Food Chemistry 413 (2023) 135596

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

The incorporation of peach gum polysaccharide into soy protein based

microparticles improves probiotic bacterial survival during simulated
gastrointestinal digestion and storage
Haodong Yao a, 1, Bu Liu a, 1, Li He b, Jielun Hu a, Huan Liu a, *
a
State Key Laboratory of Food Science and Technology, China-Canada Joint Laboratory of Food Science and Technology (Nanchang), Key Laboratory of Bioactive
Polysaccharides of Jiangxi Province, Nanchang University, Nanchang 330047, China
b
China Tobacco Jiangxi Industrial Co., Ltd., Nanchang 330096, Jiangxi, China

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

Keywords: The objective of this research was to investigate the in vitro gastrointestinal digestion and storage properties of
Probiotics Lactobacillus plantarum 550 encapsulated in soy protein isolate (SPI) and peach gum polysaccharide (PG) through
Peach gum spray drying. High survival rates (>8.1 Log CFU/g) were obtained for all encapsulation formulas containing PG.
Microencapsulation
Combination of SPI and PG showed positive effects on both gastric resistance and storage stability of cells.
Spray drying
In vitro digestion
Among the formulas tested, sample of SPI:PG = 3:1 showed the highest survival (7.88 ± 0.12 Log CFU/g),
corresponding to the strongest electrostatic interaction between SPI and PG. With PG content increasing, the
storage stability of probiotic was also enhanced, as PG could reduce the moisture content within microcapsules as
well as scavenge free radicals generated during storage. In conclusion, the current study demonstrates that SPI
combined with PG may provide effective protection to cells not only during spray drying, but also during storage
and gastrointestinal digestion.

1. Introduction effects on the host, it is necessary to ensure enough number of viable

Researches on probiotics can be traced back to more than 100 years
ago when Buster discovered lactic acid bacteria in 1857 and since then,
detailed functions of probiotics have been advanced and deepened.
According to the World Health Organization (WHO) and the United
Nations Food and Agriculture Organization (FAO), the definition of
probiotics is live microorganisms that can have a beneficial effect on the
health of the host when ingested in a certain amount. Studies in recent
years have shown that probiotics own several functions that are bene­
ficial to human health as listed: (1) treat allergies, inflammation of small
intestine and colon, and diarrhea; (2) enhance the innate immunity and
cognitive function of healthy adults; (3) inhibit pathogens from
attaching to the surface of intestinal tract and affect the secretion
products of harmful microorganisms; (4) regulate the composition of
intestinal flora; (5) metabolize carbon sources that are difficult for the
human body to digest and alleviate lactose intolerance; (6) reduce blood
sugar and blood lipids, prevent diabetes, cancer, etc. (Rudzki et al.,
2019; Sanchez et al., 2017). In order to sufficiently exert their beneficial
probiotics when consumed by consumers, and the recommendation
from the International Dairy Federation was at least 6 Log CFU/g
(Ouwehand & Salminen, 1998). This requirement making the addition
of probiotics into foods faces severe challenges. As during processing,
transportation and storage, cell damage can be easily caused by oxygen,
heat stress, mechanical damage and high osmotic pressure stress leading
to the loss of probiotic viability. To resolve these above-mentioned
challenges, microencapsulation technology has thus been widely
applied.
Among the wall materials used for probiotic microencapsulation,
proteins and polysaccharides are regarded as the most commonly used
ones (Sun et al., 2007; Hu et al., 2023). As for proteins, they own well
film-forming properties, however, tend to aggregate and are sensitive to
pepsin in gastric juices (Moakes et al., 2015). Polysaccharides own well
acid resistance properties and are difficult to degrade in the stomach,
which can improve the compactness of the microcapsules and endow the
probiotic microcapsules with targeted release effect. However, due to
the limitations of poor film forming property and the formation of large

* Corresponding author.
E-mail address: liu_huan2011@hotmail.com (H. Liu).
1
Authors contributed equally and should be recognized as co-first author.

