International Dairy Journal 20 (2010) 393399

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International Dairy Journal

journal homepage: www.elsevier.com/locate/idairyj

Effect of deamidation by protein-glutaminase on physicochemical and functional properties of skim milk

N. Miwa a, *, K. Yokoyama b, H. Wakabayashi a, N. Nio b
a b

Food Product Application Center, Ajinomoto Co., Inc., Kawasaki, 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan Institute of Life Science, Ajinomoto Co., Inc., Kawasaki, 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan

a r t i c l e i n f o
Article history: Received 7 July 2009 Received in revised form 18 December 2009 Accepted 19 December 2009

a b s t r a c t
Protein-glutaminase (PG) is an enzyme that catalyzes the deamidation of glutamine residues in proteins, and protein deamidation is a promising method of improving solubility and other functional properties of foods. We investigated the effect of PG on skim milk proteins, nding that their deamidation degree increased dose-dependently. The solubility and relative viscosity of skim milk solution were remarkably improved as the deamidation degree increased, while its turbidity decreased until the milk became translucent; emulsifying capacity also increased, and the mean droplet diameter decreased to <4 mm. Particle size distribution analysis and transmission electron microscopy showed that smaller submicelle particles were produced in highly deamidated skim milk. These observations suggest that PG deamidation induces the dissociation of casein micelle, resulting in an increase of the oil/water surface area. This enzymatic deamidation helped to improve the functionality of skim milk by increasing the electrostatic repulsion of carboxyl groups in caseins. 2010 Elsevier Ltd. All rights reserved.

1. Introduction Deamidation has been investigated as an effective tool for protein modication. Protein deamidation is expected to improve various functionalities of food proteins and to expand their use in the food industry (Hamada, 1994; Sikorski, 2001). The conversion of protein amide groups to carboxyl groups by deamidation leads to a reduced isoelectric point (pI) due to an increase in the number of negatively charged carboxyl groups. This makes it possible for proteins to be more soluble at weak acidic pH levels. Furthermore, protein deamidation causes the exposure of hydrophobic regions through a change in protein structure and therefore improves their emulsifying properties (Hamada & Marshall, 1989). An enzymatic approach to protein deamidation has several advantages over a chemical approach because of its high specicity, mild reaction conditions and food safety. Enzymatic methods using transglutaminase (TG) (Motoki, Seguro, Nio, & Takinami, 1986; Nonaka, Sawa, Matsuura, Motoki, & Nio, 1996; Ohtsuka, Umezawa, Nio, & Kubota, 2006), peptide-glutaminase (Hamada & Marshall, 1989, 1992), a combination of pH and protease (Matsudomi, Tanaka, Kato, & Kobayashi, 1986) and protein-glutaminase (PG) are known to be useful for the deamidation of food proteins.

* Corresponding author. Tel.: 81 44 223 4174; fax: 81 44 246 6081. E-mail address: noriko_miwa@ajinomoto.com (N. Miwa). 0958-6946/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2009.12.015

Of these, the PG method appears to be the most attractive for practical use because it does not cause any side reactions, such as cross-linking by TG or peptide hydrolysis by protease. PG, a new enzyme that catalyzes the deamidation of protein (as shown in Fig. 1), was discovered in the culture supernatant from Chryseobacterium proteolyticum (Yamaguchi & Yokoe, 2000; Yamaguchi, Jeenes, & Archer, 2001), and the action of PG has been investigated for several food proteins (Gu, Matsumura, Yamaguchi, & Mori, 2001; Yong, Yamaguchi, Gu, Mori, & Matsumura, 2004; Yong, Yamaguchi, & Matsumura, 2006). The major milk proteins, caseins and whey proteins carry out many important functions in the manufacture of dairy products. In milk, caseins, which account for about 80% of the total protein content, exist primarily in the form of association colloids called casein micelles that range in size from 50 to 300 nm (Fox, 2003). With respect to the enzymatic deamidation of milk proteins, Motoki et al. (1986) found that the deamidation of alpha s1-casein by TG improves solubility at weak acidic pH levels, and more than 2-fold even in the presence of CaCl2. Additionally, Hamada and Marshall (1992) reported that heat and proteolysis in casein deamidation using peptide-glutaminase and partial protein hydrolysis prior to enzymatic treatment increase the deamidation of casein amide residues. Previous research on the basic characteristics of PG has demonstrated that alpha- and beta-casein are excellent substrates of PG (Yamaguchi et al., 2001), and Gu et al. (2001) reported that the molten globule state of alpha-lactalbumin, a milk

