Innovative Food Science and Emerging Technologies 68 (2021) 102639

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Innovative Food Science and Emerging Technologies

journal homepage: www.elsevier.com/locate/ifset

Continuous production of tempe-based bioactive peptides using an

automated enzymatic membrane reactor
Azis Boing Sitanggang *, Julius Sumitra , Slamet Budijanto
Department of Food Science and Technology, IPB University, Kampus IPB Darmaga, Bogor 16680, Indonesia

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

Keywords: The utilization of bioactive peptides from food-grade raw materials has received increasing interest, especially
AICE inhibitor for the production of functional foods. Within this study, a combination of fermentation and in vitro papain
Antioxidant hydrolysis was devised to produce bioactive peptides exhibiting antioxidant property and angiotensin I-con­
Bioactive peptides
verting enzyme (AICE) inhibitory activity. The utilization of an automated membrane reactor system developed
Enzymatic membrane reactor
Papain
in this study could facilitate the continuous hydrolysis of tempe flour-rich in protein under constant flux, thus
Tempe constant residence time operation. The optimum operating conditions were: [E]/[S] = 10% (w/v) and τ = 9 h.
With a 10-kDa membrane filtration, the resulted antioxidant capacity and AICE inhibition were 0.033 mg AEAC/
mL and 50.9%, respectively. Further separation of permeate with a 2-kDa membrane cut-off leveled off the
antioxidant capacity but increased the AICE inhibitory activity (i.e., Reduction of IC50 of ~40%). By this, the
obtained IC50 for AICE was 0.08 mg protein/mL, and this value was comparable with the IC50 values reported in
the literature.
Industrial relevance: The production of bioactive peptides from soybean through a combination of fermentation
and in vitro enzymatic hydrolysis is limitedly reported. In this work, fermented soybean (tempe) was further
hydrolyzed continuously using an automated membrane reactor. The hydrolysis under optimum operating
conditions could increase the antioxidant activity and AICE inhibition of permeate. This work is expected to
increase the commercialization of tempe-based food products by using safer technological approaches (i.e.,
fermentation and membrane technology).

1. Introduction
Tempe (Indonesian spelling), referred to as tempeh, is a soybean-
based fermented food widely consumed in Indonesia (Astuti, Meliala,
Dalais, & Wahlqvist, 2000). Tempe is popular as nutritious non-meat
protein food, especially in the USA, Japan, and Europe (Nout & Kiers,
2005). Tempe has received increased interest, and numerous studies
have demonstrated tempe as an attractive fermented food related to the
prevention of various chronic diseases (Mani & Ming, 2017). Tempe has
been reported to have a potent antioxidant capacity, antimicrobial ac­
tivity, and positive effects on intestinal microbiota, due to a high content
of isoflavone, folate, vitamin B12, and other compounds (Cao, Green-
Johnson, Buckley, & Lin, 2019). On the other hand, however, tempe
still has several drawbacks in terms of consumer acceptance. The bitter
taste and unpleasant odor are the two main drawbacks of tempe. These
have caused tempe commercialization in the market, limited only as a
whole food. Therefore, there is a need to provide an alternative
technology that can utilize tempe for producing functional ingredients.
Bioactive peptides are peptides having 2–20 amino acids within the
structures of the parent proteins (Agyei, 2015; Sanjukta & Rai, 2016).
These peptides must be released from parent proteins in order to have
health-beneficial activities, at least in three ways: (i) through in vivo
hydrolysis by gastrointestinal proteolytic enzymes, (ii) using microbial
fermentation, and (iii) through in vitro hydrolysis by the actions of
microorganism- or plant-based proteases (Banan-Mwine Daliri, Oh, &
Lee, 2017; Korhonen & Pihlanto, 2006; Liu & Zhao, 2010). In such,
bioactive peptides have physiological functionalities, especially for
cardiovascular system (antioxidative, antithrombotic, and antihyper­
tensive), nervous system, gastrointestinal system (mineral binding,
antimicrobial activity), and immune system (immunomodulatory)
(Chalamaiah, Keskin Ulug, Hong, & Wu, 2019; Korhonen & Pihlanto,
2006; Sanjukta & Rai, 2016; Tanzadehpanah, Asoodeh, Saberi, & Cha­
mani, 2013).
The protein content in soybean is approximately 30–48% (dry basis,

* Corresponding author.
E-mail address: boing.lipan@apps.ipb.ac.id (A.B. Sitanggang).

