Influence of succinylation on physicochemical property of yak casein micelles

Accepted Manuscript Influence of succinylation on physicochemical property of yak casein micelles Min Yang, Jitao Yang, Yuan Zhang, Weibing Zhang PII: DOI: Reference:

S0308-8146(15)00919-X http://dx.doi.org/10.1016/j.foodchem.2015.06.030 FOCH 17712

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Food Chemistry

Received Date: Revised Date: Accepted Date:

4 March 2015 9 June 2015 10 June 2015

Please cite this article as: Yang, M., Yang, J., Zhang, Y., Zhang, W., Influence of succinylation on physicochemical property of yak casein micelles, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.06.030

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Influence of succinylation on physicochemical property of yak casein micelles Min Yang

a,b*

, Jitao Yanga, Yuan Zhang a, Weibing Zhangb,c

a

College of Science, Gansu Agricultural University, Lanzhou, China

b

Functional Dairy Product Engineering Lab of Gansu Province, Lanzhou, China

c

College of Food Science and Engineering, Gansu Agricultural University, Lanzhou, China

Abstract Succinylation is a chemical-modification method that affects the physicochemical characteristics and functional properties of proteins. This study assessed the influence of succinylation on the physicochemical properties of yak casein micelles. The results revealed that surface hydrophobicity indices decreased with succinylation. Additionally, denaturation temperature and denaturation enthalpy decreased with increasing succinylation level, except at 82%. The buffering properties of yak casein micelles were affected by succinylation. The results revealed that chemical modification contributed to a slight shift of the buffering peak towards a lower pH value and an markedly increase of the maximum buffering values of yak casein micelles at pH 4.5–6.0 and pH <3. Succinylation increased yak casein micellar hydration and whiteness values. The findings obtained from this study will provide the basic information on the physicochemical properties of native and succinylated yak casein micelles. *

Corresponding author: Min Yang

Address: College of Science, Gansu Agricultural University, Lanzhou, China E-mail: [email protected] (M. Yang) Tel.: 008613893272871

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Keywords: Yak casein micelles; Succinylation; Physicochemical properties; Surface hydrophobicity; Thermal properties; Buffering property

Chemical compounds studied in this article: Succinic anhydride (CID: 7922) Casein (CID: 73995022)

1. Introduction Caseins are valuable milk components. Caseins are commonly used in the food, chemical, cosmetic, and pharmaceutical industries due to their textural, sensory, and nutritional properties. Yak (Bos grunniens) caseins, consumed by inhabitants of the Tibetan plateau, are widely used in the production of high quality food ingredients, soaps, glues, leather-polishing reagents, and clothing (Li, et al., 2010). It has been reported that yak caseins have higher micellar calcium and lower inorganic phosphorus than cow caseins (Cui, Liu, Qu, Dong, Cao, & Ma, 2012; Wang, Liu, Wen, Zhang, Guo, & Ren, 2013; Zhang, Ma, Wu, Fei, Yang, & Wan, 2010). Furthermore, yak casein micelles have different composition, size, and hydration properties compared to cow casein micelles (Wang, et al., 2013; Yang, Zhang, Wen, Zhang, & Liang, 2014). The functional properties of proteins are strongly related to their physicochemical properties. To improve the physicochemical and functional properties of caseins, chemical modification methods (e.g., succinylation) are commonly used. The changes in casein functional properties with succinylation have been reported (Lakkis & Villota, 1992; Santos & Tomasula, 2000; Strange, Holsinger, & Kleyn, 1993; Yang, Shi, Wang, Liu, Wen, & Ren, 2014). According to several studies, succinylation