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PG

Protein

+ H2O

Protein Glu C OO

+ NH4+

enzyme, and then cooled in ice. The control sample was similarly treated without PG. 2.4. Deamidation degree Glutamine (Gln) residues and glutamic acid (Glu) residues of the proteins in skim milk were measured using an Amino Acid Analyzer L-8500 (Hitachi, Tokyo, Japan) after enzymatic protein hydrolysis by the method described by Grifn, Wilson, and Lorand (1982). Twenty mg of freeze-dried skim milk sample was dissolved in 3 mL of 0.1 M borate buffer (pH 8.0). Complete protein hydrolysis was achieved by pronase (Roche Diagnostics GmbH, Mannheim, Germany), leucine amino-peptidase (Sigma L-5658; Sigma Chemical Co., St. Louis, MO, USA), prolidase (Sigma P-6675) and carboxypeptidase A (Sigma C-9268). The reaction mixture after hydrolysis was ultraltrated using Ultrafree molecular mass 5000 Da cut-off membranes (Millipore, Bedford, MA, USA). The ltrate was subjected to amino acid analysis. The difference in the number of Gln residues before and after PG reaction was calculated. The deamidation degree was expressed as the ratio of deamidated residues to total glutamine residues in skim milk proteins. 2.5. Measurement of turbidity and viscosity PG-treated skim milk samples were stored at 25  C for 60 min, at which point turbidity was measured at 600 nm using a spectrophotometer (Shimadzu Corporation, Kyoto, Japan) and 10-mm quartz cell. Samples were diluted with simulated milk ultraltrate (<5000 Da). Measurements were performed in triplicate. The viscosity of the skim milk samples was measured by sine-wave vibro viscometer (SV-10; A&D Company, Ltd., Tokyo, Japan) with a waterjacket assembly (AX-SV-37, A&D Company, Ltd., Tokyo, Japan). This viscometer measures viscosity by detecting the drive current necessary to resonate the two sensor plates at constant low frequency of 30 Hz. All measurements were carried out in duplicate at 25  C. 2.6. Analysis of milk serum fraction by ultracentrifugation Soluble casein and whey proteins were dened as those that did not sediment from the milk during ultracentrifugation at 100,000 g for 60 min at 25  C in a Hitachi P40ST-1572 ultracentrifuge and the associated 70P-72 rotor (Hitachi Koki Co., Ltd, Tokyo, Japan). Weighed aliquots of skim milk samples (10 mL) were transferred to centrifuge tubes and ultracentrifuged. The milk serum was carefully removed from the pellets for further analysis pez-Fandin o, De la Fuente, Romas, & Olano, 1998). The ultra(Lo centrifuge experiments were conducted in duplicate for each milk sample. The total nitrogen (TN) of the milk samples and the supernatants obtained by centrifugation were determined by the Kjeldahl (1883) method. Soluble nitrogen, i.e., the TN of the supernatants, was expressed as a percentage of the TN of the milk sample. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out following the method described by Laemmli (1970) with 520% polyacrylamide gradient gel. The protein bands were xed and stained using a solution of Coomassie Brilliant Blue R-250. The total calcium of the skim milk samples and the casein-bound calcium in milk serum were measured by o-cresolphthalein complexion (OCPC) method using the Calcium-C-test (Wako Pure Chemical, Osaka, Japan). To determine the concentration of nonprotein-bound calcium in milk serum, the ultracentrifugal supernatant was ultraltrated by Ultrafree-LC centrifugal lter units with molecular mass cut-off at 5000 Da (Millipore, Bedford, MA, USA). The

Gln C NH2 O

Fig. 1. Reaction scheme of protein-glutaminase (PG).

