LWT - Food Science and Technology 148 (2021) 111725

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LWT
journal homepage: www.elsevier.com/locate/lwt

Whey protein isolate-lignin complexes as encapsulating agents for

enhanced survival during spray drying, storage, and in vitro gastrointestinal
passage of Lactobacillus reuteri KUB-AC5
Phùng Diệp Huy Vũ , Akkaratch Rodklongtan , Pakamon Chitprasert *
Biotechnology of Biopolymers and Bioactive Compounds Special Research Unit, Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok,
10900, Thailand

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

Keywords: This study aimed to investigate the effect of encapsulant weight ratios of whey protein isolate (WPI)-lignin
Antioxidant complex (10:0, 9.5:0.5, 9:1, 8.5:1.5, and 8:2) on survival of Lactobacillus reuteri KUB-AC5 during spray drying,
Encapsulation storage, and passage through simulated gastrointestinal tract. Change in the secondary structure of WPI indicated
Lipid peroxidation
its interaction with lignin. Higher lignin amounts resulted in higher yields and lower moisture contents. After
Malondialdehyde
Probiotic
spray drying and 2-month storage, the highest and lowest malondialdehyde contents were found in 10:0 and 9:1
formulations, respectively. After drying, the lowest and highest numbers of viable cells, 8.70 and 9.34 log CFU/g,
were found in 10:0 and 9:1 formulations, respectively. After storage at 4 and 37 ◦ C, the lowest and highest
survival rates were also observed in these two formulations. At higher lignin concentrations, inactivation rate
constants were reduced. After passage through simulated gastrointestinal tract, the highest and lowest cell
viability was observed in 9:1 formulation and free cells, respectively. The results showed that microencapsulation
using WPI-lignin complexes at 9:1 ratio offered the best protection. Lignin improved survival of spray-dried
probiotics through improving the structure and antioxidant properties of WPI.

1. Introduction around a core, live microorganisms or sensitive compounds, in order to

Spray drying is a preferred drying method for converting a bacterial
suspension into a powder on a commercial scale due to its low cost and
high productivity. However, there remain challenges in obtaining a
high-quality spray-dried probiotics because a high-temperature process
can damage bacteria. During drying, cells suffer from thermal, osmotic,
oxidative, and dehydration stresses, inducing injury to the cell wall and
multicellular components, especially cytoplasmic membranes, nucleic
acids and ribosomes, and eventually leading to a decrease in cell survival
(Khem, Woo, Small, Chen, & May 2015). During subsequent storage,
free radical-induced lipid peroxidation gives rise to structural change in
the cell membranes, resulting in a continuous decrease in cell viability
and cell degeneration (Liu, Gu, Liao, & Yu, 2014; Reusser, 1963; Teix­
eira, Castro, & Kirby, 1996). After being ingested, probiotics encounter a
strong acid in the stomach and bile salt in the small intestine (Darjani,
Nezhad, Kadkhodaee, & Milani, 2016). Therefore, a suitable strategy
must be employed to protect probiotics against these stresses.
Microencapsulation is a process of forming a functional barrier
preserve biological, chemical, and physical properties of the core against
undesired reactions (Rodrigues, Cedran, Bicas, & Sato, 2020; Zhang
et al., 2020). Spray drying is considered as one of the most commonly
employed method for microencapsulation (Rodrigues et al., 2020). A
key success factor in microencapsulation by spray drying is the selection
of an encapsulant with desirable properties (Geranpour, Assadpour, &
Jafari, 2020). Whey protein isolate (WPI) recovered from cheese
manufacturing waste, is an interesting encapsulant because it was found
effective in protecting probiotic viability during spray drying (Khem,
Small, & May 2016; Ying et al., 2013) and passage through gastroin­
testinal tract (Picot & Lacroix, 2004). However, it showed weakness in
maintaining the viability of spray-dried bacteria during storage (Wang,
Lin, & Zhong, 2020) due to its poor moisture barrier property (Silva,
Mauro, Goncalves, & Rocha, 2016) and low antioxidant activity (Jia
et al., 2020).
A practical approach to overcome such disadvantages of using WPI
as an encapsulant is complexation with a hydrophobic compound with
antioxidant properties. For example, the hydrophilic nature of WPI

* Corresponding author.
E-mail address: pakamon.c@ku.ac.th (P. Chitprasert).

