Maltodextrin or gum Arabic with whey proteins as wall-material blends increased the stability and physiochemical characteristics of mulberry microparticles

Food Bioscience 31 (2019) 100445

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Maltodextrin or gum Arabic with whey proteins as wall-material blends increased the stability and physiochemical characteristics of mulberry microparticles

T

Ibrahim Khalifaa,c, Mengli Lia, Torkun Mameta, Chunmei Lia,b,

⁎

a

College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, China Key Laboratory of Environment Correlative Food Science, Huazhong Agricultural University, Ministry of Education, Wuhan, Hubei, 430070, China c Food Technology Department, Faculty of Agriculture, Benha University, Toukh, Qalyubia, 13736, Egypt b

ARTICLE INFO

ABSTRACT

Keywords: Gum Arabic Maltodextrin Whey proteins Mulberries Morus australis poir

The efficiency of various encapsulating wall-materials on the stability and the characteristics of polyphenols in mulberry juice microparticles were studied. The ternary mixtures of maltodextrin (MD), gum Arabic (GA), and whey proteins (WP) were used as wall-materials and mulberry juice polyphenols were used as the core using a simplex-lattice experimental design. The mixture was then spray dried and the physicochemical properties of the powder were measured. In addition, the storage stability of polyphenols in both powder and reconstituted juice were measured. The WP-based samples had the highest powder yield and smallest particles using scanning electron microscopy. MD led to higher solubility, hygroscopicity, color stability, and anti-α-glucosidase activity. The combined wall-materials, especially, WP with GA or MD, increased the polyphenols stability and their antioxidant capacity during storage better than their individual counterparts. It was determined that the binary mixtures of proteins and carbohydrates gave better properties for manufacturing mulberry juice microparticles.

1. Introduction Mulberry (Morus australis Poir), which belong to the genus Morus and the family Moraceae, are broadly cultivated in different climatic zones (Butkhup, Samappito, & Samappito, 2013). Along with their good flavor, mulberry have significant beneficial health effects, such as antioxidant, anticancer, hypolipidemic, and hypoglycemic effects, mainly due to their high polyphenols (Khalifa, Zhu, Li, & Li, 2018a). Mulberry are juicy fruits and mostly consumed freshly. Due to their short harvesting season and storage sensitivity; processing of mulberry fruits to shelf-stable products, such as juice, jam and powder is generally needed. However, the polyphenols are generally unstable during food processing with heat (Yu et al., 2014). Thus, enhancing the retention of mulberry polyphenols after processing may be beneficial. Spray drying microencapsulation is a technique used for converting the fresh fruits to powders because of the short time of contact with drying medium and the high rate of evaporation. This method gives high quality particles with relatively low costs compared to other conventional drying methods (Moreno et al., 2016). It is also an efficient method for polyphenols stabilization which extend their industrial applications. Due to the

high content of low molecular weight substances, such as monosaccharides and organic acids, that cause sticky problems during spray drying (Jafari, Assadpoor, He, & Bhandari, 2008), encapsulation of pure juice directly to microparticles is not easily achieved. To overcome these challenges, certain wall-materials are simultaneously incorporated with sugar-abundant juice before atomisation to avoid the stickiness to the wall chamber with the particles. Typical wall-materials for encapsulation include maltodextrins (MD), gum Arabic (GA), and whey proteins (WP) (Santiago et al., 2016). MD is a low-cost material with a mild flavor; however, it has low emulsifying capacity and protective effects (Jafari et al., 2008). GA has reasonable emulsifying properties with acceptable protecting effects, but its viscosity at high concentrations limits its industrial applications (Risch & Reineccius, 1988). WP is a functional protein with excellent drying aid properties including high yield, smooth flow, and shielding effects (Bazaria & Kumar, 2016), whereas high proportions lead to color fading. Because a single wall-material does not meet the requirement of high powder recovery with satisfactory quality characteristics (Moser et al., 2017), combining wall-materials may be beneficial. Mulberry juice powders dried with only MD or GA carriers showed

⁎ Corresponding author. Lab 315 Food Chemistry, College of Food Science and Technology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, Wuhan, Hubei, 430070, China. E-mail address: [email protected] (C. Li).

https://doi.org/10.1016/j.fbio.2019.100445 Received 5 August 2018; Received in revised form 2 August 2019; Accepted 10 August 2019 Available online 11 August 2019 2212-4292/ © 2019 Elsevier Ltd. All rights reserved.

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Abbreviations AOA AUC BI C CV CGE C3R DSC ΔE* FD

FTIR GAE GA MD ORAC PCA PC1 PC2 TE TMA TPC WP

Antioxidant activity Area under the curve Browning index Chroma Coefficient of variation Cyanidin 3-glucoside equivalents Cyanidin 3-rutinoside Differential scanning calorimetry Color changes Freeze drying

acceptable physical properties (Fazaeli, Emam-Djomeh, Kalbasi Ashtari, & Omid, 2012), but the author used a high ratio (16%) of wall-materials and the polyphenols retention of the powders was not considered. Because a low wall-material ratio is nutritionally preferred (Ferrari, Germer, & de Aguirre, 2012), it was hypothesized that blending with different types of wall-materials might not only increase their efficiency but also reduce their feed ratio as well as protect the polyphenols of the particles. Therefore, the purpose of this study was to measure the applicability of low concentrations of MD, GA, and WP mixtures with the mulberry juice polyphenols. The physical, functional, and morphological indices of 10 experimental runs using the simplex-lattice procedure were determined. The storage stability of mulberry microparticle polyphenols and reconstituted juice-like models was also determined.