whey proteins, affects the reactivity of PG. Nevertheless, few studies have been carried out on the reaction of PG to whole proteins in liquid milk. PG is expected to contribute to improving the quality of various dairy products such as yoghurt, cheese, acid milk drinks, etc. Therefore, it is important to examine the inuence of deamidation by PG on milk proteins, including caseins, which have a micellar structure. The purpose of the present study was to investigate the effect of PG deamidation on the physicochemical properties of milk proteins, focusing particularly on structural changes in casein micelles in the reconstituted skim milk often used in dairy products. 2. Materials and methods 2.1. Enzyme preparation PG (EC.3.5.1.44) derived from C. proteolyticum was produced by Corynebacterium glutamicum, according to the method previously described by Kikuchi, Itaya, Date, Matsui, and Wu (2008). The specic activity of puried PG is approximately 120 units per 1 mg protein. The method of PG assay using Z-Gln-Gly (Peptide Laboratory, Osaka, Japan) as a substrate (Yamaguchi and Yokoe, 2000) was modied as follows: 10 mL of enzyme solution was added to 100 mL of 176 mM phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly. After incubating at 37  C for 10 min, 100 mL of 12% (v/v) trichloroacetic acid (TCA) solution was added to stop the reaction. Enzyme concentration was diluted to 0.05 mg mL1 with 20 mM phosphate buffer (pH 6.0). After centrifugation for 5 min at 10,600 g at 4  C, ammonia released from deamidated glutamine residues in the supernatant was measured by following NADH-glutaminate dehydrogenase activity using an F-kit (Roche Diagnostics GmbH, Mannheim, Germany). Protein concentration was measured by the Bradford assay using Coomassie brilliant blue 5 solution (Nakarai Tesque, Kyoto, Japan) and bovine serum albumin (Pierce, Rockford, IL, USA) as a standard. One unit of enzyme was dened as the amount that released 1 mM of ammonia per min under the above conditions. 2.2. Skim milk preparation Low-heat skim milk powder (protein content 35%, w/w, as dened by the manufacturer) was obtained from Yotsuba Milk Products Co., Ltd. (Hokkaido, Japan). Experimental skim milk samples were prepared by reconstituting the skim milk powder to 10% (w/v) total solids in distilled water. The samples were allowed to equilibrate overnight at 5  C with gentle stirring. The skim milk was stirred for 2 h at 22  C before the experiments. 2.3. PG treatment of skim milk samples Skim milk samples preheated to 50  C for 5 min were incubated with 0.39, 0.78, 1.8, 3.9 and 18 units of PG per 1 g milk protein (the unit is abbreviated as u gp1) for 90 min at 50  C. After incubation, all samples were heated in boiling water for 10 min to inactivate the

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Released NH 3 (mmol L-1)

80 16 12 8 4 0 0 5
10 15 20

70 60 50 40 30 20 10 0

2.7. Size distribution measurements The particle size distribution of untreated and PG-treated skim milk samples was measured using a dynamic light-scattering particle size distribution analyzer (Zetasizer Nano; Malvern Instruments Ltd, Worcs., England). All samples were diluted 3-fold with the simulated milk ultraltrate. All measurements were performed at 22  C in triplicate. For transmission electron microscopy (TEM) materials, untreated skim milk and PG (18 u gp1)-treated skim milk were prepared. One mL of each sample was mixed with the same quantity of 2.45% (v/v) solution of glutaraldehyde in 0.05 M cacodylate buffer for 2 h at room temperature to x. Fixed milk samples were then diluted 1:1000 with distilled water and placed on a carbon support lm to dry. After the platinumcarbon (PtC) replica was shadowed by electron beam evaporation, microstructure, particle size and particle distribution studies were performed by TEM (JEM-1011; JEOL, Tokyo, Japan). All sample preparation was performed at room temperature. 2.8. Emulsifying properties Emulsifying capacity was determined by the turbidimetric method described by Pearce and Kinsella (1978). To prepare emulsion, the lyophilized skim milk powders were reconstituted to 1% (w/v) total solids in distilled water and the dispersed sample (3 mL) and soybean oil (1 mL) were then sonicated with Handy Sonic (Tomy Seiko Co., Ltd, Tokyo, Japan) at output 9 for 30 s at room temperature. Emulsifying capacity was represented as absorbance at 500 nm of the emulsions diluted 1:500 using 0.1% SDS. The particle size distribution of the resultant emulsions was measured using a laser diffraction particle size analyzer (LA500; Horiba, Ltd, Kyoto, Japan). Experiments were performed in triplicate on three independent occasions. 3. Results 3.1. Deamidation of skim milk by PG Fig. 2 shows the amount of ammonia released from 10% (w/v) skim milk incubated with various amounts of PG, and deamidation degrees. The ammonia released from skim milk increased with increasing doses of PG. At doses of PG up to 3.9 u gp1, the deamidation degree increased to approximately 60% in proportion to the enzyme dosage. From 3.9 u gp1 to 18 u gp1, the deamidation degree increased gradually, reaching about 80%. The amount of ammonia in the milk solution then reached approximately 18 mM L1. These results showed that skim milk proteins are welldeamidated by PG and that 0.5 mM of ammonia per 1 g milk protein was released when the skim milk was incubated with enough enzyme to saturate the reaction. 3.2. Turbidity of skim milk by PG