https://doi.org/10.1016/j.lwt.2021.111725
Received 2 November 2020; Received in revised form 12 May 2021; Accepted 13 May 2021
Available online 18 May 2021
0023-6438/© 2021 Elsevier Ltd. All rights reserved.
P. Diệp Huy Vũ et al.
limiting its ability to provide an effective moisture barrier was signifi­
cantly decreased by incorporating curcumin into WPI matrices (Kevij,
Salami, Mohammadian, & Khodadadi, 2020). Enhanced storage stability
could be achieved by adding an antioxidant to an encapsulant to reduce
oxidative stress. For instance, Nag and Das (2013) showed that fortifi­
cation of vitamin E as antioxidant in reconstituted whole milk improved
the stability of freeze-dried Lactobacillus casei during 20-week storage
period at 25 ◦ C. However, vitamin E and other low molecular weight
natural antioxidants are unstable when exposed to heat and oxygen
during spray drying (Saldanha do Carmo et al., 2017). Therefore, a
thermostable and hydrophobic antioxidant might be an ideal alternative
to replace low molecular weight antioxidants for combination with WPI.
Lignin has attracted a lot of attention for providing resistance against
thermo-oxidative degradation of food products due to its low cost and
low toxicity. It is a highly branched hetero-phenolic compound con­
sisting of p-coumaryl alcohol, coniferyl alcohol, and syringyl alcohol.
The phenolic hydroxyl group has been reported to mainly contribute to
antioxidant activity of lignin due to the ability of hydrogen atoms to
scavenge free radicals (Liao, Latif, Trache, Brosse, & Hussin, 2020).
Azadfar, Gao, & Chen (2015) revealed that alkali lignin had a compa­
rable antioxidant activity compared to 3,5-di-tert-butyl-4-hydrox­
ytoluene (BHT), a widely used antioxidant in oils, fats, and processed
foods. Lignin has been successfully incorporated into other biopolymers
to improve not only antioxidant activity, but also moisture barrier
property and thermal stability of polymer materials for food packaging
applications (Aadil, Prajapati, & Jha, 2016; Shankar, Reddy, & Rhim,
2015). Furthermore, lignin can retard the oxidation of oxygen-sensitive
food products and extend their shelf life. For example, gelatin-lignin
complexed films were found to prevent lipid oxidation of salmon fil­
lets under high-pressure cooking (Ojagh, Núñ;ez-Flores,
López-Caballero, Montero, & Gómez-Guillén, 2011). However, to the
best of our knowledge, no previous studies have been conducted using
WPI-lignin complexes for microencapsulation of probiotics to improve
cell viability during and after spray drying.
The aim of this work was to investigate the possibility of using WPI
combined with lignin in microencapsulation of probiotic Lactobacillus
reuteri KUB-AC5 via spray drying. The influence of lignin addition on the
secondary structure and thermal stability of WPI was evaluated using
Fourier transform infrared spectroscopy and differential scanning calo­
rimetry, respectively. The effect of WPI to lignin ratios on yields and
moisture contents was examined. The antioxidant function of lignin in
reducing lipid peroxidation of microcapsules was quantified by
measuring malondialdehyde contents. The viability of spray-dried pro­
biotics after 2-month storage at 4 and 37 ◦ C as well as under simulated
gastrointestinal conditions was determined to prove the protective role
of the WPI-lignin complexes.
2. Materials and methods
2.1. Materials
WPI was purchased from Power Corporation (Bangkok, Thailand).
Alkaline lignin was obtained from Sigma-Aldrich (St. Louis, MO, USA).
De Man, Rogosa and Sharpe (MRS) was purchased from Difco™ (Sparks,
MD, USA). 2,2′ -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)
(ABTS), 6-hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic acid
(Trolox), pepsin from porcine gastric mucosa, pancreatin from porcine
pancreas, and bile extract porcine were obtained from Sigma-Aldrich
(St. Louis, MO, USA). All chemicals were analytical reagent grade.
2.2. Spray drying of encapsulants
A 10 g of WPI and lignin mixtures at 10:0, 9.5:0.5, 9:1, 8.5:1.5, and
8:2 wt ratios were dissolved in 100 mL deionized water under stirring at
600 rpm for 60 min. The solutions were spray-dried using a Mini Spray
Dryer B-290 with a two-fluid nozzle (BÜCHI Labortechnik AG, Flawil,
2
LWT 148 (2021) 111725
Switzerland). Operating conditions were as follows: 140 ◦ C inlet tem­
perature, 79 ◦ C outlet temperature, 6 mL/min feed flow rate, and 35 m3/
h air flow rate.
2.2.1. Fourier-transform infrared (FTIR) analysis
FTIR spectra were recorded using FTIR spectrometer (Bruker Tensor
27 FT-IR spectrometer, USA) in an attenuated total reflectance mode in a
4000–400 cm− 1 wave number (Rodklongtan & Chitprasert, 2017). A
total of 60 scans was performed with 4 cm− 1resolution. The spectra were
baseline corrected to obtain the amide I band in 1700–1600 cm− 1. A
Fourier self-deconvolution of overlapping bands and a Gaussian
curve-fitting of deconvoluted band contours as a linear combination of
Gaussian-shaped component bands was performed using Origin 8.0
software (OriginLab, Northampton, UK). The percentage of each struc­
ture was calculated by comparing the relative area of its respective
component band with the total area of all band components.
2.2.2. Differential scanning calorimetry (DSC) analysis
DSC analysis was performed using Mettler Toledo DSC-1 analyzer
(STARe System, Mettler-Toledo International Inc., Greifensee,
Switzerland). Approximately 5 mg of samples was weighed into
aluminum pans with lids. Data were collected at 10 ◦ C/min heating rate
over a temperature range from − 50 to 250 ◦ C under nitrogen atmo­
sphere with 20 mL/min flow rate.
2.2.3. Antioxidant activity analysis
Antioxidant activity was determined by ABTS radical cation
(ABTS•+) scavenging assay. An ABTS•+ stock solution was prepared by
mixing 7 mmol/L ABTS with 2.45 mmol/L potassium persulfate and
incubated at room temperature for 16 h in the dark. The stock solution
was diluted with phosphate buffer saline (pH 7.4) to obtain an absor­
bance 0.700 ± 0.02 at 734 nm. Samples were mixed with the ABTS•+
working solution (1:1000 v/v) and incubated for 6 min before mea­
surement. Results were expressed as mg of Trolox equivalent (TE)/g
sample.
2.3. Preparation of L. reuteri KUB-AC5
Probiotics were cultured following the method of Rodklongtan and
Chitprasert (2017). Frozen cultures were streaked on MRS agar plates
containing 0.6% (w/v) calcium carbonate and incubated at 37 ◦ C for 48
h. A single colony was sub-cultured twice in 5 mL MRS broth at 37 ◦ C for
12 h. It was scaled up by transferring to 100 mL MRS broth. The sus­
pension was centrifuged at 2370×g for 15 min at 4 ◦ C followed by
washing twice with 0.85% (w/v) sodium chloride.
2.4. Spray drying of probiotics
Cell pellet was dispersed in WPI-lignin solutions with stirring at 600
rpm for 15 min. The suspension was spray-dried using the conditions
stated in 2.2.
2.4.1. Yield and moisture content
Yields were calculated using equation (1):
Yield (g powders/100 g of total solid materials) = (WP/WS) × 100 (1)
where WP and WS are the dry weights (g) of powders and total solid
materials, respectively.
Moisture contents were determined according to AOAC (2005).
Briefly, 2 g samples were placed in aluminium pans and dried at 105 ◦ C
until constant weight. The residual moisture content was expressed as
percentage of the initial weight.
2.4.2. Scanning electron microscopy (SEM)
Morphologies were examined using SEM (JSM-6610LV, JEOL,
P. Diệp Huy Vũ et al.
Japan) at 15 kV accelerating voltage. Samples were mounted on an
aluminum specimen stub. The stub was sputter-coated with a thin gold
layer using an ion sputtering coater (IB2, Giko Engineering, Japan).
