Origin of Hypoglycemic Benefits of Probiotic-Fermented Carrot Pulp

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Food and Beverage Chemistry/Biochemistry

The origin of hypoglycemic benefits of probiotic-fermented carrot pulp Yujun Wan, Hui-Fang Shi, Rou Xu, Junyi Yin, Shao-Ping Nie, Tao Xiong, and Ming-Yong Xie J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06976 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 7, 2019

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Journal of Agricultural and Food Chemistry

The origin of hypoglycemic benefits of probiotic-fermented carrot pulp Yu-Jun Wan, Hui-Fang Shi, Rou Xu, Jun-Yi Yin*, Shao-Ping Nie, Tao Xiong, Ming-Yong Xie* State Key Laboratory of Food Science and Technology, China-Canada Joint Lab of Food Science and Technology (Nanchang), Nanchang University, 235 Nanjing East Road, Nanchang 330047, China.

*Corresponding authors: M. Y. Xie: E-mail: [email protected]. Tel / Fax: 0086-791-88305860. J. Y. Yin: E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT:

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It has been found that probiotic-fermented carrot pulp has a beneficial effect in reducing

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blood glucose, more so than unfermented pulp. This paper explores the reason for this by

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looking at fermentation-induced changes in nutritional components and hypoglycemic effects

5

of its polysaccharides. Micronutrients content showed minor changes except for titratable

6

acidity. Fat and protein decreased, while total carbohydrates increased. These polysaccharides

7

are pectinic, and the number of total polysaccharides rose after fermentation. Scanning electron

8

microscopy (SEM) showed the morphology changed from filamentous solid to spiral. The

9

molecular weight of water-soluble polysaccharide (WSP) diminished after fermentation, while

10

those of acid-soluble and alkali-soluble polysaccharides increased. The WSP had stronger

11

hydroxyl radical scavenging activity in vitro, and WSP from probiotic-fermented carrot pulps

12

showed better hypoglycemic effects than WSP from non-fermented carrot pulps in animal

13

experiments. Thus, the fermentation-induced improvement in diabetes control from fermented

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carrot pulp probably arises from its WSP.

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KEYWORDS:

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Carrot; Diabetes; Nutritional component; Polysaccharides; Probiotics fermentation; Structural characteristic.

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1. INTRODUCTION

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Carrot (Daucus carota L.) contains many nutritional components, particularly rich in

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carbohydrate and carotenoid.1 These various components indicate to health-related functional

21

properties, such as antioxidant, anticancer, enhancing body immunity, vitamin A supplements.2-

22

3

23

properties of its ingredients.4-5 This process may affect the viscosity, titratable acidity and

24

carbohydrate (polysaccharide) of the ingredients.6-8

There is considerable evidence that probiotic fermentation can enhance the functional

25

Many bioactive compounds from food play an important role in body health. Polyphenols

26

can have beneficial effects in gut microbiota and neuromodulation;9 vitamins can effectively

27

regulate the metabolism.10 Certain polysaccharides can also function as bioactive

28

macromolecules with several advantages and benefits for body health,11-12 and these functions

29

depend on the polysaccharide structure. For example, the treatment of diabetes has been found

30

to depend on the structure of polysaccharide, the lower molecular and more homogeneous

31

polysaccharide from natural material had higher scavenging rate of reactive oxygen species

32

(mainly including peroxides, superoxide, hydroxyl radical, and singlet oxygen) than other

33

polysaccharides from the same material.13 This can protect cells against impairments to blood-

34

sugar regulation.14 So, it is useful to characterize the structure of polysaccharide to improve

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knowledge of the hypoglycemic function.

