FOOD AND BIOPRODUCTS PROCESSING 9 0 ( 2 O I 2 ) 4 4 9 - 4 5 2

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Food and Bioproducts Processing

ELSEVIER
journal homepage: www.elsevier.com/locate/fbp

IChemE

Role of actinidin in the hydrolysis of the cream milk proteins

Ivana Puglisi, Goffredo Ptrone^ Angela R. Lo Piero'*'^
Dipartimento di Scienze delle Produzioni Agrarie e Alimentari (DISPA), Facolt di Agraria, Uniuersit degli Studi di Catania, 95123 Catania, Italy

A B S T R A C T

Gream from milk is commonly used as the starting material of variety food products such as cream cheese, fat added cheeses, butter and buttermilk. The protein content of cream is only about 2% but strongly affects the quality of fat containing dairy products especially cheeses which are frequently eharacterized by an unsatisfactory texture. In this work, the suitability of the actinidin to function in a "fat-supplemented" environment was tested and the SDS-PAGE profile of cream treated with the enzyme was also analyzed. The results show that aetinidin is able to degrade all the cream proteins to smaller than 29.0 kDa fragments suggesting a novel utilization as preparatory treatment in the production of milk derived products characterized by a reduced content of undesirable proteins. 2011 The Institution of Ghemical Engineers. Published by Elsevier B.V. All rights reserved. Keyujords: Actinidin; Fat; Gream; SDS-PAGE; Milk fat globule membranes (MFGM)

1.

Introduction

Gream is a dairy product obtainable by physical separation from milk in the form of an emulsion of fat in skimmed milk. The fat content of cream varies from 10% to 50%, whereas there are only 2% proteins comprising those in the milk fat globule membranes (MFGM) and caseins and whey proteins (Morin et al, 2007). As presented schematically in Fig. 1, cream represents the starting material of a number of milk derived products assigned to the food industry. The addition of cream to milk or curd in order to optimize the cheese fat content represents a common practice in the manufacture of several dairy products such as cheddar (Nair et al., 2004) and Halva cheeses (Kurultay et al., 2008). Therefore, it has been used to optimize the cheese fat content in the manufacture of both pizza and cheddar cheeses (Govindasamy-Lucey et al., 2006; Nair et al., 2004), or in cream cheese production (Monteiro et al., 2009). Mainly, the cream is churned into butter releasing an aqueous phase called buttermilk as a co-product (Fig. 1). The protein content of buttermilk resembles that of skimmed milk (caseins and whey proteins), and also contains a large fraction of MFGM material (Gorredig & Dalgleish, 1998). Similarly to cream, buttermilk has been also proposed as a milk ingredient to increase the fat content of manufactured cheeses (Gorredig & Dalgleish, 1998). However, the presence

of MFGM proteins negatively affects the rennet gel properties, in particular the firmness and the set-to-cut time of rennet gels (Morin et al., 2008; Govindasamy-Lucey et al, 2006). Buttermilk has recently gained much attention as a substrate for the production of functional phospholipid concentrates because of the healthy properties of these MFGM components (Govindasamy-Lucey et al., 2006). By contrast, the large fraction of the MFGM proteins, of both cream and buttermilk, have a lower functional value than that exhibited by caseins and whey proteins. Hence, several processes have been proposed to separate the phospholipids from the protein components of MFGM, a lot of them showing drawbacks such as the low value of protein by-product or the excessive length of the separation procedures (Britten et al., 2008). Recently, a washing treatment was reported which removes the MFGM proteins and caseins from cream. The effects of this treatment upon the cream milk positively developed into the further steps of the butter making process leading to the production of a buttermilk fraction with increased purity of phospholipids (Britten et al., 2008). In a previous work, two kiwi fruit proteases were purified from Actinidia chinensis and their enzymatic properties, substrate specificities and pH-activity profiles have been determined (Sugiyama et al., 1997). The interest toward actinidin as an enzyme for the hydrolysis of food proteins has recently increased. Kaur et al. (2010a) showed that actinidin

* Corresponding author. Tel.: +39 0 95 7580238; fax: +39 0 95 7141581. E-mail address: rlopiero@unict.it (A.R. Lo Piero). Received 19 July 2011; Received in revised form 21 November 2011; Accepted 22 November 2011 ' 'These authors contributed equally to this work. 0960-3085/$ - see front matter 2011 The Institution of Ghemieal Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.fbp.2011.11.008

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Milk
Centrifugal separation

2.2.

