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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 ACCEPTED MANUSCRIPT
1 Moringa extract enhances the fermentative, textural, and bioactive properties of

2 yogurt

4 Ting Zhanga,1, Chang Hee Jeonga,1, Wei Nee Chenga, Hyojin Baea, Han Geuk Seoa,

5 Michael C. Petriellob, and Sung Gu Hana*

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

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8 University, Seoul 05029, Korea
b
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Department of Animal and Food Sciences, University of Kentucky, Lexington,

10 KY40536, USA

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14 Running Title: Bioactive properties of moringa-supplemented yogurt

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18 *Corresponding author: Sung Gu Han, PhD

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

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20 University, Seoul 05029, Korea

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

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22 These authors contributed equally to this work

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24 Abstract

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

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

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

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

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

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

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

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

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

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34 radical-scavenging activity of ME yogurt increased up to 40% in a dose-dependent
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35 manner during the 21 days of cold storage. Sensory testing showed that the addition of

36 0.05% ME to yogurt did not negatively influence the overall acceptability of the product,
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37 compared to the control. The addition of ME to yogurt decreased the oxidative stress

38 and increased the expression of antioxidant proteins in human colon cells. Thus, ME-
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39 fermented yogurt maintains the sensory acceptability and exerts positive health benefits
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40 because of increased LAB proliferation and enhanced antioxidant properties.

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41 Keywords: Moringa, Yogurt, Dairy products, Antioxidant activity, Lactic acid bacteria

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44 1. Introduction

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

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

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

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

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

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

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51 2013). Yogurt is a coagulated milk product obtained by lactic acid bacteria (LAB)

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

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

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decreasing cholesterol absorption, and reducing blood pressure) because of the high
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55 concentration of LAB (Arief & Taufik, 2016). In particular, Lactobacillus acidophilus

56 exerts probiotic effects such as enhancing lactose digestion, lowering serum cholesterol
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57 levels, and preventing cancer. Because of its multiple probiotic effects, L. acidophilus
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58 was reported to be utilized in dairy product, such as Minas cheese and dairy desert
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59 (Lollo et al., 2015; Moura et al., 2016). Streptococcus species was reported to improve

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

62 Moringa oleifera (moringa), a native Indian tree, has been used for medical use
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63 and food. Because almost every part of moringa contains bioactive components, it has
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64 been used as a herbal medicine and as a valuable food resource (Salem, Salama,

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

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

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

68 inflammatory components and contain antitoxic and antioxidant components (Salem,

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

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70 employed in various forms of traditional medicine. For example, moringa has been used

71 in traditional medicine to treat diseases and symptoms, including inflammation,

72 infectious diseases, cardiovascular, gastrointestinal, hematological, and hepatorenal

73 disorders (Sreelatha & Padma, 2009).

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

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

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

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

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

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

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80 moringa during yogurt fermentation.
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81 Thus, this study aimed to evaluate the bioactive properties and quality

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

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83 0.2%, w/v). The ME-supplemented yogurt was evaluated for (1) changes in pH and

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

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85 capacity (WHC), and color; (3) change in pH, viscosity, antioxidant activity, and total
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86 phenolic compound content during the 21 days of cold storage; (4) sensory properties;
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87 and (5) antioxidant effects on human colon cells.

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89 2. Materials and methods

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90 2.1. Materials

91 Skimmed milk powder for producing yogurt was purchased from Seoul Dairy

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

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

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

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

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96 Bile salts and M17 broth were purchased from Oxoid Ltd., (Basingstoke,

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

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

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

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

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

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

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

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

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

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106 Chemical and Metals Co., Ltd., (Siheung, Korea). RPMI-1640 medium was
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107 purchased from Lonza (Walkersville, USA). Fetal bovine serum (FBS) was

108 obtained from Atlas Biologicals (Ft. Collins, CO, USA). Phosphate-buffered saline
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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),
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111 NAD(P)H quinone dehydrogenase 1 (NQO1), glyceraldehyde 3-phosphate

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112 dehydrogenase (GAPDH), and goat anti-rabbit IgG-HRP were purchased from
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113 Santa Cruz Biotechnology (Santa Cruz, CA, USA).

