Properties of soluble dietary fiber-polysaccharide from papaya peel obtained through alkaline or ultrasound-assisted alkaline extraction

Accepted Manuscript Title: Properties of Soluble Dietary Fiber-Polysaccharide from Papaya Peel Obtained Through Alkaline or Ultrasound-Assisted Alkaline Extraction Authors: Weimin Zhang, Guanglin Zeng, Yonggui Pan, Wenxue Chen, Wuyang Huang, Haiming Chen, Yuansong Li PII: DOI: Reference:

S0144-8617(17)30537-4 http://dx.doi.org/doi:10.1016/j.carbpol.2017.05.030 CARP 12316

To appear in: Received date: Revised date: Accepted date:

24-1-2017 23-4-2017 8-5-2017

Please cite this article as: Zhang, Weimin., Zeng, Guanglin., Pan, Yonggui., Chen, Wenxue., Huang, Wuyang., Chen, Haiming., & Li, Yuansong., Properties of Soluble Dietary Fiber-Polysaccharide from Papaya Peel Obtained Through Alkaline or Ultrasound-Assisted Alkaline Extraction.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.05.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Properties of Soluble Dietary Fiber-Polysaccharide from Papaya Peel Obtained Through Alkaline or Ultrasound-Assisted Alkaline Extraction Weimin Zhang a, Guanglin Zeng a, Yonggui Pan a, Wenxue Chen a, Wuyang Huang b, Haiming Chen a,*, Yuansong Li b,* a

College of Food Science and Technology, Hainan University, Haikou, Hainan

570228, China b

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

China

* Corresponding author: Dr. Haiming Chen Tel: 086-898-66256495 Fax: 086-898-66256495 Email: [email protected]

Prof. Yuansong Li Tel: 086-898-66256495 Fax: 086-898-66256495 Email: [email protected]

Highlights 

Soluble dietary fiber was recovered from the peel of papaya.



Alkaline and ultrasound-assisted alkaline extraction method were used.



The composition, structure and properties of the extracts were compared.



u-SDF exhibited higher thermal stability.



u-SDF exhibited higher water- and oil-holding, and swelling capacities than a-SDF.

Abstract Soluble dietary fiber (SDF) from the peel of papaya (Carica papaya Linn.) was recovered through alkaline extraction (alkaline-extracted SDF, a-SDF) and ultrasound-assisted alkaline extraction (ultrasound-treated SDF, u-SDF) processes, and the composition, structure and properties of the extracts were compared. The optimal parameters for obtaining the maximum extraction yield of u-SDF were evaluated through response surface methodology. Under optimal conditions, the maximum yield of u-SDF was 36.99%, and u-SDF had a lower total amino acid content but a higher essential amino acid (16.18%) than a-SDF. A monosaccharide analysis indicated that the primary sugars in a-SDF and u-SDF were neutral sugars and pectic saccharides, respectively. An X-ray diffraction analysis confirmed that u-SDF was less crystalline than a-SDF. Moreover, a thermal analysis indicated that u-SDF exhibited higher thermal stability. In addition, u-SDF exhibited higher water-holding, oil-holding and swelling capacities than a-SDF. These results indicate that papaya peel is a potential inexpensive source of natural dietary fiber and a potential functional food ingredient. Keywords: Carica papaya, Soluble dietary fiber, Ultrasound-assisted alkaline extraction, Optimization, Comparison.

1. Introduction Papaya (Carica papaya Linn.), which belongs to the Caricaceae family, is native to tropical America and has been disseminated throughout the world (Teixeira da Silva et al., 2007). In addition to its striking aroma and high vitamin content, the fruit of C. papaya Linn. is attractive to consumers because it contains numerous carotenoids, specifically β-carotene, lycopene, and anthraquinones glycoside, and hence possesses medicinal properties, including anti-inflammatory, hypoglycemic, anti-fertility, abortifacient, hepatoprotective, and wound-healing properties. Its antihypertensive and antitumor activities have also recently been established (Almora, Pino, Hernandez, Duarte, González & Roncal, 2004; Yogiraj, Goyal, Chauhan, Goyal & Vyas, 2014). In addition to the fruit, papaya stems and leaves are also used in the formulation of cosmetics and medications (Teixeira da Silva et al., 2007). However, the peel of papaya (~20% of the fruit weight), an abundant by-product, is primarily used in animal feed or fertilizer and has a few other commercial uses. Moreover, papaya peel is highly biodegradable due to its high concentrations of dietary fiber (DF), saccharides, mineral substances and proteins. Therefore, the peel of papaya is perishable without treatment, resulting in not only a waste of resources but also environmental pollution. Consequently, numerous studies have attempted to utilize this waste as a source of pectin (Koubala, Christiaens, Kansci, Van Loey & Hendrickx, 2014; Boonrod, Reanma & Niamsup, 2006), protease (Chaiwut, Pintathong & Rawdkuen, 2010), carboxymethyl cellulose (Rachtanapun, 2009) and other high-added-value compounds (Parniakov, Barba, Grimi, Lebovka & Vorobiev, 2014;

