Food Bioscience 43 (2021) 101259

Contents lists available at ScienceDirect

Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

Antioxidant activity of Bifidobacterium animalis MSMC83 and its application

in set-style probiotic yoghurt
Porntipha Vitheejongjaroen a, Praphaiphan Kanthawang b, Fabien Loison c, d, e,
Yamaratee Jaisin f, Ulisa Pachekrepapol g, Malai Taweechotipatr h, i, *
a
Biomedical Sciences Program, Srinakharinwirot University, Bangkok, 10110, Thailand
b
Research and Development, CP-Meiji Co., Ltd, Nong Khae, Saraburi, 18230, Thailand
c
Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand
d
Systems Biology of Diseases Research Unit, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand
e
Current Address: Department of Pathology, Harvard Medical School, Department of Lab Medicine, Children’s Hospital Boston, Boston, MA, 02115, USA
f
Department of Pharmacology, Srinakharinwirot University, Bangkok, 10110, Thailand
g
Division of Food Science and Nutrition, Faculty of Agricultural Product Innovation and Technology, Srinakharinwirot University, Nakhon Nayok, 26120, Thailand
h
Department of Microbiology, Faculty of Medicine, Srinakharinwirot University, Bangkok, 10110, Thailand
i
Center of Excellence in Probiotics, Srinakharinwirot University, Bangkok, 10110, Thailand

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

Keywords: This study aims to investigate the antioxidative activities of probiotics derived from healthy human infants and to
Probiotics develop probiotic yoghurt. Viable cells, cell free supernatant and intracellular cell free extract of MSMC83
Antioxidant exhibited antioxidant activities. MSMC83 was assigned to Bifidobacterium animalis with 99% similarly based on
Bifidobacterium
16 S rRNA gene sequence analysis. B. animalis MSMC83 showed high survival rate in simulated gastrointestinal
Yoghurt
Physicochemical
conditions, a substantial adherence to Caco-2 cells and production of bile salt hydrolase. Set-style yoghurt fer­
mented with B. animalis MSMC83 and yoghurt starter cultures was produced. Microbiological and physico­
chemical properties of yoghurt were investigated during refrigerated storage. Fermentation time of probiotic
yoghurt was 4.5 h compared to 6.0 h found in regular yoghurt. The viable count of B. animalis remained at 8.53
log CFU/g after 4-week storage at 4 ◦ C. The probiotic yoghurt possessed firmer texture and lower syneresis
compared to the yoghurt fermented with starter cultures. Our results support the use of B. animalis MSMC83 as
probiotic culture in the development of yoghurt with antioxidative property.

1. Introduction 2020). Therefore, increase in antioxidants is considered vital in the

reduction of oxidative damage and the risk factor of metabolic diseases
Free radicals are highly unstable biomolecules and derived either (Terahara et al., 2001).
from normal essential metabolic processes in cells or stimulation from Probiotics have become important in health care, foods, animal feeds
extrinsic factors (Lobo et al., 2010). They cause oxidative stress and lead and other agricultural products. In 2014, the International Scientific
to senescence and aging of cells, which are linked to metabolic syn­ Association for Probiotics and Prebiotics (ISAPP) defined probiotics as
drome, cancer, Alzheimer’s disease and aging (Liguori et al., 2018). “live microorganisms that, when administered in adequate amounts,
Reactive oxygen species (ROS), including hydroxyl radical (HO.-), confer health benefit to the host” (Hill et al., 2014). The cell free su­
hydrogen peroxide (H2O2), and superoxide radicals (O−2 ), are the most pernatant (CFS) is the metabolites secreted from probiotics, which is
important forms of free radicals and are generated continuously in the defined as postbiotics, while paraprobiotics refer to intracellular cell
mitochondria of growing cells (Sena & Chandel, 2012). Among them, free extract (Zendeboodi et al., 2020). The most common probiotic
hydroxyl radical is the most reactive and directly damages DNA, pro­ microorganisms are lactic acid bacteria (LAB) and bifidobacteria. In the
teins, and lipids. Antioxidants, synthesized from cells and obtained from human body, probiotic microorganisms are found in the gastrointestinal
foods, reduce free radical and prevent oxidative stress (Feng & Wang, tract as part of the normal microbiota. They adhere and colonize the

* Corresponding author. Department of Microbiology, Faculty of Medicine, Srinakharinwirot University, Bangkok, 10110, Thailand.
E-mail address: [email protected] (M. Taweechotipatr).

https://doi.org/10.1016/j.fbio.2021.101259
Received 4 May 2021; Accepted 18 July 2021
Available online 19 July 2021
2212-4292/© 2021 Elsevier Ltd. All rights reserved.
P. Vitheejongjaroen et al. Food Bioscience 43 (2021) 101259

