Hovenia dulcis polysaccharides: Influence of multi-frequency ultrasonic extraction on structure, functional properties, and biological activities

Journal Pre-proof Hovenia dulcis polysaccharides: Influence of multi-frequency ultrasonic extraction on structure, functional properties, and biological activities

Bing Yang, Yuxin Luo, Qunjun Wu, Qiong Yang, Jianquan Kan PII:

S0141-8130(19)39854-X

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.006

Reference:

BIOMAC 14310

To appear in:

International Journal of Biological Macromolecules

Received date:

1 December 2019

Revised date:

29 December 2019

Accepted date:

2 January 2020

Please cite this article as: B. Yang, Y. Luo, Q. Wu, et al., Hovenia dulcis polysaccharides: Influence of multi-frequency ultrasonic extraction on structure, functional properties, and biological activities, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2020.01.006

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© 2018 Published by Elsevier.

Journal Pre-proof Hovenia dulcis polysaccharides: Influence of multi-frequency ultrasonic extraction on structure, functional properties, and biological activities

Bing Yanga,b, Yuxin Luoa,b, Qunjun Wuc, Qiong Yangc, Jianquan Kana,b,*

College of Food Science, Southwest University, 2 Tiansheng Road, Beibei,

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a

Laboratory of Quality & Safety Risk Assessment for Agro-products on Storage and

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b

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Chongqing 400715, PR China

Taijiyuan Biotechnology co., Ltd., Hi-tech Development Zone 725700, Xunyang, PR

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China.

*Corresponding author: Jianquan Kan

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c

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Preservation (Chongqing), Ministry of Agriculture, Chongqing 400715, PR China

College of Food Science, Southwest University, No. 2 Tiansheng Road, Beibei District, Chongqing, 400715, P. R. China

Tel: +86-23-68250375; Fax: +86-23-68251947

E-mail address: [email protected] (J. Kan)

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Journal Pre-proof Abstract: The directional effect of single-frequency ultrasonic was the cause of the low extraction yield of polysaccharide macromolecule. Thus, a possible solution was to use multi-frequency ultrasonic technology to improve the yield of polysaccharide. Single-frequency (SF), dual-frequency (DF), and three-frequency (TF) ultrasonic extraction were applied to extract polysaccharides of Hovenia dulcis (HDPs). A maximal polysaccharide extraction yield (9.02 ± 0.29%) was gat using the dual-frequency ultrasonic with optimized DF conditions comprising 58.00 ℃, 33.00

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min, 28&40 kHz.

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The three HDPs were compared for their physicochemical, rheological, and functional

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properties, and their antioxidant activities. DF-HDPs contain higher uronic acid than

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SF-HDPs and TF-HDPs. Rheological tests indicated that the HDPs had excellent colloid properties and a promising potential to serve as a thickener, gelatinizer, and

lP

stabilizing agent in the food industry. Moreover, the DF-HDPs exhibited a notable oil

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holding capacity (3.92 ± 0.04 g oil/g), foaming capacity (35.26 ± 0.47%), and emulsion capacity (43.96 ± 0.67%). Compared to the SF- and TF-HDPs, the

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DF-HDPs had superior antioxidant activities. In conclusion, a better extraction method (dual-frequency ultrasonic extraction) was achieved.

Keywords: Hovenia Dulcis; Multi-frequency ultrasonic assisted extraction; Characterization; Functional property; Antioxidant activity

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1. Introduction Traditional methods for extracting organic compounds include well-established techniques that use an extraction solvent under heat and,or agitation. Modernization has sought to improve the extraction efficiency of these outdated extraction methods via more advanced technologies, including high-power ultrasonic, which leverages the energy released from rupturing cavitation bubbles to ensure that the extraction

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solvent penetrates deeply into the sample matrix, thus enhancing interfacial mass

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transfer[1]. A multi-frequency ultrasonic reactor ensures strong mode interference,

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thereby inhibiting directional effects in the reactor. By contrast, a single-frequency

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ultrasonic reactor imparts directional variation in the ultrasonic field, resulting in uneven energy dissipation in the reaction medium and limited extraction yield[2]. For

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this reason, multi-frequency ultrasound is typically preferred and is an essential tool

and

leaching[3-5].

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in many applications, including medicine, recalcitrant organic pollutant degradation, Nevertheless,

previous

reports

have

demonstrated

that

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single-frequency ultrasonic reactors can extract plant polysaccharides such as Ligusticum chuanxiong, Hohenbuehelia serotine, and Nephelium lappaceum L.[6-8]. This work thus aims to apply multi-frequency ultrasound to explore how polysaccharide properties vary according to their extraction by single-frequency (SF), dual-frequency (DF), and three-frequency (TF) ultrasound. Hovenia dulcis is a tall deciduous tree belonging to buckthorn family Hovenia Thunb. The edible portion is a peduncle (inflorescence axis) rather than the fruit, and the peduncle is fleshy, with many branches that are not straight, often in the shape of a T-shaped or a swastika-shaped. H. dulcis is also known as a honey tree, Chicken feet pear, Wood coral, etc. due to the unique shape of the peduncle. H. dulcis has been 3

Journal Pre-proof used as a traditional Chinese medicine for thousands of years, and also plays a vital role in traditional medicine in Korea and Japan. In recent years, the chemical and nutrient components, pharmacological activities, and product development of H. dulcis were studied. Pharmacological studies have shown that the crude substances and related chemical components from peduncle, seeds, leaves, and other parts have the effects of hangover and liver protection, anti-lipid oxidation, anti-fatigue, anti-tumor, and lowering blood pressure[8-12]. However, studies on the potential

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biological activities of H.dulcis polysaccharide is rarely reported.