2.4.3. Malondialdehyde (MDA) content
MDA was measured according to the method of Rodklongtan and
Chitprasert (2017). A powder was dissolved in 9 mL deionized water and
the suspension (1 mL) was mixed with 1 mL of 20% (w/v) SDS for 10
min. Then, 2 mL 2-thiobarbituric acid (TBA) solution, consisting of 15%
(w/v) trichloroacetic acid and 0.5% (w/v) 2-thiobarbituric acid, was
added. The mixture was maintained at 90 ◦ C for 1 h. It was allowed to
cool to room temperature and ethanol (2 mL) was added. The suspension
was centrifuged at 4000×g for 15 min. The supernatant was filtered with
a 0.22-μm nylon membrane. MDA content was analyzed using a
high-performance liquid chromatography (HPLC) with fluorescence
detection. The filtrate (20 μL) was injected into an HPLC system
comprising a solvent delivery pump (Alliance® HPLC - e2695 Separa­
tions Module, Waters, MA, USA), a reversed-phase column (AcclaimTM
120, 120 Å, C18 5 μm, 4.6 × 150 mm, Phenomenex, CA, USA), and a
fluorescence detector (JASCO, FP-2020 plus, Tokyo, Japan). A detector
wavelength was set at excitation 532 nm and emission 553 nm. A mobile
phase was a mixture of 0.05 mol/L methanol/potassium phosphate
(45:55, v/v) buffer (pH 6.8) and a flow rate was 0.8 mL/min.
2.4.4. Cell viability after spray-drying and during storage
Powders were packed in aluminum-laminated bags and stored at 4
and 37 ◦ C. Viable counts were determined before storage and at 2-week
intervals for 2-month storage. Approximately 1 g was dispersed in 25 mL
of normal saline. Then, it was 10-fold serially diluted and subsequently
pour-plated on MRS agar. The plates were incubated at 37 ◦ C for 24 h.
Colonies formed were reported as colony-forming units (CFU)/g. The
logarithmic value of residual cell counts after a given storage period (NT)
was fitted with a first-order kinetic model shown in equation (2) (Moats,
1971):
NT = N0 + kT (2)
where N0 represents an initial logarithmic number of viable bacteria, k is
an inactivation rate constant (log CFU/week) calculated from an abso­
lute value of a regression coefficient of each regression line, and T is a
storage period (day).
2.4.5. Cell viability under simulated gastrointestinal conditions
Viability of fresh and spray-dried probiotics under simulated
gastrointestinal conditions was assessed following the method of Gebara
et al. (2013), and Picot and Lacroix (2004) with modifications. Simu­
lated gastric juice (SGJ) was prepared by dissolving potassium chloride
(1.12 g/L), sodium chloride (2.0 g/L), calcium chloride (0.11 g/L), po­
tassium phosphate monobasic (0.4 g/L), and pepsin (0.26 g/L) in
deionized water, followed by adjusting pH to 1.73 by adding 1 mol/L
HCl. Then, it was filtered with a 0.22-μm membrane. Simulated intes­
tinal juice (SIJ) was prepared by dissolving pancreatin (0.5% w/v) and
bile salts (0.3% w/v) in deionized water, followed by adjusting pH to 7
with 1 mol/L NaHCO3. Approximately, 1 g powder or 1 mL fresh cell
concentrate was added into 29 mL SGJ. The samples were incubated at
37 ◦ C and 100 rpm. A 1 mL aliquot was removed to assess the viable cells
after 0, 30, 60, 90, and 120 min incubation. After centrifugation at
2370×g for 15 min at 4 ◦ C, cells were transferred to 29 mL SIF. A 1 mL
aliquot was taken to determine the viability at 1, 2, 3, and 6 h.
2.5. Statistical analysis
Data represented means ± standard deviations of triplicate mea­
surements. Statistical analyses were performed using a one-way analysis
of variance (ANOVA), followed by Fisher Least Significant Difference
(LSD) test using Minitab 16 statistical software (Minitab Inc., PA, USA).
3
LWT 148 (2021) 111725
Differences were considered significant at p < 0.05.
3. Results and discussion
3.1. FTIR spectra
FTIR analysis was carried out to evaluate the effect of WPI-lignin
complexation on the conformational change in WPI molecules at the
secondary structure level. Spectra of all samples in the 4000–400 cm− 1
wavenumber range are shown in Fig. 1a. The amide I band (1700–1600
cm− 1) was selected as a target band for monitoring the protein confor­
mation and interaction because it is the most sensitive spectral region to
the secondary structure among nine characteristic infrared absorption
bands of protein (Kong & Yu, 2007). It arises mostly (80%) from C– –O
stretching vibrations on peptide backbone, influenced by the strength of
hydrogen bonds and the interactions between amide units (Mahato
et al., 2010). As shown in Fig. 1b, the amide I band of WPI (10:0) at
1633 cm− 1 shifted to 1639, 1643, 1645, and 1649 cm− 1 in the presence
of lignin (9.5:0.5, 9:1, 8.5:1.5, and 8:2, respectively). The shift of amide I
bands to higher wavenumbers known as red shifts with lower ampli­
tudes suggested that protein became increasingly disordered (Zadeh,
O’Keefe, & Kim, 2018). Results from the quantitative analysis of the
secondary structure by FTIR deconvolution and curve fitting are shown
in Fig. S1. Major structures of pure WPI were β-sheet (32.30%) and
α-helix (30.51%), considered ordered conformations, and the rest
(disordered conformations) were random coil, β-turn and β-antiparallel
(Table 1). The shift of these conformations was used to determine the
effects of complexation with lignin on the amide I peaks. It was observed
that the ordered conformations shifted toward lower wavenumbers with
the increase in lignin content, whereas the disordered conformations
shifted toward higher wavenumbers (Fig. 2). The shift toward lower
wavenumbers could be due to the increase in hydrogen bondings be­
tween the phenolic hydroxyl groups of lignin and the carbonyl groups of
the peptide chains. An overall change in the secondary structural com­
positions of WPI due to complexation with lignin could be described
using the relative content (R) of the ordered and disordered conforma­
tions (Meng & Li, 2021; Oliviero, Verdolotti, Di Maio, Aurilia, & Ian­
nace, 2011). The R values were significantly decreased with increasing
lignin content up to 9:1. The increased proportion of the disordered
regions indicated lignin-induced partial unfolding of the protein struc­
ture (Zhang et al., 2014). Similar results for a significant decrease in
α-helix and β-sheet and a remarkable increase in β-turn and random coil
contents of WPI due to complexation with polyphenols were reported by
Meng and Li (2021). Besides hydrogen bondings, hydrophobic in­
teractions might contribute to the structural change. Hydrophobic in­
teractions occurred between the aromatic rings of the lignin subunits
and the hydrophobic residues (benzene and aliphatic side chains) of the
amino acids (Ozdal, Capanoglu, & Altay, 2013). The spectral change
accompanying the hydrophobic interactions was monitored in the re­
gion of 3000–2800 cm− 1, representing CH2 antisymmetric and sym­
metric stretching vibrations (Fig. 1b). The CH2 bands of WPI shifted to
higher wavenumbers upon lignin complexation.
3.2. DSC thermograms
Thermal analysis was performed to determine complexation between
WPI and lignin. The thermogram of WPI showed a broad endothermic
peak at 150.21 ◦ C (Tpeak), representing the melting temperature (Fig. 3,
Table 2). The broadness of the melting peak was indicative of a multi­
component nature of WPI. The DSC data for the WPI-lignin complexes
showed the shift in the melting peak to higher temperature from 150.21
to 163.86 ◦ C. As a higher melting temperature is an indicator of a more
stable material, the WPI-lignin microcapsules with higher lignin con­
tents showed higher thermal stability. In addition, the specific enthalpy
(ΔH) increased from − 45.27 to − 113.15 J/g upon increasing lignin
content. This indicated the enhanced thermal stabilizing effect of lignin
P. Diệp Huy Vũ et al. LWT 148 (2021) 111725