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Carrot pulp fermented by Lactobacillus plantarum NCU 116 has been found to have better

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function properties than the unfermented pulp,15-16 especially in regulating ability of glucose

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and lipid metabolism in type II diabetes rats.17-18 This study aims to see if there are benefits in

39

controlling hyperglycemia in carrot pulp after fermentation with probiotics, and, if these are 3



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found, to determine which component(s) of the fermented pulp are responsible for these

41

beneficial effects. The structure of its constituent polysaccharides was measured with UV/vis

42

spectroscopy, Fourier transform infrared spectroscopy (FT-IR), multiple-detector size-

43

exclusion chromatography (SEC) and high performance liquid chromatography (HPLC). The

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functional properties measured were effects of the pulp on fasting blood glucose (FBG), serum

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insulin and serum lipids in streptozotocin (STZ)-induce type II diabetic rats. This is the first

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systematic study of the impact of probiotic fermentation on the nutritional components and its

47

hypoglycemic effect of carrot.

48 49

2. METHODS

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2.1. Chemicals and materials

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2.1.1. Reagents

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Non-fermented carrot pulps (NFCP) and probiotic-fermented carrot pulps (PFCP) were

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provided by Kuangda Biotech Co. (Nanchang, Jiangxi, China)19 (Fermentation conditions: The

54

sterilized carrot pulps were inoculated with L. plantarum NUC116 and fermented at 37 °C for

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24 h. After that, the fermented carrot pulps were sterilized at 105 °C for 20 s and packaged).

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Vitamin standards (VB1, VB2, VB6, VC, VE, niacin, nicotinamide, pantothenic acid and beta-

57

carotene) were purchased from the Aladdin Industrial Corporation (Shanghai, China). Folic

58

acid kit was from R-Biopharm AG Co. (Darmstadt, Germany). Dietary fiber kit was from

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Megazyme (Bray, Ireland). Chromatographic grade mobile phases (Methanol, acetonitrile,

60

chloroform and others) were from Merck Co. (California, USA). Monosaccharide standards (L-

61

fucose (Fuc), L-rhamnose (Rha), D-arabinose (Ara), D-galactose (Gal), D-glucose (Glu), D4


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xylose (Xyl), D-mannose (Man), D-fructose (Fru), D-glucuronic acid (GlcA), D-galacturonic

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acid (GalA)) and dextran standards (T-50, T-80, T-150), streptozocin were obtained from

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Sigma Chemical Corp. (St. Louis, USA) or Merck Corp. (Darmstadt, Germany). A one-touch

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glucometer (Accuchek Performa) was acquired from Roche Diagnostics, (Mannheim,

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Germany). Assay kits for determination of insulin and glycogen were purchase from the

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JianChen Bioengineering Institute (Nanjing, China). All other reagents used were of analytical

68

grade unless otherwise specified.

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2.1.2. Animals

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Fifty male Wistar rats, weighing around 180-200 g were purchased from Shanghai Slaccas

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Laboratory Animal Company Limited (Certificate Number SCXK (hu) 2012-0002, Shanghai,

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China). These rats were housed under a controlled environment, which was12 h light-dark cycle

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and free access to diet and water at 25 ± 1 °C. The animal test was conducted following the

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internationally accepted ethical principles for laboratory animal (NIH Publication No. 85-23,

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revised 1996), as well as the international rules for animal experiments.

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2.1.3. Animal model for type II diabetes mellitus (T2DM) and dietary treatments

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The T2DM animal model was induced by using the methods of Zhu et al. with minor

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modifications.20 Briefly, after a week to adaptation, all the rats were fed with high fat diet except

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10 rats which were set as blank control.18 After 8 weeks of high fat diet feeding, these 40 rats

80

were fasted overnight and injected with streptozotocin (pH: 4.5, 0.1 M cold citrate buffer) at a

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dose of 28 mg/kg body weight. The rats of the blank control group were injected with 0.1 M

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cold citrate buffer in the tail vein. Hyperglycemia was assessed at 10 days after STZ induction

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with measuring the fasting blood glucose (FBG) level of rat’s blood samples. When the FBG 5



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concentrations were higher than 11 mM, these rats could be considered as type II diabetic rats.21

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These 40 rats were divided into four groups (n = 10) as follows: model control group (untreated

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diabetic rats), metformin-treated group (MTG) (rats treated with 100 mg/kg metformin, and the

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equivalent in adult (60 kg) was about 0.5 g (2 tablets of metformin) per day according to the

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method from FDA),22 WSP-n group (rats treated with high dose of WSP-n with 400 mg/kg and

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the equivalent in adult (60 kg) was about 200 g NFCP per day), WSP-p group (rats treated with

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a high dose of WSP-p with 400 mg/kg and the equivalent in adult (60 kg) was about 200 g

91

PFCP per day).

