Influence of phenolic compounds on physicochemical and functional properties of protein isolate from Cinnamomum camphora seed kernel

Food Hydrocolloids 102 (2020) 105612

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Influence of phenolic compounds on physicochemical and functional properties of protein isolate from Cinnamomum camphora seed kernel Xianghui Yan a, b, c, Shibo Liang a, b, d, Ting Peng a, b, d, Guohua Zhang a, b, d, Zheling Zeng a, b, c, *, Ping Yu a, b, c, **, Deming Gong b, c, e, Shuguang Deng b, c, f a

State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, China Jiangxi Province Key Laboratory of Edible and Medicinal Resources Exploitation, Nanchang University, Nanchang, 330031, China School of Resource and Environmental and Chemical Engineering, Nanchang University, Nanchang, 330031, China d School of Food Science and Technology, Nanchang University, Nanchang, 330031, China e New Zealand Institute of Natural Medicine Research, 8 Ha Crescent, Auckland, 2104, New Zealand f School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, AZ, 85284, USA b c

A R T I C L E I N F O

A B S T R A C T

Keywords: Cinnamomum camphora seed kernel Protein isolate Protein-phenolic complex Structure change Functional property

Cinnamomum camphora seeds are abundant in China. For high value utilization of Cinnamomum camphora seed kernel (CCSK) protein, the effects of phenolic compounds on physicochemical and functional properties of protein isolate (PI) from CCSK were investigated in this study. A total of 12 phenolic compounds in CCSK have, for the first time, been tentatively identified by HPLC-ESI-QTOF-MS2. PI had a higher level of protein content compared to that of protein-phenolic complex (PPC). The formation of PPC resulted in a reduction of free sulfhydryl groups content and surface hydrophobicity as well as an increase of particle size and zeta potential. Fourier-transform infrared spectroscopy showed that the contents of secondary structure of PI and PPC were not obviously different. However, a remarkable increase in β-sheet and a decrease in random coil of PPC compared to PI were found by circular dichroism. Differential scanning calorimetry analysis revealed that PPC was thermally more stable than that of PI. PI and PPC exhibited superior essential amino acid composition and similar protein subunits. Furthermore, PPC exhibited higher solubility, better foaming and emulsifying properties than those of PI. These results suggested that the protein products from CCSK may be used as natural functional materials in the food industry.

1. Introduction Cinnamomum camphora seeds are abundant in China, with an annual yield of more than 1 million tons. People from south of the Yangtze river in China have been consuming Cinnamomum camphora seed kernel (CCSK) like soybean and peanut since 1960s. CCSK is rich in mediumchain oil and protein, with the contents of 48.24–62.60% and 17.15–26.33%, respectively. Since 2009, we have studied the extraction technology, structure and physiological effects of Cinnamomum cam­ phora seed kernel oil (CCSKO), and found that CCSKO, the only natural medium-chain oil discovered in the world, improved the lipid and glucose metabolism disorder in SD rats (Fu et al., 2015, 2016b; Fu, Zeng, Zeng, Wang, & Gong, 2016a). However, to our knowledge, there are no studies on the isolation, structure and biological activity of CCSK

protein. With the increasing interest on renewable and sustainable protein resource of plant origin (Rodrigues, Coelho, & Carvalho, 2012), the protein products isolated from CCSK are expected to be applied to food industry by studying their physicochemical and functional properties. The traditional alkaline-solution and acid-precipitation technique is typically used to isolate proteins from plants, but the technique also facilitates the extraction of phenolic compounds (Hernandez-Jabalera et al., 2015). In this procedure, a portion of phenolic compounds may bind with proteins through covalent bond in the presence of oxygen (Kroll, Rawel, & Rohn, 2003). Specifically, these highly reactive phenolic compounds could covalently combine with sulfhydryl (-SH) and amino (-NH2) groups of proteins at high pH values (Pierpoint, 1969). Studies have shown that individual phenolic compounds, such as

* Corresponding author. State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang, 330047, China. ** Corresponding author. State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang, 330047, China. E-mail addresses: [email protected], [email protected] (Z. Zeng), [email protected] (P. Yu). https://doi.org/10.1016/j.foodhyd.2019.105612 Received 25 August 2019; Received in revised form 19 November 2019; Accepted 18 December 2019 Available online 19 December 2019 0268-005X/© 2019 Elsevier Ltd. All rights reserved.

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anthocyanin, chlorogenic acid and ( )-epigallocatechin-3-gallate (EGCG) affected the physicochemical and functional properties of pro­ teins by covalent modification (Lu et al., 2018; Sui et al., 2018; Wei, Yang, Fan, Yuan, & Gao, 2015). However, considering the structural diversity of phenolic compounds among plants, the interaction behavior of protein and phenolic compounds could be more complex than origi­ nally expected, especially in food systems. Proteins as valuable food ingredients mainly provide structure to foods and many other functional properties (Ali, Homann, Khalil, Kruse, & Rawel, 2013). It has been proposed that elucidation of structure-function relationship of protein is a prerequisite for devel­ oping new food materials (Fukuda, Maruyama, Salleh, Mikami, & Utsumi, 2008). Similarly, the formation of protein-phenolic complex could directly change the structure of protein, and thus affect their functions, including solubility, foaming and emulsifying properties (Foegeding, Plundrich, Schneider, Campbell, & Lila, 2017). Neverthe­ less, there is very limited information available on the structure and functions of natural protein-phenolic complex isolated from plant-based foods. Therefore, this study was aimed to develop CCSK protein for high value utilization. For that, protein-phenolic complex (PPC) and protein isolate (PI) were prepared from CCSK, and the effect of phenolic com­ pounds on physicochemical and functional properties of protein were investigated. The amino acid composition of PI and PPC were also determined. These results would provide us with more information to evaluate the protein products prepared from CCSK as potential food materials.

