Composition, antioxidant, and anti-biofilm activity of anthocyanin-rich aqueous extract from purple highland barley bran

Journal Pre-proof Composition, antioxidant, and anti-biofilm activity of anthocyanin-rich aqueous extract from purple highland barley bran Yongzhu Zhang, Yanfei Lin, Lu Huang, Mekonen Tekliye, Hafiz Abdul Rasheed, Mingsheng Dong PII:

S0023-6438(20)30169-9

DOI:

https://doi.org/10.1016/j.lwt.2020.109181

Reference:

YFSTL 109181

To appear in:

LWT - Food Science and Technology

Received Date: 11 August 2019 Revised Date:

14 February 2020

Accepted Date: 15 February 2020

Please cite this article as: Zhang, Y., Lin, Y., Huang, L., Tekliye, M., Rasheed, H.A., Dong, M., Composition, antioxidant, and anti-biofilm activity of anthocyanin-rich aqueous extract from purple highland barley bran, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/ j.lwt.2020.109181. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

1

Composition, antioxidant, and anti-biofilm activity of anthocyanin-rich aqueous extract

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from purple highland barley bran

3

Yongzhu Zhanga, Yanfei Lina, Lu Huangb, Mekonen Tekliyea, Hafiz Abdul Rasheeda,

4

Mingsheng Donga*

5

a

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Nanjing, Jiangsu 210095, PR China

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b

8

Province, China.

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*Corresponding author:

College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang Road,

Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nan Jing, Jiangsu

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Tel: +86 25 84396989

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Fax: +86 25 84399090

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E-mail address: [email protected]

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Abbreviations

14

LC, liquid chromatography; MS, mass spectrometry; PHBB, purple highland barley bran;

15

DPPH,

16

2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; FRAP, ferric reducing

17

antioxidant power; RP, reducing powder; Vc, ascorbic acid; ACE, anthocyanin crude extract;

18

AAE, anthocyanin-rich aqueous extract; TAC, total anthocyanin content; UPLC,

19

ultra-performance liquid chromatography; MIC, minimal inhibitory concentration; CLSM,

20

confocal laser scanning microscopy; DAPI, 2-(4-amidinophenyl)-6-indolecarbamidine

21

dihydrochloride.

2,2-diphenyl-1-picrylhydrazyl;

TPTZ,

1

2,4,6-tris(2-pyridyl)-s-triazine;

ABTS,

22

Abstract

23

Anthocyanin-rich cereal grains have attracted considerable attention recently due to their

24

health benefits in humans. In this work, purple highland barley bran (PHBB) from the Tibetan

25

Plateau in China was evaluated. Results showed that the anthocyanins in PHBB were easily

26

extracted by water in 10 min, and demonstrated good stability under high extraction

27

temperature. The extracts were further purified by AB-8 resin for composition and antioxidant

28

analysis, and the concentration factor increased by about 62-fold in the final purified powder.

29

Anthocyanins in PHBB were analyzed by liquid chromatography-mass spectrometry

30

(LC-MS), and findings showed a complex and high acylated anthocyanin profile with

31

cyaniding malonyl glucoside making up 73.50±3.49% of the total anthocyanin content (TAC)

32

for the first time. Anthocyanin-rich aqueous extract exhibited exceptional antioxidant

33

capacities and remarkable anti-biofilm properties. Therefore, this study strongly suggested

34

that the extracts of PHBB could be used as a high-quality natural food colorant and functional

35

ingredient.

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Keywords: Cereal grain; Stability; LC-MS; Acylated anthocyanin profile; Functional

37

ingredient

2

38

Introduction

39

Anthocyanins are a group of water-soluble pigments and widely distributed secondary

40

metabolites in plants. Many different groups of anthocyanidins exist in nature, such as

41

pelargonidin, peonidin, cyanidin and delphinidin (Kong et al., 2003). According to previous

42

studies (Li, Bao, & Wang, 2011; Bishayee et al., 2016), anthocyanins are beneficial to human

43

health because of their anti-oxidant, anti-cancer, anti-aging, and anti-inflammation effects.

44

The daily intake of anthocyanins in the diet of one adult is approximately 12.5 mg in the

45

United States (Wu et al., 2006). Fruits and vegetables are common good sources of

46

anthocyanins. Blueberry is well known for its high antioxidant activity, which is mainly

47

dependent on the abundance of total anthocyanins (Hosseinian & Beta, 2007). Red cabbage

48

also shows a high level of anthocyanins (390.6 mg/L; Chandrasekhar et al., 2012). In recent

49

years, interest in anthocyanin-rich grains has grown. Colored wheat consists of functional

50

food ingredients due to its high anthocyanins content (Abdel-Aal, Hucl, & Rabalski, 2018).