114
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115 2.2. Preparation of water extracts from moringa leaf powder

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

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

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

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

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

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

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122 Co., Ltd., Tokyo, Japan) at 50°C, and the residue was freeze-dried and stored at -

123 80°C until use.

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125 2.3. Preparation of yogurt and yogurt supernatant

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

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127 al., 2015; Cruz et al., 2013). Briefly, Yogurt was produced by adding ME (0.05%, 0.1%,

128 and 0.2%, w/v) and skimmed milk powder (12%, w/v) to distilled water, followed by

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

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with the starter culture (7.06 log CFU/ml; 2.5%, v/v) containing S. thermophilus, L.

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

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132 reached 4.6, and then stored at 4ºC. Un-inoculated yogurt was used as the control. The
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133 samples were collected at 6 h post fermentation as well as at 1, 7, 14, and 21 days of

134 refrigerated storage. For preparing yogurt supernatant, 10 g of yogurt samples were
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135 centrifuged at 4,330 × g for 5 min at 4ºC, and then, the supernatants were re-centrifuged

136 at the same conditions. The supernatants were stored at -80ºC until use.
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137
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138 2.4. Kinetic parameters

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139 The kinetic parameters of acidification were estimated as described previously

140 (Jeong et al., 2018). The acidification rate (Vmax) was calculated as the time-dependent
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141 variation in pH (dpH/dt), and expressed as 10-3 pH units/min. At the end of

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142 fermentation, the following kinetic parameters were calculated: (1) tmax (h), time at

143 which Vmax was reached; (2) tpH 5.0 (h), the time required to reach a pH of 5.0; and (3) tf

144 (h), the time required to complete the fermentation.

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146 2.5. Color, pH, and viscosity measurements

147 The color parameters (L*, a*, and b*) of the yogurt samples were measured using a

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148 colorimeter (Konica Minolta, Inc., Tokyo, Japan) (Cappato et al., 2018). Different

149 values represent different colors: L*, darkness-lightness (0~100); a*, greenness-redness

150 (-60~60); b*, blueness-yellowness (-60~60). The pH value of the yogurt samples was

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

152 Schwerzenbach, Switzerland) at room temperature. The viscosity of yogurt samples was

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153 determined during the 21 days of cold storage and measured with a DV-E viscometer

154 (Brookfield, Toronto, Canada).

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155

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156 2.6. Microbial properties of yogurt during fermentation

157 Microbial count was determined after subsequent serial dilution using selected

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158 medium agar plates for LAB during fermentation (Batista et al., 2015). S. thermophilus
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159 was grown on M17 agar containing 0.5% lactose; L. acidophilus was grown on MRS

160 agar containing 0.15% filter-sterilized bile salts (0.1 g/mL); and B. longum was grown
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161 on MRS-LP agar [MRS agar supplied with 0.3% filter-sterilized lithium chloride,

162 0.05% filter-sterilized L-cysteine hydrochloride (0.1 g/mL), and 0.9% filter-sterilized
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163 sodium propionate]. LAB were enumerated on selective medium agar plates after
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164 aerobic (S. thermophilus and L. acidophilus)/anaerobic (B. longum) incubation at 37°C
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165 for 48 h. The number of colony-forming units (CFUs) on plates containing 30–300

166 colonies (0, 2, 4, and 6 h) was determined, and the number of cells was expressed as log
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167 CFU/mL of fermented milk.

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169 2.7. Determination of syneresis and WHC

170 Syneresis and WHC was measured as described previously with slight

171 modifications (Joung et al., 2016). About 10 g of yogurt sample was centrifuged at 500

172 × g for 4, 6, and 8 min, and then, the clear supernatant was poured off and weighed.

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174 2.8. Determination of radical scavenging activity and total phenolic content

175 DPPH and ABTS radical-scavenging activities were measured as described

176 previously (Jung et al., 2016). DPPH reagent (0.1 mM) was mixed with yogurt

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

178 temperature. DPPH reagent added in ethanol was served as a control. Calculation of

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179 DPPH scavenging activity was as follows:

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

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181 Abs is the absorbance read at 515 nm.