92

2.2. Changes of nutrient properties of PFCP and NFCP

93

In our previous study,18 PFCP had better effects on controlling symptoms in diabetic rats.

94

So, some components may be changed during the fermentation process to endow PFCP with an

95

improved hypoglycemic effect. Various nutritional components were measured to compare the

96

difference between NFCP and PFCP.

97

2.2.1. Total fat, protein and titratable acidity contents

98 99 100

Total fat, protein and titratable acidity compositions of PFCP and NFCP were determined following AOAC methods 963.15, 2001.11 and ISO 750: 1998, respectively.23-24 2.2.2. Minerals

101

Minerals analysis was performed on an Optima 5300 DV ICP-OES (Perkin-Elmer, USA)

102

with a cross-flow nebulizer at the Analytical Instrumentation Center of Nanchang University

103

(Jiangxi, China), where the samples were digested in a mixture solution of HNO3 and HClO4

104

(4:1, w/w).25 The mineral contents were quantified through a calibration curve according to

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standard curves. 6


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2.2.3. Vitamins

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Vitamin of PFCP and NFCP were measured by the Institute for Food Control (Jiangxi, China).

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VB1, VB2, VB6, niacin and nicotinamide, pantothenic acid, VC and VE were determined

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following methods of National Standard for Food Safety of the People’s Republic of China (GB

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5413.11-2010, GB 5413.12-2010, GB 5413.13-2010, GB 5413.15-2010, GB 5413.17-2010,

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GB 5413.18-2010, GB 5413.9-2010). Folic acid was measured by using folic acid kit. Total

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carotenoid was tested according to the ultraviolet spectrophotometry method described by

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Wellburn.26

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2.2.4. Dietary fiber

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The dietary fiber content in PFCP and NFCP were tested by Megazyme total dietary fiber kit

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according to AOAC official method 985.29, 991.42, 991.43 and 993.19.

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2.3. Polysaccharide characterization

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2.3.1. Extraction of the polysaccharide

119

Polysaccharides were extracted according to the procedure shown Figure 1. Briefly, the

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NFCP or PFCP was extracted with deionized water at 100 °C for 2 h. The extraction procedure

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was implemented twice, then water extracts were filtered and concentrated at 60 °C under

122

reduced pressure. The solution was precipitated using ethanol at a final concentration of 80%

123

at 4 °C overnight. After centrifugation, the precipitate was washed with acetone and then diethyl

124

ether. The precipitate was redissolved in deionized water and treated by the Sevag method to

125

remove protein.27 Then, it was dialyzed against deionized water for 2 days. Finally, the liquid

126

was lyophilized in a freeze drier (Labconco Corporation, USA) to obtain water-soluble

127

polysaccharide from NFCP (WSP-n) or water-soluble polysaccharide from PFCP (WSP-p). 7



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The residue was twice extracted with 0.05 M cyclohexane-diamine-tetraacetic acid (CDTA)

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buffer solution at 28 °C.28 After filtration, CDTA extract was disposed as water extracts to

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collect CDTA-soluble polysaccharide from NFCP (CSP-n) or CDTA-soluble polysaccharide

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from PFCP (CSP-p). Then, the residue of previous step was extracted twice with 0.05 M

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Na2CO3 solution at 4 °C. After filtration, the Na2CO3 extract was processed as above to give

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Na2CO3-soluble polysaccharide from NFCP (NSP-n) or Na2CO3-soluble polysaccharide from

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PFCP (NSP-p).