51

Both the anthocyanin-rich extracts of purple corn and red rice have been used as functional

52

raw material and natural colorant (Siebenhandl et al., 2007). Colored barley germplasm,

53

including hulled and unhulled barley, was also found to own a high level of anthocyanins,

54

which mainly existed in the bran fraction (Kim et al., 2007; Van Hung, 2016). Nevertheless,

55

compared with colored rice and wheat, purple highland barley in China has drawn less

56

attention by the scientific community due to cultural eating habits.

57

Highland barley, known as Qingke in China, is the main staple food crop in the

58

Qinghai-Tibet Plateau, which is located at high altitudes of 4200-4500 m above sea level (Liu

59

et al., 2013). It is applied as an important food crop for human consumption, as brewing

60

material, and as food source (Yang et al., 2013). Bran of highland barley, which is considered

61

waste in the area of the Qinghai-Tibet Plateau, is often produced as a by-product of milling in

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the production of refined grains. Early research mainly focused on the health benefits of 3

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bioactive phytochemical components in highland barley, such as phenolic compounds and

64

fiber components, especially β-glucan (Du, Zhu, & Xu, 2014; Zhu, Du, & Xu, 2015; Liu et

65

al., 2018). In recent years, growing attention has been paid to purple highland barley due to its

66

abundant anthocyanin content in the bran (Van Hung, 2016). However, the composition of

67

anthocyanins and its contribution to the health benefits of purple highland barley also remain

68

poorly understood.

69

In this study, the anthocyanin composition in PHBB was identified by LC-MS. The

70

antioxidant activities and anti-biofilm properties were quantified, and the anthocyanins in

71

PHBB were extracted using only acid water for the first time. Several key factors of water

72

extraction, such as solid-liquid ratio, extraction duration, extraction temperature, pH,

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ultrasonic power, and extraction times, were investigated to achieve a high anthocyanin yield.

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This study aimed to encourage the reuse and consumption of purple barley bran and explore

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the potential uses of anthocyanin in PHBB as a natural functional food ingredient or colorant.

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1. Materials and methods

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2.1. Chemicals

78

The

standards

of

cyaniding-3,5-di-glucoside,

cyaniding-3-galactoside,

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pelargonidin-3-glucoside, and peonidin-3-glucoside were purchased from Polyphenols

80

Laboratories

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2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)

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diammonium salt (ABTS), ascorbic acid (Vc), and 2-(4-amidinophenyl)-6-indolecarbamidine

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dihydrochloride (DAPI) were all purchased from Sigma-Aldrich Chemical Co. (St. Louis,

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MO, USA). All other chemicals and reagents were of analytical grade and purchased from

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Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

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2.2. Bacterial strains and culture conditions

(Sandens,

Norway).

2,2-diphenyl-1-picrylhydrazyl

4

(DPPH),

87

The test organism Pseudomonas aeruginosa PAO1 and Salmonella enterica ATCC10398

88

were supplied by Jiangsu Collaborative Innovation Center of Meat Production and

89

Processing, Quality and Safety Control, Jiangsu Province, China. Bacteria were first cultured

90

at 37 ℃ for 24 h. Cells were allowed to expand under the same condition for 14 h and used to

91

determine biofilm formation.

92

2.3. Preparation of PHBB

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Purple highland barley used in the present study was from the Tibetan Plateau (provided

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by Tibet Tianhe Industrial Limited by Share Ltd, China). The bran fraction for the extraction

95

of anthocyanins was prepared by a TM-5 laboratory-scale pearler (Satake Corp., Hiroshima,

96

Japan). The purple barley was pearled to remove about 30% of their outer kernel layers by

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machine. The bran fraction passed through a 50-mesh to remove small flour particles and

98

stored at −20℃ for further analysis.

99

2.4. Determination of variables of anthocyanin extraction

100

According to previously published report (Silva et al., 2017), several key independent

101

factors were selected to determine the optimum extraction condition for the anthocyanins in

102

PHBB by varying a single parameter at a time while the others remained constant. The

103

single-factor experiments were conducted as follows: solid-liquid ratio (1/10-1/60 g/mL) with

104

fixed extraction duration (30 min), temperature (40 °C), pH (3.0), ultrasound power (200 W),

105

and extraction times (1 time); extraction duration (10-150 min) with fixed solid-liquid ratio

106

(1/40 g/mL), temperature (40 °C), pH (3.0), ultrasonic power (200 W), and extraction times (1

