LWT - Food Science and Technology 135 (2021) 110061

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

Selected probiotic lactic acid bacteria isolated from fermented foods for
functional milk production: Lower cholesterol with more
beneficial compounds
Jirayu Jitpakdee a, Duangporn Kantachote a, *, Hiroshi Kanzaki b, Teruhiko Nitoda b
a
Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, 90112, Thailand
b
Graduate School of Environmental and Life Science, Faculty of Agriculture, Okayama University, Okayama, 700-8530, Japan

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

Keywords: Two lactic acid bacteria (LAB) were isolated from fermented foods and identified as Pediococcus pentosaceus
γ-aminobutyric acid ENM104 and Lactobacillus plantarum SPS109. ENM104 reduced cholesterol at 7.53 ± 1.78% in basal medium
Angiotensin-converting enzyme (ACE) containing 100 μg/ml cholesterol, while SPS109 released γ-aminobutyric acid (GABA) at 1157.01 ± 4.76 μg/ml
Antioxidants
in MRS containing 5 mg/ml monosodium glutamate (MSG). They hydrolysed skim milk, deconjugated glyco­
Lactobacilli
Pediococci
cholic acid (GCA), taurocholic acid (TCA) and taurodeoxycholic acid (TDCA) as bile salts and tolerated simulated
gastrointestinal juice, including H2O2. None performed any haemolysis. ENM104 cultured in MRS reduced
cholesterol at 15.34 ± 1.12% (initial content, 100 μg/ml) at 72-h and produced intracellular cholesterol oxidase
at 8.73 ± 2.00 mU/mg protein. Milk fermentation using the two strains as starters in single cultures and a co-
culture showed that the co-culture (1:1) was the most effective. At 72 h, cholesterol in milk fermented with
the co-culture was significantly reduced cholesterol at 10.98 ± 3.80%, while GABA content increased to 4.47 ±
0.04 μg/ml and angiotensin-converting enzyme (ACE) inhibition increased to 57.63 ± 2.97%. Based on 2,2′ -
azinobiz(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-2-picrylhydrazyl (DPPH) and oxygen
radical absorbance capacity (ORAC) assays, all antioxidant activities significantly increased. Fermented milk
using probiotic LAB ENM104 and SPS109 has the potential to be an approved functional dairy product.

1. Introduction neurotransmission in the sympathetic nervous system, a diabetic sup­

The formation of clogged arteries due to high blood cholesterol in­
creases the risk of heart disease. In 2017, 4.3 million people died from
hypercholesterolemia: a 20.8% increase since 1990 (Virani et al., 2020).
There is also a positive correlation between cholesterol content and
blood pressure, and the use of LAB is an attractive way to reduce both
risk factors. LAB are “generally recognised as safe (GRAS)” to use as
dietary additives to support the consumer’s health. Some LAB can be
probiotic candidates and also provide bioactive compounds such as
γ-aminobutyric acid (GABA), riboflavin and bioactive peptides to
transform certain foods and beverages into functional foods (Daliri, Lee,
Kim, & Oh, 2018; Ge, Zhang, Corke, & Gan, 2020; Ratanaburee, Kant­
achote, Charernjiratrakul, Penjamras, & Chaiyasut, 2011). GABA is
produced by the transformation of glutamic acid (GA) using glutamate
decarboxylase (GAD). It was reported to be a primary inhibitor of
pressant, an antihypertensive and an aid to emotional control (Die­
z-Gutiérrez, San Vicente, Luis, Villarán, & Chávarri, 2020). Certain LAB,
including Lactobacillus, Lactococcus, Pediococcus and Weissella, produced
angiotensin-converting enzyme (ACE) inhibitors, in fermented foods i.e.
dairy and soybean products, to down-regulate hypertension (Barla et al.,
2016; Daliri et al., 2018). Some LAB strains have also been reported to
improve antioxidant activity by antioxidative peptides in fermented
foods and beverages (Kantachote, Ratanaburee, Hayisama-ae, Sukhoom,
& Nunkaew, 2017; Panchal, Hati, & Sakure, 2020). LAB for use as
starters in commercial applications can be safely isolated from fer­
mented foods. For example, Pediococcus pentosaceus HN8 and Lactoba­
cillus namurensis NH2 were added to Thai fermented pork for GABA
enrichment and cholesterol reduction (Ratanaburee, Kantachote, Char­
ernjiratrakul, & Sukhoom, 2013a).
Various food products are derived from milk. Cheeses, butter and

* Corresponding author.
E-mail addresses: jirayu.jitpakdee@gmail.com (J. Jitpakdee), duangporn.k@psu.ac.th (D. Kantachote), hkanzaki@okayama-u.ac.jp (H. Kanzaki), nitoda@
okayama-u.ac.jp (T. Nitoda).

