Increases in Phenolic, Fatty Acid, and Phytosterol Contents and

Subscriber access provided by Kaohsiung Medical University

Bioactive Constituents, Metabolites, and Functions

Increases of Phenolics, Fatty Acids, Phytosterols, and Anticancer Activity of Sweet Potato after Fermented by Lactobacillus acidophilus Yixiao Shen, Haiyan Sun, Haiying Zeng, Witoon Prinyawiwatkul, Wenqing Xu, and Zhimin Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05414 • Publication Date (Web): 03 Mar 2018 Downloaded from http://pubs.acs.org on March 5, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Journal of Agricultural and Food Chemistry

Increases of Phenolics, Fatty Acids, Phytosterols, and Anticancer Activity of Sweet Potato after Fermented by Lactobacillus acidophilus Yixiao Shen†, Haiyan Sun‡, Haiying Zeng∥, Witoon Prinyawiwatukul†, Wenqing Xu† and Zhimin Xu†,*

Running title: Bioactives and anti-cancer of fermented sweet potato †

School of Nutrition and Food Sciences

Louisiana State University Agricultural Center Baton Rouge, USA ‡

Key Laboratory of Life Resources of Shaanxi Province,

Shaanxi University of Technology, Hanzhong, China ∥

School of Liquor and Food Engineering

Guizhou University Guiyang, China.

Authors Shen and Sun contributed equally to this work. *Corresponding author Tel: 225-578-5280. Fax: 225-578-5300. E-mail:[email protected]

ACS Paragon Plus Environment


1

Abbreviations:

2

DMSO, Dimethyl sulfoxide;

3

DW, dry weight;

4

FBS, fetal bovine serum;

5

FID, flame ionization detector;

6

FW, fresh weight;

7

GC, gas chromatography;

8

HPLC, high performance liquid chromatography;

9

MS, mass spectrometry;

10

PBS, phosphate buffered saline;

11

LR, lipophilic extract of raw sweet potato;

12

LF, lipophilic extract of fermented sweet potato;

13

HR, hydrophilic extract of raw sweet potato;

14

HF, hydrophilic extract of fermented sweet potato

15

1


Page 2 of 30

Page 3 of 30


16

Abstract

17

Changes of phenolics, fatty acids, and phytosterols in the sweet potato (SP) fermented

18

by Lactobacillus acidophilus were evaluated and compared with its raw and boiled SPs.

19

The differences of profiles and levels of phenolics between raw and boiled SPs were not

20

as significant as their differences from fermented SP. The level of caffeic acid or 3,5-

21

dicaffeoylquinic acids in fermented SP increased to more than 4 times higher than raw or

22

boiled SP. Two phenolics, p-coumaric acid and ferulic acid which were not detected in

23

either raw or boiled SP were found in fermented SP. The level of each fatty acid or

24

phytosterol increased in fermented SP, but decreased in boiled SP.

25

hydrophilic and lipophilic extracts obtained from both of raw and fermented SPs, the

26

hydrophilic extract of fermented SP exhibited the highest capability in inhibiting cancer

27

cell PC-12 proliferation. However, each of the extracts had very low cytotoxicity to

28

normal monkey kidney cell growth.

29

Lactobacillus acidophilus significantly increased free antioxidant-rich phenolics and

30

enhanced inhibit cancer cell proliferation activity without cytotoxicity to normal cells.

Among the

The results indicated that fermented SP by

31 32

Key words: sweet potato; fermentation; phenolics; phytosterol; fatty acids; proliferation

33

inhibition.

2



34

INTRODUCTION

35

Sweet potato (Ipomoea batatas (L.)) is an important agricultural crop rich in dietary

36

fiber and vitamins. It is becoming one of the most promising economic commodities due

37

to its versatility, high production yield and strong resistance in various environmental

38

conditions.1 The compositions of sweet potatoes after being boiled, steamed, baked or

39

roasted have been well evaluated.2 It can also be used for the production of ethanol.3

40

However, composition changes, especially for phenolics, fatty acids, and phytosterols in

41

fermented sweet potato by lactic acid bacteria Lactobacillus acidophilus have not been

42

documented. Fermentation is a popular food processing method which provides the

43

preservation and organoleptic properties for foods such as cheese, vinegar, wine and

44

soybean curd with special flavors, aromas and textures.4 Generally, most bioactive

45

compounds in sweet potato are in bound form present in flesh and considered to be

46

gradually metabolized in the intestine which contains different varieties of probiotic

47

floras.5

48

compounds to increase their bioavailability via fermentation, meanwhile they could

49

reduce sugar content in the fermented foods. It has been reported that a number of people

50

with digestion problems such as small intestinal bowel infection or colonic infection are

51

not able to metabolize and absorb those nutrients in sweet potato.6 Therefore, the

52

fermentation by Lactobacillus acidophilus could be considered as a pre-digestion

53

processing method which could increase free form bioactive compounds in sweet potato