human gastrointestinal tract and tolerate low pH and bile salt. They forming units (CFU)/mL. Viable cells were re-suspended in phosphate
obtain generally recognized as safe (GRAS) status from the United States buffered saline (PBS; pH 7.2) after centrifugation (4000×g, 5 min, 4 ◦ C)
food and drug administration (Choi et al., 2005). Probiotics have for later use. For the preparation of the cell free supernatant (CFS), the
different beneficial properties on human health depending on the spe­ probiotic isolates were re-suspended in MRS broth and incubated at
cific characteristics of each strain. Some probiotic strains have been 37 ◦ C for 24–48 h under anaerobic conditions. The supernatants were
shown to possess antioxidative activity and reduce free radical accu­ collected after centrifugation (4000×g, 5 min, 4 ◦ C) and sterilized
mulation by modulating the redox status of the host via antioxidant filtration through a 0.22 μm pore-size filter. The resulting supernatants
system (Wang et al., 2017). Bifidobacterium animalis MG741, from were used as the cell free supernatant. The intracellular cell free extract
human origins, showed antioxidant activities of 38.2 ± 1.6% in 2, (ICFE) was prepared by resuspending cell pellets in deionized water with
2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging (Kim et al., lysozyme (1 mg/mL) at 37 ◦ C for 30 min. Ultrasonic disruption was
2020). Moreover, the milk fermented with B. animalis ssp. Lactis had performed in an ice bath with five 1-min intervals (power 25–30%,
higher antioxidant property compared to milk fermented with starter Sonoplus HD 2070; Bandelin, Germany). Cell debris was removed by
cultures (Li et al., 2019). Many clinical studies showed a positive impact centrifugation at 8000×g for 10 min at 4 ◦ C. The resulting cleared su­
of probiotic supplementation. Daily administration of probiotic pernatant was used as the ICFE. The CFS and ICFE were stored at − 20 ◦ C
improved antioxidative capacity and anti-inflammation, and reduced until use.
depression symptoms of myocardial infarction patients (Moludi et al.,
2019). Moreover, Lactobacillus sporogenes GBI-30 combined with pre­
2.3. Antioxidant activities of probiotic isolates
biotics consumption for 8 weeks improved glycemic status, lipid profile,
and biomarkers of oxidative stress in type 1 diabetes mellitus patients
Antioxidant activities of probiotic isolates were evaluated using
(Zare Javid et al., 2020).
three different methods, DPPH radical scavenging, hydroxyl radicals
Yoghurt is a dairy product, fermented and acidified by addition of a
scavenging activity, and dichlorofluorescein (DCF) assay on colon car­
starter culture containing Streptococcus thermophilus and L. delbrueckii
cinoma cell lines (Caco-2). Each method was repeated three times.
ssp. Bulgaricus. Milk is an excellent precursor for bacterial growth and
can ensure the survival of probiotics during fermentation and storage
2.3.1. Evaluation of antioxidant activity by DPPH radical scavenging
because of buffering capacity of milk (Hadjimbei et al., 2019). In addi­
method
tion, yoghurt is considered a healthy product and receives a positive
The free radical scavenging activity of the three samples (viable cell,
reputation in the minds of consumers. Research over the past decades
CFS, ICFE) of probiotic isolates were analyzed using DPPH (Sigma
has demonstrated that addition of probiotics in yoghurt improves its
Aldrich Co., St. Louis, MO, USA) according to the modified method of
functionality (Soni et al., 2020). The application of L. casei Zhang
Xing et al. (2015). In brief, 0.2 mM solution of DPPH in 95% (v/v)
reduced fermentation time and syneresis, and improved texture, rheo­
ethanol was prepared. Samples (50 μL) were mixed with 100 μL of DPPH
logical properties and bacterial viability (Bai et al., 2020). The cow’s
solution into a 96 microculture plate. The microplate was vortexed and
milk and soymilk yogurts fermented with starter cultures and B. ani­
incubated for 30 min at room temperature (22–25 ◦ C) in the dark, and
malis subsp. Lactis, L. acidophilus, and L. rhamnosus required shorter
the supernatant was obtained by centrifugation at 4000×g at 25 ◦ C for
fermentation time to reach pH 4.5, and their physicochemical properties
10 min. The absorbance of the supernatant at 517 nm was measured.
were significantly improved compared to yoghurts fermented with
Ascorbic acid (1% w/v; Sigma Aldrich) was used as a positive control.
starter cultures alone (Cui et al., 2021). Therefore, yoghurt with anti­
The percentage of scavenging activity was calculated according to the
oxidant activity can be obtained by addition of probiotics. The effects of
equation:
probiotic application on the yoghurt matrix and stability of the culture
during storage, however, have to be characterized. scavenging activity (%) = (1 – (As– Ab) / Ac) x 100 (1)
The objectives of this study were to identify and screen probiotic
strains from healthy human infants with antioxidant activity using Where As was the absorbance of samples, Ab was the same amount of
DPPH radical scavenging capacity, hydroxyl radical scavenging capacity water and sample mixture, and Ac was the absorbance of the blank
and reduction of intracellular free radical level in Caco-2 cells. Potential control.
probiotic properties including resistance to simulated gastrointestinal
conditions, adhesion property on Caco-2 cells, and bile salt hydrolase 2.3.2. Evaluation of antioxidant activity by hydroxyl radicals scavenging
activity were also evaluated. The candidate probiotic identified was activity
used for milk fermentation. Microbiological and physicochemical The hydroxyl radical scavenging activity was measured based on the
properties of the resulting yoghurt during refrigerated storage were Fenton reaction as previously described by Wang et al. (2015) with
investigated. minor modifications. The Fenton reaction was freshly prepared by
mixing 1.0 mL of 0.5 mM FeSO4, 0.5 mL of 0.435 mM brilliant green
2. Materials and methods (Sigma Aldrich) and 0.5 mL of samples (viable cells, CFS and ICFE), or
deionized water used as a negative control. The reaction was initiated by
2.1. Probiotic isolates the addition of 0.75 mL of H2O2 (3% w/v). The mixture was incubated at
room temperature for 20 min after which absorbance (624 nm) was
Probiotics were derived from healthy human infants and were measured. Brilliant green was used as a blank and 1% (w/v) ascorbic
maintained as frozen stocks (− 80 ◦ C) (ethical approval number acid was used as a positive control. The hydroxyl radicals scavenging
SWUEC37/2551). Fifty probiotic isolates were randomly selected and activity was calculated as below:
grown on de Man, Rogosa, Sharpe (MRS) agar (HiMedia Lab., India) at
scavenging activity (%) = ((As–Ac) / (Ab – Ac)) x 100 (2)
37 ◦ C for 24–48 h under anaerobic conditions (10% O2; 10% CO2; 80%
N2) and were sub-cultured three times before use in subsequent Where As was the absorbance of samples, Ab was the absorbance of the
experiments. blank control, andA c was the same amount of water and sample
mixture.
2.2. Preparation of viable cells, cell free supernatant and intracellular cell
free extract 2.3.3. Quantification of intracellular ROS by dichlorofluorescein assay
The colorectal adenocarcinoma cell lines (Caco-2) were obtained
The number of probiotic isolates was adjusted to 9.0 log colony from the American Type Culture Collection (ATCC HTB-37) and were