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In recent years, plant-derived polysaccharides have come to be widely appreciated

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for their antioxidant, bacteriostasis, anti-tumor, antiviral, immunomodulatory,

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anti-inflammatory, anti-radiation, probiotic and anti-diabetic effects. These products are particularly attractive because they are sourced naturally, and they impart less

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toxicity and fewer side effects than synthetic antioxidants do. Natural plant-sourced

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polysaccharides also have wide-ranging prospective applications in functional food, biological medicine, pharmaceuticals, and other fields due to their useful functional

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characteristics such as strong hydrophilicity, ideal rheological properties, and the ability to act as gelling, foaming, emulsifying, and thickening agents. Owing to the many benefits of H. Dulcis polysaccharides (HDPs), this work describes HDPs extraction via multi-frequency ultrasound to explore how different frequencies affect the polysaccharides’ biochemical, functional characteristics. To this end, this work demonstrates HDPs extraction using a multi-frequency ultrasonic reactor. The effects of varied ultrasonic frequencies on the HDPs’ structural characteristics, rheological properties, functional properties, and antioxidant activities are considered, along with the broader implications for potential HDPs applications.

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Journal Pre-proof 2. Materials and methods 2.1. Materials Hovenia dulcis was picked from Taiji yuan Biotechnology Co., Ltd., in November 2018. A powder was prepared by mixing the H. dulcis in tap water, followed by drying via heat pump, crushing, and sieving (bore diameter 250 µm). The powder was stored at 25 ℃.

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2.2. HDPs extraction pretreatment

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The crushed H. dulcis is immersed in petroleum ether(30-60 ℃) for 24 h, during

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which it is continuously stirred. Then it was put in 80% ethanol and treated with hot

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water for 24 h. After pretreatment, the samples were filtered, dried, and screened, and

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2.3. Experimental method

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stored for later use[13].

2.3.1 Multi-frequency ultrasonic extraction

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The sample (5 g) was combined with 0.1 L double distilled water in a 0.25 L conical flask. Extraction was performed with a multi-frequency ultrasonic reactor (Five Pines, Jiangsu Univ. Biotechnology Co., Ltd. Jiangsu, China.). The samples were extracted via a water bath maintained at 200 W 50 ℃ for 30 min twice in different ultrasonic frequencies. Following ultrasonic, the supernatant was collected by centrifugation. The supernatant was concentrated to 25 mL in a vacuum, mixed with 100 mL anhydrous ethanol, and then the mixed solution was placed in a 4 ℃ environment for 12 h. After resting, the mixed solution was centrifuged to precipitate the polysaccharide, and the Savage method was used to remove the protein. Then it was washed sequentially with anhydrous ethanol, absolute ether, and acetone. Finally, 5

Journal Pre-proof the precipitation is redissolved with an appropriate amount of deionized water, dialyzed for 48 h, and freeze-fry. The lyophilized products prepared by single-frequency, dual-frequency, and three-frequency ultrasound were designated SF-HDPs, DF-HDPs, and TF-HDPs, respectively. The following equation calculated the

polysaccharide

extraction

weight of dried HDPs (g)

w

(%).Yield (%, w) = weight of pretreated 𝐻.𝐷𝑢𝑙𝑐𝑖𝑠 powder (g) × 100

(1)

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2.3.2 Experimental design

yield

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The optimal condition of single-frequency ultrasonic extraction was 40 KHz, 200

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W, at 55 ℃ for 35 min according single-factor experimental (Fig. S1. Seen supplementary materials); The extraction frequency of three-frequency ultrasonic was

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20&28&40 KHz under fixed conditions (200 W ultrasound power, at 55 ℃ for 35 min.

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Data not show). Meanwhile, the optimal condition of dual-frequency ultrasonic extraction was obtained by the response surface method.

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The rough range of the extraction factors (X1: temperature, X2: time, and

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X3:dual-frequency ) was determined according to the results of the single-factor experiment (data not shown). The three-level-three-factor Box–Behnken design (BBD) was introduced to optimize polysaccharide extraction. The whole design consisted of 17 experimental points, as shown in Table S1a (seen Supplemental material).

2.4. Physicochemical properties analysis 2.4.1. Chemical composition The content of total carbohydrate and uronic acid were assessed via the phenol-sulfuric acid colorimetric method and the 3-phenylphenol method, respectively[14]. The protein content was measured by Bradford assay with BSA as the standard[15]. 6

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2.4.2. Physicochemical characterization The molecular weight distribution (MWD) of the HDPs was measured using high-performance-size-exclusion-chromatography (HPSEC) on an Agilent 1260 HPLC system (Agilent Corporation, USA)[16]. The monosaccharides comprising the polysaccharides were labeled with PMP according to a previously reported method[13]. The Fourier-transform infrared spectroscopy (FT-IR), TGA, and XRD of

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HDPs were determined using the same methods by Yang, et al.[13].

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2.5. Rheological properties

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The rheological properties measurements of the aqueous solution of HDPs using an MCR 302 (Anton Paar GmbH, Graz, Austria) equipped with a cylinder (double gap),

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the experimental method described by Yang et al.[13].