https://doi.org/10.1016/j.foodchem.2023.135596
Received 14 August 2022; Received in revised form 21 January 2023; Accepted 27 January 2023
Available online 9 February 2023
0308-8146/© 2023 Elsevier Ltd. All rights reserved.
H. Yao et al.
pores between molecular chains, polysaccharides are generally unable
to provide enough protective effect to probiotics. Therefore, combina­
tion of proteins and polysaccharides has gained high attention to ensure
probiotic microcapsules with higher encapsulation yield and better
protective effect as compared to either protein or polysaccharide alone
(Yuan, Hu, He, Hu, & Liu, 2023). For instance, Vu, PDH et al. applied
WPI combined with lignin to encapsulate Lactobacillus reuteri KUB-AC5
using spray drying technology and found significantly higher survival
rates of cells during spray drying (increased from 8.70 Log CFU/g to
9.34 Log CFU/g), storage (increased from 8.43 Log CFU/g to 9.10 Log
CFU/g) and in vitro gastrointestinal digestion (about increased from 7.7
Log CFU/g to 8.4 Log CFU/g) (Diệp Huy Vũ et al., 2021). Han et al.
(2020) prepared calcium-alginate (Ca-Alg) coated WPI microcapsules
for the protection and delivery of Lactobacillus delbrueckii subsp. bul­
garicus (L. bulgaricus) and Lactobacillus paracasei subsp. Paracasei
(L. paracasei) and the results showed that after gastrointestinal diges­
tion, increased survival of up to 3.43 Log CFU/g was found compared
with that in free cells. Hugues-Ayala et al. (2020) coated Lactobacillus
rhamnosus GG using alginate capsules with regular buttermilk proteins
and found that the survival of probiotics after freeze-drying was
significantly higher in protein coated microcapsules (8.91 ± 0.03 Log
CFU/g) than that in alginate alone sample (7.67 ± 0.09 Log CFU/g).
Although positive effects have been proved by the combination of pro­
teins and polysaccharides, however, nowadays the available species of
both proteins and polysaccharides are limited and thus seeking novel
potential wall materials has gained high attention.
Peach gum polysaccharide (PG) is a kind of acidic polysaccharide
with a highly branched molecular structure and is composed of (1 → 3)-
linked β-D-Galp units in the main-chain and arabinogalactan in the side
chains (Simas-Tosin et al., 2009). PG owns well functions of improving
immunity (Bouaziz et al., 2016; Song & Du, 2010; Wei et al., 2019; Wu
et al., 2017), anti-tumor, scavenging free radicals, anti-aging, anti-
infection, lowering blood fat, lowering blood sugar, etc. (Bouaziz et al.,
2016; Song & Du, 2010; Wei et al., 2019; Wu et al., 2017). Zhu et al.
(2018) developed a stable and pH-responsive semi-interpenetrating
hydrogel network structure consisting of PG and Auricularia polytricha
β-glucans and found that the release of encapsulated Lactobacillus plan­
tarum CICC20264 and Lactobacillus salivarius CICC23174 in the simu­
lated gastric fluid were both below 2 Log CFU/g. Besides, in stimulated
intestinal condition, viable cells were progressively increased corre­
sponding with the degradation of hydrogels, and finally, the complete
release activity of probiotics was reached up to 7 Log CFU/g after 10.25
h incubation, which strongly indicated the potential function of PG
based hydrogels in intestinal targeted delivery (Zhu et al., 2019).
In our previous study, the gastric resistance and storage properties of
Lactobacillus zeae LB1 were enhanced by using protein-based probiotic
microparticles incorporated with gum arabic (Liu et al., 2016). As the
carboxyl group carried by the gum arabic molecule can form a strong
hydrogen bond with the amide and carboxyl group of the protein
molecule, the addition of gum arabic thus enhanced the strength of the
polymer network, inhibited the dissolution of microparticles, ultimately
retained and protected the cells within the polymer matrix. The chem­
ical composition of PG is similar to that of gum arabic with lower con­
tent of galactose, rhamnose, uronic acid, and higher content of
arabinose. Moreover, compared with gum arabic, the yield of PG
worldwide is much higher corresponding with lower price and cost.
However, few studies were reported using PG as wall material to
encapsulate probiotic bacteria. Whether applying PG into protein matrix
can offer well protection to probiotic bacteria during gastric digestion
and storage as compared to that in gum arabic is still of much concern
and worth in-depth further study. Therefore, the purpose of this study
was to investigate the effects of incorporating PG into SPI based pro­
biotic microparticle matrix on the gastrointestinal digestion, storage and
thermal resistance properties of probiotic bacteria and the correspond­
ing mechanisms behind.
2
Food Chemistry 413 (2023) 135596
2. Materials and methods
2.1. Materials
Lactobacillus plantarum 550 was purchased from Sichuan Gaofuji
Biological Technology Co., ltd (Chengdu, China), Porcine pepsin (10000
U/mg), pancreatin and trypsin (250 U/mg), SPI and de Man, Rogosa,
and Sharpe (MRS) broth were purchased from Beijing Solarbio Science
& Technology Co., ltd. (Beijing, China). Bile salt was purchased from
Macklin Biochemical Technology Co., ltd. (Shanghai, China). PG was
obtained from Dingli Rubber Industry Co., ltd. (Taian, China). All other
chemical reagents used in this study were of analytical grade or better,
used without further purification.
2.2. Zeta potential measurement
SPI solution (2.0 wt%) was prepared by dispersing SPI into 0.01 M
phosphate buffer (pH 7.0) and stirred at 600 rpm for 2 h at room tem­
perature. PG solution (2.0 wt%) was prepared by dispersing PG into
deionized water and stirred at 600 rpm for 2 h at room temperature,
then adjusted to pH 7.0 using 0.1 M NaOH. The SPI solution concen­
tration was fixed at 1.0 (wt%) and the PG solution concentration was
varied to achieve the initial SPI-PG ratio at 1:0, 3:1, 1:1, 1:3 and 0:1. The
pH of the biopolymer mixture was then adjusted from 7.0 to 2.5 by the
addition of 0.1 M HCl and measured the zeta potential value (every 0.25
pH value, from pH 5.0 to 3.0) using Zetasizer Nano-ZS 90 (Malvern
Instruments, Worcestershire, UK).
2.3. Bacterial cell preparation
The preparation of the bacterial cell was determined according to the
published method with some modifications (Cavender et al., 2021). A
tube of a mixture of bacterial liquid and glycerol solution stored at
− 20 ◦ C was thawed at room temperature and activated twice in 35 mL
MRS broth medium at 37 ◦ C. After the activation, culture cells were
harvested after 22 h by centrifugation (4000 rpm, 10 min), washed, and
re-suspended in 0.9 % saline to obtain a final concentration above 10
Log CFU/mL.
2.4. Microencapsulation of bacterial cells
The complex coacervation method combined with the spray drying
method was used to prepare probiotic microcapsules. The preparation
processes of the microcapsules were as follows: (1) prepare SPI-PG
mixed wall material solutions with different ratios (1:0, 3:1, 1:1, 1:3
and 0:1) and keep the total solid content at 5 % (w/v) (pH 3.75); (2)
place the above solution in a refrigerator at 4 ◦ C overnight to fully hy­
drate; (3) Add 2 mL of concentrated cell solution to the above solution
with continuous stirring for 30 min; (4) spray dry the mixed solution by
spray dryer (B-290, BUCHI, Switzerland) and collect the dry powder.
The spray drying conditions were set as follows: inlet temperature was
140 ◦ C, outlet temperature was constant at 75 ◦ C, pump efficiency was
30 %, and gas flow rate was 30 m3/h.
2.5. Enumeration of Lactobacillus plantarum 550
After diluting the probiotic bacteria suspension with normal saline
(0.9 % sodium chloride) to a suitable concentration range, take 0.1 mL
and evenly spread it on the MRS agar medium. The plates were incu­
bated in an anaerobic incubator at 37 ± 1 ◦ C for 36 h. The count of
viable probiotic cells was expressed as Log colony-forming units per
gram (Log CFU/g). However, before enumeration, to ensure the com­
plete release of the entrapped bacteria from inside the microcapsules,
the samples (0.1 g) were re-suspended in 15 mL of phosphate buffer (0.2
mol/L, pH 7.2) followed by dispersion using a high-speed disperser for
10 min at 4000 rpm. All the experiments were performed in triplicate.
H. Yao et al. Food Chemistry 413 (2023) 135596