54

and improve their bioavailability with reducing of sugar content.7

The floras assist in releasing and bio-transforming those health benefit

55

Lactobacillus acidophilus is one of the dominant probiotic floras in the human

56

gastrointestinal tract.8 It plays an important role in generating antimicrobial peptides,

3


Page 4 of 30

Page 5 of 30


57

improving gastrointestinal barrier function and competing with pathogens for epithelial

58

adherence.9 Lactobacillus acidophilus is mainly used in dairy products and reported to

59

have health beneficial effects including the prevention of diarrhea, stimulation of the

60

immune system, anti-inflammation of intestinal disorders, etc.8

61

acidophilus can also convert some carbohydrates to organic acids and has cinnamoyl

62

esterase and decarboxylase activities for releasing phenolic compounds from the

63

fermented food matrix.10 Sweet potato is rich in free sugar and other carbohydrates and

64

an ideal substrate to be fermented by Lactobacillus acidophilus. The fermentation could

65

release the bound bioactive compounds and produce free secondary metabolites with high

66

bioactivity and bioavailability. Therefore, the levels and profiles of antioxidant phenolics,

67

fatty acids, and phytosterols in the fermented sweet potato were evaluated and compared

68

with the original raw sweet potatoes.

69

inhibiting pheochromocytoma derived cancer cell (PC-12) and its cytotoxicity to normal

70

monkey kidney cell (CV-1) were studied in this study as well. The results of this study

71

could provide a new utilization method for sweet potato through the fermentation by

72

Lactobacillus acidophilus. Compared with raw sweet potato, the fermented sweet potato

73

could have a higher level of free bioactives with high bioavailability which enhances the

74

overall health promoting and anticancer capabilities.

Lactobacillus

The capability of fermented sweet potato in

75 76

MATERIALS AND METHODS

77

Chemicals and materials

78

purchased from Fisher Chemicals (Fair Lawn, NJ, USA). Acetone and ethyl acetate were

79

purchased from Macron (Charlotte, NC, USA) and EM Science (Gibbstown, NJ, USA),

HPLC grade acetonitrile, acetic acid and hexane were

4



Page 6 of 30

80

respectively.

Standards of phenolic acids, fatty acids, and phytosterols as well as

81

derivatization reagents trimethylsilyl imidazole (TMSIM) and BCl3-methanol were

82

purchased from Sigma-Aldrich (St. Louis, MO, USA). Frozen cultures of Lactobacillus

83

acidophilus LA-K (Chr. Hansen’s Laboratory, Milwaukee, WI) were stored at -35°C

84

before use. Pheochromocytoma derived cancer cell (PC-12) and normal monkey kidney

85

cell (CV-1) lines were purchased from ATCC® (Manassas, VA, USA). Fetal bovine

86

serum (FBS), dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS), cell titer

87

blue and antibiotic (penicillin–streptomycin) were ordered from Invitrogen (Grand Island,

88

NY, US).

89

Garnet sweet

90

Fermentation of sweet potato mash and microbiological analysis

91

potatoes (Ipomoea batatas (L.)) were purchased in a local grocery store (Baton Rouge,

92

LA, USA). They were cleaned, peeled and cut into 1 cm cubes and then ground by a

93

kitchen blender with sterilized water at the ratio of 2:1 (w/v). Then, the sweet potato

94

mash (30 g) was added in each of nine flasks (250 mL). Three of the flasks were used for

95

each of the raw, boiled, or fermented sweet potato group. For the boiled sweet potato

96

group, the mash in flask was boiled on a hot plate stirrer (PC-351 Corning, Corning, NY,

97

USA) for 15 min with stirring by a magnetic stirring bar.

98 99

The three flasks used as the fermented sweet potato group were autoclaved before inoculation of Lactobacillus acidophilus solution.

The solution was prepared by

100

suspending 2 milliliters of freshly thawed Lactobacillus acidophilus (LA-K) culture in 20

101

mL of sterilized phosphate buffer (10 mM, pH 7.0). Then, the flasks were incubated at

102

37oC for 24 h fermentation. The pH of the fermented sweet potato mash was measured

5


Page 7 of 30


103

using a pH meter (Orion 3 star Benchtop; Thermo Orion, Beverly, MA, USA). The

104

populations (CFU/mL) of Lactobacillus acidophilus before and after the fermentation

105

were enumerated using direct plate count on Lactobacilli MRS agar.

106 107

Extraction and determination of phenolics, fatty acids, and phytosterols in sweet

108

potatoes

109

potato mash was extracted with 20 mL of methanol three times at 60°C for 20 min. After

110

the methanol layers were combined, the methanol solvent was evaporated by a vacuum

111

centrifuge evaporator (Labconco, Kansas City, MO, USA).

