Complex Coacervation of Soy Proteins, Isoflavones and Chitosan

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Article Complex Coacervation of Soy Proteins, Isoﬂavones and Chitosan

Yu-Hsuan Hsiao 1, Sheng-Yang Hsia 2, Yin-Ching Chan 3,† and Jung-Feng Hsieh 1,2,*,†

1 Ph.D. Program in Nutrition & Food Science, Fu Jen Catholic University, Taipei 24205, Taiwan; [email protected] 2 Department of Food Science, Fu Jen Catholic University, Taipei 24205, Taiwan; [email protected] 3 Department of Food and Nutrition, Providence University, Shalu District, Taichung 43301, Taiwan; [email protected] * Correspondence: [email protected]; Tel.: +886-2-2905-2516; Fax: +886-2-2905-3622 † These authors contributed equally to this work.

Received: 23 May 2017; Accepted: 14 June 2017; Published: 20 June 2017

Abstract: In this study, the chitosan-induced coacervation of soy protein-isoflavone complexes in soymilk was investigated. Most of the soymilk proteins, including β-conglycinin (7S), glycinin (11S), and isoflavones, were found to coacervate into the soymilk pellet fraction (SPF) following the addition of 0.5% chitosan. The total protein in the soymilk supernatant fraction (SSF) decreased from 18.1 ± 0.3 mg/mL to 1.6 ± 0.1 mg/mL, and the pH values decreased slightly, from 6.6 ± 0.0 to 6.0 ± 0.0. The results of SDS-PAGE revealed that the 7S α0, 7S α, 7S β, 11S A3, and 11S acidic subunits, as well as the 11S basic proteins in the SSF, decreased to 0.7 ± 0.5%, 0.2 ± 0.1%, 0.1 ± 0.0%, 0.2 ± 0.2%, 0.2 ± 0.2% and 0.3 ± 0.2%, respectively. We also found that isoflavones in the SSF, including daidzein, glycitein, and genistein, decreased to 9.6 ± 2.3%, 5.7 ± 0.9% and 5.9 ± 1.5%, respectively. HPLC analysis indicated that isoflavones mixed with soy proteins formed soy protein-isoflavone complexes and were precipitated into the SPF by 0.5% chitosan.

Keywords: β-conglycinin; glycinin; isoﬂavones; chitosan; coacevation

1. Introduction Chitosan is a naturally non-toxic, modiﬁed, food-compatible polymer that is a cationic polysaccharide produced by the deacetylation of chitin [1]. The structure of chitosan comprises units of D-glucosamine and (1,4)-linked 2-amino-deoxy-β-D-glucan that form a linear polysaccharide [2]. This polysaccharide has been employed in the food industry to improve the safety and shelf-life of food products [3]. The protein-polysaccharide interactions play an essential structure-controlling role in foods [4]. Chen et al. reported that chitosan can be used as a coagulant in the preparation of cheese, as it induced destabilization and coagulation of casein micelles via chitosan-milk protein interactions [5]. No et al. suggested that chitosan could to be used as a coagulant to interact with soy proteins in soymilk to form insoluble complexes in the preparation of tofu [6]. Tofu is a gel-like soybean food, and its preparation generally includes the coagulation of soymilk followed by molding and pressing. Soymilk is a colloidal solution that contains 3.6% protein, 2.9% carbohydrate, 2.0% fat and 0.5% ash [7]. Among the soy proteins, 7S and 11S are the two major proteins in soymilk [8]. The 7S protein is a trimeric protein composed of three subunits (i.e., α, α0, and β subunits) with molecular weights of approximately 76, 70 and 53 kDa, respectively [9]. The 11S protein is hexameric and consists of acidic and basic polypeptides and consists of ﬁve subunits: A1aB1b, A2B1a, A1bB2, A3B4 and A5A4B3 [10]. These 7S and 11S proteins are the major materials involved in gelling and coagulation following the addition of coagulant during the production of tofu.