https://doi.org/10.1016/j.ifset.2021.102639
Received 12 November 2020; Received in revised form 10 February 2021; Accepted 10 February 2021
Available online 16 February 2021
1466-8564/© 2021 Elsevier Ltd. All rights reserved.
A.B. Sitanggang et al.
db) (Syah et al., 2015). With this amount, soybean is a good source of
parent proteins to produce bioactive peptides through different ap­
proaches mentioned previously. Singh, Vij, and Hati (2014) has
reviewed the functional significances of bioactive peptides derived from
soybean. These include antihypertensive, antioxidative, antiobesity,
immunomodulatory, anti-diabetic, hypocholesterolemic, and anti­
cancer. Gibbs, Zougman, Masse, and Mulligan (2004) reported angio­
tensin I-converting enzyme (AICE) inhibitory, anti-thrombotic, and
antioxidant properties of bioactive peptides isolated from soy-fermented
foods, like natto and tempe. Rho, Lee, Chung, Kim, and Lee (2009) also
demonstrated the production of AICE inhibitory peptide from fermented
soybean extract.
During soybean fermentation, the proteins are only partially hy­
drolyzed. It is due to the mass-transfer limitation of proteases to access
parent proteins (i.e., inside bean kernels). Additionally, most of the
extracellular proteases only cleave glycoproteins and phosphoproteins
(Gibbs et al., 2004). Herein, the fermentation of soybean (tempe), and
followed by in vitro hydrolysis, is considered as an apt method to pro­
duce small molecular weight bioactive peptides.
In our previous study, we reported a combined method between
fermentation and in vitro hydrolysis for producing tempe-based bioac­
tive peptides (Sitanggang, Lesmana, et al., 2020). The hydrolysis of
tempe flour using papain and followed by 5-kDa membrane filtration
obtained the highest bioactivities (i.e., antioxidant activity, AICE-,
α-glucosidase-inhibitory activity). Additionally, the influence of mo­
lecular weight on the bioactivities of peptides was also demonstrated.
The smaller membrane cut-off used for sieving the peptides, the higher
the bioactivities obtained (Ajibola, Fashakin, Fagbemi, & Aluko, 2011;
Sonklin, Laohakunjit, & Kerdchoechuen, 2018). The limitation in our
previous study was that tempe flour was hydrolyzed batch-wise
(Sitanggang, Lesmana, et al., 2020). The batch reaction is often
considered unproductive due to a time-consuming intermittent stop
between operation cycles (Sitanggang, Drews, & Kraume, 2016; Ubilla
et al., 2020). To our best of knowledge, continuous production of
bioactive peptides in membrane reactors is mainly applied to milk
proteins (Korhonen & Pihlanto, 2006). It is due to the high solubility of
milk proteins. Within this study, an attempt to continuously hydrolyze
tempe flour-rich in protein to produce bioactive peptides was realized.
The automated membrane reactor system developed in this work could
circumvent the limitations of batch-wise production of bioactive pep­
tides (i.e., low productivity and enzyme utilization). Permeate exhibit­
ing ACE inhibition and antioxidant activity is of interest because these
bioactivities are dominating in tempe-based peptides (Sitanggang, Les­
mana, et al., 2020). This work is expected to increase the commercial­
ization of tempe-based functional ingredients by using safer
technological approaches (i.e., fermentation and membrane
technology).
2. Materials and methods
2.1. Materials
Soybean was purchased from Koperasi Tempe Indonesia (KOPTI),
Bogor, Indonesia. Technical grade hexane (80% v/v) and pure water
(water one™) were purchased from Sentral Kimia and Jayamas Medica
Industri, Indonesia, respectively. Folin–Ciocalteu reagent, methanol pro
analysis, 2,2-diphenyl-1-picrylhydrazyl (DPPH), bovine serum albumin
(BSA), NaOH, HCl, Na2CO3 were purchased from Sigma-Aldrich, Inc.
(Singapore). Hippuryl-histidine-leucine (HHL) was from Bachem AG
(Switzerland). The 4-(2-Hydroxyethyl)-1-piperazine ethanesulfonic acid
(HEPES) was purchased from Shanghai Chaining Chemicals Co., Ltd.
(China). Papain (100,000 U/g), and angiotensin I-converting enzyme
(AICE) were purchased from, Xi’An Saiyang Bio-Technology Co., Ltd.
(China), and Xi’An Saina Biological Technology Co., Ltd. (China),
respectively. The UF flat-sheet polyethersulfone (PES) membranes
(molecular weight cut-off/MWCO of 2-, 4-, 5-, and 10-kDa) were
2
Innovative Food Science and Emerging Technologies 68 (2021) 102639
Fig. 1. Automated enzymatic membrane reactor (EMR) (No. 1 = Nitrogen (N2),
2 = pressure reducer, 3 = PPP-MPPES, 4 = substrate tank, 5 = reactor, 6 = UF
membrane, 7 = analytical balance, 8 = water bath system, 9 = PC with Lab­
VIEW program).
purchased from Microdyn-Nadir GmbH (Germany).
2.2. Tempe production and preparation of tempe flour-rich in protein
The fermentation of soybean was conducted according to our pre­
vious study (Sitanggang, Lesmana, et al., 2020). Soybean (soybean-to-
water ratio = 1:5 (w/w)) was boiled at 90–95 ◦ C for 90 min. The
fermentation was performed by inoculating the starter culture (also
known as ‘laru’, composed mainly by Rhizopus spp., and growth media)
as much as 2.0 g per kg soybean, at 32 ◦ C, relative humidity of 75% for
six days. The resulted tempe was cut into dices (3 × 0.5 × 0.5 cm), dried
in a tray dryer (Terara Seisakusho C. Ltd. No 4-60SP, Japan) at 70 ◦ C for
7 h. The dried tempe was then comminuted using FFC 23 pin disc-mill
(Agowindo-Maksindo, Indonesia).
Tempe flour was defatted using technical grade hexane following
Rosset, Acquaro, and Beléia (2014). The defatted tempe flour was
dispersed in distilled water with a ratio of 1:10 (w/v), and the pH was
adjusted to ~8 using 2.0 N NaOH. The solution was stirred at 200 rpm
for 2 h at 30 ◦ C. For facilitating phase separation, the mixture was
allowed to stand for 24 h. The upper phase containing dissolved proteins
was withdrawn, and its pH was brought down to the protein’s isoelectric
point (pI = ~ 4.5) using 1.0 N HCl. The slurry at the bottom of the
mixture was obtained, neutralized with 2.0 N NaOH, and finally dried in
a freeze dryer (Liu et al., 2008). The resulted solid was milled and sieved
(Θ = 80 Tyler mesh) to obtain tempe flour-rich in protein.
2.3. Enzymatic membrane reactor (EMR)
The enzymatic membrane reactor (EMR) was constructed in parallel
(n = 2, Fig. 1). The upper and bottom part of the reactor (5) and sub­
strate tank (4) were made of 304 stainless steel. The reactor body was
made of borosilicate glass (wall thickness = 3 mm, diameter = 48 mm,
height = 72 mm). The reactor volume was 9 × 10− 2 L, with a D/d ratio
of 1.175 and the maximum pressure stability of 6.0 bar. The UF mem­