https://doi.org/10.1016/j.lwt.2020.110061
Received 5 May 2020; Received in revised form 11 August 2020; Accepted 11 August 2020
Available online 17 August 2020
0023-6438/© 2020 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
J. Jitpakdee et al.
whey protein are popular with consumers and that popularity is re­
flected in the market. In 2019, 852 million tonnes of milk were sold
worldwide (FAO, 2019). The application of potent probiotic LAB to
dairy products should produce functional foods that can reduce
cholesterol and supplement diets with GABA. Moreover, LAB can in­
crease the value of functional foods by supplying anti-hypertensive ACE
inhibitors to reduce blood pressure, and antioxidative compounds that
combat reactive oxygen species (ROS) and free radicals. ROS promotes
chronic diseases such as atherosclerosis and cardiovascular diseases.
Amongst ROS, the hydroxyl radical, derived from H2O2, was the most
active species, causing DNA lesions and protein oxidation (Imlay, 2015).
Hence, this research aimed to isolate LAB from Thai fermented foods and
select the cholesterol-lowering and GABA-producing strains. Potent LAB
strains were identified and evaluated for cholesterol degradation, ACE
inhibition and antioxidant activities. Moreover, the ability of probiotic
properties to survive crossing the biological barrier was established to
ensure that the obtained dairy products could be considered functional
when the selected strains were applied as starters.
2. Materials and methods
2.1. Chemicals used
ABTS, DPPH, cholesterol, cholesterol-PEG600, 85% glycerol, GA,
GCA, glycodeoxycholic acid (GDCA), TCA, TDCA, (±)-6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), glyceryl tribu­
tyrate, pepsin, pancreatin and GABA were from Sigma (USA). Bromoc­
resol purple (BCP), bromocresol green, de Man, Rogosa and Sharpe
(MRS) and silica gel 60 F254 were from Merck (Germany). Bile acids
(Oxoid, France), soluble starch (Difco, USA) and skim milk (HiMedia,
India) were also used.
2.2. Isolation of LAB
Sixty samples of various fermented foods and raw milk were
collected from several regions of Thailand. To obtain safe LAB as normal
flora, products were chosen that were known to be safe in regular con­
sumption. Each sample was serially diluted in 0.1% (w/v) peptone
water. Solid samples were first homogenised in a stomacher (Seward,
UK) at 230 rpm for 30 s. Using a pour plate technique, LAB were isolated
from appropriate dilutions of samples on MRS agar fortified with 400
mg/L BCP. The criteria for isolation were yellowish colonies on MRS
that were Gram positive and catalase negative. All stock cultures of LAB
isolates were stored in 20% (v/v) glycerol in distilled water at − 80 ◦ C,
while working cultures were stabbed in MRS agar and kept at 4 ◦ C.
2.3. Screening of cholesterol-lowering LAB
All LAB isolates were primarily screened by incubation in MRS broth
for 18 h. After twice washing with normal saline solution (NSS),
screened isolates were suspended in NSS adjusted to OD660 at 1.0 as
inoculums. This protocol was used for inoculum preparation for all ex­
periments in this study. A 3% (v/v) inoculum of each isolate was culti­
vated at 35 ◦ C for 72 h in basal medium at pH 7.0 ± 0.2 composed of 1.0
g yeast extract, 2.0 g K2HPO4, 1.0 g polysorbate 80, 2.0 g (NH4)2SO4,
0.2 g MgSO4⋅7H2O, 0.04 g MnSO4⋅H2O in 1 L distilled water (Yehia,
Hassanein, & Ibraheim, 2015) fortified with 100 μg/ml cholesterol, the
approximate cholesterol content specified in commercial cow’s milk
products and found in raw milk samples. Each isolate was streaked on
MRS agar to check its survival. They were secondarily screened in the
same medium without yeast extract and checked by streaking as pre­
viously described. Tertiary screening confirmed cholesterol degradation
based on proliferation determined by viable count after 72 h incubation
in basal medium fortified with 100 μg/ml cholesterol and in basal me­
dium without cholesterol as a control. Cholesterol loss was then inves­
tigated by inoculating a 3% inoculum into basal medium containing 100
2
LWT 135 (2021) 110061
μg/ml cholesterol-PEG600, while un-inoculated medium served as the
control. After incubation at 35 ◦ C for 72 h, cell pellets were separated
from supernatants by centrifugation at 5000×g for 10 min and twice
washed with NSS. Cells were disrupted by suspension in 1 ml hexane
using ultra-sonication with glass beads (Sonics, USA) at 60% amplitude
with a 25 s cycle-on and a 5 s cycle-off for 10 min before cell debris was
removed by centrifugation at 12,000×g at 4 ◦ C for 10 min. Cholesterol in
cell-free supernatants and cell pellets was detected by spectrophotom­
etry (OD570) (Tomaro-Duchesneau et al., 2014). Cholesterol loss was
determined from the following formula, in which A was cholesterol in
cell-free supernatant after incubation, B was cholesterol of the control
and C was cholesterol in cell pellets:
B − [A + C]
% Cholesterol loss = × 100%
B
2.4. Screening of GABA-producing LAB
A 3% inoculum of each isolate was inoculated in MRS fortified with
20 mg/ml MSG and incubated at 35 ◦ C for 24 h. Cell-free supernatants
were spotted on silica gel F254 with controls containing MRS, MRS+20
mg/ml MSG, and a mixture of 20 mg/ml MSG and 20 mg/ml GABA
(Hiraga, Ueno, & Oda, 2008). GABA-producing LAB strains were iden­
tified by computing retardation factors (Rf).
To determine MSG and GABA contents, a 3% inoculum was inocu­
lated into MRS with 5 mg/ml MSG and incubated at 35 ◦ C for 72 h,
sampling every 24 h. The cell-free supernatant was harvested and ana­
lysed by high-performance liquid chromatography (Populin, Moret,
Truant, & Conte, 2007). Conversion efficiency was then computed based
on MSG and GABA contents (Ratanaburee et al., 2011).
2.5. Identification of selected LAB
Selected LAB strains were identified by sugar fermentation using the
API 50 CHL kit (BioMérieux, France). Raw data was encrypted by api­
web™ identification software (V5.1) (BioMérieux, France) and 16S
rRNA genes of selected LAB were sequenced (Ratanaburee, Kantachote,
Charernjiratrakul, & Sukhoom, 2013b). Forward and reverse primers for
amplification were 27F 5′ -AGAGTTTGATCCTGGCTCAG-3′ and 1492R
5′ -GGTTACCTTGTTACGACTT-3′ , respectively. The reaction contained
10 μM of each primer mixed with 3 μl genomic DNA, 2.5 unit/μl Hot­
StarHiFidelity polymerase (Qiagen, Germany), 5x nitrogenous-base
buffer, and the total volume of 70 μl was made up with RNase-free
water. Templates were multiplied by denaturing for 15 s at 94 ◦ C,
annealing for 1 min at 46.4 ◦ C, and extension for 1 min 45 s at 72 ◦ C.
After purification (Biofact, South Korea), sequences were compared by
nBLAST with entries in the NCBI database (https://blast.ncbi.nlm.nih.
gov/Blast.cgi), and a phylogenetic tree was established using the
Jukes-Cantor nucleotide model of the neighbour-joining statistical
method.
2.6. Examination of probiotic properties of selected LAB
Basic properties of selected LAB were examined to prove their pro­
biotic properties in vitro (Ratanaburee et al., 2013b). Main nutrient
hydrolysis was evaluated on 2 g/L starch agar, 10 g/L tributyrin agar
and 2 g/L skim milk agar. Bile salt hydrolase activity was assessed on
MRS agar supplemented with 5 mg/ml each of GCA, GDCA, TCA, and
TDCA. The transparent zones surrounding colonies were observed. A 10
μl aliquot of each inoculum was spotted onto the test media and incu­
bated at 37 ◦ C for 24 h. The selected strains were tested against bio­
