Removal of bound polyphenols and its effect on antioxidant and prebiotics properties of carrot dietary fiber

Food Hydrocolloids 93 (2019) 284–292

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Removal of bound polyphenols and its effect on antioxidant and prebiotics properties of carrot dietary fiber

T

Shuai Liua, Mengyun Jiaa, Jiajun Chena, Haisheng Wanb, Ruihong Donga, Shaoping Niea, Mingyong Xiea, Qiang Yua,∗ a b

State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, China School of Food Science and Technology, Nanchang University, Nanchang, 330031, China

ARTICLE INFO

ABSTRACT

Keywords: Carrot dietary fiber Bound polyphenols Antioxidant Prebiotics

The present work was designed to investigate the effect of bound polyphenols on antioxidant and prebiotics properties of carrot dietary fiber (CDF). The polyphenols were removed from CDF by alkaline extraction to obtain dephenolized dietary fiber (CDF-DF). The optimized conditions of extracting polyphenols from CDF were solid-liquid ratio of 1:8.5, the treat temperature of α-amylase 66 °C, the addition of protease of 0.2% and the treat temperature of amyloglucosidase 60 °C, respectively. The FT-IR spectroscopy illustrated that the major functional group of CDF-DF and CDF shared most of the key features, despite changes in the amplitude of some functional groups. Scanning electron microscopy (SEM) was further performed demonstrating no significant structure differences between CDF-DF and CDF. Moreover, the antioxidant properties, including superoxide anion radical, hydroxyl radical and DPPH radical scavenging activity, of CDF-DF were significantly lower than that of CDF. In addition, CDF was more conducive for the growth of Lactobacillus rhamnosus as compared with the CDF-DF. In summary, the above results concluded that there was no significant change of the dietary fiber after removal of bound polyphenols, and the bound polyphenols contributed significantly to the antioxidant and prebiotics properties of dietary fiber from carrot.

1. Introduction Dietary Fiber (DF) is a broad category of non-digestible food ingredients. According to the unifying definition by the commission of Codex Alimentarius in 2009, the major components of dietary fiber are non-starch polysaccharides (NSP), fructans (inulin and fructoligosaccharides), resistant starch and lignin. Based on the solubility to water, DF can be divided into two categories: soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) (Dhingra, Michael, Rajput, & Patil, 2012; Vitaglione, Napolitano, & Fogliano, 2008). In recent years, there has been an increasing interest in DF due to its health-promoting features. Experimental and epidemiological studies have correlated consumption of DF with many health benefits, such as regulation of intestinal transit, lower incidences of cardiovascular diseases and colorectal cancer (Anderson et al., 2010; Rodríguez, Jiménez, Fernández-Bolaños, Guillén, & Heredia, 2006; Xiang et al., 2018; Yu et al., 2015). The mechanism by which DF exerts its protective effects seems to be associated with the gut microbiota (Lee & Hase, 2014). The fermentation of DF could promote the positive change of the bacterial composition in the gut, accompanied with enhancing bile acid deconjugation, production of short chain fatty acids (SCFAs), and modulation of inflammatory bioactive

∗

substances (Verspreet et al., 2016). Polyphenols consist of a large variety of compounds in plants that share one or more phenol groups, including flavonols, flavones, isoflavones, stilbenes and phenolic acids, etc. Polyphenols are the most commonly consumed dietary antioxidants, which have potent antioxidant and free-radical scavenging properties that protect against oxidative damage to important biomolecules. Recent trends in polyphenols research have led to a proliferation of studies with respect to the qualitative and quantitative analysis (Burgos-Edwards, Jiménez-Aspee, Thomas-Valdés, Schmeda-Hirschmann, & Theoduloz, 2017; Zhang et al., 2018), bioaccessibility and bioavailability (Yang, Jayaprakasha, & Patil, 2018; Zhong, Sandhu, Edirisinghe, & Burtonfreeman, 2017), and the health effects derived from polyphenols intake (Scalbert, Manach, Morand, Rémésy, & Jiménez, 2005b). Mounting studies have reported that polyphenols have positive effects on anti-inflammatory, cardiovascular and neurodegenerative diseases, as well as cancers (Scalbert, Johnson, & Saltmarsh, 2005). Current research on the polyphenols mostly focuses on the extractable polyphenols (EPP) that can be extracted with aqueous–organic solvents. However, there is another fraction of polyphenols with significant value remains in the residues after the extraction, named non-extractable

Corresponding author. State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang, 330047, China. E-mail address: [email protected] (Q. Yu).

https://doi.org/10.1016/j.foodhyd.2019.02.047 Received 21 August 2018; Received in revised form 19 February 2019; Accepted 25 February 2019 Available online 26 February 2019 0268-005X/ © 2019 Elsevier Ltd. All rights reserved.