Fig. 1. The effect of pH on the zeta potential of SPI-PG mixtures with different
SPI to PG ratios (1:0, 3:1, 1:1, 1:3, 0:1).
Fig. 2. Survival of Lactobacillus plantarum 550 encapsulated in SPI-PG mi­
crocapsules with different ratios (1:0, 3:1, 1:1, 1:3, 0:1) during simulated
Table 1
gastric (1–2 h) and intestinal (2–6 h) digestions. Free: free bacteria without
Physicochemical properties and thermal stability of probiotic microcapsules
microencapsulation.
after spray drying and during heat treatment.
Samples Viability Yield Moisture Viability loss (Log ΔH (J/
(%) (%) (g/100 g) CFU/g) g)

65 ◦ C 75 ◦ C
30 min 10 min

Free cell undetected / / 9.65 ± 9.65 ± /

0.34a 0.34a
SPI:PG 3.23 ± 20.03 5.01 ± 2.12 ± 1.47 ± 86.87
= 1:0 0.03b ± 0.89a 0.01a 0.041b 0.007b
SPI:PG 14.81 ± 23.63 4.79 ± 2.49 ± 1.79 ± 93.89
= 3:1 1.08c ± 0.11b 0.04b 0.018c 0.035c
SPI:PG 23.98 ± 30.96 4.59 ± 2.62 ± 2.36 ± 123.08
= 1:1 0.11d ± 0.78c 0.13c 0.036c 0.024b
SPI:PG 37.20 ± 36.09 4.32 ± 3.45 ± 3.15 ± 139.51
= 1:3 0.42e ± 0.20d 0.07d 0.034d 0.004d
SPI:PG 64.78 ± 43.68 4.14 ± 3.59 ± 3.98 ± 142.36
= 0:1 1.08f ± 0.08e 0.02d 0.013e 0.003e

Results were expressed as mean ± standard deviations, in the same column for
each sample with different superscripts are significantly different (p < 0.05).