logical barriers in simulated gastrointestinal juice using an inoculum
containing roughly 10 log CFU/ml. Each harvested inoculum was first
suspended in a gastric juice composed of MRS broth and 3 mg/ml pepsin
with pH at 2.0 ± 0.1 and microaerobically incubated at 37 ◦ C for 2 h.
Cell pellets were then harvested and suspended in intestinal juice based
J. Jitpakdee et al.
on MRS broth containing 1 mg/ml pancreatin and 3 mg/ml bile acids at
pH 8.0 ± 0.1 for 6 h. Viable cells were counted at 0, 2, 3 and 6 h of
incubation. Tolerance of H2O2 was tested using cell pellets washed twice
with 0.1 M phosphate buffer saline (PBS) at pH 7.4 and suspended in the
same buffer to attain a cell density of roughly 8 log CFU/ml. Then, 1 mM
H2O2 was added into the cell suspension. A suspension without H2O2
served as the control. All culture tubes were incubated at 35 ◦ C for 24 h
with sampling every 4 h to monitor survival. Haemolysis was investi­
gated to ensure their safety by streaking onto blood agar and incubated
for 48 h to observe for any haemolytic reaction. Method of cell surface
hydrophobicity was modified from Ratanaburee et al. (2013b) as hexane
was replaced by xylene. Cholesterol-lowering activity was determined
by growing a 3% inoculum in MRS broth fortified with 100 μg/ml
cholesterol-PEG600 at 35 ◦ C for 72 h with harvesting every 24 h.
Cholesterol content was determined as previously described.
2.7. Evaluation of cholesterol oxidase activity
Cholesterol-lowering LAB candidates were evaluated for cholesterol
oxidase activity. In short, 3% inoculums were microaerobically incu­
bated at 35 ◦ C for 72 h in 50 ml of MRS broth fortified with 100 μg/ml
cholesterol. Cultures were sampled every 24 h. Cell pellets were washed
twice with 0.1 M PBS at pH 7.4, suspended in 1.5 ml deionised water,
disrupted by ultra-sonication and managed as previously described.
Both the cell pellets and culture supernatants were evaluated for
cholesterol oxidase activity (Kiatpapan, Yamashita, Kawaraichi, Yasuda,
& Murooka, 2001), 1 unit represented production of 1 μmol H2O2 per
min. Protein concentration was measured by the Bradford assay (Kruger,
2003) to find specific enzyme activity.
2.8. Application of selected LAB as starter(s) to produce functional milk
To prepare milk for fermentation, commercial pasteurised milk was
sterilised by autoclaving at 121 ◦ C for 15 min. The experimental design
comprised four sets including an unseeded control, an ENM104 single
culture, an SPS109 single culture, and a co-culture of the two at a ratio of
1:1. A 3% inoculum was applied to each single culture set, while the co-
culture set was made up of 1.5% inoculums of each strain. All sets were
incubated at 35 ◦ C for 72 h and sampled every 24 h.
Various functional properties were monitored. pH value was
measured by pH meter (Horiba, Japan), while viable count was detected
in re-formulated MRS medium replacing glucose with sucrose to
distinguish Pediococcus from Lactobacillus. Pediococcus sp. were indi­
cated by bluish-white-pin-pointed colonies present after 48–72 h while
Lactobacillus sp. colonies were indicated by their bright yellow colour
after 24 h. Cholesterol reduction was monitored as previously described.
For the determination of GA and GABA, milk was first extracted (Abu­
bakr, Hassan, Imdakim, & Sharifah, 2012). Conversion efficiency was
calculated as previously described. ACE inhibition was also determined
from extracted milk (Nakano et al., 2006). Agglutinated casein was
removed by centrifugation at 10,000×g for 10 min and the supernatant
was used to determine antioxidant activity in terms of trolox equiv­
alent/ml (μmol TE/ml) using DPPH (Banerjee, Dasgupta, & De, 2005),
ABTS (Re et al., 1999) and ORAC (Ou, Hampsch-Woodill, & Prior,
2001).
2.9. Statistical analysis
Data from three biological replicates of all experiments were ana­
lysed with the R programme (version 3.5.2). One-way ANOVA with
factorial analysis was used. Significant differences were appraised at
critical points (p < 0.05) and differentiated by Tukey’s honestly signif­
icant difference (HSD) multiple comparison.
3
LWT 135 (2021) 110061
3. Results
3.1. Isolation and selection of cholesterol-lowering and GABA-producing
LAB strains
A total of 244 LAB isolates were isolated from 39 samples of various
fermented foods, including raw milk. In basal medium that had
cholesterol as a source of carbon and energy, primary screening found
139 LAB strains. However, only ten LAB isolates were able to survive
secondary screening. Based on cell proliferation in tertiary screening for
cholesterol degradation, only three LAB strains showed significantly
higher populations (p < 0.05) in treatment than the control. The log
value of the count of CFU/ml of strain ENM104 was 7.54 ± 0.08
compared with 7.21 ± 0.09 for its control. This strain also produced the
highest cholesterol reduction at 7.53 ± 1.78% (p < 0.05). Therefore, it
was selected for further studies.
The ten selected LAB that passed secondary screening were further
investigated to obtain GABA-producing LAB. However, only strain
SPS109 showed GABA-producing activity with a detected Rf of 0.32
combined with a small Rf of MSG at 0.20. This strain was incubated in
MRS fortified with 5 mg/ml MSG and analysed by HPLC for GABA
production. GABA was detected at 1157.01 ± 4.76 μg/ml with a con­
version efficiency of 37.73 ± 0.10% at 72 h (Table 1). This strain was
also chosen for further studies.
3.2. Identification of selected LAB
The two selected LAB (ENM104 and SPS109) received from the
screenings were evaluated by biochemical and molecular methods.
From 49 sugar fermentations for their differences, ENM104 produced
positive results for D-xylose and D-tagatose, while SPS109 was positive
for D-mannitol, D-sorbitol, methyl-αD-mannopyranoside, D-lactose, D-
melibiose, D-sucrose, D-melezitose, D-raffinose and potassium gluconate.
Strain ENM104 was identified as Pediococcus pentosaceus with a highest
similarity of 99.9%, while strain SPS109 was closest to Lactobacillus
plantarum at 99.9% similarity.
The molecular relationship between ENM104 and SPS109 strains
and other closely similar species was illustrated by a phylogenetic tree
based on 16S rRNA genes (Fig. 1). ENM104 and SPS109 strains were
closest to P. pentosaceus DSM20336T and L. plantarum DSM20174T,
respectively, with similarities of 99.65 and 99.93%. The cholesterol-
lowering strain identified as P. pentosaceus ENM104 (accession num­
ber: MN915130) was isolated from fermented pork sausage from Muk­
dahan province, Thailand, while the GABA-producing strain identified
as L. plantarum SPS109 (accession number: MN915131) was isolated
from fermented fish from Narathiwat province, Thailand.
3.3. Evaluation of probiotic properties
The probiotic characteristics of the two strains were presented in
Table 2. Both selected LAB strains showed γ-haemolysis and hydrolysed
Table 1
GABA production by strain SPS109 incubated for 72 h in MRS broth containing
5 mg/ml monosodium glutamate (MSG).
Incubation time Concentration (μg/ml) % Conversion
(h) efficiency
MSG GABA
0 5028.50 ± 7.48 a 0 ± 0.00 d 0 ± 0.00 d
24 4096.10 ± 462.11 ± 5.27 c 15.07 ± 0.15c
19.17 b
48 3339.55 ± 9.46 c 911.98 ± 16.28 29.74 ± 0.58b
b
72 3097.25 ± 6.51 d 1157.01 ± 4.76 37.73 ± 0.10a
a
Values are given as means ± standard deviation (n = 3). Different lowercase
letters in each column represent significant differences (p < 0.05).
J. Jitpakdee et al. LWT 135 (2021) 110061

