Immunomodulatory Effects of Enzymatic-Synthesized Î±

Subscriber access provided by UNIV OF DURHAM

Food and Beverage Chemistry/Biochemistry

Immunomodulatory Effects of Enzymatic Synthesized #Galactooligosaccharides and Evaluation of the Structure-activity Relationship Zhuqing Dai, Wanyong Lyu, Xiaoli Xiang, Yuhong Tang, Bing Hu, Shiyi Ou, and Xiaoxiong Zeng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01939 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 8, 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 36

Journal of Agricultural and Food Chemistry

Immunomodulatory Effects of Enzymatic Synthesized α-Galactooligosaccharides and Evaluation of the Structure-activity Relationship

Zhuqing Dai,†,‡,ǁ Wanyong Lyu,§,ǁ Xiaoli Xiang,† Yuhong Tang,† Bing Hu,† Shiyi Ou, ¶

†

and Xiaoxiong Zeng†,*

College of Food Science and Technology, Nanjing Agricultural University, Nanjing

210095, People’s Republic of China ‡

Institute of Farm Product Processing, Jiangsu Academy of Agricultural Sciences,

Nanjing 210014, People’s Republic of China §

Nutrition and Food Branch of China Association of Gerontology and Geriatrics,

Beijing 100050, People’s Republic of China ¶

Department of Food Science and Engineering, Jinan University, Guangzhou

510632, Guangdong, People’s Republic of China

*To whom correspondence should be addressed. Tel & Fax: +86 25 84396791; E-mail: [email protected] (X. Zeng). ǁ These authors contributed equally to this study and share first authorship. 1

ACS Paragon Plus Environment


Page 2 of 36

1

ABSTRACT

2

In this study, α-galactooligosaccharides (α-GOSs) were synthesized by using

3

galactose as the substrate and α-galactosidase from Aspergillus niger as the catalyst.

4

In the reaction, synthesized products of U1, U2, U3 and U4 were detected by HPLC.

5

By mass spectrometry, NMR and 1-phenyl-3-methyl-5-pyrazolone derivatization, U1

6

was

7

α-D-Galp-(1→2)-α-D-Gal, α-D-Galp-(1→3)-α-D-Gal, α-D-Galp-(1→4)-α-D-Gal;

8

U2 was identified to be α-D-Galp-(1→6)-α-D-Gal; U3 was the mixture of

9

galacto-trisaccharides linked by one α-(1→6)-glycosidic linkage and one other

the

mixture

of

disaccharides

linkage;

of

was

α-D-Galp-(1→1)-α-D-Gal,

10

α-glycosidic

11

α-D-Galp-(1→6)-α-D-Galp-(1→6)-α-D-Gal. Afterwards, the synthesized α-GOSs

12

(U1, U2, U3, U4 and their mixture) as well as α-GOSs (manninotriose, stachyose,

13

ciceritol and verbascose) obtained from natural materials were used as subjects to

14

evaluate their immunomodulatory effects in vitro by culturing mouse macrophage

15

RAW264.7. The results showed that α-GOS with higher degree of polymerization

16

had better immunomodulatory activity, while to a certain extent, α-GOS linked with

17

α-(1→6)-galactosidic linkage showed better immunomodulatory effect.

18

KEYWORDS: α-Galactooligosaccharide; Enzymatic synthesis; Immunomodulatory

19

effect; Structure-activity relationship

U4

2


identified

as

Page 3 of 36


20

INTRODUCTION

21

α-Galactooligosaccharides (α-GOSs) are typical functional oligosaccharides that

22

comprised of 2-10 monosaccharide units with α-galactosidic linkages. A variety of

23

beneficial health effects has been reported for this group of compounds, such as

24

regulating gut microflora, reducing the risk of cancer, enhancing cardiovascular

25

function, improving immune activities, maintaining urinary tract health, modulating

26

inflammatory response, lowering blood pressure and blood lipids, resisting bacterial

27

and virus and preventing osteoporosis.1-5 Currently, most of the well documented

28

α-GOSs are present in natural legumes or Chinese herbal medicines.2,6 Thus, most of

29

the reported α-GOSs are extracted from raw materials, and there is relatively little

30

research on their enzymatic synthesis.

31

GOSs can be divided into β-GOSs and α-GOSs, both considered to be prebiotics.

32

Meanwhile, it has become a mature technology for commercial production of

33

β-GOSs by enzymatic synthesis. Related reports have been widely published, for

34

example, using lactose as the substrate and β-galactosidase as the catalyst have

35

become a typical method to synthesize β-GOSs.7,8 It has been reported that different

36

sources of β-galactosidase could mediate the formation of different types of

37

β-glycosidic bond. For example, β-galactosidase from Aspergillus oryzae catalyzed

38

the systhesis of β-GOSs with β-(1→6)-glycosidic linkge; β-galactosidase from

39

Bifidobacterium

40

β-(1→3)-glycosidic linkge; β-galactosidase from Bacillus circulans mediated the

41

synthesis of β-GOSs with β-(1→4)-glycosidic linkge.9-11 Compared to this, the study

bifidum

catalyzed

the

production

3


of β-GOSs

with


42

of enzymatic synthesis of α-GOSs is much less than that of β-GOSs.12-16 For

43

example, raffinose was synthesized with sucrose and galactose as the substrates with

44

α-galactosidase from Absidia corymbifera as the catalyst.13 It has been reported that

45

α-galactosidase from A. niger APC-9319 could effectively catalyze a reverse

46

reaction of galactose to produce α-GOSs.14 The α-GOSs contained 58%

47

α-galactobiose, 28% α-galactotriose, and 14% oligosaccharides larger than

48

α-galactotriose. However, the specific structures of α-GOSs were unknown in the

49

study. Therefore, α-GOSs were synthesized and characterized in this study to further

50

understand their biological function.

51

The human immune system is an important defense system that maintains the

52

integrity of the body and prevents the body from being invaded by microorganisms.

53

Macrophages are important immune cells of the innate immune system of the body,

54

and have various immune effects such as phagocytosis of foreign substances,

55

secretion of immune factors and processing of antigens. A series of immune

56

functions of macrophages are mainly achieved by the release of proinflammatory or

57

chemical agents such as nitric oxide (NO), prostaglandin E2 (PGE2), interferon (IFN)

58

and interleukin (IL).17-19 The release of these proinflammatory factors is also an

59

important way to maintain self-stabilization when the body is injured.20 Functional

60

oligosaccharides have been reported to promote the proliferation of bifidobacterium

61

and other probiotics while inhibit the growth of pathogen.2,3,21 Their prebiotic

62

function may play an important role in regulating the immune response.3,22-25

63

However, most of the existing studies of α-GOSs used mixture of α-GOSs extracted 4


Page 4 of 36

Page 5 of 36


64

from natural materials, which lacked the inquiry of structure-activity relationship.