2.6. Determination of microencapsulation viability

The viable cell counts encapsulated in microcapsules were deter­

mined according to the published method (Han et al., 2020) with some
modifications. Briefly, 0.10 g microcapsules were mixed with 14.9 mL
0.2 M sodium citrate di-hydrate solution (pH 7.2–7.4) at room temper­ Fig. 3. The survival loss of Lactobacillus plantarum 550 in microcapsules with
ature until it completely dissociated. The cell viability after encapsula­ different wall materials during storage at 4 ℃.
tion was calculated using the following equation.
Viability % = (N/N0 ) × 100 (1) Y% = (m1 /m0 ) × 100 (2)

N ——the number of viable probiotics cells after spray drying.
N0 ——the number of viable probiotics cells before spray drying.
The absolute difference of the measurement results obtained from
the same sample under repeated conditions shall not exceed 5 % of the
arithmetic mean.
2.7. Determination of microencapsulation yield
Y ——the yield of microencapsulation;
m1 ——the mass of the microcapsules after spray drying.
m0 ——the mass of wall materials before spray drying.
The absolute difference of the measurement results obtained from
the same sample under repeated conditions shall not exceed 5 % of the
arithmetic mean.

The yield of microencapsulation was determined using the method 2.8. Moisture content
with some modifications (Jun-xia et al., 2011). The yield of microcap­
sules was calculated by the following formula: The moisture content was determined as described by the Associa

