ACCEPTED MANUSCRIPT

Accepted Manuscript

Moringa extract enhances the fermentative, textural, and bioactive

properties of yogurt

Ting Zhang, Chang Hee Jeong, Wei Nee Cheng, Hyojin Bae, Han Geuk Seo, Michael
C. Petriello, Sung Gu Han

PII: S0023-6438(18)30961-7
DOI: https://doi.org/10.1016/j.lwt.2018.11.010
Reference: YFSTL 7574

To appear in: LWT - Food Science and Technology

Received Date: 21 June 2018

Revised Date: 29 October 2018
Accepted Date: 4 November 2018

Please cite this article as: Zhang, T., Jeong, C.H., Cheng, W.N., Bae, H., Seo, H.G., Petriello,
M.C., Han, S.G., Moringa extract enhances the fermentative, textural, and bioactive properties
of yogurt, LWT
- Food Science and Technology (2018), doi:
https://doi.org/10.1016/j.lwt.2018.11.010.

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 .Moringa extract enhance the fermentative, textural, and bioactive properties of yogurt

a,1 a,1 a a a
Ting Zhang , Chang Hee Jeong , Wei Nee Cheng , Hyojin Bae , Han Geuk Seo ,
b a
Michael C. Petriello , and Sung Gu Han *

a
Department of Food Science and Biotechnology of Animal Resources, Konkuk

University, Seoul 05029, Korea

b
Department of Animal and Food Sciences, University of Kentucky, Lexington,

KY40536, USA

Running Title: Bioactive properties of moringa-supplemented yogurt

*Corresponding author: Sung Gu Han, PhD

Department of Food Science and Biotechnology of Animal Resources, Konkuk

University, Seoul 05029, Korea

Tel: +82-2-450-0526; Fax: +82-2-455-1044; E-mail: hansg@konkuk.ac.kr

1
These authors contributed equally to this work

1
 ACCEPTED MANUSCRIPT

Abstract
Yogurt is a fermented dairy food produced by lactic acid bacteria (LAB). Moringa

oleifera is known for its bioactive properties. The aim of this study was to evaluate the

effects of moringa on the fermentation, quality characteristics, and bioactive properties

of yogurt. Yogurt was supplemented with 0–0.2% moringa extract (ME; hot water

extract, 100°C, 30 min) and fermented using mixed starter cultures (Streptococcus

thermophilus, Lactobacillus acidophilus, and Bifidobacterium longum). Addition of ME

to yogurt significantly accelerated the rate of fermentation by promoting growth of LAB.

ME reduced syneresis up to 21% and enhanced the water-holding capacity by 17%. The

viscosity of 0.2% ME yogurt was approximately 5-fold higher than control yogurt and

radical-scavenging activity of ME yogurt increased up to 40% in a dose-dependent

manner during the 21 days of cold storage. Sensory testing showed that the addition of

0.05% ME to yogurt did not negatively influence the overall acceptability of the product,

compared to the control. The addition of ME to yogurt decreased the oxidative stress

and increased the expression of antioxidant proteins in human colon cells. Thus, ME-

fermented yogurt maintains the sensory acceptability and exerts positive health benefits

because of increased LAB proliferation and enhanced antioxidant properties.

Keywords: Moringa, Yogurt, Dairy products, Antioxidant activity, Lactic acid bacteria
 ACCEPTED MANUSCRIPT
1. Introduction

Yogurt has excellent health benefits and high nutritional quality because of its

protein, lipid, vitamin, and mineral contents (Sidira et al., 2017). However, consumers

are concerned about the excessive intake of dietary fat because of its association with

diseases, including obesity, cancer, and diabetes (Ferrão et al., 2016). In developed

countries, skimmed milk has normally been used by yogurt manufacturers for producing

low-fat or non-fat yogurt (Srisuvor, Chinprahast, Prakitchaiwattana, & Subhimaros,

2013). Yogurt is a coagulated milk product obtained by lactic acid bacteria (LAB)

fermentation (Batista et al., 2015). It has a significant therapeutic value, and exerts

beneficial health effects (e.g., preventing intestinal disorders and chronic diseases,

decreasing cholesterol absorption, and reducing blood pressure) because of the high

concentration of LAB (Arief & Taufik, 2016). In particular, Lactobacillus acidophilus

exerts probiotic effects such as enhancing lactose digestion, lowering serum cholesterol

levels, and preventing cancer. Because of its multiple probiotic effects, L. acidophilus

was reported to be utilized in dairy product, such as Minas cheese and dairy desert

(Lollo et al., 2015; Moura et al., 2016). Streptococcus species was reported to improve

the nutritional contents of fermented products and digestibility in human gut (Jung et al., 2016).

Moringa oleifera (moringa), a native Indian tree, has been used for medical use

and food. Because almost every part of moringa contains bioactive components, it has

been used as a herbal medicine and as a valuable food resource (Salem, Salama,

Hassanein, & El Ghandour, 2013). Moringa leaves are a rich source of phenols,

proteins, minerals, and vitamins, as well as good sources of phytonutrients (Saini,

Sivanesan, & Keum, 2016). Moreover, the leaves have high amounts of anti-

inflammatory components and contain antitoxic and antioxidant components (Salem,

Salama, Hassanein, & El Ghandour, 2013). Therefore, the extracts from these leaves are
 ACCEPTED MANUSCRIPT

employed in various forms of traditional medicine. For example, moringan has been usedin
traditional medicine to treat diseases and symptoms, including inflammation,

infectious diseases, cardiovascular, gastrointestinal, hematological, and hepatorenal

disorders (Sreelatha & Padma, 2009).

In response to consumer demands and to improve the bioactive properties of dairy

products, yogurt is fortified with fruits, vegetables, or herbs during fermentation

(Chouchouli et al., 2013; Kiros, Seifu, Bultosa, & Solomon, 2016; Muniandy, Shori, &

Baba, 2016). In recent studies, moringa was added to dairy products such as cheese, and

the addition of moringa exerted health-promoting properties (Salem, Salama,

Hassanein, & El Ghandour, 2013). However, only few studies discuss the application of

moringa during yogurt fermentation.

Thus, this study aimed to evaluate the bioactive properties and quality

characteristics of yogurt supplemented with moringa extract (ME; 0.05%, 0.1%, or

0.2%, w/v). The ME-supplemented yogurt was evaluated for (1) changes in pH and

microbial properties during yogurt fermentation; (2) syneresis rate, water-holding

capacity (WHC), and color; (3) change in pH, viscosity, antioxidant activity, and total

phenolic compound content during the 21 days of cold storage; (4) sensory properties;

and (5) antioxidant effects on human colon cells.