2.6. Functional properties assay

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2.6.1. Water holding capacity and oil holding capacity The water holding capacity and oil holding capacity of the HDPs were measured according to a previously reported method[17].

2.6.2. Foaming qualities The foaming capacity (FC) and foaming stability (FS) of the HDPs were determined by a method previously reported[18]. Suspensions containing HDP concentrations of 1.0%, 2.0%, 3.0%, and 4.0% (w/v) were homogenized for 2 min to add air and make foaming. FC was measured immediately after whipping, while FS was determined 60 min afterward. The FC and FS were computed using the following 7

Journal Pre-proof equations: FC(%) =

Intial foam volume × 100 Total suspension volume

(2)

FS(%) =

Final foam volume × 100 Total suspension volume

(3)

2.6.3. Emulsion properties assay

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The emulsion capacity (EC) and emulsion stability (ES) of the HDPs were measured according to a previously reported method[17]. 10 mL of HDP solution (1,%

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2%, 3%, or 4%, w/v) was mixed with commercial soybean oil (3 mL) at room

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temperature (25 ± 2 ℃), and the mixture was homogenized for 60 s and then

Emulsion volume × 100 Total volume

(4)

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EC(%) =

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centrifuged at 1000 g for 10 min. The EC was computed as follows:

Each emulsion sample was heated and maintained at 80 °C for 30 min before being

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cooled back down to approximately room temperature. The samples were then

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centrifuged at 1000 g for 10 min. The ES was computed as follows: ES(%) =

Final emulsion volume × 100 Total volume

(5)

2.7. Antioxidant activity analysis of different HDPs The HDPs’ scavenging activities concerning the DPPH, hydroxyl, and superoxide anion radicals were measured via a method that has been previously described[14]. The Fe2+ chelating activity was also evaluated according to a well-established procedure[19].

2.8. Statistical analysis 8

Journal Pre-proof All measurements were performed in triplicate, and discrepancies were analyzed using analysis of variance (ANOVA) with SPSS 20.0 (IBM SPSS Statistics for Windows, IBM Corp., USA), with contrast tests applied to the data to determine differences among the means. The criterion for statistical significance was p < 0.05. Results are expressed as the mean ± standard deviation (SD).

3.1. HDPs extraction by multi-frequency ultrasonic

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3. Results and discussion

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An analysis of variance was made to assess the quality of the fitting model (Table

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S1a and Table S1b). In this model, the quadratic function model for the extraction of DF-HDPs was extremely significant with a small model (p < 0.0001) and satisfying

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measurement coefficient (R2 = 0.9813). The model had significant first-order linear

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effect (X2, X3: p< 0.0001, 0.0024) and second-order quadratic effect (X1,X2 ,and X3, both p< 0.0001), while X1X2, X1X3 and X2X3 had significant interaction effect (p <

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0.0001). Additional, the pure error result of the noise was not significant because of

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the lack of fit F-value (4.49) and p-value (0.0905). A relatively low value of C.V. value (2.76%) meant that the general availability and accuracy of the polynomial model are adequate.

The regression model’s significance and lack of fit are non-significant, indicating that the regression equation can better reflect the actual relationship between the three variable factors (X) and a response value (Y). The following quadratic polynomial equation was obtained by multiple regression analysis of experimental data: Y= -115.86 + 0.4796A + 4.0922B – 0.3073C + 5.4E-003AB – 0.0255AC + 0.0645BC – 0.0105A2 – 0.0388B2 – 0.4998C2

The 3D surface diagram shows the type of relationship and interaction between the independent and dependent variables. The independent variable X1-X2, X1-X3, and 9

Journal Pre-proof X2-X3 showed quadratic function effect (p < 0.05) on the yield of polysaccharide while another independent variable was fixed at 0 levels. The yield of DF-HDPs was greatly affected by temperature variation. The yield of DF-HDPs increased with increasing ultrasonic time (25.00 to 35.50 min) and temperature (50.00 to 57.00 ℃) until reaching a maximum level with other independent variables fixed at 0 levels (Fig. 1a). In a word, with the extension of extraction time (25.00-35.50 min) and increase of extraction temperature (50.00-57.00 °C), the ultrasonic dual-frequency (28&40

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KHz) toward the optimal yield direction, DF-HDPs yield continues to improve. The observed values of the yield of polysaccharide were in good agreement with

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the predicted values (Fig. 1d). The residual normal probability graph and the model

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normality hypothesis are mutually verified (Fig. 1e). The normal distribution of the

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residuals is satisfied. This also indicates that the design optimization of RSM extraction of polysaccharide is accurate and applicable. Meanwhile, the influence of

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the independent variable on the response at the midpoint of the design space was studied (Fig.1f). The higher the slope, the more sensitive the response (polysaccharide

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yield) to this independent variable. According to the perturbation plot, the degree of quadratic effect of each variable is different, but the curve change of variable X2 (time)

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is the most significant.

To verify the accuracy of the model, the experiment was carried out under the optimal condition of 33 min, 58 C, and 28&40 KHz. The mean yield of HD-HDPs (9.09 ± 0.29%, n=3) was obtained under this condition. The deviation from the predicted value is 1.31%, indicating that the model can be useful to optimize the extraction of DF-HDPs.