1 1
Fig. 1. FTIR spectra of spray-dried WPI-lignin complexes at different weight ratios: (a) spectra in 4000–400 cm− and (b) spectra in 3000–2800 cm− and
1700–1600 cm− 1.

Table 1
Composition (%) of secondary structural elements and ratio of ordered to disordered phases (R) of spray dried WPI-lignin (LN) complexes at different weight ratios.
WPI-LN (w: β-Sheet (1610–1640 Random coil (1641–1648 α-Helix (1650–1660 β-Turn (1660–1680 β-Antiparallel (1680–1692 R
w) cm− 1) cm− 1) cm− 1) cm− 1) cm− 1)

10:0 32.30 ± 0.99b 18.19 ± 0.27a 30.51 ± 0.72a 10.83 ± 1.17c 8.27 ± 0.38c 1.68 ±
0.09a
b b a bc c
9.5:0.5 31.00 ± 0.71 16.39 ± 0.13 30.96 ± 1.36 12.66 ± 0.93 8.99 ± 0.41 1.63 ±
0.05a
9:1 31.59 ± 0.83b 17.69 ± 0.27ab 26.91 ± 0.13b 14.55 ± 0.78b 9.27 ± 0.38bc 1.41 ±
0.03b
8.5:1.5 31.34 ± 1.90b 14.20 ± 0.28c 27.31 ± 0.98b 16.77 ± 0.33a 10.38 ± 0.54ab 1.42 ±
0.01b
8:2 36.43 ± 2.02a 10.85 ± 1.20d 23.05 ± 0.49c 18.11 ± 0.16a 11.57 ± 0.66a 1.47 ±
0.01b

The results represent mean ± standard deviations (n = 3). a-d Means with different superscripted lower case letters in the same column represent statistical difference
(p < 0.05).

of the milk proteins (Ferraro, Madureira, Sarmento, Gomes, & Pintado,

2015). Our DSC and FTIR results showed complexation between WPI
and lignin through hydrophobic interactions and hydrogen bonding,
resulting in the enhanced thermal stability of the microcapsules.