2. Materials and methods

2.1. Materials

Skimmed milk powder for producing yogurt was purchased from Seoul Dairy

Cooperative (Seoul, Korea), and moringa leaf powder was purchased from

Philippine Moringa & More Corporation (Rizal, Philippine). The starter culture

powder for yogurt fermentation (Samik Dairy & Food Co. Ltd., Seoul, Korea)

contains S. thermophilus, Lactobacilllus acidophilus, and Bifidobacterium longum.

1
 ACCEPTED MANUSCRIPT
Bile salts and M17 broth w ere purchased from Oxoid Ltd.,
asingstoke,

Hampshire, England). Lactobacillus MRS broth was purchased from Becton

Dickinson and Company (Sparks, MD, USA). Lactose monohydrate was purchased

from Samchun Pure Chemical Co., Ltd., (Pyeongtaek, Korea). L-Cysteine

hydrochloride, lithium chloride, sodium propionate, 2,2-diphenyl-1-picrylhydrazyl

(DPPH), 2′,7′-dichlorofluorescein diacetate (DCFDA), 2,2'-azino-bis (3-

ethylbenzothiazoline-6-sulfonic acid) (ABTS), and Folin-Ciocaltaeu’s phenol

103

reagent were obtained from Sigma-Aldrich (St. Louis, MO, USA). Sodium

104

carbonate anhydrous was obtained from Shinyo Pure Chemical Co., Ltd., (Osaka,

105

Japan). Gallic acid and hydrogen peroxide (H2O2) was purchased from Daejung

106

Chemical and Metals Co., Ltd., (Siheung, Korea). RPMI-1640 medium was

107

purchased from Lonza (Walkersville, USA). Fetal bovine serum (FBS) was

108

obtained from Atlas Biologicals (Ft. Collins, CO, USA). Phosphate-buffered saline

109

(PBS), penicillin/streptomycin, and trypsin were purchased from Gibco (Grand

110

Island, NY, USA). Antibodies for nuclear factor erythroid 2-related factor 2 (Nrf2),

111
1
 NAD(P)H quinone dehydrogenase 1 (NQO1), glyceraldehyde 3-phosphate

112

dehydrogenase (GAPDH), and goat anti-rabbit IgG-HRP were purchased from

113

114

Santa Cruz Biotechnology (Santa Cruz, CA, USA).

11 2.2. Preparation of water extracts from moringa leaf powder

5

11 Boiled hot distilled water (100°C; 200 mL) was poured into a beaker
6

11 containing 10 g of moringa leaf powder, as described previously (Siddhuraju &

7

11 Becker, 2003). The beaker was covered using aluminum foil and brewed for 30 min
8

11 with stirring. The brewed mixture was cooled to 60°C, and filtered using a
9

12 Whatman No. 1 filter paper (GE Healthcare Life Sciences, Buckinghamshire, UK).
0

12 The filtered solvent was concentrated using a rotary evaporator (Tokyo Rikakikai
1

2
 ACCEPTED MANUSCRIPT
12 Co., Ltd., Tokyo, Japan) 50°C, and the residue was freeze-dried and stored at -
2
1 80°C until use.
2
3

1
2
4

1 2.3. Preparation of
2 yogurt and
5 yogurt
supernatant

1 Yogurt was
prepared as
previously
described with
minor
modifications
(Batista et

1 al., 2015; Cruz et al.,

2 2013). Briefly, Yogurt
7 was produced by
adding ME (0.05%,
0.1%,

1 and 0.2%, w/v) and

2 skimmed milk powder
8 (12%, w/v) to distilled
water, followed by

1 pasteurization at 85ºC
2 for 30 min.
9 Pasteurized yogurt
was cooled to 42ºC
and inoculated

1 with the starter culture

3 (7.06 log CFU/ml;
0 2.5%, v/v) containing
S. thermophilus, L.

1 acidophilus, and B.
3 longum. This
1 inoculated milk was
incubated at 42ºC
until the pH

1 32
1
r U e at 6 h post fermentation as well as at 1, 7, 14, and 21 ( , the time required to
e n d days of h complete the
a - ) fermentation.
c i 1 refrigerated storage. For preparing yogurt supernatant,
h n 3 10 g of yogurt samples were 1 2.5. Color, pH, and
e o 4 4 viscosity
d c measurements
u 6
1 centrifuged at 4,330 × g for 5 min at 4ºC, and then, the
4 la
. te 3 supernatants were re-centrifuged 1 The color
6 d 5 parameters (L*, a*,
, y and b*) of the
o 1 at the same conditions. The supernatants were stored at yogurt samples
a g 3 -80ºC until use. were measured
n u using a
6
d rt
w 1
t a 3
h s
e u 7
n s
1 2.4. Kinetic parameters
e
s d 3
t a 8
o s
r t 1 The kinetic parameters of acidification were
e h 3 estimated as described previously
d e
9
c
a o
1 (Jeong et al., 2018). The acidification rate (Vmax)
t n
tr 4
was calculated as the time-dependent variation in pH
4 o 0
º l.
C T 1 -3
(dpH/dt), and expressed as 10 pH units/min. At the
. h 4
e 1 end of

1 s 1 fermentation, the following kinetic parameters were

3 a 4 calculated: (1) tmax (h), time at
3 m 2
p
le 1 which Vmax was reached; (2) tpH 5.0 (h), the time
s 4 required to reach a pH of 5.0; and (3) tf
w
e 3
r
1 5
e
c 4
o 4
ll
e 1
ct 4
2
 ACCEPTED MANUSCRIPT
14 colorimeter (Konica ta, Inc., Tokyo, Japan) (Cappato al., 2018). Different
8 et
1 values represent
4 different colors: L*,
9 darkness-lightness
(0~100); a*, greenness-
redness

1 (-60~60); b*, blueness-

5 yellowness (-60~60).
0 The pH value of the
yogurt samples was

1 determined during
5 fermentation and
1 storage using a pH
meter (Mettler Toledo,