3.2. Chemical composition Extraction methods that differ according to their ultrasound frequencies are compared for their polysaccharide yields. The yield of dual-frequency ultrasound polysaccharides (DF-HDPs, 9.09 ± 0.29%) is significantly higher than those of 10

Journal Pre-proof single-frequency ultrasound polysaccharides (SF-HDPs, 6.93 ± 0.14%) and three-frequency ultrasound polysaccharides (TF-HDPs, 8.14 ± 0.05%). The results indicated that dual-frequency ultrasonic extraction of polysaccharides could significantly increase the yield of polysaccharides, which might be that dual-frequency ultrasonic extraction can increase the cavitation, thermal, and mechanical

effects

of

ultrasound,

resulting

in

the

dissolution

of

more

polysaccharides[20]. The superposition of more frequencies has both positive and

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negative impacts on cavitation, thermal, and mechanical impact. Therefore this might

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also lead to the yield of three-frequency ultrasonic extraction is lower than that of

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dual-frequency extraction.

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The total sugar contents of the SF-, DF-, and TF-HDPs are 50.81 ± 1.02%, 46.35 ± 0.75%, and 44.00 ± 0.22%, respectively (Table 1). The content of uronic acid, an

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ingredient with promising properties for hypoglycemic drugs and moisturizing

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cosmetics[21, 22], is greater in the DF-HDPs (21.19 ± 0.67%) than in the SF-HDPs (16.90 ± 0.27%) or TF-HDPs (14.58 ± 1.09%). The protein contents of the SF-HDPs,

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DF-HDPs, and TF-HDPs are 5.28 ± 0.25%, 5.06 ± 0.67%, and 4.95 ± 0.12%, respectively. These results indicate that the polysaccharides obtained by different multi-frequency ultrasonic extraction methods have different chemical components.

3.3. Characterization of polysaccharides 3.3.1. Molecular weight distributions The polysaccharides’ molecular weight distributions were measured by HPGPC (Figs. 2a-c and Table 1). The H. dulcis extracts are composed of polydisperse heteropolysaccharides with wide molecular weight (Mw) distributions. The molecular weight distribution of SF-HDPs has four sharp peaks at 24.54 × 102, 13.25 × 102, 3.64 11

Journal Pre-proof × 102, and 0.72 × 102 KDa, accounting for 2.54 ± 0.08%, 33.37 ± 0.04%, 26.91 ± 0.17%, and 37.17 ± 0.08% of the area under the distribution curve, respectively. The medium-high Mw values (0.27×102 to 41.04×102 KDa) of the SF-HDPs are higher than those of polysaccharides isolated from Zizyphus jujube (0.86 × 102 to 1.6 × 102 KDa)[23] and Fructus Jujubae (0.83 × 102 to 1.23 × 102 KDa)[24]. As shown in Fig. 2b, the Mw distribution of DF-HDPs can be divided into five sections corresponding to the five sharp peaks at 24.16 × 102, 13.82 × 102, 7.70 × 102, 4.27 × 102, and 0.69 ×

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102 KDa, which account for 2.70 ± 0.05%, 14.84 ± 0.01%, 12.77 ± 0.07%, 31.55 ±

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0.08%, and 38.16 ± 0.05% of the area under the distribution curve, respectively. These

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results confirm that the type of multi-frequency ultrasound treatment has a significant

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effect on the extracted polysaccharide components’ Mw distribution. The TF-HDP Mw distribution has three sharp peaks at 13.36 × 102, 4.61 × 102, and 0.67 × 102 KDa,

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accounting for 33.85 ± 0.12%, 30.26 ± 0.06%, and 35.89 ± 0.06% of the area under

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the distribution curve, respectively. These results indicated that the maximum molecular weight of polysaccharides decreased with the superposition of single

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frequencies, and the maximum molecular weight distribution peaks of SF, DF, and TF-HDPs were 24.54, 24.16, and 13.36 KDa, respectively. Meanwhile, the proportion of small molecular weight distribution in molecular weight of DF-HDPs (69.71%) was high than that of SF-HDPs (64.68%). The reason might be that frequency superposition enhances cavitation effect of ultrasound, leading to the serious destruction of the polysaccharide molecular chain. Interestingly, the medium-high Mw polysaccharides exhibit high solubility in water, biological absorptive ability, and biological activity[25]. Therefore, the antioxidant activities and functional characteristics of H. dulcis polysaccharides may warrant future analysis.

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Journal Pre-proof 3.3.2. Monosaccharide composition The monosaccharide compositions of the polysaccharides were analyzed by HPLC (Figs. 2e-h) and found to consist of mainly Man, Rib, Rha, GlcA, GalA, Glc, Gal, and Ara. While the polysaccharides extracted via different ultrasonic treatments are qualitatively similar, they vary according to their relative monosaccharide contents. The molar ratios of the monosaccharides comprising the SF-, DF-, and TF-HDPs were 2.91:5.80:16.47:1.29:2.74:19.08:24.58:27.14,2.80:9.69:13.11:1.83:2.62:22.01:23.86:2

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4.09, and 2.85:6.86:16.28:1.79:4.61:21.53:25.29:20.78, respectively. On a molar ratio

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basis, the most prevalent monosaccharides of three polysaccharides are Rha, Glc, and

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Gal. The monosaccharides of the three polysaccharides had the same species but

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different in their content. The maximum content of monosaccharides in SF-, DF, and TF-HDPs were galactose, glucose, and arabinose, respectively, and the content of