Calvache, Cueto, Farroni, de Escalada Pla, & Gerschenson, 2016). In addition, soluble dietary fiber (SDF) might be recoverable at acceptable yields from this DF-rich by-product. SDF is composed of carbohydrate-based polymers, including pectic substances, gums, mucilage, and some hemicelluloses, which have significant effects on human health, such as the prevention of heart disease, obesity and cancers (Elleuch et al., 2011). In addition, SDF from different agro-industrial products has different components and functions. A recent report demonstrated that SDF from orange by-products can be used as a potential fat replacer (O'Shea, Arendt, & Gallagher, 2012). Moreover, SDF has been added to various foods, including meat products (Viuda-Martos, Ruiz-Navajas, Fernández-López, & Pérez-Álvarez, 2010), breakfast cereals, bakery products (Vergara-Valencia et al., 2007), and dairy products (Sendra et al., 2008) to enhance their textural properties and promote a favorable mouthfeel (Elleuch et al., 2011). However, the components and potential values of SDF from papaya have not been evaluated. Chemical, enzymatic, and enzymatic-chemical methods are currently used for the recovery of DFs from different food sources. The processing conditions change the composition and microstructure of DFs, resulting in both desirable and undesirable effects on physicochemical and functional properties (Peerajit et al., 2012). Because of its environmentally friendly nature, ultrasonic technology has been used for research and development in the food industry in recent years (Minjares-Fuentes, et al., 2016). As ultrasonic waves pass through a liquid medium, the interactions of the

ultrasonic waves with liquids and dissolved gases lead to acoustic cavitation, which might affect the morphology and structure of the carbohydrate polymers. In addition, ultrasonic treatments have been reported to increase the extraction yield or reaction rate and to reduce the extraction time (Bagherian, Zokaee Ashtiani, Fouladitajar, & Mohtashamy, 2011). Ultrasound-assisted extraction technology has been successfully used for the extraction of components, such as polysaccharides, oils and proteins (Chen, Fu, & Luo, 2015). To date, most relevant studies have simply evaluated the yield and chemical composition of ultrasonic-treated SDF (u-SDF), whereas various other properties have been scarcely characterized and compared with those of alkaline-extracted SDF (a-SDF). We suspect that the extraction yield, structure and composition of a-SDF and u-SDF may exist differential. In addition, the properties of SDF may be also influenced on the basis of different extraction methods. The present work aimed to optimize the preparation conditions of u-SDF and to further compare the properties of u-SDF with those of a-SDF. To this end, response surface methodology was used to optimize the levels of the preparation variables to obtain the maximum yield of u-SDF. We further compared the nutritional composition (amino acids, mineral elements, neutral sugars, aldehyde acids and aminosaccharides), structure [Fourier transform infrared, (FT-IR), X-ray diffraction (XRD), and scanning electron microscopy (SEM)] and properties [thermal characteristics, water-holding capacity (WHC), oil-holding capacity (OHC) and swelling capacity (SC)] of u-SDF with those of a-SDF. This work might provide a functional food additive. 2. Materials and methods

2.1. Materials 2-Deoxy-D-glucose, myo-inositol, L-fucose (Fuc), L-rhamnose (Rha), L-arabinose (Ara),

D-xylose

(Xyl),

D-galactose

(Gal),

D-mannose

(Man),

D-galacturonic

acid

(GalA), glucosamine (GlcN), ribose (Rib), galactosamine (GalN), ferulic acid (FA), succinic acid, glacial acetic acid and methanol were purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI, USA). Ripened papaya (Carica papaya Linn.) were purchased from a local market in Haikou, Hainan Island, China. All other reagents were of analytical grade. 2.2 Preparation of SDF 2.2.1 Pretreatment of papaya peel Papaya peel was first washed with tap water to eliminate the bulk of sand and other inorganic materials. The peel was then immersed in 96% (v/v) boiling ethanol for 20 min and washed with 70% ethanol to remove low-molecular-weight sugars, organic acids, and inorganic salts and to inactivate enzymes. The residue was dried at 40°C to generate an alcohol-insoluble residue (AIR). After drying, the AIR was ground and passed through a 50-mesh standard sieve. 2.2.2 Alkaline extraction method AIR (2.0 g, dry basis) was suspended in NaOH solution (1%, w/w) with stirring (200 rpm) using a magnetic stirrer at room temperature (25°C), and the suspension was then transferred to a thermostatic water bath (50°C). After the suspension was incubated at 50°C for 30 min, the supernatant was obtained by centrifugation at 4000g for 30 min. The SDF-rich extract was precipitated by adding four volumes of ethanol

at room temperature and allowing the mixture to stand for 60 min. The precipitated SDF was recovered by centrifugation at 8000g for 20 min, and the precipitate was then collected and dried by freeze-drying. 2.2.3 Ultrasound-assisted alkaline extraction method The procedure of ultrasound-assisted alkaline extraction was performed using a UP200S ultrasonic system (Hielscher Ultrasonics GmbH, Teltow, Germany). The compact ultrasonic system is designed to be mounted on a stand and was equipped with a water bath coupled to a temperature controller (Frigiterm, J.P. Selecta, Barcelona, Spain) to maintain the desired extraction temperature. AIR (2.0 g, dry basis) was accurately weighed and transferred to an extraction tube. Based on the experimental design, a set of NaOH solutions was added to the extraction tube, and each extraction was performed under controlled ultrasound-assisted alkaline conditions. The effects of the NaOH concentration (0.6% to 3.0%), extraction temperature (30 to 80°C), extraction time (10 to 60 min), L/S ratio (10: 1 to 36: 1) and ultrasonic power (125 to 250 W) were investigated. After extraction, the suspension was centrifuged at 4000g for 30 min and then precipitated and freeze-dried as with the alkaline extraction method. 2.3 Response surface methodology Response surface methodology comprises a group of empirical techniques for evaluating the relationships among a series of controlled experimental factors and measured responses according to one or more selected criteria (Chen, Fu & Luo, 2015). The three extraction variables considered in this study were ultrasonic power,

ultrasonic time and liquid/solid (L/S) ratio (w/w), and the proper range and center-point value of these three independent variables were confirmed based on a single-factor experiment (Fig. 1). According to the Box–Behnken design, 15 experimental runs were performed, and the zero experiment was repeated five times. Table 1 Three-factor, three-level Box–Behnken design used for RSM and experimental data of the investigated response X1

X2

X3

Ultrasonic power (W)