and concentrated in a vacuum rotary evaporator at 45 � C. After dispersed in distilled water and filtered through the filter paper, the CCSK 80% (v/v) ethanol extract was freeze dried and stored at 20 � C for further analysis. In this way, defatted CCSK flour with phenolic compounds and defatted CCSK flour free of phenolic compounds were obtained as PPC and PI preparation, respectively. 2.4. HPLC-ESI-QTOF-MS2 analysis of phenolic compounds Individual phenolic compounds were analyzed using an Agilent HPLC 1260 (Agilent, Waldbronn, Germany) equipped with an auto­ sampler injector, a binary solvent delivery system, a column compart­ ment and a UV detector. Prior to analysis, the sample was filtered through a 0.22 μm Whatman Anotop filter (GE Healthcare, Germany). The chromatographic separation was carried out on an amethyst (Sepax Technology Co., Ltd., USA) C18–H reverse-phase column (250 mm � 4.6 mm � 5 μm) at 30 � C with an injection volume of 10 μL. The solvent system consisted of 0.1% (v/v) formic acid in water (A) and methanol (B). The gradient conditions were as follows: 5–10% B in 0–8 min; 10–20% B in 8–11 min; 20–35% B in 11–28 min; 35–50% B in 28–38 min; 50–70% B in 38–58 min; 70-5% B in 58–60 min. The flow rate was 1.0 mL/min and wavelength of UV detector was set at 280 nm. For identification, an AB SCIEX TripleTOF 5600 system (SCIEX, Foster City, CA, USA) was used. The MS instrument was operated with an electro­ spray source ionization (ESI) under negative mode. The major param­ eters were set as follows: ion spray voltage, 4500 V; source temperature, 550 � C; curtain gas, 35 psi; ion source gas 1, 60 psi; ion source gas 2, 60 psi; collision energy, 30 eV; scan range, 100–1500 Da. Data acquisition and processing were performed with SCIEX OS software (SCIEX). Online databases including ChemSpider (http://www.chems pider.com), MassBank (http://www.massbank.jp), HMDB (http://www.hmdb.ca/) and Phenol-Explorer (http://www.phenol-ex plorer.eu) were used to analyze the MSn data.

2. Materials and methods 2.1. Materials The Cinnamomum camphora seeds used in this study were collected from the campus of Nanchang University (Nanchang, China) in November 2017. Professor Zheling Zeng authenticated the plant and a voucher specimen (SN004618) was deposited at Chinese Virtual Her­ barium. Soybean oil was purchased from a local supermarket (Nan­ chang, China). 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB) was purchased from Shanghai Aladdin Reagent Co., Ltd. (Shanghai, China), 8-Anilino-1-naphthalenesulfonic acid (ANS) was purchased from SigmaAldrich Trading Co., Ltd. (Shanghai, China). HPLC-grade formic acid and methanol were purchased from Fisher Scientific (Pittsburgh, PA, USA). Deionized water was prepared using a Milli-Q system (Merck Millipore, USA). All other chemicals used were of analytical grade.

2.5. Preparation of PPC and PI PPC and PI were prepared from two portions as described above according to the method of Guimaraes Drummond et al. (2017) with some modifications. To obtain PPC, the defatted CCSK flour with phenolic compounds was dispersed in distilled water at a ratio of 1:15 (w/v), the pH was adjusted to 9.0 with 4.0 M NaOH. The mixture was stirred at room temperature for 30 min. After centrifuged at 3,500g for 15 min, the pH of the supernatant obtained was adjusted to 5.0 with 1.0 M HCl to precipitate protein, followed by centrifugation at 3,500g for 15 min. The pellet was recovered, resuspended in distilled water and its pH adjusted to 7.0 with 1.0 M NaOH. After centrifuged at 3,500g for 15 min to remove insoluble aggregate, the supernatant containing protein was collected. Finally, the protein sample was freeze dried and stored at 20 � C until further analysis. The defatted CCSK flour free of phenolic compounds was used to prepare PI at pH 9.0, the preparation process was the same as above.

2.2. Sample pretreatment The Cinnamomum camphora seeds were manually cleaned to remove all flesh and dried in the sun, dehulling of seeds were carried out at room temperature using a dehuller. To obtain the defatted CCSK, CCSK was crushed using a high speed pulverizer and defatted with n-hexane in a ratio of 1:3 (w/v) at room temperature. The mixture was continuously stirred for 12 h and repeated twice, then separated by an aspirator filter pump. The filtrate was discarded, the residual pellet was air-dried at room temperature in a fume hood. The dried flour was then passed through a 40 mesh screen and stored at 4 � C until further use.

2.6. Determination of chemical composition of CCSK and its products The proximate contents of moisture, lipid, ash, protein and dietary fiber of CCSK, defatted CCSK, PI and PPC were determined according to standard methods of the Association of Official Analytical Chemists (AOAC, 2006). The total phenolic contents of CCSK, defatted CCSK, PI and PPC were determined using the Folin-Ciocalteu method, and expressed as gallic acid equivalent (GAE) per g of 100 g (Degirmencio­ glu, Gurbuz, Herken, & Yildiz, 2016).

2.3. Extraction of phenolic compounds Phenolic compounds were extracted from defatted CCSK using the method of Tang, Wang, Liu, and Wang (2009) with some modifications. The defatted CCSK was dispersed in 80% (v/v) ethanol at a flour to solvent ratio of 1:20 (w/v) and stirred for 2 h at room temperature, then the mixture was centrifuged at 3,500g for 15 min. The precipitate was resuspended in the solvent and the extraction process was repeated three times, the supernatants containing phenolic compounds were combined

2.7. Determination of physicochemical properties of PI and PPC 2.7.1. Measurement of free sulfhydryl (-SH) groups content The contents of free –SH groups of PI and PPC were measured 2

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according to Beveridge, Toma, and Nakai (1974). 15 mg of each protein sample was fully dissolved in 5.0 mL Tris-Gly buffer (0.09 M glycine, 0.086 M Tris and 0.004 M EDTA, pH 8.0) containing 8 M urea, followed by adding 50 μL of Ellman’s reagent (DTNB in Tris-Gly buffer, 4 mg/mL). The mixture was incubated at room temperature for 1 h and then centrifuged at 8,000g for 15 min. The absorbance of the superna­ tant was measured at 412 nm using an UV–vis spectrophotometer (UV-1950, Purkinje General Instrument Co., Ltd., Beijing, China). The total free –SH content was expressed as μmol per gram of protein. The calculation was as the following Eq.