107

time); temperature (30°C-90 °C) with fixed solid-liquid ratio (1/40 g/mL), extraction duration

108

(10 min), pH (3.0), ultrasonic power (200 W), and extraction times (1 time); pH (0.5-7.0) with

109

fixed solid-liquid ratio (1/40 g/mL), extraction duration (10 min), temperature (80 °C),

110

ultrasonic power (200 W), and extraction times (1 time); Ultrasonic power (200-450 W) with

111

fixed solid-liquid ratio (1/40 g/mL), extraction duration (10 min), temperature (80 °C), pH 5

112

(1.0), and extraction times (1 time); extraction times (1-3 times) with fixed solid-liquid ratio

113

(1/40 g/mL), extraction duration (10 min), temperature (80 °C), pH (1.0), and ultrasonic

114

power (200 W). Concentrated hydrochloric acid (HCl) was used to adjust pH. After obtaining

115

the optimum condition for anthocyanin extraction, the bran was extracted and then the

116

mixture was centrifuged at 14, 940

117

anthocyanin crude extract (ACE). ACE was further purified by AB-8 resin and freeze-dried,

118

affording anthocyanin-rich aqueous extract (AAE) for further study. In this study, the color of

119

the supernatant and residue of the PHBB was also observed after anthocyanin extraction

120

under the optimum condition.

121

2.5. Determination of TAC

g and 4 ℃ for 20 min. The supernatant was obtained as

122

The anthocyanin crude extract (ACE) was used to determine the TAC in PHBB with the

123

method described by Ryu and Koh (2018). TAC in the extract was calculated using the

124

equation below:

125

TAC (mg/100 g) = (A M DF V 105) / (ɛ L W)

126

where A is the difference in the absorbance (A520 nm pH 1.0 − A700 nm pH 1.0) − (A520 nm

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pH 4.5 − A700

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DF is the dilution factor; V is the total volume of ACE (mL); W is the dry weight of purple

129

highland barley bran (mg); L is the optical path length (1 cm); ε is the molar absorptivity of

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cyanidin-3-glucoside (26,900 L/mol

131

2.6. Identification of anthocyanin compounds with LC-MS

nm

pH 4.5); M is the molecular weight of cyanidin-3-glucoside (449.2 g/mol);

cm).

132

The identity of the composition of AAE was carried out by the LC-MS system (G2-XS

133

QTof, Waters, USA). Separation was achieved on an ACQUITY UPLC BEH C18 column (2.1

134

mm 100 mm, 1.7 µm, USA). Elution was performed with a flow rate of 0.4 mL/min using

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the following gradient of buffer A (0.1% formic acid in water) and buffer B (0.1% formic acid

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in acetonitrile): 0-0.5 min, 5% buffer B; 0.5-15.5 min, 5%-95% buffer B; 15.5-17.5 min, 95% 6

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buffer B. 2 µL of the sample was injected into the UPLC column. The standard compounds

138

were used to quantify the different anthocyanins based on the peak areas and retention times.

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MS was carried out using an electrospray source in negative ion mode with MSe acquisition

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mode, scanning from the mass (m/z) 50-1200. The key parameters of ionization were as

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follows: capillary voltage, 2.5 kV; sample cone, 40 V; source temperature, 120 °C; and

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desolvation gas temperature, 400 °C. Leucine-enkephalin (m/z 556.2771) was used to enable

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the lock mass for recalibration. Data were collected and processed using Masslynx 4.1.

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2.7. Measurements of antioxidant activities

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Freeze-dried AAE was dissolved in 80% methanol at different concentration (3.91-4000

146

µg/mL) for antioxidant assays. The antioxidant capacity was evaluated by four assays

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described in our previous work, ABTS radical cation scavenging assay, FRAP assay, reducing

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power assay (RP) and DPPH radical scavenging assay (Xiao et al., 2015). Vc was used as a

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positive control.

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2.8. Effects of AAE on biofilm development

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2.8.1. Determination of minimal inhibitory concentration of AAE

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The minimal inhibitory concentration (MIC) of AAE against the tested pathogens S.

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enterica ATCC10398 and P. aeruginosa PAO1 was determined by the method recommended

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by the Clinical and Laboratory Standards Institute, USA (2006). In brief, 1% of tested

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pathogens (0.6 OD600 nm) were inoculated to LB medium supplemented with AAE to attain the

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final concentrations ranging from 0.125 mg/mL to 8 mg/mL and incubated at 37 ℃ for 48 h.