112

dissolved in acetone (20 mL) twice to precipitate possible protein and carbohydrate

113

contaminants. Then, the supernatants were collected and evaporated again to obtain the

114

dried hydrophilic extract for determination of phenolics. For the lipophilic extract, ten

115

grams of each of those sweet potato mesh samples was extracted by a mixture of hexane

116

and ethyl acetate (50:50; v/v) and followed the same procedure as the methanol

117

extraction above to obtain the dried extracts for determination of lipophilic fatty acids,

118

and phytosterols. A working solution (10 mg/mL) of each hydrophilic or lipophilic

119

extract was prepared by using methanol and hexane, respectively.

For the hydrophilic extract, ten grams of the raw, boiled or fermented sweet

The dried extract was

120

Phenolics were determined by an HPLC system (2690, Waters, Torrance, USA) with a

121

reversed phase column (C18, id 250×4.60 mm, 5 µm, Phenomenex, Torrance, USA) and

122

a diode array detector based on the method of Du et al.11 The hydrophilic extract

123

working solution (2 mL) was filtered by 0.45 µm microporous film before injection. The

124

concentration of each compound was calculated by its corresponding standard calibration

125

curve.

6



126

The lipophilic extract working solution (2 mL) was mixed with an internal standard

127

solution (C17:0). After hexane solvent was evaporated, 2 mL of BCl3-methanol was

128

added to carry out esterification derivatization. After incubation at 60°C for 30 min, 1

129

mL of hexane was added to the reaction solution and vortexed. The upper hexane layer

130

was collected and transferred to a clean test tube containing 0.1 g of anhydrous NaSO4 for

131

removing any moisture and then transferred to a GC vial. A GC system equipped with

132

FID detector and Supelco SP2380 (30 m × 0.25 mm) column (Bellefonte, PA, USA) was

133

employed to determine fatty acids in the samples according to the method in the study of

134

Du et al.11

135

The determination method of phytosterols in the samples was based on the study of

136

Xu and Godber.12 Two milliliter of the lipophilic extract working solution was transferred

137

to a clean test tube and dried again. Then, 200 µL of TMSIM and 50 µL of acetonitrile

138

were mixed with the extract and incubated at 65 ˚C for 30 min for derivatization. Then it

139

was extracted by 200 µL of hexane. The supernatant was transferred to a GC vial. A

140

Varian CP-3800 GC (Valnut Creek, CA, USA) was interfaced with a Saturn 2200 MS

141

and a DB-5 column (60 m × i.d. 0.25 mm and 0.25 µm thin coating film) (Supelco,

142

Bellefonate, PA, USA). The oven temperature increased from 200˚C to 280 ˚C at a rate

143

of 10 ˚C /min and was held at the final temperature for 62 min. Helium was used as a

144

carrier gas at a constant flow rate of 1.5 mL/min with a split injection mode (1: 50). The

145

injection port temperature was set at 280˚C.

146 147

Determination of effect of raw and fermented sweet potato extracts on

148

Pheochromocytoma derived cancer (PC-12) cell line and normal monkey kidney

7


Page 8 of 30

Page 9 of 30


149

(CV-1) cell line Pheochromocytoma derived cancer cell (PC-12) cell line of rat adrenal

150

medulla and normal monkey kidney (CV-1) cell line were used to determine the effect on

151

cancer cell and normal cell proliferations of each extract, respectively. The cells were

152

maintained in a CO2 incubator with 5% CO2 and 95% humidity and supplemented with

153

Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine

154

serum (FBS) and 1 % antibiotic (penicillin–streptomycin). The PC-12 or CV-1 cells

155

were plated in a 96-well plate at a density of 3.0 × 103 cells/well and incubated for 48 h.

156

The dried hydrophilic and lipophilic extracts of raw sweet potato and fermented sweet

157

potato were dissolved with 0.2 % DMSO in PBS culture media. Then, the PC-12 or CV-

158

1 cells were incubated with a series of the extracts with different concentrations for 24 h

159

at 37 °C. The cells mixed only with 0.2 % DMSO in PBS culture media were used as a

160

blank group. After incubation, the media was discarded. The cells were stained by 100

161

µL of fresh media containing 20% cell titer blue (Invitrogen, Grand Island, NY, US).

162

The fluorescence intensity of each well was obtained by a FluoStar Optima micro-plate

163

reader (BMG, Germany) at excitation/emission wavelengths of 570/615 nm to calculate

164

the cell viability in the well. The percentage of survived cells of sample group relative to

165

the blank group was used to indicate the anti-cancer capability or cytotoxicity of each

166

treatment.

167 168

Data analysis Determinations of phenolics, fatty acids, and phytosterols in the samples

169

were carried out in triplicates and expressed as means ± standard deviation by using

170

Microsoft Excel (Redmond, WA). The significant differences among treatments were

171

conducted by one-way ANOVA at P< 0.05 (SAS, 9.1.3, Cary, NY, US). The results

8



Page 10 of 30

172

from the cell culture studies were obtained from five replications for each group and

173

analyzed by GraphPad Prism (Version 6.0, GraphPad Software Inc., USA).

174

significant difference of the results between two groups was considered at p < 0.05.