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Tofu is an excellent source of isoflavones. Prabhakaran et al. reported that 1317.88 ± 6.77 µg isoflavones/g dry tofu was precipitated into tofu during the tofu-making process [11]. These isoflavones included daidzin, glycitin, genistin, daidzein, glycitein and genistein, and the contents of these isoflavones were found to be 1022.19 ± 13.2, 111.52 ± 7.8, 1049.32 ± 15.4, 37.14 ± 0.77, 37.14 ± 0.77 and 16.90 ± 0.53 µg/g, respectively. A previous study reported that isoflavones, including daidzin, glycitin, genistin, malonyldaidzin, malonylgenistin, daidzein, glycitein and genistein, bind with the 7S and 11S proteins to form 7S-isoflavone and 11S-isoflavone complexes in soymilk [12]. Moreover, the addition of polysaccharides to soymilk, such as propylene glycol alginate and chitosan, induced the coacervation of the soy protein-isoflavone complexes [13]. Soy protein-chitosan interactions have garnered considerable attention due to their influence on the structure of foods [14]. Huang et al. indicated that the complex was formed through the electrostatic + interactions between the amine groups of chitosan (–NH3 ) and the carboxyl groups of soy proteins (–COO−), with hydrogen bonding also being involved in the soy protein-chitosan interaction [15]. As mentioned above, chitosan has the potential to coacervate soy proteins. However, the effects of chitosan-induced coacervation of soy protein-isoflavone complexes have not been described. Therefore, we utilized SDS-PAGE and HPLC to analyze the effects of chitosan on the coacervation of the soy protein-isoflavone complexes. The objective of this study was to investigate the coacervation of soy protein-isoflavone complexes by chitosan in soymilk.

2. Results and Discussion

2.1. Effects of Chitosan on the Coacervation of Soy Proteins Soymilk samples were incubated with various quantities of chitosan (0.0%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%) at 30 ◦C for 1 h, whereupon we determined the total protein content in both SSF and SPF. As shown in Figure1, without the addition of chitosan, total protein levels in the SSF and SPF were 18.1 ± 0.3 and 0.4 ± 0.0 mg/mL, respectively. Previous studies have suggested that protein coacervation does not occur in soymilk samples without the addition of chitosan. However, we found that higher chitosan concentrations led to a decrease in protein content in the SSF and an increase in the protein content in the SPF. More speciﬁcally, 90.9 ± 0.4% of soy proteins were precipitated by chitosan, and the total protein concentration in the SSF decreased from 18.1 ± 0.3 mg/mL to 1.6 ± 0.1 mg/mL following treatment with 0.5% chitosan. Conversely, total protein in the SPF showed an obvious increase after the addition of 0.4% chitosan. In addition, the pH of each SSF sample treated with a different concentration of chitosan (0.0%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%) was 6.6 ± 0.0, 6.5 ± 0.0, 6.4 ± 0.0, 6.2 ± 0.0, 6.2 ± 0.0, and 6.0 ± 0.0, respectively. Our results revealed that most soy proteins coacervate at a pH of 6.0 and that the soy protein-chitosan ratio is approximately 3.6. Huang et al. previously reported that the maximum coacervation rate associated with the formation of the soy protein-chitosan complex occurred at a pH value of 6.0–6.5, 25 ◦C, and a ratio of soy proteins to chitosan of 4:1 was observed. The coacervation of soy proteins and chitosan is associated with interactions between proteins and polysaccharides [15]. Liu et al. reported that protein-polysaccharide interactions may be formed by an electrostatic attraction between the negatively charged surface of proteins and the positively charged chitosan [16]. Huang et al. further reported that these complex interactions + involved hydrogen bonding between the amine groups of chitosan (–NH3 ) and the carboxyl groups of soy proteins (COO−)[15]. Thus, the coacervation of soy proteins and chitosan is due to interactions between proteins and polysaccharides. Molecules 2017, 22, 1022 3 of 10 Molecules 2017, 22, 022 3 of 10