Fig. 1. The construction of the phylogenetic tree of LAB isolates ENM104 and SPS109 was based on 16S rRNA identification using the Jukes-Cantor nucleotide model
of neighbour-joining statistics. Notations include taxonomical names and strain codes. Accession numbers appear in parentheses. Streptococcus pneumoniae
DSM20566T (NR028665) is the chosen outgroup and evolutionary length is shown by the bar= 0.020.

casein, indicating their safety and capacity to digest protein, while the
Table 2
bile salts; GCA, TCA and TDCA were deconjugated. Both strains survived
Probiotic properties of P. pentosaceus ENM104 and L. plantarum SPS109.
the biological barrier test, surviving in log CFU/ml at 6.49 ± 0.00 and
Probiotic property LAB strain 7.63 ± 0.02 from the average initial value of 10.32 ± 0.20. ENM104 was
ENM104 SPS109 more tolerant to 1 mM H2O2 than SPS109 as a significant reduction of
Hydrolysis viable cells of the former was found after 8 h of incubation compared
Starch (2 g/L) – – with 4 h for the latter strain. No survivors were detected in ENM104 and
Glyceryl tributyrate (10 g/L) – + SPS109 after 20 and 12 h, respectively (Fig. 2). The hydrophobicity test
Casein (2 g/L) + + using xylene droplets by strains ENM104 and SPS109 were 7.66 ± 3.33
Deconjugated bile salts (each 5 mg/ml) and 27.77 ± 2.04%, respectively. After 72-h incubation in MRS broth
Glycocholic acid (GCA) + + supplemented with 100 μg/ml cholesterol, ENM104 and SPS109
Glycodeoxycholic acid – –
reduced cholesterol by 15.34 ± 1.12 and 10.05 ± 2.32%, respectively.
(GDCA)
Taurocholic acid (TCA) + +
Taurodeoxycholic acid + +
(TDCA)