Time (min)

L/S ratio

1

-1 (150)

-1 (20)

0 (15)

29.48 ± 0.54

2

-1

1 (40)

0

30.48 ± 0.31

3

1 (200)

-1

0

27.36 ± 0.26

4

1

1

0

26.95 ± 0.33

5

0 (175)

-1

-1 (10)

17.20 ± 0.12

6

0

-1

1 (20)

24.83 ± 0.58

7

0

1

-1

20.88 ± 0.05

8

0

1

1

27.82 ± 0.06

9

-1

0 (30)

-1

22.20 ± 0.20

10

1

0

-1

19.59 ± 0.17

11

-1

0

1

33.71 ± 0.46

12

1

0

1

32.81 ± 0.33

13

0

0

0

37.13 ± 1.01

14

0

0

0

38.71 ± 0.52

15

0

0

0

34.12 ± 0.83

Runs

a

Yield (%)a

Mean (n = 3) ± SD The Design Expert 8.0.6 software package (Stat-Ease, Inc., Minneapolis, MN, USA)

was used to establish the mathematical progress. In developing the regression equation, the test factors were coded according to the equation

𝑥𝑖 =

𝑋𝑖 −𝑋𝑖𝑥 ∆𝑋𝑖

, (1)

where xi is the coded value of the ith independent variable, Xi is the real value of the ith independent variable, 𝑋𝑖𝑥 is its value in the center point of the interval and ΔXi is the step change value. In addition, 𝑌 = 𝑏0 + ∑𝑖 𝑏𝑖 𝑥𝑖 + ∑𝑖 ∑𝑗 𝑏𝑖𝑗 𝑥𝑖 𝑥𝑗 + ∑𝑖 𝑏𝑖𝑖 𝑥𝑖2 , (2) where Y represents the dependent variable, b0 is the constant coefficient, bi, bii and bij represent the model coefficients of the linear, quadratic and interaction effects of the variables, respectively, and xi and xj are the coded independent variables. 2.4 Component analysis The concentrations of neutral sugars, aldehyde acids and aminosaccharides were determined by HPLC using a Waters 2690 HPLC system (Waters Inc., Milford, MA, USA) equipped with a C18 column (250 × 4.6 mm2, 4 µm, GraceSmart™, Deerfield, IL, USA). The mobile phase was composed of sodium phosphate buffer (100 mM, pH 6.7, A) and acetonitrile (B). The gradient elution conditions were 85% A and 15% B for 10 min, 83% A and 17% B for 20 min, 80% A and 20% B for 5 min, and 85% A and 15% B for 5 min. All determinations were performed at a temperature of 30°C and a flow rate of 1 mL/min. The free amino acid content was measured using the modified method described by Kim et al. (2013) with an L-8800 automatic amino acid analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan) equipped with a 4.6-mm (ID) × 60-mm ion-exchange column (Hitachi High-Technologies Corporation, Tokyo, Japan). The measurement conditions were as follows: buffer flow rate of 0.4 mL/min, reagent

flow rate of 0.35 mL/min, reactor heater temperature of 135°C, column temperature of 75°C, auto-sampler temperature of 5–8°C, sample injection volume of 20 µL, and detection wavelength of 570 nm (for proline) or 440 nm (for all other amino acids). An external standard was used to calculate the concentration of each amino acid. The mineral concentrations were measured according to the method described by Tabarsa, Rezaei, Ramezanpour, Waaland, and Rabiei (2012) with a flame atomic absorption spectrophotometer (Philips, PU 9400, USA) equipped with single hollow-cathode lamps for each element and an air-acetylene burner. The mineral concentrations were quantified using calibration curves from various standards. To determine the biomolecules, the samples (500 mg) were dissolved in 1 M hydrochloric acid, and filtered, and the resulting volume was then increased to 100 mL with distilled water. The obtained solution was stored and used for analysis. For trace element measurements, a dried sample (1 g) was dissolved in mixtures consisting of 10 mL of 63% HNO3 and 5 mL of 37% HCl, and the sample volumes were increased to 100 mL with distilled water for the determination. 2.5 Characterization FT-IR analyses were performed at room temperature using a PerkinElmer Spectrum RXIFT-IR spectrometer (PerkinElmer Instruments, USA). The sample powder was blended with KBr powder (1:10, w/w) and pressed into tablets, and the spectra were obtained over the range of 4000 to 400 cm-1 with a resolution of 8 cm-1. The homogeneity and morphology of the SDF samples were observed using a scanning electron microscope (Quanta 200, FEI Company, USA). The samples were

mounted onto bronze stubs and coated with a gold layer. The images were collected at an accelerating voltage of 10.0 kV. Micrographs were recorded at 400× and 3000× magnifications to ensure clear images. XRD patterns were recorded using an X-ray diffractometer (D/Max-200, Rigaku Denki Co., Ltd., Tokyo, Japan). The instrument was equipped with a theta compensating slit and a monochromatic Cu-Kα radiation source with a wavelength of 0.1542 nm and operated at 30 mA and 30 kV. The diffraction angle (2θ) was scanned from 10° to 70° at a rate of 3°/min. The crystallinity percentages were calculated using the Hermans–Weidinger method (Hermans & Weidinger, 1961). The thermal analysis of a-SDF and u-SDF was performed using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) techniques according to Khatkar, Barak, and Mudgil (2013) with minor modifications. The TGA of a-SDF or u-SDF (6.0 mg) was performed in a nitrogen atmosphere using a thermogravimetric analyzer (Pyris 1 TGA, PerkinElmer, USA) at a heating rate of 10°C/min over a temperature range from 30 to 600°C. DSC analysis was performed on a differential scanning calorimeter (DSC-204F1, Netzsch Group, Selb, Bavarian, Germany). A sample (5 mg) was placed into an aluminum pan, and immediately covered with a punched aluminum cover. An empty aluminum pan was considered as reference. All the DSC measurements were performed at a heating rate of 10 °C /min using nitrogen as purge gas at a flow rate of 50 mL/min. The change in heat flow of different samples was analyzed over a temperature range of 50–300 °C. The WHC was conducted according to the method described by Mateos-Aparicio,