65

Therefore, both synthesized α-GOSs and natural α-GOSs (manninotriose, stachyose,

66

ciceritol and verbascose) from raw materials were used as subjects to evaluate their

67

immunomodulatory activities in the present study. This study was aimed to provide a

68

theoretical basis for further study of the immunomodulatory activity of α-GOSs and

69

the structure-activity relationship.

70

MATERIALS AND METHODS

71

Materials and Chemicals. α-D-galactosidase from A. niger DS was got from

72

Amano Enzyme Co., Ltd. (Nagoya, Japan). p-Nitrophenyl-α-D-galactopyranoside

73

(Galα-pNP), 1-phenyl-3-methyl-5-pyrazalone (PMP), galactose and activated

74

charcoal (Darco G-60, 100 mesh) were purchased from Sigma-Aldrich Chemical Co.

75

(St. Louis, MO, USA). Celite 535 was purchased from Fluka Co. (Buchs,

76

Swiltzerland). Acetonitrile (grade of high performance liquid chromatography

77

(HPLC)) was purchased from Hanbon Science and Technology Co., Ltd. (Jiangsu,

78

China). Membrane filters (0.45 µm) were obtained from Millipore Co. (Bedford,

79

MA, USA). All other chemicals were analytical grade.

80

Assay of α-D-Galactosidase Activity. The α-D-galactosidase activity was

81

measured according to previous report,26 and one unit of enzyme activity was

82

defined as the amount of enzyme (product weight) hydrolyzing 1 µmol of pNP from

83

Galα-pNP per minute. In brief, the reaction was carried out in 2.0 mL solution of 1.0

84

mM Galα-pNP (dissolved in pH 5.5 sodium acetate buffer) with an appropriate

85

amount of enzyme. After incubated at 37 ℃ for 10 min, the reaction was stopped 5



86

by adding 8.0 mL 0.5 M Na2CO3 solution and the absorbance (Abs) at 405 nm of

87

reaction mixture was measured spectrophotometrically. As a result, the activity of

88

α-D-galactosidase was 9.48 U/mg.

89

Synthesis of α-GOS. Galactose was dissolved in distill water and treated under

90

125 ℃ and 110 kPa conditions for 20 min, affording a 96% supersaturated

91

galactose solution. The galactose solution was then transferred to a 60 ℃ water

92

bath and cooled to the reaction temperature. A certain amount of α-D-galactosidase

93

(35U/g galactose) dissolved in pH 5.5 sodium acetate buffer was added and the

94

galactose solution was adjust to a concentration of 90%. The reaction was then

95

activated at 60 ℃ once the enzyme was added, and the reaction was monitored by

96

interval sampling and analysis with HPLC. For termination the reaction, the reaction

97

mixture was heated for 10 min under 100 ℃. All assays were run in triplicate.

98

Chromatographic Determination of Reaction Products. An Agilent 1100 series

99

HPLC system with a refraction index detector (RID) was used to analyze the

100

reaction mixture. The separation of sugars was completed on a Sugar-D column (4.6

101

mm × 250 mm, Nacalai Tesque Inc., Tokyo, Japan). The mobile phase was

102

acetonitrile-water (75:25, v/v) at a flow rate of 1.0 mL/min. Injection volume was 20

103

µL. Standard sugars were used to identify the peaks by comparing the retention

104

times and to quantify the reaction products by an external calibration curve using its

105

corresponding standard solutions. The synthetic yield was defined as the percentage

106

of the amount of produced products to the amount of galactose added. The standard

107

sugars were isolated and purified from reaction mixture as described below. 6


Page 6 of 36

Page 7 of 36


108

Purification and Characterization of Synthesized α-GOSs. The reaction solution

109

was centrifuged at 5000 g for 10 min, the resulting supernatant was filtered through

110

a 0.45 µm filter and then loaded onto an activated charcoal-Celite preparative

111

column (3.0 × 50 cm, Yamazen Co., Osaka, Japan). The column was eluted with a

112

linear gradient ethanol-water solution (from 0 to 20%) at a flow rate of 4 mL/min.

113

The eluent was collected (10 mL/tube) and monitored by phenol-sulfuric acid

114

method. Those with sugars were further analyzed by HPLC, collected separately and

115

concentrated by rotate evaporation. Briefly, galactose was eluted at 4-5% (v/v)

116

aqueous ethanol solution, U1 was eluted with 9-10% aqueous ethanol solution, U2

117

was eluted with 8% aqueous ethanol solution, U3 was eluted with 10-11% aqueous

118

ethanol solution and U4 was eluted with 13% aqueous ethanol solution. As some

119

fractions could not be completely separated by activated charcoal-Celite preparative

120

column, they were further separated by polyacrylamide Biogel P-2 column (1.5 × 90

121

cm, BioRad, Richmond, USA). The column was eluted by ultrapure water at a flow

122

rate of 0.25 mL/min, and the eluates were treated as mentioned above. As results,

123

four fractions were collected, concentrated and frzzed-dried, affording U1, U2, U3

124

and U4 as white powder.

125

The structures of synthesized products were identified by mass spectrometry

126

with an electrospray ionization source (ESI-MS) and nuclear magnetic resonance

127

spectroscopy (NMR). The Mariner system 5304 mass spectrometer (Applied

128

Biosystems, Foster City, CA, USA) was used to obtain MS spectra by direct

129

injection. The Bruker Avance DRX-500 spectrometer (Bruker, Karlsruhe, Germany) 7



130

was used to record 1H and

13

131

D2O. The residual solvent signal was used as internal standard. The chemical shifts

132

(δ) are given in ppm, and coupling constants (J) are given in Hz.

C NMR spectra at 300 K. Samples were dissolved in

133

Analysis of PMP Derivatives. The four synthetic products (U1, U2, U3 and U4)

134

were further analyzed by PMP derivatization according to reported method.27 Briefly,

135

100 µL of 5 mg/mL product solution was mixed with 100 µL of 0.6 M NaOH

136

solution, half of the mixture was then derivatized with 100 µL of 0.5 M PMP

137

solution (dissolved in methanol) for 2 h at 70 ℃. After derivatization, 0.3 M HCl

138

solution was used to neutralize the reaction solution. The resulting solution was

139

concentrated by rotate evaporation to dryness, and the residue was dissolved with

140

distilled water and chloroform was added to extract the water phase. After filtered

141

through a 0.45 µm membrane, the water phase was analyzed by an Agilent HPLC

142

system equiped with Eclipse Plus C18 column (4.6 × 250 mm, 5 µm, Agilent) and

143

diode array detector at a flow rate of 1.0 mL/min at 30 ℃. The mobile phase was

144

consisted of 83% phosphate-buffered saline (PBS, 0.1 M, pH 6.7) and 17%

145

acetonitrile (v/v).

146

Cell Culture. The murine macrophage RAW264.7 cells were cultured in complete

147

DMEM (supplemented with 10% newborn calf serum, 100 IU/mL penicillin, and

148

100 IU/mL streptomycin) and incubated in a humidified 5% CO2 incubator at 37 ℃.

149

The adherent cells were kept for cell passage and used for further culture when the

150

cells were spread over 80% of the bottom of the culture dishes.