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polyphenols (NEPP), which has been long neglected (Perez-Jimenez, DiazRubio, & Saura-Calixto, 2013). NEPP mainly associated with macromolecules in the food matrix, such as DF and protein. During the ingestion process, NEPP could pass through the stomach and small intestine with scarcely release from the food matrix regardless of the acidic pH and digestive enzymes. Upon reaching the colon nearly intact, NEPP will be fermented by the colonic microflora and release substances into the colon with specific health benefits, mainly relating to the antioxidant property (González-Sarrías, Espín, & Tomás-Barberán, 2017). In general, dietary fiber and polyphenols are usually studied separately because of differences in their chemical structures, physicochemical and biological properties, and metabolic pathways (Saura-Calixto, 2011). Bound polyphenols, which are also referred to as NEPP, integrated in the dietary fiber had not been fully investigated. Recently, some researchers proposed that bound polyphenols should be included as components of dietary fibers (Goñi, Díaz-Rubio, Pérez-Jiménez, & Saura-Calixto, 2009; Saura-Calixto, 2012). There are scientific evidences suggesting that the significant contribution of bound polyphenols to the health-related properties attributed to DF (Palafox-Carlos, Ayala-Zavala, & González-Aguilar, 2011; Vitaglione et al., 2008). Thus, it is worth to consider whether the benefits traditionally attributed only to DF are due to the association of both components. Carrot is a popular consumed vegetable favored by people as a good source of natural antioxidants (Chantaro, Devahastin, & Chiewchan, 2008; Prakash, Jha, & Datta, 2004). However, many by-products of carrot, such as skin and residue, which are rich in dietary fiber and polyphenols, are not fully utilized and usually discarded. The aim of this study was to optimized the removal of polyphenols from carrot dietary fiber, and investigate the effect of bound polyphenols on the antioxidant and prebiotics properties of carrot dietary fiber.

modified. In short, 200 mg of CDF were placed in dark-coloured screw-cap bottles with NaOH (2 M, 5 mL). The head-space of the bottles was flushed with N2 to remove the air. The liquid was slowly stirred by a rotor on magnetic stirrer for 4 h at room temperature and in the absence of light. Then samples were acidified with HCl (6 M) to pH 1.5–2 and extracted with 60% ethanol (twice volumes) for 30 min. After centrifuging (4800 r/min, 10 min), the substrate that was washed with distilled water, ethanol (95%), and acetone (95%) was vacuum filtered stored in a refrigerator at −20 °C and supernatants were used to determine the phenolic content. 2.4. Determination of total phenolic content Total phenolic content of CDF alkaline extracts were determined by Folin-Ciocalteu method (Singleton & Rossi, 1965). In this method, 200 μL supernatants prepared above was mixed with 200 μL of Folin-Ciocalteu reagent without light for 30s. Then 600 μL of Na2CO3 (20%) was added into the mixture and measured at 765 nm with microplate reader (Varioskan Flash, Thermo, America) after 30 min against distilled water as a blank. Ferulic acid (0–100 μg/mL) was used as a standard to make standard curve (y = 0.01 x + 0.0138, R2 = 0.999) and the results were shown as milligrams of ferulic acid equivalent (FAE) per gram of sample (mg FAE/g dry sample). 2.5. Experimental design and data analysis Optimization of polyphenols extraction from CDF was carried by response surface methodology (RSM). A four-factor and a three-level facecentered cube design (FCD) consisting of twenty-nine experimental runs was employed including five replicates at the center point. Because of extraneous factors, the effects of unexplained variability in the observed response were minimized by randomizing the order of experiments (LiyanaPathirana & Shahidi, 2005), the treat temperature of α-amylase (X2, °C), the addition of protease (X3, %), the treat temperature of amyloglucosidase (X4, °C) while response variable was the content of polyphenols (Y, %). The response surface regression procedure of statistical analysis system (SAS) and Design Expert software (Version 8.0) were used to analyze the experimental data. A second-order polynomial regression model was assumed for predicting individual Y variables. The model used in the response surface analysis was as follows:

2. Materials and methods 2.1. Samples and chemicals Carrot was purchased from local market in Nanchang, Jiangxi province. Carrot was dried in electric thermostat blast drying oven (DGG-9140B, Senxin, Shanghai, China) at 45 °C and ground to pass a 0.15 mm screen and stored at −20 °C before experiments. Heat-stable α-amylase (Aladdin reagent Co., USA), protease (Pangbo Bioen-gineering Co., Nanning, China) and amyloglucosidase from Aspergillus niger were used to prepare carrot dietary fiber (CDF). Foli-Ciocalteu (Li Da Bioengineering Co., Shanghai, China) and ferulic acid were used to measure the content of phenolic. DPPH (1, 1-diphenyl-2-picrylhydrazyl), pyrogallol (1, 2, 3-Benzenetriol), salicylic acid and ferrous sulfate were applied for antioxidant potential evaluation. MRS broth (Huankai Microbial Sci. & Tech. Co., Ltd., Guangdong, China) was used to culture Lactobacillus rhamnosus. Ethanol, distilled water acetone, and ethyl acetate were used for sample preparation.

4

Y=

0

+

4 i Xi

i= 1

+ i=1

2 ii X i

4

+

ij Xi Xj i
(1)

where Xi and Xj are the independent variables; β0, βi (i = 1, 2,...,k), βii (i = 1, 2,...,k), and βij (i = 1,2,...,k; j = 1,2,...,k) are the regression coefficients for intercept, linear, quadratic, and interaction terms, respectively and Xi and Xj are the independent variables. 2.6. Structural determination of CDF and CDF-DF

2.2. Preparation of CDF

2.6.1. FT-IR spectroscopy 1 mg dry sample was mixed with 0.1 g KBr and pressed into a pellet under ambient conditions in the mid-infrared area. The FT-IR spectrum of the samples with KBr pellet was immediately measured (wave number range 4000–400 cm−1).

CDF was prepared based on enzymatic method (Guo & Beta, 2013) with modifications. Briefly, 1 g of sample was subject to sequential enzymatic digestion by heat-stable α-amylase (50 μL, pH 7), protease (pH 7, 60 °C water bath for 30 min) and amyloglucosidase (300 μL, pH 4.5) to remove starch and protein. After centrifuging (4800 r/min, 10 min), the substrate that had been washed with distilled water, ethanol (95%), and acetone (95%) was vacuum filtered and stored in a refrigerator at −20 °C. The contents of total dietary fiber (TDF), protein and starch were measured by AOAC Official Method 985.29, AOAC Official Method 2001.11 and AOAC Official Method 996.11, respectively.

2.6.2. Scanning electron microscopy (SEM) The morphology of CDF and CDF-DF were recorded according to the method (Saha, Balakrishnan, & Ulbricht, 2007). In short, dry sample was prepared with sputter coating using scanning electron microscope (JSM 6701F, Jeol, Japan), and then shot in enlargement multiple 1000.

2.3. Preparation of dephenolized carrot dietary fiber (CDF-DF)

2.7. Determination of antioxidant activity of CDF and CDF-DF

Alkaline hydrolysis was applied to release ester linked phenolic from CDF to prepare CDF-DF (Guo & Beta, 2013). The method was slightly

2.7.1. Preparation of sample solution 4 g of sample were soaked with 400 mL of distilled water for 1 h and 285

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A

C

B

D

Fig. 1. Effect of various factors on polyphenols content. (A) The effect of solid-liquid ratio on polyphenols. (B) The effect of the temperature of α-amylase on polyphenols. (C) The effect of the addition of protease on polyphenols. (D) The effect of the temperature of amyloglucosidase on polyphenols. These data are the means ± SEM of polyphenols content in CDF.

heated to 70 °C for 20 min. Then, the mixed solution was emulsified in homogenizer (MODEL, Wodi Automation Equipment Co., Shanghai, China) for 5 min. The emulsion was configured into 0, 1, 2, 4, 6, 8, 10 mg/mL solutions, respectively.