Mateos-Peinado and Ruperez (2010) with some modifications. Briefly, 0.50 g of SDF (dry basis, W1) was accurately weighed and then well mixed with distilled water (20 mL). After equilibration at 25°C for 24 h, the slurry was centrifuged at 4000g for 10 min, and the supernatant was then removed. The weight of the residue was recorded (W2). The WHC was calculated as the amount of water retained by the sample (g/g dry weight) as follows: WHC (g/g) =

𝑊2 −𝑊1 𝑊1

. (3)

The OHC was determined in triplicate according to the method described by Wang et al. (2015) with slight modifications. Briefly, 0.50 g of SDF (dry basis, M1) was accurately weighed and mixed with 5 mL of olive oil. The sample was allowed to equilibrate at 4°C for 1 h and was then centrifuged at 4000g for 15 min. After the supernatant was decanted, the residue was collected and weighed (M2). The OHC was calculated as the ratio of the quantity of oil to the initial dry weight of the residue as follows: OHC (g/g) =

𝑀2 −𝑀1 𝑀1

. (4)

2.6. Statistical analyses The data were statistically analyzed using IBM SPSS statistical software (version 21.0, SPSS Inc., Chicago, IL, USA). Design Expert 8.0.6 software was used for analyzing the experimental data. The differences between means were assessed by analysis of variance (ANOVA) with Duncan’s test using a significance level of p < 0.05. 3. Results and discussion

3.1. Single-factor experiment and model fitting Taking the extraction yield of u-SDF from papaya (Carica papaya Linn.) peel as the assessment index, a single-factor experiment combined with response surface methodology was performed to optimize the extraction conditions. The factors and levels of the Box–Behnken central composite design (Table 1) were confirmed by testing several variables as the single factors, including NaOH concentration (%), ultrasound power (W), ultrasonic time (min), temperature (°C) and L/S ratio (w/w) (Fig. 1). The effect of the NaOH concentration on the SDF yield was determined in the range of 0.5 to 3.0%. The SDF yield first increased to its maximum level as the NaOH concentration was increased to 1.0% and then decreased with further increases in the NaOH concentration (Fig. 1A). Both the mass transfer rate and the density of the solvent might be affected by the temperature. The analysis of the effect of temperature revealed that the yield presented a modest increase as the temperature was increased from 30 to 40°C and a relatively rapid increase as the temperature was increased from 40 to 50°C. The increase of yield might be due to increases in the transfer rate as the temperature increased from 30 to 50°C (Fig. 1B). Further increases in the temperature, however, rapidly decreased the yield from 26.71 ± 0.42% to 21.94 ± 0.44%. This result might be due to a decrease in the carrying capacity, which is positively correlated with the density of the solvent obtained with increases in the extraction temperature. The extraction time is also an important variable because SDF dissolution requires a certain time, but thermal degradation could occur if the

extraction time is too long. In the present study, six extraction times (10, 20, 30, 40, 50, and 60 min) were tested to evaluate the impact of the extraction time on the SDF yield. As shown in Fig. 1C, an extraction time of 20-30 min is key for controlling the SDF yield. The extraction time should not exceed 30 min because other extraction times will have adverse impacts on the extraction of SDF. The yield of SDF also first increases and then decreases with increases in the L/S ratio (w/w). As shown in Fig. 1D, the highest yield was obtained with an L/S ratio of 15. Under appropriate ultrasonic conditions, the cell wall can be destroyed by ultrasonic waves, leading to more soluble substances (SDF). However, SDF might be degraded under high-intensity waves. As shown in Fig. 1E, an intensity of 180-200 W is suitable for SDF extraction. 36

28

a

A b

26

32

bc 28

c

Yield (%)

Yield (%)

a

B

c

24

b

b

24

cd

c d

d 22

20 0.5

1.0

1.5

2.0

2.5

3.0

30

40

NaOH concentration (%) 26

a

C

26

b

24

22

20

16

24

b Yield (%)

Yield (%)

b

18

c

c

D

20

60

70

80

a

ab bc

22

bc bc

20

c

18 10

10

50

Temperature (℃)

30

40

Time (min)

50

60

15

20

25

L/S ratio (g/mL)

30

35

26

a

E

a Yield (%)

24

ab b

b

22

b

20 120

160

200

240

Ultrasonic power (W)

Fig. 1. Effect of different extraction variables [(A) NaOH concentration, (B) extraction temperature, (C) extraction time, (D) L/S ratio, and (E) ultrasound power] on the extraction yield of u-SDF. Table 2 Significance of the regression equation coefficientsa Source

df

Model X1

-

9 ultrasonic

1

power

Sum

of

Mean

F-value

P-value

Significanceb

Squares

Square

553.2081

61.4676

10.6084

0.009070

***

10.4882

10.4882

1.8101

0.2363

*

X2 - time

1

6.5885

6.5885

1.1371

0.3350

*

X3 - L/S ratio

1

193.0613

193.0613

33.3194

0.002194

***

X1X2

1

0.4970

0.4970

0.08578

0.7814

*

X1X3

1

0.7310

0.7310

0.1262

0.7369

*

X2X3

1

0.1190

0.1190

0.0205

0.8916

*

X12

1

12.5744

12.5744

2.1701

0.2007

*

X22

1

143.7888

143.7888

24.8157

0.004170

***

X32

1

220.6499

220.6499

38.0808

0.001627

***

Residual

5

28.9713

5.7943

Lack of Fit

3

18.0964

6.0322

1.1094

0.5063

*

Pure Error

2

10.8749

5.4374

14

582.1794

Cor Total 2

2

r = 0.9502, adj r = 0.8607, cv = 8.53%, adeq precision = 15.3512. a

b

df, degrees of freedom; cv, coefficient of variation. *No significant difference (p > 0.05). **Significantly different (p < 0.05).