Fourier-transform infrared spectroscopy Gallic acid equivalents Gum Arabic Maltodextrin Oxygen radical absorbance capacity assay Principal component analysis First principal component Second principal component Trolox equivalents Total monomeric anthocyanins Total phenolic content Whey proteins

(Zhengzhou, Henan, China). The HPLC grades of Folin-Ciocalteu reagent, gallic acid, 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), methanol, formic acid, and sodium fluorescein were bought from the Aladdin Co. (Shanghai, China). The HPLC grades of 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH●), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) salts (ABTS+●), trolox, α-glucosidase (50 U mg−1), and p-nitrophenyl-α-Dglucopyranoside were purchased from the Shyuanye Co., Ltd. (Shanghai, China). The analytical grades of citric acid, isopropanol, methanol, sodium carbonate, potassium bromide, and potassium chloride, sodium acetate, and sodium persulfate were bought from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Deionized water (Direvt-u8, Millipore Corp., Bedford, Massachusetts, USA) was used. 2.2. Preparation of mulberry juice microparticles

2. Materials and methods

The standard methods of the Association of Official Analytical Chemists were used to measure the total sugar (Official Method 923.09), reducing sugar (Official Method 925.35), and titratable acidity (Official Method 942.15) of raw mulberry juice (AOAC, 2000). The total phenolic content (TPC), total monomeric anthocyanins (TMA), antioxidant activity (AOA), and anti-α-glucosidase of the raw juice and particles were also determined as described later (section 2.4). Mulberry juice microparticles were prepared using spray drying with different wall-materials. A simplex-lattice design using the statistical software (section 2.6) with 3 compounds (MD, GA, and WP), 3 interior points, and 1 central point, was studied using 10 experimental runs (Table 1). These wall-materials were selected because of their higher recovery and solubility than 8 other wall-materials (soybean proteins, β-cyclodextrin, starch, octenyl succinic anhydride starch,

2.1. Raw materials and reagents Raw Chinese mulberry juice (Morus australis Poir.) was obtained in October 2016 from the Guangdong Baosangyuan Health Food Co., Ltd. (Guangzhou, Guangdong, China). The total soluble solids of the juice were 7.75 Brix. The liquid juice arrived at the laboratory of the College of Food Science and Technology, Huazhong Agricultural University (Wuhan, Hubei, China) in sealed packages. The juice was initially preserved at −18 ± 1 °C for 2 wk during the preliminarily experiments and then thawed by leaving the package at 25 °C for 2 h. Whey proteins, gum Arabic, maltodextrin, soybean proteins, β-cyclodextrin, corn starch, octenyl succinic anhydride starch, chitosan, xanthan gum, guar gum, and tara gum powders (≥95%) were purchased from the Henan Co., Ltd.

Table 1 Simplex-lattice design of mulberry juice microparticles spray dried with the aid of different encapsulating wall-materials and their physicochemical characteristics; (Mean ± SD), n = 3. Experimental design/technological and physical properties Particles size

Moisture (%)

Dispersion (%)b

Mean (μm)

1.4 5.3 3.3 4.9 1.7 2.8 0.5 4.1 1.2 1.3

49 34 31 34 27 13 11 19 29 11

± ± ± ± ± ± ± ± ± ±

3g 1f 1e 2f 1d 1b 0.1a 1c 1de 0.1a

2.2 4.0 5.4 3.7 2.9 3.7 4.7 3.5 4.0 5.0

± ± ± ± ± ± ± ± ± ±

0.5a 0.5bc 0.2d 0.4bc 0.3ab 0.4bc 0.1cd 0.2b 0.1bc 0.4d

Run Hygroscopicity (%)

20 25 13 15 19 24 16 21 23 14

± ± ± ± ± ± ± ± ± ±

Solubility (%)

2bc 1e 1a 1a 2b 1de 1a 2bcd 1cde 2a

97 80 79 69 93 72 62 95 84 60

± ± ± ± ± ± ± ± ± ±

2d 2c 2c 1b 2d 1b 1a 1d 1c 1a

A, b, c, …: Means with the same letter in the same column are not significantly different (p ≥ 0.05). a The yield was done one time without replicates. b Calculated as the coefficient of variation of the mean particles size. c Central points. 2

Yelida (%)