3
H. Yao et al.
Fig. 4. FTIR spectra of probiotic microcapsules with different wall materials.
tion of Official Analytical Chemists (AOAC, 1995). Briefly, samples were
placed in an oven at 105℃ until constant weight was obtained. Moisture
content was obtained by calculating % loss based on dry weight.
m1 − m2
Moisture content (%) = × 100
m1 − mo
m1 ——the mass of the sample and the oven before drying, in grams
(g);
m2 ——the mass of the sample and the oven after drying, in grams
(g);
m0 ——the mass of the oven, in grams (g).
2.9. Fourier transform infrared (FT-IR) analysis
The microcapsule powder (2 mg) was mixed with KBr (198 mg) and
ground into a fine powder with an agate mortar. Subsequently, a tablet
press is used to compress the ground powder into transparent flakes. The
FT-IR spectrum of each sample was acquired with an FT-IR spectrometer
(Nicolet iS10, Thermo Fisher Scientific, America) according to the
method described by Guo et al. with some modifications (Guo et al.,
2021). The spectra data of each sample were obtained at the wave­
number ranging from 400 to 4000 cm− 1 with air spectra subtracted. The
scan speed was set at 0.2 cm/s with a resolution of 4 cm− 1.
2.10. Storage
The storage stability of probiotics encapsulated in microcapsules was
determined according to the published method with some modifications
(Liu et al., 2015). The absolute difference of the measurement results
obtained from the same sample under repeated conditions shall not
exceed 5 % of the arithmetic mean. Use a disperser to disperse for 10 min
at 4000 rpm to completely release the probiotics from the microcap­
sules. After serial dilution 10 times, the bacterial suspension was spread
on the plate of the MRS agar medium.
2.11. Heat treatment
The tolerance of free and encapsulated probiotics under heat treat­
ment was evaluated according to the published method with some
modifications (Ahmad et al., 2019). Briefly, we transfer 1 mL of free or 1
g encapsulated probiotics to 9 mL of sterile water preheated to 65 ℃ for
30 min or transfer to 75 ℃ sterile water for 10 min. After cooling to
room temperature, use a high-speed disperser to de-capsulate the sample
Food Chemistry 413 (2023) 135596
at 4000 rpm for 10 min, and then use the method in Section 2.5 to spread
the plate for counting.
2.12. Survival of free and encapsulated probiotics during digestion
The viability of free and spray-dried probiotics under simulated
gastrointestinal conditions was assessed following the method of Liu
et al., and Gebara et al. with some modifications (Gebara et al., 2013; Liu
et al., 2016). Brief, dissolve sodium chloride (2 g/L) and pepsin (3.2 g/L)
in sterilized ultrapure water, then adjust the pH of the solution to 2.0
with 1 M hydrochloric acid to prepare simulated gastric juice (SGJ).
Dissolve pancreatin (1 g/L) and porcine bile salt (0.8 g/L) in sterile 0.2
M PBS (pH 7.20) to prepare simulated intestinal juice (SIJ). The SGJ and
SIJ were preheated in a 37 ◦ C water bath for 10 min and then filtered and
sterilized with a 0.22 μm membrane. Adding 0.1 g microcapsules or 0.1
mL free cells (above 9 Log CFU/mL) into a test tube containing 9.9 mL of
sterilized SGJ, then vortex to mix the solution thoroughly, place the test
tube on the constant temperature at 37 ℃ and shaker at 180 rpm. To
determine the survival rate of microencapsulated probiotics after 30, 60,
90, and 120 min of incubation in the simulated gastric digestion stage,
an aliquot of 1 mL was removed into 9 mL 0.2 M PBS and homogenized
for 10 min at 4000 rpm and then plated on MRS agar after isocratic
dilution with 0.9 % sodium chloride, the probiotic cells were enumer­
ated in three replications using the method described in the previous
section. After gastric digestion, the pH of the digestive juice in the test
(3) tube was adjusted to 7.0 with 1 M sodium hydroxide immediately, and
then 10 mL of SIJ was added to simulate intestinal digestion. To deter­
mine the survival rate of the probiotics in the microcapsules during 1, 2,
3, and 4 h of intestinal digestion, an aliquot of 1 mL was removed into 9
mL 0.2 M PBS and homogenized for 10 min at 4000 rpm and then plated
on MRS agar after isocratic dilution with normal saline (0.9 % sodium
chloride), the probiotic cells were enumerated in three replications
using the method described in the previous section.
2.13. Scanning electron microscopy (SEM) analysis
Take the appropriate amount of microcapsule powder and stick them
on the scanning electron microscope observation table evenly (S-2150,
HITACHI, Tokyo, Japan). Sputter gold on the surface of the microcap­
sules with an ion sputtering coating machine, about 10 nm later. Put the
processed sample in the sample box, the acceleration voltage is 5 kV, and
upon the microstructure of the probiotic microcapsules at different
multiples. The powders were systematically observed at a magnification
between 500, 2000, 5000, and 20,000x.
2.14. DSC analysis
The DSC (DSC 8000, PerkinElmer, Massachusetts, USA) analysis of
the microcapsule was performed with a differential scanning calorim­
eter. Took 10–15 mg of each sample and seal it in an aluminum pan and
load the DSC. Under nitrogen flow, the sample was heated from 25 ◦ C to
180 ◦ C at a rate of 10 ◦ C/min. All measurements are performed on an
empty spot, and the heat flow is recorded as a function of temperature.
2.15. Statistical analysis
Data were reported as mean ± SD for triplicate determinations. The
statistical significance was analyzed by one-way ANOVA and Tukey’s
test using SPSS software (version 21.0, IBM software, Chicago, IL, USA).
A Pearson correlation analysis was also conducted using SPSS software.