1 Schwerzenbach,
5 Switzerland) at room
2 temperature. The
viscosity of yogurt
samples was

1 determined during the

5 21 days of cold storage
3 and measured with a
DV-E viscometer

1 (Brookfield, Toronto,
5 Canada).
4

1
5
5

1 2.6. Microbial
5 properties of
6 yogurt during
fermentation

1 Microbial count was

determined after
subsequent serial
dilution using
selected

1 medium agar plates for

5 LAB during
8 fermentation (Batista et
al., 2015). S.
1
t p s was grown on MRS m fications (Joung et al.,
h h o 2016). About 10 g of
e il 1 agar containing 0.15% filter-sterilized bile salts (0.1 d yogurt sample was
r u 6 g/mL); and B. longum was grown i centrifuged at 500
m s 0
o 1 × g for 4, 6, and 8 min,
1 on MRS-LP agar [MRS agar supplied with 0.3% filter- 7 and then, the clear
1 w supernatant was poured
6 sterilized lithium chloride, 2
5 a off and weighed.
1
9 s
g 1
r 1 0.05% filter-sterilized L-cysteine hydrochloride (0.1
7
o 6 g/mL), and 0.9% filter-sterilized
w 3
2
n
o 1 sodium propionate]. LAB were enumerated on selective
n
6 medium agar plates after
M
1 3
7
a 1 aerobic (S. thermophilus and L. acidophilus)/anaerobic
g 6 (B. longum) incubation at 37°C
a 4
r
c 1 for 48 h. The number of colony-forming units (CFUs)
o
6 on plates containing 30–300
n
ta 5
i
n 1 colonies (0, 2, 4, and 6 h) was determined, and the
i 6 number of cells was expressed as log
n 6
g
0. 1 CFU/mL of fermented milk.
5 6
%
l 7
a
1
ct
o 6
s 8
e;
L 1 2.7. Determination of syneresis and WHC
. 6
a 9
c
i
d 1 Syneresis and WHC was measured as
o 7 described previously with
p slight
0
h
il
1 1
u
7
2
 ACCEPTED MANUSCRIPT
17 2.8. Determination of ical scavenging activity and phenolic content
4 total
1 DPPH and ABTS
radical-scavenging
activities were
measured as
described

1 previously (Jung et al.,

7 2016). DPPH reagent
6 (0.1 mM) was mixed
with yogurt

1 supernatant in a 96-
7 well plate and then
7 allowed to react in the
dark for 30 min at
room

1 temperature. DPPH
7 reagent added in
8 ethanol was served as
a control. Calculation
of

1 DPPH scavenging
7 activity was as
9 follows:

1 DPPH scavenging
8 activity (%) = [1 -
0 (Abssample /
Abscontrol)] × 100%,

1 Abs is the absorbance

8 read at 515 nm.
1

1 ABTS reagent (14.8

8
mM) was mixed with
2
5 mM potassium
1
8 persulfate (1:1, v/v)
3
and left in the dark

for 16 h at room

temperature. The

1
Ai w bsorbance 0.700 ± 0.05 at 734 nm before use. Samples m rograms of gallic acid
i equivalent (GAE) per
B l a
+ c milliliter.
of yogurt supernatant were mixed with ABTS
T u t
solution in a 96-well plate and then allowed to react in 1 2.9. Sensory
S t e 9 evaluation

r + 8
e the dark for 15 min at room temperature. ABTS
+
1 Sensory analysis
d solution added in ethanol was served as a control.
was used to
t evaluate the
+ differences between
Calculation of ABTS scavenging activity was as
o the yogurt samples
s w
follows:
o i +
ABTS scavenging activity (%) = [1 - (Abssample /
a
l t Abscontrol)] × 100%,

u h 1 Abs is the absorbance read at 734 nm.

8
t
9
i d
1 The total phenolic content was determined using the
o i 9 Folin-Ciocalteau’s phenol reagent
0
n s
t 1 with some modifications (Cho et al., 2017). Briefly, 30
9 µL of yogurt extract diluted with
wi 1

a l 1 120 µL of distilled water was mixed with 30 µL of

9 Folin-Ciocalteau’s phenol regent and
s l
2
e
1 30 µL of sodium carbonate (Na2CO3) in a 96-well
d d 9 plate, which was then left in the dark
1 8 3
8 7
1 for 30 min at room temperature. The absorbance of the
4
9 sample was read at 595 nm.
1
1 8 4
8 8
5 1 Gallic acid (10–60 µg/g) was used as the standard. The
9 results were expressed as
1 5
8
6 1
197
9
1 6
2
 ACCEPTED MANUSCRIPT
20 by 30 untrained panelists using a 9-point hedonic scale as et al., 2016). Panelist
0
20 ages ranged from 20 to 35 years old. Panelists were recruited from students
1 majoring

20 Food Sciences in Konkuk University (Seoul, Korea). Thirty panelists (12 male
2 and 18

20 female) were divided into 3 groups (12 panelists with aged 20-24; 13 panelists
3 with

20 aged 25-30; 5 panelists with aged 31-35). Specifically, for the quantification of
4 the

20 descriptive attributes of the yogurt samples, an equal amount of sucrose (2%) was
5 added

20 to the yogurt to reduce the bitterness of ME. Specific indicators, i.e., sweetness,
6

20 sourness, bitterness, flavor, acidic aroma, texture (feeling in the mouth), viscosity,
7 and

20 overall acceptability, were also estimated. Panelists were asked to rate the samples
8 by

20 using the words “low” (1–3), “medium” (4–6), and “high” (7–9) (with 1 being
9 “bad”

21 and 9 being “excellent”) for each yogurt sample. Sensory evaluation was
0 conducted in

21 individual booths to prevent rate score bias. Scores are presented as the mean ±
1 standard

21 error. Yogurt was served in paper cups codified with three digits. The panelists
2 used

21 water to clean their palates between samples.

3

21
4

21 2.10. Cell culture and protein expression

5

21 The human colorectal cell line HT-29 was cultured in RPMI-1640 medium
6
1
 d
2 s
1 u 21 atmosphere containing 5% CO2 at 37°C. Cells were grown to 90% confluency
7 p 8 and
p
le 21 synchronized overnight in a medium containing 1% FBS before initiating
m 9 treatment.
e
n 22 Nrf2 and NQO1 expression was determined in HT-29 cells by using SDS-PAGE
te
d 0 and
w
22 Western blotting, as described previously (Jeong, Seok, Petriello, & Han, 2017).
it
h 1
1
0 22 Briefly, cells were lysed in RIPA buffer with 50 mM Tris (pH 8.0), 150 mM
% 2 NaCl, 1%

F 22 Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and a protein inhibitor
B 3 mixture
S
a 22 (2 µg/mL aprotinin, 10 µg/mL leupeptin, 1 µg/mL pepstatin A, 1 mM PMSF, 5
n
4 mM
d
1
22 EDTA, 1 mM EGTA, 10 mM sodium fluoride and 1 mM sodium orthovanadate).
%
p 5
e
n
ic
il
li
n
/s
tr
e
p
t
o
m
y
ci
n
i
n
a
h
u
m
i
d
if
ie
2
 ACCEPTED MANUSCRIPT
22 Supernatants were collecte 1,000 × g for 10 min at 4°C.
6 d after centrifuging lysed cells at
TM
Protein concentration was determined with Pierce BCA Protein Assay Kit
22
7
22 (Thermo Scientific, Rockford, IL, USA). Protein samples (30 µg) were separated
8 by

22 SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were

9

23 blocked with 3% non-skim milk buffer and incubated with primary antibody for
0

23 overnight at 4°C. After washing, membranes were incubated with secondary

1
®
23 antibody for 1.5 hour at room temperature and visualized using Pierce ECL
2
Western Blotting

23 Substrate (Thermo Scientific, Rockford, IL, USA). The expressions of the

3 proteins were

23 quantified using ImageJ and normalized to GAPDH.