PG (u gp -1)
Fig. 2. Inuence of PG on the release of ammonia and the deamidation degree in skim milk samples. Control sample without PG was also incubated at 50  C for 90 min and subsequently heated in boiling water. The error bars represent standard deviations between duplicate measurements.

degree. This decrease in turbidity suggests a disruption of casein micelles which are the main light-scattering particles in skim milk. 3.3. Viscosity of skim milk by PG Casein micelles are the primary contributors to the viscosity of skim milk (Walstra & Jenness, 1984). To investigate the effect of PG on the physical properties of skim milk, the viscosity was measured using the sine-wave vibro viscometer at 25  C. Relative viscosity increased corresponding to the deamidation degree (Fig. 3b). The viscosity of a highly deamidated skim milk sample was 2.9 mPa, which is approximately 1.7 times higher than that of the control sample. 3.4. Solubility of skim milk proteins To study the effect of deamidation by PG on casein micelle form, we determined the level of nonsedimentable nitrogen in casein micelle-free milk serum obtained by the ultracentrifugation of skim milk samples. An increase in the soluble nitrogen in supernatants was observed in proportion to the deamidation degree. When the deamidation degree was more than approximately 60%, the amount of soluble nitrogen reached almost 100% (Fig. 3c). This is due to the solubilization of casein micelles by deamidation of PG, which can be conrmed by SDS-PAGE (Fig. 4). It also showed that no observable change of molecular weight occurred in either case, though the bands corresponding to alpha- and beta- casein were observed to be more separate with the increase in the deamidation degree, and the position of the alpha-lactalbumin and beta-lactoglobulin bands was also slightly shifted to the upper side when the deamidation degree was greater than 75%. This observation indicates that not only caseins but also main whey proteins, alphalactalbumin and beta-lactoglobulin are deamidated by high dosage of PG. Further, the slight change in the SDS-PAGE pattern by PG is attributable to the inhibition of SDS binding to proteins because of the increase in the negative charge by deamidation. 3.5. Calcium

Deamidation of skim milk by PG causes a change in appearance: as the PG reaction proceeds, the milk becomes translucent. Fig. 3(a) shows that the turbidity at 600 nm of skim milk treated with PG decreases with the increase in the deamidation degree. Turbidity decreased signicantly in proportion to the deamidation degree up to 34%, above which it decreased gradually up to nearly 0. The visible change had occurred remarkably at 60% or more of the deamidation

Calcium exists in three forms in milk: free calcium (calcium ion), calcium complexed with inorganic anions such as phosphate and citrate, and calcium bound to casein (Neville & Watters, 1983). The sum of the rst two is characterized as soluble calcium because casein-associated calcium can be precipitated by ultracentrifugation. In the present study, to investigate the effect of deamidation

Deamidation degree (%)

ltrate was used for calcium analysis. All samples were acidied with 1 M HCl to solubilize all types of calcium, and ltrated before analysis. All measurements were carried out in duplicate or triplicate in order to ensure their accuracy.

20

90

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15 Turbidity (600nm)

10

0 0 20 40 60 80 100

Deamidation degree (%)

3.5 Viscosity (mPa s) 3.0 2.5 2.0 1.5 1.0 0

b
Fig. 4. SDS-PAGE prole of proteins in skim milk incubated with PG (Lanes 27) and supernatants of skim milk samples obtained by ultracentrifugation (Lanes 813). Lane 1: low molecular mass marker (94, 67, 43, 30, 20 and 14 kDa); lanes 37: skim milk samples with PG (deamidation degree 0%, 9%, 15%, 34%, 59% and 75%); lanes 813: supernatant from skim milk samples treated with PG (deamidation degree 0%, 9%, 15%, 34%, 59% and 75%).