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2.3.2. Physicochemical properties

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Total sugar content was analyzed by the phenol sulfuric acid assay using D-glucose as

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standard.29 D-glucuronic acid was applied as the standard by a total uronic acid content

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determination following the sulfate-carbazole method.30

139

2.3.3. Monosaccharide compositions

140

Polysaccharides were hydrolyzed with 2M H2SO4 at 100 °C for 4 h in an oil bath.31 Then,

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samples were diluted with ultra-pure water. Finally, monosaccharide and uronic acid

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composition of samples were analyzed by using Dionex™ ICS-5000 (Thermo Fisher

143

Corporation, USA).

144

2.3.4. SEC analysis of polysaccharides

145

SEC separates by molecular size, the hydrodynamic radius Rh. Parameters including dn/dc

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(the change in refractive index with polymer concentration), weight distribution w(logRh)

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(which is the relative weight, not molecular weight, of molecules of size Rh) and overall weight-

148

average molecular weight average (M – w) were measured with SEC-MALLS following the

8


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procedure as mentioned at supporting information. SEC data was analyzed following the

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method described by Gaborieau et al.32

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2.3.5. FT-IR

152

FT-IR spectra of polysaccharides were recorded from 4000 to 400 cm-1 in a Thermo Nicolet

153

5700 infrared spectrograph (Thermo Fisher Corporation, USA). Samples were dried prior to

154

tableting with KBr powder.

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2.3.6. Scanning electron microscopy (SEM)

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Polysaccharides were dissolved with deionized water at the final concentration of 1 mg/mL.

157

Then, the solutions were freeze dried, and morphology of polysaccharides were characterized

158

by scanning electron microscopy (JEOL Ltd, Tokyo, Japan) at room temperature with an

159

acceleration voltage of 5 kV.

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2.4. Hypoglycemic effects

161

As mentioned in introduction, the hydroxyl radical scavenging activity involved in the ability

162

for regulating body-sugar.14,

33

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polysaccharide can be used to give a preliminary evaluation of the hypoglycemic activity. Then,

164

the most effective part can be chosen to carry out in vivo experiment for a more accurate result.

165

2.4.1. Hydroxyl radical scavenging activity

Therefore, the hydroxyl radical scavenging activity of

166

The scavenging activity of these polysaccharides were compared according to the method

167

described by Brand-Williams et al. with minor modifications.34 Briefly, samples were dissolved

168

in water under various concentrations (0.5, 1.0, 2.0, 4.0 mg/mL), respectively. Scavenging

169

ability was calculated by detecting absorbance at 510 nm using the equation:

170

Scavenging effect (%) = (1- (As – As0)/ A0)  100% 9



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A0 means the absorbance of blank (water with H2O2), As means the absorbance of sample, As0

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means the absorbance of control (water with sample).

173

2.4.2. FBG levels of each groups

174

Blood samples of each groups were withdrawn from the rats’ tail vein after 12 h fasting, and

175

the blood glucose were measured immediately by a glucometer during the experimental period.

176

The serum samples at the 4th week were used to measure the fasting serum insulin (FINS),

177

insulin resistance (HOMA-IR) and insulin sensitivity index (QUICKI). The HOMA-IR and

178

QUICKI were calculated by using the following equations.35-36

179

HOMA-IR = (CFINS  CFBG) / 22.5

180

QUICKI = 1 / (log CFINS + log CFBG)

181

2.4.3. Serum lipid profile test

182

High density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C),

183

triglycerides (TG) and total cholesterol (TC) of the 4th week serum samples were determined

184

with automatic biochemistry analysis apparatus.

185

2.5. Statistical analysis

186

The results were expressed as mean ± standard deviations (SD). Data was analyzed by t-tests

187

to compare two groups with SPSS 22.0 (Chicago, IL, USA) and one-way analysis of variance

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(ANOVA) with Duncan’s multiple range test was conducted for comparing the difference

189

among multiple groups. The value of p < 0.05 was taken as statistically significant.