***Extremely significantly different (p < 0.01). Based on the results from the single-factor experiments, 15 experiments were

designed and performed via a BBD design of RSM, which was applied to optimize the ultrasonic-assisted extraction conditions, and the results are listed in Table 1. The experimental factor codes and results were evaluated by regression analysis and tested for significance (Table 2). The extraction yield could be explained by the following quadratic regression [Eq. (5)]: Y = 36.65333 – 1.145X1 + 0.9075X2 + 4.9125X3 – 1.845417X1X1 – 0.3525X1X2 + 0.4275X1X3 – 6.240417X2X2 – 0.1725X2X3 – 7.730417X3X3 (5) where Y is the yield of u-SDF, and X1, X2 and X3 are the coded variables. ANOVA was performed to evaluate and screen the effects of the significant variables in linear and quadratic forms, and the results are summarized in Table 2. The F-value and P-value of the model were 10.61 and 0.0091, respectively. According to Atkinson and Donev (1992), higher F-values and lower p-values indicate that the corresponding variables are more significant. In addition, the ANOVA regression model (p < 0.01) and the r2 value (0.9502) demonstrated that the quadratic model was significant for estimating the u-SDF yield. The lack-of-fit was non-significant (p > 0.05), indicating that yield of u-SDF can be accurately predicted using the quadratic model. Values of p less than 0.05 and 0.01 indicate that model terms are significant and extremely significant, respectively. In this case, the coefficients X3, X2X2 and X3X3 were found to be significant model terms, and the other coefficients were insignificant (p > 0.05). The lack-of-fit was not significant relative to the pure error because the corresponding F-value was 1.1094. In addition, the value of the determination coefficient (r2) was 0.9502, which implies that 95.02% of the variation could be

explained by the model. The relatively low values of the coefficient of variation (8.53%) and adeq precision (15.3512) indicate a good model fit. The three-dimensional (3D) response surface, as an essential part of the regression equation, can vividly expound the interactions between two variables and allow determination of the optimal levels of these two variables (Fig. 2). As shown in Fig. 2A, a slight interaction was obtained between ultrasonic power (X1) and ultrasonic time (X2). At an ultrasonic time of zero, the yield of u-SDF changed within a small range (31-33%). In contrast, a marked change in the u-SDF yield was obtained by changes in the ultrasonic time at a constant ultrasonic power. These results suggest that X1X2 was not significant. As revealed by Figs. 2B-2C, the L/S ratio (X3) strongly affected the u-SDF yield, and the effect of X3 was significant. The optimal reaction conditions for the maximal u-SDF yield (ultrasonic time of 30.76 min, L/S ratio (w/w) of 16.55, and ultrasonic power of 175 W) were obtained from the regression equation [Eq. (5)]. The maximal predicted u-SDF yield under the aforementioned conditions was 36.99%.

A

B

C

Fig. 2. Response surface plots for (A) Y = f(X1, X2), (B) Y = f(X1，X3), and (C) Y = f(X2,X3). Table 3 Amino acid and mineral element contents (mg/g dry weight) of a-SDF and u-SDF a-SDF (mg/g dry weight)

u-SDF (mg/g dry weight)

Threonine Valine Methionine Isoleucine Leucine Phenylalanine Lysine Tryptophan Histidine Arginine a

0.220 0.222 0.034 0.008 0.047 0.007 0.036 0.096 0.032 0.040

0.179 0.219 ND 0.004 0.005 0.001 0.028 0.062 0.020 0.029

NEAAs Serine Glutamic acid Glycine Alanine Cysteine Aspartic acid Tyrosine

ND 0.178 0.226 0.221 0 3.350 0.002

ND 0.115 0.170 0.155 0 2.078 0.003

EAAs

Proline 0.013 TAAs 4.732 EAAs/TAAs (%) 15.68 Mineral elements (mg/g dry weight) Na** 8.37 × 104 Mg** 1634 K** 5.22 × 104 Ca** 1172 P** 11.70 Fe* 68.12 Mn* 76.11 Cr* 0.34 Cu* 15.69 Zn* 11.28 As* 0.12 Se* 0.055 Cd 0.013 Pb 0.13 Hg 0.001 TMs 1.39 × 105

0.010 3.078 17.77 9.45 × 104 1960 4.75 × 104 1464 112.51 77.09 93.86 0.53 11.23 18.48 0.12 0.10 0.019 0.17 0.001 1.46 × 104

* Essential trace elements. ** Main minerals. EAAs: essential amino acids; NEAAs: non-essential amino acids; TAAs: total amino acids; TMs: total minerals; ND: not detected; a-SDF: soluble dietary fiber prepared using the alkaline extraction method; u-SDF: soluble dietary fiber prepared using the ultrasound-assisted method. a

Essential only in certain cases.