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ABTS reagent (14.8 mM) was mixed with 5 mM potassium persulfate (1:1, v/v) and left

183 in the dark for 16 h at room temperature. The ABTS+ solution was diluted with distilled

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184 water to absorbance 0.700 ± 0.05 at 734 nm before use. Samples of yogurt supernatant
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185 were mixed with ABTS+ solution in a 96-well plate and then allowed to react in the dark

186 for 15 min at room temperature. ABTS+ solution added in ethanol was served as a
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187 control. Calculation of ABTS+ scavenging activity was as follows:

188 ABTS+ scavenging activity (%) = [1 - (Abssample / Abscontrol)] × 100%,

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189 Abs is the absorbance read at 734 nm.

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190 The total phenolic content was determined using the Folin-Ciocalteau’s phenol reagent
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191 with some modifications (Cho et al., 2017). Briefly, 30 µL of yogurt extract diluted with

192 120 µL of distilled water was mixed with 30 µL of Folin-Ciocalteau’s phenol regent and
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193 30 µL of sodium carbonate (Na2CO3) in a 96-well plate, which was then left in the dark
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194 for 30 min at room temperature. The absorbance of the sample was read at 595 nm.

195 Gallic acid (10–60 µg/g) was used as the standard. The results were expressed as

196 micrograms of gallic acid equivalent (GAE) per milliliter.

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198 2.9. Sensory evaluation

199 Sensory analysis was used to evaluate the differences between the yogurt samples

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200 by 30 untrained panelists using a 9-point hedonic scale (Dantas et al., 2016). Panelist

201 ages ranged from 20 to 35 years old. Panelists were recruited from students majoring

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

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

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

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

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

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

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

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

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210 and 9 being “excellent”) for each yogurt sample. Sensory evaluation was conducted in
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211 individual booths to prevent rate score bias. Scores are presented as the mean ± standard

212 error. Yogurt was served in paper cups codified with three digits. The panelists used
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213 water to clean their palates between samples.

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215 2.10. Cell culture and protein expression

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216 The human colorectal cell line HT-29 was cultured in RPMI-1640 medium
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217 supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified

218 atmosphere containing 5% CO2 at 37°C. Cells were grown to 90% confluency and
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219 synchronized overnight in a medium containing 1% FBS before initiating treatment.

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220 Nrf2 and NQO1 expression was determined in HT-29 cells by using SDS-PAGE and

221 Western blotting, as described previously (Jeong, Seok, Petriello, & Han, 2017).

222 Briefly, cells were lysed in RIPA buffer with 50 mM Tris (pH 8.0), 150 mM NaCl, 1%

223 Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and a protein inhibitor mixture

224 (2 µg/mL aprotinin, 10 µg/mL leupeptin, 1 µg/mL pepstatin A, 1 mM PMSF, 5 mM

225 EDTA, 1 mM EGTA, 10 mM sodium fluoride and 1 mM sodium orthovanadate).

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226 Supernatants were collected after centrifuging lysed cells at 21,000 × g for 10 min at

227 4°C. Protein concentration was determined with PierceTM BCA Protein Assay Kit

228 (Thermo Scientific, Rockford, IL, USA). Protein samples (30 µg) were separated by

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

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

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

232 for 1.5 hour at room temperature and visualized using Pierce® ECL Western Blotting

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233 Substrate (Thermo Scientific, Rockford, IL, USA). The expressions of the proteins were

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quantified using ImageJ and normalized to GAPDH.

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236 2.11. Determination of antioxidant effects
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237 The intracellular presence of reactive oxygen species (ROS) was measured using

238 DCF-DA, a fluorescent dye. Briefly, HT-29 cells were grown until 70–80% confluency
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239 in six-well plates. The cells were pretreated with yogurt supernatant (100 µL/mL) for 16

240 h, followed by H2O2 (500 µM) for 2 h, to intentionally produce oxidative stress. This
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241 concentration of H2O2 was selected based on previous studies using HT-29 cells
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242 (Najgebauer-Lejko, Sady, Grega, & Walczycka, 2011). Cells were then incubated with
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243 DCF-DA (at a final concentration of 10 µM for 30 min). The six-well plates were

244 evaluated under an Olympus IX71 fluorescence microscope, and the images were
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245 digitally captured using an Olympus DP71 camera and DP controller software
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246 (Olympus Optical Co. Ltd, Tokyo, Japan).