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Figure 1. Changes in the total protein content of soymilk resulting following the addition of various Figure 1. Changes in the total protein content of soymilk resulting following the addition of various quantities of chitosan (0.0%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%). Samples were incubated at 30 °C for 1 quantities of chitosan (0.0%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%). Samples were incubated at 30 ◦C for 1 h. h. ○: soymilk supernatant fraction (SSF); ●: soymilk pellet fraction (SPF). - - -: pH value (SSF). Vertical : soymilkFigure supernatant1. Changes in fractionthe total (SSF);protein content: soymilk of soymilk pellet fractionresulting (SPF). following - - -: the pH addition value (SSF).of various Vertical bars represent standard deviations. bars# representquantities standardof chitosan deviations. (0.0%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%). Samples were incubated at 30 °C for 1 h. ○: soymilk supernatant fraction (SSF); ●: soymilk pellet fraction (SPF). - - -: pH value (SSF). Vertical 2.2. Analysisbars of representthe Effects standard of Chitosan deviations. on Soy Proteins Using SDS-PAGE 2.2. Analysis of the Effects of Chitosan on Soy Proteins Using SDS-PAGE Glycinin globulin (11S) and β-conglycinin globulin (7S) are the two major proteins in soymilk Glycinin2.2. Analysis globulin of the Effects (11S) of and Chitosanβ-conglycinin on Soy Proteins globulin Using (7S) SDS-PAGE are the two major proteins in soymilk [17]. [17]. Soymilk samples were incubated using various concentrations of chitosan (0.0%, 0.1%, 0.2%, Soymilk samplesGlycinin were globulin incubated (11S) and using β-conglycinin various concentrations globulin (7S) are of chitosan the two major (0.0%, proteins 0.1%, 0.2%, in soymilk 0.3%, 0.4% 0.3%, 0.4% or 0.5%). The results of SDS-PAGE (Figure 2) indicate that the concentrations of the or 0.5%).[17]. TheSoymilk results samples of SDS-PAGE were incubated (Figure using2) indicate various thatconcentrations the concentrations of chitosan of (0.0%, the subunits 0.1%, 0.2%, (7S α0, subunits (7S α’, 7S α, 7S β, 11S A3, 11S acidic, and 11S basic) in the SSF decreased as the concentration 7S α,0.3%, 7S β, 0.4% 11S A3,or 0.5%). 11S acidic, The results and 11Sof SDS-PAGE basic) in the (Figure SSF decreased2) indicate asthat the the concentration concentrations of of chitosan the of chitosan increased (Figure 2A). Furthermore, a marked decrease in the concentration of proteins increasedsubunits (Figure (7S α2A).’, 7S Furthermore,α, 7S β, 11S A3, a 11S marked acidic,decrease and 11S basic) in the inconcentration the SSF decreased of proteinsas the concentration was observed was observed at a chitosan concentration of 0.4%. Similarly, the quantities of soy protein subunits at a chitosanof chitosan concentration increased (Figure of 0.4%. 2A). Similarly, Furthermore, the quantitiesa marked decrease of soy protein in the concentration subunits mentioned of proteins above mentionedwas observed above increasedat a chitosan in concentration the SPF as ofthe 0.4%. concentration Similarly, the of quantities chitosan of increasedsoy protein (Figuresubunits 2B). increased in the SPF as the concentration of chitosan increased (Figure2B). Therefore, our results Therefore,mentioned our resultsabove increasedsuggest inthat the a SPFchitosan as the concentrationconcentration ofof chitosan at least increased 0.4% could (Figure cause 2B). the suggest that a chitosan concentration of at least 0.4% could cause the agglomeration of soy protein agglomerationTherefore, of our soy results protein suggest subunits, that includinga chitosan theconcentration 7S α’, 7S αof, 7Sat βleast, 11S 0.4% A3, 11Scould acidic, cause and the 11S subunits, including the 7S α0, 7S α, 7S β, 11S A3, 11S acidic, and 11S basic subunits, resulting in the basic subunits,agglomeration resulting of soy in protein the transformation subunits, including of SSF the to 7S SPF. α’, 7S α, 7S β, 11S A3, 11S acidic, and 11S transformationbasic subunits, of SSF resulting to SPF. in the transformation of SSF to SPF.

Figure 2. Cont.

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Figure 2. Changes in the SDS-PAGE profiles of soymilk following the addition of different amounts Figure 2. Changes in the SDS-PAGE proﬁles of soymilk following the addition of different amounts of of chitosan (0.0%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%). Samples were incubated at 30 °C for 1 h. (A) chitosan (0.0%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%). Samples were incubated at 30 ◦C for 1 h. (A) soymilk soymilk supernatant fraction (SSF); (B) soymilk pellet fraction (SPF); M: protein marker. supernatant fraction (SSF); (B) soymilk pellet fraction (SPF); M: protein marker.

Densitograms corresponding to the SDS-PAGE analysis of the soymilk samples treated with various quantities of chitosan are shown in Figure3. When 0.4% chitosan was added, the intensities of total soy protein subunits sharply decreased. Moreover, at the highest concentration of chitosan tested 0 (0.5%), the 7S αFigure, 7S 2.α Changes, 7S β, in 11S the A3,SDS-PAGE 11S acidic, profiles of and soymilk 11S following basic subunits the addition were of different decreased amounts in the SSF to 0.7 ± 0.5%, 0.2of± chitosan0.1%, 0.1(0.0%,± 0.1%,0.0%, 0.2%, 0.2 0.3%,± 0.2%, 0.4%, or 0.2 0.5%).± 0.2% Samples and were 0.3 incubated± 0.2%, at respectively30 °C for 1 h. (A (Figure) 3A). soymilk supernatant fraction (SSF); (B) soymilk pellet fraction (SPF); M: protein marker.

Figure 3. Densitograms corresponding to the SDS-PAGE analysis of soymilk proteins containing various concentrations of chitosan (0.0%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%). Samples were incubated at 30 °C for 1 h. (A) soymilk supernatant fraction (SSF); (B) soymilk pellet fraction (SPF).