Survival in simulated gastrointestinal juice (log CFU/ml) in MRS broth*

Gastric juice 0 h incubation 10.18 ± 0.02 b 10.46 ± 0.04 a
Gastric juice 2 h incubation 6.20 ± 0.12 f 7.39 ± 0.12 d
Intestinal juice 0 h 6.29 ± 0.10 ef 7.48 ± 0.10 cd
incubation
Intestinal juice 3 h 6.35 ± 0.01 ef 7.54 ± 0.01 cd
incubation
Intestinal juice 6 h 6.49 ± 0.00 e 7.63 ± 0.02 c
incubation

Haemolysis γ-haemolysis (no γ-haemolysis (no

reaction) reaction)

Cell surface hydrophobicity 7.66 ± 3.33% 27.77 ± 2.04%

Cholesterol reduction (%) in MRS broth with added 100 μg/ml cholesterol

0 h incubation 0 ± 0.00 d 0 ± 0.00 d

24 h incubation − 4.41 ± 2.75 e − 2.65 ± 1.44 de
48 h incubation 14.88 ± 0.49 ab 10.89 ± 1.06 bc
72 h incubation 15.34 ± 1.12 a 10.05 ± 2.32 c

* Gastric juice: 3 mg/ml pepsin, pH 2, intestinal juice: 1 mg/ml pancreatin and 3

mg/ml bile salts.
(+) indicates a positive result and (− ) a negative result. Values are given as
means ± standard deviation (n = 3). Different lowercase letters in each column
represent significant differences (p < 0.05). Fig. 2. Effect of 1 mM H2O2 on population of P. pentosaceus ENM104 and
L. plantarum SPS109, NS indicates no significant difference in their pairings.