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248 2.12. Statistical analysis

249 Data were expressed as the mean ± standard error of the mean (SEM). Statistical

250 significance was determined using the SPSS-PASW statistics software version 18.0 for

251 Windows (SPSS, Chicago, IL) by one-way ANOVA; the groups were compared using

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252 Tukey’s test. A probability value of <0.05 was considered statistically significant.

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254 3. Results and discussion

255 3.1. Acidity- and fermentation-related kinetic parameters during

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

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257 0.2%) was measured during fermentation. The yogurt was incubated at 42°C until

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

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

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260 significantly shorter at all ME concentrations, compared to the control yogurt (Fig.

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

262

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yogurt fermentation (Table 1). The addition of ME significantly accelerated yogurt
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263 fermentation in a concentration-dependent manner (P < 0.05). It increased Vmax in a

264 dose-dependent manner by 34% (0.05% ME), 39% (0.1% ME), and 48% (0.2%
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265 ME), compared to the control yogurt. The shortest Tmax was arrived within 2 h of
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266 fermentation (1.77–1.9 h), while the control sample took 2.63 h. The TpH 5.0 and Tf
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267 were shortened in ME-supplemented yogurt, particularly in case of 0.2% ME-

268 supplemented yogurt. The components present in moringa might be responsible for
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269 the enhanced fermentation activity. Moringa contains organic acids, phenolic acids,

270 and flavonoids (Rodríguez-Pérez, Mendiola, Quirantes-Piné, Ibáñez, & Segura-

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271 Carretero, 2016). These phytochemical components are considered to promote the
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272 growth of LAB and cause an accelerated drop in pH. In a previous study, yogurt

273 supplemented with herbs such as peppermint, dill, and basil increased the count of

274 LAB and contributed to the rapid drop in pH (Joung et al., 2016). Addition of green

275 tea powder also contributed to the accelerated drop in pH (Jeong et al., 2018).

276 These results indicate that the addition of moringa can enhance the metabolic

277 activities of LAB through potential prebiotic roles during yogurt fermentation.

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278

279 3.2. Growth of LAB during fermentation

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

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

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

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

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

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

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

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

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288 after 6 h (Fig. 2B). Like the two LAB, the growth of B. longum was highly increased in
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289 the yogurt supplemented with 0.2% ME, compared to control (Fig. 2C). At the end of

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

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

292 B. longum were significantly higher only in 0.2% ME-supplemented yogurt, compared
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293 to the control yogurt (P < 0.05). Similar findings have been reported in yogurts
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294 supplemented with plant substances. The addition of persimmon leaf powder and white
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295 mulberry leaf extracts to yogurt has shown to have increased the counts of S.

296 thermophilus and Lactobacillus during the 12 h of fermentation (Joung et al., 2016). In
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297 addition, cardamom-supplemented yogurt has showed increasing count of

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

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

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

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

302 acidophilus were above 106 CFU/mL, which is a standard requirement of viable LAB

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

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304 components of ME such as polyphenols. In fact, ME contains dietary polyphenolic

305 substances (Siddhuraju & Becker, 2003), which could have promoted the rate of

306 fermentation of LAB. The higher rate of metabolism and survival of LAB should be

307 associated with this accelerated fermentation. Similar to our results, yogurt

308 supplemented with soybean, which contains polyphenolic compounds, showed higher

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309 LAB counts, compared to the control yogurt, by enhancing the viability of probiotics

310 (Shori, 2013). In addition, polyphenols prevented potential spoilage by inhibiting the

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311 growth of spoilage microorganisms such as yeasts and molds during fermentation

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

313 effects by promoting the growth of LAB during yogurt fermentation.