WP

GA

MD

51 41 72 47 47 50 65 45 64 62

0.0 0.0 1.0 0.33 0.17 0.17 0.66 0.0 0.5 0.5

0.0 1.0 0.0 0.33 0.17 0.66 0.17 0.5 0.0 0.5

1.0 0.0 0.0 0.33 0.66 0.17 0.17 0.5 0.5 0.0

1 2 3 4c 5 6 7 8 9 10

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chitosan, and xanthan, guar, and tara gums) which were tested in preliminarily experiments (data not shown). The integration rate was calculated based on the total soluble solids of raw mulberry juice to the total solids of the wall-materials (1:0.25). Thus, 1.95 g of each wallmaterial was blended with 100 mL of raw juice to achieve the lowest applicable blending rate (1:0.25) based on preliminarily experiments (data not shown). The final core and the wall-material mixtures were mixed using a magnetic stirrer (Sh-4c, Huanghua Faithful Instrument Co., Ltd., Huanghua, Hebei, China) at 800 rpm, 28 °C, and 5 min, and then homogenized using the Omni-mixer homogenizer (JHG, Jinshanzhangyan Homogenizer Co., Ltd., Shanghai, China) at 30000 rpm and 28 °C for 3 min. The feed mixture was microencapsulated using a laboratory-scale spray dryer (YC-1000, Shanghai Pilotech Instrument Co., Ltd., Yancheng, Jiangsu, China) coupled with a two-fluid nozzle atomizer (diameter 1.0 mm), inlet and an outlet temperature of 110 and 85 ± 1 °C, respectively. The feed solution was pumped into the drying chamber using a peristaltic pump (OLF-550-4, Zhejiang Shengyuan Co., Ltd., Taizhou, Zhejiang, China) using the fan frequency of 35 Hz, compressor air pressure of 0.2 MPa, and feed flow rate of 11.5 mL min−1. The particles were collected from the collection cyclone, manually sealed in polyethylene pouches (Qingdao Hiprove Medical Technologies Co., Ltd., Qingdao, Shandong, China), and stored in a desiccator containing silica gel at 25 °C for a maximum of 6 d. Mulberry juice was lyophilized (FD) in a laboratory-scale freeze dryer (LGJ-12, Songyuanhuaxing Technology Co., Ltd., Beijing, China) at −50 °C to a constant weight as a control sample.

The color changes (ΔE*) was calculated using the equation of Silva, Stringheta, Teófilo, and de Oliveira (2013):

2.4. Retention of polyphenols and their functional properties 2.4.1. Quantitative analysis of mulberry particle polyphenols TPC was determined using the method of Khalifa, Barakat, El‐Mansy, and Soliman (2016), based on the reduction of Folin-Ciocalteu reagent colored complexes at λ = 765 nm (Multiskan GO, Microplate reader, Thermo Fisher, Waltham, Massachusetts, USA). In brief, 200 μL of each sample was mixed with 1 mL of Folin-Ciocalteu reagent, and 1 mL of 7.5% sodium carbonate was added after 5 min. Then, 1.5 mL of distilled water was added and the mixture was incubated at 25 °C for 60 min. TPC was measured based on the standard curve of gallic acid, for Y = 0.013x + 0.103 (R2 = 0.99) and the results were expressed as mg of gallic acid equivalents (mg GAE g−1). The method of Lako et al. (2007) was used to determine TMA using a pH-differential method with potassium chloride (pH 1.0, 0.025 M) and sodium acetate (pH 4.5, 0.4 M) buffers. Briefly, 0.4 mL of each sample was mixed with 3.6 mL of corresponding buffers and read against a blank at λ = 510 and 700 nm. TMA were calculated as mg cyanidin 3-glucoside equivalents (mg CGE g−1) using the following equations:

2.3.1. Particles yield, moisture, solubility, and hygroscopicity The method of Jayasundera, Adhikari, Howes, and Aldred (2011) was used to measure the particle yield after spray drying as a percentage of total solids collected to the total solids in the feed suspension. The moisture was measured by drying the samples in an oven (DHG9140A, Shanghai Jinghong Co., Ltd., Shanghai, China) at 105 °C to reach a constant weight using the method of Caliskan and Dirim (2016). The method of Cortes-Rojas and Oliveira (2012) was used to determine the solubility index. A 0.5 g sample and 50 mL deionized water were magnetically stirred, kept at 37 °C for 30 min, and centrifuged at 9000×g (6300 rpm in a M2 rotor, Model D3024R centrifuge, Scilogex Co., Ltd., Hartford, Connecticut, USA) at 37 °C for 15 min using 50 mL centrifuge tubes (CLS430897, Millipore Sigma, Burlington, Massachusetts, USA). The supernatant was dried at 105 °C to a constant weight. The solubility index (%) was calculated as the ratio of the solids mass of supernatant and original powder. The method of Caparino et al. (2012) was used to determine the hygroscopicity by storing the samples (0.5 g) in a desiccator using a saturated NaCl solution (75% relative humidity) at 25 °C for 7 d. The samples were weighed and the hygroscopicity was expressed as a percentage.

A= (A510

TMA =

100 BI =

(

b a

(a + 1.75L ) (5.645L + a 3.012b )

0.17

(2)

)

(A510

(A× MW×DF)×100 /MA

A700)pH4.5

(5) (6)

2.4.2. Thermal stability of particles X-ray diffractograms were obtained using a diffractometer system (D-8, Bruker Corp., Karlsruhe, Baden-Württemberg, Germany) in which the X-ray source was Cu Kɑ (λ = 1.5418 Å) radiation with 45 kV and 40 mA, measured at angle 2ɵ. The relative intensity was plotted versus the angle 2ɵ. Differential scanning calorimetry (DSC) was also done using the method of Rutz et al. (2013) using a VP-Capillary-DSC (Q20, Thermal Analysis Corp., New Castle, Delaware, USA) at a rate of 10 °C min−1, between 45 and 280 °C, and a nitrogen flow of 40 mL min−1. The heat flow values (mW mg−1) versus temperature (°C) were plotted. The lyophilized particles and the wall-materials were used as a control. Fourier-transform infrared spectroscopy (FTIR) analysis was done using the method of Tao et al. (2017) using a Nicolet 470 (Thermo Fisher) using a sample:KBr disc at a ratio of 1:99. The spectra were obtained in the transmission mode from 4000 to 400 cm−1 with a resolution of ± 2 cm−1, 21 scans min−1, 64 times for each spectrum.

(1) 1

A700)pH1.0

Where A = absorbance, MW = molecular weight (449), DF = dilution factor, and MA = molar extinction coefficient of CG (26,900).