3.3. Antioxidant activity

Oxidative stress-induced lipid peroxidation is the primary cause of

Fig. 2. Amide I peak positions in FTIR spectra of spray-dried WPI-lignin
complexes at different weight ratios for β-sheet ( ), random coil ( ),
α-helix ( ), β-turn ( ) and β-antiparallel ( ).
on the WPI structures. These results are in agreement with a previous
report that the complexation between major milk protein components
(β-lactoglobulin and α-lactalbumin) and rosmarinic acid, a bioactive
plant phenolic compound, contributed to the increased thermal stability
cell injury and cell death of gut-inhabiting probiotics with oxygen-
sensitive nature upon drying and storage (Teixeira et al., 1996). On
the contrary, probiotics possessing high antioxidant capacity can survive
bactericidal actions of free oxygen radicals by using their effective redox
systems associated with antioxidant enzymes and oxidative damage
repair systems (Feng & Wang, 2020; Stecchini, Torre, and Munari
(2001). A possible approach to temporarily reducing the oxygen level
and improving the survival of oxygen-sensitive probiotics during spray
drying is the addition of an antioxidant in an encapsulant (Celik &
O’Sullivan, 2013; Ghandi, Powell, Howes, Chen, & Adhikari, 2012). In
this research, lignin was used to enhance the antioxidant ability of WPI.
WPI alone presented the lowest antioxidant activity and its complexes
with higher amounts of lignin showed higher antioxidant activity
(Table 3). Non-etherified phenolic groups, ortho-methoxy groups, hy­
droxyl groups, and double bonds between the outermost carbon atoms
in the side chains of lignin can scavenge free radicals through hydrogen
atom transfer and single electron transfer mechanisms (Dizhbite, Tely­
sheva, Jurkjane, & Viesturs, 2004). The richness of these functional
groups makes lignin attractive for applying as a natural antioxidant for
stabilization of probiotics.

4
P. Diệp Huy Vũ et al. LWT 148 (2021) 111725

Fig. 3. DSC thermograms of spray dried WPI-lignin complexes at different weight ratios.

Table 2 Table 4
DSC data, peak temperature (Tpeak), onset temperature (Tonset), endset temper­ Yields and moisture contents of L. reuteri KUB-AC5 microencapsulated with WPI-
ature (Tendset), and enthalpy change (ΔH) for spray dried WPI-lignin (LN) com­ lignin (LN) complexes at different weight ratios.
plexes at different weight ratios. WPI-LN (w: Yield (g powder/100 g to total solid Moisture content (g/
WPI-LN (w:w) Tpeak (◦ C) Tonset (◦ C) Tendset (◦ C) ΔH (J/g) w) materials) 100 g)

10:0 150.21 148.33 159.03 − 45.27 10:0 55.18 ± 0.34a 2.94 ± 0.47a
9.5:0.5 150.44 148.55 157.60 − 88.97 9.5:0.5 58.16 ± 0.54b 2.64 ± 0.01ab
9:1 155.43 153.81 162.17 − 77.85 9:1 61.29 ± 1.04b 2.28 ± 0.24b
8.5:1.5 161.94 159.88 170.36 − 90.53 8.5:1.5 62.91 ± 0.14c 2.01 ± 0.11b
8:2 163.86 163.18 169.84 − 113.15 8:2 65.08 ± 0.99d 2.04 ± 0.12b

Table 3
Antioxidant activity of spray dried WPI-lignin (LN) complexes at
different weight ratios.
WPI-LN (w:w) TEAC (mg TE/g sample)
10:0 7.57 ± 0.15 e
9.5:0.5 42.19 ± 0.60d
9:1 65.98 ± 1.44c
8.5:1.5 87.25 ± 0.02bc
8:2 102.07 ± 1.95b
0:10 548.08 ± 17.76a
The results represent mean ± standard deviations (n = 3). a-
d
Means with different superscripted lower case letters in the same
column represent statistical difference (p < 0.05).
3.4. Yield and moisture content
High yield (>50 g/100 g of total solid materials) and low moisture
content (<4 g/100 g) are generally desirable characteristics indicative of
spray drying performance, process economics, and final product quality.
Several parameters influence yields and moisture contents of spray dried
microcapsules and one such parameter is an encapsulant. The yields and
moisture contents of the spray-dried probiotics formulated with WPI and
its complexes with lignin at different ratios ranged from 55.18 to 65.08
g/100 g of total solid materials and 2.05–2.94 g/100 g, respectively
(Table 4). Upon increasing lignin content, the yield increased, while the
moisture content decreased. The results suggested that the addition of
lignin resulted in high drying efficiency. This can be explained by the
fact that lignin has a lower water binding ability than WPI and the
structure of lignin facilitates the removal of water from the droplets
during the contact with hot air. Lignin, a phenolic polymer, is a smaller
molecule (the molecular weight = 10 kDa) compared to WPI (the mo­
lecular weight of its main components, β-lactoglobulin = 18.3 kDa and
The results represent mean ± standard deviations (n = 3). a-d Means with
different superscripted lower case letters in the same column represent statistical
difference (p < 0.05).
α-lactalbumin = 14.4 kDa) (de Morais et al., 2020). All spray dried
probiotic microcapsules had satisfactory yields and moisture contents,
complying with standard acceptable values for spray-dried products.
3.5. Surface morphology
The majority of the WPI microcapsules (Fig. 4a) were spherical in
shape and polydispersed in size. The broad size distribution is probably
due to the fact that WPI contains a number of protein constituents, i.e.
β-lactoglobulin (70.2%), α-lactalbumin (14.1%), bovine serum albumin
(8.2%), immunoglobulins (6.5%), and lactoferrin (1.0%) (Tavares,
Croguennec, Carvalho, & Bouhallab, 2014). Both large and small WPI
microcapsules presented a smooth surface. Fragmentation of the thin
microcapsule walls was observed in a few large particles, indicating that
droplets expanded due to thermal expansion of water vapour inside at
the early stage of drying, technically known as the ballooning phe­
nomenon (Sheu & Rosenberg, 1998). The resulting enlarged solid wall
had low mechanical strength; thus, explosion occurred leading to
microcapsule fragmentation and pore formation after drying (Sheu &
Rosenberg, 1998). The fragility of the WPI wall might result from a low
solid content (only 10%). These surface defects can ruin protective
functions of the microcapsules against harsh conditions that can cause
stress and damage to the cells. In the case of the small WPI microcap­
sules, almost all of their surfaces exhibited several deep dents, which
could be associated with a rapid crust formation and then uneven sur­
face shrinkage of the microcapsules prior to onset of expansion due to
high drying rates of the droplets (Sheu & Rosenberg, 1998). In addition,
no visible pores and cracks were observed in the small microcapsules.
When the microcapsules were prepared with the mixtures of WPI and
lignin at the ratios of 9.5:0.5, 9:1, 8.5:1.5, and 8:2, no distinct difference