4
J. Jitpakdee et al.
3.4. Evaluation of cholesterol oxidase activity
Due to its greater ability to reduce cholesterol (Table 2), only the
ENM104 strain was evaluated for intracellular and extracellular
cholesterol oxidase activity in MRS broth containing 100 μg/ml
cholesterol. Since the cell-free supernatant exhibited no cholesterol
oxidase activity, the enzyme was not present extracellularly. The highest
specific enzyme activity shown by cells from the pellets was 8.73 ± 2.00
mU/mg protein at 72 h (Table 3).
3.5. Application of selected LAB as starter(s) to produce functional milk
The changes in parameters monitored during milk fermentation
showed significant differences (p < 0.05) compared with the uninocu­
lated control set (Fig. 3). At 72 h, the co-culture set had significantly
reduced pH (p < 0.05) to a low of 5.32 ± 0.01 compared with the control
at 6.52 ± 0.02 (Fig. 3A). The dominant LAB population in the milk at 72
h was strain SPS109 in both single and co-culture sets at 8.29 ± 0.01 and
8.27 ± 0.04 log CFU/ml, respectively (Fig. 3B). The population of
ENM104 in the single culture set (7.41 ± 0.04 log CFU/ml, 25.6 ± 0.21
× 106 CFU/ml) was significantly higher than its population in the co-
culture set (6.93 ± 0.01 log CFU/ml, 8.57 ± 0.21 × 106 CFU/ml) at
72 h. The single ENM104 set showed the highest reduction of cholesterol
(15.71 ± 0.64%) at 72 h followed by the co-culture set (10.98 ± 3.80%)
(Fig. 3C). All inoculated sets had significantly increased ACE inhibition
(p < 0.05) at 72-h (Fig. 3D). Single culture sets of ENM104 and SPS109
increased ACE inhibition to 51.93 ± 4.54 and 56.02 ± 3.27%, respec­
tively, while the co-culture showed an increase to 57.63 ± 2.97%. At
approximately 43.46 ± 1.14 and 4.41 ± 0.01 μg/ml, respectively, GA
and GABA contents in the control set showed no significant difference
throughout the 72 h (Table 4). Hence, GABA production by sets was in
the order of SPS109 > co-culture > ENM104 > control. Antioxidant
activity was reported in μmol TE/ml. Detected by ABTS assay, the
antioxidant activity of the single ENM104 set reached a peak at 0.14 ±
0.02 at 24 h and at 48 h (0.09 ± 0.01) detected by DPPH assay
(Fig. 4A–B). In the ORAC assay, the co-culture reached a peak of 2.58 ±
0.26 at 24h with no significant difference compared to both single cul­
tures (Fig. 4C).
4. Discussion
To identify LAB that could reduce cholesterol intake, particularly if
consumed in functional milk products, cholesterol-lowering LAB were
isolated from fermented foods that contained high levels of cholesterol.
After three screening stages, three LAB strains were proven to use
cholesterol as a source of carbon and energy in the absence of other
compounds for both sources. A significant increase in proliferation of
viable cells was observed after 72 h in basal medium supplemented with
100 μg/ml cholesterol. Strain ENM104 produced the highest cholesterol
reduction at 7.53 ± 1.78%. Amongst ten LAB strains screened for GABA
production, only L. plantarum SPS109 was effective, converting roughly
Table 3
Cholesterol degradation by P. pentosaceus ENM104 in MRS broth supplemented
with 100 μg/ml cholesterol
Incubation time Intracellular cholesterol oxidase
(h)
Enzyme activity Protein content Specific enzyme activity
(mU) (mg) (mU/mg)
0 0.09 ± 0.01 b 0 ± 0.00 c NA
24 0.09 ± 0.02 b 0.14 ± 0.01 a 0.67 ± 0.12 c
48 0.25 ± 0.09 b 0.13 ± 0.01 a 2.00 ± 0.73 b
72 0.79 ± 0.18 a 0.09 ± 0.01 b 8.73 ± 2.00 a
Values are given as means ± standard deviation (n = 3). Different lowercase
letters in each column represent significant differences (p < 0.05). NA: not
available.
5
LWT 135 (2021) 110061
38% in MRS broth containing 5 mg/ml MSG (Table 1) compared with
L. plantarum L2A21R1, which converted only 3.12% in the same medium
containing 30 mg MSG/ml (Ribeiro, Domingos-Lopes, Stanton, Ross, &
Silva, 2018). Therefore, SPS109 was not only a cholesterol degrader but
also a significant GABA producer.
Both biochemical (API 50 CHL kit) and molecular tools were used to
identify the two selected LAB candidates. Both methods identified the
strains as P. pentosaceus ENM104 and L. plantarum SPS109 (Fig. 1).
Strain ENM104 was isolated from fermented ground pork that normally
presents the same flora as the raw ground pork. SPS109 was isolated
from a Thai fermented fish that normally contains the same microbiota
as Puntius gonionotus. The results indicate that both selected LAB strains
were related to their habitats: one to the high cholesterol environment in
pork and the other to the high protein source of GA in fish.
The probiotic properties of both LAB candidates were assessed by the
hydrolysis of the casein component in milk. Therefore, their application
in the fermentation of dairy products was plausible. This was not sur­
prising because amylase activity was absent in both strains and lipase
activity was only observed in SPS109 (Table 2). It should be noted that
proteolysis activity provides some advantages for studying ACE inhibi­
tion, which is popular in fermented milk as an anti-hypertensive agent
(Georgalaki et al., 2017). Both strains produced bile salt hydrolases. This
function indicates the gastrointestinal tolerance required by cells to
survive in intestinal juice and the two strains could cope with most bile
salts except GDCA. Possibly, GDCA may be toxic for these cells. When
their survival in gastrointestinal juice was tested, both strains were able
to survive 8 h of incubation. From their initial log CFU/ml count of
roughly 10.32, viable cells decreased about 3 log cycles for both
ENM104 (6.49 ± 0.00) and SPS109 (7.63 ± 0.02). This result was in
agreement with reports of other LAB such as Pediococcus, Lactobacillus
kefiri, Weissella sp. and Enterococcus durans (Mercha et al., 2020; Rata­
naburee et al., 2013b; Yusuf, Nuraida, Dewanti-Hariyadi, & Hunaefi,
2019). This suggests that both strains in the present study have potential
as probiotic candidates. Both selected strains are potential antioxidants
as they were tolerant to 1 mM H2O2 after 8 h compared with 0.4 mM for
other LAB (Shi et al., 2019). Tolerance to oxidative stress enables bac­
teria to survive and produce their beneficial properties when applied as
starters. They performed non-haemolytic. However, to confirm their
safety as starters, their entire genomes must be investigated to ensure
that there are no signs of virulence factors or antibiotic resistance genes
in their genomes. As cell surface hydrophobicity may supply to adhesion
of probiotics to host cells for maintenance in gastrointestinal tract;
however, probiotic LAB including commercial strains showed low per­
centages of cell hydrophobicity (this study, Ratanaburee et al., 2013b;
Huang et al., 2020). This suggests that our LAB strains might be po­
tential probiotics.
The cholesterol-lowering property of the two strains was evaluated
in 100 μg/ml cholesterol-fortified MRS and 100 μg/ml cholesterol-
fortified basal medium. The higher number of viable cells of ENM104
in cholesterol-fortified media at 72 h produced a cholesterol reduction
approximately two times greater in MRS broth than in basal medium.
This greater cholesterol reduction could be caused by a higher cell
proliferation in the enriched medium as not only could more cholesterol
be degraded but also more could be bio-accumulated. Significant
intracellular cholesterol oxidase activity was found in ENM104 after 48
h incubation. Therefore, the enzyme was generated in the stationary
phase as an inducible enzyme. Moreover, the period of enzyme activity
directly correlated with the period of cholesterol reduction (Table 3 and
Fig. 3C). Cholesterol oxidase activity indicates degradation of choles­
terol by the conversion of hydroxyl groups to carbonyl groups in 4-cho­
lesten-3-one. This strain produced no extracellular cholesterol oxidase,
which explains why it showed slow growth in the basal medium that
contained only cholesterol as a carbon source. The activity of ENM104
was lower than the activity of Lactobacillus helveticus CD6 (68 U/mg
protein) in 1 mg/ml cholesterol-fortified MRS (Ahire et al., 2012).
Possibly, the higher cholesterol concentration resulted in more
J. Jitpakdee et al. LWT 135 (2021) 110061