8000 (PerkinElmer Co., Ltd. Massachusetts, USA) using a previous method (Cao & Xiong, 2017) with some modifications. Briefly, 5–10 mg of each protein sample was accurately weighed and hermetically sealed in an aluminum sample pans, followed by heating from 10 to 110 � C in a rate of 10 � C/min. The thermal parameters Tonset (onset temperature), Td (denaturation temperature), Tendset (end-temperature) and ΔH (enthalpy change) were determined. 2.7.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) SDS-PAGE was performed using a Mini-PROTEAN Tetra Electro­ phoresis System equipped with TGX Stain-Free™ FastCast™ Acrylamide Kit (Bio-Rad Laboratories, California, USA). According to the manufac­ turer’s instruction, a SDS-Tris-glycine discontinuous buffer for SDSPAGE was prepared. 1 mg/mL of each protein solution was mixed with 5 � sample loading buffer in a ratio of 4:1 (v/v), followed by heating at 95 � C for 5 min. After the sample solution was cooled to room temperature, each lane of the gel was loaded with 20 μL of the protein solution, electrophoresis was conducted at 200 V for 40 min. After electrophoresis, the gel was stained with 0.1% (w/v) Coomassie Brilliant Blue (R-250) for 0.5 h and destained with 10% (v/v) acetic acid for 12 h. A ChemiDoc Touch Imaging System (Bio-Rad Laboratories, California, USA) was used to photograph the gel image.

-SH (μmol/g) ¼ (73.53 � A412 � D)/C Where A412 is the absorbance at 412 nm; C is the sample concentration in mg/mL; D is the dilution factor; 73.53 is derived from 106/(1.36 � 104); 1.36 � l04 is the molar absorptivity and l06 is for the conversion factor from the molar basis to the μmol/ml basis and from mg solids to g solids. 2.7.2. Determination of surface hydrophobicity (H0) The H0 of PI and PPC were measured using ANS as a fluorescence probe based on the method of Haskard and Li-Chan (1998). Briefly, 0.05–0.25 mg/mL of sample solutions and 8 mM ANS solution with 10 mM PBS buffer (pH 7.2–7.4) were prepared, respectively. After that, 4 mL of sample solution was mixed with 20 μL of ANS solution. The fluorescence intensity (FI) was recorded with an excitation wavelength of 390 nm and the emission wavelength of 350–550 nm, the excitation and emission slit widths were both 2.5 nm, the FI at 470 nm was used. The mixture without protein sample was used as a blank. H0 was expressed as the slope of the linear regression of the FI against the protein concentration.

2.8. Analysis of amino acid composition of PI and PPC The amino acid compositions of PI and PPC were analyzed according to Malomo and Aluko (2015). Approximately 20 mg of each protein sample was fully hydrolyzed with 10 mL of 6.0 M HCl at 110 � C for 24 h under a nitrogen atmosphere. After that, an amino acid auto-analyzer S-433D (Sykam Scientific Instrument Co., Ltd., Beijing, China) was used, results were expressed as g of amino acid/100 g of protein.

2.7.3. Particle size and zeta potential measurements The particle size and zeta potential of PI and PPC were measured using a Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire, UK), using the method by Sui et al. (2018). For particle size determi­ nation, the refractive indexes for the protein particles and the dispersion medium were 1.46 and 1.33, respectively. Prior to analysis, the samples were diluted to 0.2 mg/mL with 10 mM PBS buffer (pH 7.2–7.4).

2.9. Determination of functional properties of PI and PPC 2.9.1. Protein solubility The solubility of PI and PPC were determined using the method of H M. Rawel, D. Czajka, S. Rohn, & J. Kroll (2002) with some modifications. Briefly, 50 mg of each protein sample was dissolved completely in distilled water, the pH values of the solutions were adjusted to 3–11 with 1.0 M HCl or 1.0 M NaOH. After centrifuged at 10,000g for 10 min at 4 � C, the protein content of the supernatant was determined by Bradford protein assay method (Bradford, 1976), and bovine serum albumin was used as a standard.

2.7.4. Determination of secondary structure by Fourier-transform infrared spectroscopy (FTIR) The freeze-dried sample flour (2–3 mg) of PI and PPC were mixed with KBr powder in a ratio of 1:100 (w/w) and recorded at a range of 4000 to 400 cm 1 at room temperature by an Nicolet 5700 FTIR spec­ trometer (Thermo Nicolet Co.,USA). The spectral region between 1600 and 1700 cm 1 was selected and processed according to the modified method of Li, Du, Jin, and Du (2012). The data were analyzed using the OMNIC 8.0 Software (Thermo Fisher Scientific Inc., Waltham, MA, USA) and PeakFit 4.12 Software (SPSS Inc., Chicago, IL, USA).

2.9.2. Foaming properties The foaming properties of PI and PPC were determined according to the method of Bandyopadhyay and Ghosh (2002) with some modifica­ tions. Briefly, 100 mg of each protein sample was dissolved completely in 10 mL (V) distilled water. The pH values of solution were adjusted to 3–11 with 1.0 M HCl or 1.0 M NaOH, then homogenized using a T18 digital ULTRA-TURRAX (IKA, Germary) at the speed of 13,600 rpm for 2 min at room temperature. The volumes of the foams after homogeni­ zation were recorded at 0 min (V0) and 10 min (V10), respectively. The foaming capacity (FC) and foaming stability (FS) were calculated with the following Eq.