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314
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315 3.3. Measurement of yogurt color

316 The color of ME-supplemented yogurt (0.05, 0.1 and 0.2%) was analyzed on day 1
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317 post fermentation (Table 2). The L*, a*, and b* values represent changes from darkness

318 to lightness, greenness to redness, and blueness to yellowness, respectively. No

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319 significant changes were observed in the L* value. The a* value significantly changed
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320 in 0.1–0.2% ME-supplemented yogurt, compared to the control. The b* value

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321 significantly changed from 9.83 to 15.5, depending on the concentration of ME.

322 Overall, the data showed decreased redness and increased yellowness in ME-
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323 supplemented yogurt without any changes in lightness.

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324

325 3.4. Syneresis and WHC of yogurt

326 The physical properties of yogurt, such as syneresis and WHC, are important

327 because these characteristics can limit the shelf-life and acceptability of products (Kiros

328 et al., 2016; Sidira et al., 2017). In the current study, both syneresis and WHC were

329 analyzed on day 1 post fermentation. Syneresis decreased up to 21% in 0.2% ME-

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330 supplemented yogurt (Fig. 3A). WHC increased up to 17% with the addition of ME in a

331 concentration-dependent manner (Fig. 3B). Syneresis is dependent on the WHC of

332 proteins; also, the type and content of proteins are important (Ünal & Akalin, 2013).

333 Actually, milk proteins can behave as a thickener and help increase the apparent

334 viscosity of yogurt (Srisuvor et al., 2013). Therefore, our results showed that ME

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335 supplementation of yogurt led to lower syneresis values and higher WHC, suggesting

336 improved viscosity. This could be attributed to some interactions between the

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337 components of ME and the proteins in the yogurt. The yogurt gel matrix seemed to

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increase by the addition of ME, thereby being able to hold more yogurt serum, as shown

339 by the WHC values obtained in the current study.

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340
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341 3.5. pH and viscosity of yogurt

342 During the 21-day-long cold storage at 4°C, the pH of each yogurt sample was
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343 markedly decreased (Fig. 4A). The pH was 4.46–4.47 at day 1 (Fig. 4A), which

344 consistently decreased to 4.18–4.20 during the 21-day storage period because of post-
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345 acidification of yogurt. Addition of ME did not influence the pH of the yogurt (Fig.
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346 4A). In previous studies, probiotic yogurt supplemented with spices such as cardamom,
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347 nutmeg, and cinnamon also showed a similar trend of pH changes during the storage

348 period (Illupapalayam et al., 2014). Our data indicate that the addition of ME to yogurt
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349 do not further influence the quality of yogurt during the 21 days of cold storage.
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350 Viscosity has a large impact on the quality of liquid and semisolid foods (Karaman

351 et al., 2014). Overall, the viscosity decreased along the storage period (Fig. 4B).

352 However, at certain time points, the ME-supplemented yogurt showed significantly

353 higher viscosity than the control yogurt (P < 0.05). In particular, the viscosity of 0.2%

354 ME-supplemented yogurt was approximately 5-fold higher than that of the control

355 yogurt during the storage period (Fig. 4B). The addition of fruit or plant substances

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356 often increases the viscosity of yogurt products. Similar to our data, the addition of

357 cupuassu to yogurt improved viscosity and texture during storage (Costa et al., 2015).

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

359 interactions. In addition, moringa contains abundant polyphenolic compounds, as shown

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

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

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

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

364

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

365 viscosity scores during sensory evaluation (Table 3).

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366
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367 3.6. Antioxidant activity and total phenolic contents of yogurt

368 The antioxidant activity of yogurt samples was determined using two different
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369 assays. ME-supplemented yogurt showed a significantly higher antioxidant activity,

370 compared to the control yogurt during all storage periods in both DPPH and ABTS
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371 radical-scavenging assays (Fig. 5A and B). ME-supplemented yogurt showed

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372 concentration-dependent antioxidant activity by both radical-scavenging assays. In

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373 particular, 0.2% ME-supplemented yogurt had the highest antioxidant activity

374 (73.32% ± 0.5% in DPPH and 86.29% ± 0.17% in ABTS assays), compared to the
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375 control yogurt (29.15% ± 1.74% in DPPH and 34.20% ± 0.28% in ABTS assays) at
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376 day 21 of storage. Therefore, it seems that ME-supplemented yogurt maintains a

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

378 The total phenolic content of ME supplemented yogurts was also significantly

379 higher than that of control yogurt during the 21 days of cold storage (Fig. 6). It

380 increased in the ME-supplemented yogurt in a concentration-dependent manner.