20

40

60

80

100

samples, casein-bound calcium in serum reached more than about 1000 mg L1, corresponding to about w80% of the total calcium. This observation is consistent with the increase in soluble casein in the supernatants of skim milk samples treated with PG. 3.6. Casein micelle size

Deamidation degree (%)

120 Total nitrogen (%) 100 80 60 40 20 0 0 20 40 60 80 100

To understand better the effect of deamidation by PG on casein micelle structure in skim milk, casein micelle size distributions were examined. Fig. 6 shows the patterns of casein micelle size distribution in skim milk samples with the different deamidation degree, 0%, 15%, 34%, 59% and 75%. The data represent the mean of triplicate samples and the graph is a representative example. In the untreated skim milk sample (with the deamidation degree 0%), the particle size distribution has a single peak with a mean size of approximately 164 nm. The size distribution patterns were little affected by the deamidation degree up to 34%; however, a signicant peak shift to

Deamidation degree (%)

Fig. 3. (a) Turbidity at 600 nm of skim milk samples, (b) Relative viscosity of skim milk samples, (c) Soluble nitrogen in supernatants obtained from ultracentrifuged skim milk samples (100,000 g for 60 min at 25  C). All samples were incubated with various amounts of PG at 50  C for 90 min. Control sample was treated in the same way. The error bars represent standard deviations between duplicate measurements.

1500

1250

Ca concentration (mg L )

-1

1000

by PG on the distribution of calcium in skim milk, the colloidal (casein-bound) and soluble calcium concentrations were evaluated. It was found that the total calcium concentration in any skim milk sample was 1238 83 mg L1 (31 mM), and this value agrees well with the extensive published literature (Udabage, McKinnon, & Augustin, 2000). On the other hand, the calcium concentration in the ultracentrifugal supernatants, serum calcium increased as the deamidation degree increased (Fig. 5). But the calcium in their ultraltrates (<5000 Da), which contain free and colloidal calcium, was largely unaffected by PG treatment; it remained approximately 230 mg L1. Therefore, casein-bound calcium which is calculated as the difference between total serum calcium and calcium in serum ultraltrates (<5000 Da) increased with the increase in the deamidation degree. The point is that in highly deamidated skim milk

750

500

250

15

34

59

75

Deamidation degree (%)

Fig. 5. Calcium concentration in milk serum obtained by ultracentrifugation (-), and ultraltrate from milk serum (<5000 Da) ( ). Mean values represent triplicate analysis and error bars represent standard deviation between duplicate experiments.

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14 12

control sample was 150200 nm, while the particles in the skim milk sample highly deamidated by PG were w40 nm. 3.7. Emulsication properties The functional properties of proteins are highly related to their molecular structure (Dickinson & McClements, 1995). There has been considerable interest in the effect of enzymatic deamidation on the functionalities of food protein. We focused here on emulsifying properties. As shown in Fig. 8, emulsifying capacity, which is expressed as the absorbance of diluted emulsion at 500 nm, increased as the deamidation degree increased. The analysis of emulsion particle size distribution showed that the mean particle size decreased to less than 4 mm depending on the deamidation degree (Fig. 8). These results mean that deamidation by PG improves the emulsion capacity of skim milk solution. 4. Discussion Caseins in milk exist as a suspension of colloidal particles called casein micelles, which are held together by calcium ions and hydrophobic interactions. In this work, we investigated the effect of deamidation by PG on physicochemical and functional properties of skim milk and further considered the inuence of enzymatic deamidation on casein micelle structure. The important ndings in this study are: (1) the deamidation of skim milk proteins by PG, (2) the change of casein micelle structure caused by PG and (3) the improvement of emulsifying properties of skim milk by PG. Therefore, we discuss these points in this order below. First, we revealed that PG catalyzes Gln deamidation of proteins in skim milk. Casein accounts for approximately 80% of the protein in milk and approximately 20% of that in whey. Previous reports have shown that casein in reagent grade is highly susceptible to PG

Volume (%)

10 8 6 4 2 0

*
1 10 100 1000 10000

Size (nm)
Fig. 6. Size distribution of casein micelles in skim milk treated with PG. Deamidation rate 0% (), 15% (6), 34% ( ), 59% ( ) and 75% (>).