151

Assay of RAW264.7 Cell Viability. The RAW264.7 cell viability was measured 8


Page 8 of 36

Page 9 of 36


152

according to the MTT-based colorimetric method.28 Briefly, the RAW264.7 cells

153

were seeded into a 96-well flat-bottom plate (100 µL/well) when suspended at a

154

density of 1 × 106 cells/mL in DMEM. They were incubated under 5% CO2 at 37 ℃

155

for 12 h. The nonadherent cells were washed with PBS twice. After that, 100 µL of

156

different samples dissolved in DMEM with a series of concentrations (final

157

concentrations of 25.0, 50.0, 100.0, 200.0 and 400.0 µg/mL) were added to each well,

158

respectivrly. Positive control (10.0 µg/mL LPS) and blank control (complete DMEM

159

alone) were also added. The cells were incubated as mentioned above for 12, 24, 36

160

and 48 h, respectively. After each incubation, each well was added 200 µL of MTT

161

solution (0.5 mg/mL) and then incubated further at 37 ℃ for 4 h. Finally, 150 µL of

162

DMSO was added to each well to dissolve formazan crystals. A Synergy 2

163

Multimode Microplate Reader (BioTeK Instruments, Inc., Winooski, VT, USA) was

164

used to measure the Abs at 570 nm. The cell viability rate was calculated according

165

to the formula below:

166

Cell viability = Abssample/Absblank control

167

Assay of Phagocytosis of RAW264.7 Cells. The cell phagocytosis was measured

168

according to the reported method29 with modification. The prior stage culture of

169

RAW264.7 cells was done as described above. The samples at concentrations of 25.0,

170

50.0, 100.0, 200.0 and 400.0 µg/mL were added to a 96-well plate (100 µL/well).

171

Positive control (10.0 µg/mL LPS) and blank control (complete DMEM alone) were

172

also added. The plate was incubated at 37 ℃ in a 5% CO2 incubator for 24 h.

173

Subsequently, each well (100 µL/well) was added with 0.075% of neutral red 9



174

solution and further incubated for 1 h. Then, the wells were washed with PBS twice

175

to remove excess neutral red. After that, each well was added with 100 µL of cell

176

lysate (0.1 M glacial acetic acid:ethanol=1:1, v/v), and the plate was incubated

177

overnight at 37 ℃. Finally, the Abs at 540 nm of each well was determined and the

178

phagocytosis index was calculated by using the following equation:

179

Phagocytosis index = Abssample/Absblank control

180

Assay of NO and Cytokines. The NO amount released by peritoneal macrophages

181

was measured by an NO assay kit from Nanjing Jiancheng Bioengineering Institute

182

(Nanjing, China). The levels of cytokines (IL-6, IL-1β and IFN-γ) were measured by

183

using ELISA kits (Nanjing Jiancheng Bioengineering Institute) according to the

184

manufacturer’s protocol.

185

Statistical Analysis. The data are expressed as means ± SD and subjected to

186

one-way analysis of variance (ANOVA). Duncan’s new multiple range test was

187

performed to determine the significant difference using SPSS 18.0 software (SPSS

188

Inc., Chicago, IL, USA).

189

RESULTS AND DISCUSSION

190

Synthesis and Characterization of α-GOS. As shown in Figure 1, 4 peaks for

191

synthesized products were observed in the HPLC chromatogram by using galactose

192

as the substrate and α-galactosidase as the catalyst. The products were isolated from

193

the reaction mixture by using activated charcoal-Celite column and Biogel P-2

194

column to afford fractions of U1, U2, U3 and U4, respectively. Purified U1, U2, U3

195

and U4 were used to draw the standard curves by HPLC according to the peak area 10


Page 10 of 36

Page 11 of 36


196

and sample concentration. The concentration of each synthetic product was

197

calculated from the standard curve. As a result, the synthetic yield was 25%. Among

198

the synthesized products, U1 accounted for 32%, U2 accounted for 40%, U3

199

accounted for 15% and U4 accounted for 13%. The yield of α-GOSs synthesis by

200

enzymatic method is usually less than 30%. For example, in a enzymatic reaction

201

using α-galactosidase from Lactobacillus reuteri as the catalyst, a maximum yield of

202

26% (w/v) trisaccharide was achieved using melibiose as the substrate.16 Improved

203

enzymatic synthesis for higher yields needs to be further investgated.

204

The MS analysis of synthetic products is shown in Figure 2. The mass spectra of

205

U1 and U2 showed significant peak at m/z 365 for [M+Na]+, indicating that their

206

molecular weights were 342 with disaccharide structure. The mass spectra of U3 and

207

U4 gave peak at m/z 538.9 for [M+Cl]-, implying their molecular weights were 502.9

208

with trisaccharide structure. In general, the shift of α-pyranoside bond on the C-1 is

209

more than δ 5.0 ppm. Meanwhile, the coupling constant J of α-pyranoside bond is

210

usually between 2-4 Hz, while the β-pyranoside bond is usually between 7-9 Hz,

211

which will be able to distinguish between the two different types of linkage. The

212

shift of the C displacement in 13C NMR is also different. If substitution occurs, the

213

C-1 anomeric carbon shifts its chemical shifts from δ 90-95 ppm to δ 97-103 ppm,

214

the C-6 anomeric carbon shifts its chemical shifts from δ 60-65 ppm to δ 67-70

215

ppm.10 As shown in Figure 3, the characteristic signals of U2 at δ 5.27 ppm (J = 3.8

216

Hz) and 5.02-4.97 ppm (J = 3.48 Hz) in the 1H NMR spectrum indicated that the

217

galactosyl residues were connected through α-linkage. Furthermore, in the 13C NMR 11



218

spectrum of U2, there was only one signal peak of C-6 at δ 61.18 ppm. Therefor, U2

219

was identified to be α-D-Galp-(1→6)-α-D-Gal. Similar to U2, the 1H NMR

220

spectrum of U4 (Figure 4) showed the characteristic chemical shift signals of

221

galactose residues at δ 5.27 ppm (J = 3.8 Hz), 5.02 ppm (J = 3.3 Hz) and 4.98 ppm

222

(J = 3.7 Hz), indicating that the glycosidic bonds of U4 were also α-linkages.

223

Meanwhile, according to its

224

61.19 ppm, U4 was identified as α-D-Galp-(1→6)-α-D-Galp-(1→6)-α-D-Gal. The

225

results indicated that the synthesized products were mainly linked with

226

α-(1→6)-glycosidic linkage, which is consistant with previous report.16 However,

227

the 1H and

228

(shown in supplementary material, Figures S1 and S2), suggesting that they were

229

not single compound. Therefor, the PMP derivatization was carried out to further

230

determine their compositions.

