).
2.3. The Different types of ATPS
Binary solutions of either two polymers (typically polyethylene
glycol, PEG, and dextran) or a polymer and an inorganic salt
(phosphates, citrates or sulfates) constitute the most commonly

employed form of an ATPS (Iqbal et al., 2016). Some ionic
liquids and short chain alcohols form another type of ATPS
(Asenjo and Andrews, 2012; Grilo et al., 2016; Hatti-Kaul,
2000, 2001; Molino et al., 2013; Ruiz-Ruiz et al., 2012; van Berlo
et al., 1998) while micellar and reverse micellar forms of ATPSs
are constituted by ionic and/or nonionic surfactants (Liu et al.,
1998; Ruiz-Ruiz et al., 2012; Xiao et al., 2000). Moreover, an
ionic liquid (cholinium cation and anion) – polymer (polypro-
pylene glycol) ATPS has recently been successfully employed
for protein separation with demonstrated high recyclability
(Lee et al., 2017).
2.4. Advantages of using ATPS
Conventional protein separation processes, especially chro-
matographic techniques, are expensive, time-consuming, and
cannot be scaled up easily for industrial operation. Moreover,
such processes require a higher number of intermittent steps
which not only increase the operational cost but also results in
loss of certain quantity of the desired product (Phong et al.,
2018). ATPS, on the other hand, possess a number of proce-
dural and monetary advantages vis-
a-vis protein separation
and purification. Specifically, ATPS is more energy-efficient
and economical, highly selective, simple to operate, offers
speedy separation (Goja et al., 2013; Quental et al., 2015; Raja
et al., 2011; Rito-Palomares, 2004; Soares et al., 2015; Zhao
et al., 2014) and a bio-compatible environment (Agasøster,
1998; Albertsson, 1986), and has enormously low interfacial
tension (Albertsson, 1986) and no downstream pollution
(Hatti-Kaul, 2000).
3. ATPS for protein purification in the food industry
The intricate structure of proteins is a hindrance in efforts
towards its purification (Grilo et al., 2016; Silva and Franco,
2000). The industrial separation and purification of proteins
cannot efficiently be achieved through conventional separation
techniques like chromatography (Iqbal et al., 2016) and organic
solvents mediated extraction (Asenjo and Andrews, 2011;
Carlson, 1988). Furthermore, protein’s functionality depends
on its intricate three dimensional structure while extreme tem-
perature and pH can irreversibly mutilate its structure resulting
in proteins with no function, and hence, no worth.
Due to its mild operating conditions and ease of scaling it
up, ATPS offers a more viable and reliable alternative for the
industrial processing of proteins. Moreover, as it is regarded as
an authentic, reliable and reproducible analysis and isolation
tool, it can help a great deal in the design, manufacture and
marketing of food products as well as substances required in
Table 2. The different low-calorie protein sweeteners and taste-modifiers.
Protein Source
Thaumatin Thaumatococcus danielli Benth
Monellin Dioscoreophyllum cumminsii Diels
Mabinlin Capparis masakai Levl
Pentadin Pentadiplandra brazzeana Baillon
Brazzein Pentadiplandra brazzeana Baillon
Curculin Curculingo latifolia
Miraculin Synsepalum dulcificum
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 3167
the production of such foodstuffs. The composition of ATPS
(polymers and/or salt used) determines the partitioning behav-
ior of proteins between the two phases (Asenjo and Andrews,
2011). The upper less-polar hydrophobic phase (generally
PEG) will normally be rich in proteins while for the separation
of proteins from each other, some alterations in the molecular
weight of the polymer and/or the ionic strength of the salt will
be needed (Iqbal et al., 2016).
In fact, this technique has been put to use by various
researchers to achieve the separation, concentration and purifi-
cation of proteins, and their results are quite promising. In the
following paras, we have tried to compile some of the most
noteworthy and important progress in this direction in a hope
that it will provide a reference source for endeavoring research-
ers in this field.
3.1. Low-calorie protein sweeteners and taste-modifiers
There has been a global increase in the incidence of diabetes
and obesity lately (Levine et al., 2003). A number of synthetic
sweeteners, such as AcesulfameK, Saccharin, Cyclamate, and
Aspartame, have been available globally to patients with sugar
consumption related disorders but their use is potentially asso-
ciated with bladder-cancer, mental sicknesses, psychological
issues, brain-tumors and heart-failures (Cohen, 2001; Hagiwara
et al., 1984; Kanarek, 1994; Nabors, 1988; Weihrauch et al.,
2001). Since protein, unlike sucrose, do not petition an insulin
requirement, it can act as a health-friendly alternative to
sucrose for the diabetics and obese in particular, and the rest in
general. Hitherto seven proteins, Brazzein (Ming and
Hellekant, 1994), Thaumatin (Wel and Loeve, 1972), Monelin
(Inglett and May, 1969), Curculin (Harada et al., 1994),
Mabinlin (Liu et al., 1993), Miraculin (Takahashi et al., 1990)
and Pentadin (Faus, 2000), have been identified as potential
sweeteners and/or taste-modifiers which are all extracted from
tropical rainforest plants (Table 2).
3.1.1. Thaumatin
The low-calorie protein sweetener and taste-modifier, thauma-
tin, from the katemfe fruit, Thaumatococcus daniellii, is a
healthy substitute for sucrose, and, in fact, is approximately
2000–3000 times sweeter than the later (O’Neil, 2015). Since it
is digestible as any other protein, it is safe for human consump-
tion. Higginbotham and co-workers (Higginbotham et al.,
1983) found it non-hazardous and raised no objection to its use
as a sweetener. The West Africans have been using thaumatin
as sweetener for centuries (O’Neil, 2015). In fact, it has been
sanctioned to be utilized as a sweetener and taste-enhancer in
Location Sweetness-factor Molecular Mass (kDa) ATPS Purification
West Africa 3000 22.2 Yes
West Africa 3000 10.7 No
China 100 12.4 No
West Africa 500 12.0 No
West Africa 2000 6.5 No
Malaysia 550 24.9 No
West Africa — 98.4 Yes
3168 B. M. KHAN ET AL.
numerous regions of the world (Zemanek and Wasserman,
1995).