2.3.2. Instrumental color The particle color was measured using a Chromameter (CR-410, Konica Minolta, Inc, Tokyo, Japan) using the CIELAB scale (L*, a*, and b*) calibrated by a standard white tile. The chroma (C), hue angle (°), and browning index (BI) were calculated using the equations of Caliskan and Dirim (2016):

Hue angle (°) = tan

(4)

2.3.3. Particle size and morphology The particle size was measured using the method of Du et al. (2014), using a Malvern laser diffraction particle size analyzer (APA 2000, Malvern Instruments, Malvern, Worcestershire, West Midlands, UK), using isopropanol as a dispersing medium. Particle size dispersion was defined as the ratio of the standard deviation to the mean of each particle size distribution (Lacerda et al., 2016), which was statistically calculated along with estimating the coefficient of variation (CV) using Minitab, ver. 16 (Minitab, Inc, State College, Pennsylvania, USA). The morphology was done using the method of Fazaeli et al. (2012). The particles were attached to stubs using double-sided adhesive and coated by gold under vacuum using a sputter coater (MC1000, Hitachi Co., Ltd., Atlanta, Georgia, USA) at a coating rate of 0.51 A°/s, 3–5 mA, 1 V, and 0.08–0.09 mbar for 180 s. The stubs were observed in a scanning electron microscope (SU8010, Hitachi Co., Ltd.) operated at 10 kV and 50 pA with a magnification of 1000 × .

2.3. Characterization of particles

C = (a 2 + b 2)1/2

2 1/2

2

E* = [( L )2 + ( a ) + ( b ) ]

0.31 (3) 3

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2.4.3. Antioxidant potential analysis The antioxidant activity (AOA) was measured using DPPH●, ABTS+●, and oxygen radical absorbance capacity (ORACFL) assays. The DPPH● procedure described by Khalifa, Barakat, El‐Mansy, and Soliman (2017) was based on the DPPH● solution absorbance decreases at λ = 515 nm. The DPPH radical scavenging percentage was calculated from a trolox standard curve of Y = 0.001x + 0.657 (R2 = 0. 99) and expressed as trolox equivalents (mmoL TE g−1). The ABTS+● activity was measured and expressed as mmoL TE g−1 from a standard curve of Y = −0.008x + 1.574 (R2 = 0.99). This method is based on the spectrophotometric measurement (λ = 734 nm) of the scavenging ability of the blue-green colored ABTS radicals. The ABTS solution was prepared by mixing 7 mM ABTS radicals with 2.45 mM potassium persulfate at a ratio of 2:1. The solution was kept in the dark for 12–16 h and the absorbance measured at λ = 734. The ORACFL is based on the ability of the samples to scavenge peroxyl radicals in a solution, as described by Ou, Hampsch-Woodill, & Prior (2001). Fluorescence was measured every 5 min for 45 min at λex = 493 and λem = 515 nm. The ORACFL results were calculated using the area under the curve (AUC):

AUC = 5 +

f5 f f f f + 10 + 15 + 20 + … 45 f0 f0 f0 f0 f0

from the PubChem database (PubChem: 441674), while the α-glucosidase structure (3w38) was acquired from the protein data bank (Research Collaboratory for Structural Bioinformatics, RCSB) (https://www.rcsb. org). Briefly, the water molecules were removed virtually, and the hydrogen atoms were added to the α-glucosidase structure. The docked complex was optimized according to its fit within the receptor pocket, for discrete and a continuum of hydrophobicity, van der Waals interaction, Hbonding, electrostatics, and entropy. The libdock algorithm was used for the docking (Khalifa, Nie, Ge, Li, & Li, 2018b; Khalifa, Peng, Jia, Li, Zhu, Yu-juan, et al., 2019). From the docking results, the best docked model was chosen to represent its most favorable binding mode based on their binding score (the libdock score of the best model was 132, which indicates the relative binding affinity). A score above 100 is preferred. 2.5. Storage stability of mulberry polyphenols The fluctuations in TMA, TPC, and their AOA (DPPH● value) during storage was studied either in reconstituted juice or mulberry powder models compared to raw and lyophilized juice. After sealing the samples, they were kept at 4 and 25 ± 1 °C and measured every 3 and 10 d for a total of 18 and 60 d for liquid and particle samples, respectively. The juice was reconstituted with the same total soluble solids of raw mulberry juice, and residual insoluble particles were removed before testing as previously described (2.4.1 and 2.4.3).

×5

(7)

Where F0 = the initial fluorescence before AAPH addition at times of f0, f5, f10, .., f45 fluorescence at time 0, 5, 10, .., 45 min, respectivly. The relative capacity was calculated by deducting AUC of samples from AUC of deionized water as a blank, based on the TE standard curve of Y = 0.001x + 70.05 (R2 = 0. 99).

2.6. Statistical analysis Statistica software, ver. 10 (StatSoft Inc, Palo Alto, California, USA) was used to design and fit the experiment. The principal component analysis was done using Minitab. Quality indices and storage stability were compared using two-way ANOVA followed by Tukey multiple comparison post-test using the Statistical Package for the Social Sciences software, ver. 19 (International Business Machines Corp., New York, New York, USA). Results were reported as the mean ± standard deviation and p values ≤ 0.05 were regarded as significant.