13

13

C NMR spectrum, only one signal peak of C-6 at δ

C NMR spectra of U1 and U3 showed miscellaneous information

231

PMP can react with reducing sugars under mild conditions and the products have

232

no stereoisomer with a strong UV absorption at 245 nm. After derivatized by PMP,

233

the hydrophobicity of reduced sugar improved while the derivative is charged and

234

can be separated well afterwards. As shown in Figure 5, U2 and U4 showed only

235

one peak after derivatization (the retention time at 15.6 min for PMP, the retention

236

time at 43 min for galactose), while U1 had four peaks (except for the overlapped

237

peak with U2). Since α(1→5)-glycosidic linkage can not be formed, it is therefore

238

assumed

239

α-D-Galp-(1→2)-α-D-Gal, α-D-Galp-(1→3)-α-D-Gal, α-D-Galp-(1→4)-α-D-Gal.

that

U1

was

the

mixture

of

12


α-D-Galp-(1→1)-α-D-Gal,

Page 12 of 36

Page 13 of 36


240

U3 showed two peaks after PMP derivatization (except for the PMP peak), as the

241

α-(1→6)-glycosidic bond occupied the highest proportion in the products, U3 was

242

speculated as the mixture of galacto-trisaccharides linked by a α-(1→6)-glycosidic

243

bond and an other type α-glycosidic linkage.

244

Effect of α-GOS on Macrophage Viability. The cell viability of macrophage is

245

an indicator of immune activation and is the basis for subsequent experiments. The

246

effects of different treatment groups on cell viability are shown in supplementary

247

material (Table S1), and the cell proliferation index reached the maximum when

248

treated for 24 h in almost all treatment groups. After 24 h, the cell proliferation index

249

decreased with the increase of culture time. Therefor, the treatment time was set at

250

24 h for the following experiments. When compared with different concentrations,

251

the cell proliferation index reached the maximum at concentration of 100 or 200

252

µg/mL. By comparing the effect of different samples, it was found that all α-GOSs

253

treatments promoted the proliferation of RAW264.7 cells, while no significant

254

differences was observed between differnet α-GOS treatments.

255

Effect of α-GOS on Macrophage Phagocytosis. Activated macrophages not only

256

involve in specific and non-specific immune regulation, but also play an important

257

role in the connection between the two immune responses. The macrophage

258

phagocytosis is a significant sign of macrophage activation.30 As shown in Table 1,

259

when the concentrations of different α-GOS treatments were less than 200 µg/mL,

260

the phagocytosis index increased with the increase of the sample concentrations,

261

showing a dose-dependent relationship. Comparing different α-GOS treatments on 13



262

cell phagocytosis at the concentration of 200 µg/mL, all the α-GOS treatments

263

showed higher phagocytosis index than positive control, while ciceritol and

264

verbascose exhibited significant higher phagocytosis than other α-GOSs (p < 0.05).

265

The results indicated that ciceritol and verbascose had better effects in promoting

266

macrophage phagocytosis than other structure of α-GOS.

267

Effects of α-GOS on Production of NO and Cytokines. Once activated, the

268

macrophages will release NO and a large number of cytokines, such as IFN and

269

PGE2, which play an important role in against exogenous pathogens and

270

anti-cancer.31 NO is a neurotransmitter modulator that acts as a vasodilator as well as

271

pathogens resistant. NO is produced by L-arginine under the action of inducible

272

nitric oxide synthase (iNOS).32 It has been reported that the immune reponses of

273

anti-tumor, antiviral and inflammatory modulation are closely linked to the release of

274

NO.33 As shown in Table 2, the treatment of α-GOSs at concentration of 200 or 400

275

µg/mL showed better effect on reseasing NO. The effect of α-GOSs on promotion of

276

NO also exhibited a dose-dependent manner. When compared the treatment groups,

277

the levels of NO in α-GOSs groups were lower than those of positive control at low

278

concentrations. However, when the treatment concentration reached 200 µg/mL,

279

they showed similar effect as positive control. Among the α-GOS treatments, U4,

280

stachyose and verbascose significantly protomted the release of NO than other

281

α-GOS groups (p < 0.05). For the four synthesized products, U2 showed

282

significantly higher level of NO than U1 at 100 and 400 µg/mL concentrations; U4

283

had significantly higher level of NO than U3 at 200 and 400 µg/mL concentrations 14


Page 14 of 36

Page 15 of 36


284

(p < 0.05).

285

Cytokines are small molecule proteins that are immunologically regulated by

286

immune cells. TNF-α and IL-1β are considered as "early response cytokines".

287

Upregulation of these cytokine on the inner cell wall may contribute to the migration

288

of phagocytic cells to the injury site.34,35 In addition, IFN-γ may promote antigen

289

presentation, cytokine production, the lethal effect of monocyte-macrophages and

290

natural killer cells.36 The effects of different treatments on TNF-α, IL-1β and IFN-γ

291

secretion are shown in Table 3-5. The levels of TNF-α, IFN-γ and IL-1β in α-GOS

292

treatment groups were significantly higher than those of blank control. Besides, all

293

treatment groups showed the highest levels of cytokines when treated at

294

concentration of 200 µg/mL. When the concentration was less than 200 µg/mL, the

295

levels of TNF-α, IFN-γ and IL-1β increased with the increase of treatment

296

concentratios, showing a dose-dependent relationship. The results showed that the

297

production of TNF-α in U3, U4, stachyose and verbascose groups was significantly

298

higher than that of other treatment groups (p < 0.05). Stachyose, ciceritol and

299

verbascose treatments significantly promoted the secretion of IL-1β compared with

300

other groups (p < 0.05). Meanwhile, U4, stachyose and verbascose exhibited better

301

effect in promoting the release of IFN-γ. Comparing the effects of four synthesized

302

products on production of TNF-α, IFN-γ and IL-1β, it revealed that U2 showed

303

better effects than U1, and U4 showed better effects than U3. However, the

304

differences were not statically significant in some cases.

305

In this study, it was found that α-GOSs significantly promoted macrophages 15



306

phagocytosis and secretion of NO, TNF-α, IFN-γ and IL-1β when treating

307

macrophages RAW264.7 in vitro. It also showed a dose-dependent manner when the

308

concentration less than 200 µg/mL. The results indicated that α-GOSs had positive

309

immune regulation functions, which is consistant with our previous report.37 Related

310

reports also indicated that oligosaccharides promoted the secretion of these cytokines

311

in macrophages, and this immune regulation function was mainly through the

312

activation of a series of signaling pathways like toll-like receptor, nuclear factor and

313

the mitogen-activated protein kinase pathway.25,38-40 The prebiotic effect of GOSs

314

has been well studied and it has been reported that the degree of polymerization (DP),

315

the type of linkage, and the monosaccharide composition are the three factors

316

affecting their probiotic activity.41 In this study, we demnonstread that DP and the

317

type of linkage also affected greatly the immunomodulatory effects of α-GOSs.