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The biomass (total viable cell density) of the tested pathogens was measured by the plate

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count method. The MIC was the lowest concentration of AAE, which showed the complete

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inhibition of visible growth of the tested strain (Zhang et al., 2014). All further experiments

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were performed only at sub-MIC of AAE.

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2.8.2. Anti-biofilm properties of AAE 7

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The effect of AAE on biofilm formation was quantified with the method described by

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Zhang et al. (2014) with minor modifications. In brief, 1% of an overnight culture of tested

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bacteria (0.4 OD600 nm) was inoculated to LB medium supplemented with a series of different

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AAE concentrations (0.125, 0.5, 1, 2, and 4 mg/mL). 200 µL of the medium above was added

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to each well of 96-well plates. The plates were kept at 37 ℃ for 24 h without shaking. After

167

incubation, 50 µL of suspension culture was removed for the determination of total biomass,

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and then the tested pathogen cells, which not attached to the biofilm in the well, were

169

removed by deionized water for three times. Methanol was used to remove the anthocyanins

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attached to the biofilm surface and avoid detachment of the biofilm during the staining and

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rinsing steps (Ommen, Zobek, & Meyer, 2017). The biofilms were stained with 225 µL of 1%

172

crystal violet per well for 10 min and washed three times by deionized water to remove excess

173

dye. The dye attached to biofilm was solubilized with 200 µL of 95% ethanol. The biomass of

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biofilm was represented as the OD measured at 595 nm.

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2.9. In-situ visualization of biofilm

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The biofilms were observed under a CLSM (Leica TCS SP8; Leica Microsystems,

177

Heidelberg, Germany) as described previously (Singh et al., 2017). First, 1% of an overnight

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culture of the tested bacteria was inoculated to fresh LB medium supplemented with or

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without AAE (4 mg/mL). Second, about 2 mL of cell suspensions was dispensed into the

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wells of a 6-cell plate containing a glass slide (1 1 cm). After 48 h of incubation at 37 ℃, the

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glass slides were carefully washed with PBS and stained with 1 µg/mL DAPI. Stained glass

182

slides were washed with PBS to remove excess stain and then observed by CLSM.

183

2.10. Statistical analysis

184

All the experiments were performed in triplicate, and measurements were reported as the

185

mean ± standard deviation (SD). Statistical analysis was performed by applying single-factor

8

186

ANOVA in SPSS. The significance of the difference was determined by the Duncan test.

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Treatment effects were considered significant at p < 0.05.

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2. Results and discussion

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3.1. Optimization of extraction conditions

190

As shown in Fig. 1, both solid-liquid ratio and temperature had significant effects (p <

191

0.05) on the TAC of PHBB. TAC increased with the solid-liquid ratio ranging from 1/10 g/mL

192

to 1/40 g/mL and then remained constant (Fig. 1A). Thus, a large volume of solvent was more

193

effective than a small volume to dissolve solute, resulting in a high extraction yield. TAC also

194

increased with increasing temperature from 30 °C to 80 °C, which suggested that a high

195

temperature facilitated solvent diffusion and mass transfer. Interestingly, no significant

196

decrease in TAC was observed with the high extraction temperature at 90 °C (Fig. 1C). Ryu

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and Koh (2018) found that the optimized extraction temperature for the TAC of black

198

soybeans is 56.8 °C by using response surface methodology (RSM). Zou et al. (2011)

199

demonstrated that the optimum temperature obtained by RSM for anthocyanin extraction from

200

mulberry is 43.2 °C. The optimum extraction temperature (80 °C) of anthocyanins in this

201

work was higher compared with that reported above. Cacace and Mazza (2003) reported that

202

excessive temperature can lead to anthocyanin degradation. Therefore, anthocyanins in PHBB

203

might have comparative stability under high extraction temperature. As shown in Fig. 1D,

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TAC suffered a significant decrease at high pH 7.0, maintained steady at pH from 2 to 4, and

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slightly increased at pH 1. It has been reported that anthocyanins were unstable and easy to be

206

degraded at pH values higher than 7 (Castañeda-Ovando et al., 2009). Our results indicated

207

that anthocyanins in PHBB should be extracted under acidic condition.

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By contrast, no significant effects on TAC were observed with extraction duration,

209

ultrasonic power, and extraction times (Figs. 1B, E, and F). TAC remained steady with

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extraction duration ranging from 10 min to 150 min (Fig. 1B). The extraction time of the TAC 9

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in blueberry ranged from 10 min to 50 min, and the optimum condition was found at 40 min

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(Jiang et al., 2017). According to the report of Zou et al. (2011), the optimum extraction time

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of anthocyanins in mulberry is 40 min. In this study, the extraction time of anthocyanins was

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only 10 min, which indicated that the anthocyanins in PHBB were easily extracted by the

215

water. The result was also supported by the study of ultrasonic power (Fig. 1E). Moreover,

216

approximately 92% of TAC in PHBB was obtained after extraction for the first time (Fig. 1F).