Table 1 Independent variables and their coded and actual values used for optimization. Independent variable

Solid-liquid ratio The treat temperature of α-amylase The addition of protease The treat temperature of amyloglucosidase

Units

°C % °C

Symbol

X1 X2 X3 X4

Coded levels −1

0

1

1:5 60 0.1 50

1:10 70 0.2 60

1:15 80 0.3 70

2.7.2. DPPH radical scavenging activity Free radical scavenging activities of CDF and CDF-DF were determined by using a stable DPPH radical following a previously reported method (Pérez-Jiménez et al., 2008). The method has a slight change. Briefly, freshly prepared solution of DPPH (0.2 mM, 2.0 mL) and sample (1.0 mL) were added for 30 min, followed by centrifugation (5000 r/min) for 5 min, and the absorbance was measured at 517 nm. 286

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Table 2 Four-factor, three-level face-centered cube design used for RSM. Standard order

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

X1

X2 (°C)

0(1:10) −1(1:5) 0(1:10) 0(1:10) 1(1:15) 0(1:10) 0(1:10) −1(1:5) 0(1:10) 1(1:15) −1(1:5) 0(1:10) 1(1:15) 0(1:10) 0(1:10) 1(1:15) 0(1:10) −1(1:5) 0(1:10) 0(1:10) 1(1:15) 1(1:15) −1(1:5) 0(1:10) −1(1:5) 0(1:10) 0(1:10) 0(1:10) 0(1:10)

−1(60) 0(70) 1(80) 0(70) 1(80) 1(80) 0(70) 0(70) 0(70) 0(70) 0(70) −1(60) 0(70) 1(80) 0(70) −1(60) 1(80) −1(60) −1(60) 0(70) 0(70) 0(70) 0(70) 0(70) 1(80) −1(60) 0(70) 0(70) −1(60)

X3 (%)

−1(0.1) 1(0.3) 1(0.3) −1(0.1) 0(0.2) 0(0.2) 1(0.3) 0(0.2) 0(0.2) 0(0.2) 0(0.2) 0(0.2) 0(0.2) 0(0.2) 0(0.2) 0(0.2) −1(0.1) 0(0.2) 0(0.2) 0(0.2) −1(0.1) 1(0.3) −1(0.1) −1(0.1) 0(0.2) 1(0.3) 0(0.2) 0(0.2) −1(0.1)

X4 (°C)

0(60) 0(60) 0(60) 1(70) 0(60) −1(50) −1(50) −1(50) 0(60) 1(70) 1(70) −1(50) −1(50) 1(70) 0(60) 0(60) 0(60) 0(60) 1(70) 0(60) 0(60) 0(60) 0(60) −1(50) 0(60) 0(60) 0(60) 0(60) 0(60)

Absorbance (Abs)

0.316 0.335 0.331 0.271 0.291 0.190 0.254 0.263 0.289 0.348 0.246 0.296 0.324 0.248 0.285 0.386 0.217 0.256 0.335 0.306 0.373 0.211 0.227 0.291 0.327 0.220 0.321 0.371 0.359

Table 4 Estimated regression model of relationship between response variables (yield of polyphenols) and independent variables (X1, X2, X3, X4).

The content of polyphenols (mg(FAE)/ 10 g) 24.965 26.458 26.161 21.203 22.828 14.495 19.784 20.592 22.704 27.530 19.132 23.306 25.575 19.313 22.366 30.674 16.748 20.006 26.532 24.131 29.667 16.228 17.573 22.836 25.823 17.036 25.344 29.502 28.479

MSc

F-value

P-value

X1 X2 X3 X4 X1X3 X1X4 X2X3 X2X4 X3X4 X12 X22 X32 X42

102.59 74.49 0.23 0.72 0.98 0.15 1.34 4.05 13.57 236.74 95.63 42.09 40.04

1 1 1 1 1 1 1 1 1 1 1 1 1

102.59 74.49 0.23 0.72 0.98 0.15 1.34 4.05 13.57 236.74 95.63 42.09 40.04

51.43 37.34 0.11 0.36 0.49 0.077 0.67 2.03 6.8 118.68 47.94 21.1 20.07

< 0.0001 < 0.0001 0.7396 0.5579 0.4948 0.7857 0.427 0.176 0.0206 < 0.0001 < 0.0001 0.0004 0.0005

c

Sums of squares. Degree freedom. Mean square.

following equation:

WOH (%) = (1

AHx

AH0 + AHf ) × 100 Ax0

(3)

Where AHx is the absorbance of sample and reagent solution, AH0 is the absorbance of sample and distilled water, AHf is a blank control group for incomplete extraction of extracted phenolic and Ax0 is the absorbance of the control.