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3. RESULTS and DISCUSSION

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3.1. Nutrient characteristics of PFCP and NFCP

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3.1.1. Total fat, protein, titratable acidity and dietary fiber contents

194

Table 1 shows some nutritional properties of PFCP and NFCP. Total fat and protein

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decreased after fermentation, presumably because they were consumed to provide energy for

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the growth and reproduction of lactic acid bacteria.37 A significant increase in titratable acidity

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was observed after fermentation, from 0.06 to 0.18 mg/mL, which suggested that the acetate

198

kinase route for fermentation was activated.38 The content of dietary fiber, including total

199

dietary fiber (TDF), soluble dietary fiber (SDF) and insoluble dietary fiber (IDF), showed an

200

upward trend after fermentation. This trend in fermented products has been reported

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previously,39 and ascribed to the result of microorganism fermentation.

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3.1.2. Minerals

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All content of minerals showed significantly different (p < 0.05) between PFCP and NFCP

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except for Ca and P (Table 2). Most minerals were increased after fermentation, especially the

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amount of K and Na, reaching 2145 and 667 mg/kg, respectively, a result of probiotic

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metabolism. The literature suggests that this is probably because the fortification and

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dephytinization during fermentation with probiotics.40 In contrast, the contents of Fe and Zn

208

fallen slightly during the process, presumably because these were absorbed to stimulated the

209

growth of probiotic strains.41

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3.1.3. Vitamins

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It is noticeable that almost all kinds of vitamins showed significantly different (p < 0.05)

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after fermentation process except pantothenic acid, shown in Table 3. There were large 11



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decreases in the amounts of VB2, niacin, VB1, niacinamide, VE and VC in PFCP, which had

214

fallen by 100%, 60.07%, 44.76%, 26.57%, 12.85% and 6.24%, respectively. On the other hand,

215

VB6, folic acid and total carotenoid were gradually increased in PFCP, reaching 180.90 μg/100g,

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14.33 μg/100g and 184.33 mg/100g respectively. The increases of VB6 and folic acid were

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attributed to the biosynthesis of probiotics.42-43 However, although total carotenoid increased

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after this process, there was not sufficient evidence to prove that probiotics could synthesis

219

carotenoid during fermentation. This was perhaps because of the colorimetric reaction in the

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test for total carotenoid.

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3.2. Change of basic structural characteristics of polysaccharides

222

Many nutritional components changed during fermentation. There is evidence that

223

polysaccharides, the bioactive macromolecules, play a positive role in controlling diabetes.33,

224

44

225

fermentation. The change in content of polysaccharides here is however insufficient to

226

understand the functional difference without further information. It is necessary to compare the

227

change in the basic structure of polysaccharides to explain this.

228

3.2.1. Yield, total sugar, total uronic and monosaccharide compositions of polysaccharides

The analysis of nutritional component also illustrated the change in the polysaccharides after

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Table S1 illustrates the basic structural characteristics of polysaccharides extracted from

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PFCP and NFCP. It is apparent that the number of total polysaccharides increased after

231

fermentation, including a trend to increase in dietary fiber (including total dietary fiber (TDF),

232

soluble dietary fiber (SDF) and insoluble dietary fiber (IDF)) in NFCP and PFCP. The content

233

of total sugar decreased gradually after fermentation, from 81.76 to 76.52% in WSP, from 69.80

234

to 64.59% in CSP and from 75.90 to 70.99% in NSP. The amount of total uronic acid increased 12


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slightly. The results of monosaccharide compositions also showed a similar trend. All these

236

polysaccharides are typical pectic polysaccharides,45 mainly containing galactose, galacturonic

237

acid and arabinose as monomer units. Combining the results of chromogenic reaction and

238

monosaccharide and uronic acid compositions, it suggested that the lactic acid bacteria mainly

239

use neutral sugar for its metabolism during fermentation.

240

3.2.2. Molecular size and weight distributions

241

The dn/dc value, needed for calculating the molecular weight, is shown in Table S2. The

242

SEC data showed that all polysaccharides extracted from PFCP and NFCP had three

243

components (Figure. 2 and Table S2), as is common in natural polysaccharides.46 The size

244

maximum of WSP and both the M – w in each region and the overall M – w diminished noticeably

245

after fermentation, but those of CSP and NSP increased significantly.