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P. Vitheejongjaroen et al. Food Bioscience 43 (2021) 101259

cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Thermo prepared. The Caco-2 cells were seeded at a concentration of 1 × 105
Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% heat- cells/well in 24-well standard tissue culture plates and cultured for 14 ±
inactivated fetal bovine serum (Gibco) and antibiotics (penicillin 100 U/ 1 days prior to use. The cell culture was washed twice with PBS (pH 7.2)
mL and streptomycin 100 μg/mL Gibco) in a humidified atmosphere and placed in serum-free and antibiotics-free medium at least 1 h before
with 5% CO2 at 37 ◦ C. All cell culture experiments were carried out the adhesion assay. The probiotic strain was grown in MRS broth at
when the culture was approximately 60–80% confluent. 37 ◦ C overnight and resuspended in DMEM (9.0 log CFU/mL). There­
The intracellular ROS level was measured by the oxidation of 2,7- after, 1 mL probiotic strain suspension was added to each well and
dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma Aldrich) as incubated for 1 h at 37 ◦ C in a humidified atmosphere containing 5%
described by Hou et al. (2019) with minor modifications. Briefly, Caco-2 (v/v) CO2. After incubation, the cells were washed thrice with PBS to
cell suspension (2.4 × 104 cells/well, 100 μL/well) was seeded on a remove non-adherent bacteria, and lysed by addition of 0.5% (v/v)
black 96-well microplate and incubated at 37 ◦ C for 24 h. Then, cells Triton® X-100 (Merck) for 10 min. The lysed cells were plated on MRS
were treated with the samples of probiotic isolates for 24 h. Finally, agar and incubated at 37 ◦ C for 48 h under anaerobic conditions.
hydrogen peroxide (H2O2; Merck, Darmstadt, Germany) was added to L. rhamnosus GG (LMG 18243) was used as a positive control. The
the final concentration of 500 μM for 2 h to induce oxidative stress. adhesion percentage was then calculated as:
Subsequently, the cells were stained with DCFH-DA (25 μM final con­
centration), incubated for 30 min at 37 ◦ C, and washed with PBS. After % adhesion= (A b / Ac) x 100 (4)
removal of the DCFH-DA excess, fluorescence was measured using a Where Ab was the adhesion of viable bacteria cells, and Ac was the total
microplate reader at excitation and emission wavelengths of 485 and number of bacteria added.
535 nm, respectively. Cells treated with DCFH-DA and H2O2 were used
as a positive control while cells treated with DCFH-DA and PBS used to 2.3.8. Bile salt hydrolase activity
measure the baseline ROS level. The integrated areas of the sample (S) Bile salt hydrolase (BSH) activity was adapted from Hernán­
and control curves (C) were calculated, and the cellular antioxidant dez-Gómez et al. (2021). Briefly, the probiotic strain was cultured under
activity was calculated using the following formula: anaerobic conditions at 37 ◦ C for 24 h. The final concentration of 1 ×
cellular antioxidant activity (unit)= (1-(S/C)) × 100 (3) 109 CFU/mL was obtained and spotted on MRS agar plates supple­
mented with 0.5% (w/v) sodium salt of tauro-deoxycholic acid (TDCA;
Sigma Aldrich) and 0.037% (w/v) CaCl2. After incubation at 37 ◦ C for
72 h, the BSH activity was determined by measuring the precipitation
2.3.4. Genotypic characteristics of selected probiotic strain zone surrounding the colony.
Probiotic isolates were identified using 16 S rRNA gene sequencing.
The 16 S rRNA genes were selectively amplified from purified genomic
2.4. Optimization of yoghurt with selected probiotic strain
DNA using the polymerase chain reaction (PCR) with two primers as 20
F (5′ -AGTTTGATCCTGGCTC-3′ ) and 1530 R (5′ -AAGGAGGTGATCC
The mixed culture of S. thermophilus and L. delbrueckii subsp. bul­
AGCC-3ʹ). The quality of the PCR fragments was examined by agarose
garicus (YoFlex–YC-X11) was purchased from Chr. Hansen (Hørsholm,
gel electrophoresis and purified using a PCR Clean-Up & Gel Extraction
Denmark). Each strain was isolated and grown in MRS broth. Pasteur­
Kit (GeneDireX Inc., USA). Sequencing was carried out by Macrogen Inc.
ized milk, kindly provided by CP-Meiji (CP-Meiji Co. Ltd., Thailand),
(Seoul, Korea) and sequences were aligned with National Center for
was heated to 85 ◦ C for 30 min before yoghurt preparation. The selected
Biotechnology Information (NCBI) GenBank database and EzTaxon
probiotic B. animalis MSMC83, S. thermophilus and L. delbrueckii subsp.