SSa

DFb

MSc

F-value

P-value

Model Residual Lack of fit Pure error Cor.total

507.72 27.93 22.14 5.79 535.65

14 14 10 4 28

36.27 1.99 2.21 1.45

18.18

< 0.0001

1.53

0.3623

2.7.4. Superoxide anion radical scavenging activity The non-enzymatic generation of superoxide anion was measured according to the method (Robak & Gryglewski, 1988). The sample solution (0.1 mL) was mixed with 1 mL Tris-HCl (pH8.2, 50 mM) at 25 °C for 20 min. After 5 min incubation with 0.1 mL pyrogallol solution (10 mM), the absorbance was measured at 325 nm. The hydroxyl radical scavenging activity (W0) was calculated according to the following equation:

W0 (%) = (1

ASx

AS0 + ASf ) × 100 Ax0

(4)

Where ASx is the absorbance of the samples; AS0 is the absorbance of the blank and Ax0 is the absorbance of the control; supernatant of CDF-DF instead of samples (ASf) is a blank control group for incomplete extraction of extracted phenolic.

R2 = 0.9479; Radj2 = 0.8957; CV.% = 6.14. a Sums of squares. b Degree freedom. c Mean square.

2.8. In vitro fermentation of CDF and CDF-DF by Lactobacillus rhamnosus

DPPH radical scavenging activity (Wd) was calculated according to the following equation:

AD0 + ADf ) × 100 Ax0

DFb

b

Source

ADx

SSa

a

Table 3 Analysis of variance for the fitted quadratic polynomial model of extraction of polyphenols.

Wd (%) = (1

Source

2.8.1. Probiotics preparation Lactobacillus rhamnosus was subcultured three times overnight in MRS broth to make the starter culture inocula. The broth cultures were washed thrice with 10 mL of 0.9% NaCl aqueous solution and finally resuspended in 0.9% NaCl to obtain a bacterial suspension of approximately 7 log colony-forming units (cfu) mL−1.

(2)

Where ADx is the absorbance of sample and DPPH, AD0 is the absorbance of sample and distilled water, ADf is the absorbance of DPPH and supernatant of CDF-DF and Ax0 is the absorbance of distilled water and DPPH.

2.8.2. Fermentation of CDF and CDF-DF The fermentations of CDF and CDF-DF were prepared by mixing 2 g of sample with 100 mL of distilled water, 1 g of peptone, 0.8 g of beef extract, 0.4 g of yeast, 0.2 g of ammonium ferric citrate, 0.1 mL of Tween 80, 0.15 g of dipotassium hydrogenphosphate, 0.01 g of magnesium sulphate, 0.004 g of manganese sulphate, 0.3 g of sodium acetateanhydrous and 1 mL of bacterial suspension using a laboratory shaker (MS2 minishaker, IKA, Germany). All of the fermentation cultures were incubated at 37 °C. Samples were collected before and after fermentation for measurements of pH and enumeration of bacteria at 4, 8, 12, 24 and 36 h, respectively.

2.7.3. Hydroxyl radical scavenging activity Hydroxyl radical scavenging capacity of CDF and CDF-DF was evaluated according to the method (Yin, Nie, Zhou, Wan, & Xie, 2010). In short, 1 mL sample solutions were mixed with 1 mL FeSO4 (9 mM) and 1 mL H2O2 (8.8 mM) at 37 °C for 10 min, then adding 1 mL ethanol solution of salicylic for 10 min. The absorbance was measured at 550 nm after centrifugation (5000 r/min) for 5 min. The hydroxyl radical scavenging activity (WOH) was calculated according to the 287

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A

B

B

B

B

B

C

D B

E

F

Fig. 2. Response surface plot and contour plot of variables and their mutual interactions. (A) Solid-liquid ratio and the treat temperature of α-amylase. (B) The addition of protease and the treat temperature of α-amylase. (C) Solid-liquid ratio and the addition of protease. (D) The treat temperature of α-amylase and the treat temperature of amyloglucosidase. (E) Solid-liquid ratio and the treat temperature of amyloglucosidase. (F) The addition of protease and the treat temperature of amyloglucosidase on the yield of polyphenols.