3.2. Composition analysis The amino acid and mineral contents of a-SDF and u-SDF were evaluated, and the results revealed that a-SDF contains high levels of most essential and non-essential amino acids. As shown in Table 3, the contents of 10 essential amino acids (Thr, Val, Met, Ile, Leu, Phe, Lys, His, Try and Arg) and eight non-essential amino acids (Ser, Glu, Gly, Ala, Cys, Asp, Tyr and Pro) were determined. In a-SDF, valine had the highest concentration among the essential amino acids, followed by threonine,

tryptophan, leucine, arginine, lysine, methionine, histidine, and isoleucine, whereas phenylalanine had the lowest concentration. Aspartic acid was the non-essential amino acid with the highest concentration in a-SDF. Ultrasound strongly affected the type and quantity of amino acids in a-SDF. The total amino acid (TAA) contents in a-SDF and u-SDF were 4.732 and 3.078 mg/g, respectively, suggesting that ultrasonic conditions yielded a lower TAA content than the traditional extraction method. However, the percentage of essential amino acids in u-SDF (16.18%) was higher than that in a-SDF (14.16%). In fiber pectin of tomato pomace, the essential and non-essential amino acids constitute 38.2% and 61.7% of TAAs (Namir, Siliha, & Ramadan, 2015). Minerals are one of the main nutritional components of a-SDF. The main minerals (Ca, K, Na, P and Mg) and essential trace elements (Fe, Cu, Zn, Cr, As, Se and Mn) are very important in biological processes (Gorinstein et al., 2001). In this study, 15 mineral substances in a-SDF and u-SDF were detected. As shown in Table 3, the total mineral (TM) contents of a-SDF and u-SDF were 1.39 × 105 and 1.46 × 105 mg/kg, respectively. In addition, almost all essential minerals were found at a higher concentration in u-SDF than in a-SDF. The mineral with the highest concentrations in a-SDF was Na (8.37 × 104 mg/kg), followed by K (5.22 × 104 mg/kg), Mg (1.63 × 103 mg/kg) and Ca (1.17 × 103 mg/kg). K and Mg play an important role in the prevention of coronary atherosclerosis by controlling life-threatening arrhythmias. In addition to forming a major part of the human skeleton, Ca is an important element for maintaining the proper function of the myocardium and heart vessels (Baxter,

Sumeray, & Walker, 1996; Gorinstein et al., 2001). a-SDF was also found to be have high concentrations of essential trace elements, particularly Mn (76.11 mg/kg), Fe (68.12 mg/kg), Cu (15.69 mg/kg) and Zn (11.28 mg/kg). Mn, Fe and Cu are very effective in the prevention and treatment of atherosclerosis and the corresponding complications. Fe is also an integral part of hemoglobin and is used for treating some forms of anemias (Kiechl, Willeit, Egger, Poewe, & Oberhollenzer, 1997). Zn is required by the body to maintain a sense of smell and a healthy immune system, build proteins, trigger enzymes, and create DNA. A deficiency in essential trace elements can lead to physiological imbalance and physical illnesses (Luo, et al., 2013). u-SDF can be used as a potential mineral-fortifying additive instead of inorganic zinc products, which have a low absorption rate. Table 4 shows the molar ratios of neutral sugars and aldehyde acids in and properties of a-SDF and u-SDF. The primary component of a-SDF was found to be neutral sugars, particularly Glc (31.94 ± 4.19%), which was found at a lower concentration in a-SDF than in SDF from guava peel (~42.3%) (Jiménez-Escrig, Rincón, Pulido, & Saura-Calixto, 2001). This result suggests that glucose oligosaccharides and dextrins, which consist of glucose units, might be the main compounds in a-SDF. After the ultrasonic treatment, the molar ratio of Glc decreased substantially to 15.49 ± 0.85%. Ultrasonic treatments have been reported to accelerate saccharification and degradation reactions (Chen, Huang, Fu, & Luo, 2014). Therefore, glucose oligosaccharides and dextrins might have been degraded during

Table 4 Molar ratio of neutral sugars and aldehyde acids in and properties of a-SDF and u-SDF Properties (g/g)

Chemical composition (mol%)

Sample

Man

Ara

Rib

Rha

Gal

Glc

GalA

GlcA

GlcN

WHC

OHC

SC

a-SDF

8.95±0.63a

7.23±0.37a

0.88±0.22a

7.28±0.81a

17.46±0.74a

31.94±4.19a

15.63±0.32a

7.49±0.20a

3.15±0.25a

4.93±0.10a

1.15±0.09a

4.05±0.18a

u-SDF

7.52±0.15b

6.74±0.33b

2.35±0.16b

7.80±0.56b

11.12±1.29b

15.49±0.85b

27.36±2.81b

13.79±0.18b

4.94±0.55b

5.26±0.15b

1.40±0.12b

4.54±0.21b

*The values represent the means from triplicate measurements ± standard deviation. Values in the same column with different letters are significantly different (p < 0.05). (a-SDF: soluble dietary fiber prepared using the alkaline extraction method; u-SDF: soluble dietary fiber prepared using the ultrasound-assisted method; Man: mannose; Gal:

galactose; Ara: arabinose; Glc: glucose; Xyl: xylose; GlcA: glucuronic acid; Rha: rhamnose; GalA: galacturonic acid; GlcN: glucosamine; Rib: ribose; WHC: water-holding capacity; OHC: oil-holding capacity; SC: swelling capacity).

the ultrasonic-assisted extraction of u-SDF. In addition, the covalent linkages between pectin and non-pectic polysaccharides can be broken by ultrasonic waves (Wang et al., 2016). As a result, the content of GalA, which constitutes the backbone of the homogalacturonan and rhamnogalacturonan regions of pectin, increased substantially, from 15.63 ± 0.32% to 27.36 ± 2.81%, after ultrasonic treatment. The content of GlcN was lower in a-SDF than in u-SDF. 3.3 Structure analysis The functional groups and their bonding configurations in a-SDF and u-SDF were elucidated by FT-IR spectra, as shown in Fig. 3A. The general spectral profiles of a-SDF and u-SDF were similar with the exception of some characteristic bands. The strong, broad absorption at 3443 cm−1, which corresponds to the stretching of O-H groups, mainly from pectin (galacturonic acid) and hemicellulose (xylose, mannose, galactose and arabinose), and the two weak absorption bands at 2936 and 2865 cm−1, which originate from C–H stretching (Cui, Phillips, Blackwell, & Nikiforuk, 2007), indicate the presence of the typical structure of polysaccharide compounds (Yan, Ye, & Chen, 2015). The bands in the range 1200–1800 cm-1 indicate the stretching modes of carboxyl groups from GalA of pectin and methoxy glucose uronic acid of hemicellulose. Specifically, the vibration in the ranges 1745–1750 and 1616–1634 cm-1 is assigned to esterified and ionized carboxyl groups, respectively (Pappas et al., 2004). In contrast, no obvious vibration signal in the range 1745–1750 cm-1 of the spectra was observed, indicating that the pectin in a-SDF and u-SDF is low-methoxyl pectin. The breakage of ester bonds can be explained by the principle of ester