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improves the solubility, emulsification, and foaming characteristics of caseins. Additionally, casein micellar solvation increases and zeta potential decreases after succinylation (Vidal, Marchesseau, & Cuq, 2002). Lakkis and Villota (1992) reported that the surface hydrophobicity of caseins, bovine serum albumin, and whey protein isolates increases and the apparent thermal transition temperature of α-globulin decreases with increasing succinylation (Zaghloul & Prakash, 2002). In addition, succinylation decreases the zeta potential of β-lactoglobulin and increases the viscosity of Brazil nut kernel globulins (Caillard, Boutin, & Subirade, 2011; Ramos & Bora, 2005). Even though studies have focused on the effect of succinylation on the physicochemical properties of cow caseins and other proteins, there is little evidence on the effects of succinylation on yak caseins. In previous studies, we assessed the effects of succinylation on yak and cow caseins functional properties and concluded that differences in composition and size of yak casein micelles affect the succinylation process (Yang, et al., 2014). The objective of this study was to assess the effect of succinylation on the physicochemical properties of yak casein micelles. Seven succinylation levels were evaluated. A detailed analysis of the effects of succinylation on the surface hydrophobicity, thermal properties, buffering capacity, hydration, and color of yak casein micelles is presented. The findings obtained from this study will provide the basic information on the physicochemical properties of native and chemically-modified yak casein micelles. The succinylated yak caseins could be used as one of the high quality protein materials in chemical industry.

2. Materials and methods 2.1. Materials

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Yak milk used in this study was collected from Tianzhu grassland, on the Qinghai-Tibetan Plateau, in northwest China. After milking, 0.02% (w/v) sodium azide was added to inhibit bacterial growth. The samples were then put in sterile plastic bottles and stored in a box filled with ice. The samples were transported to the laboratory within 6 h. Yak milk was defatted twice by centrifugation (TDD5M, Changsha Pingfan Instrument Co. Ltd., Changsha, China) at 4,000 × g for 10 min at 20 °C. The skim milk was centrifuged using a Beckman Optima XL-100K refrigerating ultracentrifuge (Beckman Coulter, USA) at 120,000× g for 40 min at 20 °C (Françoise, Kablan, Kamenan, & Lagaude, 2009). The supernatant was removed, and the firm pellet at the bottom was the casein micelles. The pellet was freeze dried to constant weight using vacuum freeze-drying machine (GLZ-0.4, Su Yuan Zhong Tian scientific Inc., Beijing, China). Total nitrogen and casein nitrogen contents in the dried pellet were tested by the method of Li et al. (2011). Non casein nitrogen content was the difference between total nitrogen content and casein nitrogen content. It was shown that total nitrogen content in the dried pellet was 91.17±0.31% (w/w) and non casein nitrogen content was 0.71±0.03% (w/w).

2.2. Chemical modification of yak casein micelles Chemical modification of yak casein micelles followed the method of Yang, Cui, Fang, Shi, Yang and Wang (2015). Dried caseins were dissolved in distilled water at 20 mg/mL by constant mixing at 3,000 rpm (40°C). The pH of the solution was kept at 7.0 with 1 M NaOH. After caseins were completely dissolved, the pH of the solution was adjusted to 8.0-9.0 with 1 M NaOH, and the succinic anhydride was added under constant stirring for 50 min at 40°C. Succinic anhydride was added at

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different succinic anhydride/caseins ratios: 0.02:1, 0.04:1, 0.06:1, 0.08:1, 0.1:1, 0.2:1 and 0.6:1 (g/g). The pH of the solution was maintained at 8.0-9.0 with 1 M NaOH. After the reaction, the pH was adjusted to 7.0 with 1 M HCl. The solution was dialyzed against distilled water for 48 h at 4°C in a dialysis bag (Oso-T8280, MD25, 8000-14000, Union Carbide Corporation, Danbury, USA). Then part of solution was freeze dried to constant weight and considered as modified caseins, and the remainder was centrifuged in a Beckman Optima XL-100K ultracentrifuge (rotor 70Ti; Beckman Coulter, USA) at 120,000g for 40 min at 20°C. The pellet obtained by ultracentrifugation was weighed and then freeze-dried to constant weight. The hydration of casein micelles was expressed as g H2O per g dry pellet. Caseins hydration (g H2O / g caseins) =(wet pellet – dry pellet) / dry pellet (1)

2.3. Determination of the degree of modification The degree of modification was determined by the o-phthalaldehyde (OPA) method (Yang et al., 2014). In this experiment, 3 mL of caseins solution (0.2 mg/mL) was mixed with 3 mL of OPA, which was prepared according to the method reported by Dinnella et al.(2002). After 2 min, absorbance was measured at 340 nm in a 1-cm length quartz cell (UV-2100 spectrophotometer; Beijing Beifen-Ruili Analytical Instrument Co., Ltd., Beijing, China). The number of amino groups was calculated from an L-leucine standard curve. The percentage of amino group-modified caseins was calculated using the following formula (Dinnella, Gargaro, Rossano, & Monteleone, 2002), Modification degree (%)=(No−Nm)/No×100

(1)

where N0 and Nm are the number of free amino groups in the unmodified and modified caseins, respectively.