4

23
5

23 2.11. Determination of antioxidant effects

6

23 The intracellular presence of reactive oxygen species (ROS) was measured

7 using

23 DCF-DA, a fluorescent dye. Briefly, HT-29 cells were grown until 70–80%
8 confluency

23 in six-well plates. The cells were pretreated with yogurt supernatant (100 µ L/mL)
9 for 16

24 h, followed by H2O2 (500 µM) for 2 h, to intentionally produce oxidative stress.

0 This

24 concentration of H2O2 was selected based on previous studies using HT-29 cells
1

24 (Najgebauer-Lejko, Sady, Grega, & Walczycka, 2011). Cells were then incubated
2 with

1
2 D re
4 C
24 evaluated under an Olympus IX71 fluorescence microscope, and the images were
3 F
- 4
D
A 24 digitally captured using an Olympus DP71 camera and DP controller software
( 5
at
a 24 (Olympus Optical Co. Ltd, Tokyo, Japan).
fi 6
n
al 24
c
7
o
n 24 2.12. Statistical analysis
c
e 8
n
tr 24 Data were expressed as the mean ± standard error of the mean (SEM).
at 9 Statistical
i
o 25 significance was determined using the SPSS-PASW statistics software version
n 0 18.0 for
o
f 25 Windows (SPSS, Chicago, IL) by one-way ANOVA; the groups were compared
1 1 using
0
µ
M
f
o
r
3
0
m
i
n
).
T
h
e
si
x
-
w
el
l
p
la
te
s
w
e
2
 value of <0.05 was considered
ACCEPTED MANUSCRIPT
25 Tukey’s test. A probability tistically significant.
2
25 3. Results and discussion
25 4
3

25 3.1. Acidity- and fermentation-related kinetic parameters during

5

25 fermentation The pH of the yogurt supplemented with ME (0%, 0.05%, 0.1%,

6 and

25 0.2%) was measured during fermentation. The yogurt was incubated at 42°C until
7

25 the pH dropped to 4.6. Supplementation with ME did not affect the initial pH of
8 the

25 yogurt, which ranged from 6.25 to 6.40 (Fig. 1). Fermentation time was
9

26 significantly shorter at all ME concentrations, compared to the control yogurt

0 (Fig.

26 1). Additionally, acidification-related kinetic parameters were monitored during

1

26 yogurt fermentation (Table 1). The addition of ME significantly accelerated

2 yogurt

26 fermentation in a concentration-dependent manner (P < 0.05). It increased Vmax

3 in a

26 dose-dependent manner by 34% (0.05% ME), 39% (0.1% ME), and 48% (0.2%
4

26 ME), compared to the control yogurt. The shortest Tmax was arrived within 2 h
5 of

26 fermentation (1.77–1.9 h), while the control sample took 2.63 h. The TpH 5.0
6 and Tf

26 were shortened in ME-supplemented yogurt, particularly in case of 0.2% ME-

7

26 supplemented yogurt. The components present in moringa might be responsible

8 for

26 9
1
t gu ez-Pérez, Mendiola, Quirantes-Piné, Ibáñez, & Segura-
h M
e o 27 Carretero, 2016). These phytochemical components are considered to promote
ri 1 the
e n
n g 27 growth of LAB and cause an accelerated drop in pH. In a previous study, yogurt
h a 2
a c
n o 27 supplemented with herbs such as peppermint, dill, and basil increased the count
c n
e ta 3 of
d i
n 27 LAB and contributed to the rapid drop in pH (Joung et al., 2016). Addition of
f s 4 green
e o
r r 27 tea powder also contributed to the accelerated drop in pH (Jeong et al., 2018).
mg 5
e a
n n 27 These results indicate that the addition of moringa can enhance the metabolic
t ic 6
a a
t ci 27 activities of LAB through potential prebiotic roles during yogurt fermentation.
i d
7
o s,
n p
h
a e
c n
t o
i li
v c
i a
t ci
y d
. s,

2 a
7 n
0 d
fl
a
v
o
n
o
i
d
s
(
R
o
d
rí
2
 ACCEPTED MANUSCRIPT
278

27 3.2. Growth of LAB during fermentation

9

28 Yogurt was inoculated with the starter culture (2.5%) containing S. thermophilus, L.
0

28 acidophilus, and B. longum, and fermented at 42°C for 6 h. The viable cell counts of S.
1

28 thermophilus and L. acidophilus were 7.78–8.88 and 5.81–7.35 log CFU/mL (0.2%
2

28 ME), respectively (Fig. 2A and Fig. 2B). The viable cell counts of S. thermophilus were
3

28 dynamically higher at 2 h in 0.1–0.2% ME-supplemented yogurt compared to the

4

28 control, and 0.2% ME-supplemented yogurt achieved the highest viable LAB count at 6
5

28 h (Fig. 2A). The viable cell count of L. acidophilus was higher at 4 h in ME-
6

28 supplemented samples, and continued to remain higher, compared to the control yogurt,
7

28 after 6 h (Fig. 2B). Like the two LAB, the growth of B. longum was highly increased in
8

28 the yogurt supplemented with 0.2% ME, compared to control (Fig. 2C). At the end of
9

29 fermentation, the growth of L. acidophilus was significantly higher in ME-

0

29 supplemented yogurt (0.05%, 0.1%, and 0.2%), and the growth of S. thermophilus and
1

29 B. longum were significantly higher only in 0.2% ME-supplemented yogurt, compared

2

29 to the control yogurt (P < 0.05). Similar findings have been reported in yogurts
3