bioinformatics software (Kim et al., 2013).
bulgaricus were each inoculated at 9.0 log CFU/mL at the same time.
A phylogenetic tree was constructed by the neighbor-joining method
Yoghurt inoculated with starter culture alone was used as a control.
(Saitou & Nei, 1987) using MEGA version X. Bootstrap resampling
Fermentation was performed at 43 ◦ C until pH 4.60 was reached, and the
analysis of 1000 replicates was performed to estimate the confidence
decrease in pH level was monitored using a pH meter (ORION 3-star;
levels of the tree topologies. Bacillus subtilis (AJ276351) was used as
Expotech, USA). The yoghurt samples were analyzed weekly during 4
outgroup.
weeks of storage at 4 ± 1 ◦ C. The viability of microorganisms, physi­
cochemical properties including pH changes, texture, syneresis, rheo­
2.3.5. Evaluation of general probiotic properties of selected probiotic strain
logical properties, and apparent viscosity were determined.
General probiotic properties including resistance to simulated
2.7 Viability of microorganisms in yoghurts.
gastrointestinal conditions, and adhesion property on Caco-2 cells were
Viability of S. thermophilus and L. delbrueckii subsp. bulgaricus during
assessed. Each method was performed in duplicate.
storage were evaluated on MRS agar plates incubated at 37 ◦ C for 48 h
under anaerobic condition. The enumeration of B. animalis MSMC83 was
2.3.6. Resistance to simulated gastrointestinal conditions
performed on Bifidus selective medium (BSM) agar with BSM supple­
The survival of probiotic strain with antioxidant activity in simulated
ment (Sigma Aldrich) under anaerobic condition at 37 ◦ C for 48 h. The
gastrointestinal conditions was investigated as described previously by
number of microorganisms was expressed as log CFU/g.
Ladda et al. (2015). Briefly, MRS broth was adjusted to either pH 2.0, pH
3.0, or pH 4.0 with addition of 1 M HCl for acid conditions or supple­
mented with bovine bile at 0.3% (w/v) or 0.8% (w/v) for bile salt 2.5. Physicochemical analysis of yoghurt
conditions. The probiotic strain was grown in MRS broth at 37 ◦ C
overnight, and a 0.1 mL aliquot containing 9.0 log CFU/mL was inoc­ 2.5.1. Measurement of pH value
ulated in different pH and bile conditions. The samples were left for 3 h The pH level of yoghurts during storage were determined using a pH
at 37 ◦ C. Viability was quantified by counting colonies after spreading meter. The yoghurts were slightly vortexed before the measurement.
cells on MRS agar plate at 37 ◦ C for 24–48 h. The viable cell counts were
expressed as log CFU/mL. 2.5.2. Firmness analysis
Yoghurt samples formed in a glass container (52 mm in diameter
2.3.7. Adhesion property on Caco-2 cells until a height of 50 mm) were analyzed for firmness using a compression
Adherence was performed as previously described by Bustos et al. test carried out with a TA. XT plus Texture Analyzer (Stable Micro
(2012) with some modifications. A monolayer of Caco-2 cells was Systems, Surrey, UK) equipped with a 20 mm acrylic cylinder probe
(Krasaekoopt et al., 2004). The test speed was 1 mm/s with the

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P. Vitheejongjaroen et al. Food Bioscience 43 (2021) 101259

penetration depth of 10 mm. Firmness was defined as the peak of

compression force (g). The experiments were repeated three times.

2.5.3. Measurement of syneresis

Syneresis of different yoghurts was determined as described by
Vivar-Quintana et al. (2006). Briefly, 30 g yoghurts formed in centrifuge
tubes were spun at 680×g at 4 ◦ C for 10 min. The supernatant was
weighed and syneresis was calculated according to the equation:

syneresis (%) = (weight of supernatant / total weight of milk) x 100 (5)

2.5.4. Measurement of rheological properties

The determination of rheological properties was performed using a
rheometer (HAAKE Mars 40, Thermo Fisher Scientific Inc., Karlsruhe,
Germany) according to the method proposed by Sah et al. (2016) with
some modifications. A plate and cone geometry (60 mm diameter) with
1 mm gap were used. The measurement was performed at 4 ◦ C. The
sample was stirred and loaded to the rheometer. The pre-sheared process
was applied for 30 s at a shear rate of 500 s − 1, and the sample was left
for equilibration for 300 s. The frequency sweep was performed with a
strain of 0.5% and the frequency ranged from 0.1 to 10 Hz. The storage
modulus (G’) and the loss modulus (G”) were reported at a frequency of
1 Hz.