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2.4. Surface hydrophobicity Surface hydrophobicity was determined using ANS, according to the method of Kato and Nakai (1980) with slight modifications. In brief, 8 mL casein solutions of ten various concentrations (0.001-0.01 mg/mL) in distilled water (pH 7.0) were prepared. 200 µL of ANS (8.0mM) in distilled water was added to each casein concentration, and the mixtures were shaken in a vortex mixer for 5 s. Fluorescence intensity was measured at wavelengths of 390 nm (excitation) and 470 nm (emission) using a RF-5301PC luminescence spectrometer (Japan Shimadzu Company) at room temperature (25°C), with a constant excitation and emission slit of 5 nm. The fluorescence intensity for each sample with probe was recorded and the initial slope of the fluorescence intensity versus casein concentration plot was calculated by linear regression analysis and used as an index of hydrophobicity.

2.5. Differential scanning calorimetry (DSC) DSC experiments were performed on a Pyris Diamond TG/DTA thermal analyzer (Perkin Elmer), according to the procedure of Meng and Ma (2001) with some modifications. Approximately 10.0 mg of caseins samples were accurately weighed into aluminum pans, and 10µL 50 mM phosphate buffer (pH 7.0) was added. The pans were hermetically sealed and heated in the calorimeter from 25 to 200°C at a rate of 5°C/min. A sealed empty pan was used as a reference. Peak or denaturation temperature (Td) and enthalpy change of denaturation (∆H) were computed from the thermograms by the Universal Analyzer 2000 software, version 4.1 D (TA Instrument-Waters LLC, USA). All experiments were conducted in triplicate.

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2.6. Acid-Base Buffering Property The acid-base titrations were performed using the method described by Lucey (1993). Titrations were performed in triplicate on 25 mL solutions of 8 mg/mL for each casein sample at room temperature (25°C) on a 794 Basic Titrino Autotitrator (Metrohm, Herissau, Switzerland). The rate of titration was 0.2 mL/min. The samples were titrated from the initial pH of 7.0±0.1 to 2.5 with 0.1 M HCl. Buffering index values (dB/dpH) were calculated according to the formula of Raouche, Dobenesque, Bot, Lagaude and Marchesseau (2009) and plotted as a function of pH: dB/dpH = [(volume of acid) × (normality factor)] / [(volume of sample) × (∆pH)] (2)

2.7. Color Determination CIE color system (CIELAB) coordinates were measured to characterize the color of caseins protein products. The product was put into an empty and dry weighing bottle, in which the sample surface was flat and horizontal. Chroma Meter HP-200 (Henan xiongdi instrument co., LTD, China) was used to measure the CIELAB coordinates of each caseins protein product according to the method of Wu et al. (2011). Measurements were performed in triplicate. The three attributes of color in CIE color system were L*, a* and b*, where L* was the lightness variable proportional to value in the Munsell system, and a* and b* were chromatic coordinates designating positions on a red/green and yellow/blue axis, respectively (+a = red,-a = green; +b = yellow,-b = blue). The W value representing the whiteness of the protein samples was calculated as follows: W=100-[(100-L*) 2+ a*2+ b*2]1/2

(3)

2.8. Statistical Analysis

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All data were expressed as mean ± standard deviation (SD) from at least three independent trials. The differences were assessed by one-way analysis of variance (ANOVA) and Duncan’s multiple range tests. Statistical significance was set at p < 0.05. PASW Statistics 18.0 software (SPSS Inc., Chicago, IL, US) and Origin 8.0 (OriginLab Corporation, Northampton, MA, US) were used to analyze the data.