29 supplemented with plant substances. The addition of persimmon leaf powder and white
4
1
29 mulberry leaf extracts to yogurt has shown to have increased the counts of S.
5

29 thermophilus and Lactobacillus during the 12 h of fermentation (Joung et al., 2016). In

6

29 addition, cardamom-supplemented yogurt has showed increasing count of

7

29 Bifidobacterium during storage (Illupapalayam, Smith, & Gamlath, 2014). In fact, to

8

29 obtain the desired bioactive effects in human body, the probiotic bacteria should be
9

30 available in sufficient numbers. Our data showed that ME accelerates the growth of
0

30 LAB during yogurt fermentation. In particular, the counts of S. thermophilus and L.

1
6
30 acidophilus were above 10 CFU/mL, which is a standard requirement of viable
2
LAB

30 count in dairy products. The increased growth of LAB can be attributed to the
3

2
 ACCEPTED MANUSCRIPT
30 components of ME such polyphenols. In fact, ME dietary polyphenolic
4 contains
3 substances
0 (Siddhuraju & Becker,
5 2003), which could
have promoted the rate
of

3 fermentation of LAB.
0 The higher rate of
6 metabolism and
survival of LAB
should be

3 associated with this

0 accelerated
7 fermentation. Similar
to our results, yogurt

3 supplemented with
0 soybean, which
8 contains polyphenolic
compounds, showed
higher

3 LAB counts,
0 compared to the
9 control yogurt, by
enhancing the viability
of probiotics

3 (Shori, 2013). In
1 addition, polyphenols
0 prevented potential
spoilage by inhibiting
the

3 growth of spoilage
1 microorganisms such
1 as yeasts and molds
during fermentation

3 (Georgakouli et al.,
1 2016). Overall, our
2 data indicate that
moringa extract has
prebiotic

3 314
1
3

1
 e w 3 The color of ME-supplemented yogurt (0.05, 0.1 3 et al., 2016; Sidira et
f t 1 and 0.2%) was analyzed on day 1 2 al., 2017). In the
f h current study, both
e o 6 8
syneresis and WHC
c f were
t L 3 post fermentation (Table 2). The L*, a*, and b* values
s A 1 represent changes from darkness
3 analyzed on day 1 post
B 7 2 fermentation.
b d Syneresis decreased
9
y u 3 to lightness, greenness to redness, and blueness to up to 21% in 0.2%
ri 1 yellowness, respectively. No ME-
p n
8
r g
o y
3 significant changes were observed in the L* value. The
m o
o g 1 a* value significantly changed
t u 9
i rt
n f 3 in 0.1–0.2% ME-supplemented yogurt, compared to the
g e 2 control. The b* value
r
0
t m
h e
3 significantly changed from 9.83 to 15.5, depending on
e n
ta 2 the concentration of ME.
g ti 1
r o
o n. 3 Overall, the data showed decreased redness and
2 increased yellowness in ME-
3 M
3.3. 2
1 e
5 a 3 supplemented yogurt without any changes in lightness.
s
u 2
r 3
e
m 3
e 2
n 4
t
o 3 3.4. Syneresis and WHC of yogurt
f 2
y
5
o
g
3 The physical properties of yogurt, such as syneresis
u
r 2 and WHC, are important
t 6
c
o 3 because these characteristics can limit the shelf-life and
l 2 acceptability of products (Kiros
o 7
r
2
 ACCEPTED MANUSCRIPT
33 supplemented yogurt 3A). WHC increased up to 17% with the addition of ME in a
0 (Fig.
3 concentration-dependent manner (Fig. 3B). Syneresis is
3 dependent on the WHC of
1

3 proteins; also, the type and content of proteins are

3 important (Ünal & Akalin, 2013).
2

3 Actually, milk proteins can behave as a thickener and

3 help increase the apparent
3

3 viscosity of yogurt (Srisuvor et al., 2013). Therefore,

3 our results showed that ME
4

3 supplementation of yogurt led to lower syneresis values

3 and higher WHC, suggesting
5

3 improved viscosity. This could be attributed to some

3 interactions between the
6

3 components of ME and the proteins in the yogurt. The

3 yogurt gel matrix seemed to
7

3 increase by the addition of ME, thereby being able to

3 hold more yogurt serum, as shown
8

3 by the WHC values obtained in the current study.

3
9

3
4
0

3 3.5. pH and viscosity of yogurt

4
1

3 42

1
D w hich v iscosity of 0.2%

3 m 3 consistently decreased 3 ME-supplemented yogurt was approximately 5-fold

4 a 4 to 4.18–4.20 during the 5 higher than that of the control
3 r 4 21-day storage period 4
k because of post-
e
3 yogurt during the storage period (Fig. 4B). The addition
d 3 acidification of yogurt.
Addition of ME did 5
l of fruit or plant substances
4
y
5 not influence the pH of 5
d the yogurt (Fig.
e
c 3 4A). In previous
r 4 studies, probiotic
e yogurt supplemented
6
a with spices such as
s cardamom,
e
d 3 nutmeg, and cinnamon
( 4 also showed a similar
F trend of pH changes
7
i during the storage
g.
4 3 period (Illupapalayam
A 4 et al., 2014). Our data
). indicate that the
8
T addition of ME to
h yogurt
e
p 3 do not further
H 4 influence the quality of
w yogurt during the 21
9
a days of cold storage.
s
4. 3 Viscosity has a
4 large impact on the
6 quality of liquid
– and semisolid foods
4 (Karaman
.4
7 3 et al., 2014). Overall,
at 5 the viscosity decreased
d along the storage
1
a period (Fig. 4B).
y
1 3 However, at certain
( 5 time points, the ME-
F supplemented yogurt
2
i showed significantly
g.
4 3 higher viscosity than
A 5 the control yogurt (P <
), 0.05). In particular, the
3
2
 ACCEPTED MANUSCRIPT
35 often increases the viscosity of yogurt products. Similar tor data, the addition of
6
35 cupuassu to yogurt improved viscosity and texture during storage (Costa et al.,
7 2015).

35 The authors found that increased viscosity of yogurt was due to protein-
8 polyphenol

35 interactions. In addition, moringa contains abundant polyphenolic compounds, as

9 shown

36 by the total phenolic content assay (Fig. 6). Because these polyphenols possess a
0

36 significant affinity to proteins, it can form complexes with milk proteins such as
1 casein

36 (Vital et al., 2015). This complex can contribute to the observed decrease in
2 syneresis

36 and increase in the WHC of ME-supplemented yogurt (Fig. 3A and B). Therefore,
3 our

36 viscosity data suggest that ME supplementation can contribute to good texture and
4

36 viscosity scores during sensory evaluation (Table 3).