Figure 3. Densitograms corresponding to the SDS-PAGE analysis of soymilk proteins containing Figure 3. Densitogramsvarious concentrations corresponding of chitosan (0.0%, to the0.1%, SDS-PAGE 0.2%, 0.3%, 0.4% analysis or 0.5%). of Samples soymilk were proteins incubated containing at various concentrations30 °C for 1 h. (A of) soymilk chitosan supernatant (0.0%,0.1%, fraction 0.2%, (SSF); 0.3%, (B) soymilk 0.4% pellet or 0.5%). fraction Samples (SPF). were incubated at 30 ◦C for 1 h. (A) soymilk supernatant fraction (SSF); (B) soymilk pellet fraction (SPF).

Molecules 2017, 22, 022 5 of 10 Molecules 2017, 22, 1022 5 of 10 Densitograms corresponding to the SDS-PAGE analysis of the soymilk samples treated with various quantities of chitosan are shown in Figure 3. When 0.4% chitosan was added, the intensities 0 In addition,of total soy the protein intensities subunits of sharply the 7S decreased.α , 7S α Mo, 7Sreover,β, 11S at the A3, highest 11S acidic, concentration and 11Sof chitosan basic subunits increased markedlytested (0.5%), in the the 7S SPFα’, 7S upon α, 7S β the, 11S addition A3, 11S acidic, of 0.4% and 11S chitosan basic subunits (Figure were3B). decreased Yuan et inal. the reported that chitosanSSF to is 0.7 positively ± 0.5%, 0.2 ± charged 0.1%, 0.1 ± at 0.0%, a slightly 0.2 ± 0.2%, acidic 0.2 ± 0.2% pH and (<6.5) 0.3 ± 0.2%, and respectively that the carboxyl (Figure 3A). groups of In addition, the intensities of the 7S α’, 7S α, 7S β, 11S A3, 11S acidic, and 11S basic subunits proteins hadincreased a distributed markedly negative in the SPF chargeupon the that addition could of be0.4% shown chitosan by (Figure the zeta-potential 3B). Yuan et al. reported distribution [18]. The coacervationthat chitosan results is positively also described charged the at a solubility slightly acidic of the pH 7S(<6.5) and and 11S that proteins the carboxyl following groups the of addition of chitosan.proteins Yuan had et a distributed al. discussed negative the charge coacervate that could be of shown the 7Sby the protein zeta-potential and 11Sdistribution protein [18]. combined with chitosanThe bycoacervation turbidimetric results analysisalso described individually. the solubility They of the suggested 7S and 11S that proteins interactions following betweenthe the addition of chitosan. Yuan et al. discussed the coacervate of the 7S protein and 11S protein combined chitosan-inducedwith chitosan 7S proteins by turbidimetric and the analysis chitosan-induced individually. They 11S suggested proteins that become interactions stronger between at pH the values of 6.0 and 6.5,chitosan-induced respectively [19 7S]. proteins Nevertheless, and the chitosan-induced the results of 11S this proteins study become suggest stronger that theat pH 7S values protein of and 11S proteins coacervate6.0 and 6.5, byrespectively the addition [19]. Nevertheless, of 0.5% chitosan the resu andlts of athis decrease study suggest in pH that to the 6.0 7S± protein0.0. and 11S proteins coacervate by the addition of 0.5% chitosan and a decrease in pH to 6.0 ± 0.0. 2.3. HPLC Analysis of Isoflavones in Soymilk 2.3. HPLC Analysis of Isoflavones in Soymilk Eight isoflavonesEight isoflavones (Figure 4A)(Figure were 4A) identified were identified in soymilk in soymilk by HPLC, by andHPLC, the and HPLC the chromatographHPLC of soymilkchromatograph is presented inof Figuresoymilk4 isB. presented For this in investigation, Figure 4B. For wethis separatedinvestigation, the we eight separated major the isoflavoneseight in soymilk samplesmajor isoflavones into two in types: soymilk glycones samples andinto aglycones.two types: glycones The glycone and aglycones. isoflavones The includeglycone daidzin isoflavones include daidzin (peak 1), glycitin (peak 2), genistin (peak 3), malonyldaidzin (peak 4), (peak 1), glycitinand malonylgenistin (peak 2), genistin (peak 5). (peakThe aglycones 3), malonyldaidzin isoflavones were (peakdaidzein 4), (peak and 6), malonylgenistin glycitein (peak 7), (peak 5). The aglyconesand genistein isoflavones (peak were8) [12]. daidzein (peak 6), glycitein (peak 7), and genistein (peak 8) [12].

Figure 4. HPLCFigure 4. chromatograms: HPLC chromatograms: (A) ( isoﬂavoneA) isoflavone standards; standards; (B ()B isoflavones) isoﬂavones extracted extracted from soymilk. from soymilk.