2.4.4. Anti-α-glucosidase activity The inhibition of α-glucosidase activity of diluted reconstituted mulberry juice was measured using the method of Yu et al. (2014). Briefly, 50 μL reconstituted mulberry juice diluted 40 times by deionized water was added to the 96-well plates (16196-1SA, Qingdao Co., Ltd., Qingdao, Shandong, China) containing 100 μL of α-glucosidase solution (1 U mL−1, dissolved in 0.1 M sodium phosphate buffer, pH 6.9). After preincubation in the microplate reader (Thermo Fisher) at 25 °C for 10 min, 50 μL of 5 mM p-nitrophenyl-α-D-glucopyranoside solution in 0.1 M sodium phosphate buffer (pH 6.9) was added to each well. Then, the absorbance (λ = 405 nm) was measured after incubating at 25 °C for another 3 min. The anti-α-glucosidase activity was calculated as a percentage of inhibition by deionized water as a control. In addition, a docking study was used to explain the anti-α-glucosidase activity of the major anthocyanins in the mulberry microparticles using Discovery Studio software ver. 2.5 (Accelrys Software Inc, San Diego, California, USA). The cyanidin 3-rutinoside (C3R) structure was obtained

3. Results and discussion 3.1. The combined wall-materials increased the physiochemical and technological characteristics of mulberry microparticles more than the individual blends Mulberry juice had TPC and TMA with values of 1.84 ± 0.02 × 103 mg GAE L−1 and 3.7 ± 0.1 × 102 mg CGE L−1, respectively. The antioxidant and anti-α-glucosidase activities of mulberry juice were about 17 ± 1 mmoL TE L−1 and 52 ± 1%,

Table 2 Effect of different encapsulating wall-material blends on the color parameters of different mulberry microparticles; (Mean ± SD), n = 3. Instrumental color parametersb ΔE*

Run Hue angle (o)

BI c

8.7 ± 0.1 14 ± 0.1e 23 ± 0.1h 14 ± 0.2e 12 ± 0.1d 19 ± 0.1g 17 ± 0.2f 3.3 ± 0.3a 7.5 ± 0.2b 13 ± 0.1e

23 21 15 18 19 19 17 16 20 21

± ± ± ± ± ± ± ± ± ±

g

0.1 0.1f 0.1a 0.1d 0.1e 0.1e 0.1c 0.4b 0.1ef 0.2f

e

0.2 ± 0.1 0.4 ± 0.1f −6.7 ± 0.1a 1.1 ± 0.1g −0.2 ± 0.1de −3.1 ± 0.1c 0.7 ± 0.1g −5.1 ± 0.3b −0.4 ± 0.1d 2.6 ± 0.1h

C 17 18 17 15 15 20 15 13 15 16

b* ± ± ± ± ± ± ± ± ± ±

e

a* d

0.1 0.1e 0.1e 0.2c 0.1c 0.1f 0.1c 0.2a 0.1b 0.1d

0.1 ± 0.1 0.2 ± 0.1e −2.1 ± 0.1a 0.5 ± 0.1g −0.1 ± 0.1cd −1.1 ± 0.1b 0.2 ± 0.1f −1.1 ± 0.1b −0.1 ± 0.1c 0.8 ± 0.1h

17 18 17 15 15 20 15 13 15 16

L* ± ± ± ± ± ± ± ± ± ±

e

0.1 0.1e 0.1e 0.1c 0.1c 0.1f 0.1c 0.2a 0.1b 0.1d

51 57 64 59 56 67 62 48 52 57

± ± ± ± ± ± ± ± ± ±

0.1b 0.1d 0.1f 0.1e 0.1c 0.1g 0.2f 1a 0.2b 0.1d

1 2 3 4a 5 6 7 8 9 10

A, b, c, …: Means with the same letter in the same column are not significantly different (p ≥ 0.05). a Central points. b Some parameters were obtained directly from the colorimeter (L*, a*, and b*), and the others were calculated and compared to their values in lyophilized raw mulberry juice. 4

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Fig. 1. Digital snapshots of different mulberry microparticles and their reconstituted juice, encapsulated using MD, GA, WP, and their mixtures as encapsulating wallmaterials, n = 1.

Fig. 2. Scanning electron micrographs of some selected mulberry juice microparticles encapsulated with combined wall-materials compared with the freeze-dried particles with a magnification of 1000 × , n = 1.

5

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Fig. 3. TPC (A), TMA (B), X-ray diffractograms (C), DSC thermograms (D), and FTIR spectra (E and F) of mulberry particles encapsulated with different wallmaterials.

respectively. Mulberry juice also had total sugar, reducing sugar, and titratable acidity as citric acid equivalents with values of 4.5 ± 0.2, 3.0 ± 0.1, and 0.8 ± 0.1%, respectively. Mulberry particles cannot be recovered without incorporation of wall-materials, and large deposits were formed at the cyclone walls using the standard spray drying conditions. Thus, addition of suitable wall-materials with appropriate concentrations before feeding is necessary to increase the yield without negative effects on the particle characteristics. The recovery values were carrier-dependent (Table 1). Relative high yields (> 60%) were observed in the WP-based runs, such as R#3, 7, and 9. However,

particle recovery of all GA-based runs were below 50%, which is regarded as the minimum criterion for satisfactory drying in laboratory scale dryers (Bazaria & Kumar, 2016). Mulberry juice is a sugar-abundant juice with adhesive behavior during spray drying (Fang & Bhandari, 2012; Sánchez et al., 2014), explaining the low recovery of some runs. The higher dried recovery using proteins was probably attributed to the surface activity properties of WP (preferential migration to the air/water interface), which could form a glassy film around the mulberry droplets and mitigate their contact with the chamber wall. These recovery values were similar to that of jussara pulp (61%) 6