3. Results and Discussion 3.1. Degree of succinylation The degree of succinylation of yak caseins is shown in Table 1. The degree of succinylation increased with increasing succinic anhydride. This result was in agreement with those obtained in succinylated cow caseins, Brazil nut kernel globulin, lablab bean protein concentrate, soy protein, canola 12S globulin, and mung bean protein isolate (Achouri & Zhang, 2001; El-Adawy, 2000; Gruener & Ismond, 1997; Olayide S. Lawal, 2005; Ramos & Bora, 2005; Yang, et al., 2014). The succinylation degree of mung bean protein was approximately 80%, with a succinic anhydride/protein ratio of 0.6:1 (g/g) (El-Adawy 2000). The succinylation degree of soy protein hydrolysate was 18.3% and 29.6%, with a succinic anhydride/protein ratio of 10% and 20%, respectively (Achouri & Zhang, 2001). The succinylation degree of canola 12S globulin was 3% and 48% with a succinic anhydride/protein ratio of 0.02 and 0.1, respectively (Gruener and Ismond 1997). The succinylation degree of proteins mentioned above were all lower than that of yak caseins micelles with same succinic anhydride/protein ratio. The different modification degree of proteins at similar succinic acid/protein ratios could be attributed to differences in protein structure, especially the nature of the surrounding amino acid residues, and experimental conditions.

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3.2. Surface hydrophobicity Surface hydrophobicity indices of native and chemically-modified yak casein micelles are shown in Figure 1. Surface hydrophobicity decreased (p < 0.05) with increasing succinylation level. Even though succinylation induced substantial changes in the spatial structures of yak casein micelles (e.g., loose casein micelles structures, resulting in the exposure of more tryptophan residues to the polar environment), both steric hindrance and electrostatic repulsion prevented the negatively charged hydrophobic ANS probe from approaching and binding to the hydrophobic sites in the succinylated caseins (Knopfe, Schwenke, Mothes, Mikheeva, Grinberg, Görnitz, et al., 1998; Yang, et al., 2015). Paulson and Tung (1987) reported that increasing succinylation levels contributed to an increase in charge frequency and electronegativity and to a decrease in surface hydrophobicity of proteins. Therefore, casein micelles were more hydrophilic with increasing chemical modification. The surface hydrophobicity of succinylated faba bean legumin and canola 12S globulin showed the same changing trend as that of yak casein micelles (Gruener & Ismond, 1997; Knopfe, et al., 1998). However, contradictory results have been reported in succinylated bovine serum albumin, caseins, and whey protein isolates (Lakkis & Villota, 1992), possibly due to different conformational protein structures and exposure of hydrophobic residues.

3.3. Thermal properties The thermal properties of succinylated yak casein micelles were evaluated in this study. Denaturation temperature and enthalpy changes are summarized in Table 1. With increasing succinylation, the Td value of yak casein micelles decreased. It was

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reported that proteins with a high proportion of hydrophobic residues or with more compact structures have high Td values (Arntfield, Murray, & Ismond, 1986). Succinylation leads to the link of hydrophilic carboxy group on the surface of the casein micelles (Yang, et al., 2015). Furthermore, the association between the bulky, negatively charged succinyl groups and the lysine residues of caseins induced electrostatic repulsions and steric hindrance, resulting in the looser structure than native yak casein micelles. The enthalpy changes of proteins represent the proportion of non-denatured protein in a sample or content of ordered structure (Arntfield & Murray, 1981). Total enthalpy changes of yak casein micelles were markedly affected by succinylation (Table 1). With increasing succinylation up to 70%, ∆H values of yak caseins decreased, indicating casein denaturation and unfolding, which was in agreement with the Td results. At the highest succinylation level, ∆H increased sharply, with a value higher than that of native caseins. The structure of the sub-micelles may remain intact at 82% level of succinylation, and sub-micelles had more regular structures (Yang, et al., 2015). Furthermore, a large number of hydrogen bonds could be formed among sub-micelles or intra- and intermolecular of caseins with 82% succinylation, contributing to high ∆H values.

3.4. Buffering property The buffering curves obtained upon acid titration from pH 7.0±0.1 to 2.5 are shown in Figure 2. The buffering peak was at pH 4.5–6.0 and pH <3. The pH of the buffering peak and maximum buffering value of yak casein micelles with different succinylation degree were showed in Table 1. Succinylation induced a slight shift of the buffering peak towards a lower pH value and a markedly increase of the maximal