5

36
6

36 3.6. Antioxidant activity and total phenolic contents of yogurt

7

36 The antioxidant activity of yogurt samples was determined using two different
8

36 assays. ME-supplemented yogurt showed a significantly higher antioxidant

9 activity,

37 compared to the control yogurt during all storage periods in both DPPH and
0 ABTS

37 radical-scavenging assays (Fig. 5A and B). ME-supplemented yogurt showed

1

37 concentration-dependent antioxidant activity by both radical-scavenging assays.

2 In
1
 cti vity
3 p
7 a 37 (73.32% ± 0.5% in DPPH and 86.29% ± 0.17% in ABTS assays), compared to the
3 rt 4
ic
u 37 control yogurt (29.15% ± 1.74% in DPPH and 34.20% ± 0.28% in ABTS assays)
la 5 at
r,
0. 37 day 21 of storage. Therefore, it seems that ME-supplemented yogurt maintains a
2
% 6

M 37 significantly higher antioxidant activity during the 21-day-long cold storage.

E 7
-
s 37 The total phenolic content of ME supplemented yogurts was also
u 8 significantly
p
p 37 higher than that of control yogurt during the 21 days of cold storage (Fig. 6). It
le 9
m
e 38 increased in the ME-supplemented yogurt in a concentration-dependent manner.
n
0
te
d
38 Addition of 0.2% ME showed up to 1.95-fold higher total phenolic content,
y
o 1 compared
g
u
rt
h
a
d
t
h
e
h
i
g
h
e
st
a
n
ti
o
x
i
d
a
n
t
a
2
 ACCEPTED MANUSCRIPT
38 to the control yogurtg 21 days. The higher totalc content was
2 durin phenoli

3 along the 21 days.

8
3

3 Taken together, the increased antioxidant activity of

8 ME-supplemented yogurt was
4

3 probably because of the phenolic compounds from

8 moringa leaf extract. In fact,
5

3 moringa leaves are known to be rich in bioactive

8 components. For example, moringa
6

3 contains quercetin and kaempferol, which are reported

8 to exhibit strong antioxidant
7

3 properties (Tumer, Rojas-Silva, Poulev, Raskin, &

8 Waterman, 2015; Wang et al., 2017).
8

3 Thus, the higher antioxidant activity in ME-

8 supplemented yogurt was most likely
9

3 because of the herbal phytochemical content (e.g.,

9 phenolic compounds) of ME. These
0

3 data indicate that moringa is a good source for

9 producing bioactive yogurt products.
1

3
9
2

3 3.7. Sensory evaluation

9
3

1
3 T i mproved quality
h 3 acidic aroma, texture
e 9 (feeling in the mouth), 4 characteristics without generating significant negative
5 viscosity, and overall 0 sensory properties.
r acceptability are 5
e
s 3 listed in Table 3. 4
u 9 Overall, the addition of 0
l 6 ME resulted in
t increased bitterness, 6
s viscosity,
4 3.8. Antioxidant effects of yogurt on human cells
o 3 and texture, but 0
f 9 decreased overall 7
s 7 acceptability,
e sweetness, sourness,
n flavor, and acidic
s
o 3 aroma (Table 3). In
r 9 particular, the addition
y 8 of ME to yogurt
caused a significant
e
v 3 decrease in the flavor
a 9 score, compared to the
l 9 control yogurt (P <
u 0.05). These results
a
t 4 were probably because
i 0 of the bitter taste and
o 0 herbal flavor of
n moringa. However, the
,
i 4 addition of 0.05% ME
n 0 to yogurt did not
c 1 significantly influence
l the overall
u acceptability,
d
i 4 compared to the
n 0 control yogurt. Based
g 2 on the sensory
evaluation data
s obtained in this
w
e 4 study, 0.05% ME
e 0 supplementation could
t 3 be useful for the
n production of ME-
e
s 4 supplemented yogurt
s 0 because this
, 4 concentration showed
2
 ACCEPTED MANUSCRIPT
40 Excessive ROS production can increase cellular e stress, and damage the
8
40 cells and tissue, which can result in diseases such as inflammatory bowel
9 disease (Jung,

41 Nam, & Park, 2005). The levels of ROS such as H2O2 have been measured
0 in cell

41 culture studies as an indicator of oxidative stress, because of its

1 characteristics such as

41 high stability and permeability through the cell membrane (Lee, Choi, & Bae,
2 2013). In

41 the current study, human colorectal epithelial cells HT-29 were intentionally
3 exposed to

41 H2O2 to induce cellular oxidative stress. HT-29 cells were pretreated with
4 yogurt

41 supernatants for 16 h, followed by treatment with H2O2 (500 M) for 2 h.

5 The intensity

41 of fluorescent light indicates the amount of H2O2 in cells. Cellular H2O2

6 level

41 significantly decreased in the cells pretreated with yogurt extracts (Fig. 7A).
7 All yogurt

41 supernatant samples exert a strong antioxidant effect against H2O2-induced

8 cellular

41 oxidative stress. The antioxidant effects increased with increasing

9 concentrations of ME

42 (Fig. 7A). Previous studies have shown that extracts from moringa leaves can
0 decrease

42 intracellular ROS production in human cells (Jung, 2014; Shori & Baba,
1 2013).

42 Antioxidant derivatives such as bioactive polyphenols in moringa have

2 attracted special

42 attention because of their protective effects in the human body against

3 oxidative stress

42 and related diseases (Singh et al., 2009; Sreelatha & Padma, 2009). It has
4 been strongly
1
 ot ecting the
4 s
2 u 42 integrity of polyphenols (Fernandez & Marette, 2017). Yogurt samples not
5 g 6
g
e 42 supplemented with ME also decreased the level of cellular H2O2 (Fig. 7A).
st 7 Microbial
e
d 42 cells have many antioxidant defense mechanisms whose specific role is to
t
h 8 remove or
at
42 inactivate any ROS to protect the biological system (Stecchini, Del Torre, &
t
h 9 Munari,
e
d 43 2001). In fact, these data are in agreement with our radical scavenging
ai 0 activity results,
r
y 43 wherein the control yogurt also showed radical-scavenging activity (Fig. 5).
m 1
at
ri 4 A cellular transcription factor, Nrf2, is activated in response to oxidative
x
3 stress in
c
a 2
n
e 43 cells and animals. Upon binding to the antioxidant response element in the
n 3 nucleus, the
h
a
n
c
e
a
n
ti
o
x
i
d
a
n
t
a
ct
i
v
it
y
b
y
p
r
2
43
4 transcription of downstrea ACCEPTED MANUSCRIPT
e increased,
m targetthereby
genes such as
NQO1 can b