Lipid assay

Lipid content in cream was measured by the phosphovanillin method described in Zoellner and Kirsch (1962).
Butter
Cream
C\rurning

2.3.

Proteolytic activity assay

Skim milk
Acid coagulation

Cen trifugal sep aration

Buttermilk

Cream cheese

Washed cream
churning

Addition to milk or curd

Prote n-reduced butter Protein-reduced buttermilk

Addition to milk or curd

Fat added cheese

Fig. 1 - Schematic representation of milk-derived products.

can provide enhanced digestion of sodium caseinate, beef muscle protein, and, to some extent, soy protein isolate under gastric digestion conditions. Furthermore, kiwifruit extract containing actinidin was found to enhance the digestion of whey protein isolate as well as zein, gluten, and gliadin under simulated gastric conditions (Kaur et al., 2010b). More recently, the cysteine protease actinidin purified from kiwifruits was characterized in view of its possible suitability as a coagulant enzyme (Lo Piero et al., 2011). The data showed that actinidin exhibits the ability to form milk clots in which the casein coagulum is separated away from the whey proteins. The enzyme turns out to be fully compatible with the physical-chemical conditions utilized during cheese manufacture (40-42 C, sub acid pH values). The C/P ratio is crucial to evaluate the degree of the clotting activity of the enzyme (C) compared to the extent of the detrimental general proteolytic activity (P) which is normally responsible for the bitterness found in cheeses obtained by plant rennet (Lo Piero et al., 2011). Actinidin shows a C/P value (1.1) as high as that reported for calf chymosin. In this work, we investigated the effect of cream milk upon the enzyme activity as well as the ability of actinidin to degrade the cream milk proteins. The results suggest that actinidin might be utilized in the application of an novel enzymatic approach directed to eliminate the MFGM protein residues from cream and its derivatives.

The proteolytic activity of the enzyme was assayed using total casein, a-casein, -casein, K-casein (all from Sigma, St. Louis, MO, USA) as substrates as described in Lo Piero et al. (2002). The assay mixture (1 mL) contained 20 g/1 of the substrates dissolved in 67 mM NaH2PO4 pH 7.2 and 2.5 mM DTT. The effect of the cream (12% in fat) was monitored by adding the appropriate amount of commercially available cream, ranging from 0.1 to 5% in fat, to the standard assay mixture. The samples were incubated at 55 C for 20 min, then the reaction was stopped by adding 1.5 mL of 50% (g/1) TCA. After TCA precipitation, the supernatant was recovered by centrifugation at 9,000 g for 10 min in a benchtop centrifuge ALC PK 121 R (ALC, New Jersey, USA). The absorbance of supernatant was measured at 280 nm using a Shimadzu UV-VIS 1240 spectrophotometer (Shimadzu Corporation, USA). The enzyme activity was expressed in a unit defined as the amount of enzyme that yielded a 0.001 absorbance change per min at 280 nm. The hydrolysis against cream proteins was measured by incubation of the enzyme with an increasing amount of cream ranging from 0.01% to 0.1% in protein content as determined by the method described in Lowry et al. (1951). All the experiments were repeated four times on independent enzyme preparations and the standard deviation was calculated by the average of the four experiments. 2.4. Electrophoretic analysis of the actinidin action upon cream proteins The hydrolysis of cream proteins was achieved by incubation of 0.065 mg of proteins with actinidin (12 ig) for 30 min at 50 C (final volume 0.3 mL) in 67 mM NaH2PO4 pH 7.2. After incubation, the samples were prepared for SDS-PAGE by adding an equal volume of double-concentrated loading buffer (4% SDS, 30% sucrose, 0.12% Tris pH 8.0, 0.042 mM DTT, 2% bromophenol blue). SDS-PAGE (12.5% slab gels) was performed according to the Laemmli method (1970). The gel was stained with a solution containing 50% methanol, 7.5% acetic acid and 0.2% Comassie R-250. This was followed by repeated washes in 15% methanol and 7.5% acetic acid. All the experiments were repeated four times on independent enzyme preparations.
3.