2.7.5. Determination of secondary structure by far-UV circular dichroism (CD) The far-UV CD spectra of PI and PPC were measured using a JASCO J815 spectropolarimeter (JASCO Corporation, Tokyo, Japan) according to the method by Yang et al. (2017) with some modifications. Briefly, the protein samples were diluted with 10 mM PBS buffer (pH 7.2–7.4) to adjust the protein concentration to 0.1 mg/mL. Far-UV CD spectra were recorded at the range of 190–250 nm at room temperature in a quartz cuvette of 10 mm optical length. The values of scanning speed, spectral resolution, and bandwidth were 200 nm/min, 0.5 nm and 2.0 nm, respectively. The data were analyzed using the SELLON3 program in CDPro Software.

FC (%) ¼ (V0–V)/V � 100; FS (%) ¼ (V10/V0) � 100.

2.9.3. Emulsifying properties The emulsifying properties of PI and PPC were determined by the method of Zhong et al. (2012) with some modifications. 15 mL of each protein solution (2 mg/mL) was prepared. The pH values of the solutions

2.7.6. Determination of thermal properties The thermal properties of PI and PPC were determined using a DSC 3

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were adjusted to 3–11 with 1.0 M HCl or 1.0 M NaOH, followed by adding 5 mL of soybean oil. The mixture was homogenized using a T18 digital ULTRA-TURRAX (IKA, Germany) at a speed of 13,600 rpm for 2 min at room temperature. Then 50 μL of the emulsion was pipetted from the bottom of each container and dispersed in 5 mL of 0.1% (w/v) SDS solution at 0 and 10 min, respectively. The absorbance of each sample was obtained at 500 nm using an UV–vis spectrophotometer (UV-1950, Purkinje General Instrument Co., Ltd., Beijing, China), with 0.1% (w/v) SDS as a blank control. The absorbance measured immediately (A0) and after 10 min (A10) were recorded to calculate emulsifying activity index (EAI) and emulsion stability index (ESI) with the following Eq.

significant (p < 0.05) effect on protein purity. The notable change in protein content of PI compared to PPC was similar to the observation of flaxseed protein products reported by Guimaraes Drummond et al. (2017). As the study of Malik et al. (2017), the reduction of protein content may be caused by the interaction of phenolic compounds with protein at alkaline pH during extraction process. The total phenolic contents in CCSK and defatted CCSK were 0.97 � 0.03% and 2.12 � 0.07%, respectively. The CCSK with a high content of phenolic com­ pounds could be developed as potential new natural ingredients. Extraction of defatted CCSK with 80% (v/v) ethanol reduced the phenolic content in PI by 92.23% as compared to PPC. Phenolic com­ pounds were reported to be readily oxidized to quinones under alkaline conditions to further covalently bind to protein (Kroll et al., 2003). Therefore, the preparation of PPC in the present study was considered as covalent complex of protein and phenolic compounds.

EAI (m2/g) ¼ (2 � 2.303 � A0 � DF)/(c � φ � 10000); ESI (%) ¼ (A10/A0) � 100 Where A0 and A10 represent the absorbance at 500 nm measured immediately and 10 min after emulsion formation, respectively. DF is the dilution factor (101), C refers to the protein concentration (g mL 1) before emulsification, and φ is the oil volume fraction (v/v) of the emulsion (φ ¼ 0.25).

3.2. Identification of phenolic compounds in CCSK For investigating the chemical structure of phenolic compounds extracted from CCSK, HPLC-ESI-QTOF-MS2 was used to study the mass spectral fragmentation pattern. According to their retention times (tR), exact mass, MS spectrum, MS/MS spectrum and by comparing with online mass databases, a total of 12 phenolic compounds were tenta­ tively identified for the first time (Table 2, Fig. 1). Considering the fragmentation pattern of Peaks 10 and 11 did not match any references, they were temporarily named as unknown compounds. It was b ovious that the labeled peaks tentati vely identified corresponded to glycosylatedformsofphenoliccompounds.Forexample,Peaks1,2,3,4,5,7and9 withtherespectivemolecularionsatm/z432,477,315,343,461,355and385 [M-H] producedMS2fragmentionsatm/z270,315,153,181,299,193and223 [M-H-C6H10O5] ,respectively,indicatingthatthelossof162Da(hexosyl)from their molecular ions. These compounds (1, 2, 3, 4, 5, 7, 9) were tentatively identified as pelargonidin 3-O-glucoside, isorhamnetin 3-O-glucoside, hydroxytyrosol 1-O-glucoside, domesticos ide, verbasoside, 1-O-feruloylglu­ coseisomer,1-O-sinapoylglucose,respectively.Moreover,Peak12displayed the respective molecular ion at m/z 441 [M-H] and MS2 fragment ions at m/z 330, 205, and 139, which corresponded to lusitanicoside by comparison with online mass databases. Peaks 6 and 8 with the respective molecular ions at m/z 371 and 401 [M-H] , were tentatively identified as dihydroferulic acid 4-Oglucuronide and 4-hydroxy-5-(30 ,50 -dihydroxyphenyl)-valeric acid-Oglucuronide,respectively.

2.10. Statistical analysis All the experiments were replicated in triplicate and the results are presented as means � standard deviation (SD). Figures were made by Origin 9.0 Software. One-way analysis of variance (ANOVA), followed by Tukey’s test for pairwise multiple comparisons was used to assess statistically significant differences between the results at a significance level of p < 0.05 or p < 0.01 using SPSS Statistics 22 Software. 3. Results and discussion 3.1. Chemical composition of CCSK and its products The contents of protein, moisture, ash, lipid and dietary fiber of CCSK, defatted CCSK, PI and PPC were measured in this study. As shown in Table 1, CCSK contained 19.34 � 0.15% protein, 4.81 � 0.02% moisture, 1.95 � 0.01% ash, 59.34 � 0.10% lipid and 10.91 � 0.09% dietary fiber. The results showed that CCSK was rich in protein and lipid, and could be developed for high value utilization. In defatted CCSK, high contents of protein (49.61 � 0.36%) and dietary fiber (29.78 � 0.14%) and a low level of lipid (5.66 � 0.23%) were observed. For PPC and PI, the protein contents were 77.93 � 0.42% and 95.15 � 0.43%, respec­ tively, indicating that the presence of phenolic compounds had a