brane (6) having an effective membrane area of 1.24 × 10− 3 m2 was
placed at the bottom of the reactor. The nitrogen (N2) (1) was used to
pump the substrate from the substrate tank to the reactor. The release of
nitrogen was controlled by means of MPPES-3-1/4–6-010 proportional
pressure regulator (Festo SE & Co. KG, Germany) (3). Permeate was
collected in a container that was located on the analytical balance (7).
The automation developed in this study was to bring the process
variable flux JPV as close as possible to a set flux JSV, thus resulted in a
A.B. Sitanggang et al.
small control error (Sitanggang et al., 2016; Sitanggang, Drews, &
Kraume, 2015). A program was realized using Laboratory Virtual In­
strument Engineering Workbench software (LabVIEW, National In­
struments, USA) where the proportional-integral-derivative (PID)
controller was embedded (Sitanggang, Drews, & Kraume, 2014a), and
tuned according to Kuhn (1995). To facilitate this automation, the data
acquisition (DAq) was supported by compact DAQ USB chassis (cDAQ)-
9174, NI 9263, and NI 9201 (National Instruments, USA).
2.4. Membrane cleaning
The membrane was cut with a diameter of 48 mm. The cleaning was
done by soaking the membrane in 0.1% (w/v) NaOH for 15 min. The
membrane was rinsed and placed at the bottom of the reactor prior to
the filtration of pure water at a constant transmembrane pressure ∆P
(TMP) of 0.5 bar for 20 min.
2.5. Papain filtration
The tempe flour-rich in protein was continuously hydrolyzed using
freely-dispersed papain. Herein, the selection of membrane’s MWCO has
to ensure complete rejection of the enzyme molecules. The filtration of
papain was performed using two MWCOs (i.e., 5- and 10-kDa). Papain
(2.5% (w/v)) was dissolved in 0.01 M phosphate buffer (pH 6.8) and
subject to the filtration at a constant flux. The enzyme’s activity in
permeate was measured, and the rejection R was calculated as follows
(Eq. 1):
( )
Ui− Up
R = × 100% (1)
Ui
where Ui and Up were enzyme’s activity (U/g) at initial and in the
permeate, respectively.
2.6. Continuous production of tempe-based bioactive peptides
The substrate was prepared by dissolving tempe flour-rich in protein
with 0.01 M phosphate buffer (pH 6.8) with a ratio of solid-to-buffer was
1:100 (w/v) in a beaker glass. The solution was rested for about 15 min,
and two-third of the substrate solution from the upper part was further
utilized, thus leaving coarse suspension at the bottom of the beaker
glass. The substrate was then placed in the substrate tank (4) and the
reactor (5, see Fig. 1). The reaction was carried out at a temperature T of
40 ◦ C, and agitation N of 300 rpm. Within this study, the influences of
enzyme-to-substrate ratio (i.e., [E]/[S] = 5, 10 and 15%) and residence
time (i.e., τ = 5, 7 and 9 h) on the production of bioactive peptides were
investigated. For analyses, samples were withdrawn intermittently
(every one hour) from the permeate pipe and also from the container
placed on the analytical balance. The sample from the container was
only taken once, at the end of the operation. This sample was considered
to represent the cumulative or overall reaction performance. In addition
to this, permeate obtained from the optimum operating conditions was
filtered subsequently using 5-, 4- and 2-kDa PES membrane. The half-
maximal inhibitory concentration (IC50) of the resulted permeate was
measured especially for the antioxidant activity and angiotensin I-con­
verting enzyme inhibitory activity.
For continuous operation where the density does not change in the
system, the hydraulic residence time τ is calculated as (Eq. 2):
VR
τ= (2)
V̇
where VR = reactor volume (L) and V̇ = permeate volumetric flow rate
(L/h). During the continuous production of bioactive peptides, the
reactor was fully filled with substrate, and the reactor volume was
maintained constant. Herein, permeate flux J (L/m2.h) was dependent
on the volumetric flow rate V̇ and effective membrane area Am (m2) (Eq.
3
Innovative Food Science and Emerging Technologies 68 (2021) 102639
3):
V̇
J= (3)
Am
From Eq. (2) and Eq. (3), one could see that controlling flux invariant
during the operation would also maintaining residence time constant
because the reactor volume and the effective membrane area did not
change respective to the time.
2.7. Measurement of enzyme’s activity
A total of 5.0 mL of casein (0.65% w/v) was poured in a 15-mL
centrifuge tube and added with 1.0 mL of papain (0.5% w/v). The so­
lution was vortexed and incubated for 10 min at 37 ◦ C. Furthermore, 5.0
mL of 0.5 M trichloroacetic acid were added into the reacting solution to
stop the hydrolysis. The mixture was centrifuged at 1000 xg (Hermle Z
383 K, Wehingen, Germany), at 25 ◦ C for 10 min. A 1.0-mL of the su­
pernatant was withdrawn and added with 5.0 mL of 0.5 M sodium
carbonate solution and 1.0 mL of Folin-Ciocalteu (1:5 v/v). The mixture
was vortexed and incubated for 30 min at 37 ◦ C. The absorbance was
monitored at a wavelength of 660 nm using a Genesys™ 140 UV–Vis
Spectrophotometer (USA). Tyrosine (0–60 ppm) was used to construct
the standard curve (y = 0.0143× + 0.0079, R2 = 0.992). One unit of
activity (U) was defined as the amount of tyrosine (in micromoles)
released during the proteolytic action of papain on the casein per minute
(Cupp-Enyard, 2008).
2.8. Measurement of protein content
The protein content of tempe flour-rich in protein was measured
according to the standard methods from the Association of Official
Agricultural Chemists (AOAC). In addition to this, the protein content of
permeate was monitored following Lowry’s method (Lowry et al., 1951)
in which bovine serum albumin (0–150 ppm) was used to construct the
standard curve (y = 0.0054× + 0.0384, R2 = 0.992).
2.9. Analysis of total phenol
A 0.2-mL sample was poured into the test tube and added with 1.8
mL of fresh Folin-Ciocalteau reagent. The mixture was incubated at
room temperature for 6 min. A total of 1.8 mL of Na2CO3 (6% w/v) was
added to the solution prior to the incubation for 90 min in a dark place at
room temperature. The absorbance was measured at a wavelength of
725 nm. Gallic acid (0–200 ppm) was used to construct the standard
curve (y = 0.0047× + 0.009, R2 = 0.997). Total phenol was expressed as
milligram gallic acid equivalent (mg GAE) per milliliter sample.
2.10. Measurement of antioxidant capacity
A 0.3-mL of the sample was added with 0.7 mL of distilled water, and
3.0 mL of 120 μM 2,2-diphenyl-1-picrylhydrazyl (DPPH). The vortexed
solution was incubated for 30 min in a dark room at room temperature.