The partitioning behavior of thaumatin was investigated by
Cascone et al. 1991. The lysed E. coli cells were used as a source
for the isolation of this protein in two distinct ATPSs (PEG –
dextran and PEG – potassium phosphate) wherein PEG –
potassium phosphate ATPS was found more efficient. The
molecular weight of PEG, amount of NaCl (supplementary salt
used) and pH of the system were found to affect the partition
coefficient, K, with low molecular weight PEG and high NaCl
concentration favoring the partition of thaumatin. A single-
step 20-fold purification and a significantly high recovery yield
(90-95%) for thaumatin was reported (Cascone et al., 1991).
In contrast to the aforementioned study, a relatively recent
work reports that the PEG concentration does not affect thau-
matin partition in a significant way (Ahmad et al., 2008). Even
this study, however, testifies the importance of salt concentra-
tion vis-
a-vis thaumatin partitioning in ATPS with high salt
concentration enhancing the partition coefficient by sevenfold.
A PEG – sodium sulfate ATPS worked more proficiently in this
regard than its counterpart (PEG – potassium phosphate)
revealing a partition coefficient of 24.1 and a recovery of 96.0%
at optimum conditions. Another important point highlighted
in this study nullifies an interacting effect of polymer and salt
concentration on thaumatin partition and suggests that the
individual concentration variations of the two are the sole fac-
tor in this regard (Ahmad et al., 2008).
A PEG – salt ATPS (sodium sulfate in particular) with high
salt concentration (phase forming and supplementary salt) and
low molecular weight polymer, hence, can be regarded as the
best means for thaumatin extraction and purification (Table 3).
Nevertheless, some more work in this regard is required as the
available text in this direction is scarce and relatively outdated.
Having said that, new studies might base their work on the
aforementioned findings to minimize the efforts needed to
attain the goal of devising an industrially feasible ATPS for
thaumatin separation and recovery.
3.1.2. Miraculin
A taste-modifier protein alters the bitter taste enabling it to be
perceived as sweet by modifying sweet taste receptors upon
binding them (Gerritsen, 2001; Theerasilp et al., 1989). Such
unusual taste modifying activity is traditionally associated with
red berries of an indigenous West African plant, Synsepalum
dulcificum (Wong and Kern, 2011). Actually, a constituent pro-
tein of this “miracle fruit,” miraculin, is responsible for its
Table 3. A compilation of the utilization of ATPS for thaumatin separation.
ATPS Results Reference
PEG – dextran and PEG – potassium phosphate ATPS was (Cascone
PEG – potassium found more efficient; 60-fold et al., 1991)
phosphate increase in partition coefficient; low
molecular weight PEG and high NaCl
concentration favored the partition;
20-fold purification; 90–95% yield
PEG – sodium sulfate PEG – sodium sulfate ATPS worked (Ahmad et al.,
and PEG – more proficiently; high salt 2008)
potassium concentration enhances the partition
phosphate coefficient; partition coefficient of
24.1; recovery of 96.0%
Table 4. Conventional methods used for miraculin purification.
Procedure Result Drawback
Organic solvents fails to yield the tiresome
mediated protein in its
purification pure form
Ion exchange yields pure expensive; cannot easily be scaled
chromatography protein up for industrial application
Ammonium-sulfate recovered protein the need for ultra-filtration after
fractionation is highly pure fractionation with ammonium-
trailed by CM- sulfate and the two
Sepharose ion chromatography procedures
exchange and make this process ineffective,
ConA-Sepharose expensive and time-intensive;
affinity cannot easily be scaled up for
chromatographic industrial application
steps
Immobilized metal the process is expensive; cannot easily be scaled
affinity simple and up for industrial application
chromatography effective
taste-modifying ability (Zhang and Sun, 2001). Miraculin’s his-
tidine residues are considered to be involved in its taste-altering
effect, though its exact mechanism of action is hitherto
unknown (Ito et al., 2010; Paladino et al., 2010). Chromato-
graphically extracted miraculin (20mg) can significantly dimin-
ish lemon’s sour taste and, hence, expressively enhance its
sweetness (Giroux and Henkin, 1974). Owing to its potential,
there have been continuing efforts to fully characterize miracu-
lin’s structure and its mechanism of action but the currently
employed purification processes for miraculin are both expen-
sive, and labor- and time-intensive (Table 4).
Efforts have been put into force for organic solvents medi-
ated purification of miraculin (Inglett et al., 1965), however,
this process is not only tiresome but also fails to provide the
protein in its pure form. This protein was first obtained in its
pure form by utilizing the ion exchange chromatographic tech-
nique (Kurihara and Beidler, 1968), and later another proce-
dure (ammonium-sulfate fractionation trailed by CM-
Sepharose ion exchange and ConA-Sepharose affinity chro-
matographic steps) was reported for 97% purification of mirac-
ulin (Theerasilp and Kurihara, 1988). In spite of the high purity
of the final product, the need for ultra-filtration after fraction-
ation with ammonium-sulfate and the two chromatography
procedures make this process ineffective, expensive and time-
intensive. Immobilized metal affinity chromatography has been
lately utilized for the purification of this protein from miracle
fruit and transgenic tomato (Duhita et al., 2009; Duhita et al.,
2011). Despite being simple and effective, separations through
chromatography, however, are not only expensive but also can-
not easily be scaled up for industrial application.