3.3. Effect of phenolic compounds on physicochemical properties 3.3.1. Free sulfhydryl (-SH) contents of PI and PPC The content of free sulfhydryl (-SH) groups was measured to evaluate the tertiary structure and the effect on functional properties of PI and PPC. As shown in Table 3, the free –SH groups content of PPC was significantly (p < 0.05) lower than that of PI. Similarly, the reduction of free –SH groups content in bovine milk proteins and ( )-epi­ gallocatechin-3-gallate (EGCG) covalent complex was observed as compared to the control protein (Wei et al., 2015). The same result was also found in the interaction of soy protein with several phenolic com­ pounds such as chlorogenic acid, gallic acid, caffeic acid and quercetin (H. M. Rawel, D. Czajka, S. Rohn, & J. Kroll, 2002). These results indi­ cated that the presence of phenolic compounds in protein solution showed strong reactivity with cystein residue under oxygen and alkaline conditions. Moreover, it could more directly provide the reason for the reduction of free –SH groups content by covalent C–S bonds formed between protein and phenolic compounds.

Table 1 Chemical composition (%) of CCSK, defatted CCSK, PI and PPCa. Samples

Proteinb

Moisture

Ash

Lipid

Dietary fiber

Phenolic content

CCSK

19.34 � 0.15d

4.81 � 0.02a

0.97 � 0.03c

49.61 � 0.36c

3.74 � 0.03b

59.34 � 0.10a 5.66 � 0.23b

10.91 � 0.09b

DefattedCCSK

29.78 � 0.14a

2.12 � 0.07b

PPC

77.93 � 0.42b

1.08 � 0.01c

NDc

2.23 � 0.06c

2.19 � 0.04a

PI

95.15 � 0.43a

0.81 � 0.03d

1.95 � 0.01b 2.14 � 0.01a 1.02 � 0.02c 0.87 � 0.04d

ND

0.32 � 0.02d

0.17 � 0.02c

c

ND, not determined. Data are expressed as means � SD (n ¼ 3). Values with different letters (a, b, c, d) in the same column in each sample were significantly different (p < 0.05). a CCSK, Cinnamomum camphora seed kernel; PI, protein isolate; PPC, proteinphenolic complex. b N (%) � 6.25.

3.3.2. Surface hydrophobicity (H0) of PI and PPC The H0 value showed the number of hydrophobic groups exposed on the surface of protein, and provided information of the change in conformation and functionality of protein (Li et al., 2019). As shown in Table 3, the H0 values of protein were significantly (p < 0.05) decreased 4

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Table 2 Tentative identification of phenolic compounds in CCSKa by HPLC-ESI-QTOFMS2. Peak

tR (min)

Molecular Formula

[MH] (m/ z)

MS/ MS (m/z)

Error (ppm)

270, 162 315, 162 162, 153, 135, 123 181, 163, 135, 119 299, 221, 179, 161, 131 163, 119 193, 149 193, 149

0.1

223, 193, 179, 149, 121 549, 387, 372, 265, 162 212, 176, 162 330, 205, 139

0.4

Dihydroferulic acid 4-O-glucuronide 1-O-Feruloylglucose isomer 4-Hydroxy-5-(30 ,50 dihydroxyphenyl)valeric acid-Oglucuronide 1-O-Sinapoylglucose

1.0

Unknown 1

0.4

Unknown 2

0.4

Lusitanicoside

1

14.79

C21H21O10

432

2

14.98

C22H22O12

477

3

15.26

C14H20O8

315

4

17.00

C15H20O9

343

5

17.29

C20H30O12

461

6

17.96

C16H20O10

371

7

20.46

C16H20O9

355

8

20.46

C17H22O11

401

9

22.91

C17H22O10

385

10

27.64

C28H38O12

565

11

27.95

C28H40O12

567

12

35.74

C21H30O10

441

a

0.5 0.0

Tentative identification

Domesticoside

0.8

Verbasoside

0.6 0.3

Samples

-SH (μmol/ g)

H0

Particle size (nm)

PI

17.13 � 0.66a 12.54 � 0.39b

194.44 � 5.15a 133.81 � 8.98b

64.30 � 0.73b

28.10 � 1.85b

86.30 � 3.80a

35.00 � 0.69a

PPC

Pelargonidin 3-Oglucoside Isorhamnetin 3-Oglucoside Hydroxytyrosol 1-Oglucoside

0.2

0.5

Table 3 The free sulfhydryl (-SH) groups content, surface hydrophobicity (H0), particle size and zeta potential of PI and PPCa. Zeta potential (mV)

a

PI, protein isolate; PPC, protein-phenolic complex. Data are expressed as means � SD (n ¼ 3). Values with different letters (a, b) in the same column in each sample were significantly different (p < 0.05).

et al. (2018) reported that the formation of whey protein-caffeic acid complex showed significant lower H0 value than that of whey protein. In addition, the remarkable decrease in the H0 value from PI to PPC caused by phenolic compounds corresponded to the decrease in free –SH groups content. 3.3.3. Particle size and zeta potential of PI and PPC The information of particle size and size distribution may reflect the structure of protein and thus link to the functional properties of protein. As shown in Fig. 2, both PI and PPC mainly exhibited bimodal distri­ bution, one at around 15 nm and the other at around 100 nm. These results indicated that the protein has a non-uniform particle size dis­ tribution, which was consistent with the observation from soy protein isolate and its covalent complex with anthocyanins (Sui et al., 2018). As compared to PI, the two peaks of PPC shifted to larger sizes, which could be considered as an indication of the formation of a protein-phenolic complex. Specifically, the particle sizes of PI and PPC were 64.30 � 0.73 and 86.30 � 3.80 nm, respectively (Table 3). The fact that the protein-phenolic covalent complex increased the particle size of protein agreed with the observation by Pham, Wang, Zisu, and Adhikari (2019a), who found that the complex of flaxseed protein isolate and phenolic compounds had a larger particle size than that of native flax­ seed protein. The zeta potential of PI and PPC were 28.10 � 1.85 and 35.00 � 0.69 mV, respectively (Table 3), indicating that the absolute zeta po­ tential of PPC was significantly (p < 0.05) higher than that of PI due to the presence of phenolic compounds. The result was also in good agreement with the study by Pham et al. (2019a). It has previously been suggested that the positively charged groups of protein were neutralized

CCSK, Cinnamomum camphora seed kernel.