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The color observation of supernatant and residue of bran also showed a small amount of

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anthocyanin left after extraction for the first time by the water (Fig. 2). The residue of bran

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was further treated by amylase, protease, and xylanase, and no improvement in TAC was

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detected (data not shown). Hence, the optimal extraction conditions for anthocyanins in

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PHBB were solid-liquid ratio (1/40 g/mL), extraction duration (10 min), temperature (80 °C),

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pH (1.0), ultrasonic power (200 W), and extraction times (1 time). Our results demonstrated

223

that anthocyanins in PHBB were easily extracted by acid water.

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3.2. Composition of anthocyanins in PHBB

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The anthocyanin extract of PHBB from the Tibetan Plateau in China was characterized

226

based on the MS properties and the retention times of components separated by

227

ultra-performance liquid chromatography (UPLC). Six numbers of anthocyanin compounds in

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PHBB were found in this study (Table 1). They were all derived from anthocyanidins and

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sugar through a glycoside bond. The dominant anthocyanidins in PHBB were cyanidin,

230

pelargonidin, and peonidin, and the sugars were glucose and galactose. Cyanidin malonyl

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glucoside came first at 73.50±3.49% followed by cyanidin-3-galactoside (19.24±1.23%),

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cyanidin acetyl galactoside (2.66±0.24%), and cyanidin di-glucoside (2.15±0.15%; Table 1).

233

The results revealed that cyanidin was the dominant anthocyanin pigment forming a covalent

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bond with glucose and galactose, which made up approximately 98% of TAC in PHBB (Table

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1). Previous studies have shown that cyanidin anthocyanins are widespread in many fruits, 10

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vegetables, and colored grains such as sugar beet molasses (Chen, Zhao, & Yu, 2015),

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mulberry (Liu et al., 2004), and purple wheat (Abdel-Aal et al., 2016). Nevertheless, no

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anthocyanin compounds in purple barley from Canada were detected chemically or visually

239

from the color of the methanolic extracts (Bellido and Beta, 2009). Cyanidin 3-glucoside was

240

the most abundant anthocyanin in purple hulled and unhulled barley, whereas delphinidin

241

3-glucoside in the blue and black barley (Kim et al., 2007). The difference in the total content

242

and composition of anthocyanins was also determined between colored corns (Abdel-Aal,

243

Young, & Rabalski, 2006). The content and composition of anthocyanins in plants could be

244

affected by many factors such as edaphic factors, including environmental factors (soil and

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climate), genotype, and crop year within the same variety. Moreover, other anthocyanins such

246

as pelargonidin-3-glucoside and peonidin glucoside were found in AAE of PHBB but at small

247

concentrations.

248

Three anthocyanin compounds were detected with two to three isomers in AAE of PHBB

249

due to the sensitivity and selectivity of LC-MS (Table 1). Although cyanidin di-glucoside

250

content was 2.15±0.15% of TAC, three isomers were detected with one ion pair of 611 m/z

251

and 287 m/z, which could be positional or structural isomers due to differences in hexose type

252

and/or position. For this compound, the ion 611 m/z was the parent ion obtained after

253

cyanidin di-glucoside protonation. The daughter ions were 287 m/z and 449 m/z, which were

254

the parent ion minus two and one glucose moieties. The content of cyanidin di-glucoside

255

isomers was 267.60±18.34 mg/100 g in AAE of PHBB. Cyanidin di-glucoside is frequently

256

detected in foods such as purple sweet potato (Zhang et al., 2016) and red rice (Abdel-Aal,

257

Young, & Rabalski, 2006), which demonstrated that it is one common anthocyanin compared

258

with the others.

259

Peonidin glucoside was also present in two isomeric forms. The total concentration of the

260

two isomers was 191.09±21.85 mg/100 g in AAE of PHBB (Table 1). This pigment was also 11

261

found in other colored grains, such as pigmented rice (Samyor, Das, & Deka, 2017) and black

262

bean (Chen et al., 2018). The notable antioxidant and anti-inflammatory properties of

263

peonidin-3-glucoside have been reported (Hu et al., 2003). The growth of human tumor cells

264

was significantly suppressed with pure peonidin-3-glucoside by inhibiting the G2/M phase of

265

the cell cycle and inducing the apoptosis of HCT116 colon and HS578T breast cells (Chen et

266

al., 2005).