The figures were drawn by Origin Pro software (version 8.0, Stat-Ease
Company, Minnesota, USA).
4
H. Yao et al.
Fig. 5. Scanning electron microscopy (SEM) images of microcapsules with different wall materials. 20,000×, 5000×, 2000×, and 500× are the magnification times.
3. Results and discussion
3.1. Optimization of pH conditions for the complex coacervate formation
Fig. 1 showed the variation in zeta-potential of SPI and PG with
different ratios as affected by pH. Taking into account the intolerance of
Lactobacillus plantarum 550 to highly acidic or alkaline environment, we
selected a relatively mild pH range from 3.0 to 5.0. The zeta potential
value of all the samples decreased with the increase of pH. For SPI only
sample, it was positively charged among pH < pI (4.35) which can
attribute to the protonation of the amino moieties (–NH2+) and the
lower of the pH, the stronger of the intensity of charge, while for pH > pI
(4.35), adverse situation was found which can be due to the ionization of
the carboxylic moieties (–COOH) (Malhotra & Coupland, 2004). At pH
= pI (4.35), the charges from protonation of amino moieties equaled to
that of ionization of carboxylic moieties, SPI was insoluble and precip­
itated and zeta potential of 0 mV was presented. As for the PG only
5
Food Chemistry 413 (2023) 135596
sample, the negatively charged property was found throughout the
whole pH range and the higher of the pH, the stronger of the intensity. At
a given pH, with the increase of PG ratios, a reducing trend was found in
the value of zeta potential. The formation of complex coacervates and
the corresponding physicochemical properties highly rely on the pH and
ratio between protein and polysaccharides. Researchers have illustrated
that microcapsules prepared around the critical complex coacervate
condition, in which the zeta potential of the composite wall materials
was 0 mV, own the strongest electrostatic interaction between protein
and polysaccharide (Abid et al., 2018). Thus, with the purpose of
maximum combination between SPI and PG molecules through elec­
trostatic interaction, pH = 3.75 was finally applied afterward at which
the zeta potential values of both SPI and PG alone reached relative
maximum and at this pH, the zeta potential of SPI: PG = 3:1 equaled to
zero representing a critical and complete electrostatic interaction
occurred between SPI and PG molecules.
H. Yao et al.
3.2. Physicochemical properties of microcapsules after spray drying
The encapsulation viability and yields of Lactobacillus plantarum 550
during spray drying with different wall materials were shown in Table 1
and for all the samples tested, the numbers of viable cells inside were
higher than the recommendation of 6–7 Log CFU/g by the International
Dairy Federation. For SPI only sample (SPI: PG = 1:0), the encapsulation
viability was only 3.23 % corresponding with the lowest yield of 20.03
%, while for PG only sample (SPI: PG = 0:1), both highest values of
encapsulation viability (64.78 %) and yield (43.68 %) were found. With
the ratios of PG increased within the wall material, both the encapsu­
lation viability and yield increased significantly which could attribute to
the fact that PG owns the ability to replace water molecules on the
surface of the cell membrane thus preserving and stabilizing the struc­
ture of the cell membrane during spray drying (Pandey & Mishra, 2021).
Furthermore, with the increase in PG ratio, the specific enthalpy (ΔH)
also raised from 86.87 to 142.36 J/g (Table 1), which indicated that the
addition of PG into the SPI matrix was beneficial to the enhancement of
thermal stability leading to the higher viability of cells in microcapsules
(Khodaei et al., 2020).
The shelf life of probiotic microcapsules is directly related to the
moisture content in dry products (Santana et al., 2016) and with the
purpose of maintaining well storage stability and preventing agglom­
eration or recrystallization, moisture content values vary between 4 and
5 g/100 g were recommended (Ghandi et al., 2012). In this study, the
moisture contents of all the spray-dried probiotic microcapsules were
within the range of 4.14–5.01 g/100 g and with the ratio of PG
increasing, the moisture content decreased significantly, which could
attribute to the fact that PG had a lower water binding ability than SPI
and the structure of PG facilitated the removal of water from the droplets
during the contact with hot air (Diệp Huy Vũ et al., 2021). Besides, with
the water contents decreasing, higher yields after spray drying can be
found correspondingly. For probiotic microcapsules, water inside can
act as a strong plasticizing agent affecting its glass transition tempera­
ture (Santana et al., 2016). The higher of the moisture content inside, the
lower of the glass transition temperature presents accompanied by more
microcapsules sticking to the wall of the drying chamber and finally
leading to the reduction of yield in microcapsule powder collected.
3.3. The survival of cells in microcapsules during simulated
gastrointestinal digestion
Probiotics are commonly very sensitive to the high acid and bile salt
concentration environment in the human gastrointestinal tract. Thus,
after being ingested by consumers, probiotics will be largely inactivated
and cannot exert their beneficial effects on the host. To explore the
survival rate of free and encapsulated probiotic cells in the gastroin­
testinal tract, in vitro digestion tests were thus applied (Fig. 2). As shown
in Fig. 2, free cells lost all their activity in only 0.5 h of gastric digestion.