5
P. Diệp Huy Vũ et al.
Fig. 4. SEM micrographs of L. reuteri KUB-AC5 microencapsulated with WPI-lignin complexes at different weight ratios: (a) 10:0, (b) 9.5:0.5, (c) 9:1, (d) 8.5:1.5, (e)
8:2, and (f) lignin.
between their surface topography (Fig. 4b, c, d, and e, respectively) and
that of the WPI microcapsules was visually detected. Fig. 3f represents
the cell-loaded lignin microcapsules prepared with the same solid con­
tent. The lignin microcapsules appeared to be more perfectly spherical
and smaller than the WPI microcapsules. The surfaces were smooth and
free of wrinkles. Only a few small and relatively shallow dents were
observed in a few particles. These findings suggested that the lignin
matrix had higher mechanical strength compared to the WPI matrix. The
lignin microcapsules allowed water vapour expansion, while they still
retained their spherical shape under the given spray drying condition.
3.6. Lipid peroxidation
To assess the effect of the encapsulant on cell protection against
oxidative damage during spray drying and subsequent storage, the
concentration of MDA, a widely used biomarker for lipid peroxidation,
was measured. After spray drying, the 10:0 formulation had the highest
MDA concentration, followed by the 9.5:0.5, 8:2, 8.5:1.5, and 9:1 for­
mulations (Fig. 5). The results indicated that during cell exposure to hot
air stream, lignin had a greater ability to inhibit free radical-induced
oxidation than WPI, consistent with the TEAC results. However, the
Fig. 5. MDA concentrations of L. reuteri KUB-AC5 microencapsulated with WPI-lignin complexes at different weight ratios after spray drying ( ) and 2-month
storage at 4 ◦ C ( ) and 37 ◦ C ( ).
6
LWT 148 (2021) 111725
level of oxidative stress at high lignin concentration (2%) was higher
than that at low lignin concentrations (1 and 1.5%). It is well-known that
high temperature applied during spray drying promotes cell membrane
fluidity and therefore, enhancing the interaction between fatty acids in
the phospholipids of the cell membrane and the functional groups,
mainly –OH, –CO, and –COOH, in the phenolic units of lignin (Gyawali
& Ibrahim, 2014; Yang et al., 2016). This possibly induced more severe
membrane damage and increased lipid peroxidation in the case of high
lignin concentration. After 2-month storage at 4 ◦ C, the MDA value of
only the 10:0 formulation significantly increased (p < 0.05), whereas
those of the others were relatively constant. The results implied that
lipid peroxidation occurred with the cells encapsulated with WPI during
storage even in the dried state and at low temperature, but the presence
of lignin (≥0.5%) retarded the MDA formation. At high storage tem­
perature (37 ◦ C), MDA increased more rapidly, especially in the 10:0
formulation. Similarly, Shafiei et al. (2014) reported that high storage
temperature at 35 ◦ C enhanced the formation of reactive oxygen species
and the extent of oxidative damage of freeze-dried Acetobacter senegal­
ensis compared to low storage temperature (4 ◦ C). Nevertheless, it is
important to note that only the sample in the presence of 2% lignin
stored at 37 ◦ C did not show any statistically significant changes in the
P. Diệp Huy Vũ et al. LWT 148 (2021) 111725