Wang et al. previously reported on the content of each isoﬂavone in 5.5 L soymilk; speciﬁcally, the daidzin, glycitin, genistin, malonyldaidzin, malonylgenistin, daidzein, glycitein, and genistein amounts were 65.6, 18.9, 90.2, 15.3, 48.5, 15.1, 7.9 and 19.7 mg, respectively [20].

2.4. HPLC Analysis of the Effects of Chitosan on the Isoflavones To understand the content of isoflavones transforming, we generated HPLC chromatographs to determine the isoflavone content in SPF treated with various concentrations of chitosan (0.0%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%), the results of which are shown in Figure5. As previously mentioned, soy proteins in the SSF were found to coacervate into the SPF following the addition of 0.4% chitosan. Molecules 2017, 22, 022 6 of 10

Wang et al. previously reported on the content of each isoflavone in 5.5 L soymilk; specifically, the daidzin, glycitin, genistin, malonyldaidzin, malonylgenistin, daidzein, glycitein, and genistein amounts were 65.6, 18.9, 90.2, 15.3, 48.5, 15.1, 7.9 and 19.7 mg, respectively [20].

2.4. HPLC Analysis of the Effects of Chitosan on the Isoflavones To understand the content of isoflavones transforming, we generated HPLC chromatographs to determine the isoflavone content in SPF treated with various concentrations of chitosan (0.0%, 0.1%, Molecules 2017, 22, 1022 6 of 10 0.2%, 0.3%, 0.4% or 0.5%), the results of which are shown in Figure 5. As previously mentioned, soy proteins in the SSF were found to coacervate into the SPF following the addition of 0.4% chitosan. SimilarSimilar results results were were obtained obtained when when quantifying quantifying the the isoﬂavone isoflavone content content that that had had transformed transformed into into SPF SPF byby the the addition addition of of 0.4% 0.4% chitosan chitosan (Figure (Figure5). 5).

FigureFigure 5. 5.Changes Changes inin thethe percentage percentage of of isoﬂavone isoflavone content content (%) (%) in in soymilk soymilk samples samples with with various various quantitiesquantities of of chitosan chitosan (0.0%, (0.0%, 0.1%,0.1%, 0.2%,0.2%, 0.3%, 0.4% or or 0.5%). 0.5%). Samples Samples were were incubated incubated at at30 30 °C◦ forC for 1 h. 1( h.A) ( Asoymilk) soymilk supernatant supernatant fraction fraction (SSF); (SSF); (B (B) )soymilk soymilk pellet pellet fraction (SPF).(SPF). VerticalVertical bars bars represent represent standardstandard deviations. deviations.

