Abstract and keywords
Abstract (English):
Introduction. The article provides a review of technologies for membrane fractionation of various hydrolyzed food substrates in membrane bioreactors (MBR). In food industry, MBRs are popular in functional food production, especially in the processing of whey, which is a very promising raw material due to its physicochemical composition. Study objects and methods. The research was based on a direct validated analysis of scientific publications and featured domestic and foreign experience in MBR hydrolysis of protein raw material. Results and discussion. The MBR hydrolysis of proteins combines various biocatalytic and membrane processes. This technology makes it possible to intensify the biocatalysis, optimize the use of the enzyme preparation, and regulate the molecular composition of hydrolysis products. The paper reviews MBRs based on batch or continuous stirring, gradient dilution, ceramic capillary, immobilized enzyme, etc. Immobilized enzymes reduce losses that occur during the production of fractionated peptides. Continuous MBRs are the most economically profitable type, as they are based on the difference in molecular weight between the enzyme and the hydrolysis products. Conclusion. Continuous stirred tank membrane reactors have obvious advantages over other whey processing reactors. They provide prompt separation of hydrolysates with the required biological activity and make it possible to reuse enzymes.

Milk proteins, whey proteins, hydrolysis, membranes, enzymes, membrane reactor, substrate
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Balanced diet and natural food quality are the most
important issues of contemporary food science [1–4].
Environmental pollution and such diet-related diseases
as hypertension, diabetes, allergies, etc., require new
types of diet and functional products [5–8]. Modified
milk and whey proteins can serve as basic components
of functional foods [9–13]. Enzymatic hydrolysis of
dairy proteins is the most popular method of whey
modification, which makes it possible to impart
additional functional and technological properties,
e.g. emulsifying, foaming, antioxidant, antihypertensive,
immunomodulatory, etc. [14, 15].
Whey proteins and their hydrolysates possess high
nutritional value, which makes them the most promising
components for diet therapy products. Whey proteins
owe their useful functional properties to bioactive
peptides [16, 17]. Bioactive peptides are amino acid
sequences, encoded in the primary structure of native
proteins. A protein hydrolyzate contains a mix of
biologically active and inactive peptides, in addition to
non-hydrolyzed proteins. Fractioning can isolate certain
biologically active peptide fractions from hydrolysates.
Fractioning relies on such membrane separation
processes as ultrafiltration and microfiltration [18–22].
Membrane separation means that two or more
components are separated through a membrane that
acts as a selective semipermeable barrier that partially
or completely stops one or more substances. The
retained components produce retentate, while those that
pass through the membrane form permeate [23, 24].
Membrane processes have several advantages over
other separation methods. First of all, they require less
energy than evaporation or distillation. Second, they
demonstrate high selectivity and are easy to scale.
Finally, they are material friendly, which is a very
important factor for food industry [24]. 

Development and design of new membrane
bioreactors (MBR) is one of the most promising
and dynamic areas of industrial biotechnology.
MBR technology combines various membrane and
biochemical separation processes, the latter being
induced by a catalyst of biological origin, i.e. an enzyme.
The main advantage of MBR enzymatic hydrolysis
is that it saves expensive enzyme preparations and
regulates the molecular composition of hydrolysis
products by combining membranes with a recommended
molecular weight cut-off [18].
Unfortunately, contemporary food industry uses
only about 50% of the whey produced worldwide,
which means that the task of whey recycling is yet to be
solved. This issue remains controversial and requires
comprehensive research. The present review describes
how various whey processing MBRs can increase the
value of whey components [25].
The present research was based on a direct validated
analysis and featured the most recent domestic and
foreign publications on protein hydrolysis in various
membrane reactors.
Figure 1 illustrates two most common membrane
reactors (MBR). In the first type, the membrane
controls the mass transfer of the substrate and
enzyme preparation to and from the reactor module,
thus producing an indirect effect on the hydrolytic
degradation of the substrate (Fig. 1a). In the other
type, the reaction occurs at the membrane level and
complements the regulation of substrate and enzyme
mass transfer [26, 27]. Complex as it is, MBRs of the
second type makes it possible to control proteolysis at
the cellular level (Fig. 2b) [26, 27].