MDA concentration, indicating that 2% lignin was the most effective in
protecting the cells from oxidative stress during storage due to the
highest TEAC value.
3.7. Cell viability during spray drying and storage
Table 5 shows that the 9:1 formulation provided the highest viable
count of 9.34 log CFU/g after spray drying from the initial count of 9.36
log CFU/mL, followed by the 8.5:1.5, 8:2, 9.5:0.5, and 10:0 formula­
tions. WPI alone exhibited the worst protective effect during cell expo­
sure to hot air, which could be attributed to its lowest thermal stability.
However, the 8:2 formulation showing the highest thermal stability and
antioxidant activity did not provide the highest cell survival, which is
possibly due to too high concentration of lignin. A negative effect of 2%
lignin on cell survival may be related to its ability to destroy the cell
membrane, generate MDA, and eventually lead to cell death during
spray drying. It is evident from the MDA results that the MDA concen­
tration in the 2% lignin treatment was higher than that in the 1 and 1.5%
lignin treatments, but lower than that in the 0.5% lignin treatment and
the control (WPI alone).
After 2-month storage, the viable counts decreased in all treatments.
The highest cell number was found in the 9:1 microcapsule, followed by
8.5:1.5, 8:2, 9.5:0.5, and 10:0 microcapsules for both temperatures. The
index of the rate of viability loss was presented in terms of an inacti­
vation rate constant. High R2 and low root mean squared error (RMSE)
values indicate the goodness of fit of a model (Zhao, Chen, Zhao, He, &
Yang, 2020). The 8:2 microcapsule had the lowest inactivation rate
constant, while the WPI microcapsule without lignin had the highest
values for both storage temperatures. There seemed to be an inverse
relationship between the antioxidant activity of the encapsulants and
the inactivation rate constant. Moreover, there was a strong inverse
linear relationship between the remaining cell numbers and the MDA
concentrations with R2 of 0.9888 and 0.9985 for all tested microcapsules
stored at 4 and 37 ◦ C, respectively (Fig. S2). These findings confirm the
antioxidant role of lignin in reducing lipid peroxidation reported to be a
possible mechanism of cell death during storage (Ananta, Volkert, &
Knorr, 2005). Similar results were obtained by using a commonly used
antioxidant, vitamin E, to maintain the viability of spray-dried Lacto­
bacillus casei CRL 431 during 20-week storage period at 25 ◦ C (Nag &
Das, 2013) and the viability of spray-dried Lactobacillus rhamnosus GG
during 24-week storage period at 4 and 25 ◦ C (Ying et al., 2011). The
great impact of storage temperature on cell survival can also be observed
in Table 5, where the higher the storage temperature, the faster the cells
died during storage. At 4 ◦ C storage, only a small reduction of viable
cells of all formulations was found, remaining over 8.43 log CFU/g. As
can be expected from the Arrhenius theory, the adverse effect of high
storage temperature (37 ◦ C) led to lower probiotic viability. The effect of
storage temperature on the survival rate of the spray dried probiotics
found in this study is in agreement with the previous results of Vanden
Braber et al. (2020) and Agudelo, Cano, González-Martínez, and Chiralt
(2017). The lower survival rates of the microcapsules stored at 37 ◦ C
might be related to higher MDA contents, resulting from more extensive
damage of the cell membrane. Complexation between WPI and lignin
partially counteracted the harmful effect of lipid peroxidation acceler­
ated by high temperature. However, high lignin concentrations resulted
in the adverse effects to the cells as discussed above. The results showed
that 1% was an appropriate concentration for complexation with WPI. In
addition, the results suggested that cold storage is necessary for
extending the shelf life of spray-dried probiotics, although it signifi­
cantly increases product costs.
3.8. Cell viability under simulated gastrointestinal conditions
Besides the ability to preserve cell stability during production and
storage, microcapsules must have the ability to protect cells from the
harsh environment of gastrointestinal tract so cells can survive and
reach the intestine in adequate numbers. This study investigated the
viability of free and microencapsulated L. reuteri KUB-AC5 under
simulated gastrointestinal conditions. The number of the viable cells
decreased continuously from 9.2 log CFU/g in the beginning to 7.5 log
CFU/g after 120 min exposure to SGF (Fig. 6). The loss in viability
during incubation in the gastric juice was likely due to the acidic con­
ditions (pH 1.73). When encapsulated in WPI, the viable count was
higher (8.1 log CFU/g). The protective effect of WPI microcapsules ob­
tained by spray drying was observed by other authors for different
probiotics. Puttarat, Thangrongthong, Kasemwong, Kerdsup, & Tawee­
chotipat (2021) observed that free cells of L. reuteri TF-7 decreased by
3.8 log CFU/g after exposure to gastric juice at pH 2 for 2 h and by 2.4
log CFU/g when microencapsulated in WPI. Lactobacillus acidophilus
NRRL-B 4495 in a free form presented a significant reduction of 2.7 log
cycles, whereas the cells microencapsulated in WPI had only 0.81 log
cycles decrease after 3 h incubation in gastric juice at pH 3 (Çabuk &
Harsaa, 2015). The buffering capacity of WPI might be responsible for
reducing the negative effect of SGF on the cells (Rodrigues et al., 2011).
Another possible explanation is that β-lactoglobulin and α-lactalbumin
in WPI were resistant to pepsinolysis due to their compact globular
structures (Halabi, Croguennec, Bouhallab, Dupont, & Deglaire, 2020).
A smaller decline in the viability was observed for the formulations of
9:1 and 9.5:0.5. This implied that complexation with lignin caused an
increase of protein unfolding, resulting in a reduction in protein di­
gestibility. This result is consistent with the findings of a previous study
(Stojadinovic et al., 2013) that complexation of beta-lacoglobulin and
polyphenols enhanced protein stability to digestion by pepsin at the
extreme pH of simulated gastric fluids. Additionally, Petzke, Schuppe,

Table 5
Viability and inactivation rate constant of L. reuteri KUB-AC5 microencapsulated with WPI-lignin (LN) complexes at different weight ratios after spray drying and 2-
month storage at 4 and 37 ◦ C.
WPI:LN Cell number after spray Storage at 4 ◦ C Storage at 37 ◦ C
(w:w) drying (log CFU/g)
Cell number (log k (log CFU/ RMSE R2 Cell number (log k (log CFU/ RMSE R2
CFU/g) week) CFU/g) week)