Aqueous two phase system can easily be used for the indus-
trial separation and purification of this important protein with
the least monetary and resource investment. In the available lit-
erature, we can find one promising study (He et al., 2015)
wherein extremely pure miraculin (94.8% purity) with a recov-
ery rate of 22% was purified using a reverse aqueous two phase
micellar system (ATPS-RM). Surfactants in polar-solvents are
used to stabilize “water-in-oil” micro-emulsion drops in an
ATPS-RM. The ATPS-RM (sodium di (2- ethylhexyl) sulfosuc-
cinate/isooctane) was employed for the purification of miracu-
lin from the miracle-fruit. The study suggested that the most
suitable conditions for the purification of miraculin in an
 CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 3169

ATPS-RM are 0.1 mole per liter sodium di (2- ethylhexyl) sul-
fosuccinate/isooctane added to the crude extract at pH 8 (for-
ward extraction), and 0.5 mole per liter NaCl at pH 11
(backward stripping) (He et al., 2015). The “forward extrac-
tion” in an ATPS-RM refers to the initial extraction of the pro-
tein from water into the reversed-micellar phase, while
“backward stripping” is a term reserved for the final extraction
of the reversed-micellar phase with another water phase for the
recovery of the protein (Dekker et al., 1989).
Though this study offers promising results in reference to
the potential industrial purification of miraculin using an
ATPS-RM but this is the only study in this regard to be found
in the available literature. Further studies, henceforth, are
required for validation of these results and for more complete
characterization of the system conditions for miraculin’s purifi-
cation. Moreover, some efforts for its purification using simpler
forms of ATPSs will prove beneficial for the broad-range accep-
tance of ATPS’s industrial utility in this regard. Having said
that, this study, nonetheless, can be regarded as a giant leap for-
ward in the right direction. Furthermore, here we yet again wit-
ness the positive effect of supplementary salt concentration
(NaCl in this study) on protein purification.
Thaumatin and miraculin are, so far, the only sweet and
taste-altering proteins whose purification efficiency has been
checked using ATPS. As such proteins represent a health-
friendly alternative of sugar in the food and brewery industry,
it is pertinent to devise ways for their feasible and efficient
extraction and purification. Since ATPS offers extraction and
primary purification in a single step while having the ability to
be scaled up easily, it is by far the most suitable technique for
this purpose. Therefore, more efforts in characterizing the par-
tition of the other members of sweet and/or taste-altering pro-
teins using ATPS is required.
3.2. Bovine serum albumin (BSA)
Food emulsions are commonplace in the food industry. A con-
coction of two liquids (e.g., water and oil) brought about by
splitting the molecules in each into extremely fine small drop-
lets so that they will stick together is referred to as an emulsion.
Margarine, salad dressings, milk, ice cream, sausages, sauces,
and mayonnaise are some examples of food emulsions. The
industrial scale packaging and manufacture of these emulsions
require emulsifiers for stabilizing the mixture and to avoid the
separation of the different ingredients from each other (IFT,
2013). As proteins are amphipathic and malleable, they provide
more stability to emulsions in comparison with non-protein
amphiphiles (Dickinson and Leser, 2007). Bovine serum albu-
min (BSA) is a globular protein whose potential as an emulsi-
fier in the food industry has been proved (Haque and Kinsella,
1988).
More intricate procedures incurring higher expenses are
required to recover and purify BSA (Virtuoso et al., 2010).
ATPS, due to its simplicity of handling and lower costs, can be
used for the scaled up recovery and separation of this important
protein (Table 5). BSA has been investigated for its phase-sepa-
ration behavior in ATPS (dodecyltriethylammonium bromide
– sodium dodecylsulfate) which was successful in achieving its
separation from lysozyme while not affecting their final activi-
ties in any way (Xiao et al., 2000). Its partitioning efficiency has
also been determined using PEG – dextran ATPS (Diamond
and Hsu, 1990) and PEG – phosphate ATPS (Chow et al.,
2015). An auspicious 90.8% yield (partition coefficient of 2.4)
in the upper-phase was reported for the PEG – phosphate
ATPS with PEG (33.0% w/w), phosphate (8.0% w/w), NaCl
(8.5% w/w), and the pH set at 9.0 (Chow et al., 2015). The addi-
tion of the supplementary salt yet again shows marked
improvement in reference to protein purification using ATPS.
Yet another study points towards the effect of salt concen-
tration (phase forming sodium citrate) on the partition of
BSA, suggesting that salt interaction dehydrates the protein
and hence exposes its hydrophobic zones resulting in
enhanced protein-polymer interactions, and eventually
improved extraction (Virtuoso et al., 2010). Similar effect of
the phase forming salt (potassium phosphate) on BSA parti-
tion behavior in PEG – potassium phosphate ATPS was
reported earlier in 2007 (Faravash et al., 2007). Likewise, the
explicit salt-protein interactions and the potential variance
across the two phases created by the addition of salt has been
attributed as the cause behind the improved partitioning of
proteins in ATPS (Settu et al., 2015). Settu and coworkers
employed PEG – poly acrylic acid (PAA) ATPS and found
that the addition of 1M NaCl to PEG – PAA sytem
(PEG4000) which is operated at 0 C and pH 8 yields the best
results in relation to BSA partitioning (Settu et al., 2015). In
addition, low molecular weight polymer has been found to

Table 5. Different types of ATPSs employed for BSA purification.

ATPS Results Reference

dodecyltriethylammonium
bromide – sodium
dodecylsulfate
PEG – dextran
PEG – KH2PO4
Polyethylene oxide
(PEO) – sodium citrate
PEG – potassium phosphate
PEG – poly acrylic acid
PEG – potassium orthophosphate
successfully separated BSA from lysozyme while not affecting its final activity; the phase (Xiao et al., 2000)
separation depended on the concentration and molar ratio of mixed surfactants
characterized the partition behavior in reference to polymer molecular weight, protein-water, protein- (Diamond and Hsu,
polymer and polymer-water interaction, and the electrostatic potential difference between the 1990)
phases
90.8% yield; partition coefficient of 2.4; addition of NaCl showed marked improvement in reference (Chow et al., 2015)
to protein purification
partition coefficient increased from 0.125 to 0.440; salt concentration affected the partition (Virtuoso et al., 2010)
Concentration of phase-forming salt influenced partitioning; (Faravash et al., 2007)
salt-protein interactions and the potential variance across the two phases created by the addition (Settu et al., 2015)
of salt has been attributed as the cause behind the improved partitioning
low molecular weight polymer was found to work better (Selvakumar et al., 2012)
3170 B. M. KHAN ET AL.

work better in the context of BSA recovery and ease of operat-
ing the two phase system (Selvakumar et al., 2012).