The absorbance was measured at 515 nm. The antioxidant capacity was
expressed in milligram ascorbic acid equivalent antioxidant capacity
(mg AEAC) per milliliter or gram sample (y = − 0.0083× + 0.9446, R2 =
0.995) (Brand-Williams, Cuvelier, & Berset, 1995; Sitanggang, Lesmana,
et al., 2020; Sitanggang, Sinaga, Wie, Fernando, & Krusong, 2020).
2.11. Measurement of angiotensin I-converting enzyme (AICE) inhibitory
activity
A 200-μL of buffer (6.7 mM hipuryl-l-histidyl-l-leucine in 50 mM 4-
(2-hydroxyethyl) -1-piperazineethanesulfonic acid with 300 mM NaCl,
pH 8.3) was added with 100 μL of sample, and pre-incubated at 37 ◦ C for
5 min. A 30-μL AICE (0.33 U/mL) was added to the mixture before the
A.B. Sitanggang et al.
Table 1
Several characteristics of tempe flour-rich in protein.
Parameters –
Moisture content (% wb) 12.25 ± 0.24
Protein content (% db) 51.60
Antioxidant activity (mg AEAC/g) 1.61 ± 0.04
Total phenol (mg GAE/g) 27.37 ± 0.10
AICE inhibition (%) 36.46 ± 0.82
incubation for 20 min at 37 ◦ C. The reaction was stopped by the addition
of 3.0 mL of 1.0 N HCl (Guo, Pan, & Tanokura, 2009). The hippuric acid
released due to the action of ACE was extracted using 2.4 mL ethyl ac­
etate, and vortexed for two minutes (Sitanggang, Lesmana, et al., 2020).
A 1.0-mL hippuric acid containing solution was withdrawn from the
upper layer, and evaporated at 120 ◦ C for 30 min. The remaining sedi­
ment was then dissolved in 1.0 mL of distilled water, and the absorbance
was measured at a wavelength of 228 nm. The AICE inhibition was
evaluated as follows (Eq. 4):
(a − b) − (c − d)
Inhibitory act.(%) = × 100%
(a − b)
where a = absorbance of solution containing ACE without sample, b =
absorbance of solution containing ACE that has been inactivated with
HCl (without sample), c = absorbance of solution containing ACE and
sample, and d = absorbance of solution containing ACE and sample that
has been inactivated with HCl.
2.12. The calculation of papain (protein) charge at different physiological
pHs
The distribution of papain (protein) charge at different physiological
pHs was performed using a software provided at http://protcalc.source
forge.net (The Scripps Research Institute, USA). The complete amino
acid sequential composition was collected from Basic Local Alignment
Search Tool (BLAST) with P00784 entry (The Universal Protein
Resource, UniProt - www.uniprot.org) (Paul, Therrien, & Furrer, 2015;
Sitanggang et al., 2016).
2.13. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) analysis
A 50-mg of the sample was dissolved in Tris-HCl (pH 8.4). The so­
lution was incubated at 80 ◦ C for one hour and subject to centrifugation
at 16,000 xg for 25 min at 25 ◦ C. The final residue was dissolved with
distilled water to obtain a concentration of 4.0 mg/mL (Sitanggang,
Lesmana, et al., 2020). To run the gel separation, the detailed proced­
ures were adopted from literature published elsewhere (Sitanggang,
Alexander, et al., 2020). The electrophoresis apparatus (Bio-Rad,
Singapore) was used according to the manufacturer’s guide and the
analysis was started by giving a constant voltage of 70 V for approxi­
mately 3 h.
2.14. Statistical analysis
The evaluation of the results statistically was done using SPSS Soft­
ware version 23 (IBM, US). The analysis of variance (ANOVA) was
performed with a confidence level of 95%.
3. Results and discussion
3.1. Characteristics of tempe flour-rich in protein
The characteristics of tempe flour-rich in protein can be seen in
Table 1. The protein content was 51.60% (db). The antioxidant activity
and AICE inhibition were 1.61 mg AEAC/g and 36.46%, respectively. In
Innovative Food Science and Emerging Technologies 68 (2021) 102639
Fig. 2. SDS-PAGE analysis of (A) defatted soybean meal, (B) commercial SPI,
(4) (C) defatted tempe flour, and (D) tempe flour-rich in protein.
addition to this, the total phenolic content was up to 27.37 mg GAE/g.
The analysis of SDS-PAGE revealed that soybean fermentation increases
the protein degree of hydrolysis (Fig. 2). The distribution of low mo­
lecular weight peptides (i.e., <17 kDa) was higher in defatted tempe
flour (lane C) and tempe-flour rich in protein (lane D) rather than in
defatted soybean meal (lane A) and commercial SPI (lane B). The protein
bands (electrophoretograms) of higher molecular weight peptides were
also thinner in tempe flour-rich in protein and defatted tempe flour
compared to commercial soy isolate protein. Additionally, the higher
amount of free amino acids might present in both tempe derivatives that
passed the SDS gel (Zinchenko, Muranova, Melanyina, & Miroshnikov,
2019). The pooled extracellular proteases were considered to perform
proteolysis on soybean proteins leading to a higher degree of hydrolysis
(de Reu et al., 1995; Zhang et al., 2014)
3.2. Rejection of papain by UF membrane
The initial activity of papain (2.5% w/v) in 0.01 M phosphate buffer
(pH 6.8) was about 79,265 U/g. There was small papain’s activity
recovered in permeate. These activities were approximately 0.2–0.3 U/g
for both MWCOs (i.e., 5- and 10-kDa). Herein, the rejections of papain
by 5- and 10-kDa PES membrane were identical, more than 99% (see
Fig. 3a). A complete rejection of papain by 5-kDa PES membrane and its
utilization for the production of sesame protein hydrolysate has also
been reported elsewhere (Das, Bhattacherjee, & Ghosh, 2009). As
common to the constant flux filtration, to compensate membrane
fouling, TMPs increased over time during papain filtrations using both
MWCOs. In addition to this, since papain has a molecular weight of
approximately 23 kDa (Muharram & Abdel-Kader, 2017), a higher
fouling propensity with a smaller membrane cut-off was evident. TMP
by 5-kDa filtration was still higher than 10-kDa regardless of the dif­
ference in set flux value (JSV). Herein, 10-kDa PES membrane was
selected for further investigations.
3.3. Robustness of the control system
The PID controller introduced in the reactor system was tuned
following Kuhn (1995) under the normal setting (Sitanggang et al.,
2016). The controller gain K was about 0.036, while the integral TI and
derivative time TD were 0.468 and 0.118 min, respectively. This setting
was tested to filter the substrate solution (1% w/v) at varying fluxes (JSV
= 10 ➔ 25 ➔ 15 L/m2.h) using 10-kDa PES membrane. As seen in Fig. 4,
although an overshoot was found in process variable flux JPV as an initial
response of the controller, the control error could be minimized over
4
A.B. Sitanggang et al. Innovative Food Science and Emerging Technologies 68 (2021) 102639