The

175 176

RESULTS AND DISCUSSIONS

177

Profiles and levels of phenolics in raw, boiled and fermented sweet potatoes As

178

shown in Table 1, the pH of sweet potato mash after fermentation drop to 3.45 from 6.20,

179

because of the production of organic acids through the enzymatic conversion of

180

carbohydrates by Lactobacillus acidophilus.3 The bacteria can take advantage of glucose

181

in sweet potato to produce lactic and acetic acids which were mainly responsible for

182

lowering the pH after fermentation.13 The protein and carbohydrates in sweet potato also

183

provided a sufficient source of nitrogen and carbon for the bacteria proliferation. The

184

viable cell counts of Lactobacillus acidophilus in the fermented sweet potato increased to

185

7.48 ± 0.67

186

indicated that sweet potato mash could be a suitable substrate for the fermentation by

187

Lactobacillus acidophilus.

×

108 from 3.80 ± 0.01

×

106 CFU/mL after incubation at 37oC for 24 h. It

188

The main phenolics in sweet potato are chlorogenic acid and its related derivatives.

189

The raw sweet potato contained five major phenolics including chlorogenic acid, caffeic

190

acid, and 4,5-dicaffeoylquinic, 3,5-dicaffeoylquinic and 3,4-dicaffeoylquinic acids. The

191

concentrations of the phenolics in raw, boiled, and fermented sweet potatoes are shown in

192

Table 2. In raw sweet potato, 3,4-dicaffeoylquinic acid (29.12 ± 1.68 mg/100g DW) was

193

the dominant phenolic, while the other phenolics were in a range of 2.39 mg/100g DW

194

for caffeic acid to 5.00 mg/100g DW for 4,5-dicaffeoylquinic acid. With the involvement

9


Page 11 of 30


195

of fiber and starch in raw sweet potato flesh, some phenolics could be in bound and

196

conjugated form which usually have lower bioavailability than free form.14 Generally, the

197

traditional cooking method, boiling, is believed to be able to disrupt the fiber or starch

198

matrix to release bound or conjugated form phenolics.15 As shown in Table 2, the levels

199

of most phenolics in the boiled sweet potato were significantly higher than those in raw

200

sweet potato, except 3,4-dicaffeoylquinic acid.

201

dicaffeoylquinic acid had been present in free form in raw sweet potato and degraded

202

during boiling treatment. The increase of other phenolics may be due to a significant

203

amount of their bound phenolics were released and convert to free form by heat treatment,

204

although some of their free form phenolics could also be degraded at the same time.16

It was suggested that most of 3,4-

205

In this study, it was found that the increases of most of the phenolics in fermented

206

sweet potato are much higher than boiled sweet potato after the fermentation by

207

Lactobacillus acidophilus (Figure 1 & Table 2). For example, the level of caffeic acid or

208

3,5-dicaffeoylquinic acid in fermented sample was three times higher that in raw sweet

209

potato or boiled sweet potato (Table 2). Furthermore, p-coumaric acid and ferulic acid

210

which were not detected in either raw or boiled sweet potato were produced to 26.42 ±

211

2.01 and 19.10 ± 1.87 g/100g DW in the fermented sweet potato, respectively.

212

indicated that the fermentation effectively released bound phenolics through the enzymes

213

of Lactobacillus acidophilus which are able to utilize or break down the fiber or starch

214

matrix of sweet potato. It was reported that lower pH of fermented sweet potato could

215

also assist in the release of phenolics which have ester and ether bonds with plant cell

216

wall.17 Compared with boiling treatment, undesirable thermal degradation of phenolics is

217

totally avoided during the fermentation. Therefore, the bound phenolics were largely

10


It


218

converted to free form phenolics and retained in the fermented sweet potato without

219

thermal degradation.

220

balancing gut microbiota, inhibiting pathogenic infection, lowering blood cholesterol and

221

reducing the risks of colon cancer.18 The fermentation of Lactobacillus acidophilus

222

occurring in the intestine of humans may also improve the release and bioavailability of

223

bound phenolics in consumed food fiber.

There are evidences that Lactobacillus acidophilus help in

224

The biotransformation of chlorogenic acid to caffeic acid by Lactobacillus acidophilus

225

during fermentation was confirmed by using cholorgenic acid only in MSR media for 24

226

h in this study. Similar result has been reported in the study of Bel-Rhlid et al, in which,

227

Lactobacillus johnsonii NCC 533 could convert chlorogenic acid to caffeic acid in green

228

coffee extract.19 The biotransformation might be due to that Lactobacillus acidophilus

229

contains chlorogenate esterase and hydroxycinnamate decarboxylase which exhibit high

230

catalytic activity on aromatic compounds such as chlorogenic acid.19

231

Generally, ferulic and p-coumaric acid are hydroxycinnamic acids which are mostly in

232

bound form as ester-linked with polysaccharides, organic acids, lipids or protein of plants

233

in the cell wall.20 After the fermentation, the two bound phenolics significantly changed

234

to free form and became the major phenolics in fermented sweet potato. It has been

235

reported that Lactobacillus species produces active extracellular cellulolytic enzymes

236

such as feruloyl esterases.21 Thus, it could de-esterify dietary fiber and release free ferulic

237

acid and p-coumaric acid from plant cell wall. Some clinic studies have demonstrated

238

free caffeic acid was more readily absorbed in the stomach and small intestine than

239

chlorogenic acid, while ferulic acid and p-coumaric could be bioavailable in blood

240

plasma.22 Therefore, the releasing of bound phenolics in sweet potato by fermentation,

11


Page 12 of 30

Page 13 of 30


241

especially caffeic acid, ferulic acid and p-coumaric acid would enhance their

242

bioavailability to the human body.