4 producing antioxidant effects (Han, Han, Toborek, &

3 Hennig, 2012). To identify the
5

4 intracellular mechanisms associated with ME-

3 supplemented yogurt, the expression of
6

4 Nrf2 and NQO1 in the human colorectal epithelial cells

3 HT-29 was measured. Cells
7

4 were treated with yogurt for 18 h before harvesting

3 whole cell lysate. Both Nrf2 and
8

4 NQO1 were markedly increased by the treatment of

3 cells with ME-supplemented yogurt
9

4 (Fig. 7B). The upregulation of Nrf2 and NQO1 seems to

4 be more closely associated
0

4 with ME components because the control yogurt

4 showed only mild increase in the
1

4 levels of these proteins. Previous studies have shown

4 that the extracts of moringa leaf
2

4 and seed significantly elevated the expression of Nrf2

4 and its target gene NQO1 (Jaziri,
3

4 Slama, Mhadhbi, Urdaci, & Hamdi, 2009; Jung et al.,

4 2005). Taken together, ME-
4

4 supplemented yogurt can provide beneficial health

4 effects by the suppression of cellular
5

4 46
1
 o x u sion
x p
i r 4 This study showed
d e that the addition of
a s ME to yogurt can
t si accelerate yogurt
i o
v n 4 fermentation rate by
e o 5 promoting the growth
f of LAB. In addition,
s N 1
during the 21 days of
t rf
r 2 4 cold storage, ME-
e a 5 supplemented yogurt
s n showed higher
s d 2
viscosity and free
N radical-
t Q
h O 4 scavenging activity,
r 1 5 compared to the
o i control group.
u n 3
Increased expression
g h of the
h u
m 4 antioxidant proteins,
i a 5 namely, Nrf2 and
n n NQO1, was shown in
c c 4
human colon cells.
r o The
e l
a o 4 results of sensory
s r 5 acceptability of ME-
e e supplemented yogurt
d ct 5
showed no significant
al
e 4 negative effects,
5 particularly in 0.05%
4 e ME yogurt, compared
6
4 p to the control yogurt.
7 it In
h
4
el 4 conclusion, ME-
ia 5 supplemented yogurt
4 l
7 can provide positive
8 c
health benefits by
el promoting
ls
. 4 the proliferation of
5 LAB and antioxidant
8 properties without a
4.4 C
significant sacrifice of
4 o
9 n
cl
2
 ACCEPTED MANUSCRIPT
45 sensory acceptability. work highlights the usefulness oforinga to produce
9 This m
4 bioactive yogurt
6 products.
0

4
6
1

4 Acknowledgements
6
2

4 This study was

6 supported by the
3 Konkuk University in
2018.
4
6
4

4 Conflict of interest
6
5

4 The authors declare

6 no conflicts of
6 interest.

4
6
7

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yoghurt.
58 International Journal of Dairy Technology, 66(2), 264-272.
1
58 Vital, A. C. P., Goto, P. A., Hanai, L. N., Gomes-da-Costa, S. M., de Abreu Filho, B. A.,
2 Nakamura, C. V.,
58 et al. (2015). Microbiological, functional and rheological properties of low fat yogurt
3
58 supplemented with Pleurotus ostreatus aqueous extract. LWT-Food Science and
4 Technology,

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585 64(2), 1028-1035.
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58 Wang, Y., Gao, Y., Ding, H., Liu, S., Han, X., Gui, J., et al. (2017). Subcritical ethanol
6 extraction of
58 flavonoids from Moringa oleifera leaf and evaluation of antioxidant activity. Food
7 Chemistry,
58 218, 152-158.
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58
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59 Figure Legends
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59
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59 Fig. 1. pH changes in the yogurt supplemented with 0–0.2% (v/v) moringa extract (ME)
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59 during fermentation. Values are presented as the mean ± standard error of the mean
5

59 (SEM; n = 3). 0% ME, yogurt fermented without ME (control); 0.05% ME, yogurt
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59 fermented with 0.05% ME; 0.1% ME, yogurt fermented with 0.1% ME; 0.2% ME,
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59 yogurt fermented with 0.2% ME

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59
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60 Fig. 2. Growth of lactic acid bacteria during yogurt fermentation. Values are presented
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60 as the mean ± SEM (n = 3). Growth of (A) S. thermophilus, (B) L. acidophilus, and (C)
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60 B. longum during fermentation. *Significantly different compared to the control (P <

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60 0.05). 0% ME, yogurt fermented without ME (control); 0.05% ME, yogurt fermented
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60 with 0.05% ME; 0.1% ME, yogurt fermented with 0.1% ME; 0.2% ME, yogurt
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60 fermented with 0.2% ME

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60
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60 Fig. 3. Changes in the (A) syneresis and (B) water-holding capacity of the yogurt
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60 8
su pplemented with 0–0.2% ME during fermentation. Values are presented as the mean ±

60 SEM (n = 3). *Significantly different compared to the control (P < 0.05). 0% ME,
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61 yogurt fermented without ME (control); 0.05% ME, yogurt fermented with 0.05% ME;
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61 0.1% ME, yogurt fermented with 0.1% ME; 0.2% ME, yogurt fermented with 0.2% ME
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61
2

61 Fig. 4. Changes in the (A) pH value and (B) viscosity of the yogurt supplemented with
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61 0–0.2% ME during fermentation. Values are presented as the mean ± SEM (n = 3).
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61 Different lowercase letters represent statistical difference observed on the same day (P <
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61 0.05), whereas different uppercase letters represent statistical differences observed over
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61 the storage period (P < 0.05). 0% ME, yogurt fermented without ME (control); 0.05%
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61 ME, yogurt fermented with 0.05% ME; 0.1% ME, yogurtmented
fer with 0.1% ME; 0.2%
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61 ME, yogurt fermented with 0.2% ME
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62
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62 Fig. 5. Antioxidant activity of yogurt supernatant. (A) DPPH and (B) ABTS radical-
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62 scavenging activities of the yogurt supernatant supplemented with 0–0.2% ME

2 during

62 refrigerated storage. Values are presented as the mean ± SEM (n = 3). Different
3

62 lowercase letters represent statistical differences observed on the same day (P <
4 0.05),

62 whereas different uppercase letters represent statistical differences observed during

5 the

62 storage period (P < 0.05). 0% ME, yogurt fermented without ME (control); 0.05%
6 ME,