Fig. 3. (a) Rejection of papain by 5- and 10-kDa PES membrane, and (b) filtration behavior of papain (2.5% w/v) at constant fluxes, thus residence times (τ for 5-kDa
membrane = 3 h and JSV = 22.73 L/m2.h, τ for 10-kDa membrane = 2 h and JSV = 34.09 L/m2.h).

time by less than 5%. In addition to this, the controller output (i.e., TMP)
was not jerky. A similar investigation has been reported elsewhere,
where the open-loop tuning was utilized to tune the PID setting, and also
resulted in a control error of less than 5% (Sitanggang, Drews, &
Kraume, 2014b). Conclusively, the developed PID controller in this
study could be used to facilitate a constant flux operation during the
continuous hydrolysis of tempe flour-rich in protein for producing
bioactive peptides.

3.4. Influence of enzyme-to-substrate ratio for producing bioactive

peptides

The influence of enzyme-to-substrate ratio [E]/[S] was investigated

Fig. 4. Robustness of PID controller during the filtration of tempe flour-rich in
protein (15% w/v) at varying fluxes (JSV: 10 ➔ 25 ➔ 15 L/m2.h). PID setting: K
= 0.036, TI = 0.468 min and TD= 0.118 min, 10-kDa PES membrane.
at three different levels (i.e., 5, 10, and 15%). The total phenol and
protein concentration insignificantly changed in every hour of sampling
for the three levels of [E]/[S]. The antioxidant activity and AICE inhi­
bition of permeate at 10% [E]/[S] were slightly higher than that of 5 and
15% (see Fig. 5).
The total phenols, protein concentrations, antioxidant activities, and
AICE inhibitions between the initial substrate and cumulative permeate
are shown in Table 2. The total phenol and protein concentration of the

Fig. 5. The influence of enzyme-to-substrate ratio [E]/[S] on total phenol, protein content, antioxidant activity and AICE inhibition during continuous hydrolysis of
tempe flour-rich in protein. Reacting conditions: [S] = 1% (w/v), τ = 5 h (JSV = 13.64 L/m2.h), pH 6.8, N = 300 rpm, T = 40 ◦ C, 10-kDa PES membrane.