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(Lacerda et al., 2016), but higher than that of bayberry (55%) (Fang & Bhandari, 2011) and beetroot juice powders (55%) (Bazaria & Kumar, 2018). Mulberry powder using 6% MD and 2% GA at an inlet air temperature of 130 °C had a high yield (82%), while 8% MD at inlet air temperature of 110 °C had a low yield (45%) (Fazaeli et al., 2012). In addition, the other characteristics of the particles including moisture, solubility, and hygroscopicity, which are related to shelf life, wettability, and stickiness, are indicators to the drying efficiency (Bazaria & Kumar, 2016; Kuck & Noreña, 2016). As shown in Table 1, these properties were carrier-dependent. Particles had an acceptable solubility and hygroscopicity. Generally, MD and GA based runs showed the highest solubility and hygroscopicity, probably due to their abundant –OH–residues, which facilitated their moisture absorption from the surroundings (Tonon, Brabet, Pallet, Brat, & Hubinger, 2009). Conversely, both of these properties were bottomed with WP-runs, especially the solubility, probably due to the low solubility and high viscosity of proteins at high temperatures (Cano-Chauca, Stringheta, Ramos, & Cal-Vidal, 2005). The hygroscopicity was negatively correlated with the wall-material concentrations (Bazaria & Kumar, 2018), explaining the high hygroscopicity of some runs (Fazaeli et al., 2012). The particles had a suitable moisture, suggesting their storage ability although ˂5% is preferred (Can, Guzel, & Ak, 2016). The moisture of these particles is lower than that of blackberry particles dried with a low concentration of MD (Fang & Bhandari, 2012), suggesting the importance of the blending of wall-materials. Particles color is another parameter which influences the marketing values of the powder. The individual based-runs changed the particle redness more than their multiple blends (Table 2). MD led to a significant decline in L* and a* values (R#8) compared with GA and WP, agreeing with the blackberry powder values (Ferrari et al., 2012). Meanwhile, integration of WP with MD and GA reduced the ΔE* in R#3, 7, 9, and 10, respectively. Color of the reconstituted juice from WP-runs was darker than that of others (Fig. 1), probably due to the different mechanism of the particle development during spray drying (Fang & Bhandari, 2012). Hue angle (°) and the chroma (C) values indicate the color intensity and saturation. The C values were positively correlated with the a* values, signifying its relation with the anthocyanins of the particles. The multiple runs, especially R#8 and 9, had the closed C values with the lyophilized juice (C = 10.6), signifying the color intensity of the combined wall-materials. The mixed wall-materials, especially R#8, 9, and 10, had the closed color indices with the raw mulberry juice, mainly due to the hypochromic effects of the wallmaterials (Lacerda et al., 2016; Tonon et al., 2009). Particles size dispersion is an essential index due to its influence on the processing, handling, and shelf life of fruit powders (Tonon et al., 2009). The size of the particles was carrier-dependent. Its diameter varied significantly (Table 1). Particle diameter and size dispersion were weakly correlated (r = 0.6, p ≤ 0.05, n = 10), suggesting that the runs which provide the small particles were better than the others (Lacerda et al., 2016). Generally, the individual wall-material based runs, especially MD, led to higher particles diameter and size dispersion than the combined wall-materials (Fig. S1). On the other hand, the WPbased runs, especially R#9 and 10, showed the smallest size, which also had acceptable physicochemical characteristics as previously discussed. The combined wall-material based runs were compared with the lyophilized particle as a control to observe the morphological characterization of the particles. All particles had smooth surfaces without crevices and/or interruptions (Fig. 2). This might explain why these runs limited the polyphenol degradation. The lyophilized particles were irregular flake particles, while spray drying microparticles were spherically shaped. The microparticles of R#8 and 4 showed a rough surface with dents and slight invaginations. A typical microparticle with surface roughness was observed, probably due the low drying temperature used in the spray drying, which retained the moisture of the microparticles surface, and deflated and shriveled as it cool down (Lacerda et al., 2016). Bridges linking microparticles and condensed

Fig. 4. Effect of different encapsulation wall-materials on the AOA (DPPH●, ABTS●+, and ORACFL assays) of mulberry microparticles. Results were surface fitted based on the simplex-lattice experimental design.

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Fig. 5. Anti-α-glucosidase activity fitted surfaces of mulberry microparticles (mean ± SD), n = 3. The schematic diagram generated using 2D- and 3D-features of the interactions between C3R and α-glucosidase and its neighboring residues.

agglomerates were observed in the particles of MD-based runs, agreeing with the results of particles size and hygroscopicity. Microparticles of R#9 and 10 with high proportions of WP showed the smoothest surface, mainly due to the higher flexibility of this protein, again highlighting the features of these runs. Thus, based on the aforementioned characteristics, the WP-based combined runs, such as R#7, 9, and 10, might be the best to use for the production of mulberry microparticles production. Although R#8 also had acceptable physicochemical features, its hygroscopicity needs to be improved.