246

3.2.3. FT-IR

247

The FT-IR spectra (Figure 3) profiled the characteristic absorption peaks of polysaccharides

248

extracted from NFCP and CFCP. The two sets of spectra were similar. The absorption peaks

249

near the region of 3400 cm-1 were due to the hydroxyl stretching vibration of polysaccharides,

250

and the bands between 3000 and 2800 cm-1 usually indicate C-H stretching vibrations. The

251

weaker absorption near 2300 cm-1 was ascribed to the presence of environmental CO2 during

252

the test. The absorption near 1700 cm-1 signified a trace of -COOH in the samples. The

253

absorption peaks near 1620 cm-1 and 1400 cm-1 were the stretching and bending vibrations of

254

the N-H bond. The absorption peaks near the region of 1200 cm-1 correspond to the stretching

255

vibration of the C-H band of polysaccharides. The band between 1160 and 950 cm-1 was

256

attributed to the characteristic absorption of a pyranoid ring.47 Those absorption peaks were not 13



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changed after fermentation suggested that fermentation has no effect on certain functional

258

groups. However, the intensity of most absorption band was clearly changed, indicating the

259

differences in the proportion of these functional groups. These changed were attributed to

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fermentation with probiotics through the influence on the number of uronic acid and neutral

261

sugar content.48-49

262

3.2.4. SEM

263

Figure 4 compares the SEM micrographs of polysaccharides extracted from PFCP and NFCP.

264

The polysaccharides of NFCP were seen to be both filamentous and lamellar, but the

265

polysaccharides had more spiral and winding morphologies after fermentation. This

266

phenomenon indicates the different structure of polysaccharides from PFCP and NFCP.

267

3.2.5. Hydroxyl radical scavenging ability

268

Reactive oxygen species include peroxides, superoxide, hydroxyl radical, and singlet oxygen.

269

The previous study showed that reactive oxygen species scavenging was beneficial in

270

controlling diabetes.14 Figure 5 shows that WSP had the highest effects on scavenging hydroxyl

271

radical, followed by CSP. WSP-p had stronger hydroxyl radical scavenging ability than WSP-

272

n, which indicates better performance against cell impairments and improving blood-sugar

273

regulation.14,

274

making up over 96% of the total polysaccharides. So, it is worthwhile evaluate the

275

hypoglycemic effect of WSP-n and WSP-p by conducting animal tests.

276

3.3. Hypoglycemic effects of WSP-n and WSP-p

277

3.3.1. Hypoglycemic effect of WSP-n and WSP-p

33

At the same time, WSP was dominant in the polysaccharides from carrot,

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The fasting or postprandial blood glucose increasing is the main identifier of diabetes. The

279

main aim of a diabetes drug is to reduce the blood sugar. Figure 6 shows the FBG of each group

280

during the 4 weeks. Comparing with blank group during the 4 weeks, it is apparent that the

281

STZ-induced diabetic rats showed significant increases (p < 0.01) in FBG level. In this period,

282

compared with the model group, the FBG of all treatment groups showed a downward trend. It

283

indicates WSP-n, WSP-p and metformin had a positive effect on reducing FBG of diabetic rats.

284

The result also showed that WSP-p performed better than WSP-n in this aspect. Although the

285

diabetic rats’ model was induced successfully using STZ. The treatment effect of MTG group

286

was ineffective in this experiment according to the FBG results. Compliance may be one of the

287

reasons from the literature.50

288

3.3.2. Hypolipidemic effect of WSP-n and WSP-p

289

TC, TG and LDL-C always increase in the diabetic condition, and HDL-C showed a

290

downward trend in the condition. These blood lipids are also considered to the risk factors for

291

some heart disease.44,

292

experimental period. The concentration of LDL-c, TC and TG in the serum of diabetic rats

293

showed an upward trend compared with blank group. By contrast, the HDL-c component

294

showed the opposite trend. At the same time, all treatment groups were efficacious for

295

regulating blood lipid levels.