2.8.3. Enumeration of bacteria The viability of the starter culture inocula and the numbers of cells in the cultures after fermentation were ascertained by traditional plate counts on MRS agar. All agar plates were incubated for 3 d at 37 °C.

the temperature of α-amylase, the addition of protease and the temperature of amyloglucosidase in the extraction solution were determined as the main variables affecting the extraction efficiency. Hence, we performed the response surface methodology (RSM) to further evaluate the optimal conditions for extracting polyphenols from CDF. In the first step of optimizing the extraction of CDF, the lower, middle, and upper layers of these four independent variables were selected from the levels based on the preliminary experiments. Fig. 1(A) showed the effect of solid-liquid ratio on extraction of polyphenols. We could find that the solid-liquid ratio (1:5 to 1:15) was favorable for producing polyphenols. The effect of the temperature of α-amylase on extraction of polyphenols was displayed in Fig. 1(B). It was apparent that the treat temperature of α-amylase of 60–80 °C was sufficient to obtain polyphenols. Fig. 1(C) presented effect of the addition of protease on extraction of polyphenols. It can be seen from Fig. 1(C) that the addition of protease of 0.1–0.3% was better to remove polyphenols. The effect of the temperature of amyloglucosidase on extraction polyphenols were displayed in Fig. 1(D). It showed that the temperature of amyloglucosidase (50–70 °C) was favorable for extraction of polyphenols.

2.8.4. pH of fermentation solution pH was measured in samples of fermented CDF and CDF-DF by Φ 32 pH-meter (Mettler Toledo, Switzerland). 2.9. Statistical analysis Values are expressed as means ± SEM (standard error of the mean). One-way analysis of variance followed by the Student-NewmanKeuls test was used to determine the statistical significance between various groups. A value of p < 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. Selection of lower, middle and upper levels of the design variables for removal of polyphenols from CDF

3.2. Optimization of extraction conditions of polyphenols

Efficiency of the extraction of compound is generally influenced by several factors (Ilaiyaraja, Likhith, Sharath Babu, & Khanum, 2015; Karazhiyan, Razavi, & Phillips, 2011). In this study, solid-liquid ratio,

3.2.1. Predicted model and statistical analysis The four independent variables and lower, middle and upper design 288

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Table 5 The predicted and experimental value of response at the optimum conditions.

Optimum conditions (predicted) Modified conditions (actual)

Solid-liquid ratio

α-amylaetemperature

The addition of protease

amyloglucosidase temperature

The content of polyphenols(mg (FAE)/ 10 g)

8.50 8.50

66.02 66.00

0.2 0.2

59.82 60.00

30.10 33.03

points for RSM in coded and natural values were shown in Table 1. The design matrix and the corresponding results of RSM experiments to determine the effects of the four independent variables were shown in Table 2. Through multiple regression analysis on the experimental data, the model for the predicted response Y could be expressed by the following quadratic polynomial equation (in the form of actual values):

conditions and the result (Table .5) revealed that the experimental values (33.03 mg (FAE)/10 g) were reasonably close to the predicted values confirming the validity and adequacy of the predicted models. In addition, the measured values laid within a 95% mean confidence interval of the predicted value for the yield of phenolic compounds. The result confirmed the predictability of the model for the extraction of polyphenols from carrot dietary fiber in the experimental condition used. Moreover, the contents of TDF, protein and starch in CDF were 94.27%, 0.21% and 0.032%, respectively. Meantime, the results of protein and starch content values in CDF-DF were 0.20% and 0.030%, respectively. It could be found the content of starch and protein had no significant changes and very low in CDF and CDF-DF.

Y = −167.73291 + 2.15X1 + 4.04X2 − 37.79X3 + 1.97X4 + 0.04X1X2 − 0.99X1X3−3.91 × 10−3X1X4 + 0.58X2X3 + 0.01X2X4+ 1.84X3X4 − 0.24X12 − 0.04X22 − 254.72X32 − 0.02X42 Statistical testing of the model was operated by analysis of variance (ANOVA). The ANOVA for the fitted quadratic polynomial model of extraction of polyphenols were shown in Table 2. The quadratic regression model manifested that the value of the determination coefficient (R2) was 0.9479, which implied that 94.79% of the variations could be explained by the fitted model. For a good statistical model, R2adj should be close to R2. As shown in Table 3, R2adj that was 0.8957, which implied that only less 5.0% of the total variations were not explained by the model. It also indicated that a high degree of correlation between the observed and predicted values. The coefficient of variation (CV) were 6.14% < 10% indicated a better reliability of the experiments values (Montgomery, 1991). If the F-value becomes greater and the p-value becomes smaller, the corresponding variables would be more significant. Values of p-value less than 0.05 indicated model terms were significant. So, the F-value (F = 18.18) and p-value (p < 0.0001) of model implied this model was significant. Significance of the model was also decided by lack-of-fit test. As shown in Table 3, F-value and pvalue of the lack of fit were 1.53 and 0.3623, respectively, which implied that it was not significant and a 36.23% chance could occur due to noise. The significance of each coefficient was determined using F-value and p-value. The results were presented in Table 3, two independent variables (X1, X2) and four quadratic terms (X12, X22, X32 and X42) significantly affected the yield of polyphenols, and the interaction between X1 and X2 and X3 and X4 were significant as well (p < 0.05). The above results indicated that the independent factor X1, X2, X12 and X22 were the most significant factors on the experimental yield of polyphenols(See. Table 4).