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uronic acid was also different, and different monosaccharide composition affect the

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functional properties and antioxidant capacity of polysaccharides. These results manifested that different multi-frequency ultrasound extraction methods had certain

3.3.3. FT-IR

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promoting effects on some parts of monosaccharides

The SF-HDPs, DF-HDPs, and TF-HDPs generally have similar spectral features except for some characterized bands that differ in absorbance and wavenumber (Fig. 2o). The typical characteristic peaks of polysaccharides appear at approximately 3390, 2930, and 1421 cm-1 [17]. The strong signals at 3390 cm-1 are put down to the stretching vibrations of polysaccharide hydroxyl groups, while the weak signals at 2930 cm-1 are indicative of the C-H stretching of polysaccharide methylenes. The absorption bands in the 1500-1750 cm-1 region issue from C=O bending and symmetric stretching vibrations. The characteristic strong absorption peaks at 1421 13

Journal Pre-proof cm-1 are indicative of C-H bending. Combined these results indicate that the SF-, DF-, and TF-HDPs are acidic polysaccharides [26]. This observation is consistent with the presence of uronic acid in the HDPs. The absorption peak (S=O stretching vibrations) at 1249 cm-1 confirmed the existence of trace sulfuric acid radicals. The relative weakness of the peaks may be attributed to the sulfuric acid radicals’ destruction by ultrasound. The signals at 1000-1200 cm-1 demonstrate the existence of C-O-C and C-O-H bonds, which are a feature of pyranose. The bands at approximately 830 cm-1

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indicate the presence of α-configurations[27]. There was no significant influence on

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the distribution of sugar rings and glycosidic bonds in polysaccharides by the change

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of ultrasonic extraction frequency, as evidenced by the similar spectra observed for

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3.3.4. TGA and XRD

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polysaccharides achieved by different ultrasonic frequency extraction methods.

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The TGA curves (Figs. 2i-k) of the polysaccharides are similarly shaped. The first mass loss occurs between 25 and 110 ℃ and is mainly due to the vaporization of

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adsorbed and structural water. Above 150 ℃, the devolatilization of polysaccharides leads to mass loss, with the thermal decomposition occurring mostly between 200 and 520 ℃. When the system temperature rises to 520 ℃, the residual masses of the SF-, DF-, and TF-HDPs are approximately 44%, 26%, and 44%, respectively. Finally, as the temperature increased further, the residual masses of the three HDPs remained almost entirely unchanged. Therefore, the degradation behavior and thermal stability analyses indicate that all of the polysaccharides are comprised of a variety of complex polymers with different structures. In the highest temperature region, ranging from about 520 to 700 ℃, the weight of the HDPs gradually decreases. X-ray diff raction is a powerful method to evaluate the crystalline characteristics of 14

Journal Pre-proof the polysaccharides. Figs. 2l-n demonstrate that diffraction peaks are detected at 2θ = 20.04, which indicates that the extracted HDPs are semi-crystalline polymers, as is consistent with the literature[15]. This character directly affects the elasticity, expansibility, strength of extension, dissolvability, and other physical properties of the HDPs.

3.4. Rheological properties

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3.4.1. Effect of polysaccharide concentration on apparent viscosity

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The apparent viscosities of the HDPs were tested for sensitivity to polysaccharide

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concentrations ranging from 0.1%-3.0%. The results, displayed in Figs. 3a-c, indicate

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that the polysaccharides’ apparent viscosity and mass concentration increase together for all samples, since high concentrations generate strong interactions among

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polysaccharide chains and increased polymerization[28]. Moreover, the DF-HDPs

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exhibit superior thickening behavior compared to the TF-HDPs and SF-HDPs. All of the samples with concentrations in the tested range display reduced viscosity with

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increasing shear rate, which indicates that the HDPs present a shear-thinning behavior of typical non-Newtonian fluids[29]. The behavior of shear-thinning mainly attributed to entanglements among macromolecular colloidal particles was made up of giant chain molecules, which can prompt a higher apparent viscosity. This viscosity is usually generated under static or low flow rate conditions. Nevertheless, as the shear rate increases, the disordered macromolecular polymers line up in the direction of flow, weakening their interactions, and ultimately leading to a viscosity reduction[30]. Among differently prepared samples of similar concentrations, the DF-HDPs display the highest apparent viscosity, while the TF-HDPs exhibit the lowest. The shear-thinning behavior of the HDPs may be of great interest for foodstuff processing 15

Journal Pre-proof applications, such as the manufacture of solidified fermented milk. Overall, the apparent viscosities of all of the polysaccharide solutions diminish slowly with increased shear rate (10-1000 s-1) before stabilizing, and exhibit fluid properties which are consistent with rheological characteristics reported for the polysaccharides of Polygonatum cyrtonema Hua[28].

3.4.2. Effect of NaCl on the apparent viscosity of 1.0% HDP solutions

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To measure the polysaccharides’ functional rheological properties, it is important to

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identify the effect of ion concentration on the viscosity of polysaccharide solution.