BSA, due to its utility as an alternate emulsifier, is getting
attention from the food industry, while its traditional isolation
and recovery procedures are regarded as inefficient and costly.
The extensive literature available on the separation and purifi-
cation of this protein in various types of ATPSs is a good omen
for its acceptance and widespread use in the food industry.
However, the secondary structural modifications of the parti-
tioned BSA in PEG – potassium phosphate ATPS reported by
(Faravash et al., 2007) must be taken into account and further
studies entailed for complete description of such an effect and
possible remedial measures undertaken. Nonetheless, the
enhanced partitioning of BSA in the presence of supplementary
salt is evident from the available literature in this regard.
3.3. Lysozyme
Some proteins also find use in the food industry as preserva-
tives. Lysozyme, a representative of such proteins, is found in
tears, mucous, human milk, and egg albumin. Various antibac-
terial properties have been attributed to this protein. Although,
the highest concentration of lysozyme can be found in tears,
hen egg albumin is usually favored for its large-scale extraction.
It has been reported as preservative for many foodstuffs
(Mecitoglu et al., 2006), such as sausages, fish cakes, bacon,
fresh vegetables, fish, meat, fruits, tofu bean curd, parched milk
compositions, oysters, shrimp, cheese, etc. (Proctor et al.,
1988). Lysozyme can be extracted using various techniques like
ion-exchange-chromatography (Jiang et al., 2001), gel-filtra-
tion-chromatography (Islam et al., 2006), dye-binding-chroma-
tography (Tejeda-Mansir et al., 2003), membrane-separation
(Chiu et al., 2007), reverse-micelles (Noh and Imm, 2005),
magnetic-citation-exchange (Safarik et al., 2007), and ethanol-
precipitation (Gemili et al., 2007; Jin et al., 2009). But due to
the associated inefficiency and unviability, most of them are
not employed for industrial purification of this enzyme (Lu
et al., 2013).
ATPS, because of its inherent advantages, has been produc-
tively utilized for the extraction of lysozyme from hen egg-
white (Table 6). An ATPS (dodecyltriethylammonium bro-
mide – sodium dodecylsulfate) was successfully employed for
the separation of lysozyme from BSA, and for the
determination of its phase partitioning behavior without
affecting its final activities in any way (Xiao et al., 2000). Simi-
larly, its partitioning efficiency has also been determined using
PEG – dextran ATPS (Diamond and Hsu, 1990). In one study,
a yield of 70% was associated with an enzymatic activity of
39,500 U.mg¡1 (Su and Chiang, 2006). The ability of this pro-
tein for preferential interaction with hydrophobic-polymers,
PEG for example, has also been reported (Franco et al., 1996).
Similarly, such interactions have been regarded as the main
reason behind lysozyme phase-partitioning in polymer – salt
ATPS (Johansson et al., 2008). A significantly higher yield
(85%) and activity (32,300 U.mg¡1) was reported in
EO50PO50 – K2HPO4 ATPS (Dembczy nski et al., 2010a;
Dembczy nski et al., 2010b). In another study, lysozyme was
separated from conalbumin in two ATPSs (PEG – citrate and
PEG – sulfate) wherein the earlier partitioned to the polymer-
rich phase and the later to the salt-rich phase in both systems
(de Sousa et al., 2009).
Lysozyme is the most hydrophobic of all the proteins pres-
ent in egg-white (Franco et al., 1996; Su and Chiang, 2006). A
more prominent effect of hydrophobic interactions was
observed in PEG – citrate ATPS in comparison to PEG – phos-
phate and PEG – sulfate ATPSs (Andrews et al., 2005). Another
testimony regarding the superior partition-coefficient of lyso-
zyme in PEG – citrate ATPS than that in PEG – sulfate ATPS
(de Sousa et al., 2009) is also visible in the available literature.
The biodegradability and nontoxicity of citrates makes PEG –
citrate ATPS environment-friendly, hence, increasing its suit-
ability for biomaterial extraction (Perumalsamy and
Murugesan, 2006). A promising activity-yield (103%), purifica-
tion-factor (21.1) and specific-activity (31,100 U.mg¡1) has
been reported for lysozyme in PEG (18%, w/w) – potassium cit-
rate (16%, w/w) ATPS at pH 5.5 and 30 C in the presence of
KCl (3.7%, w/w) as supplementary salt (Lu et al., 2013).
ATPS, PEG – citrates in particular, has proven potential to
replace the currently practiced inefficient and economically
unviable techniques. The available literature points towards the
superiority of using citrates as phase-forming salt instead of
phosphates and sulfates. Moreover, the hydrophobic interac-
tions between lysozyme and PEG have been regarded as the
main factor influencing its partition. In addition, the effect of
phase-forming salts as well as that of the supplementary salt
(KCl) is evident from research communications in this regard.

Table 6. Purification of lysozyme through different types of ATPSs.