3.4. The FT-IR spectrum of CDF and CDF-DF FT-IR spectra of CDF and CDF-DF were illustrated in Fig. 3. It could be found that most characteristic bonds of CDF and CDF-DF were similar in FT-IR spectra, such as bands at 3437 cm−1 (OeH stretching vibration in hemicellulose and cellulose), 2937 and 2897 cm−1 (CeH stretching vibration of the methylene group of the saccharide), 1628 cm−1 (characteristic absorption peak of C=O in lignin benzene ring). (Fringant, Tvaroska, Mazeau, Rinaudo, & Desbrieres, 1995; Kačuráková, Ebringerová, Hirsch, & Hromádková, 2010; Kacurakova, Belton, Wilson, Hirsch, & Ebringerova, 1998; Tul'Chinsky, Zurabyan, Asankozhoev, Kogan, & Khorlin, 1976). On the other hand, the disappearance of the band at 1748 cm−1was observed, which is C=O stretching modes of ester group, it may be due to the destruction of the ester bond between polyphenols and CDF. Moreover, 1259 and 793 cm−1 were also disappeared, which referred to the characteristic absorption peak of CeO in hydroxybenzene, the bending vibration absorption peak of CeH in benzene ring, respectively, proving that the polyphenols were released in the process of alkali treatment. In summary, these results revealed that there were no major functional group transformations during the progress of polyphenols removal.

3.2.2. Analysis of response surface The relationship between independent and dependent variables was indicated by the three-dimensional representation of the response surfaces and the two-dimensional contour plots generated by the model (shown in Fig. 2). Response surface plots were plotted between two independent variables while keeping the other independent variables at the zero coded level. Fig. 2(A) exhibited the effect of solid-liquid ratio (X1) and the treat temperature of α-amylase (X2) on yield of polyphenols. There was a significant interaction between solid-liquid ratio (X1) and the treat temperature of α-amylase (X2). The effect of the addition of protease (X3) and the treat temperature of amyloglucosidase (X4) on yield of polyphenols was shown in Fig. 2(F) and they were significant interaction. 3.3. Verification experiments In order to verify the predictive capacity of the model, the predicting optimum response values were tested using the selected optimal

Fig. 3. FT-IR spectrum of CDF and CDF-DF. 289

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(p < 0.05). Hydroxyl radical scavenging activity of CDF and CDF-DF was shown in Fig. 5(B). CDF possessed hydroxyl radical scavenging activity with the percentage between 24.83 and 62.95%, while CDF-DF with values ranging from 3.32 to 16.75%. The hydroxyl radical scavenging activity of CDF was significantly higher than that of CDF-DF in dose dependent manner. These results were consistent with the result of Fig. 5(A) since it has been reported that hydroxyl radical scavenging activity can be formed by the reaction of superoxide with hydrogen peroxide in the presence of metal ions (Macdonald, Galley, & Webster, 2003). DPPH has been used extensively as a free radical to evaluate reducing substances (Cotelle et al., 1996). Fig. 5(C) showed the DPPH radical scavenging activity of CDF and CDF-DF. Following the addition of CDF and CDF-DF, the increase in the DPPH radical scavenging activity was recorded, CDF with the values of from 31.96 to 60.91%, CDFDF with values ranging from 19.52 to 29.43%. From these data, it was apparent that the CDF has a higher (p < 0.05) ability to inhibit DPPH radical than the CDF-DF. To comprehensively evaluate the antioxidant capacity of compound, different methods with different action mechanisms are necessary to be used (Pérez-Jiménez et al., 2008; Wang, Hu, Nie, Qiang, & Xie, 2015). In the present study, we combined more than one method, i.e. superoxide anion radical, hydroxyl radical and DPPH radical scavenging activity, in order to determine the antioxidant capacity of samples in vitro. Our experiment data demonstrated that the antioxidant capacity of CDF was significantly higher than that of CDF-DF. The decline of antioxidant capacity of CDF-DF may be due to the removal of polyphenols from CDF. Overall, these results indicated that the bound polyphenols significantly contributed to the antioxidant capacity of CDF.