After encapsulation, the survival rates of cells significantly increased,
which could attribute to the compact structure in microcapsules that
reduced the diffusion of acids into the microcapsules and thus enhanced
the acid resistance of cells (Divya & Nampoothiri, 2015). Compared with
the SPI only sample, the addition of PG (SPI: PG = 3:1 and SPI: PG = 1:1)
significantly increased the cell survival after simulated gastrointestinal
digestion. During gastric digestion (pH = 2.0), SPI (pI = 4.35) was
positively charged while PG was negatively charged, thus electrostatic
interaction occurred between each other leading to a more compact
structure within microcapsules and providing extra protection for the
entrapped probiotic cells. Similar results can also be found in Liu et al.
(2016) who encapsulated Lactobacillus zeae LB1 using gum arabic and
NaCas at a ratio of 3:1 (w:w) and significant lower cell viability loss of
1.20 Log Log CFU/g was found compared with that in NaCas only
sample (2.50 Log CFU/g), which could also attribute to the fact that
carboxyl groups carried by gum arabic molecules could form strong
interaction with the amide and carboxyl groups of NaCas molecules.
6
Food Chemistry 413 (2023) 135596
However, with the ratio of PG further increased (SPI: PG = 1:3 and
SPI: PG = 0:1), the survival of cells after gastrointestinal digestion
decreased gradually, which could be due to the high solubility of PG that
accelerated the swelling and disintegration of microcapsules. Among all
the samples, the survival rate in SPI: PG = 3:1 retained the maximum
activity of 7.88 Log CFU/g after the whole gastrointestinal digestion,
which could attribute to the strongest and complete electrostatic inter­
action between SPI and PG (as shown in Fig. 1, the zeta potential value
of SPI: PG = 3:1 equaled to zero) that formed the most compact structure
inside the microcapsule.
3.4. The survival of cells in microcapsules during heat treatment
Pasteurization is a generally applied sterilization process in food
industry, especially for dairy foods. However, probiotics are commonly
very sensitive to thermal treatment leading to a large reduction in
viability, which has become a tough challenge for food manufacturers
(Capra et al., 2004). To simulate the effect of the typical pasteurization
process on the survival of cells, we applied the microcapsule samples at
65 ◦ C for 30 min or 75 ◦ C for 10 min, respectively (Table 1). After heat
treatment, free cells lost all the viabilities. For probiotic microcapsule
samples, enormous improvements in survival rates were found, how­
ever, with the concentration of PG increased, the viability losses of cells
increased significantly in both heat conditions (p < 0.05). Although the
specific enthalpy (ΔH) increased with the rising of PG ratio which
indicated an enhanced thermal stabilization effect on the structure of
microparticles, the increased viability losses may attribute to the fact
that as in ultrapure water (pH = 6.5), both SPI and PG were negatively
charged (Fig. 1) leading to the rapid disintegration of microcapsules and
release of cells. On the other hand, as PG owns well solubility in aqueous
solution, the addition of PG to SPI based microcapsules may also
accelerate the dissolution of composite particles which limited the
protective effect of microcapsules.
3.5. Storage properties of probiotic microcapsules
Fig. 3 showed the survival loss of Lactobacillus plantarum 550 in
microcapsules with different wall materials during storage at 4 ℃. It can
be seen that with the increase in storage time, the viability loss of cells in
all samples increased. For SPI only sample, the survival losses of pro­
biotics were the highest at each sampling time point, which increased
from 0.19 Log CFU/g at 2 weeks to 0.93 Log CFU/g at 8 weeks. After
adding PG into the SPI matrix, however, the viabilities of probiotic
bacteria in all composite samples were significantly enhanced, which
could attribute to the filling effect of PG that compensated the pores of
the microcapsules and isolated the probiotics from the external envi­
ronment. Furthermore, with the content of PG increased within the wall
material of microcapsules, the probiotic bacteria viabilities were further
improved during storage (p < 0.05). According to Liu et al. (2016), the
gap between the Tg of wall material and the storage temperature can be
an indicator of the storage property of encapsulated probiotics and the
larger of the temperature gap, the better of the storage property. As
shown in Table 1, with the contents of PG increased, the moisture
content of microcapsules after spray drying decreased, which could thus
generate higher Tg that further increased the viscosity of the matrix,
slowed down the molecular mobility within microcapsules, and hin­
dered the occurrence of several diffusion-controlled reactions (such as
oxidation) (Laine & Heinonen, 2008; Liu et al., 2016). Besides, during
storage, the structure of the cell membrane would be injured by the free
radical-induced lipid peroxidation resulting in the degeneration of the
cell and a dramatic decrease in viability (Diệp Huy Vũ et al., 2021). PG
has been reported of owning the potential to scavenge free radicals (Yao
et al., 2013), therefore, with the ratio of PG increased, the ability of wall
materials to scavenge free radicals generated during storage increased,
which thus further improved the storage stability of encapsulated pro­
biotic bacteria.
H. Yao et al.
3.6. FT-IR spectra analysis
Fig. 4 showed the FT-IR spectra of probiotic microcapsules with
different SPI and PG ratios. For all the samples, the peak at 3413–3419