243 244

Profiles and levels of fatty acids and phytosterols in raw, boiled, and fermented

245

sweet potatoes The fatty acid compositions of the three sweet potatoes are listed in Table

246

3. In raw sweet potato, the most abundant fatty acid was linoleic acid (C18:2n-6) (85.67±

247

5.56 mg/100g DW), then followed by palmitic acid (C16:0), myristic acid (C14:0),

248

linolenic acid (C18:3n-3) and stearic acid (C18:0) which were a range from 47.18 ± 3.24

249

to 17.96 ± 0.61 mg/100g DW. However, the level of each fatty acid significantly

250

decreased in boiled sweet potato, especially for unsaturated fatty acids, linoleic and

251

linolenic acid. Similar results were reported in the study of Longo et al. that heat

252

treatment (60-100 °C) resulted in degradation of unsaturated fatty acids in tomatoes.23

253

After the fermentation, linolenic acid (171.07 ± 12.77 mg/100g DW) or each of other

254

fatty acids which ranged from 42.86 ± 0.87 to 109.29 ± 3.74 mg/100g DW in fermented

255

sweet potato was approximately more than two times higher, compared with raw sweet

256

potato. In addition to free form fatty acids, some fatty acids also bind with phytosterols,

257

glycerols, phospholipids and sugar-containing glycolipids in plants as important

258

membrane constituents in the chloroplasts and mitochondria.24 The increase of the fatty

259

acids in fermented sweet potato might result from the enzymatic action based on

260

desertification of bound fatty acids through the fermentation to release palmitic, myristic

261

and stearic acids.25 Since unsaturated fatty acids are important to brain cells growth,

262

neurological development and cognitive function, the accumulation of those fatty acids in

12



263

fermented sweet potato would have higher health promoting function than raw or boiled

264

sweet potato.26

265

Although three phytosterols, campesterol, stigmasterol and β-sitosterol slightly

266

decreased in sweet potato after boiled due to thermal degradation, they significantly

267

increased in the fermented sweet potato (Table 3). As phytosterols have been reported to

268

inhibit intestinal cholesterol absorptions and control cholesterol concentrations in plasma

269

due to they have similar chemical structure of cholesterol, the increases of the

270

phytosterols in fermented sweet potato would also improve the function of lowering

271

serum cholesterol level.27

272

In general, as Lactobacillus acidophilus is a probiotic bacteria species which could

273

help in reducing overgrowth of pathogens, stimulating an immune response, improving

274

the blood lipid metabolism or potentially killing cancer cells in the human gastrointestinal

275

tract.28 The fermented sweet potato could be considered as an ideal processed food source

276

to enhance bioavailability of the bioactive compounds of sweet potato and deliver

277

probiotic Lactobacillus acidophilus at the same time. Based on the results above, the

278

hydrophilic and lipophilic raw (HR and LR) and fermented sweet potato extracts (HF and

279

LF) were selected to evaluate their effects on the pheochromocytoma derived cell (PC-12)

280

and normal monkey kidney cell (CV-1) proliferations.

281 282

Effect of raw and fermented sweet potato extracts on proliferation of

283

pheochromocytoma cells (PC-12) and normal monkey kidney cells (CV-1) of raw

284

and fermented sweet potato extracts The inhibitory effect on cancer cell proliferation

285

of hydrophilic and lipophilic extracts of raw sweet potato versus fermented sweet potato

13


Page 14 of 30

Page 15 of 30


286

were evaluated by using pheochromocytoma cancer cell (PC-12) model.

Figure 2a

287

demonstrates that the hydrophilic extracts of raw (HR) and fermented (HF) sweet potato

288

had a dose-dependent inhibition mode on PC-12 cell proliferation.

289

concentration increased to 0.2 mg/mL, the survival rate of the cancer cells dropped to

290

9.5% in HF treatment, while it was 63.2% in HR treatment (Figure 2a). When the

291

concentration of HF increased to 0.5 mg/mL, only 2.1% of the cancer cells was survived,

292

compared with the control, while the survival rate in HR treatment was 30.1% (Figure 2a).