(Kuck & Noreña, 2016). The combined wall-materials significantly increased the polyphenols retention. Moreno et al. (2016) observed that a low ratio of WP gave a high TPC recovery from grape marc extracts. These results were lower than that of blueberry dried with the aid of an ultrasonic nozzle (81–98%) (Tatar, Cengiz, Sandıkçı, Dervisoglu, & Kahyaoglu, 2016), and higher than that of pomegranate (53–83%) powders with the same wall-materials (Santiago et al., 2016). As observed in Fig. 3B, the use of MD and GA (R#8) as wall-materials led to higher anthocyanins retention (91%) than the other formulas. Meanwhile, the individual wall-materials had higher TMA instability than the combined one. The presence of MD in R#1, 5, 8, and 9 increased the stability of TMA compared with the other wall-materials, mainly due to its high solubility (section 3.1). Moreover, MD helps to prevent anthocyanins transformation to other unstable forms by reducing reactant mobility and complexing the flavylium cation form (Estupiñan, Schwartz, & Garzón, 2011; Moser et al., 2017). These results were highly correlated with the results of ΔE* and visual color screening (Fig. 1). Similar results with acai, blackberry, and jussara powders were previously reported (Ferrari et al., 2012; Lacerda et al., 2016; Tonon et al., 2009). However, these values are slightly lower than that of bayberry powder (94%) (Fang & Bhandari, 2012), probably

3.2. Combining wall-materials increased the stability of mulberry polyphenols and their functional properties after spray drying Encapsulated juice-polyphenols had higher stability than the polyphenol-extracts due to their high stability (Moser et al., 2017; Santiago et al., 2016). Generally, TPC in the individual runs were lower than that of the combined ones, as shown in Fig. 3A. The lowest TPC was observed in R#3, whereas the highest TPC was observed in R#10 consisting of WP and GA simultaneously, agreeing with recent results (Can et al., 2016; Tonon et al., 2009). The protective effect of GA was increased in the presence of proteins, allowing an effective encapsulation 8

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Fig. 6. TMA, TPC, and their DPPH● values of mulberry microparticles (A) and reconstituted juice (B) during storage, influenced by wall-material combinations (mean ± SD), n = 3. The different letters indicate a significant difference at p ≤ 0.05.

due to the variation of anthocyanins concentration in the core materials, spray drying conditions, and wall-material concentrations. TMA in the mulberry microparticles was higher than that of TPC; however, TMA are a subset of TPC and these results therefore are of concern. Although similar results were previously reported (Li, Chen, & Fu, 2016; Yang, Liu, Zhang, Jin, & Li, 2016; Jiang & Nie, 2015), who did not discuss these conflicting results. These conflicting results may be due to the interaction ability between wall-materials and –OH groups of polyphenols which could reduce its reduction ability toward FolinCiocalteu reagents and led to a decrease in TPC. This might suggest that the methodology needs to be improved. As shown in Fig. 3C, the multiple wall-materials increased the thermal stability of mulberry microparticles more than their individual analogues. A halo was seen in all diffractograms, especially in the individual runs such as R#1 and 3, mainly due to their amorphous state which was decreased in the presence of the combined wall-materials. The absence of the endothermal peaks in each run showed that the mulberry polyphenols were encapsulated in the wall-materials (Fig. 3D). Meanwhile, the absence of new peaks in the combined carrier-based runs suggested that an interaction occurred between core and wall-materials, thus changing their thermal stability (Bazaria & Kumar, 2016). As shown in Fig. 3E and F, the FTIR analysis showed that the wall-materials and mulberry

polyphenols could bind together. For instance, mulberry polyphenols peaks (around 1500 cm−1, aromatic structures of polyphenols) became more intensive after encapsulation, suggesting the successful integration of mulberry polyphenols into the wall-materials (Tao et al., 2017). Moreover, the combined carriers-based runs had more intensive polyphenols-related peaks, explaining the shielding effect of the multiple wall-materials. As observed in Fig. 4, the AOA stability was carrier-dependent, fluctuating from 46 ± 2 to 95 ± 1% compared to the lyophilized particles, representing the protective effects of the wall-materials on the antioxidative polyphenols. R#1 had a slightly lower AOA than the other runs, representing the limited effect of MD on AOA deterioration. It was reported that the use of MD as a wall-material did not expand the cellular antioxidant of grape marc extract compared with WP (Moreno et al., 2016). However, integration of MD and the other wallmaterials increased its antioxidant activity. The highest AOA stability was observed in R#4. Furthermore, it was shown that runs with WP and/or GA alone or simultaneously as wall-materials (R#9 and 10) had the highest AOA (DPPH● and ABTS●+ values), because of their electron-donating ability (Gad et al., 2011). The different results of different methods could be explained by the different mechanisms reflected in the different assays (Kuck & Noreña, 2016). 9

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Fig. 7. PCA of different indices of mulberry microparticles (A), polyphenols stability during storage of the reconstituted juice (B), and mulberry microparticles (C).

The α-glucosidase is a main intestinal enzyme associated with the postprandial rise in blood glucose. Anthocyanins were shown to be the most effective polyphenols in mulberry fruits on α-glucosidase (Jin,

Yang, Ma, Cai, & Li, 2015; Yu et al., 2014). Generally, WP and GA combination-based runs resulted in the highest reduction of anti-αglucosidase activity. The highest retention was observed with MD-based 10