cm− 1 was the result of the overlap of –OH stretching vibration absorp­
tion peak associated with the –NH stretching vibration absorption peak,
the peaks at 2930–2932 cm− 1 and 1403–1418 cm− 1 were allocated to
the C–H stretching (Duhoranimana et al., 2017; Pal et al., 2019). For
SPI only sample, it is noticeable that the peaks at 1653, 1542, and 1230
cm− 1 were the amides I, II and III band absorption peaks of protein
respectively (Fathi et al., 2019; Kong & Yu, 2007). For PG only sample,
the peaks at 1730 and 1620 cm− 1 were the asymmetric stretching vi­
bration absorption peaks of –COO and the peak at 1417 cm− 1 was the
symmetric stretching vibration absorption peaks of –COO (Zhu et al.,
2019). Besides, characteristic arabinogalactan group and both α- and
β-configurations of aldopyranoses could be found at peaks of 1039 cm− 1,
845–833 cm− 1, and 901–897 cm− 1 respectively (KacÆuraÂkovaÂ,
2000; Shan et al., 2015). With the addition of PG into SPI, the amide I
group (1653 cm− 1) slightly moved to 1622 cm− 1, the amide II group
with N–H bending vibration and C–N stretching vibration (1541 cm− 1)
and amide III group (1230 cm− 1) gradually decreased and completely
disappeared in SPI: PG = 1:3, which could attribute to the reaction be­
tween –NH3+ groups in SPI and –COO groups in PG (Naderi et al., 2020).
Espinosa-Andrews et al. (2010**) also found that after coacervating gum
arabic and chitosan, the characteristic absorption peak of the carbonyl-
amide region disappeared. Apart from the shifting of characteristic ab­
sorption peaks, the addition of SPI also impressed the absorption peaks
of PG, for instance, the symmetric –C– – O stretching vibration (1730
cm− 1) of PG moved slightly to the amide I group (1653 cm− 1).
3.7. SEm
Fig. 5 showed the morphology of probiotic microcapsules prepared
with different ratios of SPI and PG. The free cell was not observed on the
surface of all samples. For SPI only sample, the shape of microparticles
was close to spherical with serious rough ravines and pores existing on
the surface. As in an aqueous solution, SPI molecules are prone to form
large aggregates with a conformation of vesicles or solid which are
combined with each other leading to the formation of a rough surface (Li
et al., 2010). After adding PG into SPI matrix, the serious rough grooves
and pores phenomenon on the surface of microcapsules disappeared and
replaced by a smoother and denser surface. As at pH 3.75, unfolded PG
molecular chain own strong negative charges (Fig. 1), which would be
adsorbed and wound on the surface of the large aggregate formed by SPI
through electrostatic interaction, making up for the rough grooves and
pores on the surface of SPI based microcapsules, leading to the surface of
microcapsules smoother. Besides, the storage stability of probiotic mi­
crocapsules corresponded well with the surface structure of microcap­
sules, as fewer pores on the surface of the microcapsules and the
smoother of the surface, the better of the storage stability presented,
which could be due to the fact of separating the probiotics inside the
microcapsules from the unfavorable factors of the external environment
(Mao et al., 2019).
4. Conclusion
This study demonstrated that the addition of PG to SPI encapsulation
formula could effectively maintain the survival of L. plantarum 550
during gastric digestion and storage. For probiotic microcapsules pre­
pared by coacervation method, ratio of wall materials under critical
complex coacervate condition (zeta potential = 0 mV) could contribute
to higher viability during gastrointestinal digestion, as at this moment,
the strongest and complete electrostatic interaction occurred between
the biopolymers that formed the most compact structure inside the
microcapsule (SPI:PG = 3:1). Besides, our results also confirmed the
importance of moisture content to the survival rate of cells during
7
Food Chemistry 413 (2023) 135596
storage: the higher of the moisture content, the lower of the cell
viability. The formula developed in this research holds potential for
application to a broad range of probiotics, and further investigation and
optimization of this formula could lead to commercial products.
CRediT authorship contribution statement
Haodong Yao: Conceptualization, Software, Writing – original draft,
Methodology, Investigation, Visualization, Data curation, Formal anal­
ysis. Bu Liu: Data curation, Software, Formal analysis, Methodology,
Visualization, Writing – review & editing. Li He: Resources, Project
administration, Funding acquisition, Resources, Conceptualization,
Validation, Supervision. Jielun Hu: Resources, Project administration,
Funding acquisition, Resources, Conceptualization, Validation, Super­
vision. Huan Liu: Conceptualization, Methodology, Writing – review &
editing, Project administration, Funding acquisition, Resources,
Conceptualization, Validation, Supervision.
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.
Data availability
Data will be made available on request.
Acknowledgment
The work was supported by the National Natural Science Foundation
of China (31901648, 32222065, 31960464), Key Research and Devel­
opment Program of Jiangxi Province (20224BBF62002), “Double
Thousand Plan” of Jiangxi province (jxsq2018106029), “Jing Gang
Scholars” of Jiangxi province (QNJG2018007), Cultivation of National
Science and Technology Award Project (20192AEI91004), Central
Government Guide Local Special Fund Project for Scientific and Tech­
nological Development of Jiangxi Province” (20212ZDD02008,
20221ZDD02001).
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