Fig. 3. Time course in milk using starters

P. pentosaceus ENM104 and L. plantarum SPS109
either in single cultures or a co-culture (1:1): (A)
pH, (B) LAB population, (C) cholesterol reduction
and (D) ACE inhibition. An asterisk (*) indicates
significant differences (p < 0.05), while NS in­
dicates no significant difference. The asterisks in the
pH and ACE inhibition indicate points of compari­
son between the control and other treatment sets. In
the LAB population, the asterisk indicates points of
comparison between single and co-culture sets,
while in the cholesterol reduction, the asterisk in­
dicates points of comparison among all sets.

72-h fermentation, the pH of the ENM104 single set had only slightly
Table 4
decreased as no lactose fermentation occurred based on the API 50 CHL
GABA production during milk fermentation by individual starters (P. pentosaceus
test. However, SPS109 did use lactose, as pH significantly decreased (p <
ENM104 and L. plantarum SPS109) and a co-culture (1: 1).
0.05) in both the single set SPS109 and the co-culture set (Fig. 3A).
Treatment Concentration (μg/ml) % Conversion
Modified MRS agar was used to distinguish both strains in the co-culture
efficiency
Glutamic acid GABA set and the dominant population in log CFU/ml was SPS109 (8.27 ±
Control 0h 43.39 ± 0.38 ab 4.39 ± 0.01 e 0 ± 0.00 e 0.04) rather than ENM104 (6.93 ± 0.01) (Fig. 3B). These figures re­
24 h 44.47 ± 0.40 a 4.41 ± 0.02 de 0.05 ± 0.12 de flected the rapid consumption of lactose as a carbon source for growth
48 h 41.89 ± 0.38 ab 4.40 ± 0.00 e 0.02 ± 0.03 e by SPS109, which resulted in the lower pH. Among all inoculated sets,
72 h 44.09 ± 0.39 a 4.42 ± 0.04 de 0.10 ± 0.09 de
only the ENM104 single set significantly reduced cholesterol content
ENM104 0h 43.39 ± 0.38 ab 4.39 ± 0.01 e 0 ± 0.00 e and there was no significant change of cholesterol in the control at 72 h
24 h 44.62 ± 0.37 a 4.47 ± 0.00 cd 0.26 ± 0.04 cd (Fig. 3C). This was supported by the higher population of ENM104 in the
48 h 42.29 ± 0.41 ab 4.49 ± 0.03 bc 0.32 ± 0.05 bc
72 h 43.75 ± 0.15 a 4.49 ± 0.02 bc 0.32 ± 0.04 bc
single culture set than the co-culture set. The results imply that strain
ENM104 used cholesterol as a carbon by producing cholesterol oxidase
SPS109 0h 43.36 ± 0.42 ab 4.39 ± 0.01 e 0 ± 0.00 e
to degrade cholesterol, and used casein to produce hydrophobic pep­
24 h 41.07 ± 2.18 abc 4.54 ± 0.02 ab 0.49 ± 0.02 ab
48 h 39.83 ± 2.06 bcd 4.53 ± 0.01 0.44 ± 0.06 abc tides, possibly to cope with cholesterol micelle to reduce cholesterol
abc content in fermented milk (Rendon-Rosales et al., 2019). Additionally,
72 h 37.66 ± 1.31 cd 4.58 ± 0.00 a 0.61 ± 0.05 a casein digestion by LAB may produce some bioactive peptides to support
Co- 0h 43.39 ± 0.38 ab
4.39 ± 0.01 e
0 ± 0.00 e inhibition of ACE (Hagi, Kobayashi, & Nomura, 2016), and this was
culture 24 h 40.91 ± 1.09 abc 4.51 ± 0.00 bc 0.38 ± 0.05 bc observed in all inoculated sets in this study. There was no significant
48 h 37.91 ± 0.92 cd 4.51 ± 0.00 bc 0.37 ± 0.04 bc change in the control (Fig. 3D). ACE inhibition significantly increased in
72 h 36.44 ± 0.95 d 4.52 ± 0.00 0.42 ± 0.05 abc
abc all inoculated milk samples (p < 0.05) compared with the control set,
and ACE inhibition was greater at 72 h fermentation in all fermented
Values are given as means ± standard deviation (n = 3). Different lowercase milk sets in a range of 52–58%. This finding was in accordance with a
letters in each column represent significant differences (p < 0.05). report that P. pentosaceus SDL1409 in fermented soybean produced low
molecular weight peptides (≤7 kDa) that showed ACE inhibition of 65.1
cholesterol oxidase production. ± 0.78% (Daliri et al., 2018). Therefore, the use of potent LAB strains as
Since both strains were able to utilise casein, they were used indi­ starters in the development of antihypertensive functional foods is not
vidually as starters for milk fermentation and also in a co-culture. After