ATPS Results Reference

dodecyltriethylammonium separated lysozyme from BSA (Xiao et al., 2000)

bromide – sodium dodecylsulfate
PEG – potassium citrate
PEG – citrate, PEG – phosphate
and PEG – sulfate
PEG – sodium sulfate
EO50PO50 – K2HPO4
PEG – citrate and PEG – sulfate
PEG – sodium polyacrylate
PEG – dextran
activity-yield (103%); purification-factor (21.1); specific-activity (Lu et al., 2013)
(31,100 U.mg¡1); addition of supplementary salt enhances partitioning
PEG – citrate system was more efficient; hydrophobic interactions influenced (Andrews et al., 2005)
partitioning
yield of 70%; enzymatic activity of 39,500 U.mg¡1 (Su and Chiang, 2006)
85% yield; 32,300 U.mg¡1 activity (Dembczynski et al., 2010a;
Dembczynski et al., 2010b)
PEG – citrate system was more effective; separated from conalbumin (de Sousa et al., 2009)
Partitioning was found to be determined by the hydrophobic and (Johansson et al., 2008)
electrochemical (salt) effects
partitioning efficiency was determined vis-
a-vis polymer molecular weight, (Diamond and Hsu, 1990)
protein-water, protein-polymer and polymer-water interaction, and
the electrostatic potential difference between the phases

3.4. Trypsin
Another enzymatic protein, trypsin, enjoys enormous utility in
food processing and food engineering. This serine protease is
found in the small intestine of humans where it helps in break-
ing down food proteins. It has found use in food processing
wherein it is employed to enhance the function (emulsification,
solubility, foaming and gelling) and nutrition of dietary pro-
teins. Trypsin also has proven ability to enhance digestibility of
proteins, to yield bioactive peptides, and to diminish allergens
concentration in foodstuffs (Yu and Ahmedna, 2012). More-
over, it is also used as a nutritive-supplement in the food
manufacturing sector (Tubio et al., 2009).
Trypsin has been conventionally extracted and purified
through ion-exchange and affinity chromatographic techni-
ques. These purification methods, however, are costly, time-
intensive and unable to be scaled up with ease (Perez et al.,
2015). In addition, extracted trypsin is accompanied by another
serine protease (chymotrypsin). Since both proteins demon-
strate analogous physical and chemical properties, a number of
time consuming chromatographic procedures are required to
separate trypsin from chymotrypsin (Johnson et al., 2002).
Hence, they do not represent the techniques of choice for tryp-
sin purification on commercial scale, thereby needed to be
replaced by a more suitable and feasible alternative like ATPS
(Table 7).
A PEG – sodium phosphate system has been employed for
analyzing the phase-separation behavior of trypsin (Klomklao
et al., 2010). ATPS has been productively utilized for the extrac-
tion of this protease from albacore tuna spleen and the
extracted enzyme was able to hydrolyze white shrimp shells,
thereby offering an inexpensive method for the isolation of
shell’s carotenoprotein (Poonsin et al., 2017). A PEG – sodium
citrate ATPS, due to its superior partitioning effect and envi-
ronment friendly nature, has been reported as a better alterna-
tive to the usually employed PEG – phosphate system for the
separation and purification of trypsin (Perez et al., 2015; Tubio
et al., 2009). A three-fold purification with a yield of 60% was
observed in the upper phase of PEG – sodium citrate system
when the polymer was used in a molecular weight of 3350 at
pH 5.2 and the concentration (w/w) of NaCl (supplementary
salt) was maintained at 7% (Tubio et al., 2009). Similarly, a two
and half-fold purification with a promising recovery (99.7%)
Table 7. A comparison of various ATPSs employed for trypsin purification.
ATPS Results Reference
PEG – sodium specific activity (30.05 U.mg¡1); (Klomklao et al.,
phosphate purification (27.3 fold) 2010)
PEG – NaH2PO4 Purity increased by 5.5 times; 71.92% (Poonsin et al.,
recoverey 2017)
PEG – sodium three-fold purification; yield of 60%; (Tubio et al.,
citrate addition of NaCl improved purification 2009)
PEG – sodium two and half-fold purification; recovery (Perez et al.,
citrate (99.7%); addition of NaCl improved 2015)
purification
PEG – dextran partitioning efficiency was determined (Diamond and
vis-
a-vis polymer molecular weight, Hsu, 1990)
protein-water, protein-polymer and
polymer-water interaction, and the
electrostatic potential difference
between the phases
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 3171
has been testified for trypsin in the top phase of an ATPS with
identical composition (polymer and salt) when the supplemen-
tary salt, NaCl, was added in a weight by weight concentration
of 3.3% (Perez et al., 2015). The phase-separation behavior of
this important protein has been fully investigated by Diamond
and Hsu, 1990. They used an ATPS comprising PEG – dextran
for this purpose and reported promising results in the context
of industrial processing of trypsin (Diamond and Hsu, 1990).
Since the conventional extraction procedures fail to effi-
ciently separate trypsin from chymotrypsin and to purify it on
commercial scale, ATPS needs to be employed for this purpose.
Moreover, ATPS comprising of a polymer and salt offers more
efficient purification of this protease. Specifically, citrates are
recommended to be used as the phase forming salts due to their
non-hazardous nature and their superior partitioning ability in
comparison to phosphates and sulfates. Furthermore, adding
an inert supplementary salt, NaCl for example, significantly
boosts the partitioning and recovery of trypsin in an ATPS.
3.5. Invertase
The suitability of ATPS as a separation and purification system
for another food industry’s favorite protein, invertase, has also
been determined in a number of research endeavors (Table 8).
It can well be regarded as a major breakthrough because of
invertase’s importance in the food industry as a key enzyme in
the processing of various foodstuffs. In particular, it is utilized
in the candy-making industry for making candy-liquid-centers,
cr
eme-eggs, chocolate-covered-cherries, fondant-candies, and
other confectioneries. Invertase breaks down sucrose into fruc-
tose and glucose, known as “invert sugar” or “inverted sugar
syrup,” a vital ingredient in candy manufacturing and market-
able baking due to its ability in keeping baked-stuff moist over
extended times (Labau, 2017). In addition, it is extensively uti-
lized in the manufacture of synthetic honey, jams, liquid-sugar,
and non-crystallizing creams (Guimar~aes et al., 2007; Kotwal
and Shankar, 2009; Marquez et al., 2008). There are no safety
concerns associated with the use of this enzyme as it finds place
on the “generally recognized as safe (GRAS)” list of US FDA
(US-FDA, 2018).
Traditionally, this industrially important enzyme has been
purified using various forms of chromatographic techniques,
Table 8. Reported types of ATPS for the purification of invertase.