293

Thus, the greater inhibitory capability of HF may be contributed by the higher levels of

294

free form phenolics or other bioactives generated by the fermentation. For example, the

295

total level of phenolics including caffeic acid, p-coumaric acid, ferulic acid and 3,5-

296

dicaffeoylquinic acid in fermented sweet potato was much higher than that in raw sweet

297

potato (Table 2). Similar result was found in the study of Rocha et al., which reported

298

that caffeic acid, 3,4-, 3,5- and 4,5- dicaffeoylquinic acid as well as ferulic and p-

299

coumaric acid could inhibit colon cancer cell lines including RKO, HT-29 and Caco-2.29

300

However, the difference between the lipophilic extracts of raw (LR) and fermented (LF)

301

sweet potatoes in inhibiting PC-12 cell proliferation was not as significant as the

302

hydrophilic extracts (Figure 2b). Although the concentration of fatty acids concentration

303

in fermented sweet potato was much higher than that in raw sweet potato, they were not

304

the primary factors responsible for the anticancer potential. Thus, the LR or LF extract

305

treatment exhibited relatively weaker anticancer potential than either HR or HF extract

306

treatment.

As the extract

307

The interaction of lipophilic and hydrophilic bioactive compounds in inhibiting cancer

308

cell relies on their different accessibilities and inhibition pathways. Generally, lipophilic

14



309

compounds could readily cross the lipid bilayers of cell membranes by simple diffusion

310

or special membrane proteins for transferring such non-polar solutes, while most

311

hydrophilic compounds have difficulties in penetrating the cell membrane.30 However, at

312

a physiologic pH level, multiple hydroxyl groups in phenolics could interact with the

313

polar head groups of phospholipids at membrane surface via hydrogen bonds formation.31

314

It would then improve the penetration of the phenolics by deprotonation of hydroxyl

315

groups and modify the membrane structure and fluidity.32 Compared with the control

316

group shown in Figure 2c, the apoptotic progress of cancer cells was induced and

317

accelerated by the hydrophilic fermented sweet potato extract. It resulted in the cell

318

shrinkage and marginated nucleus indicated by the arrows in Figure 2d.

319

The advantage of applying plant-derived bioactive extracts as anticancer medicines is

320

that the extracts have lower cytotoxicity and side effect compared with other anticancer

321

treatments such as chemotherapeutic agents or radiation. In this study, the monkey

322

kidney cell lines (CV-1) were used for assessing the influences or toxicities of the sweet

323

potato extracts on normal cells growth. At a relatively low concentration of 0.02 mg/mL

324

of HR or HF treatment, they could slightly increase the growth of CV-1 normal cells

325

(Figure 3). Even at the concentration of 0.5 mg/mL which HF had the most effective

326

inhibition to the PC-12 cancer cells, only 24.74% of CV-1 normal cells were inhibited,

327

while the levels of other treatments were all below that level (Figure 3). It indicated that

328

hydrophilic sweet potato extracts had high anticancer potential but low cytotoxicity on

329

normal cells. It could be explained by the different energy metabolisms between cancer

330

and normal cells. Generally, normal cells with competent mitochondria undergo Krebs

331

Cycle metabolic pathway and generate the majority of ATP for cell growth.33 However,

15


Page 16 of 30

Page 17 of 30


332

most cancer cells have dysfunction in mitochondria and rely more on the glycolytic

333

pathway in cytosol to generate the metabolic intermediates and ATP.34 Thus, the key

334

enzymes in glycolytic pathway have been considered as the potential therapeutic targets

335

for inhibition of cancer cell proliferation. The non-covalent interactions with proteins

336

and phenolics have been reported as the inhibitors of the key enzymes in glycolytic

337

pathway, such as glucosidases, xanthine oxidase and PKM2 activities, to further perturb

338

proliferation of the cancer cells.33 For lipophilic bioactives, such as phytosterols, they

339

could interrupt balance between cancer cell proliferation and apoptosis by the

340

interference with protein phosphatase 2A (PP2A) in sphingomyelin cycle and block the

341

cell cycle at G0/G1 phase in prostate cancer, hepatocyte, and breast cancer cell lines.35, 36

342

In summary, this study revealed that increases of hydrophilic phenolics and lipophilic

343

fatty acids and phytosterols in the sweet potato after fermented by probiotic Lactobacillus

344

acidophilus LA-K. Also, the fermented sweet potato extracts, especially the hydrophilic

345

extract exhibited higher capability than raw sweet potato in inhibiting the

346

pheochromocytoma cancer cells (PC-12) proliferation, while it had very low cytotoxicity

347

to the normal monkey kidney cells (CV-1).

348

contributed by the high level of free form phenolics in fermented sweet potato, which

349

deactivate the key enzymes of glycolytic pathway in the energy metabolism of cancer

350

cells but had less influence on the Krebs Cycle pathway for normal cells. Therefore, the

351

fermentation by Lactobacillus acidophilus is a new biotechnological approach for sweet

352

potato to increase its nutritional value and bioavailability of various bioactive compounds.

353

The fermented sweet potato or its extract could be used as an assistant treatment with low

354

side effect to cooperate with chemotherapeutic drugs or other cancer remedy treatments.