6
J. Jitpakdee et al.
Fig. 4. Antioxidant activity of LAB detected by different methods: (A) ABTS, (B) DPPH and (C) ORAC. Data were obtained from fermentation of milk for 72 h using
starters of P. pentosaceus ENM104 and L. plantarum SPS109 as single cultures and in a co-culture (1:1). Asterisks (*) indicate significant differences (p < 0.05), while
NS indicates no significant difference. The asterisks in the DPPH and ORAC indicate points of comparison between the control and other treatment sets, while in the
ABTS, the asterisks and NS show points of comparison amongst sets.
without promise.
GABA content in all inoculated sets significantly increased, while no
significant change was found in the control (Table 4). This indicates that
the starters converted GA to GABA, as the highest GABA production
(4.57 ± 0.01 μg/ml) occurred in the SPS109 set that exhibited higher
levels of proteolysis. The GABA content in commercial soy milk con­
taining added GABA from germinated rice at 13.19 ± 2.32 μg/ml
(average of two brands sold in Thailand) was higher than all the fer­
mented milk samples in this study. However, increasing the GABA
content in fermented milk should be possible by adding MSG as a sub­
strate and pyridoxial-5′ -phosphate (PLP) as a cofactor of GAD (Die­
z-Gutiérrez et al., 2020). For instance, Lactococcus lactis L-571 produced
GABA at 1153.1 ± 13.5 μg/ml in milk supplemented with 3 mg/ml of
glutamate and 100 μM PLP (Santos-Espinosa et al., 2020). This indicates
that different genera of LAB and different substrates, including supple­
mentations, affected GABA content. It should be noted that in the pre­
sent study, the GABA conversion efficiency in fermented milk was much
lower than in MRS broth (Tables 1 and 4). This suggests that the key
factor in obtaining a high GABA conversion efficiency is GAD activity,
and GAD activity is affected by factors of pH, temperature, glutamic acid
and PLP (Cui, Miao, Niyaphorn, & Qu, 2020). In MRS broth and fer­
mented milk at similar pH and incubating temperatures, insufficient
glutamic acid was the main factor as its initial content was only 43
μg/ml in the fermented milk compared with 5 mg/ml MSG in the MRS
broth.
Antioxidant activity significantly increased in all inoculated sets
compared with the control set, indicating the roles of the starters in
producing antioxidants. It was noted that these results were similar to
the results obtained from MRS broth fermentation (data not shown). It
seems that ENM104 provided better results for antioxidant activity on
the basis of ABTS, DPPH and ORAC assays (Fig. 4A–C). This behaviour
reinforced the performance of the co-culture set. Recently, antioxidative
peptides were reported to increase antioxidant activity in goat’s milk
(Panchal, Hati, & Sakure, 2020; Panchal, Hati, Darji, & Prajapati, 2020).
This report suggests that proteolytic activity by both starters in cow’s
milk produced bioactive peptides, acting not only as ACE inhibitors but
also as antioxidants.
Overall results indicate that the most effective fermentation to pro­
duce functional milk was with the co-culture set of ENM104 and SPS109
that reduced cholesterol using cholesterol oxidase, bioaccumulation and
de-conjugation of bile salts. Also, their proteolytic activity led to
significantly increase ACE inhibition and antioxidant activities. How­
ever, GABA content should be improved in further research.
7
LWT 135 (2021) 110061
5. Conclusions
P. pentosaceus ENM104 and L. plantarum SPS109 isolated from Thai
fermented foods are probiotic candidates that respectively reduce
cholesterol and produce GABA. Both strains have great potential as
starters to produce functional fermented milk with lower cholesterol
content, higher GABA content, greater ACE inhibition, and increased
antioxidant activities.
CRediT authorship contribution statement
Jirayu Jitpakdee: Methodology, Validation, Formal analysis,
Investigation, Writing - original draft. Duangporn Kantachote:
Conceptualization, Methodology, Validation, Writing - review & editing,
Supervision. Hiroshi Kanzaki: Methodology, Formal analysis. Ter­
uhiko Nitoda: Methodology, Investigation.
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.
Acknowledgements
This work was supported by the Faculty of Science Research Fund,
Prince of Songkla University (PSU), Contract number 1-2559-02-016
and also by the Graduate School, PSU.
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