Conversely,Conversely, when when 0.4% 0.4% chitosan chitosan was was added added to to soymilk, soymilk, the the isoflavone isoflavone content content in in the the SSF SSF began began toto decrease. decrease. When When 0.5% 0.5% chitosan chitosan was was added added to to the the SSF, SSF, the the content content of of each each isoflavone, isoflavone, including including daidzin,daidzin, glycitin,glycitin, genistin,genistin, malonyldaidzin,malonyldaidzin, malonylgenistin,malonylgenistin, daidzein,daidzein, glycitein,glycitein, andand genistein,genistein, decreaseddecreased to to 21.021.0 ± 0.0,0.0, 2.7 2.7 ± 0.0,± 0.0, 12.4 12.4± 0.0,± 21.10.0, ± 21.10.2, 25.3± 0.2, ± 0.1, 25.3 0.2 ± 0.0,0.1, 0.0 0.2 ± 0.0± 0.0,and 0.00.1 ±± 0.00.0 μg/mL, and 0.1 ± 0.0 µg/mL, respectively. These results reveal that the isoflavone content in the SSF decreases with the addition of chitosan. Conversely, the concentration of each isoflavone in the SPF increased with the concentration of chitosan. Specifically, the percentage of isoflaovnes in the SSF, including daidzin, glycitin, genistin, malonyldaidzin, malonylgenistin, daidzein, glycitein, and genistein decreased to 57.9 ± 0.8%, 59.4 ± 1.2%, 40.6 ± 0.9%, 61.6 ± 0.8%, 46.4 ± 1.8%, 9.6 ± 2.3%, 5.7 ± 0.9% and 5.9 ± 1.5%, respectively, when treated with 0.5% chitosan (Figure5A). The content of glycone isoflavones (peaks 1–5) decreased by approximately 40–60%, and the content of aglycone isoflavones Molecules 2017, 22, 022 7 of 10 respectively. These results reveal that the isoflavone content in the SSF decreases with the addition of chitosan. Conversely, the concentration of each isoflavone in the SPF increased with the concentration of chitosan. Specifically, the percentage of isoflaovnes in the SSF, including daidzin, glycitin, genistin, malonyldaidzin, malonylgenistin, daidzein, glycitein, and genistein decreased to Molecules 2017, 22, 1022 7 of 10 57.9 ± 0.8%, 59.4 ± 1.2%, 40.6 ± 0.9%, 61.6 ± 0.8%, 46.4 ± 1.8%, 9.6 ± 2.3%, 5.7 ± 0.9% and 5.9 ± 1.5%, respectively, when treated with 0.5% chitosan (Figure 5A). The content of glycone isoflavones (peaks 1–5)(peaks decreased 6–8) decreased by approximately by approximately 40–60%, 90–95%. and th Therefore,e content theof isoflavoneaglycone isoflavones content increased (peaks in6–8) the decreasedSPF with theby approximately addition of 0.4% 90–95%. chitosan Therefore, and increased the isoflavone markedly content with theincreased addition in the of 0.5%SPF with chitosan the addition(Figure5 B).of 0.4% chitosan and increased markedly with the addition of 0.5% chitosan (Figure 5B). TheThe effects effects of of chitosan chitosan on on the the coacervation coacervation of of isoflavones isoflavones were were also also evaluated. evaluated. The The isoflavones isoflavones extractedextracted from from soymilk soymilk were were incubated incubated with with 0.0–0.5 0.0–0.5%% of of chitosan. chitosan. However, However, no no significant significant changes changes inin the the amounts amounts of of daidzin, daidzin, glycitin, glycitin, genistin, genistin, malonyldaidzin, malonyldaidzin, malonylgenistin, malonylgenistin, daidzein, daidzein, glycitein, glycitein, andand genistein genistein in in the the supernatant supernatant were were observed observed (Figure (Figure 6).6 ).This This result result indicated indicated that that isoflavones isoflavones did notdid immediately not immediately coacervate coacervate with chitosan. with chitosan. As previously As previously mentioned, mentioned, isoflavones, isoflavones, including including daidzin, glycitin,daidzin, genistin, glycitin, malonyldaidzin, genistin, malonyldaidzin, malonylgenistin, malonylgenistin, daidzein, glycitein, daidzein, and glycitein,genistein, andwere genistein, isolated fromwere the isolated dried 7S from and the 11S dried proteins 7S and [12]. 11S These proteins isoflavones [12]. bind These to isoflavones the 7S and 11S bind proteins to the to 7S form and 7S- 11S isoflavoneproteins to complexes form 7S-isoflavone and 11S-isoflavone complexes andcomplexes. 11S-isoflavone These complexes.soy protein-isoflavone These soy protein-isoflavone complexes then coacervatecomplexes into then the coacervate SPF following into the the SPF addition following of chitosan. the addition The presence of chitosan. of soy The protein-isoflavone presence of soy complexesprotein-isoflavone suggests complexes that soy proteins suggests and that isoflavone soy proteinss are and coprecipitated isoflavones areinto coprecipitated tofu during the into tofu- tofu makingduring theprocess. tofu-making process.

Figure 6. Changes in the isoflavone contents with different amounts of chitosan (0.0%, 0.1%, 0.2%, Figure 6. Changes in the isoflavone contents with different amounts of chitosan (0.0%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%) at 30 °C for 1 h. Vertical bars represent standard deviations. 0.3%, 0.4% or 0.5%) at 30 ◦C for 1 h. Vertical bars represent standard deviations. 2.5. Reaction Scheme for the Effects of Chitosan on Soy Proteins and Isoflavones 2.5. Reaction Scheme for the Effects of Chitosan on Soy Proteins and Isoflavones A reaction scheme based on the results of this study is presented in Figure 7. This scheme A reaction scheme based on the results of this study is presented in Figure7. This scheme illustrates the effects of chitosan on the coacervation of individual soy proteins. The mechanism by illustrates the effects of chitosan on the coacervation of individual soy proteins. The mechanism which chitosan induced the coacervation of soy proteins involves two steps, as follows: (1) the by which chitosan induced the coacervation of soy proteins involves two steps, as follows: (1) the hydrophobic regions of protein molecules in the native state are exposed to the outside environment hydrophobic regions of protein molecules in the native state are exposed to the outside environment by by heat denaturation; (2) by adding chitosan, the chitosan is induced under a pH value of heat denaturation; (2) by adding chitosan, the chitosan is induced under a pH value of approximately approximately 6.0 to be positively charged. The negative charge of the carboxyl groups of soy 6.0 to be positively charged. The negative charge of the carboxyl groups of soy proteins forms a proteins forms a complex with the positively charged amine groups of chitosan through an complex with the positively charged amine groups of chitosan through an electrostatic interaction [21]. electrostatic interaction [21]. When charge balance occurs, the soy proteins and chitosan molecules When charge balance occurs, the soy proteins and chitosan molecules coacervate through hydrophobic coacervate through hydrophobic interactions [19]. Furthermore, hydrogen bonding is also involved interactions [19]. Furthermore, hydrogen bonding is also involved with coacervation [15]. Finally, with coacervation [15]. Finally, the isoflavones induced by chitosan in soymilk coacervation are the isoflavones induced by chitosan in soymilk coacervation are coprecipitated with proteins, and hydrophobic interactions cause most aglycones to coacervate with soy proteins, while only half of the glycones coacervate with the 7S and 11S proteins. Molecules 2017, 22, 022 8 of 10