318

When comparing the immunoregulatory function of α-GOSs with different

319

structures, it was found that verbascose showed the best effects, followed by

320

stachyose. In structure, verbascose and stachyose were both raffinose family

321

oligosaccharides, while verbascose has five monosaccharide units and stachyose has

322

four monosaccharide units. These results indicated that α-GOSs with higher DP

323

could exhit better immunomodulatory effect. It has also been reported that α-GOSs

324

with higher DP released more TNF-α.42 While comparing the effects of four

325

synthesized products on production of TNF-α, IFN-γ and IL-1β, U2 had better

326

effects than U1, U4 had better effects than U3. It is shown to a certain extent that

327

α-GOS with α-(1→6)-glycosidic linkage is more effective. However, this 16


Page 16 of 36

Page 17 of 36


328

phenomenon needs further investigation. Among these α-GOSs, U4, manninotriose

329

and ciceritol had similar structure that they all comprised of three monosaccharide

330

units with two galactose and one different monosaccharide end. Comparing the

331

effects of these three α-GOSs, U4 showed the best effects on NO, TNF-α and

332

IFN-γ promotion, indicating that oligosaccharides with galacose unit exibited better

333

immunomodulatory effect. However, manninotriose and ceritol secreted more IL-1β

334

than U4, which is contrary with previous results. Thus, the effect of monosaccharide

335

composition on immunomodulatoty activity needs to be further investigated.

336

In conclusion, α-GOSs were synthesized by using galactose as the substrate

337

under supersaturated concentration and A. niger α-galactosidase as the catalyst.

338

Among them, U1 was the mixture of disaccharides of α-D-Galp-(1→1)-α-D-Gal,

339

α-D-Galp-(1→2)-α-D-Gal, α-D-Galp-(1→3)-α-D-Gal, α-D-Galp-(1→4)-α-D-Gal;

340

U2 was α-D-Galp-(1→6)-α-D-Gal; U3 was the mixture of galacto-trisaccharides

341

linked by a α-(1→6) glycosidic bond and a other type of α-glycosidic linkage; U4

342

was α-D-Galp-(1→6)-α-D-Galp-(1→6)-α-D-Gal. Both the newly synthesized and

343

the naturally occurring α-GOSs exhibited immunomodulatory effects by promoting

344

the secreation of NO and immune cytokines. Furthermore, α-GOSs with higher DP

345

exhibited better immune regulation activity.

346

ASSOCIATED CONTENT

347

Supporting information

348

Figures for 1H and

349

treatments on RAW 264.7 cell proliferation at different treatment times.

13

C NMR spectra of U1 and U3. Table for effects of different

17



350

AUTHOR INFORMATION

351

Corresponding Author

352

*Phone: +86-25-84396791. E-mail: [email protected] (X Zeng).

353

ORCID

354

Xiaoxiong Zeng: 0000-0003-2954-3896

355

Funding

356

This work was partly supported by Grants-in-Aid for scientific research from the

357

National Natural Science Foundation of China (31171750), a project funded by the

358

Priority Academic Program Development of Jiangsu Higher Education Institutions

359

(PAPD) and a grant funded by Jiangsu Key Laboratory of Quality Control and

360

Further Processing of Cereals & Oils.

361

Notes

362

The authors declare no competing financial interest.

363

REFERENCES

364

(1) Li, T.; Lu, X.; Yang, X. Stachyose-enriched α-galacto-oligosaccharides

365

regulate gut microbiota and relieve constipation in mice. J. Agric. Food Chem. 2013,

366

61, 11825-11831.

367

(2) Abou Hachem, M.; Fredslund, F.; Andersen, J. M.; Larsen, R. J.; Majumder,

368

A.; Ejby, M.; Van Zanten, G.; Lahtinen, S. J.; Barrangou, R.; Klaenhammer, T.;

369

Jacobsen, S.; Coutinho, P. M.; Lo Leggio, L.; Svensson, B. Raffinose family

370

oligosaccharide utilisation by probiotic bacteria: insight into substrate recognition,

371

molecular architecture and diversity of GH36 α-galactosidases. Biocatal. 18


Page 18 of 36

Page 19 of 36


372

Biotransfor. 2012, 30, 316-325.

373

(3) Martinez-Villaluenga, C.; Frias, J.; Vidal-Valverde, C. Alpha-galactosides:

374

Antinutritional factors or functional ingredients? Crit. Rev. Food Sci. Nutr. 2008, 48,

375

301-316.

376

(4) Yang, X.; Zhao, Y.; He, N.; Croft, K. D. Isolation, characterization, and

377

immunological effects of α-galacto-oligosaccharides from a new source, the herb

378

Lycopus lucidus Turcz. J. Agric. Food Chem. 2010, 58, 8253-5258.

379 380

(5) Moreno, F. J.; Sanz, M. L. Food oligosaccharides: production, analysis and bioactivity. John Wiley & Sons: 2014.

381

(6) Horbowicz, M.; Obendorf, R. L. Seed desiccation tolerance and storability:

382

Dependence on flatulence-producing oligosaccharides and cyclitols-review and

383

survey. Seed Sci. Res. 1994, 4, 385-405.

384

(7) Sangwan, V.; Tomar, S. K.; Singh, R. R. B.; Singh, A. K.; Ali, B.

385

Galactooligosaccharides: Novel components of designer foods. J. Food Sci. 2011,

386

76, R103-R111.

387

(8) Yu, L.; O'Sullivan, D. J. Production of galactooligosaccharides using a

388

hyperthermophilic β-galactosidase in permeabilized whole cells of Lactococcus

389

lactis. J. Dairy Sci. 2014, 97, 694-703.

390 391

(9) Otieno, D. O. Synthesis of β-galactooligosaccharides from lactose using microbial β-galactosidases. Compr. Rev. Food Sci. Food Saf. 2010, 9, 471-482.

392

(10) Li, W.; Xiang, X.; Tang, S.; Hu, B.; Tian, L.; Sun, Y.; Ye, H.; Zeng, X. X.

393

Effective enzymatic synthesis of lactosucrose and its analogues by β-D-galactosidase 19



394 395

from Bacillus circulans. J. Agric. Food Chem. 2009, 57, 3927-3933. (11)

Panesar, P. S.; Panesar, R.; Singh, R. S.; Kennedy, J. F.; Kumar, H.

396

Microbial production, immobilization and applications of β-D-galactosidase. J.

397

Chem. Technol. Biot. 2006, 81, 530-543.

398 399 400 401

(12) Katrolia, P.; Rajashekhara, E.; Yan, Q. J.; Jiang, Z. Q. Biotechnological potential of microbial α-galactosidases. Crit. Rev. Biotechnol. 2014, 34(4), 307-317. (13) Baik, S. Synthesis of raffinose by fungal α-galacotosidase from Absidia corymbifera. Food Sci. Biotechnol. 2010, 19, 83-87.

402

(14) Yamashita, A.; Hashimoto, H.; Fujita, K.; Okada, M.; Mori, S.; Kitahata, S.

403

Reverse reaction of Aspergillus niger APC-9319 α-galactosidase in a supersaturated

404

substrate solution: Production of α-linked galactooligosaccharide (α-GOS). Biosci.

405

Biotechnol. Biochem. 2005, 69, 1381-1388.