hydrolyzation under alkaline conditions. In addition, ultrasound treatment can cleave ether linkages, as reported by Sun and Su (2004). The wavelength range 950–1200 cm-1 is considered the “fingerprint” region of carbohydrates because it enables identification of the major chemical groups (Chen, Fu, & Luo, 2015). However, the effect of ultrasonic waves on the structure of SDF observed in this study was not significant. XRD was used to further evaluate the crystallinity changes and thus detect the aggregation state of the a-SDF and u-SDF molecules. The XRD patterns of the SDFs obtained using both extraction methods are shown in Fig. 3B. The analysis of both patterns revealed two characteristic crystalline peaks at 12.82° and 22.51° 2θ for both a-SDF and u-SDF and one noncrystalline peak at 34.68° 2θ for a-SDF. These results suggest that a-SDF and u-SDF show the coexistence of crystalline and non-crystalline states, which is typical of cellulose I crystallinity. The pattern of u-SDF was similar to that of a-SDF, which indicates that ultrasonic waves did not change the crystal type of SDF. However, the degree of crystallinity of a-SDF (20.17%) was significantly (p < 0.05) higher than that of u-SDF (14.61%). The results suggest that the crystal structure of SDF was severely, but not entirely, damaged by ultrasound, and that the crystalline structure shifted from ordered to less ordered, and even disordered, which is characteristic of an amorphous area. Moreover, the interactive force between the SDF molecules become weaker. Therefore, the SDF tissue became loose after ultrasonic treatment. Ultrasonic treatment not only helps decrease the degree of polymerization of SDF but might also be effective for improving the solubility,

swelling, and water- and oil-holding capacities. A similar result was obtained by Wang et al. (2016) with ultrasound-treated pectin.

Fig. 3. FT-IR spectra (A) and XRD pattern, relative crystallinity (B) of a-SDF and u-SDF.

Fig. 4. TGA (A) and DSC (B) analysis of a-SDF and u-SDF. 3.4. Thermal analysis The thermal stability of a polymer is an important property that could make the material goof for food applications, in which the material is thermally processed through unit operations, such as sterilization and baking. TGA and DSC were performed to evaluate the thermal behaviors of a-SDF and u-SDF and to further confirm the effect of ultrasound treatment on the thermal stability of SDF. TGA was

performed at the temperature range of 30 to 600°C. As shown in Fig. 3C, significant changes in both SDFs mainly occurred at three temperature ranges (30–200°C, 200–500°C and 500–600°C), and SDF presented different degradation rates at these three temperature ranges. These results demonstrate that the types of pyrolysis material obtained at these different temperature ranges are different. The maximum weight loss was obtained in the range 200–300°C. The analysis of the first temperature range, 30-200°C, revealed that devolatilization occurred at 120°C, yielding a mass loss of approximately 21%, which can be attributed to the evaporation of absorbed water from the samples. This result suggests that a-SDF had more absorbed water than u-SDF. a-SDF demonstrated rapid weight loss (~60%) in the second temperature range (200–500°C). The relative intensities of the peaks can be related to the polysaccharide pyrolytic decomposition of the global quantities of pectic polysaccharides and hemicelluloses present in SDF (Carrier, et al., 2011; Wang, et al., 2016). As the pyrolytic temperature increased to the third temperature range (500–600°C), slow mass was observed, likely due to the thermal decomposition of char. The results of the analyses of the second and third ranges showed that the weight loss (%) of a-SDF was higher than that of u-SDF by more than 20%, indicating that u-SDF had a better thermal stability than a-SDF. Therefore, the processing temperature should be less than 200°C to avoid changing the performance indicators of SDF. A DSC analysis was performed to further elucidate the thermal transition of a-SDF and u-SDF, and the DSC curves of a-SDF and u-SDF (Fig. 4B) showed two obvious

endothermic peaks at 100 and 155°C and one exothermal peak at 230°C. At the first temperature range, the endothermic peak transition was in the range 50-135°C with a maximal peak at approximately 100°C (onset 50°C, offset 100°C), which could be ascribed to the evaporation of unbound water and the phase transition of pectin with absorbed water from a crystalline to an amorphous structure (Slavov, Panchev, Kovacheva & Vasileva, 2016). The second endothermic transition of both samples occurred at the same temperature (155°C), which could be due to the evaporation of bound water in a-SDF and u-SDF. However, the intensity of heat flow observed with a-SDF was significantly (p < 0.05) higher than that found for u-SDF, indicating that a-SDF had a higher content of bound water, which is consistent with the TGA results. Moreover, the higher intensity of heat flow also indicated that a-SDF had a lower thermal stability (Osorio, Carriazo & Barbosa, 2011). The exothermic peak shown in the DSC curve corresponds to the ongoing thermal and oxidative decomposition of the polymer and the vaporization and elimination of volatile products. The pyrolysis of polysaccharides is initiated by the random breakdown of glycosidic bonds followed by further decomposition (Mudgil, Barak & Khatkar, 2012). Both a-SDF and u-SDF had an exothermal peak at 230°C, which is likely attributed to the pyrolysis peak of pectin (Wang et al., 2016). The intensity of heat flow obtained for a-SDF was slightly lower than that found for u-SDF, which is consistent with the pectin content, as shown in Table 4. In agreement with our results, previous studies have shown that ultrasonicated lignin and cellulose show higher thermal stability than corresponding samples without ultrasonic treatment (Sun & Tomkinson, 2002; Tsalagkas, Lagaňa,