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This is especially important because charged molecules’ ionic strength can prompt

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them to act as highly viscous materials. On the other hand, positive ions may reduce repelling forces and molecular expansion among polysaccharide chains, which could

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significantly reduce viscosity. As shown in Figs. 3d-f, adding Na+ ions to the HDPs

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solutions noticeably impacts apparent viscosity, which decreases with increasing shear rate within the range of 0.01-1000 s-1. While increasing Na+ concentration reduces the

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apparent viscosity of the DF- and TF-HDPs, the effect on the SF-HDP solution is less consistent, exhibiting an increased apparent viscosity for a 0.5 mol/L Na+ solution and decreased apparent viscosities for 0.1 mol/L and 1.0 mol/L Na+ solutions. The predominant trend may be explained due to a salting-out effect induced by the NaCl, which reduces polysaccharide solution concentration and ultimately decreases apparent viscosity. It is also possible that the polysaccharides in the SF-HDPs, DF-HDPs, and TF-HDPs undergo varying degrees of molecular expansion in solution when ions are present[27].

3.4.3. Linear viscoelastic region measurements of polysaccharides 16

Journal Pre-proof Determining the linear viscoelastic region is important to ensure that the samples are not easily destroyed. HDPs of varying concentrations (0.5%, 1.0%, and 2.0%) were measured under stress from 0.1%-1000% oscillation strain. The results shown in Figs. 3g-i indicates that a 1% shape change can serve as a suitable dynamic oscillation condition for measuring each sample’s strain performance.

3.4.4. Oscillatory measurements of polysaccharides

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Based on the measured linear viscoelastic regions of the HDPs, G´ (storage

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modulus) and G´´ (loss modulus) were measured under 1% oscillation intensity. As

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depicted in Figs. 3j-r, the HDPs display solid and liquid characteristics[29]. Similar to

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other common polymer solutions, the G´ and G´´ of the HDPs increase with increasing oscillation frequency. The G´ is lower than G´´ for all of the polysaccharide

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solutions at low frequency, which indicates that the polysaccharide solutions have a

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predominantly viscous nature. As the frequency increases, the G´´ of all of the samples gradually grows stronger than G´, signaling that the polysaccharide solutions

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possess mostly elastic characteristics[31]. The cross-frequency values demonstrate the viscoelastic behavior of the polysaccharides, with lower cross-frequency values correlated to greater viscoelasticity[32]. Since the crossover values decrease with increasing polysaccharide concentration, solutions with higher polysaccharide concentrations are more elastic. The 1.0%- and 2.0%-concentrated TF-HDP solutions exhibit lower cross-frequency values compared to the SF-HDPs and DF-HDPs. On the other hand, among the solutions with polysaccharide concentrations of 0.5%, the DF-HDPs display the lowest cross-frequency values. Overall, these results demonstrate that it is possible to achieve both high viscosity and elasticity in plant-based polysaccharide solutions, which may be beneficial to applications in food 17

Journal Pre-proof processing.

3.5. Functional properties 3.5.1. Water holding capacity and Oil holding capacity Materials with high water-holding capacities (WHCs) are prized within many food applications, where they may be used as gelatinizer, stabilizers, and texture modifiers. The water holding capacity of the SF-HDPs is 1.76 ± 0.02 g water/g, which is

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significantly lower than those of the DF-HDPs (1.90 ± 0.02 g water/g) and TF-HDPs

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(1.84 ± 0.02 g water/g). The WHCs of the HDPs are higher than what has been

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reported for polysaccharides sourced from some other plants, such as pistachios (1.46

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± 0.62 g water/g)[33]. The OHC of material can serve as a metric for predicting the adsorption of organic compounds. This quantity is 3.92 ± 0.04 g oil/g for the

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DF-HDPs, which is significantly higher than those of the SF-HDPs (2.99 ± 0.01 g

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oil/g) and TF-HDPs (3.46 ± 0.05 g oil/g). Admittedly, these OHC are higher than the OHC reported for Rosa roxburghii Tratt fruit polysaccharides (3.29 ± 0.38 g oil/g)[34].

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However, the relatively high OHC of the DF-HDPs nonetheless indicates a strong potential to serve as stabilizers in high-fat foods.

3.5.2. Foaming qualities

Polysaccharides can be used as gelling or thickening agents for stabilizing dispersions, which can enhance the flavor, texture, and shelf life of food[35]. Therefore, it is important to understand the foaming qualities of the HDPs if they are to be of service to application in the food industry. According to Figs. 4a-b, the HDPs achieve slight foaming at 1% polysaccharide concentration, but the foam is breakable and dissipates nearly completely within half an hour. The foaming capacity (FC) and 18

Journal Pre-proof foaming stability (FS) are improved with increasing polysaccharide concentration. This may be attributed to the increased ability of HDPs to form reticular structures at higher concentrations, prompting greater stability of the interfacial film. When the concentrations of HDP are 3% and 4%, the DF-HDPs display the greatest FC and FS, which are significantly higher than those of the other two polysaccharides at the same concentration. The excellent FC and FS observed in 3%- and 4%-concentrated DF-HDP solutions indicate that the DF-HDPs have good potential as foaming agents

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in the food industry.

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3.5.3. Emulsion properties

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Some reports have indicated that the emulsion capacity (EC) and emulsion stability (ES) of polysaccharides largely depend on their rheological characteristics and

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molecular compositions, which generally include both complex composite sugar

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molecules and hydrophobins[36]. As indicated in Figs. 4c-d, the HDPs in this study exhibit increasing EC and ES values with increased concentration. The emulsion

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properties of the DF-HDPs are superior to those of the SF-HDPs or TF-HDPs at the same concentration. An EC value of 43.96 ± 0.67% is achieved for a DF-HDP solution of 4% concentration, indicating the potential of DF-HDPs as suitable emulsifiers in the food industry.