406

(15) Nakai, H.; Baumann, M.; Petersen, B. O.; Westphal, Y.; Hachem, M. A.;

407

Dilokpmol, A.; Duus, J.; Schols, H. A.; Svensson, B. Aspergillus nidulans

408

α-galactosidase of glycoside hydrolase family 36 catalyses the formation of

409

α-galacto-oligosaccharides by transglycosylation. FEBS J. 2010, 277, 3538-3551.

410

(16) Tzortzis, G.; Jay, A. J.; Baillon, M. L.; Gibson, G. R.; Rastall, R. A.

411

Synthesis of α-galactooligosaccharides with α-galactosidase from Lactobacillus

412

reuteri of canine origin. Appl. Microbiol. Biotechnol. 2003, 63, 286-292.

413

(17)

Schmidt, H. H. H. W.; Walter, U. NO at work. Cell 1994, 78, 919-925.

414

(18)

Lorsbach, R. B.; Murphy, W. J.; Lowenstein, C. J.; Snyder, S. H.;

415

Russell, S. W. Expression of the nitric oxide synthase gene in mouse macrophages 20


Page 20 of 36

Page 21 of 36


416

activated for tumor cell killing — Molecular basis for the synergy between

417

interferon-γ and lipopolysaccharide. J. Biol. Chem. 1993, 268, 1908-1913.

418 419 420 421 422

(19)

Akira, S.; Uematsu, S.; Takeuchi, O., Pathogen recognition and innate

immunity. Cell 2006, 124, 783-801. (20)

Laskin, D. L.; Pendino, K. J. Macrophages and inflammatory mediators

in tissue injury. Annu. Rev. Pharmacol. Toxic. 1995, 35, 655-677. (21)

Ishizuka, S.; Iwama, A.; Dinoto, A.; Suksomcheep, A.; Maeta, K.;

423

Kasai, T.; Hara, H.; Yokota, A. Synbiotic promotion of epithelial proliferation by

424

orally ingested encapsulated Bifidobacterium breve and raffinose in the small

425

intestine of rats. Mol. Nutr. Food Res. 2009, 53, S62-S67.

426

(22)

Macfarlane, G. T.; Steed, H.; Macfarlane, S. Bacterial metabolism and

427

health-related effects of galacto-oligosaccharides and other prebiotics. J. Appl.

428

Microbiol. 2008, 104, 305-344.

429

(23)

Jeurink, P. V.; van Esch, B. C. A. M.; Rijnierse, A.; Garssen, J.;

430

Knippels, L. M. J. Mechanisms underlying immune effects of dietary

431

oligosaccharides. Am. J. Clin. Nutr. 2013, 98, 572S-577S.

432

(24)

Vulevic, J.; Drakoularakou, A.; Yaqoob, P.; Tzortzis, G.; Gibson, G. R.

433

Modulation of the fecal microflora profile and immune function by a novel

434

trans-galactooligosaccharide mixture (B-GOS) in healthy elderly volunteers. Am. J.

435

Clin. Nutr. 2008, 88, 1438-1446.

436 437

(25)

Ortega-Gonzalez, M.; Ocon, B.; Romero-Calvo, I.; Anzola, A.; Guadix,

E.; Zarzuelo, A.; Suarez, M. D.; de Medina, F. S.; Martinez-Augustin, O. 21



438

Nondigestible oligosaccharides exert nonprebiotic effects on intestinal epithelial

439

cells enhancing the immune response via activation of TLR4-NFκB. Mol. Nutr. Food

440

Res. 2014, 58, 384-393.

441

(26)

Manzanares, P.; de Graaff, L. H.; Visser, J. Characterization of

442

galactosidases from Aspergillus niger: purification of a novel α-galactosidase

443

activity. Enzyme Microb. Tech. 1998, 22, 383-390.

444

(27)

Yuan, Q. X.; Zhao, L.; Cha, Q.; Sun, Y.; Ye, H.; Zeng, X. X. Structural

445

characterization and immunostimulatory activity of a homogeneous polysaccharide

446

from Sinonovacula constricta. J. Agric. Food Chem. 2015, 63, 7986-7994.

447

(28)

Luo, J.; Liu, J.; Ke, C.; Qiao, D.; Ye, H.; Sun, Y.; Zeng, X. X.

448

Optimization of medium composition for the production of exopolysaccharides from

449

Phellinus baumii Pilát in submerged culture and the immuno-stimulating activity of

450

exopolysaccharides. Carbohyd. Polym. 2009, 78, 409-415.

451

(29)

Zhang, W.; Yang, J.; Chen, J.; Hou, Y.; Han, X. Immunomodulatory and

452

antitumour effects of an exopolysaccharide fraction from cultivated Cordyceps

453

sinensis (Chinese caterpillar fungus) on tumour-bearing mice. Biotechnol. Appl.

454

Biochem. 2005, 42, 9-15.

455

(30)

Chen, X.; Lu, J.; Zhang, Y.; He, J.; Guo, X.; Tian, G.; Jin, L. Studies of

456

macrophage immuno-modulating activity of polysaccharides isolated from

457

Paecilomyces tenuipes. Int. J. Biol. Macromol. 2008, 43, 252-256.

458 459

(31)

Karnjanapratum, S.; You, S. Molecular characteristics of sulfated

polysaccharides from Monostroma nitidum and their in vitro anticancer and 22


Page 22 of 36

Page 23 of 36


460 461 462 463 464 465 466 467

immunomodulatory activities. Int. J. Biol. Macromol. 2011, 48, 311-318. (32)

Boyle, J. J. Macrophage activation in atherosclerosis: pathogenesis and

pharmacology of plaque rupture. Curr. Vasc. Pharmacol. 2005, 3, 63-68. (33)

Mayer, B.; Hemmens, B. Biosynthesis and action of nitric oxide in

mammalian cells. Trends Biochem. Sci. 1997, 22, 477-481. (34)

Laskin, D. L.; Laskin, J. D. Role of macrophages and inflammatory

mediators in chemically induced toxicity. Toxicology 2001, 160, 111-118. (35)

Commins, S. P.; Borish, L.; Steinke, J. W. Immunologic messenger

468

molecules: Cytokines, interferons, and chemokines. J. Allergy Clin. Immun. 2010,

469

125, S53-S72.

470

(36)

Riquelme, P.; Tomiuk, S.; Kammler, A.; Fandrich, F.; Schlitt, H. J.;

471

Geissler, E. K.; Hutchinson, J. A. IFN-γ-induced iNOS expression in mouse

472

regulatory macrophages prolongs allograft survival in fully immunocompetent

473

recipients. Mol. Ther. 2013, 21, 409-422.

474

(37) Dai, Z.; Su, D.; Zhang, Y.; Sun, Y.; Hu, B.; Ye, H.; Jabbar, S.; Zeng, X. X.

475

Immunomodulatory activity in vitro and in vivo of verbascose from mung beans

476

(phaseolus aureus). J. Agric. Food Chem. 2014, 62, 10727-10735.