Such MBRs are called biocatalytic because the
membrane itself acts as a catalyst. They are based on
continuous stirring: the product either passes through
the membrane, which retains the enzyme and returns it
to the reactor, or remains in the membrane module. The
biocatalyst is immobilized and separated by a membrane
in the reaction vessel [26, 28]. As a rule, the membrane
immobilizes the enzymes on membranes because
biomolecules are covalently attached to the surface of
the carrier. As a result, the system is more stable, and
the microreactor can be reused while the enzyme is no
longer active. The covalent attachment of enzymes to
solid substrates is very strong and increases the service
life of the microreactor and immobilized enzymes [29].
The numerous advantages of these MBRs make them
an alternative to simple bioreactors. The most important
advantage is that the catalyst (enzyme) can be recovered
and reused in a continuous system, which increases
the efficiency of the process. The yield rises, while the
expensive enzyme preparation is spared, which lowers
the cost of the final product. In addition, the selective
removal from the reaction medium is continuous,
and the supply of the reagent to the catalytic reaction
medium is easy to control [26].
Ultrafiltration is the most common separation
process used in this type of MBR. Unfortunately,
polarization remains its main disadvantage:
eventually, the membrane pores get clogged. Nearly
all membrane filtration processes gradually decrease,
as trapped particles accumulate on the surface of the
а) membrane bioreactor б) biocatalytic membrane reactor
Figure 1 Schematic illustration of membrane reactors
Biocatalyst that passed along
the membrane
Biocatalyst segregated with
the membrane
Built-in biocatalyst
Gelated biocatalyst
Adsorption Ionic bond
Covalent bond Cross-linking
Molecular recognition
Bound biocatalyst
Ryazantseva K.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 271–280
membrane. The rate depends on the operation type
of the membrane, the nature of the flow, the pore size,
and charge of the membrane. The flow decreases
because of certain physical or chemical interactions
that occur between the interface of the membrane
and the components of the feed stream. The formation
rate of the surface layer has to be controlled, as it
keeps accumulating on the side of the membrane that
experiences excess pressure. No pre-treatment can
prevent clogging, and the membrane has to be cleaned
regularly [26].
In a biocatalytic MBR, the membrane not only
separates but also catalyzes. The enzyme enters the
membrane matrix and is immobilized there (Fig. 1b),
increasing its stability, which is another advantage
of this type of MBR [30]. Immobilization increases
the stability of enzymes during storage, namely, their
resistance to changes in temperature and pH [31].
In their study of continuous MBRs, Wang et al.
focused on transglutaminase, which was covalently
bound to the surface of the polyethersulfone
membrane. The enzyme cross-linked α-lactalbumin
and β-lactoglobulin, thereby retaining them on the
membrane [32]. Using transglutaminase for enzymatic
modification of milk protein can prevent protein loss
during whey processing and increase the biological
value of the product [33]. During whey ultrafiltration,
α-lactalbumin and β-lactoglobulin can pass through the
membrane under transmembrane pressure, in which
case they block the pores or penetrate into the filtered
solution. As a result, β-lactoglobulin is the main cause of
membrane clogging during whey filtration [34–36].
Wang et al. studied an enzymatic MBR with
transglutaminase, its efficiency, the catalysis of protein
crosslinking, and its separation from whey. The protein
recovery rate reached 85%, but it decreased over time,
as did the relative membrane flow, probably, following
the decrease in enzymatic activity on the membrane
surface after 1365 min of continuous operation. The
overall specific performance of the enzyme bound
membrane was about 50% less than that of the pure
polyethersulfone membrane. Wang et al. concluded
that the efficiency failed because of the repulsion forces
that appeared between the cross-linked proteins and the
membrane [32].
Vasileva et al. studied β-galactosidase that was
covalently bound by glutaraldehyde to the surface of the
modified polypropylene membrane. They determined
the optimal hydrolysis conditions for lactose in a batch
MBR: enzyme activity 13.6, temperature 40°C, pH 6.8,
time 10 h. The scientists compared the resulting
degree of hydrolysis with that obtained by a free nonimmobilized
enzyme. The immobilized enzyme method
proved 1.6 times more effective than the one based
on a free enzyme, as the immobilized enzyme itself
was twice as stable as the free enzyme. The resulting
immobilized β-galactosidase/polypropylene membrane
system was used to obtain glucose-galactose syrup from
whey waste. Vasileva et al. carried out hydrolysis of
whey lactose in a MBR using an immobilized enzyme
and a spiral membrane. The optimal membrane surface
and the whey flow rate were 100 cm2 and 1.0 mL/min,
respectively. After 10 h, the lactose hydrolysis reached
91%. After cycle 20, the yield was 69.7% [37].