The great anticancer potential may be

16



355

Acknowledgements

356

This study was supported by the Key Technologies Research and Development Program

357

Agriculture of Shaanxi Province (2017NY-117) and Louisiana State University

358

Agricultural Center (NIFA Project No. LAB94334).

17


Page 18 of 30

Page 19 of 30


References: (1) Wu, Q.; Qu, H.; Jia, J.; Kuang, C.; Wen, Y.; Yan, H., Gui, Z. Characterization, antioxidant and antitumor activities of polysaccharides from purple sweet potato. Carbohydr.Polym. 2015, 132, 31-40. (2) Tang, Y.; Cai, W.; Xu, B. Profiles of phenolics, carotenoids and antioxidative capacities of thermal processed white, yellow, orange and purple sweet potatoes grown in Guilin, China. Food Sci. Hum. Wellness. 2015, 4, 123-132. (3) Masiero, S. S.; Peretti, A.; Trierweiler, L. F.; Trierweiler, J. O. Simultaneous cold hydrolysis and fermentation of fresh sweet potato. Biomass and Bioenergy. 2014, 70, 174-183. (4) Kwon, D. Y.; Nyakudya, E.; Jeong, Y. S. Fermentation: food products. In Encyclopedia of Agriculture and Food Systems, 2nd Ed.; N. K. V. Alfen., Eds.; Oxford: Academic Press, Cambridge, MA, 2014; pp.113-123. (5) Kubow, S.; Iskandar, M. M.; Sabally, K.; Azadi, B.; Sadeghi Ekbatan, S.; Kumarathasan, P.; Das, D. D.; Prakash, S.; Burgos, G.; zum Felde, T. Biotransformation of anthocyanins from two purple-fleshed sweet potato accessions in a dynamic gastrointestinal system. Food Chem. 2016, 192, 171-177. (6) Gibson, P. R.; Varney, J.; Malakar, S.; Muir, J. G. Food components and irritable bowel syndrome. Gastroenterol. 2015, 148, 1158-1174. (7) Duangjitcharoen, Y.; Kantachote, D.; Ongsakul, M.; Poosaran, N.; Chaiyasut, C. Selection of probiotic lactic acid bacteria isolated from fermented plant beverages. Pak. J. Biol. Sci. 2008, 11, 652-655. (8) Ren, D.; Li, C.; Qin, Y.; Yin, R.; Du, S.; Ye, F.; Liu, C.; Liu, H.; Wang, M.; Li, Y.; Sun, Y.; Li, X.; Tian, M.; Jin, N. In vitro evaluation of the probiotic and functional potential of Lactobacillus strains isolated from fermented food and human intestine. Anaerobe. 2014, 30, 1-10. (9) Morrow, L. E.; Gogineni, V.; Malesker, M. A. Probiotic, prebiotic, and synbiotic use in critically ill patients. Curr. Opin. Crit. Care. 2012, 18, 186-191. (10) Barthelmebs, L.; Diviés, C.; Cavin, J. Molecular characterization of the phenolic acid metabolism in the lactic acid bacteria Lactobacillus plantarum. Dairy Sci. Technol. 2001, 81, 161-171.

18



(11) Du, L.; Shen, Y.; Zhang, X.; Prinyawiwatkul, W.; Xu, Z. Antioxidant-rich phytochemicals in miracle berry (Synsepalum dulcificum) and antioxidant activity of its extracts. Food Chem. 2014, 153, 279-284. (12) Xu, Z.; Godber, J. S. Purification and identification of components of gamma oryzanol in rice bran Oil. J. Agric. Food Chem. 1999, 47, 2724-2728. (13) Cui, L.; Liu, C. Q.; Li, D.J.; Song, J. F. Effect of processing on taste quality and health-relevant functionality of sweet potato tips. Agric. Sci. in China. 2011, 10, 456-462. (14) Khoddami, A.; Wilkes, M. A.; Roberts, T. H. Techniques for analysis of plant phenolic compounds. Molecules. 2013, 18, 2328-2375. (15) Bahado-Singh, P. S.; Riley, C. K.; Wheatley, A. O.; Lowe, H. I. C. Relationship between Processing Method and the Glycemic Indices of Ten Sweet Potato (Ipomoea batatas) Cultivars Commonly Consumed in Jamaica. J. of Nutr. Metab. 2011, 2011, 6. (16) Truong, V. D.; McFeeters, R. F.; Thompson, R. T.; Dean, L. L.; Shofran, B. Phenolic acid content and composition in leaves and roots of common commercial sweetpotato (Ipomea batatas L.) cultivars in the United States. J. Food Sci. 2007, 72, C343-349. (17) Xu, Z. Analysis methods of phenolic acids. In Analysis of antioxidant-rich phytochemicals, 1st Ed.; Xu, Z; Howard, L. R. Eds.; John Wiley& Sons. Inc.: UK, 2012; pp 69-74. (18) Huang, H.Y.; Hsieh, H.Y.; King, V. A. E.; Chi, L. L.; Tsen, J. H. To pre-challenge lactic acid bacteria with simulated gastrointestinal conditions is a suitable approach to studying potential probiotic properties. J. Microbiol. Methods, 2014, 107, 138-146. (19) Bel-Rhlid, R.; Thapa, D.; Kraehenbuehl, K.; Hansen, C. E.; Fischer, L. Biotransformation of caffeoyl quinic acids from green coffee extracts by Lactobacillus johnsonii NCC 533. AMB Express. 2013, 3, 28. (20) Szwajgier, D. Biotransformation of ferulic acid by Lactobacillus acidophilus K1 and selected Bifidobacterium strains. Acta Sci. Pol. Technol. Aliment. 2010, 9, 45-59. (21) Esteban-Torres, M.; Reveron, I.; Mancheno, J. M.; de Las Rivas, B.; Munoz, R. Characterization of a feruloyl esterase from Lactobacillus plantarum. Appl. Environ. Microbiol. 2013, 79, 5130-5136. (22) Olthof, M. R.; Hollman, P. C.; Katan, M. B. Chlorogenic acid and caffeic acid are absorbed in humans. J. Nutr. 2001, 131, 66-71.