5
A.B. Sitanggang et al. Innovative Food Science and Emerging Technologies 68 (2021) 102639

Table 2 substrate were significantly higher than that of cumulative permeates at

Phenol, protein, antioxidant capacity, and AICE inhibition between initial re­ a confidence level of 95%. The rejection of soybean isoflavones from soy
action and cumulative permeate taken in the container. Reacting conditions for residue by 20-kDa membrane by more than 40% has been reported (Li,
different treatments given in Fig. 5 and Fig. 7. Wu, Dong, & Li, 2012). Within this study, with a 10-kDa membrane,
Treatment Phenol Protein Antioxidant AICE such phenol rejection was inevitable to yield smaller total phenol in
(mg GAE/ (mg/mL) (mg AEAC/ inhibition permeate compared to the substrate. From Fig. 2, the electrophoreto­
mL) mL) (%)
gram of tempe flour-rich in protein still had parent proteins with mo­
Substrate 0.151 ± 0.161 ± 0.028 ± 36.61 ± lecular weights of more than 17 kDa. The incomplete proteolysis on the
0.001d 0.001c 0.002b 0.26a parent proteins (i.e., <100% of protein degree of hydrolysis) might lead
Permeate, 5% 0.133 ± 0.114 ± 0.026 ± 43.04 ±
[E]/[S] 0.002a 0.004b 0.001a 1.71b
to the protein rejection by 10-kDa membrane (Sun, 2011). Thus, the
10% 0.136 ± 0.115 ± 0.030 ± 46.02 ± protein concentrations in the three cumulative permeates (i.e., [E]/[S]:
0.002b 0.003b 0.001b 1.10c 5, 10, and 15%) were lower than that of the substrate.
15% 0.141 ± 0.108 ± 0.027 ± 43.25 ± The trend of antioxidant activities between substrate and the three
0.001c 0.002a 0.001c 2.40b
cumulative permeates were different from that of total phenol and
Substrate 0.153 ± 0.161 ± 0.027 ± 36.60 ±
0.002d 0.001c 0.001a 0.16a protein content. The compounds acting as antioxidants were presumably
Permeate, τ 5h 0.136 ± 0.115 ± 0.030 ± 46.02 ± not limited from the isoflavones as the hydrolysis of soy protein also
0.002a 0.003a 0.001b 1.10b produced bioactive peptides exhibiting antioxidant activity (Park, Lee,
7h 0.140 ± 0.126 ± 0.032 ± 48.43 ± Baek, & Lee, 2010). Amongst the three permeates, increasing [E]/[S] to
0.002b 0.002b 0.001c 0.86c
15% was detrimental for antioxidant activity. It could be due to exces­
9h 0.142 ± 0.128 ± 0.033 ± 50.88 ±
0.002b 0.002b 0.001c 0.95d sive hydrolysis that led to the breakdown of peptides displaying anti­
oxidant activity (Xu, Shen, Chen, Bean, & Li, 2019; Zhao, Zhao, Ahn, Jin,
Note: Different superscript letters in a column show significant difference with a
& Huang, 2019). The substrate AICE inhibition was significantly lower
confidence level of 95%.
compared to the three cumulative permeates (P < 0.05). This indicated
that hydrolysis of parent proteins by papain could release bioactive

Fig. 6. Profiles of ∆P, JSV and JPV during continuous hydrolysis of tempe flour-rich in protein at different levels of [E]/[S]: (a) 5%, (b) 10%, and (c) 15%.

Fig. 7. The influence of residence time τ on total phenol, protein content, antioxidant activity and AICE inhibition during continuous hydrolysis of tempe flour-rich
in protein. Reacting conditions: [S] = 1% (w/v), [E]/[S] = 10%, pH 6.8, N = 300 rpm, T = 40 ◦ C, 10-kDa PES membrane.