Sen et al. focused on skim milk hydrolysis in a
batch MBR using β-galactosidase immobilized on a
polyethersulfone membrane with a pore diameter of
30 kDa. The study featured aqueous solutions of
skim milk in the concentration range of 30–80 kg/m3.
The solutions underwent deproteinization through
two membrane ultrafiltration modules with pore sizes
30 kDa and 5 kDa. As a result, 95–97% of lactose
became permeate. The permeates obtained were
subjected to hydrolysis in a batch MBR equipped with
an enzyme-immobilized membrane. The enzyme was
immobilized by cross-linking on an ultrafiltration
membrane using 3 and 4% glutaraldehyde. The 4%
glutaraldehyde solution provided a greater enzyme
activity retention (94.2%) and enzyme loading (98%).
The final conversion of lactose was 45.2 and 21.4%
when β-galactosidase was immobilized with 4 and 3%
glutaraldehyde, respectively. The control experiment
with an immobilized enzyme showed a significant
decrease in the flow of pure water: 27.5 for 3%
glutaraldehyde and 67.5 for 4% glutaraldehyde [38]
When the biocatalyst is confined to the membrane
module, not the reservoir with the reagents, it is not
recirculated into the outlet flow; with that, low molecular
weight products and inhibitors leave the system directly
through the membrane. This type of MBR finds
application in bio-artificial pancreas or extracorporeal
detoxification devices [26].
Biocatalytic MBRs are undoubtedly more efficient,
since both the reaction and the separation occur
in the same membrane module. However, current
knowledge about the nanoscale processes within
the microenvironment of the membrane remains
insufficient. Equally lacking is the knowledge about
the control of continuous hydrolysis at the macroscopic
level. As a result, biocatalytic MBRs cannot be used for
commercial production [39–41].
Biocatalytic MBRs, or bioreactors, are integrated
with such membrane processes as microfiltration,
ultrafiltration, reverse osmosis, membrane extraction,
etc. They are especially effective for food and beverage
production, e.g. wine, fruit juices, milk, etc. [42, 43].
In the dairy industry, MBRs were first used to produce
low lactose milk [43]. Such MBRs are still widely used
to produce functional products for patients with lactase
deficiency. However, lactose is not the only substance
that causes milk intolerance: some people cannot absorb
high molecular proteins (≥ 5 kDa) due to inadequate
immune response. MBRs are also used to produce lowallergenic
milk [44].
MBRs are getting more popular in food industry
as a result of industrial demand for functional foods,
e.g. hypoallergenic, nutraceutical, or alternative foods,
ingredients that are part of dietary and preventive
Ryazantseva K.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 271–280
menus, etc. MBRs are actively used in whey processing.
The physicochemical composition of whey makes it
a very promising raw material for functional food
production. Whey contains 0.4–0.8% of protein and
4.4–5.5% of lactose. Whey proteins possess a good latent
potential of biofunctional properties [43].
Batch MBRs are simple enough to gain extensive use
in the production of protein hydrolysates. However, they
need a lot of enzyme, energy, and labor, which makes it
expensive [19]. American scientists from the Department
of Food Science (Pennsylvania, USA) attempted to
process food substrates using batch-type enzyme
reactors with an immobilized enzyme. They identified
a number of additional disadvantages, e.g. high losses
in the activity of the biocatalyst, the expensive enzyme
immobilization, etc. [44].
Continuous stirred tank membrane reactors
(CSTMR) are an alternative to batch MBRs. They are
based on the difference in molecular weight between
the enzyme and the hydrolysis products. CSTMRs
can separate products from the reaction medium to
increase the yield. The soluble enzyme is confined to
the retentate side of the membrane, where it comes in
contact with the substrate. CSTMRs make it possible
to reuse the enzyme and select a suitable membrane
pore size, which facilitates the control of the molecular
weight of the final product [44].