Fig. 4. SEM of CDF and CDF-DF

3.5. SEM observation of ultrafine microstructure of CDF and CDF-DF The ultrafine microstructure of CDF and CDF-DF was observed by SEM (Feng, Dou, Alaxi, Niu, & Yu, 2017). As shown in Fig. 4, it showed the structure of CDF-DF was looser than CDF, which was an evidence that the bound polyphenols were removed. Moreover, no noticeable change between CDF and CDF-DF structure was observed, demonstrating the structure of CDF-DF was not significantly destroyed during the process of releasing polyphenols by alkali treatment. 3.6. Antioxidant activity of CDF and CDF-DF Fig. 5 presented the in vitro antioxidant activity of CDF and CDF-DF, including superoxide anion radical, hydroxyl radical and DPPH radical scavenging activity. In Fig. 5(A), it showed that both CDF and CDF-DF exhibited scavenging activity on superoxide anion and the radical scavenging activity of CDF was significant than that of CDF-DF

B

A

C

Fig. 5. Antioxidant activity of CDF and CDF-DF. (A) Superoxide anion radical scavenging activity of CDF and CDF-DF. (B) Hydroxyl radical scavenging activity of CDF and CDF-DF. (C) DPPH radical scavenging activity of CDF and CDF-DF. Values are mean ± SEM of antioxidant activity of CDF and CDF-DF. *p < 0.05 versus the antioxidant activity of CDF-DF. 290

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Fig. 6. Prebiotics properties of CDF and CDF-DF after fermentation by Lactobacillus rhamnosus at different fermentation times. (A) Changes of total number of colonies in fermentation broth of CDF and CDF-DF under different fermentation time. (B) Change of pH in fermentation broth of CDF and CDF-DF under different fermentation time.

3.7. Prebiotics properties of CDF and CDF-DF The experimental data on the prebiotics properties of CDF and CDFDF was presented in Fig. 6. DF is well regarded as a kind of prebiotics that could encourage the growth of healthy bacteria in the gut. In this regard, both CDF and CDF-DF could promote the growth of Lactobacillus rhamnosus, a probiotics widely used in yogurt and dairy products. However, the prebiotics properties, including pH and the number of colonies of CDF were significantly different from those of CDF-DF. The most likely cause of the result was that bound polyphenols could be released by bacterial feruloyl esterases (BFE) from dietary fiber matrices into free forms (Topakas, Vafiadi, & Christakopoulos, 2007) and beneficial for the growth of Lactobacillus rhamnosus (Requena et al., 2010). Additionally, there was an interesting finding that CDF and CDFDF shared similar prebiotics properties at 4 and 8 h, the possible explanation for these results may be the lack of adequate amount of polyphenols released to affect the prebiotics properties of bacteria at 0–8 h. To sum up, these results confirmed the association between the bound polyphenols and prebiotics properties of carrot dietary fiber. 4. Conclusion This study focused on exploring the influence of the bound polyphenols on antioxidant and prebiotics properties of carrot dietary fiber. CDF-DF was prepared by optimized removal of the bound polyphenols from CDF. FT-IR and SEM illustrated that there were no significant structure between CDF and CDF-DF. Compared with CDF, the antioxidant and prebiotics properties of CDF-DF dramatically decreased as evidenced by superoxide anion radical, hydroxyl radical, DPPH radical scavenging activity, as well as colonies number and pH of in vitro fermentation of Lactobacillus rhamnosus. Taken together, these results suggested that the bound polyphenols play an important role in the antioxidant and prebiotics properties of dietary fiber, the underlying mechanism will be further investigated in the future work. These findings contribute to existing knowledge of dietary fiber and bound polyphenols, and provide a new insight to our understanding of dietary fiber. Acknowledgements This work was supported by the National Natural Science Foundation of China (31701603), Key Research and Development Program of Jiangxi Province of China (20171BBF60041), National Natural Science Foundation of China (31571826) and Research Project of State Key Laboratory of Food Science and Technology (SKLF-ZZA201611). References Anderson, J. W., Baird, P., Davis, R. H., Ferreri, S., Knudtson, M., Koraym, A., et al. (2010). Health benefits of dietary fiber. Nutrition Reviews, 67(4), 188–205.

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