3.6. Antioxidant activity of polysaccharides 3.6.1. DPPH radical scavenging activity The SF-, DF-, and TF-HDPs have good antioxidant activities, as is confirmed by their concentration-dependent DPPH radical scavenging activities compared to vitamin C (Fig. 5a). The DPPH radical scavenging capacity (y) and concentration (X) 19

Journal Pre-proof of the SF-, DF-, and TF-HDPs are quadratic correlated (p < 0.05), and can be expressed

by

the

formula

y=-2.427x2+18.968x+41.855

y=-3.0585x2+21.9x+35.432

(R2=0.9931),

and

(R2=0.9919),

y=-3.0319x2+21.38x+31.384

(R2=0.9913), respectively. The scavenging rates of 4%-concentrated SF-, DF-, and TF-HDPs solutions are 74.64%, 79.48%, and 69.40%, respectively. The inhibitory concentrations (IC50) of SF-, DF-, and TF-HDPs on DPPH radical scavenging capacity are 0.742, 0.456, and 1.017 mg/mL, respectively. These values indicate that

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the DPPH radical scavenging capacity of the DF-HDPs is higher than those of SF-

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and TF-HDPs. The DPPH radical scavenging capacity of polysaccharides is

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determined by their monosaccharide composition, molecular weight distribution, and

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chemical composition, because it mainly depends on the electron donor capacity of the polysaccharides. The HDPs obtained by different ultrasonic frequencies do indeed

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have different monosaccharide compositions (Fig. 2e-g) and molecular weights (Fig.

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2a-d), which may have plausibly generated variation in structural configurations, monosaccharide orders, and glucosidic bonds, all of which could contribute to

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differences in DPPH radical scavenging capacity[37]. In general, the HDPs have significant DPPH radical scavenging activity, with the DF-HDPs, in particular, showing great promise as good radical-scavengers. 3.6.2. Hydroxyl radical scavenging activity The SF-, DF-, and TF-HDPs all show a significant hydroxyl radical scavenging activity that is positively correlated with increasing concentration of polysaccharide (Fig. 5b). The polysaccharide concentration (x) and hydroxyl radical scavenging capacity (y) of the SF-, DF-, and TF-HDPs are expressed by the following quadratic function

equations,

respectively:

y=-5.0046x2+42.528x+6.1703

(R2=0.9986),

y=-4.944x2+42.3x+8.9223 (R2=0.9998), and y=-5.8655x2+46.157+0.2924 (R2=0.999). 20

Journal Pre-proof The DF-HDPs have a lower IC50 value (1.117 mg/mL) than that of the SF-HDPs (1.2000 mg/mL) and TF-HDPs (1.287 mg/mL). The maximum hydroxyl scavenging activities of the SF-, DF-, and TF-HDPs are 95.69%, 98.87%, and 90.57%, respectively. Overall, these results display that the HDPs have significant hydroxyl scavenging ability and can be used as an effective hydroxyl radical scavenger.

3.6.3. Superoxide radical scavenging activity

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As shown in Fig. 5c. Superoxide anion radical scavenging was enhanced with the

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increase of polysaccharide concentration. The superoxide radical scavenging capacity

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of the SF- and TF-HDPs are significantly lower than those of the DF-HDPs at low

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concentrations (less than 2.0 mg/mL). The scavenging rates of 0.125%-concentrated SF-, DF-, and TF-HDPs are 57.37%, 64.72%, and 55.78%, respectively. Finally, the

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result showed that the HDPs have a strong ability to scavenging superoxide anion

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radicals.

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3.6.4. Fe2+ chelation activity

The Fe2+ chelation activity (y) of the HDPs is linearly related to the concentration (X) and has a concentration dependence. For SF-, DF-, and TF-HDPs, the formula takes the form y=10.338x+7.1096 (R2=0.9641), y=15.848x+9.1963 (R2=0.9684), and y=8.9097x+5.5788 (R2=0.9708), respectively. For concentrations between 0.125 and 0.25 mg/mL, there are no significant differences observed in the HDPs’ Fe2+ chelation activity as a function of multifrequency ultrasound treatment. However, once the solution concentrations increase to 4.0 mg/mL, the chelating capacity of iron ions of the DF-HDPs reaches 68.88%, which is significantly higher than that of the SF- and TF-HDPs. The enhanced chelation of F e2+ may also be confirmed by the high uronic 21

Journal Pre-proof acid content of DF-HDPs compared to SF-, and TF-HDPs, allowing Fe and COOH to form bridges[37].

4. Conclusion In this work, Single-frequency (SF), dual-frequency (DF), and three-frequency (TF) ultrasonic extraction were applied to extract Hovenia dulcis polysaccharides (HDPs). A maximal extraction yield (9.02 ± 0.29%) was obtained using the dual-frequency

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ultrasonic with optimized DF conditions comprising 58.00 ℃, 33.00 min, 28&40 kHz.