Ewert et al. used a two-stage enzymatic membrane
bioreactor (EMBR) to obtain sodium caseinate
hydrolyzate with improved antioxidant capacity and
reduced bitterness (Fig. 2) [44]. At the first stage, sodium
caseinate was hydrolyzed at 65°C and pH 6.7 using
endopeptidase Sternzym BP 25201. The stage took 12 h
and involved hydrolysis and filtration through a ceramic
ultrafiltration membrane made of hollow fiber with a
molecular weight cut-off of 10 kDa. The antioxidant
activity of the resulting permeate increased by 33%,
compared to sodium caseinate. The volume of permeate
that left EMBR-1 was automatically compensated for by
adding a new substrate to the reactor vessel.
At the second stage, the main objective was to
remove bitterness. The hydrolysis was carried out
in EMBR-2 using Flavorzyme at 50°C and pH 6.7.
After 12 h of hydrolysis, it was filtered through a UV
polyethersulfone membrane with a molecular weight cutoff
of 10 kDa. EMBR-2 also increased the antioxidant
capacity of the permeate to its half-maximal inhibition
concentration (IC50) of 13.8 μg/mL, which was 39%
more than that of sodium caseinate. The experiment
made it possible to avoid the mutual effect of peptidases
by separating endo- and exopeptidases at the two stages
of hydrolysis. The selected conditions proved optimal
and ensured a stable production for three days. The
research featured the degree of hydrolysis of biocatalysis
products. The hydrolyzate obtained in EMBR-1 had the
following parameters: degree of hydrolysis – 8.0 ± 0.2%,
permeate – 8.7 ± 0.4%, sediment fraction – 2.9 ± 0.3%.
The permeate hydrolyzed in EMBR-2 had a degree
of hydrolysis of 21.8 ± 0.8%. The loss of enzymatic
activity in both reactor vessels was compensated by the
daily addition of the corresponding enzyme. The whole
process took 110 h [45].
Due to the applied temperature, the relative activity
of peptidase in EMBR-1 decreased to 82 ± 6.9% of its
initial value during the preliminary hydrolysis. As for
EMBR-2, its initial activity remained the same during
the preliminary hydrolysis (26–38 h) and decreased to
82% after 24 h of filtration (38–62 h). The two reactors
maintained stable conditions because the activities
were adjusted every 24 h. The experiment proved that
CSTMRs can be used for commercial production of
functional antioxidant ingredients based on sodium
caseinate [45].
Guadix et al. studied hydrolyzate production of
hypoallergenic whey [44]. The research objective was
Figure 2 Block diagram of a two-stage installation of a two-stage enzymatic membrane bioreactor with continuous hydrolysis
Enzymatic membrane bioreactor – 1
Retentate 1
Substrate Enzymatic
UV-filtration Heating
Sedimentation Sediment
Permeate 1
Enzymatic membrane bioreactor – 2
Retentate 2
Permeate 2
hydrolysis UV-filtration
Ryazantseva K.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 271–280
to create a stable long-term process for the production
of whey protein hydrolysates with low antigenicity. The
study was based on other scientific schools of continuous
hydrolysis. For instance, specialists from the University
of Illinois (USA) studied continuous hydrolysis of
soy protein from Promin-D in a CSTMR with hollow
membrane fibers. At the initial stage, the conversion rate
was 90%, which dropped to 60% after 10 h because of
the leakage of the enzyme through the membrane and
thermal deactivation. The Illinois team also studied
milk protein hydrolysis. They hydrolyzed casein with
alkalase, also in a CSTMR with hollow fibers. Their
experiments determined the efficiency of the reactor at
50 and 37°C. After a 15-h fermentative treatment, the
degree of conversion dropped from 96 to 62% at 50°C
and from 75 to 51% at 37°C. Like in the first case, the
efficiency fell down because of enzyme leakage, thermal
deactivation, and enzyme-membrane interactions.
French scientists studied the effect of operating
variables on the performance of hollow fiber CSTMRs
for hydrolysis of blood plasma proteins using alcalase.
After 35 h of operation, the permeate flow dropped due
to membrane clogging, which occurred as a result of
the polarizing layer that accumulated on the membrane
surface. Spanish and Colombian biochemists hydrolyzed
whey proteins with alcalase using the same CSTMRs
with hollow fibers. They managed to maintain an
uninterrupted process only for 7 h because of the
rapid clogging and the inactivation of enzymes. Both
the proteolysis regimes and the design features of the
membranes obviously needed correction.