381 Addition of 0.2% ME showed up to 1.95-fold higher total phenolic content, compared

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382 to the control yogurt during 21 days. The higher total phenolic content was maintained

383 along the 21 days.

384 Taken together, the increased antioxidant activity of ME-supplemented yogurt was

385 probably because of the phenolic compounds from moringa leaf extract. In fact,

386 moringa leaves are known to be rich in bioactive components. For example, moringa

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387 contains quercetin and kaempferol, which are reported to exhibit strong antioxidant

388 properties (Tumer, Rojas-Silva, Poulev, Raskin, & Waterman, 2015; Wang et al., 2017).

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389 Thus, the higher antioxidant activity in ME-supplemented yogurt was most likely

390

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because of the herbal phytochemical content (e.g., phenolic compounds) of ME. These

391 data indicate that moringa is a good source for producing bioactive yogurt products.

392

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393 3.7. Sensory evaluation

394 The results of sensory evaluation, including sweetness, sourness, bitterness, flavor,
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395 acidic aroma, texture (feeling in the mouth), viscosity, and overall acceptability are
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396 listed in Table 3. Overall, the addition of ME resulted in increased bitterness, viscosity,
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397 and texture, but decreased overall acceptability, sweetness, sourness, flavor, and acidic

398 aroma (Table 3). In particular, the addition of ME to yogurt caused a significant
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399 decrease in the flavor score, compared to the control yogurt (P < 0.05). These results

400 were probably because of the bitter taste and herbal flavor of moringa. However, the
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401 addition of 0.05% ME to yogurt did not significantly influence the overall acceptability,
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402 compared to the control yogurt. Based on the sensory evaluation data obtained in this

403 study, 0.05% ME supplementation could be useful for the production of ME-

404 supplemented yogurt because this concentration showed improved quality

405 characteristics without generating significant negative sensory properties.

406

407 3.8. Antioxidant effects of yogurt on human cells

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408 Excessive ROS production can increase cellular oxidative stress, and damage the

409 cells and tissue, which can result in diseases such as inflammatory bowel disease (Jung,

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

411 culture studies as an indicator of oxidative stress, because of its characteristics such as

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

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

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

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415 supernatants for 16 h, followed by treatment with H2O2 (500 µM) for 2 h. The intensity

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416 of fluorescent light indicates the amount of H2O2 in cells. Cellular H2O2 level

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

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418 supernatant samples exert a strong antioxidant effect against H2O2-induced cellular
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419 oxidative stress. The antioxidant effects increased with increasing concentrations of ME

420 (Fig. 7A). Previous studies have shown that extracts from moringa leaves can decrease
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421 intracellular ROS production in human cells (Jung, 2014; Shori & Baba, 2013).
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422 Antioxidant derivatives such as bioactive polyphenols in moringa have attracted special

423
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attention because of their protective effects in the human body against oxidative stress

424 and related diseases (Singh et al., 2009; Sreelatha & Padma, 2009). It has been strongly
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425 suggested that the dairy matrix can enhance antioxidant activity by protecting the

426 integrity of polyphenols (Fernandez & Marette, 2017). Yogurt samples not
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427 supplemented with ME also decreased the level of cellular H2O2 (Fig. 7A). Microbial
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428 cells have many antioxidant defense mechanisms whose specific role is to remove or

429 inactivate any ROS to protect the biological system (Stecchini, Del Torre, & Munari,

430 2001). In fact, these data are in agreement with our radical scavenging activity results,

431 wherein the control yogurt also showed radical-scavenging activity (Fig. 5).