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Experimental results indicated that while the ultrasound treatments of varying

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frequency did not change the primary HDP structures, they did affect the

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polysaccharides’ chemical compositions, molecular weight distributions, rheological properties, functional properties, and antioxidant activities. It was demonstrated that

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the SF-, DF-, and TF-HDPs were all comprised of the same monosaccharide

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components but differed according to their quantitative compositions. All three frequency-controlled extraction methods yielded polysaccharides with typical

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characteristics and excellent thermal stability. Rheological tests revealed that the HDPs exhibited excellent colloid properties and have the potential to serve as suitable thickening agents, gelling agents, and stabilizers in the food industry. Moreover, the DF-HDPs displayed attractive oil holding capacity, foaming qualities, and emulsion capacity. The antioxidant activity of the DF-HDPs was superior to those of the SFand TF-HDPs, as indicated by the DF-HDPs’ effective DPPH, hydroxyl, and superoxide anion scavenging abilities and Fe2+ chelating activity. Therefore, the appealing physical properties and antioxidant behavior of the DF-HDPs make them promising materials for many potential applications in the functional food, pharmaceutical, and cosmetic industries. Furthermore, dual-frequency ultrasound 22

Journal Pre-proof extraction can serve as a compelling alternative to conventional methods for obtaining these beneficial plant-based compounds.

Acknowledgments This research was supported by the “Study on active ingredients and hypoglycemic mechanism of selenium-rich juice of Hovenia dulcis” (4411700155, and

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4411800204).

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Journal Pre-proof European Food Research and Technology. 238 (2014) 1015-1021.

Figure captions: Fig. 1. (a–c) 3D-response surface plots of RSM. (d) Plot of predicted vs. actual; (e)

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Plot of Normal Plot of Residuals; (f) Plot of Perturbation. Fig. 2. Characterization of different HDPs. (a-c) the molecular weight distribution of

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HDPs and (d) dextran standard; (e) monosaccharide composition of Lactose (Lac)

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standard and (f-h) HDPs; (i-k) TGA curve of HDPs; (l-m) X-ray diffraction pattern of

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HDPs; (o) FT-IR spectra of HDPs.

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Fig. 3. (a-c) Variation of the apparent viscosity of different concentration HDPs with the shear rate; (d-f) Variation of the apparent viscosity of four 1% -concentrated HDPs

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with the amount of NaCl addition. (g-i) Strain sweep tests at 1.0 Hz for determining the moduli G′ and G′′ of the HDPs; (j-r) Frequency sweep tests at 1% strain for

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determining the G′ and G′′ of the HDPs. Fig. 4. (a-b) Foaming properties and (c-d) emulsifying properties of the HDPs as a function of concentration. Each value is the mean ± one standard deviation (SD) of triplicate measurements. Different letters represent significantly different results (p < 0.05). Fig. 5. Antioxidant activity of different HDPs in vitro. (a) DPPH radical scavenging activity; (b) hydroxyl radical scavenging activity; (c) superoxide anion radical scavenging activity; and (d) Fe2+ chelating activity.

26

Journal Pre-proof

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Bing Yang, Jianquan Kan: Conceptualization, Methodology, Writing- Reviewing and Editing. Bing Yang: Data curation, Writing- Original draft preparation. Bing Yang, Yuxin Luo: Conduct of experiment. Qunjun Wu, Qiong Yang: Experimental materials, Funding. Jianquan Kan: Funding., Validation.

27

Journal Pre-proof Table 1. Chemical compositions, functional properties and molecular weight distribution of soluble polysaccharide components obtained by different multi-frequency ultrasound extraction methods. Different letters are significantly different results (p < 0.05). Samples

Chemical composition Sugar content(%)

Uronic acid (%)

Function properties Protein content (%)

WHC (g/g)

OHC (g/g)

c

2.99±0.06c

SF-HDPs

50.81±1.02

16.90±0.27

5.28±0.25

1.76±0.02

DF-HDPs

46.35±0.75

21.19±0.67

5.06±0.26

1.90±0.02a

3.92±0.04a

TF-HDPs

44.00±0.22

14.58±1.09

4.95±0.12

1.84±0.02b

3.46±0.05b

TF-HDPs

Area account(%)

1

6.712±0.005

2104.341-4104.812

2.54±0.08

2

7.531±0.004

614.947-2104.341

33.37±0.04

3

9.248±0.016

196.537-614.947

26.91±0.17

4

11.394±0.011

1

6.733±0.005

2

7.475±0.004

1000.602-2094.862

14.84±0.01

3

8.253±0.004

651.627-1000.602

12.77±0.07

4

9.034±0.006

187.016-651.627

31.55±0.08

5

11.446±0.006

29.957-187.016

38.16±0.05

1

7.520±0.005

657.043-3824.514

33.85 ± 0.12

8.936±0.007

190.170-657.043

30.26 ± 0.06

11.492±0.004

23.0908-190.710

35.89 ± 0.06

2 3

-p

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Mw (kDa)

27.954-196.537

37.17±0.08

2094.862-3128.435

2.70±0.05

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DF-HDPs

Retention time (min)

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SF-HDPs

Molecular weight distribution

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Peak no.

28

Journal Pre-proof Highlights



Multi-frequency ultrasound extraction was proposed to extract polysaccharides.



Dual-frequency (DF) can significantly improve the yield of Hovenia dulcis polysaccharides (HDPs) compared with Single-frequency and Three-frequency. The structure of HDPs was examined by FT-IR, XRD and HPLC analysis.



DF-HDPs exhibited interesting oil holding capacity, foaming and emulsifying

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

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DF-HDPs can be exploited as a multi-functional additive or antioxidant agent.

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

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properties.

29

Figure 1

Figure 2ah

Figure 2io

Figure 3

Figure 4

Figure 5