A team from Taiwan managed to maintain
uninterrupted operation for 16 h. In addition to alcaslase,
they also included Flavuerzyme into the enzyme
preparation. The Laboratory of New Dairy Technologies
(France) used CSTMRs to obtain specific bioactive
peptides by hydrolysis of casein-macropeptide.
Cow’s milk whey is not the only type of whey in
such studies. Cambridge specialists studied hydrolysates
of goat whey from the point of view of the formation of
biologically active peptide compounds. Goat whey was
hydrolyzed with pepsin in an enzymatic reactor. The
ultrafiltration polymer membrane was combined with a
mineral membrane with a cut-off of 30 kDa. Peptides in
the permeate were separated by reversed-phase HPLC,
which is the most common method for separating
milk peptides [46, 47]. As β-lactoglobulin is resistant
to pepsin, most opioid and antihypertensive peptides
were derived from α-lactalbumin. Pepsin exhibited a
considerable substrate specificity; the molecular weights
of the obtained peptides ranged from dipeptides to very
large peptides with disulfide bridges (150–6900 Da). As
a result of the α-lactalbumin hydrolysis, the amount of
peptides with a molecular weight of ≤ 600 Da was 36%,
600–2000 Da – 24%, and ≥ 2000 Da – 40%.
Guadix et al. hydrolyzed diluted milk whey
concentrate (50 g protein/L) in a CSTMR at 50°C and
pH 8.5 using Protex 6L bacterial protease obtained from
Bacillus licheniformis. The design of the membrane
reactor included a 3-L vessel, an automatic controller
of pH and temperature, a recirculation pump, and
a frame membrane ultrafiltration module with a
polyethersulfone plate with an effective area of 0.07 m2
and a molecular weight cut-off of 3 kDa. The reaction
mix was continuously recirculated at a rate of 1.5 L/min
with a pump at a rate of 0–15 L/min. The pump was
installed between the reaction vessel and the inlet of the
membrane module.
As a result of membrane clogging, the permeate
flow dropped from 10 mL/min to 6.3 mL/min after
16 h. After 10 h of operation, the degree of hydrolysis
stabilized at about 80%, while the permeate flow
stabilized after 13 h. As the permeate flow decreased
during the first 13 h, the enzymes demonstrated signs
of thermal inactivation. The resulting hydrolyzate
contained peptides that consisted of four amino acids.
The content of antigenic whey protein decreased by
99.97% in the final product, which means that it can
be used in hypoallergenic diets, baby food, and enteral
feeding. However, the authors had to compensate for the
loss of enzymatic activity by feeding small amounts of
fresh enzyme [44].
O’Halloran et al. developed an EMBR in which
the whey protein isolate was subjected to enzymatic
hydrolysis to obtain antidiabetic peptides that inhibit
dipeptidyl peptidase-IV (DPP-IV). The efficiency grew
Figure 3 Method of gradient dilution feeding substrate in an enzymatic membrane reactor
Dairy protein
dilution feeding
Optimal feeding mode
Stable hydrolysis
in a continuous
stirred tank
Enzymatic efficiency
Ryazantseva K.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 271–280
by 7.2 and 8.7% when using Protamex and Korolase
2TS, respectively, compared to the standard method of
batch processing. Previously, neither of the enzymes
was considered effective for obtaining peptides with
antidiabetic activity. Protamex and Korolaza 2TS proved
capable of producing peptides that inhibit DPP-IV. The
permeate hydrolyzate obtained with Protamex showed a
33.7% higher DPP-IV inhibition value compared to the
hydrolyzate obtained using Korolase 2TS. J. O’Halloran
and colleagues proved that Protamex can be used to
produce protein substrates with antidiabetic activity [48].
Huang et al. used a CSTMR to improve the yield of
peptides that inhibit angiotensin-converting enzyme
from milk protein. The research employed a new
method of gradient dilution feeding substrate (GDFS)
(Fig. 3) [49]. The scientists compared the stability
of the hydrolysis process, enzymatic efficiency, and
kinetics of the method with the traditional modes of
feeding, when adding water after feeding the substrate,
or feeding the substrate with a constant concentration.