ATPS Results Reference
PEG – Dextran Successfully determined the phase- (Diamond and Hsu,
separation behavior of invertase 1990)
PEG – sodium 5.1-fold increase in purity; 197% (Yuzugullu and
sulfate recovery; supplementary salt Duman, 2015)
favored purification
PEG – sodium yield of 90%; 5.5-fold increase in €
(Y€ucekan and Onal,
sulfate purity; supplementary salt 2011)
improved partitioning
PEG – magnesium recovery (217.7%); purity (6.2 fold); €
(Karkaş and Onal,
sulfate effect of supplementary salt on 2012)
purification was evident
PEG – potassium 60.8% recovery; refolding of 59.7% (Sanchez Trasvi~na
phosphate et al., 2015)
PEG – potassium yield (66.2%); purification (Leon Gonzalez
phosphate factor (29.5) et al., 2016)
3172 B. M. KHAN ET AL.
such as ion-exchange, gel-filtration, affinity, fast-protein-liquid
chromatography and hydrophobic-interaction chromatogra-
phy, and ammonium-sulfate precipitation (Chan et al., 1991;
Chavez et al., 1997; Ettalibi and Baratti, 1987; Madhusudhan
and Raghavarao, 2011; Moreno et al., 1990; Obenland et al.,
1993). Such techniques, despite the purity of the product, lag
behind vis-
a-vis amplification and integration of the process,
are expensive to work with, and are economically not feasible
to scale-up (Leon-Gonzalez et al., 2016).
Diamond and Hsu, 1990 determined the partitioning pat-
tern for invertase in an effort to provide a base for scaling up
ATPS for industrial scale operations. An ATPS (PEG – Dex-
tran) was used here for this purpose. They utilized an altered
form of ‘Flory-Huggins theory,’ which advocates a relation
between the natural log of protein coefficient and the differ-
ence in two phases in relation to polymer concentration. In
this direction, they related different interactions (polymer-
water, protein-water, and protein-polymer) and employed
parameters, such as protein function, polymer’s molecular
weight and electrostatic charge differences amid the two
phases. They were successful in determining the phase-separa-
tion behavior of invertase to be utilized in efforts for scaling
up ATPS for this purpose.
In an effort to purify invertase from potato tubers, a PEG –
sodium sulfate ATPS was utilized in the presence of magne-
sium sulfate (3%, w/w) as supplementary salt at pH 5.0 wherein
the purity was found to significantly increase (5.1-fold) with an
auspicious recovery of 197% (Yuzugullu and Duman, 2015).
Similarly, this enzyme has been purified from tomato using a
PEG – sodium sulfate system with a yield of 90% and a five and
half fold increase in purity when PEG3000 (15%, w/w) and
sodium sulfate (12%, w/w) were used in the presence of KCl
(5%, w/w) at pH 4.5 (Y€ €
ucekan and Onal, 2011). Invertase has
also been partitioned using a PEG – magnesium sulfate system
in the presence of MnCl2 (5%, w/w) at pH 5.0 which yielded an
extremely high recovery (217.7%) and purity (6.2 fold) (Karkaş
€
and Onal, 2012).
The bioactivity of a protein depends upon its correct folding
into distinctive secondary and tertiary structures and this rep-
resents the bottleneck in terms of industrial processing of dena-
tured proteins (Sanchez-Trasvi~ na et al., 2015). A number of
processes including dialysis, dia-filtration, gel-filtration, chro-
matography etc. are required to address this issue (Reh et al.,
2007). Since PEG has been shown to enhance accurate protein
folding (Cleland and Wang, 1990), an ATPS comprising of
PEG as the polymer may be used for industrial processing of
denatured proteins. In fact, a 60.8% recovery was obtained with
a refolding of 59.7% in a PEG – potassium phosphate system
(Sanchez-Trasvi~ na et al., 2015).
The industrial utility of an extracted enzyme can be gauged
from its bio-chemical properties after purification. Invertase’s
bioactivity and stability was determined after its partitioning in
a PEG – sodium sulfate ATPS with the optimal pH and temper-
ature turning out to be 5.0 and 50 C, respectively, in this regard
(Y€ €
ucekan and Onal, 2012). The protein established its extreme
stability at a high temperature and pH range (25 to 50 C and
4.0 to 7.5, respectively). Moreover, after storage at 4 C for two
months, it was found to have vanished just 47% of its original
enzymatic activity (Y€ €
ucekan and Onal, 2012). Such stability
and bioactivity displayed by invertase at an extensive range of
temperature and pH is a good omen for the food and beverage
industry. Invertase partitioned with a PEG – magnesium sulfate
system has also been tested for its stability and activity wherein
a pH of 5.5 and a temperature set at 60 C were found as ideal
€
in this regard (Karkaş and Onal, 2012). At a pH range of 4.0 to
7.0 and a temperature ranging from 4.0 to 50 C, it was found
highly stable retaining 95% and 98% of its original activity,
€
respectively (Karkaş and Onal, 2012).
The gains for manufacturers, and ultimately consumers
through cheap affordable foods and beverages, can be further
maximized by effectively utilizing the byproducts and wastes
from the food and beverage industry. Particularly, the yeast-
waste of brewing process accounts for the second most abun-
dant byproduct of the brewery industry (de Ara ujo et al., 2011).
Such waste is usually discarded after a few fermentation pro-
cesses so that the product may be free from the spiteful smell
and flavor. A PEG – potassium phosphate system was
employed for the recovery of invertase from brewery yeast
waste with promising results in regard to yield (66.2%) and
purification factor (29.5) of the enzyme (Leon-Gonzalez et al.,
2016).