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buffering values of yak casein micelles. Organic and inorganic phosphates buffer between pH 4.5 and 5.5; calcium citrate buffers between pH 3 and 4 (Salaün, Mietton, & Gaucheron, 2005). The buffering peak observed at pH value between 4.5 and 6.0 was due to the high solubility of caseins; the solubilization of colloidal calcium phosphate contributes to the formation of phosphate ions, which combine with H+ (Lucey, Hauth, Gorry, & Fox, 1993). These results were in good agreement with previous studies (Françoise, Kablan, Kamenan, & Lagaude, 2009; Raouche, Dobenesque, Bot, Cuq, & Marchesseau, 2008; Raouche, Dobenesque, Bot, Lagaude, & Marchesseau, 2009); however, the maximum buffering values of yak caseins were lower than those previously reported probably due to lower yak casein concentrations and mineral content differences among bovine breeds. With succinylation, the negative charges of caseins increased, resulting in the dissociation of colloidal calcium phosphate from micelles and the increase in casein micellar solubility and buffering capacity (Yang, et al., 2014). The number of amino groups decreased, while the carboxylic groups increased with succinylation, contributing to a slight shift of the buffering peak towards a lower pH value. Other buffering peaks of yak casein micelles with and without modification were observed at pH <3, and the maximum buffering values were observed pH 2.5, which were

0.019±0.001,

0.022±0.001,

0.030±0.002,

0.040±0.001,

0.042±0.001,

0,049±0.001, 0.056±0.002, and 0.063±0.001 with 0%, 9%, 19%, 33%, 44%, 59%, 70%, and 82% succinylation, respectively. Previous studies have reported that casein micelles have high buffering capacity at pH <3, which was in agreement with our results (Françoise, Kablan, Kamenan, & Lagaude, 2009; Raouche, Dobenesque, Bot, Cuq, & Marchesseau, 2008; Raouche, Dobenesque, Bot, Lagaude, & Marchesseau, 2009). With succinylation, the maximum buffering values at pH 2.5 increased due to

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the carboxylic groups on casein micelles. The pKa of amino acid carboxylic groups ranges between 2 and 3. Therefore, casein micelles had high buffering capacity at pH <3, and their values increased significantly with succinylation.

3.5. Hydration of succinylated yak caseins Succinylation contributed to an increase in yak casein hydration (Figure 3). The hydration of yak casein micelles was 2.08±0.11 g H2O per g casein. The hydration of 82% succinylated yak casein micelles had a 96% increase relative to the control (unmodified) yak casein micelles. Wang, et al. (2013) reported that the hydration of yak casein micelles was 2.16±0.19 g H2O per g casein, which was similar to our results. Water-protein interactions are mainly electrostatic interactions (Kinsella, Fox, & Rockland, 1986). The increase in hydration of modified micelles could originate from an increase in net negative charges and carboxylic groups. In addition, casein micelles dissociate after succinylation, exposing water-binding sites, which contribute to an increase in hydration. Vidal, Marchesseau, and Cuq (2002) reported that the hydration of cow casein micelles (2.81±0.01 g H2O per g protein) increased with succinylation.

3.6. Color of succinylated yak caseins The lightness of yak caseins increased at 9% succinylation and subsequently reached its lowest value at 59% succinylation (Table 2). However, the lightness of yak caseins was not significantly different at 0–33% and 19–70% succinylated levels. Red and yellow hues gradually decreased with increasing succinylation levels. Succinylated casein micelles had higher whiteness values than unmodified casein micelles. The whiteness values of yak casein micelles were not significantly different

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between 9% and 59% succinylation. Color changes in caseins were related to casein micelle dissociation, casein structure, micellar size, and density (Ahmad, Piot, Rousseau, Grongnet, & Gaucheron, 2009; Philippe, Legraet, & Gaucheron, 2005). Even though the color of yak casein micelles was not consistent with increasing succinylation, we observed that the whiteness of yak casein micelles increased with chemical modification, which is of importance in the food industry.

4. Conclusions Succinylation induced associations between the bulky, negatively charged succinyl groups and the casein lysine residues, thereby enhancing electrostatic repulsions and steric hindrance and decreasing surface hydrophobicity indices. Furthermore, the loose structure and denaturation of yak caseins originating from succinylation, also decreased denaturation temperatures. The denaturation enthalpy of yak casein micelles decreased with succinylation levels, except at 82%. The decrease of the denaturation enthalpy was attributed to the dissociation, denaturation, and conformational changes of casein micelles. Succinylation resulted in a slight shift of the buffering peak towards a lower pH value and marked increase in the maximal buffering values of yak caseins at pH 4.5–6.0 and pH <3; these changes were largely due to an increase in solubility and carboxylic group content of yak caseins. The interaction between yak caseins and water was strengthened with succinylation, resulting in an increase in hydration. The changes in yak casein micellar color with succinylation were not consistent; however, whiteness index of yak caseins increased with succinylation, making modified yak caseins more suitable for industrial applications. Acknowledgment

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This study was supported by the Postdoctoral Science Foundation of China (No. 2015M572611).