Fig. 1. HPLC chromatogram of phenolic compounds in CCSK at 280 nm.

(from 194.44 for PI to 133.81 for PPC). The result could be attributed to the reduction of exposed hydrophobic groups on the PI surface, as the phenolic compounds covalently combined with PI. It was found that the binding of 8-Anilino-1-naphthalenesulfonic acid (ANS) to the hydro­ phobic sites of protein surface were suppressed owing to the preferential binding of phenolic compounds (Yuksel, Avci, & Erdem, 2010). Pessato

Fig. 2. Particle size distribution of PI and PPC. PI ¼ protein isolate, PPC ¼ protein-phenolic complex. 5

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Food Hydrocolloids 102 (2020) 105612

by the negatively charged quinonoid forms of phenolic compounds at pH 9.0 (Sui et al., 2018). These results further confirmed the formation of PPC.

Table 4 The secondary structure content of PI and PPCa determined by FTIR.

3.3.4. FTIR analysis of PI and PPC The FTIR spectroscopy played an important role in the determination of the secondary structure change of protein. As shown in Fig. 3, the spectra of 4000–400 cm 1 were obtained, three typical protein bands, amide I (1600–1700 cm 1), amide II (1480–1580 cm 1) and amide III (1220–1300 cm 1), were observed (Zhang, Zhao, & Yang, 2015). The amide I band, between 1600 and 1700 cm 1, was the most useful characteristics in the secondary structure of PI and PPC. The strong absorption peaks at 1651 cm 1 and 1654 cm 1, for PI and PPC, – O stretching respectively, were mainly attributed to the change in C– vibration. According to a previous report (Zhao, Chen, Xue, & Lee, 2008), the bands at 1615–1637 cm 1 and 1682–1700 cm 1 were β-sheet, 1637–1648 cm 1 was random coil, 1648–1664 cm 1 was α-helix, and 1664 1682 cm 1 was β-turn, respectively. As shown in Table 4, compared with PI, PPC had slight increase in α-helix (from 21.45 to 21.84%) and random coil (from 23.59 to 23.80%), at the expense of the β-turn (from 14.47 to 13.92%). The results suggest that PI and PPC had similar secondary structure as determined by FTIR. To further determine the change in the secondary structure of protein, far-UV CD spectra were used to obtain more information on the sec­ ondary structure composition.

Samples

α-helix (%)

β-sheet (%)

β-turn (%)

Random coil (%)

PI PPC

21.45 21.84

40.49 40.44

14.47 13.92

23.59 23.80

a

3.3.5. Far-UV CD analysis of PI and PPC To further understand the effect of phenolic compounds covalent complex with protein on the change in the secondary structure of pro­ tein, the protein samples of PI and PPC were subjected to far-UV CD analysis. The major elements such as α-helix, β-sheet and random coil with characteristic CD spectra within 190–250 nm were taken into consideration. It has been reported that β-sheet reflected the stability of protein molecule, whereas α-helix, β-turn and random coil were asso­ ciated with the flexibility of protein molecule (Yong, Yamaguchi, & Matsumura, 2006). It was found that PPC induced slight increase in both positive and negative band intensity (Fig. 4). The spectra of both PI and PPC showed a positive band between 190 and 200 nm. Two major negative bands between 205 and 225 nm of PPC were observed, which was more visible than PI. This was most probably attributed to the β type, an indicator of highly ordered structure of protein (Tang et al., 2009).

PI, protein isolate; PPC, protein-phenolic complex.

Fig. 4. Far-UV CD spectra of PI and PPC. PI ¼ protein isolate, PPC ¼ proteinphenolic complex.

The fractions of α-helix, β-sheet, β-turn and random coil were sum­ marized in Table 5. As compared to PI, PPC exhibited an increase in β-sheet (from 19.81 to 21.39%) and a decrease in random coil (from 26.07 to 24.87%), whereas the changes of α-helix and β-turn contents were negligible. Similar results were reported by Pham, Wang, Zisu, and Adhikari (2019b) in a study on the covalent complex of flaxseed protein isolate and phenolic compounds. 3.3.6. Thermal properties of PI and PPC Differential scanning calorimetry (DSC) was used to study the ther­ mal stability of PI and evaluate the change of its tertiary protein. The related thermal parameters, including onset temperature (Tonset), endtemperature (Tendset), denaturation temperature (Td) and enthalpy change (ΔH) were summarized in Table 6. It was reported that Tonset and Td indicated the thermal stability of protein, and ΔH represented the extent of ordered structure of protein (Ling, Ouyang, & Wang, 2019). As shown in Fig. 5, the significant endothermic peaks containing Td of PI and PPC were observed, indicating that the protein samples were fully denatured at this temperature. As summarized in Table 5, the Tonset values were 69.53 and 76.14 � C, Td values were 86.60 and 92.96 � C, Tendset values were at 98.12 and 106.2 � C and ΔH values were 1.57 and 3.78 J/g for PI and PPC, respectively. Interestingly, a higher ΔH value of PPC was observed when compared to PI. This result showed that PPC may be thermally more stable than PI, suggestting that PPC may require more energy to unfold. This may be explained by the increase in β-sheet and decrease in random coiled structure of PI after covalent complex with phenolic compounds. However, the increases in Td and ΔH in the Table 5 The secondary structure contents of PI and PPCa determined by far-UV CD.

Fig. 3. FTIR spectra of PI and PPC. PI ¼ protein isolate, PPC ¼ proteinphenolic complex.

Samples

α-helix (%)

β-sheet (%)

β-turn (%)

Random coil (%)

PI PPC

36.38 36.63

19.81 21.39

17.74 17.11

26.07 24.87

a

6

PI, protein isolate; PPC, protein-phenolic complex.