The GDFS method showed the highest membrane
flow rate and the lowest fluctuations in the protein
concentration in the reactor. GDFS also had a higher
rate of protein hydrolysis, which increased by 67.58%.
The yield of peptides reached 138.51 g/g neutrase, and
the angiotensin-converting enzyme inhibitory activity
of hydrolysates was 0.74 mg/mL. The optimal operating
time was 720 min. The GDFS method can serve as
an alternative method for obtaining highly efficient
bioactive peptides [49].
German researchers developed a stable process for
obtaining specific hydrolysates with selected biological
properties. They developed and tested a continuous
reactor system with a ceramic capillary module
with various combinations of enzymes and protein
substrates (Fig. 4) [49]. Alcalase was immobilized on the
surface of capillaries modified with aminosilane with a
pore size of 1.5 μm. The loading capacity was 0.3 μg of
enzyme per 1 mg of capillary with a residual enzyme
activity of 43%. They tested controlled hydrolysis
of casein, sunflower, and lupine isolates. Casein
hydrolysates proved to possess the largest amount of
peptides with enhanced biological properties [50].
A continuous reactor consists of a ceramic capillary
with one enzymatic filler. The filler is made of yttriumstabilized
zirconium oxide. It is fixed in a special
stainless steel casing (Fig. 4). In a way, this system
is a plug flow reactor system. The protein solution is
pumped through the capillary module with a peristaltic
pump. The capillary module is part of the column oven,
which makes it possible to keep the temperature at 37°C.
The end of the capillary is sealed with cyanoacrylate
cement to inject the flow from the intracapillary
space into the extracapillary space. The enzyme is
immobilized on the activated surface of the ceramic
capillary with an APTES linker. The protein moves
through ceramic capillaries by forced convective flow.
The immobilization makes it possible to use the entire
available capillary surface. As a result, enzymes can
be immobilized on the inner and outer surfaces, as well
as on the pore walls. One capillary is 10 cm long and
has an outer diameter of 1.8 mm, an inner diameter of
1 mm, and an average pore size of 1.5 μm. The ceramic
capillary was replaced with a new immobilized enzyme
to prevent protein contamination. The residence time of
the substrate appeared to be inversely proportional to the
flow rate: the longer the residence time of the substrate
in the capillary filled with the enzyme, the higher the
continuous yield. These continuous reactors produced
specific peptides with the desired biologically active
properties [50].
New combined hypoallergenic functional products
need new methods of gluten reduction. For example,
MBRs can be used for wheat processing to create dairy
products fortified with vegetable protein, but with
hypoallergenic proteins and a low content of lactose
and gluten.
Merz et al. developed a 96-h continuous hydrolysis
of wheat gluten with flavurzim in an EMBR [51].
Figure 4 Capillary module that immobilizes enzymes on a ceramic substrate APTES
Protein substrate
Ceramic capillaries
APTES linker
Protein Peptides
Cyanoacrylate cement
Ryazantseva K.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 271–280
Temperature, pump load, and enzyme flow through
the membrane were the main criteria for hydrolysis
stability and direction. The scientists optimized the
hydrolysis to maximize the space-time yield. For
microbial stability, they included 8% ethanol with a
substrate concentration of 100 g/L at 37°C and pH 7.5
for 96 h (Fig. 5) [51].
A diaphragm pump (P 1) circulated one liter of
substrate. The flow rate was 3.3 L/min. Hollow fiber
ceramic membranes were 45 mm in length, 6 mm
in diameter, and 0.0085 m2 in surface area. They
performed cross-flow ultrafiltration of hydrolysates (F 1)
on a membrane with a pore size of 1, 5, or 10 kDa.
The hydrolyzate inside the reactor was stirred using
a magnetic stirrer (R 2). A constant transmembrane
pressure of 2 Bar was adjusted with a ball valve (V 1)
and measured with barometers (PI 1, PI 2). The substrate
was fed continuously using a tubular pump (P 2). The
feed container was kept in an ice bath during the entire
test [51]. This EMBR hydrolysis scheme can be costeffective
in the industrial production of hydrolysates
from grain proteins.
Russian specialists also developed a CSTMR
that produced a hydrolyzate of whey proteins with
low residual antigenicity. The installation was based
on enzyme preparation alcalase 2.4 L (Fig. 6) [52].