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runs R#1 and 8 (Fig. 5). The runs with the highest TMA showed the highest anti-α-glucosidase activity, confirming their positive correlation. The α-glucosidase inhibitory activity of polyphenols was affected by their chemical structure. The generation of polymeric phenols or small phenolics during thermal processing such as spray drying could decrease the α-glucosidase inhibitory activity. To better understand the underlying mechanism of the anti-α-glucosidase activity of mulberry anthocyanins, a docking study between cyanidin 3-rutinoside (C3R), which was the major anthocyanins of mulberries (Table S1 and Fig. S2) and α-glucosidase was done. As shown in Fig. 5, C3R could inhabit αglucosidase activity using 3 π-π interactions and 3 H-bonding forces in the side-chains of its amino acids. In agreements with the physicochemical and morphological results, the results from the polyphenols retention and their functionalities suggested that the combined WPbased runs such as R#9 and 10 are preferred. Meanwhile, R#8 and 5 could be used with further enhancements.

showing their protective effect. The location of MD-runs (R#1, 5, and 8) was in the lower right-hand quadrant, suggesting their similarity in moisture and anti-α-glucosidase activity. The R#4 and 9 were located in the upper right-hand quadrant, suggesting their similarity in yield, color indices, and polyphenols. TMA, TPC, and DPPH● of reconstituted mulberry juice were correlated with PC1 (92%) with a total variance of 99.5%. Moreover, these indices at 0 d were in the lower left-hand quadrant, except R#1, 2, and 3, mainly due to the protective effects of the multiple wall-materials. Additionally, the locations of the parameters were gradually moved with the storage (Fig. 7B), probably due to their fluctuations during storage. However, the parameters were strongly decreased from 12 d to the end of storage, explaining why they were in the lower right-hand quadrant. As for mulberry particles, with a cumulative variance contribution rate of 99.5%, no changes were observed among the runs after 10 and 20 d of storage (Fig. 7C), but a negligible difference was seen for the other periods (Moser et al., 2017).

3.3. WP-based wall-materials incorporation with carbohydrate carriers increased the storage stability of mulberry polyphenols in solid and liquid models

4. Conclusion The 10 experimental runs were measured using different parameters after spray drying and during storage to select the best consistency with acceptable physicochemical attributes. From these 10 runs, the binary and ternary mixture of encapsulation carriers generally had the best physical, morphological, and the other particle properties. Because some runs had yields ˂50, R#7, 9, and 10 were the most acceptable runs based on their non-sticky behavior, recovery, particle size, dispersion, particle morphology, solubility, and redness. Considering the bioactive stability, R#10 and 9 had the most TMA and TPC, confirming that the multiple WP-based runs are the most stable particles after spray drying microencapsulating using spray drying. The antioxidative analysis confirmed that runs R#10, 9, 8, and 4 showed high AOA. Similarly, these runs, especially R#9 and 10, had better stability of the antioxidative polyphenols than other runs during storage. Overall, mulberry juice microparticles encapsulated with wall-materials of 0.5 WP to 0.5 MD, 0.5 WP to 0.5 GA, and mixing of 0.17 MD, 0.17 GA, and 0.66 WP were finally preferred due to their desired properties. The microparticles encapsulated using this mixture might be a beneficial use of mulberry juice as a potential nutraceutical.

Anthocyanins and other polyphenols are more stable in solids than in liquid (Lacerda et al., 2016), depending on the processing and storage conditions. Anthocyanins of mulberry microparticles fluctuated non-significantly during 60 d of storage, especially in the combined wall-materials compared to the individual ones (Fig. 6A). The highest and the lowest decreasing rates were observed in R#2 and 8. Similarly, TPC steadily decreased with increased storage. However, the proteinbased runs led to higher stability than the other runs. On the other hand, the AOA, which was measured using the DPPH• assay, significantly increased initially, and it gradually descended during storage, agreeing with a recent study (Rutz et al., 2013). It was concluded that the WP-based runs gave the most stable particles during storage because of the protective layers formed around the core. The opposite results were observed for MD and GA as recently reported (Lacerda et al., 2016). The initial values of polyphenols and their AOA were carrier-dependent, with lower values than that of raw mulberry juice, mainly due to their relatively low solubility. The polyphenols and AOA (DPPH● values) strongly decreased during cold-storage (Fig. 6B). However, the polyphenols decreased faster in the raw juice than in the reconstituted once, especially from the WP-based runs. After 18 d of cold-storage, the highest TMA was observed in R#3, confirming the protective effect of WP as an encapsulating wall-material (Khalifa et al., 2018b). Taken together, the combined wall-materials based runs were high effective in protecting the polyphenols both in the mulberry juice and microparticles models.

Conflicts of interest The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript. Acknowledgment This work was supported by the National Natural Science Foundation of China with grant number 31571839 and the Chinese Scholarships Council Foundation with grant number 2016GXZ745.

3.4. Principal component analysis (PCA) PCA is used to find the relationship among variables. Thus, PCA was used to find the relationships among parameters in different runs. PCA analysis showed that the first principal component (PC1) and the second principal component (PC2) were 83 and 17%, respectively, with the cumulative variance contribution rate of 99% (> 75–85%). PC1 positively correlated with the variables: Solubility, hygroscopicity, TMA, and anti-α-glucosidase activity. The high correlation between TMA and anti-α-glucosidase activity confirmed that the anthocyanins were mainly responsible for that property, as mentioned before (section 3.2). Similarly, the soluble wall-materials such as MD gave hygroscopic particles more than the other wall-materials, explaining why hygroscopicity and anti-α-glucosidase activity were correlated with PC1. PC2 positively correlated with the variables of moisture, C, ABTS●+, a*, L*, and the yield. On the other hand, PCA showed the strong correlation between polyphenols and their AOA, agreeing with a recent study (Bao et al., 2016). Furthermore, locations of the combined wall-materials based runs and the functional properties were close (Fig. 7A), again

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