Invertase is an important enzyme in food processing and
brewing. The amount of reported works utilizing ATPS as an
alternative to the inefficient and costly procedures, currently in
vogue, highlights the utility of this system for the industrial sep-
aration and purification of this enzyme. ATPS extracted inver-
tase has been found to be highly stable retaining most of its
original activity. Furthermore, the byproducts or wastes of the
food industry and brewery can be used as an invertase source
in ATPS which will further enhance the value of such deriva-
tives and reduce the product costs of these industries. In addi-
tion, invertase partitioning in ATPS can be enhanced by the
addition of various supplementary salts. However, as discussed
earlier, citrates represent a more convenient option as a phase
forming salt due to its enhanced partitioning effect and non-
hazardous nature which warrants the characterization of inver-
tase partitioning in such an ATPS.
3.6. a-Amylase
The industrially important hydrolysis of starch into lower
molecular weight compounds like maltotriose, glucose and
maltose is catalyzed by a-amylases (Gupta et al., 2003; Kandra,
2003; Rajagopalan and Krishnan, 2008). They represent an
important group of industrial enzymes accounting for around
25% of all the commercial enzymes in the world (Rajagopalan
and Krishnan, 2008; Reddy et al., 2003). In fact, the chemically
hydrolyzed starch is now a rare sight in the respective industry
and a huge number of microbial amylases are currently being
used for the commercial starch hydrolysis (de Souza and de
Oliveira Magalh~aes, 2010). The food industry widely employs
amylases in a range of processes, for instance baked foodstuffs,
brewed beverages, gastrointestinal aids, cakes, juices and starch
syrups (Couto and Sanroman, 2006). In the baked-food indus-
try, a-amylase is used to enhance the fermentation rate and to
reduce the viscosity of dough, thereby improving the product’s
volume and appearance. Besides, the bread’s flavor, color and
toasting qualities are greatly enhanced because of the extra

sugar generated in the dough as a result of a-amylase addition.
Moreover, it also augments the softness retention of baked-
foods, enhancing their shelf-life (Gupta et al., 2003; Van Der
Maarel et al., 2002). Amylases find use in brewing for clarifying
beer and/or fruit juices, and as a pretreatment for improving
digestibility of animal feed fiber (Gavrilescu and Chisti, 2005;
Ghorai et al., 2009; Van Der Maarel et al., 2002).
Such an extended industrial demand of this enzyme calls for
an industrially feasible isolation process like ATPS (Table 9).
One such effort in this direction employed an ATPS constituted
by PEG and MgSO4 (He et al., 2005). The partitioning and
extraction of the enzyme was studied in relation to the concen-
tration and molecular weight of PEG, pH of the system, and
the concentrations of MgSO4, and NaCl. Maximum amount of
a-amylase was recovered from the lower phase, while it was
found that the extraction and partition of this protein was not
significantly affected by the concentration and molecular
weight of PEG. On the other hand, an increase in MgSO4 con-
centration slightly increased the extraction rate, and an
enhanced extraction was also noted with an increase in pH.
NaCl at a weight by weight concentration of 5%, nevertheless,
considerably enhanced the extraction rate, while the same sur-
passed 80% at NaCl concentration of above 8% (He et al.,
2005).
Similarly, another group of researchers (Zhi et al., 2004) also
testified the effect of NaCl addition on the purification of
a-amylase in PEG – citrate ATPS wherein the purification fac-
tor increased from 1.8 with 90% yield (no NaCl) to 2.0 with
80% yield (at NaCl concentration of 4%). The amplified poten-
tial variance amid the upper and lower phase brought about by
the differential segregation of NaC and Cl¡ between the two
phases may be regarded as a cause for the augmented extraction
rate. An adjustment in the concentration of NaCl can, hence, be
utilized for the efficient separation and extraction of a-amylase.
A purification factor of 5.4 has also been reported for this
enzyme in a PEG – phosphate ATPS wherein a 45.2% enzy-
matic activity was observed in the salt rich phase (Bezerra et al.,
2006). The effect of additional salt concentration on the purifi-
cation of a-amylase is also evident from the study reported by
Kammoun and coworkers (Kammoun et al., 2009), though the
supplementary salt used here was KCl instead of NaCl. Specifi-
cally they reported that the highest purification factor of 3 with
a yield of 67% was observed at 4 C when the pH was set to 6,
Table 9. Purification of a-amylase by employing different types of ATPSs.
ATPS Results Reference
PEG – MgSO4 Effect of PEG molecular weight and (He et al., 2005)
concentration on partitioning was
minimal; extraction increased with
increase in MgSO4 concentration and
pH; supplementary salt favored
purification
PEG – citrate purification factor increased from 1.8 with (Zhi et al., 2004)
90% yield (no NaCl) to 2.0 with 80%
yield (at NaCl concentration of 4%)
PEG – phosphate purification factor of 5.4; 45.2% enzymatic (Bezerra et al.,
activity 2006)
PEG – citrate purification factor of 3; yield of 67%; (Kammoun
supplementary salt improved et al., 2009)
purification
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 3173
the concentration of PEG 8000 was maintained at 18% and that
of the buffer (citrate) at 8%, and the supplementary salt (KCl)
was used in a concentration of 3% (Kammoun et al., 2009).
a-Amylase finds extensive use in the food industry and
ATPS can be employed to facilitate its cheap and immediate
availability to this sector. The effect of supplementary salt addi-
tion on its partitioning in different forms of ATPS is quite evi-
dent. Moreover, this enzyme favors the lower salt-rich phase
and does not seem to be significantly affected by PEG molecu-
lar weight and concentration in terms of its partitioning.
3.7. b-Glucanase
The cell wall of barley and other endosperm cereals is mainly
composed of b-1,3-1,4-glucan, which in turn is made up of a
monomeric glucose polymer, the components of which are irreg-
ularly linked through b-1,3- and b-1,4-glycosidic bonds (Edney
et al., 1991; Woodward et al., 1983). Because of its distinctive
configuration, the high molecular weight barley b-glucan forms
a highly viscous solution when dissolved in water. This severely
hinders the filtration process in beer formation as high molecular
weight b-glucan is produced during mashing, and might even
result in beer having glutinous precipitates (He et al., 2005).