296

3.3.3. Effect of WSP on FINS, HOMA-IR and QUICKI

51

Figure 7 shows the blood lipid change of all groups during the

297

FINS is one of the important indices to reflect the health of islet beta cell, while HOMA-IR

298

and QUICKI indices are useful for evaluating insulin resistance and insulin sensitivity

299

respectively.35, 52 Figure 8 shows that the concentration of FINS and HOMA-IR index were 15



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higher in STZ-induced diabetic rats than in normal rats. The QUICKI of blank rats were

301

significantly higher than the diabetic rats (p < 0.01). The results also show that the HOMA-IR

302

and QUICKI levels of WSP-p group were better than that of WSP-n. These indices of WSP-p

303

group showed significant differences (p < 0.05) from the model group, indicating the potential

304

effect on controlling diabetes in rats.

305

This paper explores how probiotic fermentation of carrot pulp has beneficial hypoglycemic

306

effects. It focusses on characterizing hypoglycemic, hypolipidemic effect of its WSP. After

307

fermentation with lactobacillus, the main nutrients did not change significantly, while the

308

amounts of some nutrients increased after the process. There was some change in the molecular

309

weight of polysaccharides, and a pronounced change in the solid morphology. The literature

310

suggests that the change of polysaccharide structure after fermentation is the main contributor

311

to the improved nutritional performance,14, 33 and there is evidence that polysaccharides play

312

key biological roles in this.53-54 The beneficial effect of this product on type II diabetes has been

313

explored here using an animal model. Results show that polysaccharides from PFCP have lower

314

proportions of neutral sugars and higher percentage of uronic acid than those from NFCP, which

315

probably enable the polysaccharides from PFCP to better protect cells from damage caused by

316

high blood glucose, as well as the stronger hydroxyl radical scavenging property. The two

317

abilities are effective in intervention for type II diabetes. This study confirmed that the WSP

318

from PFCP was effective in benefitting diabetic rats.

319

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Author contribution

321

Xie, Wan, Yin and Nie designed the experiment, searched the literature, analyzed and

322

interpretated data, wrote the mansuscript. Shi, Xu and Xiong contributed to data collection and

323

created the figures.

324 325

Conflict of interest

326

All authors have no personal or financial conflict of interest.

327 328

Funding

329

The authors gratefully acknowledge the financial support of this study by the National Natural

330

Science Foundation of China (31571826), Outstanding Science and Technology Innovation

331

Team Project in Jiangxi Province (20165BCB19001), Collaborative Project in Agriculture and

332

Food Field between China and Canada (2017ZJGH0102001)，Research Project of State Key

333

Laboratory of Food Science and Technology (SKLF-ZZA-201611) and Graduate Innovative

334

Special Fund Projects of Jiangxi Province (YC2016-S055 and YC2017-S068).

335 336

Acknowledgements

337

We appreciate helpful comments and language revision on the text from Professor Robert G

338

Gilbert (University of Queensland, Australia, and Yangzhou University, China).

339

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Supporting Information

341

SEC analytical procedure, summary data of structural characteristics of polysaccharides from

342

PFCP and NFCP (Table S1-S2).

343 344

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Figure Captions

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Figure 1. The procedure of sequential extraction of polysaccharides from PFCP and NFCP.

480

Figure 2. SEC weight distribution of polysaccharides from NFCP (black line) and PFCP (red

481

line).

482

Figure 3. FT-IR spectra of polysaccharides from NFCP (black line) and PFCP (red line).

483

Figure 4. SEM images of polysaccharides from NFCP and PFCP.

484

Figure 5. ROS scavenging ability of polysaccharides from NFCP and PFCP.

485

Figure 6. Effects of WSP on FBG in diabetic rats (x ± s, n=10; * means p < 0.05, ** means p

Origin of Hypoglycemic Benefits of Probiotic-Fermented Carrot Pulp

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