19


Page 20 of 30

Page 21 of 30


(23) Longo, C.; Leo, L.; Leone, A. Carotenoids, fatty acid composition and heat stability of supercritical carbon dioxide-extracted-oleoresins. Int. J. Mol. Sci. 2012, 13, 4233. (24) Baxter, H.; Harborne, J. B.; Moss, G. P. Cabonhydrates and Lipids. In Phytochemical Dictionary: A Handbook of Bioactive Compounds from Plants, 2nd Ed.; Taylor & Francis. Philadelphia, PA, USA; 1998, pp 41-50. (25) Johnsson, T.; Nikkila, P.; Toivonen, L.; Rosenqvist, H.; Laakso, S. Cellular fatty acid profiles of lactobacillus and lactococcus strains in relation to the oleic Acid content of the cultivation medium. Appl. Environ. Microbiol. 1995, 61, 4497-4499. (26) Panickar, K.S.; Bhathena, S.J. Control of fatty acid intake and the role of essential fatty acids in cognitive function and neurological disorders. In: Montmayeur JP, le Coutre J (Eds.), Fat Detection: Taste, Texture, and Post Ingestive Effects. Boca Raton: CRC Press; 2010; pp 470-474 (27) Ishida, N. A method for simultaneous analysis of phytosterols and phytosterol esters in tobacco leaves using non aqueous reversed phase chromatography and atmospheric pressure chemical ionization mass spectrometry detector. J. Chromatogr. A. 2014, 1340, 99-108. (28) Sanders, M. E.; Klaenhammer, T. R. Invited review: the scientific basis of Lactobacillus acidophilus NCFM functionality as a probiotic. J. Dairy Sci. 2001, 84, 319-331. (29) Rocha, L. D.; Monteiro, M.C.; Teodoro, A. J. Anticancer properties of hydroxycinnamic acids-a review. J Cancer Res Clin Oncol. 2012, 1, 109-121. (30) Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter, P. Membrane Transport of Small Molecules and the Electrical Properties of Membranes. In Molecular Biology of the Cell, 4th ed.; Garland Science: New York, NY, 2002; pp 1-36. (31) Manach, C.; Scalbert, A.; Morand, C., Rémésy, C.; Jiménez, L. Polyphenols: food sources and bioavailability. Am. J. Clin. Nutr. 2004, 79, 727-747. (32) Castelli, F.; Uccella, N.; Trombetta, D. Saija, A. Differences between coumaric and cinnamic acids in membrane permeation as evidenced by time-dependent calorimetry. J. Agric. Food Chem. 1999, 47, 991-995. (33) Wen, S.; Zhu, D.; Huang, P. Targeting cancer cell mitochondria as a therapeutic approach. Future Med. Chem. 2013, 5, 53-67. (34) Aslan, E.; Guler, C.; Adem, S. In vitro effects of some flavonoids and phenolic acids on human pyruvate kinase isoenzyme M2. J. Enzyme Inhib. Med. Chem. 2015, 23, 1-4.

20



(35) Awad, A. B.; Fink, C. S. Phytosterols as anticancer dietary components: evidence and mechanism of action. J. Nutr. 2000, 130, 2127-2130. (36) van Breemen, R. B.; Pajkovic, N. Multitargeted therapy of cancer by lycopene. Cancer Lett. 2008, 269, 339-351.

21


Page 22 of 30

Page 23 of 30


Table 1 pH values and colony-forming units (CFU) of Lactobacillus acidophilus in sweet potatoes before (0 h) and after (24 h) fermentation Time pH Value 6

Visible cell (x10 CFU/mL)

0h

24 h

6.20 ± 0.15 a

3.45 ± 0.02 b

3.80 ± 0.01 a

7.48 ± 0.67 b

Values with different letters in a row are significantly different at p

Increases in Phenolic, Fatty Acid, and Phytosterol Contents and

Recommend Documents