Moreover, barley-fed piglets and hatchlings may encounter diffi-
culty in feed digestion and nutrients absorption as a result of the
extraordinary duodenal viscosity caused by b-glucan (Planas,
2000). Providentially, these adverse effects can be countered by
adding an exogenous enzyme during the process of mashing or
the preparation of feed (Godfrey and Reichelt, 1982; Stone and
Clarke, 1992; White et al., 1983). b-glucanase of selected micro-
bial origin possess identical substrate affinity to that of the same
enzyme from barley, thereby capable of precise barley b-glucan
hydrolysis (He et al., 2005).
Usually for the industrial production of Bacillus b-gluca-
nase, unprocessed crude materials are used. Orthodox separa-
tion procedures like centrifugation and filtration find it difficult
to efficiently purify the enzyme because the subsequent broth is
highly viscous and contains solid residues. Moreover, b-gluca-
nase production is accompanied by a-amylase and neutral pro-
teases formation and, hence, it needs to be selectively purified.
To add further, the scaling up of these procedures is also not
feasible due to the presence of particulate matter in the slurry
(mostly bacterial cells), compressible filter-cakes and the
broth’s viscosity (Schmidt et al., 1994). ATPS, henceforth, can
offer a helping hand in the industrial separation and purifica-
tion of b-glucanase.
In fact, this technique has been successfully employed to
develop a two-step purification method for b-glucanase
wherein an enhanced specific enzymatic activity of
14027 U/mg (6.6 times higher than that of the entire broth)
with 65.3% recovery-rate was observed (He et al., 2005). A PEG
– MgSO4 ATPS was tested in relation to the effects of the
molecular weight and concentration of the polymer, and the
concentration of the phase forming salt on enzyme’s separation
and purification. The effects of the system’s pH and the concen-
tration of supplementary salt (NaCl) in this regard were also
monitored. b-Glucanase partitioning and extraction was drasti-
cally affected by PEG molecular weight with lower molecular
weight polymer resulting in higher partition-coefficients and
3174 B. M. KHAN ET AL.
extraction-rates. On the other hand, the polymer’s molecular
weight produced negligible effects in respect of a-amylase’s and
neutral proteases’ partition-coefficients and extraction-rates.
Similarly, an enhanced partition-coefficient (2.3 times) and
extraction-rate (from 26.8% to 75.7%) was observed with the
increase in PEG concentration while such an increase virtually
failed to affect the partition and extraction of a-amylase and
neutral proteases. Conversely, an increase in NaCl concentra-
tion significantly affected the partition and extraction of
a-amylase and neutral proteases while the same had minimal
effects in respect of b-glucanase partitioning and extraction
(He et al., 2005).
From this comprehensive work it is quite clear that for effi-
cient partitioning and extraction of b-glucanase, and for its sep-
aration from the co-produced a-amylase and neutral proteases
a higher concentration of low molecular weight PEG with a
proportionate concentration of NaCl work wonders in a PEG –
MgSO4 ATPS. Further studies, however, with different types of
ATPSs are needed for confirming the effects observed in this
study and to provide a solid base for the industrial purification
of this important enzyme using this system.
4. Conclusions and Recommendations
The food industry is one of the most important
manufacturing sector, not only because of its inherent sig-
nificance in catering to the dietary needs of the population
but also due to its prominence in the national economy.
The ever-growing demand from consumers and the vibrant
competition herein warrants new and efficient production
techniques to be employed. This industry widely utilizes
proteins in the form of food ingredients and supplements
as well as catalysts in a number of food processing steps.
Downstream processing for the purification of proteins is a
tedious job due to their intricate nature, and the currently
in vogue practices for this purpose, unfortunately, are both
expensive, and labor- and time-intensive in addition to
being disruptive for proteins structural conformity. ATPS
has gained a lot of interest from the scientific community
due to its mild operating conditions, high recovery yield,
ease of scaling it up, and its cost-effective and environment
friendly nature. In fact, this technique has been put to use
by various researchers to achieve the separation, concentra-
tion and purification of proteins, and their results are quite
promising. This review tried to compile some reports
regarding the use of ATPS for the separation and purifica-
tion of proteins utilized in the food industry. Some promis-
ing indications in this regard were observed along with a
few loopholes which need to be addressed before this tech-
nique can truly demonstrate its potential vis-
a-vis industrial
protein purification. All in all, a polymer – salt ATPS can
be regarded as a better option for purifying proteins. A
polymer – citrate salt system might well be recommended
based on the superior partitioning observed in such setting
and due to no environmental safety concerns associated
with citrates. Specifically, the polymer’s molecular weight
and concentration, except in a few cases, does not seem to
alter the partitioning of proteins. The choice and concentra-
tion of the phase-forming salt, on the other hand,
significantly affects this phenomenon along with the pH of
the system. Moreover, the addition of an inert supplemen-
tary salt enhances protein partitioning in an ATPS. The
amplified potential variance amid the upper and lower
phase brought about by the differential segregation of cati-
ons and anions between the two phases may be regarded as
a cause for the augmented extraction rate. Furthermore,
protein-salt interactions may result in enhanced hydropho-
bicity of proteins, thereby accelerating their partitioning.
ATPS also has the potential for improving the market value
of industrial byproducts by utilizing them as a source of
proteins to be extracted and purified for use in the food
industry. In the case of an important food enzyme, inver-
tase, it has been found that ATPS-purified enzymes are
highly stable at wide pH and temperature ranges and that
they are able to retain most of their initial bioactivity. This,
however, cannot be assumed with certainty for the other
proteins because of an absence of scientific undertakings in
this direction. Such studies where the stability and activity
of proteins partitioned in an ATPS are characterized, hence,
may be recommended.
Declaration of Interest
None to declare.
Funding
This work was financially supported by grants from the National Natural
Science Foundation of China (21476135 & 81741167), Educational Com-
mission of Guangdong Province, China (2016KZDXM014), Ocean and
Fisheries Administration Project of Guangdong Province, China (2017)
and the National Natural Science Foundation of Guangdong Province,
China (2017A030307014).
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