267

Cyanidin malonyl glucoside was the most abundant pigment in PHBB and had two

268

isomers (Table 1). This study was the first to report such a much high percentage of cyanidin

269

malonyl glucoside (73.50±3.49%) of TAC in PHBB (Table 1). Minimal attention has been

270

given to the study of the specific structural configurations of cyanidin malonyl glucoside

271

isomers and their contribution to the total content of this pigment. The acylated anthocyanins

272

in sweet potato obtained a higher stability and antioxidant activity than the corresponding

273

nonacylated ones (Terahara et al., 2004). Acylation of the anthocyanin molecule can improve

274

its stability through intramolecular and/or intermolecular co-pigmentation and self-association

275

reactions (Giusti & Wrolstad, 2003). This phenomenon may explain why the TAC of PHBB

276

could also reach a high value even when the exaction temperature was 80 ℃ (Fig. 1C). Thus,

277

the purple barley pigments from the Tibetan Plateau were a potentially high-stability natural

278

colorant for commercial food products.

279

3.3. Antioxidant properties of the anthocyanin extracts of PHBB

280

As shown in Fig. 3A, ABTS radical scavenging activity had an obvious dosage

281

peculiarity of both ACE and AAE, which was enhanced with the extract concentration

282

increased. Significant differences (p < 0.05) were observed between ACE and AAE. For

283

example, when the concentration of the extracts was 125 µg/mL, the scavenging abilities of

284

AAE reached the maximum value (83.49±0.18%) on ABTS radical, which was 5.88 times that

285

of ACE. As shown in Fig. 3B, the FRAP of both ACE and AAE was enhanced as the extract 12

286

concentration increased. AAE exhibited a significantly higher FRAP than ACE. For instance,

287

the FRAP of AAE was 1436±21 µM FeSO4 at 1 mg/mL, which was 4.59 times that of ACE.

288

Similarly, dose dependence was observed on the RP and DPPH radical scavenging activities

289

of both ACE and AAE from 3.91 µg/mL to 125 µg/mL (Figs. 3C-D). However, the DPPH

290

radical scavenging activity of AAE reduced rapidly when the concentration of the extracts

291

ranged from 125 µg/mL to 500 µg/mL. DPPH was completely scavenged by AAE, and the

292

remaining anthocyanins presented obvious absorbance at 520 nm. Anthocyanin belongs to the

293

group of flavonoids, which are part of an even larger compounds family known as

294

polyphenols (Shipp & Abdel-Aal, 2010). Several reports have demonstrated that the

295

antioxidant capacity of cereals was dependent on their phenolic compound content (Kim et

296

al., 2007; Van Hung, 2016). In the current study, the anthocyanin content of AAE was about

297

14-fold and 62-fold that of ACE and bran, respectively. Therefore, the difference in

298

antioxidant activity between ACE and AAE was mainly caused by their different anthocyanin

299

content. Abdel-Aal, Hucl, and Rabalski (2018) also reported that the purified anthocyanin

300

extract powder of purple wheat exhibits exceptionally higher antioxidant activity than that of

301

whole grain flour and bran. The antioxidant activity analysis of milled and pearled purple,

302

black, and common barley also showed that the total antioxidant activities for the bran-rich

303

fractions, the anthocyanins content of which was six times higher than that in their

304

corresponding whole kernel flours, were significantly higher than for the whole kernel flour

305

(Bellido & Beta, 2009). All the findings above demonstrated that AAE presented higher

306

antioxidant activity than ACE, mainly because of the former’s high anthocyanin content.

307

The antioxidant activity of compounds is also generally expressed as the inhibition

308

percentage of the pre-prepared free radical by antioxidants, and the EC50, a concentration

309

needed to achieve a 50% antioxidant effect, is a typically used parameter to quantify and

310

compare the antioxidant capacity of different compounds (Chen, Bertin, & Froldi, 2013; Xiao, 13

311

et al., 2015). To obtain the EC50 values in this study, each sample was measured at several

312

different concentrations, within the range of 3.91-4000 µg/mL. EC50 was calculated by

313

interpolation or extrapolation from linear regression analysis of the data obtained with the

314

dose-response effect (Xiao et al., 2015). The results were normalized and expressed as EC50

315

values (microgram extracts per milliliter) for comparison (Table 2). The effectiveness of

316

antioxidant properties was inversely related to their EC50 values. For ABTS•+ scavenging

317

activity, the AAE of PHBB was 61.16±2.60 µg/mL, which was only 7.44% of ACE

318

(822.25±80.28 µg/mL). EC50 for the RP and DPPH were estimated to be 402.83±28.53 and

319

54.99±3.86 µg/mL, which were much lower than those for ACE of PHBB (2505.83±46.26

320

and 415.93±13.66 µg/mL, respectively). As a consequence, the extracts of PHBB presented

321

exceptional antioxidant capacity due to their high anthocyanin content.