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Fig.1 Hydrophobicity indices of native and modified yak casein micelles with different succinylated degree. Note: CM is casein micelles. SCM is succinylated casein micelles with the degree of modification showed by the following numbers.

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Fig.2 Buffering curves at 25°C of native and modified yak casein micelles with different succinylated degree. Note: CM is casein micelles. SCM is succinylated casein micelles with the degree of modification showed by the following numbers.

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Fig.3 Hydration of native and modified yak casein micelles with different succinylated degree. Samples of the same structure followed by the different letter are significantly different (p < 0.05). Note: CM is casein micelles. SCM is succinylated casein micelles with the degree of modification showed by the following numbers.

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Table 1 Degree of succinylation, temperature of denaturation, denaturation enthalpy, pH of buffering peak and the maximum buffering value of yak casein micelles with different succinylated degreea Ratios of Succinic anhydride/casein (g/g) 0.00

a

Degree of succinylation (%) 0.00h

∆H（J/g）

Td（°C）

pH of

Maximum

buffering

buffering

peak

value

89.20±6.80a

111.97±4.37b

5.73±0.07a

0.008±0.001g

0.02

9.00±0.02g

86.44±4.63ab

91.86±10.38c

5.52±0.10ab

0.020±0.001f

0.04

19.38±0.02f

84.42±6.68ab

90.45±7.85c

5.35±0.18bc

0.026±0.002e

0.06

32.96±0.03e

83.52±2.70ab

92.55±7.40c

5.25±0.11bcd

0.029±0.002d

0.08

44.41±0.02d

82.93±8.66ab

93.06±3.87c

5.16±0.25cd

0.031±0.001dc

0.10

59.06±0.01c

78.48±3.94ab

81.02±3.98cd

5.04±0.17d

0.033±0.001cb

0.20

70.21±0.01b

77.22±5.97b

71.79±4.19d

5.03±0.16d

0.035±0.001b

0.60

82.26±0.01a

61.78±3.37c

126.90±9.94a

5.00±0.18d

0.042±0.002a

Values are based on triplicate measurements. Mean ± SD values are shown. Different

superscript lowercase letters in the same column indicate significant differences (p< 0.05).

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Table 2 Color measurement of native and modified yak casein micelles with different succinylated degreea Samples

L*

a*

b*

W

CM

89.88±0.49bc

2.84±0.17a

10.52±0.48a

85.12±0.19d

SCM09

90.36±0.31ab

2.78±0.18a

9.50±0.21b

86.19±0.38c

SCM19

90.03±0.25abc

2.77±0.10a

9.30±0.32b

86.08±0.17c

SCM33

89.55±0.46bc

2.72±0.31ab

9.13±0.16b

85.86±0.44c

SCM44

89.48±0.48c

2.61±0.20ab

9.02±0.11b

85.89±0.34c

SCM59

89.43±0.16c

2.58±0.39ab

8.49±0.26c

86.02±0.25c

SCM70

90.09±0.75abc

2.30±0.29bc

7.87±0.17d

87.13±0.62b

SCM82

a

90.73±0.27a 2.10±0.20c 7.48±0.34d 87.90±0.22a Values are based on triplicate measurements. Mean ± SD values are shown. Different

superscript lowercase letters in the same column indicate significant differences (p< 0.05). CM is casein micelles. SCM is succinylated casein micelles with the degree of modification showed by the following numbers.

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Highlights 1. The surface hydrophobicity of yak caseins decreased with succinylation. 2. The Td and ∆H of yak caseins decreased with succinylation, except for 82% degree. 3. The buffering peak of yak caseins shifted towards lower pH with succinylation. 4. The buffering capacity of yak caseins increased with succinylation. 5. The hydration and whiteness values of yak caseins increased with succinylation.

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