Poljanšek, Oven & Csoka, 2016).

a

a’

b

b’

Fig. 4. Microstructure of (a, a′) a-SDF and (b, b′) u-SDF. 3.5 SEM The morphologies of a-SDF and u-SDF are shown in Fig. 4. a-SDF had a block shape and a wrinkled surface with a compact texture. In addition, numerous holes and cracks were found to be spread all over the surface of a-SDF. The surface of u-SDF was much looser than that of a-SDF (Fig. 4b) and had a porous structure (Fig. 4b′). The different microstructures indicated that ultrasound irradiation might disrupt the crosslinks between polysaccharide molecules and reorganize the u-SDF matrix. 3.6. Water- and oil-holding capacities and swelling capacity The mouthfeel experience and the rheological properties of SDF-rich functional foods are closely related to the WHC, OHC and SC of SDF (Elleuch, Bedigian,

Roiseux, Besbes, Blecker, & Attia, 2011). The WHC, OHC and SC results are shown in Table 4. The WHC represents the ability of a material to retain water, including linked water, hydrodynamic water and physically trapped water, under the conditions of centrifugation or compression (Alfredo, Gabriel, Luis, & David, 2009). The initial WHC of a-SDF was 4.93 ± 0.10 g/g, which is higher than the WHCs of the SDFs from orange peel (3.63 ± 0.21 g/g), banana pseudo stem (4.71 ± 0.31 g/g), malt bagasse (3.68 ± 0.08 g/g), oat hull (2.13 ± 0.11 g/g), and rice hull (2.58 ± 0.28 g/g) (Wang, Xu, Yuan, Fan, & Gao, 2015; Jacometti et al., 2015) and lower than that of passion fiber (7.2 g/g) and chia fiber (15.41 g/g) (Alfredo et al., 2009). In addition, the WHC of u-SDF (5.26 ± 0.15 g/g) was significantly greater (p < 0.05) than that of a-SDF (4.93 ± 0.10 g/g). The ability of DF to retain oil is important for food applications, such as preventing fat loss upon cooking (Schneeman, 1999) and removing excess fat from the human body. As shown in Table 4, the OHCs of a-SDF and u-SDF were 1.15 ± 0.09 and 1.40 ± 0.12 g/g, respectively. The increased OHC of u-SDF might be due to the comparatively looser texture caused by ultrasonic treatment, as shown by SEM. A comparison of the OHC of u-SDF with the OHCs of a-SDF from other fruit- and cereal-based sources revealed that the OHC of u-SDF was lower than the OHCs of a-SDFs from orange peel fiber (1.76 ± 0.32 g/g), rice hull (1.85 ± 0.15 g/g) and malt bagasse (2.46 ± 0.27 g/g) but higher than that of oat hull (1.37 ± 0.06 g/g) (Jacometti, et al., 2015; Wang, et al., 2015). Many consumers are currently limiting the amount of

fat and calories in their diets. DFs have been used as a fat replacement to produce low-fat ground products, such as meat products (Yılmaz, 2004; Namir, Siliha, & Ramadan, 2015; Giese, 1996), ice cream (Adapa, Dingeldein, Schmidt, & Herald, 2000) and cake (Lakshminarayan, Rathinam, & KrishnaRau, 2006). Swelling is defined as the volume of a given weight of dry fiber after equilibrium has been achieved in excess solvent. u-SDF had a higher swelling value (4.54 ± 0.21 mL/g) than a-SDF (4.05 ± 0.18 mL/g). The swelling of u-SDF is similar to those reported for SDFs from tomato pomace, orange peel and malt bagasse but higher than those reported for SDF from rice bran DF (1.4 mL/g) and rice hull fiber (3.27 mL/g) (Namir, Siliha, & Ramadan, 2015; Jacometti et al., 2015; Wang et al., 2015). The high SC value indicates that foods containing a-SDF might increase the sensation of fullness, allowing the limiting of obesity by diet control without sacrificing good nutrition. Therefore, u-SDF is a potential functional food, particularly for the prevention or limiting of obesity. 4. Conclusions In this study, the extraction conditions of u-SDF were optimized by RSM, and the properties of u-SDF were compared with those of a-SDF. The results suggest that papaya (Carica papaya Linn.) peel is an ideal material for SDF recovery and that ultrasound-assisted alkaline extraction, which showed a satisfactory yield (36.99%), is feasible. Compared with a-SDF, u-SDF presents higher percentages of essential amino acids and essential trace elements. Moreover, u-SDF exhibits higher thermal stability and increased WHC, OHC and SC values, suggesting that ultrasound-assisted

alkaline extraction can effectively improve the functionalities of SDF, which might be a potential fiber-rich ingredient in functional foods. In addition, the monosaccharide analysis indicates that u-SDF primarily contains pectic saccharides, and the FT-IR analysis shows that the pectin in u-SDF belongs to low-methoxyl pectin, which presents excellent performance in gel processing with calcium ions. Therefore, u-SDF is also a potential gel. Acknowledgements This research was supported by the National Natural Science Foundation of China (31660495), Natural Science Foundation of Hainan Province of China (317002) and the Hainan University Start-up Scientific Research Projects of China (kyqd1630). Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the manuscript.

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