6
A.B. Sitanggang et al.
Fig. 8. Profiles of ∆P, JSV and JPV during continuous hydrolysis of tempe flour-rich in protein at different levels of τ: (a) 5 h (JSV = 13.64 L/m2.h), (b) 7 h (JSV = 9.74
L/m2.h), and (c) 9 h (JSV = 7.58 L/m2.h).
peptides exhibiting both antioxidant capacity and AICE inhibitory ac­
tivity (Lo & Li-Chan, 2005; Park et al., 2010). Amongst the three cu­
mulative permeates, the highest total phenol, protein concentration,
antioxidant activity, and AICE inhibition were obtained by 10% [E]/[S]
treatment. Chiang, Tsou, Tsai, and Tsai (2006) reported an optimum
[E]/[S] of 10% during the hydrolysis of soy protein isolate (SPI) using
alcalase for producing peptides exhibiting AICE inhibitory activity.
Other studies also used [E]/[S] by 2 to 10% for hydrolyzing soybean
protein using different enzymes to obtain AICE inhibitory peptides
(Chen, Okada, Muramoto, Suetsuna, & Yang, 2002; Gouda, Gowda, Rao,
& Prakash, 2006; Li, Xia, Zhang, & Li, 2018; Wu & Ding, 2001).
Fig. 6 shows the influence of [E]/[S] on TMP during the continuous
hydrolysis of tempe flour-rich in protein. A higher [E]/[S] given in the
reacting solution caused higher increase in pressure. The UF membrane
made of PES (Microdyn-Nadir GmbH, Germany) is negatively charged
(de la Torre, Harff, Lesjean, Drews, & Kraume, 2009; Puro et al., 2010;
Suárez, Díez, García, & Riera, 2012). The calculated charge of papain at
pH 6.8 (0.01 M phosphate buffer) was approximately +1. The electro­
static interaction between papain molecules and PES membrane surface
was toward adhesion, leading to the adsorption of protein molecules
onto the membrane surface (Sitanggang et al., 2016). Therefore, a
higher fouling rate was pronounced at [E]/[S] of 15%, thus having a
higher total resistance at the end of the filtration.
3.5. Influence of residence time for producing bioactive peptides
Fig. 7 shows the influence of three residence times (τ = 5, 7, and 9 h)
on total phenol, protein content, antioxidant activity, and AICE inhibi­
tion of protein hydrolysates. The total phenol, protein content, and
antioxidant capacity of permeate were relatively constant during 5 h
reaction, in which τ of 9 h prevailed as compared to the other treat­
ments. For AICE inhibition, the fluctuations were observed, especially
when the inhibition peaked at 4 h of reaction. Similar to the other three
observed parameters, the longer residence time could obtain permeate
with a higher AICE inhibition.
0.8
(a)
Antioxidant IC50 (mg/mL)
c
0.6
0.4
a a
0.2
0.0
Substrate10 kDa 5 kDa
Fig. 9. The half-maximal inhibitory concentration (IC50) of the substrate and resulted permeates (with 10-, 5-, 4-, and 2-kDa membrane cut-off): (a) antioxidant and
(b) AICE. Different letters indicate a significant difference with a confidence level of 95%.
Innovative Food Science and Emerging Technologies 68 (2021) 102639
The total phenol and protein concentration in the substrate were
higher significantly than that of the three permeates (i.e., treated with
different residence times) with a confidence level of 95%. Amongst
permeates, longer residence time obtained higher total phenol and
protein content. Higher protein content at τ = 9 h (i.e., 0.128 mg/mL)
was due to a longer time for papain to break down the peptide bonds in
tempe parent proteins. These small molecular weight peptides and free
amino acids passed through 10-kDa membrane, thus yielded in a higher
protein concentration in permeate (Wei & Chiang, 2009). As mentioned
previously, the compounds acting as antioxidants were presumably not
limited from the isoflavones. The hydrolysis of tempe protein might also
produce bioactive peptides exhibiting antioxidant activity. The antiox­
idant activities for the three permeates were significantly higher than
that of the substrate (P < 0.05). The highest antioxidant capacity was
also found at τ = 9 h, approximately 0.032 mg AEAC/mL. As a cysteine
protease-endopeptidase, papain has been reported to cleave at (hydro­
phobic amino acid)-(R/K/E/H/G/Y) in protein structures (Fernández-
Lucas, Castañeda, & Hormigo, 2017; Nath et al., 2020). The release of
low molecular peptides containing a high portion of hydrophobic amino
acids (Q, A, V, and L), aromatic amino acids (Y, H, W, and F), and
cysteine (C) has been reported to exhibit high antioxidant activity
(Sarmadi & Ismail, 2010; Zou et al., 2016). Similar to antioxidant ca­
pacity, the AICE inhibitions for the three permeates were also higher
than the substrate. The highest inhibition of 50.9% was obtained at τ =
9 h. The production of peptides having hydrophobic amino acids (W, F,
A, I, and V) at the C-terminal position and positively charged amino
acids (K, R) is considered to display higher AICE inhibitory activity
(Sanjukta & Rai, 2016; Sarmadi & Ismail, 2010). The AICE inhibition of
50.9% was the highest one found in this study by varying two operating
conditions ([E]/[S] and τ). In our previous study, the combination of
papain hydrolysis under batch operation followed by 10-kDa membrane
filtration could obtain permeate having AICE inhibition up to 57.3%
(Sitanggang, Lesmana, et al., 2020). The batch hydrolysis was per­
formed under [E]/[S] = 3.3% (w/v), [E] = 100,000 U/g solid, [S] = 10%
(w/v) and reaction time t = 4 h. The notable difference between our
0.3
(b)
d
AICE IC50 (mg/mL)
0.2
b
c
a b b
0.1 a
0.0
4 kDa 2 kDa Substrate10 kDa 5 kDa 4 kDa 2 kDa
7
A.B. Sitanggang et al. Innovative Food Science and Emerging Technologies 68 (2021) 102639