10:0 8.70 ± 0.01d 8.43 ± 0.00e 0.033 ± 0.048 ± 0.990 ± 5.67 ± 0.01d 0.349 ± 0.132 ± 0.998 ±
0.001a 0.010b 0.001b 0.007a 0.004b 0.001a
c d c
9.5:0.5 8.94 ± 0.01 8.68 ± 0.01 0.032 ± 0.035 ± 0.996 ± 6.46 ± 0.02 0.258 ± 0.225 ± 0.985 ±
0.001a 0.006b 0.005ab 0.005b 0.011a 0.007bc
9:1 9.34 ± 0.05a 9.10 ± 0.01a 0.030 ± 0.043 ± 0.998 ± 7.25 ± 0.03a 0.234 ± 0.156 ± 0.992 ±
0.001ab 0.003b 0.001a 0.010bc 0.004b 0.000ab
8.5:1.5 9.32 ± 0.01a 9.04 ± 0.03b 0.028 ± 0.098 ± 0.995 ± 7.24 ± 0.02a 0.231 ± 0.125 ± 0.996 ±
0.001b 0.010a 0.002ab 0.011c 0.020b 0.000ab
8:2 9.09 ± 0.03b 8.86 ± 0.01c 0.027 ± 0.081 ± 0.999 ± 6.85 ± 0.01b 0.230 ± 0.230 ± 0.979 ±
0.001b 0.003a 0.000a 0.014c 0.028a 0.007c

The results represent mean ± standard deviations (n = 3). a-e Means with different superscripted lower case letters in the same column represent statistical difference (p
< 0.05).

7
P. Diệp Huy Vũ et al.
Fig. 6. Viability of L. reuteri KUB-AC5 microencapsulated with WPI-lignin complexes at different weight ratios: 10:0. (
8:2 ( ) and free cells ( ) during exposure to simulated gastric fluids (SGF) and simulated intestinal fluids (SIF).
Rohn, Rawel, and Kroll (2005) reported that the digestion of WPI in rats
was obstructed by the reactions with some phenolic compounds. On the
contrary, the formulations of 8.5:1.5 and 8:2 (high lignin concentra­
tions) showed lower viability than the other three formulations and free
cells. Lignin at high concentrations might exhibit a synergistic antibac­
terial activity with acid against the cells (Juttuporn, Thiengkaew, Rod­
klongtan, Rodprapakorn, & Chitprasert, 2018).
After 1 h of a subsequent exposure to SIF, the free cells had
approximately 1.6 log CFU/g reduction in the cell viability, whereas all
microencapsulated cells had only a slight decrease. Thereafter, the
viable counts of all samples remained relatively constant. The antibac­
terial properties of bile salts in SIF led to cell membrane disruption,
protein denaturation, iron and calcium chelation and DNA oxidation,
and eventually cell death (Begley, Gahan, & Hill, 2005). However, our
previous experiment carried out in SGF and SIF separately showed that
fresh L. reuteri KUB-AC5 had the ability to tolerate bile salts and survive
in the intestine condition (Rodklongtan, La-Ongkham, Nitisinprasert, &
Chitprasert, 2014). Therefore, a sharp decrease in the cell viability of the
free cells in this experiment is probably due to the fact that the cells after
being removed from SGF are sublethally injured and more sensitive to
SIF. By the end of 6 h incubation in SIF, the viable cell numbers arranged
from the highest to the lowest were observed in the 9:1, 9.5:0.5, 10:0,
8.5:1.5, 8:2 treatments, and the free cells. Similar to the results of the
SGF test, the enhanced physical stability of WPI by complexation with
lignin at the suitable concentrations (0.5 and 1%) was beneficial for
improving cell survival in SIF.
4. Conclusion
The present study showed the potential application of lignin as a
complexing agent for WPI in microencapsulation of L. reuteri KUB-AC5.
Due to WPI-lignin interactions under the drying condition, the thermal
stability and antioxidant activity of WPI was enhanced. The probiotics
microencapsulated in the WPI-lignin complexes maintained higher
viability after drying and storage compared to those microencapsulated
in WPI alone. After sequential exposure to gastric and intestinal fluids,
8
LWT 148 (2021) 111725
), 9.5:0.5 ( ), 9:1 ( ), 8.5:1.5 ( ),
the cells microencapsulated in the complexes formulated with lignin at 1
and 1.5% had the highest survival. It is apparent from the results that
complexation with lignin improved the microencapsulating properties
of WPI, resulting in high cell survival. Nevertheless, further studies are
needed to assess the possibility of using the developed probiotics as
functional food ingredients. The addition of microencapsulated pro­
biotics to food products used to deliver probiotics is technically chal­
lenging. Microencapsulated probiotics in food matrices must have high
enough numbers of viable cells during processing, storage, and diges­
tion. The interaction between microencapsulated probiotics and food
matrices may have a positive or negative effect on cell viability. The
sensory attributes of food products containing microencapsulated pro­
biotics should be acceptable to consumers. Finally, health benefits after
regular consumption of functional foods should be assessed.
CRediT authorship contribution statement
Phùng Diệ ệp Huy Vũ: Conceptualization, Methodology, Software,
Investigation, Data curation, Writing – original draft. Akkaratch Rod­
klongtan: Software, Investigation, Data curation. Pakamon Chitpra­
sert: Resources, Supervision, Writing – review & editing, Funding
acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
This work was supported by the Kasetsart University Scholarships for
ASEAN for Commemoration of the 60th Birthday Anniversary of Pro­
fessor Dr. Her Royal Highness Princess Chulabhorn Mahidol.
P. Diệp Huy Vũ et al.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lwt.2021.111725.
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