432 A cellular transcription factor, Nrf2, is activated in response to oxidative stress in

433 cells and animals. Upon binding to the antioxidant response element in the nucleus, the

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434 transcription of downstream target genes such as NQO1 can be increased, thereby

435 producing antioxidant effects (Han, Han, Toborek, & Hennig, 2012). To identify the

436 intracellular mechanisms associated with ME-supplemented yogurt, the expression of

437 Nrf2 and NQO1 in the human colorectal epithelial cells HT-29 was measured. Cells

438 were treated with yogurt for 18 h before harvesting whole cell lysate. Both Nrf2 and

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439 NQO1 were markedly increased by the treatment of cells with ME-supplemented yogurt

440 (Fig. 7B). The upregulation of Nrf2 and NQO1 seems to be more closely associated

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441 with ME components because the control yogurt showed only mild increase in the

442

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levels of these proteins. Previous studies have shown that the extracts of moringa leaf

443 and seed significantly elevated the expression of Nrf2 and its target gene NQO1 (Jaziri,

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444 Slama, Mhadhbi, Urdaci, & Hamdi, 2009; Jung et al., 2005). Taken together, ME-
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445 supplemented yogurt can provide beneficial health effects by the suppression of cellular

446 oxidative stress through increased expression of Nrf2 and NQO1 in human colorectal
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447 epithelial cells.

448
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449 4. Conclusion

450 This study showed that the addition of ME to yogurt can accelerate yogurt
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451 fermentation rate by promoting the growth of LAB. In addition, during the 21 days of

452 cold storage, ME-supplemented yogurt showed higher viscosity and free radical-
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453 scavenging activity, compared to the control group. Increased expression of the
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454 antioxidant proteins, namely, Nrf2 and NQO1, was shown in human colon cells. The

455 results of sensory acceptability of ME-supplemented yogurt showed no significant

456 negative effects, particularly in 0.05% ME yogurt, compared to the control yogurt. In

457 conclusion, ME-supplemented yogurt can provide positive health benefits by promoting

458 the proliferation of LAB and antioxidant properties without a significant sacrifice of

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459 sensory acceptability. This work highlights the usefulness of moringa to produce

460 bioactive yogurt products.

461

462 Acknowledgements

463 This study was supported by the Konkuk University in 2018.

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464

465 Conflict of interest

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466 The authors declare no conflicts of interest.

467

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468 References

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588 218, 152-158.

589

590

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591

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592 Figure Legends

593

594 Fig. 1. pH changes in the yogurt supplemented with 0–0.2% (v/v) moringa extract (ME)

595 during fermentation. Values are presented as the mean ± standard error of the mean

596 (SEM; n = 3). 0% ME, yogurt fermented without ME (control); 0.05% ME, yogurt

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

598 yogurt fermented with 0.2% ME

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599

600 Fig. 2. Growth of lactic acid bacteria during yogurt fermentation. Values are presented

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601 as the mean ± SEM (n = 3). Growth of (A) S. thermophilus, (B) L. acidophilus, and (C)

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602 B. longum during fermentation. *Significantly different compared to the control (P <
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603 0.05). 0% ME, yogurt fermented without ME (control); 0.05% ME, yogurt fermented

604 with 0.05% ME; 0.1% ME, yogurt fermented with 0.1% ME; 0.2% ME, yogurt
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605 fermented with 0.2% ME

606
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607 Fig. 3. Changes in the (A) syneresis and (B) water-holding capacity of the yogurt
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608 supplemented with 0–0.2% ME during fermentation. Values are presented as the mean ±
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609 SEM (n = 3). *Significantly different compared to the control (P < 0.05). 0% ME,

610 yogurt fermented without ME (control); 0.05% ME, yogurt fermented with 0.05% ME;
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611 0.1% ME, yogurt fermented with 0.1% ME; 0.2% ME, yogurt fermented with 0.2% ME
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612

613 Fig. 4. Changes in the (A) pH value and (B) viscosity of the yogurt supplemented with

614 0–0.2% ME during fermentation. Values are presented as the mean ± SEM (n = 3).