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Food Hydrocolloids 102 (2020) 105612

Table 6 The thermal parameters of PI and PPCa determined by DSC. Samples

Tonset (� C)

Tendset (� C)

Td (� C)

ΔH (J/g)

PI PPC

69.53 76.14

98.12 106.20

86.60 92.96

1.57 3.78

a

PI, protein isolate; PPC, protein-phenolic complex.

Fig. 5. DSC curves of PI and PPC. PI ¼ protein isolate, PPC ¼ proteinphenolic complex.

study were inconsistent with the results reported by Ali et al. (2013), who found that the whey protein covalently modified by caffeoylquinic acids resulted in a higher Td but lower ΔH. This discrepancy may be attributed to different types of protein or phenolic compounds. It is thus proposed that the application of protein products obtained from CCSK in the food processing industry could be expanded by improving its ther­ mal stability, as the demand for plant-based protein products continues to increase. 3.3.7. SDS-PAGE analysis of PI and PPC It has been proposed that some proteins may be involved in intraand intermolecular cross-linking, when covalently modified by phenolic compounds, the latter can be confirmed by SDS-PAGE analysis under reducing conditions (H M. Rawel, D. Czajka, S. Rohn, & J. Kroll, 2002). As shown in Fig. 6, the major high intensity bands of both PI and PPC were approximately 60, 45 and 12 kDa, and no distinct change in band position of PPC was observed as compared with PI in electrophoretic pattern. As there were not increased high molecular weight fractions in PPC (Lane 2), the intermolecular cross-linking of protein did not occur. This may be due to the short reaction time between protein and phenolic compounds, which is not sufficient to cause crosslinking of protein. Additionally, it could clearly be seen that the bands of PI and PPC exhibited different widths and intensities at the same sample concen­ trations. This could be caused by other components in the protein samples, including phenolic compounds and polysaccharides, since the protein purity of PI and PPC were different.

Fig. 6. SDS-PAGE. M: Protein markers, Land 1–2: PI and PPC, respectively. PI ¼ protein isolate, PPC ¼ protein-phenolic complex.

Correspondingly, the contents of both essential amino acids (EAA) and total amino acids (TAA) in PI were significantly (p < 0.05) higher than those in PPC. This could be because the phenolic compounds present in protein sample had significant effect on protein purity, thereby affecting the amino acid composition of the protein. In addition, both Glu and Arg were the dominant amino acids in PI and PPC, which was similar to those observed in flaxseed protein isolate (Kaushik et al., 2016). Both Glu and Arg were famous as umami amino acids (Zhang et al., 2019), so the result suggested that the protein products of CCSK could be used to produce umami-rich products for the food industry. To better understand the health benefit of PI and PPC, the proportion of EAA should be considered. The ratios of EAA/TAA in PI and PPC were 39.25 � 0.30% and 39.45 � 0.55%, respectively, which were almost equal to that in soybean protein isolate (39.33%) (Chee,

3.4. Amino acid composition of PI and PPC Amino acid composition is an important indicator for assessing the nutritional and healthy value of a protein. As shown in Table 7, a total 17 amino acids were detected, 9 are essential amino acids and 8 are nonessential amino acids. The content of each amino acid in PI was signif­ icantly (p < 0.05) higher than that in PPC, except for Cys. 7

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Food Hydrocolloids 102 (2020) 105612

Table 7 Amino acid composition of PI and PPCa. Amino acid Valine (Val) Methionine (Met) Isoleucine (Ile) Leucine (Leu) Threonine (Thr) Tyrosine (Tyr) Phenylalanine (Phe) Lysine (Lys) Histidine (His) Aspartic acid (Asp) Serine (Ser) Cysteine (Cys) Glutamic acid (Glu) Glycine (Gly) Alanine (Ala) Arginine (Arg) Proline (Pro) EAAb TAAc EAA/TAA (%)

Content (g/100 g of protein) PI

PPC

3.59 � 0.23a 1.18 � 0.07a 2.77 � 0.19a 5.65 � 0.36a 2.25 � 0.17a 4.79 � 0.30a 4.07 � 0.30a 4.31 � 0.22a 3.12 � 0.24a 7.63 � 0.47a 4.43 � 0.22a 0.90 � 0.25a 17.15 � 0.90a 3.27 � 0.17a 3.39 � 0.07a 10.44 � 0.51a 1.88 � 0.04a 31.73 � 2.03a 80.81 � 4.59a 39.25 � 0.30a

2.75 � 0.24b 1.09 � 0.05b 2.14 � 0.18b 4.52 � 0.34b 1.76 � 0.17b 3.53 � 0.26b 2.97 � 0.24b 3.52 � 0.27b 2.54 � 0.25b 5.77 � 0.43b 3.44 � 0.23b 0.88 � 0.14a 13.28 � 0.97b 2.54 � 0.22b 2.60 � 0.21b 8.24 � 0.56b 1.33 � 0.07b 24.81 � 1.91b 62.89 � 4.50b 39.45 � 0.55a

Fig. 7. Solubility of PI and PPC at different pH values. Values are the means � SD of triplicate assays. Note: (*p < 0.05, **p < 0.01). PI ¼ protein isolate, PPC ¼ protein-phenolic complex.

c

TAA, total amino acids. Data are expressed as means � SD (n ¼ 3). Values with different letters (a, b) in the same row in each sample were significantly different (p < 0.05). a PI, protein isolate; PPC, protein-phenolic complex. b EAA, essential amino acids.

protein-protein interactions at the air-water interface (Tan, Mailer, Blanchard, & Agboola, 2011). The lowest FC and FS were found at pH 5 for PI and PPC, which could be because the pH value was close to the isoelectric points of PI and PPC. Furthermore, FC and FS of both PI and PPC were increased from pH 7 to 11, which could be explained by that the protein was almost completely dissolved at pH 7 to 11, and the structure of PI or PPC was fully extended and became loose when the pH value was increased, which accelerated the spread of proteins on the water/air interface. As described above, the results suggested that the foaming properties of PI and PPC were related to the protein solubility when the pH was less than 7 and depended on the pH value when the pH was greater than 7. On the other hand, the phenolic compounds covalently complexing with protein significantly (p < 0.01) increased FC and FS of PPC. The results were different from the report by Malik et al. (2017), who found that the phenolic compounds present in the sunflower seed and kernel protein isolate resulted in significant (p < 0.05) reduction of FC and FS. Compared with a previous report (Ma et al., 2018), the higher FC and FS of PPC indicated that PPC may be a suitable foaming ingredient for food products, such as bread, ice cream and whipped toppings.