322

2.4. Anti-biofilm properties of AAE

323

The AAE of PHBB exhibited considerably weak antibacterial activity against P.

324

aeruginosa PAO1 and S. enterica ATCC10398 even at 8 mg/mL. A significant (p < 0.01)

325

decrease in biofilm formation was observed when bacterial strains were grown in the presence

326

of AAE (Fig. 4). At 4 mg/mL, AAE showed a maximum of 60.21±3.70% and 47.25±3.81%

327

reduction in biofilm biomass of P. aeruginosa PAO1 and S. enterica ATCC10398,

328

respectively (Fig. 4). Thus, AAE inhibited the biofilm formation of the test strains without

329

inhibiting biomass. Gopu, Kothandapani, and Shetty (2015) also found that the methanol

330

extract of S. cumini has high anthocyanin content and can inhibit the biofilm formation of K.

331

pneumonia, especially the component malvidin pigment. Four individual anthocyanidins,

332

pelargonidin, cyaniding, and delphinidin were tested for their effects on the biofilm formation

333

of P. aeruginosa PAO1, and all exhibited obvious anti-biofilm activity (Pejin et al., 2017).

334

Therefore, high anthocyanin content highly contributes to the anti-biofilm activity of AAE.

335

2.5. In situ image analysis of bacterial biofilms by CLSM 14

336

The effect of AAE on bacterial biofilms was further analyzed by CLSM. CLSM z-section

337

analyses showed that the tested strains formed compact biofilms when grown in the absence

338

of AAE (Figs. 5 and 6). By contrast, AAE at a sub-MIC concentration of 4 mg/mL resulted in

339

thinner and looser cell aggregates instead of typical biofilm architecture (Figs.5 and 6). The

340

confocal 3D images showed that the thicknesses of biofilms formatted by P. aeruginosa PAO1

341

and S. enterica ATCC10398 in the negative control group were 44.67±5.82 µm and 6.15±1.41

342

µm, respectively. When P. aeruginosa PAO1 and S. enterica ATCC10398 were incubated

343

together with 4 mg/mL AAE, the biofilm thicknesses dropped to 20.08±4.51 and 4.13±0.96

344

µm, respectively. Besides the difference in biofilm thickness, AAE also influenced the

345

formation density of the biofilms. The CLSM 2D image at the middle position of the biofilm

346

of the tested strains showed that the biofilms of the control groups covered the entire surface

347

of the coverslips. However, in the treatment groups with 4 mg/mL AAE, CLSM assessments

348

of both bacteria exhibited a noticeable decrease in the surface coverage of coverslips and

349

bacteria density (Figs. 5 and 6).

350

More than half of the infectious diseases were related to the bacteria that proliferate by

351

forming biofilms (Husain et al. 2013). Biofilm formation is closely linked to

352

density-dependent cell-cell communication known as quorum sensing (QS), in which small

353

diffusible signaling molecules regulate the expression of various genes including virulence

354

genes (Steenackers et al., 2010; Galloway et al., 2012). The findings in this study strongly

355

suggested that the extracts of purple barley bran could be developed as a new QS inhibitor

356

and/or anti-biofilm agent to enhance shelf life and increase food safety.

357

3. Conclusions

358

This study demonstrated that PHBB from the Tibetan Plateau in China contained a high

359

content of anthocyanins, showing potential as a functional food ingredient. PHBB was easily

360

extracted by water and further processed into an anthocyanin-rich powder. PHBB exhibited a 15

361

complex anthocyanin profile with both acylated and non-acylated pigments. Cyanidin was the

362

main aglycone, glucose was the prevailing sugar, and malonyl was the predominant acyl

363

substituent. The extracts of PHBB also exhibited apparent anti-biofilm activity. The results in

364

this study strongly suggested that the extracts of PHBB could be developed as a new

365

high-quality natural food colorant and functional additive. Further research is currently

366

underway to explore potential applications and health benefits of purple barley food products.

367

Acknowledgement

368

This research was supported by the earmarked fund for Jiangsu Agricultural Industry

369

Technology System (No JATS-2018-296).

370

Conflict of interest statement

371

The authors declare that they have no competing interests.