477

(38)

Xu, X.; Wu, X.; Wang, Q.; Cai, N,; Zhang, H. X.; Jiang, Z. D.; Wan,

478

M.; Oda, T. Immunomodulatory effects of alginate oligosaccharides on murine

479

macrophage RAW264.7 cells and their structure–activity relationships. J. Agric.

480

Food Chem. 2014, 62, 3168-3176.

481

(39)

Fang, H. Y.; Chen, Y. K.; Chen, H. H.; Lin, S. Y.; Fang, Y. T. 23



482

Immunomodulatory effects of feruloylated oligosaccharides from rice bran. Food

483

Chem. 2012, 134, 836-840.

484

(40)

Dang, Y.; Li, S.; Wang, W.; Wang, S.; Zou, M.; Guo, Y.; Fan, J.; Du, Y.;

485

Zhang, J. The effects of chitosan oligosaccharide on the activation of murine spleen

486

CD11c+ dendritic cells via Toll-like receptor 4. Carbohyd. Polym. 2011, 83,

487

1075-1081.

488

(41) Oh, S. Y.; Youn, S. Y.; Park, M. S.; Baek, N. I.; & Ji, G. E. Synthesis of

489

stachyobifiose using bifidobacterial α-galactosidase purified from recombinant

490

Escherichia coli. J. Agric. Food Chem. 2014, 66, 1184-1190.

491

(42) Searle, L. E.; Jones, G.; Tzortzis, G.; Woodward, M. J.; Rastall, R. A.;

492

Gibson, G. R.; La Ragione, R. M. Low molecular weight fractions of BiMuno® exert

493

immunostimulatory properties in murine macrophages. J. Funct. Foods. 2012, 4,

494

941-953.

495

24


Page 24 of 36

Page 25 of 36


Figure Captions Figure 1. Determination of enzymatic synthesis reaction products by HPLC Figure 2. ESI-MS spectra of synthetic products of U1 (A), U2 (B), U3 (C) and U4 (D) Figure 3. 1H (A) and 13C NMR (B) spectra of product U2 Figure 4. 1H (A) and 13C NMR (B) spectra of product U4 Figure 5. HPLC chromatograms of PMP derivatives of U1, U2, U3 and U4

25



Page 26 of 36

Table 1. Effects of Different Treatments on RAW264.7 Cell Phagocytosis Index Treatments

Cell phagocytosis index 25 µg/mL

50 µg/mL

100 µg/mL

NC

1.00 ± 0.11

LPS

1.19 ± 0.02

α-GOS mixture

1.26 ± 0.09 cd, A

1.26 ± 0.03 bc, A

200 µg/mL

400 µg/mL

1.27 ± 0.07 ab, A

1.33 ± 0.11 a, A

1.26 ± 0.12 abc, A

U1

1.13 ± 0.03 a, A

1.22 ± 0.06 ab, B

1.21 ± 0.07 a, B

1.24 ± 0.05 a, B

1.19 ± 0.04 a, AB

U2

1.16 ± 0.03 abc, A

1.21 ± 0.04 ab, AB

1.23 ± 0.04 ab, AB

1.25 ± 0.08 a, B

1.22 ± 0.05 ab, AB

U3

1.15 ± 0.03 ab, A

1.21 ± 0.02 ab, B

1.24 ± 0.02 ab, BC

1.30 ± 0.06 a, D

1.27 ± 0.03 bc, CD

U4 Manninotriose

1.24 ± 0.06 bc, A

1.29 ± 0.06 c, A

1.28 ± 0.03 b, A

1.30 ± 0.03 a, A

1.27 ± 0.03 abc, A

1.19 ± 0.05 abc, A

1.21 ± 0.04 ab, AB

1.24 ± 0.06 ab, AB

1.28 ± 0.07 a, B

1.22 ± 0.03 ab, AB

Stachyose

1.16 ± 0.1 abc, A

1.17 ± 0.02 a, A

1.23 ± 0.05 ab, AB

1.28 ± 0.06 a, B

1.22 ± 0.08 ab, AB

Ciceritol

1.49 ± 0.15 e, ABC

1.54 ± 0.03 e, BC

1.60 ± 0.07 d, C

1.43 ± 0.05 b, AB

1.40 ± 0.03 d, A

1.36 ± 0.09 d, A

1.38 ± 0.04 d, A

1.46 ± 0.09 c, A

1.61 ± 0.12 c, B

1.33 ± 0.08 cd, A

Verbascose

a-d represents significant differences between different α-GOS treatments of same concentration (P < 0.05). A-D represents significant differences between different concentrations of same α-GOS treatment (P < 0.05).

26


Page 27 of 36


Table 2. Effects of Different Treatments on NO Level of RAW264.7 Cell Treatments

NO Production（µmol/L） 25 µg/mL

50 µg/mL

100 µg/mL

NC

10.4 ± 1.8

LPS

36.4 ± 2.1

α-GOS mixture

200 µg/mL

15.1 ± 0.7 ab, A

26.8 ± 0.9 bc, B

30.5 ± 2.5 bcd, C

32.4 ± 1.8 a, C

400 µg/mL

33.0 ± 1.8 ab, C

U1

12.1 ± 2.5 a, A

22.0 ± 1.2 abc, B

24.0 ± 3.7 a, B

31.1 ± 1.4 a, C

32.0 ± 2.0 a, C

U2

15.0 ± 1.7 ab, A

24.1 ± 2.5 abc, B

30.5 ± 2.5 bcd, C

34.8 ± 1.2 ab, D

37.9 ± 0.4 bc, D

U3

15.6 ± 1.5 ab, A

23.0 ± 1.7 abc, B

27.0 ± 1.1 abc, C

32.6 ± 1.3 a, D

30.9 ± 0.4 a, D

U4 Manninotriose

18.2 ± 1.6 bc, A

26.0 ± 2.1 abc, B

33.3 ± 1.2 cde, C

38.9 ± 2.4 bcd, D

40.3 ± 3.0 c, D

15.9 ± 2.2 ab, A

21.3 ± 1.9 ab, B

26.7 ± 1.0 ab, C

31.2 ± 1.0 a, D

33.5 ± 2.8 ab, D

Stachyose

17.7 ± 2.5 bc, A

25.8 ± 1.7 abc, B

34.4 ± 3.0 de, C

40.2 ± 0.3 cd, D

38.3 ± 1.5 bc, D

Ciceritol

16.7 ± 1.6 ab, A

20.8 ± 2.4 a, B

28.6 ± 2.1 abcd, C

35.9 ± 2.1 abc, D

34.3 ± 1.2 ab, D

Verbascose

22.2 ± 1.2 c, A

27.5 ± 2.6 c, B

36.9 ± 1.2 e, C

43.7 ± 3.5 d, D

37.6 ± 2.0 bc, C


27



Table 3. Effects of Different Treatments on TNF-α Levels of RAW264.7 Cell Treatments