615 Different lowercase letters represent statistical difference observed on the same day (P <

616 0.05), whereas different uppercase letters represent statistical differences observed over

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

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

619 ME, yogurt fermented with 0.2% ME

620

621 Fig. 5. Antioxidant activity of yogurt supernatant. (A) DPPH and (B) ABTS radical-

622 scavenging activities of the yogurt supernatant supplemented with 0–0.2% ME during

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

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

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625 whereas different uppercase letters represent statistical differences observed during the

626

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

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

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628 ME, yogurt fermented with 0.2% ME
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629

630
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631 Fig. 6. Total phenolic content of the 0–0.2% ME-supplemented yogurt supernatant

632 during refrigerated storage. Values are presented as the mean ± SEM (n = 3). Different
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633 lowercase letters represent statistical differences observed on the same day (P < 0.05),
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634 whereas different uppercase letters represent statistical differences observed during the
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635 storage period (P < 0.05). 0% ME, yogurt fermented without ME (control); 0.05% ME,

636 yogurt fermented with 0.05% ME; 0.1% ME, yogurt fermented with 0.1% ME; 0.2%
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637 ME, yogurt fermented with 0.2% ME

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638

639

640 Fig. 7. Antioxidant effects of the yogurt supplemented with 0–0.2% ME in the human

641 colorectal cell line HT-29. (A) Effect of yogurt supernatant against H2O2-induced

642 cellular oxidative stress. Cells were pretreated with the yogurt supernatant (100 µL/mL)

643 for 16 h, followed by exposure to H2O2 (500 µM) for 2 h. Then, the cells were stained

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644 with DCF-DA to detect intracellular ROS production. The intensity of green

645 fluorescence was observed by fluorescence microscopy. (B) Effect of yogurt supernatant

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

647 supernatant (100 µL/mL) for 18 h. Western blotting was used to study protein

648 expression. Data represent the mean ± SEM (n = 3) of three independent experiments.

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

650 without ME; 0.05% ME, yogurt fermented with 0.05% ME; 0.1% ME, yogurt

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651 fermented with 0.1% ME; 0.2% ME, yogurt fermented with 0.2% ME

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652

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Table 1. Acidification kinetic parameters of yogurt during fermentation

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ME concentrations during yogurt fermentation

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Kinetic parameters 0% 0.05% 0.1% 0.2%

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

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Tmax (h) 2.63 ± 0.45a 1.77 ± 0.11b 1.90 ± 0.21ab 1.77 ± 0.17b

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TpH5.0 (h) 4.39 ± 0.30a 2.88 ± 0.06b 2.45 ± 0.02bc 2.16 ± 0.05c

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Tf (h) 5.34 ± 0.44a 4.12 ± 0.03b 3.57 ± 0.17bc 3.19 ± 0.11c

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Vmax = acidification rate (10-3 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.
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a-d
Different letters represent statistical differences in yogurts at the same time point (p < 0.05).
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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

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ME concentrations during yogurt fermentation

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Color value 0% 0.05% 0.1% 0.2%

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L* 89.74 ± 0.11a 89.67 ± 0.02a 89.65 ± 0.23a 89.23 ± 0.15a

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a* -7.99 ± 0.08a -8.20 ± 0.07a -8.51 ± 0.01b -8.77 ± 0.03c

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b* 9.83 ± 0.12d 11.43 ± 0.14c 13.20 ± 0.11b 15.5 ± 0.03a

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a-d
Different letters represent statistical differences in each color measurements (p < 0.05).

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

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Specific indicators 0% 0.05% 0.1% 0.2%

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

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

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

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

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

Texture TE
5.6 ± 1.65a 5.9 ± 1.59a 5.8 ± 1.35a 6.0 ± 1.83a
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Viscosity 5.0 ± 1.85a 5.7 ± 1.89a 5.7 ± 1.50a 6.0 ± 1.83a
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Overall acceptability 6.2 ± 1.77a 5.9 ± 1.15a 5.3 ± 1.70b 5.3 ± 1.83b
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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).

PT
Comparison groups: 0% ME, yogurt fermented without ME (control); 0.05% ME, yogurt fermented with 0.05% ME; 0.1% ME, yogurt ferme

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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.

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

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

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- ME yogurt maintains sensory acceptability and possesses antioxidant effects.

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