Moleculescoprecipitated2017, 22, 1022with proteins, and hydrophobic interactions cause most aglycones to coacervate with8 of 10 soy proteins, while only half of the glycones coacervate with the 7S and 11S proteins.

Figure 7. Reaction scheme illustrating the effects of chitosan on the coacervation of β-conglycinin, Figure 7. Reaction scheme illustrating the effects of chitosan on the coacervation of β-conglycinin, glycinin, and isoflavones in soymilk. glycinin, and isoﬂavones in soymilk.

3. Experimental Section 3. Experimental Section 3.1. Preparation of Soymilk 3.1. Preparation of Soymilk Soybeans [Glycine max (L.) Merrill, 100 g] were washed and soaked in distilled water at 25 °C for Soybeans [Glycine max (L.) Merrill, 100 g] were washed and soaked in distilled water at 25 ◦C for 12 h. The hydrated seeds were drained and ground into homogenates in 1 L of distilled water. The 12 h. The hydrated seeds were drained and ground into homogenates in 1 L of distilled water. The raw soymilk was filtered through a cotton filter and heated in a 90 °C water bath for 1 h. The soymilk raw soymilk was filtered through a cotton filter and heated in a 90 ◦C water bath for 1 h. The soymilk was collected and stored at 4 °C. To investigate the effects of chitosan on the aggregation of soymilk was collected and stored at 4 ◦C. To investigate the effects of chitosan on the aggregation of soymilk proteins, soymilk was centrifuged at 12,000 × g for 10 min at 4 °C using a centrifugal separator to proteins, soymilk was centrifuged at 12,000× g for 10 min at 4 ◦C using a centrifugal separator to remove the lipids. The defatted filtrate was used as soymilk in subsequent experiments. remove the lipids. The defatted filtrate was used as soymilk in subsequent experiments. 3.2. Preparation of Soymilk Samples Containing Various Concentrations of Chitosan 3.2. Preparation of Soymilk Samples Containing Various Concentrations of Chitosan Chitosan oligosaccharide lactate (molecular weight: 4–6 kDa, deacetylation > 90%) was Chitosan oligosaccharide lactate (molecular weight: 4–6 kDa, deacetylation > 90%) was purchased purchased from Sigma Chemical Co. (St. Louis, MO, USA). To investigate the effects of chitosan on from Sigma Chemical Co. (St. Louis, MO, USA). To investigate the effects of chitosan on the the coacervation of soymilk proteins, soymilk samples with varying amounts of chitosan (0.0%, 0.1%, coacervation of soymilk proteins, soymilk samples with varying amounts of chitosan (0.0%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%) were incubated at 30 °C for 1 h. After incubation, the soymilk samples were 0.2%, 0.3%, 0.4% or 0.5%) were incubated at 30 ◦C for 1 h. After incubation, the soymilk samples fractionated into the soymilk supernatant fraction (SSF) and the soymilk pellet fraction (SPF) by were fractionated into the soymilk supernatant fraction (SSF) and the soymilk pellet fraction (SPF) by centrifugation for 20 min (12,000× g). The protein concentrations of the SSF and SPF samples were centrifugation for 20 min (12,000× g). The protein concentrations of the SSF and SPF samples were determined using a protein assay kit (Bio-Rad, Hercules, CA, USA). The Bio-Rad protein assay dye determined using a protein assay kit (Bio-Rad, Hercules, CA, USA). The Bio-Rad protein assay dye was diluted with 4 volumes of water; then, it was mixed with individual standards or soymilk was diluted with 4 volumes of water; then, it was mixed with individual standards or soymilk samples. samples. The absorbance at 595 nm was measured using a VersaMax™ microplate reader (Molecular The absorbance at 595 nm was measured using a VersaMax™ microplate reader (Molecular Devices Devices Corporation, Sunnyvale, CA, USA), and bovine serum albumin (Sigma Chemical Co.) was Corporation,used as the standard. Sunnyvale, CA, USA), and bovine serum albumin (Sigma Chemical Co.) was used as the standard. 3.3. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 3.3. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Soymilk samples were analyzed using SDS-PAGE, in accordance with protocol outlined by Soymilk samples were analyzed using SDS-PAGE, in accordance with protocol outlined by Hsiao et al. [13]. Electrophoresis was carried out in 1.5 mm thick gels that contained a 5% stacking Hsiao et al. [13]. Electrophoresis was carried out in 1.5 mm thick gels that contained a 5% stacking gel (w/v) and a 12.5% separating gel (w/v). For each soymilk sample, a 0.1-mL volume of sample was gel (w/v) and a 12.5% separating gel (w/v). For each soymilk sample, a 0.1-mL volume of sample mixed with 0.3 mL of buffer (2% SDS, 5% β-mercaptoethanol, 10% glycerol, 0.02% bromophenol blue was mixed with 0.3 mL of buffer (2% SDS, 5% β-mercaptoethanol, 10% glycerol, 0.02% bromophenol and 70 mM Tris-HCl, pH 6.8) and was heated to 95 °C for 5 min. The samples (4 μL) and a protein ◦ µ blueladder and were 70 mMloaded Tris-HCl, into separate pH 6.8) wells and of was the heatedgels. Following to 95 C electrophoresis, for 5 min. The the samples gels were (4 L)stained and a proteinwith Coomassie ladder were Brilliant loaded Blue into R-250. separate The stained wells of gels the were gels. imaged Following using electrophoresis, an EPSON perfection the gels 1270 were stainedimage scanner with Coomassie (Epson America Brilliant Inc., Blue Long R-250. Beach, The stainedCA, USA) gels and were analyzed imaged using using the an Gel-Pro EPSON Analyzer perfection 1270(version image 4.0, scanner Media Cybernetics, (Epson America Inc., Rockville, Inc., Long MD, Beach, USA) CA, software USA) andprogram. analyzed using the Gel-Pro Analyzer (version 4.0, Media Cybernetics, Inc., Rockville, MD, USA) software program.