Table 3 IC50 for AICE in the substrate was significantly higher than that of
IC50 values of AICE from soy-based bioactive peptides prepared with different hydrolysates filtered by different membrane cut-offs with a confidence
methods. level of 95%. The lowest cut-off (2-kDa membrane) obtained better ac­
No. Type of Reacting conditions IC50 (mg Ref. tivity to inhibit AICE (IC50 = 0.08 mg protein/mL). Permeate resulted
soybean meal protein/ from filtration using 2-kDa membrane had IC50 ~ 40% smaller than that
mL) of 10-kDa membrane. Other studies also demonstrated the increase in
1 Soybean flour Batch-wise, pepsin, 37 ◦ C, 0.01a Chen et al., AICE inhibition using a lower membrane cut-off (Chiang et al., 2006;
24 h, pH 2, [E]/[S] = 1% 2002 Sitanggang, Lesmana, et al., 2020; Yao et al., 2019). Table 3 shows soy-
2 Batch-wise, M. purpureus 0.126 Kuba, Tana,
β-conglycinin
based bioactive peptides IC50 values for AICE prepared with different
3 Glycinin acid proteinase, 37 ◦ C, 10 0.148 Tawata, &
h, pH 3.3, [E]/[S] = 100 Yasuda, methods. The types of soybean meal utilized were soybean flour, soy­
U/g 2005 bean storage protein (β-conglycinin and glycinin), and SPI. The reported
4 Glycinin Batch-wise, Aspergillus sp. 0.001b Gouda et al., IC50 values were in a range of 0.001–0.28 mg protein/mL. The AICE IC50
Amano-P (Protease P), 2006 of tempe hydrolysate filtered with 2-kDa membrane, in this study, was
37 ◦ C, 18 h, pH 8.2, [E]/
[S] = 2%
comparable with the reported IC50 values found in the literature.
5 SPI Batch-wise, pepsin, 37 ◦ C, 0.28 Lo & Li- Furthermore, most of the reported studies were performed under batch
1 h, pH 2.0, [E]/[S] = 4% Chan, 2005 operation, except that of Chiang et al. (2006). Higher productivity for an
continued with enzyme-catalyzed reaction is always one of the considerations for
hydrolysis by pancreatin,
selecting continuous operation over batch one (Chiang et al., 2006;
37 ◦ C, 2 h, pH 7.5
6 SPI Batch-wise, E.coli JM109 0.018 Kodera & Sitanggang et al., 2014b).
protease D3, 37–40 ◦ C, Nio, 2006
24 h, pH 4.5, [E]/[S] = 4. Conclusions
0.2%
7 SPI Batch-wise, alcalase, 0.078 Chiang et al.,
50 ◦ C, 24 h, pH 9, [E]/[S] 2006 Within this study, a combination of fermentation and in vitro pro­
= 2.5%, 10-kDa PES teolysis was realized to produce soy-based bioactive peptides exhibiting
membrane antioxidant acitivity and AICE inhibitory activity. The operating con­
8 SPI Continuous, alcalase, 0.09
ditions, such as [E]/[S] = 10% (w/v), and τ = 9 h were selected as the
50 ◦ C, 8 h, pH 9, [E]/[S]
= 2.5%, τ = n.a (JSV = n. optimum operating conditions for hydrolyzing tempe flour-rich in pro­
a) 10-kDa PES membrane tein using membrane reactor. With a 10-kDa membrane filtration, the
9 Tempe-flour Continuous, papain, 0.08 This study resulted antioxidant capacity and AICE inhibition were 0.033 mg AEAC/
rich in protein 40 ◦ C, pH 6.8, [E]/[S] = mL and 50.9%, respectively. Further permeate filtration with a smaller
10%, τ = 9 h (JSV = 7.58
membrane cut-off (i.e., 2-kDa membrane) reduced the antioxidant ca­
L/m2.h), N = 300 rpm, 2-
kDa PES membrane pacity. In contrast, with a 2-kDa membrane cut-off, AICE inhibition of
permeate increased which was indicated by a reduction of IC50
Note: “a” and “b” were calculated based on molecular weight of Y-L-A-G-N-Q
approximately 40%. Conclusively, in vitro hydrolysis of tempe to pro­
and V-L-I-V-P, respectively; n.a = not available.
duce functional ingredients such as bioactive peptides is amenable. This
is expected to increase the commercialization of tempe-based food
current study with the previous one (Sitanggang, Lesmana, et al., 2020) products.
was that the current substrate concentration was one-tenth of the pre­
vious study.
Fig. 8 depicts the influence of τ on TMP. In general, a shorter resi­ Declaration of Competing Interest
dence time, thus a higher convective transport, causes a higher rate of
fouling. The TMP almost reached 2.0 bar during the hydrolysis at τ = 5 h The authors declare that the research was conducted without a po­
(JSV = 13.64 L/m2.h). When the flux reduced down to 7.58 L/m2.h tential conflict of interest.
(equivalent to τ = 9 h), the final TMP was below 1.0 bar. With such
increase in TMP within 5 h of reaction (i.e., TMP < 1.0 bar), it is still
possible to perform longer hydrolysis of tempe flour-rich in protein since Acknowledgement
the pressure stability of the reactor system is up to 6.0 bar.
The authors gratefully acknowledge the Ministry of Research and
3.6. IC50 of antioxidant and AICE with different membrane cut-offs Technology/National Agency for Research and Innovation, Indonesia,
for supporting this research through the schemes of Penelitian Dasar
The protein hydrolysate produced by employing the optimum Unggulan Perguruan Tinggi (PDUPT) 4053/IT3.L1/PN/2020 and
operating conditions (i.e., [E]/[S] = 10% and τ = 9 h, 10-kDa mem­ (partially) WCU Program managed by Institut Teknologi Bandung. We
brane) was subject to further separations using 5-, 4-, and 2-kDa PES also thank Dr. Simson Tarigan (Indonesian Research Center for Veteri­
membrane. IC50 values for the antioxidant and AICE of the corre­ nary Sciences) and Ir. Oentojo Tjiptobusono for their technical support.
sponding filtrates were measured and presented in Fig. 9. IC50 for
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