2.5.5. Apparent viscosity measurements

The viscosity of yoghurt samples was measured by applying a shear
rate sweep to the same sample after 300 s equilibration. The applied
shear rate was between 0.01 and 100 s− 1. The apparent viscosity of
yoghurts was reported at 20 s− 1 (Tan et al., 2018).

2.6. Statistical analysis

Statistical analyses were performed using GraphPad Prism Software

version 5.0 (GraphPad Software, USA). The results are presented as
mean and standard deviation (SD). Differences between the means were
evaluated by one-way analysis of variance (ANOVA) with Tukey’s
multiple comparison test. Data were considered statistically significant Fig. 1. Antioxidant activity of viable cells, cell free supernatant (CFS) and
when p < 0.05. Statistical differences were indicated with different intracellular cell free extract (ICFE) of MSMC83 in DPPH (A) hydroxyl radicals
alphabetical letters. scavenging (B) assays and cellular antioxidant activity on Caco-2 cells exposed
to H2O2 (C). Antioxidant properties of MSMC83 isolated from the Thai newborn
3. Results and discussion feces. Values are expressed as mean and SD (n = 3). The different superscript
letters a-c in the same type of sample and letters A-B in the MSMC83 and MRS
broth represent statistical significance (p < 0.05).
3.1. Antioxidant activity by DPPH radical and hydroxyl radicals
scavenging activity
scavenged DPPH and hydroxyl radical. Moreover, ICFE exhibited the
The DPPH radical and hydroxyl radicals scavenging activities were highest antioxidant activity. It suggested that the antioxidation factor
used as a tool to investigate the antioxidative properties of the 50 pro­ was present in higher concentration in the cellular compartment and
biotic isolates. The isolate, MSMC83, was found to have antioxidant that the incubation time may affect the antioxidant capacity of viable
property. The results of the two radical scavenging methods showed that cells and CFS. In addition, the iron chelation states of probiotics
the viable cells, CFS and ICFE of MSMC83 exhibited antioxidant activity inhibited Fenton reaction dependent hydroxyl radical formation (Ahire
when compared to MRS. The DPPH radical scavenging activity of et al., 2013). Therefore, the antioxidant activity of probiotics depends
MSMC83 was 17.60 ± 0.66%, 38.63 ± 0.13% and 58.00 ± 0.05% for highly on strains.
viable cells, CFS, and ICFE, respectively (Fig. 1A). The DPPH radical
scavenging activity of ICFE was significantly higher than that of viable 3.2. Quantification of intracellular ROS by dichlorofluorescein assay
cells and CFS. The hydroxyl radical scavenging method gave similar
results. The hydroxyl radicals scavenging activity of MSMC83 was The cytotoxic effect of MSMC83 on Caco-2 cell line was determined
12.57 ± 0.35%, 23.39 ± 0.83%, and 42.87 ± 0.52% for viable cells, CFS, by MTT assay using viable cells, CFS and ICFE. It was found that the
and ICFE, respectively. The hydroxyl radicals scavenging activity ICFE viable cells, CFS and ICFE at 8.0 log CFU/mL final concentration did not
exhibited the highest scavenging capability (Fig. 1B). display toxicity on Caco-2 cells (data not shown). The intracellular ROS
These results revealed that MSMC83 possibly contained some po­ level was measured to evaluate the antioxidant activity of viable cells,
tential bioactive compounds, which were electron donors and effec­ CFS and ICFE on Caco-2 cells, as shown in Fig. 1C. Viable cells, CFS and
tively reacted with free radicals to convert them to more stable products ICFE showed significantly higher antioxidant activity compared to the
and terminate the radical chain reaction. Shen et al. (2011) showed that negative control (p < 0.05), with ICFE providing the strongest protec­
intact cells, culture supernatant, and ICFE of B. animalis 01 efficiently tion of nearly 80%.

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P. Vitheejongjaroen et al. Food Bioscience 43 (2021) 101259