Hydrolysis products were accumulated in an enzymatic
medium, which was followed by membrane separation
into a purified hydrolyzate (permeate) and an insoluble
residue (retentate). The experiment aimed at complete
separation of the enzyme to keep it active inside the
reactor core.
The scientists reproduced the process described
in foreign publications, i.e. protein hydrolysis,
combined with the separation of hydrolysis products on
ultrafiltration membranes. The resulting hydrolyzate
had a low solids content (1.5%). The technology proved
commercially unprofitable and expensive. The low solids
content resulted from the low cut-off of membranes
(5 and 10 kDa). In this case, a portion of hydrolysis
products was retained by the elective membranes and
remained in the concentrate. Another disadvantage
of membranes with a low molecular weight cut-off
(≤ 10 kDa) was the low filtration rate and high
transmembrane pressure. The latter triggered the
formation of a polarization layer and, eventually,
membrane clogging [52].
The molecular weight of the enzyme used for
protein biocatalysis is the most important parameter
for determining the cut-off threshold of membranes.
Alcalase, which we used for hydrolysis of whey proteins
in our research, has a molecular weight of 24–27 kDa.
Membranes with a cut-off threshold of 20 kDa could
easily separate an enzyme with such a molecular
weight [22]. Such membranes could significantly
reduce the transmembrane pressure, thus minimizing
the formation of a polarization layer and subsequent
membrane clogging.
Separate hydrolysis and filtration made it
possible to provide optimal conditions for each of the
processes (Fig. 6).
The hydrolysis was carried out under the previously
established conditions: substrate concentration – 4.5%;
enzyme concentration – 0.5%, hydrolysis temperature –
* – the gray line indicates a membrane restart, which is activated if the pressure exceeds 6 bar
Figure 5 Enzymatic membrane reactor with two stirred reactors (B 1, B 2), a water bath (W 1) with a thermostat (TIC),
a membrane pump (P 1), a feed pump (P 2), a transverse filtration unit flow (F 1), two barometers (PI 1, PI 2), level indicator (LIC),
and valves (V 1, V 2)
Product / Permeate
Ice bath
Ryazantseva K.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 271–280
65°C; hydrolysis time – 1 h. The proteolysis did not
include pH-statisation. The initial active acidity of
the reaction mix was 10. As for the molecular weight
distribution of hydrolysis products, the residues of
unhydrolyzed protein were retained during fractionation,
which decreased the hydrolyzate yield. However, a
double filtration made it possible to increase the yield of
the finished product by an average of 6%.
The whey protein hydrolyzate had the following
parameters: degree of hydrolysis – 18–25%; mass
fraction of ash – 6.5–6.9%; osmolality of a 10% solution
– 280–300 mmol/L of water; residual antigenicity –
≤ 2×10–5 of the protein mass. The resulting hydrolyzate
in the form of a 10% aqueous solution had a clear,
moderately bitter taste, without off-flavors. Its antigenic
properties make it possible to use it in therapeutic and
prophylactic functional foods based on enzymatic
protein hydrolysates [30].
In addition to batch enzymatic reactors, bioactive
peptides are obtained by a semi-continuous reaction or
a continuous reaction in an enzymatic membrane reactor
(EMBR) [31, 39, 40, 42–45].
Considering the enzymatic efficiency and cost of
enzymatic hydrolysis, continuous reaction has obvious
advantages. Hydrolysates can promptly be separated
from the substrate, the yield of biological peptides can
be significantly increased, and enzymes can be used
more than once. In addition, the production process is
quite simple, which reduces labor costs [47, 48]. As a
result, this method is popular in food industry.
Membrane reactors can process a variety of protein
food media of plant and animal origin. They have
good prospects for whey processing in functional
food production. Bioreactors can also be used for the
proteolysis of whey proteins with maximal antigenic,
antihypertensive, and antidiabetic properties.
Protein hydrolysis in continuous EMBRs is
attracting scientific attention because it can simplify
the technological process and reduce the cost of the
final product while increasing the yield, despite high
operating costs. Therefore, the need to improve and
develop these technologies is obvious.
K.A. Ryazantseva supervised the project.
E.Yu. Agarkova and O.B. Fedotova conducted the
theoretical research, processed the data, and prepared
the manuscript.
The authors declare that there is no conflict of
interests regarding the publication of this article.


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