16

372

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22

508

Figure captions

509

Fig. 1 Effects of variables in single-factor experiments on TAC. (A) solid-liquid ratio; (B)

510

extraction duration; (C) temperature; (D) pH; (E) ultrasonic power; (F) extraction times. Each

511

value was expressed as the mean ± SD (n = 3).

512

Fig. 2 Color observation of supernatant and residue of bran before or after anthocyanin

513

extraction. (A) The supernatant of the bran extracted for the first time; (B) the supernatant of

514

the bran extracted for the second time; (C) the supernatant of the bran extracted for the third

515

time; (D) untreated bran; (E) residue of the bran after extraction for the first time; (F) residue

516

of the bran after extraction for the second time.

517

Fig. 3 Antioxidant activities of anthocyanin extracts of PHBB. (A) ABTS cation radical

518

scavenging ability; (B) ferric reducing antioxidant power (FRAP); (C) reducing power (RP);

519

(D) DPPH radical scavenging activity. Each value was expressed as the mean ± SD (n = 3).

520

Fig. 4 Effects of AAE on biofilm formation in P. aeruginosa PAO1 (A) and S. enterica

521

ATCC10398 (B). Data were represented as the percentage of biofilm inhibition. Each bar

522

represents mean and standard deviations of the mean of all measurements.

523

Fig. 5 CLSM images of biofilm formation by P. aeruginosa PAO1 in the absence of AAE

524

(Control) and the presence of AAE (Treatment). (A) 2D image in grey mode at the middle of

525

the biofilm, (B) 2D image in color mode at the middle of the biofilm, and (C) 3D image of the

526

biofilm.

527

Fig. 6 The CLSM images of biofilm formation by S. enterica ATCC10398 in the absence of

528

AAE (Control) and the presence of AAE (Treatment). (A) 2D image in grey mode at the

529

middle of the biofilm, (B) 2D image in color mode at the middle of the biofilm, and (C) 3D 23

530

image of the biofilm.

24

531 532

Fig. 1

25

533 534

Fig. 2

26

535 536

Fig. 3

27

537 538

Fig. 4

28

539 540

Fig. 5

29

541 542

Fig. 6

543

30

544

Tables

545

Table 1 Characterization of anthocyanin compounds in AAE via LC-MS analysis

Component name

Retention time (min)

Number of isomers

Major ion (m/z)

Anthocyanin (mg/100g)

Cyanidin malonyl glucoside Cyanidin-3-galactoside Cyanidin acetyl galactoside Cyanidin di-glucoside Pelargonidin-3-glucoside Peonidin glucoside Total anthocyanins

3.60,3.89 2.98 7.48 1.16, 2.99, 3.54 3.37 3.57, 6.01 —

2 1 1 3 1 2 —

535/287 449/287 491/287 611/449/287 433/271 463/301 —

9141.97±433.53 2393.26±153.11 330.49±29.97 267.60±18.34 113.06±7.54 191.09±21.85 12437.48±632.69

546 547

31

Table 2 Half-efficiency concentration (EC50) of anthocyanin extracts of PHBB

548

Samples

ABTS•+ scavenging capacity

Reducing powder

DPPH scavenging capacity

ACE

822.25±80.28a

2505.83±46.26a

415.93±13.66a

AAE

61.16±2.60b

402.83±28.53b

54.99±3.86b

Vc

13.91±0.82c

63.13±0.27c

13.03±0.35c

549

a

550

initial ABTS•+, and 50% initial Fe2+ concentration. The absorbance was 0.5 for reducing power. EC50 was

551

obtained by interpolation or extrapolation from linear regression analysis of the data obtained with the

552

dose-response effect (Xiao et al., 2015). Values were presented as the mean ± SD (n = 3), Significant

553

difference (p < 0.05) was represented as different small letters within a column.

EC50 was the effective concentration of the test sample that decreased 50% initial DPPH radical, 50%

32

1. 2. 3. 4. 5.

Anthocyanins in purple highland barley bran was easily extracted by the water. Anthocyanins in purple highland barley bran possessed a good stability. Anthocyanins in purple highland barley bran was highly acylated. Anthocyanins-rich extract exhibited obvious antioxidant capacity. Anthocyanins-rich extract exhibited obvious anti-biofilm properties.

The authors declare that they have no competing interests.

The corresponding author Professor Mingsheng Dong was responsible for the experimental design and involved in the whole process of the manuscript writing. The first author Yongzhu Zhang and the second author Yanfei Lin were responsible for the experimental implementation and the manuscript writing. The third author Lu Huang took part in the experimental implementation. The fourth and fifth author Mekonen Tekliye and Hafiz Abdul Rasheed took part in the edition of the manuscript.