The different mechanisms, which involve in the radical antioxidant 3.4. General probiotic properties of selected probiotic strain
reactions are dependent on species and strains of probiotic and host
cells, and the ability of the antioxidants to be transported to cells. One of the important criteria for selection of probiotics is the toler­
Antioxidative activities of probiotic strains showed an effective protec­ ance to low pH condition in the stomach and to the bile salt secreted in
tion against oxidative damage in Caco-2 cells. Similarly, Lin and Chang the small intestine. Survival rate of B. animalis MSMC83 after 3 h
(2000) showed that the ICFE of B. longum exhibited a higher inhibition exposure to pH 2.0, 3.0 and 4.0 was 72.75%, 94.76%, and 97.74%,
of the H2O2-induced oxidative damage in intestinal cell. Our results respectively (Fig. 3A). Survival rate of B. animalis MSMC83 exposed in
indicated that MSMC83 may inhibit the production of ROS in intestinal 0.8% (w/v) bile salt after 3 h exceeded 99% as shown in Fig. 3B. The
cells induced by H2O2, so this strain could be used in formulation of food ability of B. animalis MSMC83 to adhere to Caco-2 cells was 8.99 ±
products to prevent intestinal cell damage caused by oxidative stress. 0.32% (Fig. 3C), which was higher than for the reference strain
L. rhamnoses GG, (p < 0.05). The ability of probiotics to reduce
3.3. Genotypic characteristics of probiotic isolate cholesterol are due to the production of bile salt hydrolase (Sirichotinun
et al., 2020). B. animalis MSMC83 exhibited BSH activity identified by
Comparative analysis of 16 S rRNA gene sequences confirmed the the formation of precipitation zones around colonies on TDCA plate
results from the cluster analysis and revealed that probiotic isolate was assay (data not shown). Additionally, B. animalis MSMC83 did not
99% relatively similar to Bifidobacterium animalis (GenBank ID: exhibit hemolytic activity (γ-hemolysis) and was sensitive to the
MW147142.1). A phylogenetic tree was constructed based on their 16 S Gram-positive spectrum antibiotics except vancomycin.
rRNA gene sequences utilizing the neighbor-joining method. The results Acid and bile tolerance of probiotics depend on strains and are
showed that the MSMC83 belonged to Bifidobacterium animalis subsp. related to their biological characteristics. According to Zhang et al.
lactic DSM 10140T (Fig. 2). B. animalis MSMC83 has been deposited at (2016), survival rate of Bifidobacterium spp. Ranged from 93 to 98% at
the TISTR culture collection, Thailand institute of scientific and tech­ pH 3 and from 61 to 98% in 0.3% bile salt. The acid and bile resistances
nological research (TISTR P003). of B. animalis MSMC83 isolated were both nearly 99%. Bifidobacterium
The Bifidobacterium spp. Are generally recognized as safe. In addi­ isolated from subjects of different ages also had different degrees of
tion, they are a core member of the human gastrointestinal microbiota adhesion capability. Due to aging, some of the properties necessary for
and are dominant populations in most individual, constituting >1% of their adhesion are lost. Bifidobacterium isolated from the elderly had less
the intestinal microbiota in adults and representing up to 90% of mi­ adhesion ability than those isolated from infant (He et al., 2001).
croorganisms in the fecal of healthy breast-fed infants (Penders et al., Additionally, Enterococcus, Lactobacillus and Bifidobacterium expressed
2006). Interestingly, they are among the first reported isolation of BSH activity (Begley et al., 2006). BSH is an enzyme that catalyzes the
strictly anaerobic microorganisms colonizing the gut within the first deconjugation of bile salt. BSH activity in probiotic strains is related to
week of life in full term neonates (Palmer et al., 2007). indirect lowering cholesterol level (Tsai et al., 2014). B. animalis
MSMC83 may potentially be used to reduce the risk factor of hyper­
cholesterolemia. Altogether, the results suggested that B. animalis

Fig. 2. Phylogenetic relationship between MSMC83 and members of related Bifidobacterium species based on 16 S rRNA gene sequences. The tree was constructed by
the neighbor-joining method and rooted with Bacillus subtilis DSM10. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap
test (1000 replicates) is shown next to the branches. Bootstrap percentages above 50 are given at the branching points.

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Fig. 4. Fermentation time of control yoghurt (starter cultures) and probiotic

yoghurt (starter cultures + B. animalis MSMC83).

The fermentation time was 6 h for the control sample, and 4 h 30 min for
probiotic yoghurt. The difference in fermentation time may be attrib­
uted to B. animalis MSMC83 promoting the growth of starter cultures.
The shorter fermentation time (5 h) when co-fermented with probiotics
was also observed when L. acidophilus was added (Mani-López et al.,
2014). However, some probiotic strains such as, L. rhamnosus, and
B. lactis, retarded the fermentation of yoghurt when grown with yoghurt
starter cultures (Oliveira et al., 2009). These results suggested that ac­
celeration of acidification rate in probiotic yoghurts is possibly strain
dependent.

3.6. Viability of microorganisms in yoghurt

The initial concentration of each microorganism, S. thermophilus,

L. bulgaricus, and B. animalis MSMC83 was approximately 9.0 log CFU/
mL. The viable cell counts and the number of probiotics B. animalis
MSMC83 during storage are presented in Table 1. After 4-week storage,
the viable cell counts in control yoghurt and probiotic yoghurt were 9.84
log CFU/g and 9.60 log CFU/g, respectively. In addition, the probiotics
viability remained at 8.53 log CFU/g.
The concentration of probiotics is an important parameter in final
probiotic products. Generally, probiotic products should have minimum
concentration of 6.0 log CFU/g and daily human consumption is rec­
ommended at 8.0 to 9.0 log CFU/g of probiotic microorganisms
(Kechagia et al., 2013). The viability of bifidobacteria in all the

Table 1
Viability of microorganism in control yoghurt and probiotic yoghurt during 4
Fig. 3. Acid (A) and bile salt (B) tolerance and adhesion ability to Caco-2 cells weeks of storage at 4 ± 1 ◦ C.
(C) of B. animalis MSMC83, compared with the reference strain Lactobacillus
Yoghurts Microorganism Viability of microorganism (log CFU/g)
rhamnosus GG (LGG). The letters A-C indicate statistically significant differences
at p < 0.05 within each condition of B. animalis MSMC83. Initial Week Week Week Week
1 2 3 4