the left was observed in skim milk samples with the higher deamidation degree. There were two distinct peaks in the size distribution prole of the sample treated with the deamidation degree 59%. The pattern of the sample of the deamidation degree 75% showed that the peak of the larger casein micelles (w100 nm) almost disappeared while smaller micelles with a mean size of approximately 13.5 nm increased markedly. To investigate the effect of PG on casein micelle size, especially in skim milk sample with the high deamidation degree (75%), TEM analysis was conducted. Fig. 7a,b shows the typical size distribution of casein micelles in the control (untreated) skim milk sample. On the other hand, the image of PG-reated sample reveals the complete disintegration of casein micelles into smaller particles, probably its constituent sub-micelles (Fig. 7c,d). These results indicate that PG induces the disruption of casein micelles into smaller, submicelle particles; the typical micelle diameter in the

Fig. 7. Transmission electron micrographs showing casein micelles in a control skim milk sample (a, b) and in a skim milk sample of deamidation degree 75% (c, d). (a) & (c): scale bar 1 m, (b) & (d): scale bar 0.2 m.

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1.6 1.4

9 8 7 6

1.2 1 0.8

5 0.6 0.4 0.2 0 4 3 2

15

59

75

Deamidation degree (%)

Fig. 8. Emulsifying capacity of proteins in skim milk samples treated with or without PG. Absorbance at 500 nm (histograms) of emulsions of skim milk samples, of deamidation degree 0, 15, 59 and 75% and median diameter (C) of each emulsion, measured immediately after homogenization. Mean values represent triplicate analysis and error bars represent standard deviation between three independent experiments.

and that alpha-lactalbumin, one of the major components of milk whey proteins, is less susceptible to PG because its native state has a compact globular structure (Gu et al., 2001; Yamaguchi et al., 2001). Therefore it is natural to assume that the majority of reaction products of PG in skim milk are deamidated caseins. However, whey proteins including beta-lactoglobulin and alpha-lactalbumin should be taken into consideration according to the result of SDSPAGE. Further investigation is needed to understand how much each protein in milk is deamidated by PG and its relationship with the functionality of skim milk. Secondly, we found that the deamidation of skim milk by PG gave rise to many changes in skim milk; the decrease in its turbidity depending on the dose of enzyme, the increase in its viscosity, solubility of both casein and calcium in skim milk. These observations suggests that deamidation by PG causes the disruption of casein micelle because of electrostatic repulsions, the destruction of salt bridges, eventually leading to the loose structure of the casein micelle. The mechanism of casein micelle disruption by deamidation is estimated as follows; kappa-casein situated at the surface of casein micelle, is rst deamidated by PG. The introduction of negative charge to kappa-casein by deamidation may damage the integrity of casein micelle, causing the exposure of inside casein molecules such as alpha-casein, beta-casein to the micelle surface, which increases the reactivity of them toward PG. Therefore, the reaction is limited to kappa-casein at low deamidation degree, while most of caseins (alpha-, beta-, kappa-) are fully deamidated as PG reaction proceeds well. Many methods for the disruption of casein micelles have been studied extensively (Bienvenue, Jimenez-Flores, & Singh, 2003; Desobry, Richard, & Hardy, 1994; Huppertz & De Kruif, 2006; Huppertz, Fox, & Kelly, 2004; Liu & Guo, 2008; Vaia, Smiddy, Kelly, & Huppertz, 2006; Ward, Goddard, Augustin, & McKinnon, 1997). For example, Vaia et al. (2006) has shown that increasing the milk pH reduced the turbidity of milk because of the disruption of casein micelle. They suggested that alkaline disruption of casein micelle alkaline pH was due to increases in the net-negative charge on the caseins. Although the inuence of PG treatment on the pH of skim milk solution was also studied, no signicant change of pH at room temperature was detected (Data not shown). The present results show that not increasing the milk pH but just deamidation of