62 yogurt fermented with 0.05% ME; 0.1% ME, yogurt fermented with 0.1% ME; 0.2%
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62 ME, yogurt fermented with 0.2% ME

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62
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63
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63 Fig. 6. Total phenolic content of the 0–0.2% ME-supplemented yogurt supernatant

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63 during refrigerated storage. Values are presented as the mean ± SEM (n = 3).
2 Different

63 lowercase letters represent statistical differences observed on the same day (P <
3 0.05),

63 whereas different uppercase letters represent statistical differences observed during

4 the

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 ; 0.05% ME,
6 st
3 o 63 yogurt fermented with 0.05% ME; 0.1% ME, yogurt fermented with 0.1% ME; 0.2%
5 r 6
a
g 63 ME, yogurt fermented with 0.2% ME
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e 63
ri
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d 63
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64 Fig. 7. Antioxidant effects of the yogurt supplemented with 0–0.2% ME in the
0.
0 0 human
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). 64 colorectal cell line HT-29. (A) Effect of yogurt supernatant against H2O2-induced
0 1
%
64 cellular oxidative stress. Cells were pretreated with the yogurt supernatant (100
M 2 µL/mL)
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, 64 for 16 h, followed by exposure to H2O2 (500 µM) for 2 h. Then, the cells were
y 3 stained
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64 with DCF-DA to detect intracellular ROS production. Theensity of green
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64 fluorescence was observed by fluorescence microscopy. (B) Effect of yogurt
5 supernatant

64 on the expression of Nrf2 and NQO1 in cells. The cells were treated with the
6 yogurt

64 supernatant (100 µL/mL) for 18 h. Western blotting was used to study protein
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64 expression. Data represent the mean ± SEM (n = 3) of three independent

8 experiments.

64 Different letters represent statistical difference (P < 0.05). 0% ME, yogurt

9 fermented

65 without ME; 0.05% ME, yogurt fermented with 0.05% ME; 0.1% ME, yogurt
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65 fermented with 0.1% ME; 0.2% ME, yogurt fermented with 0.2% ME
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65
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Table 1. Acidification kinetic parameters of yogurt during fermentation

ME concentrations during yogurt fermentation

Kinetic parameters 0% 0.05% 0.1% 0.2%

-3 b a a a
Vmax (10 pH 7.61 ± 0.71 10.20 ± 0.11 10.57 ± 0.98 11.24 ± 0.75

units/min) Tmax (h) a b ab b

2.63 ± 0.45 1.77 ± 0.11 1.90 ± 0.21 1.77 ± 0.17

TpH5.0 (h) a b bc c
4.39 ± 0.30 2.88 ± 0.06 2.45 ± 0.02 2.16 ± 0.05
Tf (h)
a b bc c
5.34 ± 0.44 4.12 ± 0.03 3.57 ± 0.17 3.19 ± 0.11

-3
Vmax = acidification rate (10 pH units/min); Tmax = time at which Vmax was reached; TpH5.0 = time to reach pH 5.0; and Tf = time to
complete the fermentation.

a-d
Different letters represent statistical differences in yogurts at the same time point (p < 0.05).

Comparison groups: 0% ME, yogurt fermented without ME (control); 0.05% ME, yogurt fermented with 0.05% ME; 0.1% ME, yogurt
ferme nted with 0.1% ME; 0.2% ME, yogurt fermented with 0.2% ME.
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Table 2. The color value of yogurts at day 1

ME concentrations during yogurt fermentation

Color value 0% 0.05% 0.1% 0.2%

L* a a a a
89.74 ± 0.11 89.67 ± 0.02 89.65 ± 0.23 89.23 ± 0.15
a*
a a b c
-7.99 ± 0.08 -8.20 ± 0.07 -8.51 ± 0.01 -8.77 ± 0.03
b*
d c b a
9.83 ± 0.12 11.43 ± 0.14 13.20 ± 0.11 15.5 ± 0.03
a-d
Different letters represent statistical differences in each color measurements (p < 0.05).

L*, darkness-lightness (0~100); a*, greenness-redness (−60~60); b*, blueness-yellowness (−60~60).

Comparison groups: 0% ME, yogurt fermented without ME (control); 0.05% ME, yogurt fermented with 0.05% ME; 0.1% ME, yogurt
ferme nted with 0.1% ME; 0.2% ME, yogurt fermented with 0.2% ME.
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Table 3. Sensory evaluation of yogurts at day 1

ME concentrations during yogurt fermentation

Specific indicators 0% 0.05% 0.1% 0.2%

Sweetness a a a a
5.0 ± 1.95 4.9 ± 1.67 4.2 ± 1.66 4.4 ± 1.72

Sourness a a a a
5.4 ± 2.11 4.9 ± 1.81 4.4 ± 1.23 4.8 ± 1.94

Bitterness a a a a
4.5 ± 2.70 4.4 ± 2.23 4.4 ± 1.96 5.2 ± 1.99

Flavor a b b b
6.2 ± 1.66 5.2 ± 1.88 4.6 ± 1.68 4.5 ± 1.94

Acidic aroma a a a a
5.2 ± 2.16 4.8 ± 1.68 4.6 ± 1.80 4.9 ± 2.30

Texture a a a a
5.6 ± 1.65 5.9 ± 1.59 5.8 ± 1.35 6.0 ± 1.83

Viscosity a a a a
5.0 ± 1.85 5.7 ± 1.89 5.7 ± 1.50 6.0 ± 1.83

Overall acceptability a a b b
6.2 ± 1.77 5.9 ± 1.15 5.3 ± 1.70 5.3 ± 1.83
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a.d Different letters represent statistical differences in yogurts at the same time point (p < 0.05).

The scale of sweetness, sourness, bitterness, flavor, acidic aroma, texture, viscosity and overall acceptability scores was as follows:
“low”(1-3), “medium”(4-6) and “high”(7-9).

Comparison groups: 0% ME, yogurt fermented without ME (control); 0.05% ME, yogurt fermented with 0.05% ME; 0.1% ME, yogurt
ferme nted with 0.1% ME; 0.2% ME, yogurt fermented with 0.2% ME.
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Highlights

- Addition of moringa extract (ME) to yogurt accelerated fermentation rate.

- ME reduced syneresis and enhanced the water-holding capacity of yogurt.

- Viscosity and radical-scavenging activity increased in ME yogurt over 21 days.

- ME increased the expression of antioxidant proteins in human intestinal cells.

- ME yogurt maintains sensory acceptability and possesses antioxidant effects.