MSMC83 is a probiotic strain potentially able to grow and function in Control Viable cells 9.16 9.21 9.51 9.81 9.84
yoghurt ± 0.06 ± 0.10
gastrointestinal tract and may be considered as safe regarding this
± ± ±
0.04Ac 0.05Bc 0.03Bb Aa Aa

activity. Probiotic Viable cells 9.24 9.32 9.65 9.85 9.60

yoghurt ± 0.02 ± 0.03 ± 0.07 ± 0.09 ± 0.09
Ac Ac Ab Aa Ba

3.5. Preparation of starter cultures and fermentation of milk B. animalis 9.16 9.19 9.04 8.92 8.53
MSMC83 ± ± ± ± ±
The pH profiles of the yoghurts fermented by regular yoghurt starter 0.08a 0.05a 0.08a 0.09b 0.09b
cultures (control) and yoghurt fermented by starter cultures supple­ Each value is the mean ± SD of three independent readings. The different su­
mented with B. animalis MSMC83 are shown in Fig. 4. Fermentation was perscript letters a-c in the same row and letters A-B in the same column represent
stopped at pH 4.6 b y transferring the samples to a refrigerator at 4 ◦ C. statistical significance (p < 0.05).

6
P. Vitheejongjaroen et al. Food Bioscience 43 (2021) 101259

commercial products significantly decreased in week 4 under refriger­ Table 3

ated storage conditions (Alazzeh et al., 2020). The proliferation of Firmness properties of control yoghurt and probiotic yoghurt during 4 weeks of
probiotics may be due to the release of amino and micro-peptides, which storage at 4 ± 1 ◦ C.
are nutrients that increase the viability of probiotic during storage Yoghurts Firmness (g)
(Abdel-Hamid et al., 2019). Type and acid condition of yogurt products Initial Week 1 Week 2 Week 3 Week 4
during storage and acid resistance characteristics of probiotic strains
Control 85.75 ± 87.80 ± 95.10 ± 72.35 ± 65.85 ±
may affect the viability of probiotics (Kourkoutas et al., 2005).
yoghurt 0.77Bc 0.84Bb 0.42Ba 0.77Bd 0.56Be
Probiotic 106.56 ± 114.90 ± 136.25 ± 147.55 ± 120.30 ±
yoghurt 0.79Ae 0.42Ad 0.91Ab 0.49Aa 0.14Ac
3.7. Physicochemical analysis of yoghurts
Each value is the mean ± SD of three independent readings. The different su­
3.7.1. pH levels perscript letters a-e in the same row and letters A-B in the same column represent
statistical significance (p < 0.05).
The pH levels of yoghurts during storage are shown in Table 2. The
pH values of probiotic yoghurt significantly decreased during 3 weeks of
storage (p < 0.05), and increased to reach 4.09 in week 4. The pH of Table 4
control yogurt was 4.59 at the beginning and decreased to 4.27 b y the Syneresis properties of control yoghurt and probiotic yoghurt during 4 weeks of
end of storage. This change in pH during storage is consistent with storage at 4 ± 1 ◦ C.
previous studies. Dan et al. (2019) found decreasing pH levels to 4.01
Yoghurts Syneresis (%)
after storage of 14 days in yoghurts fermented with L. plantarum P-8.
Moreover, yogurt samples containing probiotic/paraprobiotic added Initial Week 1 Week 2 Week 3 Week 4

before and after fermentation showed pH drop during refrigeration Control 8.99 ± 15.88 ± 19.79 ± 21.58 ± 22.46 ±
(Molaee Parvarei et al., 2021). The decline in pH values may be due to yoghurt 0.87Bd 0.35Bc 0.25Bb 0.52Ba 0.38Ba
Probiotic 1.33 ± 2.56 ± 3.26 ± 4.69 ± 4.99 ±
the continuous fermentation of LAB in yoghurt. When the pH dropped
yoghurt 0.41Ad 0.33Ac 0.18Ac 0.15Ab 0.10Aa
below 4.4, Streptococcus stopped growing while Lactobacillus and Bifi­
dobacterium still grew and produced amino acids, resulting in a final Each value is the mean ± SD of three independent readings. The different su­
increase in the pH of the product (Lourens-Hattingh & Viljoen, 2001). perscript letters a-d in the same row and letters A-B in the same column represent
statistical significance (p < 0.05).
Therefore, the residual acidification during storage can be attributed to
the residual enzymatic activity of microorganisms used in yoghurt
production. (Domagala, 2012). The increase in syneresis during storage may be
described as post-acidification of yoghurts even they were stored at 4 ◦ C.
3.7.2. Texture firmness Similar results were observed when other probiotic strains, L. plantarum
Texture firmness of both yoghurts was measured during storage and and B. bifidum were added to yoghurts (Soni et al., 2020).
the results are presented in Table 3. During storage, firmness of both
types of yoghurts increased significantly, but slightly decreased by the 3.7.4. Rheological properties
end of storage (p < 0.05). Probiotic yogurt had firmer texture than the The storage modulus (G′ ) and the loss modulus (G′′ ) at frequency of 1
control, which may be attributed to the ability of some LAB to produce Hz are displayed in Tables 5 and 6. The G′ values were higher than G′′
mucogenic such as exopolysaccharides, which enhanced the strength of values in both yoghurts across the entire measuring range. In probiotic
protein matrix, resulting in improved firmness during storage (Ma et al., yoghurt, the G′ value increased from a minimum of 270.17 ± 5.71 Pa at
2019). Therefore, firmness of fermented dairy products was strain week 1 to a maximum of 482.77 ± 3.65 Pa at week 4. Interestingly, the
dependent. G′ and G” of the probiotic yoghurt were significantly higher than for the
control yoghurt (p < 0.05). The storage modulus characterizes the de­
3.7.3. Syneresis gree of elasticity and the loss modulus characterizes the degree of vis­
The results of syneresis measurement of both yoghurts during storage cosity (Singh et al., 2016). Our results showed an elastic characteristic in
presented in Table 4. The syneresis values of both yoghurts significantly both yoghurts during storage. Fermentation time may be the main factor
increased with storage time (p < 0.05). The addition of B. animalis responsible for the differences in the moduli. The rate of acidification
MSMC83 in probiotic yoghurt significantly decreased the syneresis directly affects the rheological properties, and is related to the differ­
compared to the control yoghurt, even in the beginning. Long fermen­ ences in starter composition, incubation temperature, and incubation
tation time increases rearrangements of the gel network, which may time. Increasing the rate of acidification resulted in gels with higher G’
result in larger pore formation and enhanced syneresis (Peng et al., values.
2009). Syneresis is defined as the separation of aqueous phase from
continuous phase or gel network, which is a reversible indicator of the 3.7.5. Apparent viscosity
quality of yoghurt. It may be spontaneous or may occur only when the As shown in Table 7, the viscosity in both yoghurts during 4 weeks
gel is mechanically disrupted by cutting, freezing, or centrifugal force did not significantly change, although the viscosity values slightly