glutamine residues in skim milk proteins caused disruption of casein micelles due to increases in the net-negative charge on the caseins. We also found that the deamidation of skim milk by PG affected the distribution of calcium in skim milk; that is there was the increase in calcium bound to serum casein depending on the deamidation degree. Calcium phosphate in casein micelles, which are associated with casein, is called MCP or colloidal calcium phosphate. MCP plays a crucial role in maintaining micellar integrity (Holt, Davies, & Law, 1986). Horne (1998) states that MCP acts not only as a cross-link but also as a neutralizing agent that binds to negatively charged phosphoserine clusters to reduce the protein charge to the level. In this state, interactions between the hydrophobic regions of the caseins can be allowed to dominate. From the above ndings, it is suggested that the increase in carboxylic acid residues in casein micelles by PG-induced deamidation may weaken hydrophobic interactions to dominate in caseins, resulting in the dissociation of casein micelles into smaller sub-micelles. The above results were conrmed by examinations of the casein micelle size of skim milk samples by particle distribution analysis and TEM, which revealed the formation of sub-micelles which had an average size of approximately w40 nm by PG deamidation. Now, electron microscopy has been used to study the shape and structures of the micelles directly (McMahon & McManus, 1998; Schmidt & Buchheim, 1970). Electron micrographs have supported the existence of submicelle, that is, they show small, roughly spherical subunits and bumpy surface of the micelles. In this paper, our results of TEM show the presence of globular casein micelles in control skim milk and their appearance seems very similar to the model reviewed by Walstra (1990). Furthermore, the pictures also revealed that presence of smaller particles (sub-micelles) in highly deamidated skim milk, which indicates the dissociation of casein micelle induced by PG. Casein micelles are the primary contributors to the viscosity of skim milk (Walstra & Jenness, 1984). Several factors that alter the aggregation state of casein micelles, such as pH, salt balance and heat treatment, affect the viscosity of skim milk (Bienvenue et al., 2003). The present results show that deamidation by PG induces an increase in the relative viscosity of skim milk. This change occurs because of the dissociation of casein micelles, which causes the micelles to interact more frequently with each other. In skim milk, viscosity is related to the total protein volume fraction according to Eilers relationship (Desobry et al., 1994). It is assumed that the formation of disordered sub-micelles by PG-induced deamidation brings about the change in the voluminosity of micelles and alters interactions between them. Anyway from these results, it can be deduced that the enzymatic deamidation by PG has a great inuence on the casein micelle structure; as a consequence of this, the functionality of skim milk is considered to be altered. Thirdly, we discuss the emulsifying properties. Caseins are excellent emulsiers due to their exible and amphiphilic structure, which results in fast adsorption at the interface and fast lowering of interfacial tension (Dickinson, 1989). The emulsifying properties of bovine micellar casein have been reported by Haque and Kinsella (1988), who found that emulsifying capacity increases with increasing pH in milk. This has been attributed to an increase in the electrokinetic potential of casein micelles with increasing pH resulting in micelle dissociation (McMahon & Brown, 1984). In the present work, we found that the deamidation of skim milk by PG improved emulsifying capacity as the deamidation degree increased, which we attribute to the increasing of interfacial surface area caused by the extreme dissociation of casein micelles. New Glu residues introduced by PG enhance the solubilization of casein micelles, by which the original exible and amphiphilic

Mean median diameter (m)

Absorbance at 500nm

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structure of caseins achieves a better balance between the electrostatic repulsive force and steric hindrance. Furthermore, the image of the absorption layer on the droplet surface may be deduced as follows; when skim milk is not treated by PG, the intact casein micelle is attached onto the oil droplet surface without separating to sub-micelles or protein components. But when the casein micelle is well-deamidated, sub-micelles or divided casein molecules can be adsorbed to the oil droplet surface. 5. Conclusion The deamidation of skim milk by PG remarkably increased the solubility, emulsifying capacity with the deamidation degree. In the highly deamidated skim milk, the dissociation of casein micelles was promoted, resulting in an increase in the o/w surface area. The advantages of PG over other methods previously reported are edible, its high specicity, easy to control, less complicated in practical food manufacture. Therefore, PG is expected to be helpful in improving the functionalities of skim milk in the food industry. The functional properties of milk proteins determine the qualities of dairy products, such as the texture of cheese and yogurt. In addition, milk protein ingredients are used in various other processed foods, bakery products, confectionary, beverages, etc., to provide desirable functionalities. In future, more work needs to be done to understand further functional properties such as the emulsion stability and the effect of pH on emulsifying properties, whipping ability, water holding capacity to develop the application of this enzyme for processing foods. Acknowledgments The authors would like to thank Shotaro Yamaguchi of Amano Enzyme, Inc. for advice on protein-glutaminase. References
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