Ling, & Ayob, 2012). According to the recommendations of the Food and Agriculture Organization (FAO) and the World Health Organization (WHO), the protein products from CCSK could be considered as high nutritional quality proteins as the contents of most EAA in both PI and PPC exceeded the WHO/FAO requirements for adults, except for Met. Therefore, it is suggested that the protein products obtained from CCSK may be used as potential nutraceutical supplements in the food industry. 3.5. Effect of phenolic compounds on functional properties 3.5.1. Solubility of PI and PPC The solubility of protein (PS) at different pH values could be considered as a guide for protein functionality, since the foaming and emulsifying properties of the protein were significantly affected by solubility (Kinsella & Melachouris, 1976). As shown in Fig. 7, PI and PPC had minimum solubility at pH 5, which corresponded to their isoelectric point. A similar result was reported previously for sesame protein isolate (Bandyopadhyay et al., 2002). It was found that the PS of PPC was significantly (p < 0.01) higher than that of PI at pH 3 and 5 (increased by 23.99% and 242.89%, respectively), indicating that phenolic com­ pounds covalently complexed with protein increased the solubility of protein. However, the improved protein solubility in PPC compared to PI at pH 5 may in turn decrease the extraction yield of protein. Malik and Saini (2017) reported that the complex between sunflower seed protein and phenolic compounds resulted in lower solubility compared with native protein, which could be attributed to the diverse structure of phenolic compounds.

3.5.3. Emulsifying activity index (EAI) and emulsion stability index (ESI) of PI and PPC A stable emulsion was reported to have great potential as a delivery system in common foods, like milk, yogurt and ice cream (Mao, Roos, Biliaderis, & Miao, 2017). As shown in Fig. 8C, the emulsifying activity index (EAI) values of PI and PPC at various pH ranged from 49.28 to 147.32 m2/g and 61.69–173.74 m2/g, respectively. It was found that the EAI of PPC were significantly (p < 0.01) higher than those of PI at pH 9 and 11, which may be because the covalent complex of phenolic com­ pounds with protein may provide more carboxyl groups. The lower EAI under acidic conditions could be explained by Du et al. (2012), who reported that the protein-water interaction was restrained by high sur­ face hydrophobicity of protein and the hydrophobic-hydrophilic balance on the protein surface. The minimum EAI values for PI and PPC at pH 3 indicated that the emulsifying properties were not only affected by protein solubility, but also protein purity (Shevkani, Singh, Kaur, & Rana, 2015). Compared to PI, the emulsion stability index (ESI) values of PPC significantly (p < 0.05) increased at variable pH values (Fig. 8D), indi­ cating that PPC could stabilize the oil-in-water interface more effectively

3.5.2. Foaming capacity (FC) and foam stability (FS) of PI and PPC The foaming properties of protein were the functional properties which determined their application in food systems (Rodsamran & Sothornvit, 2018). Foaming capacity (FC) was thought to be related to the production of air droplet in protein, while foaming stability (FS) meant that the protein had sufficient viscosity to maintain FC and pre­ vent cracking or coalescence (Yu, Zeng, Qin, He, & Chen, 2017). As shown in Fig. 8A and Fig. 8B, both FC and FS of PPC were significantly (p < 0.01) higher than those of PI within the pH range tested, indicating that the phenolic covalent complex with protein enhanced the 8

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Food Hydrocolloids 102 (2020) 105612

Fig. 8. Foaming properties (A, B) and emulsifying properties (C, D) of PI and PPC at different pH values. Values are the means � SD of triplicate assays. Note: (*p < 0.05, **p < 0.01). PI ¼ protein isolate, PPC ¼ protein-phenolic complex.

due to higher surface charge in emulsions (Pham et al., 2019a). The minimum ESI values of PI (2.72 � 0.20%) and PPC (30.68 � 0.46%) were found at pH 5. The results were in agreement with those observed in kidney bean and field pea protein isolates (Shevkani et al., 2015). It was reported that the interaction of green tea polyphenols with dairy matrices during digestion significantly improved polyphenol stability in the intestinal phase (Lamothe, Azimy, Bazinet, Couillard, & Britten, 2014). Therefore, the results suggested that the emulsion system con­ structed by protein-phenolic complex in functional food products could improve their bioactivities.

Declaration of competing interest The authors declare no conflict of interest. Acknowledgments We are grateful to the International Science and Technology Coop­ eration Programme of China (Project No. 2011DFA32770), the Research Program of State Key Laboratory of Food Science and Technology, Nanchang University (Project No. SKLF-ZZB-201517, SKLF-ZZA201610), the Science and Technology Program of Jiangxi Province (Project No. 20143ACG70015), and the National Natural Science Foundation of China (Project No. 31701651) for financial support.

4. Conclusions In this study, the protein products, PI and PPC were successfully produced from CCSK under alkaline conditions. A total of 12 phenolic compounds in CCSK were tentatively identified by HPLC-ESI-QTOFMS2. The content of free sulfhydryl groups, surface hydrophobicity, particle size and zeta potential of PI were significantly (p < 0.05) influenced by the formation of PPC. Far-UV CD analysis showed that the presence of phenolic compounds in PI had remarkable effect on its secondary structure content. PPC exhibited a higher thermally stability than PI, as reflected by higher Tonset and Td. PI and PPC had superior essential amino acids composition and similar protein subunits. Furthermore, the formation of PPC had a positive effect on the solubility, foaming and emulsifying properties of PI, which could be developed as new food materials. These results indicated that the protein products from CCSK have the potential to be incorporated into food applications while providing health benefits.

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