Table 2 Table 5
pH value of control yoghurt and probiotic yoghurt during 4 weeks of storage at 4 Storage modulus of control yoghurt and probiotic yoghurt during 4 weeks of
± 1 ◦ C. storage at 4 ± 1 ◦ C.
Yoghurts pH value Yoghurts Storage modulus (G′ ) (Pa)

Initial Week 1 Week 2 Week 3 Week 4 Initial Week 1 Week 2 Week 3 Week 4

Control 4.59 ± 4.43 ± 4.23 ± 4.24 ± 4.27 ± Control 161.53 ± 222.17 ± 246.60 ± 287.20 ± 327.08 ±
yoghurt 0.03Ac 0.03Bb 0.04Ba 0.02Ba 0.05Ba yoghurt 2.55Be 6.57Bd 6.66Bc 1.57Bb 2.66Ba
Probiotic 4.67 ± 4.32 ± 3.84 ± 3.72 ± 4.09 ± Probiotic 270.17 ± 350.17 ± 383.27 ± 420.67 ± 482.77 ±
yoghurt 0.04Be 0.02Ad 0.04Ab 0.05Aa 0.05Ac yoghurt 5.71Ae 10.32 Ad 8.06Ac 5.40Ab 3.65Aa

Each value is the mean ± SD of three independent readings. The different su­ Each value is the mean ± SD of three independent readings. The different su­
perscript letters a-e in the same row and letters A-B in the same column represent perscript letters a-e in the same row and letters A-B in the same column represent
statistical significance (p < 0.05). statistical significance (p < 0.05).

7
P. Vitheejongjaroen et al. Food Bioscience 43 (2021) 101259

Table 6 Ahire, J. J., Mokashe, N. U., Patil, H. J., & Chaudhari, B. L. (2013). Antioxidative
Loss modulus of control yoghurt and probiotic yoghurt during 4 weeks of storage potential of folate producing probiotic Lactobacillus helveticus CD6. Journal of Food
Science & Technology, 50(1), 26–34.
at 4 ± 1 ◦ C. Alazzeh, A., Zrieq, R., Azzeh, F., Smadi, M., Sulaiman, S., & Qiblawi, S. (2020). Initial
Yoghurts Loss modulus (G′′ ) (Pa) enumeration and viability of probiotic strains in commercial yogurt products under
refrigerated conditions. International Journal of Advanced and Applied Sciences, 7,
Initial Week 1 Week 2 Week 3 Week 4 11–16.
Bai, M., Huang, T., Guo, S., Wang, Y., Wang, J., Kwok, L.-Y., & Bilige, M. (2020).
Control 44.835 ± 58.37 ± 68.32 ± 81.26 ± 92.69 ±
Probiotic Lactobacillus casei Zhang improved the properties of stirred yogurt. Food
yoghurt 4.41Be 0.69Bd 0.72Bc 1.03Bb 0.46Ba
Bioscience, 37, 100718.
Probiotic 66.46 ± 81.37 ± 95.24 ± 105.96 ± 116.84 ± Begley, M., Hill, C., & Gahan, C. G. (2006). Bile salt hydrolase activity in probiotics.
yoghurt 3.37Ac 0.86Ab 4.09Aa 8.16Aa 4.60Aa Applied and Environmental Microbiology, 72(3), 1729–1738.
Bustos, I., García-Cayuela, T., Hernández-Ledesma, B., Peláez, C., Requena, T., &
Each value is the mean ± SD of three independent readings. The different su­
Martínez-Cuesta, M. C. (2012). Effect of flavan-3-ols on the adhesion of potential
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Choi, S. S., Kang, B. Y., Chung, M. J., Kim, S. D., Park, S. H., Kim, J. S., Kang, C. Y., &
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Initial Week 1 Week 2 Week 3 Week 4 Dan, T., Chen, H., Li, T., Tian, J., Ren, W., Zhang, H., & Sun, T. (2019). Influence of
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