TNF-α production (ng/L) 25 µg/mL

50 µg/mL

100 µg/mL

200 µg/mL

400 µg/mL

62.6 ± 7.0 ab, AB

66.6 ± 3.8 abc, B

61.5 ± 3.1 bc, AB

NC

48.2 ± 4.5

LPS

61.0 ± 0.7

α-GOS mixture

58.8 ± 3.1 ab, A

61.3 ± 1.9 a, AB

U1

56.8 ± 2.2 a, AB

60.4 ± 2.3 a, BC

61.0 ± 1.3 a, BC

64.5 ± 3.3 ab, C

54.7 ± 3.9 a, A

U2

62.2 ± 2.2 cd, AB

63.3 ± 1.6 ab, AB

63.2 ± 2.4 ab, AB

64.7 ± 4.4 ab, B

59.1 ± 3.3 ab, A

U3

59.2 ± 1.4 abc, A

60.5 ± 2.2 a, A

63.4 ± 1.0 ab, A

69.8 ± 3.7 cd, B

63.0 ± 2.0 bc, A

U4 Manninotriose

63.6 ± 1.7 d, A

63.6 ± 2.7 ab, A

64.4 ± 4.0 ab, A

70.7 ± 1.5 cd, B

64.1 ± 8.9 bc, A

59.0 ± 2.4 abc, A

63.2 ± 2.8 ab, A

64.9 ± 1.6 ab, A

61.5 ± 2.9 a, A

61.3 ± 4.6 abc, A

Stachyose

65.1 ± 1.6 d, A

65.7 ± 3.5 b, A

68.1 ± 3.1 b, A

69.2 ± 0.8 bcd, A

65.2 ± 3.7 bc, A

Ciceritol

62.0 ± 2.9 bcd, A

62.2 ± 0.8 ab, A

63.1 ± 1.8 ab, A

62.4 ± 3.2 a, A

59.8 ± 1.3 abc, A

64.0 ± 2.8 d, A

67.6 ± 2.1 b, AB

69.3 ± 6.5 b, AB

72.8 ± 3.2 d, B

66.6 ± 1.2 c, AB

Verbascose


28


Page 28 of 36

Page 29 of 36


Table 4. Effects of Different Treatments on IL-1β Levels of RAW264.7 Cell Treatments

IL-1β production (ng/L) 25 µg/mL

50 µg/mL

100 µg/mL

NC

5.40 ± 0.27

LPS

7.75 ± 0.47

α-GOS mixture

7.22 ± 0.27 ab, A

7.31 ± 0.35 ab, A

200 µg/mL

400 µg/mL

7.33 ± 0.12 ab, A

7.54 ± 0.37 ab, A

7.28 ± 0.2 ab, A

U1

6.87 ± 0.18 a, A

6.92 ± 0.11 a, A

7.09 ± 0.21 a, A

7.35 ± 0.53 a, A

7.10 ± 0.43 a, A

U2

7.03 ± 0.3 a, A

7.28 ± 0.33 ab, AB

7.42 ± 0.25 ab, AB

7.56 ± 0.28 ab, B

7.18 ± 0.18 ab, AB

U3

7.19 ± 0.26 ab, A

7.24 ± 0.3 ab, A

7.49 ± 0.16 ab, AB

7.73 ± 0.15 abc, B

7.49 ± 0.09 bc, AB

U4 Manninotriose

7.44 ± 0.08 bc, A

7.47 ± 0.17 bc, A

7.71 ± 0.3 bcd, AB

7.85 ± 0.08 abc, B

7.68 ± 0.22 cd, AB

7.57 ± 0.47 bcd, A

7.86 ± 0.39 cd, A

8.03 ± 0.1 cd, A

8.10 ± 0.45 bcd, A

7.90 ± 0.11 de, A

Stachyose

7.93 ± 0.03 de, A

7.99 ± 0.09 d, A

8.12 ± 0.42 de, A

8.35 ± 0.7 cd, A

7.95 ± 0.02 de, A

Ciceritol

7.69 ± 0.14 cde, A

7.82 ± 0.23 cd, AB

7.93 ± 0.2 cd, AB

8.24 ± 0.41 cd, B

7.78 ± 0.08 cd, A

8.08 ± 0.18 e, A

8.27 ± 0.39 d, A

8.53 ± 0.47 e, A

8.70 ± 0.42 d, A

8.23 ± 0.32 e, A

Verbascose

a-e represents significant differences between different α-GOS treatments of same concentration (P < 0.05). A-D represents significant differences between different concentrations of same α-GOS treatment (P < 0.05).

29



Table 5. Effects of Different Treatments on IFN-γ Levels of RAW264.7 Cell Treatments

IFN-γ production (ng/L) 25 µg/mL

50 µg/mL

100 µg/mL

NC

81.4 ± 6.7

LPS

113.4 ± 5.5

α-GOS mixture

107.9 ± 4.2 cd, A

111.3 ± 2.7 c, AB

112.2 ± 3.9 cd, AB

200 µg/mL

400 µg/mL

119.2 ± 4.7 cde, C

114.2 ± 1.3 c, BC

U1

92.4 ± 2.1 a, A

93 ± 3.2 a, A

95.3 ± 0.3 a, A

97.1 ± 5.9 a, A

92.7 ± 0.6 a, A

U2

92.0 ± 4.7 a, A

96.4 ± 2.2 a, AB

101.5 ± 4.6 ab, B

112.2 ± 3.5 b, C

101.2 ± 1.7 b, B

U3

112.3 ± 1.7 de, A

113.3 ± 2.9 cd, A

117.3 ± 3.5 de, AB

120.2 ± 4.4 cde, B

114.6 ± 6.1 c, AB

U4 Manninotriose

115.2 ± 2.5 e, A

117.0 ± 2.6 d, AB

119.2 ± 2.1 e, AB

122.3 ± 5.9 de, B

116.6 ± 3.2 c, AB

102.8 ± 1.7 b, A

105.4 ± 2 b, AB

107.3 ± 4.6 bc, AB

115.7 ± 6.3 bcd, C

111.7 ± 4.2 c, BC

Stachyose

114.3 ± 1.1 e, A

115.8 ± 5.5 cd, A

122.4 ± 4.0 e, B

123.7 ± 0.7 e, B

114.0 ± 2.4 c, A

Ciceritol

106.3 ± 3.3 bc, A

103.6 ± 2.6 b, A

107.9 ± 2.7 bc, A

114.1 ± 2.4 bc, B

105.1 ± 4.3 b, A

Verbascose

112.9 ± 3.3 e, A

114.6 ± 4 cd, A

121.3 ± 7.9 e, AB

125.5 ± 0.2 e, B

115.5 ± 5.5 c, A

a-e represents significant differences between different α-GOS treatments of same concentration (P < 0.05). A-D represents significant differences between different concentrations of same α-GOS treatment (P < 0.05).

30


Page 30 of 36

Page 31 of 36


Figure 1

31



Figure 2

32


Page 32 of 36

Page 33 of 36


Figure 3

33



Figure 4

34


Page 34 of 36

Page 35 of 36


Figure 5

35



Table of Contents Graphic

36


Page 36 of 36

Immunomodulatory Effects of Enzymatic-Synthesized Î±

Recommend Documents