Molecules 2017, 22, 1022 9 of 10

3.4. Preparation of Isoflavone Samples Soymilk, SSF, and SPF samples were lyophilized into powders to extract isoflavones in accordance with the method described by Prabhakaran et al. [11]. Specifically, 80% methanol was added to freeze-dried samples to serve as an extraction solvent, and the solution was shaken in a vortex mixer at 30 ◦C for 2 h. After a 10 min centrifugation at 12,000× g at 4 ◦C, the supernatants (isoflavones) were subjected to high-performance liquid chromatography (HPLC) analysis.

3.5. HPLC Analysis of the Isoflavone Samples Analysis of isoflavone samples (20 µL) was performed using a method modified from Tipkanon et al. [22]. The HPLC system comprised a PU-980 pump, and a UV-970 detector (both from JASCO, Tokyo, Japan) and a C18 packed column (Mightysil RP-18 GP, 4.6 mm × 250 mm, 5 µm spherical, Kanto Chemicals, Tokyo, Japan). The mobile phase of methanol (A) and water (B) was used with a gradient elution of 25% A and 75% B that was increased to 35% A after 10 min, 50% A after 25 min, 100% A after 35 min, and 100% A after 40 min. The flow rate was 0.5 mL/min with the column maintained at a temperature of 30 ◦C. Detection was conducted at a wavelength of 260 nm. Eight commercial isoflavones were analyzed as the standards. Daidzin, glycitin, genistin, daidzein, glycitein and genistein were purchased from LC Laboratories (Woburn, MA, USA), while malonyldaidzin and malonylgenistin were purchased from Nacalai Tesque, Inc. (Kyoto, Japan).

3.6. Statistical Analysis SPSS statistical software (version 10.0.7C, SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Data were expressed as the means ± standard deviations. The statistically signiﬁcant differences between treatments were determined using one-way ANOVA followed by Duncan’s multiple range test. Three determinations for each treatment were performed, and the signiﬁcance level was set at p < 0.05.

4. Conclusions This study examined the effects of chitosan on the coacervation of β-conglycinin, glycinin and isoﬂavones in soymilk. The results of SDS-PAGE clearly demonstrate that most of the 7S (α0, α, and β), 11S acidic (A3) and 11S basic protein subunits in soymilk coacervate by the addition of 0.5% chitosan. Moreover, HPLC analysis indicates that 90–95% of the aglycones (including daidzein, glycitein and genistein) had interacted with the 7S and 11S proteins. These isoﬂavones were also coprecipitated into SPF by 0.5% chitosan.

Acknowledgments: We thank Fu Jen Catholic University for their grant support (A0105014). Author Contributions: Jung-Feng Hsieh and Yin-Ching Chan designed and performed experiments. Yu-Hsuan Hsiao and Sheng-Yang Hsia analyzed the experimental data. Corresponder, Jung-Feng Hsieh, wrote and revised this manuscript. All authors read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are not available.

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