Results and discussion

2. 2.1.

Materials and methods Enzyme preparation

Actinidin was purified as described in Lo Piero et al. (2011). The protein content of the enzyme preparations was routinely measured by the Bradford method (1976), using bovine serum albumin as the standard.

Fig. 2 shows that the enzyme retains the proteolytic activity at around 100% of the original value for all caseins supplied either as a mixture or as single fractions, thisfindingsuggesting the suitability of actinidin to degrade milk caseins also in a "cream-supplemented" environment. In the case of casein the enzyme activity increased up to 130% along with the fat percentage. The data suggest that fat might determine a more stable three-dimensional conformation of the enzyme so that the hydrolytic action upon -casein is enhanced. This hypothesis seems to be supported by the comparison of the result with those obtained in Lo Piero et al. (2011) showing that pasteurized whole milk, probably due to a higher fat content, is the preferred substrate rather than the pasteurized

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MFGM proteins

a-casein -casein
2.5 i 3.5

K-casein 20.1 kDa

Fat content (%)

Fig. 2 - Effect of cream upon the actinidin activity using total casein (), a-casein (A), -casein (A) and K-casein (O) as substrates. The activity of the puried enzyme (10 ng) was measured in standard buffer (67 mM NaH2P04, pH 7.2) supplemented with increasing concentrations of cream's fat ranging between 0 and 5%. The error bars represent the standard deviation (N = 4). semi-skimmed milk. The proteolytic activity of actinidin was also measured using increasing concentrations of cream proteins up to 0.1% supplied as commercial cream (Fig. 3). The results showed that the rate of hydrolysis increases along with cream's protein content and reaches a maximum between 0.02 and 0.04%. The further increment in cream protein content determines a sharp decrease in the enzyme activity reaching at 0.1% a value more than three times lower than that gained at the maximum peak of the curve (Fig. 3). Therefore, calculating the percentage of cream fat corresponding to that of cream proteins, the enzymatic inhibition obtained with the addition of 0.1% in cream protein occurs at 1.56% of cream fat. As shown in Fig. 2, this fat concentration does not affect the enzyme activity measured either with total casein or with all its single fractions. Consequently, the observed inhibition of actinidin activity might be attributed to the protein component of cream milk. Therefore, the global analysis of the results suggests that the enzyme inhibition due to the protein component of cream (Fig. 3) is not achieved in a medium also containing caseins (Fig. 2) which probably are able to compete with cream proteins for the active site of the enzyme.

14.2 kDa

Fig. 4 - Analysis of the SDS-PAGE profiles of cream milk treated with actinidin (12 (Ag). 10 fjig of each sample was loaded onto 12.5% slab gel. Lane 1, molecular weights standard; lane 2, treated cream and lane 3,control cream.

0.02

0.04

0.06

0.08

O.I

Protein content (%)

Fig. 3 - Measurement of enzyme activity using the protein of the cream as substrate. The activity of the puried actinidin (10 |xg) was measured in standard buffer (67 mM NaH2P04, pH 7.2) against increasing concentrations of cream's protein ranging between 0.01 and 0.1%. The error bars represent the standard deviation (N = 4).

In Fig. 4 is shown the SDS-PAGE profile of the proteolytic fragments obtained treating the cream proteins with actinidin. The electropherogram of untreated cream (Fig. 4, lane 3) shows the presence of seven main bands, among them the bands corresponding to a-casein (32.9 kDa), -easein (30.7 kDa) and K-casein (25.7 kDa) are clearly distinguishable (Lo Piero et al., 2011). The other bands correspond to proteins whose estimated molecular weights are 14.0 kDa, 18.0 kDa, 53.0 kDa and 80.0 kDa, respectively (Fig. 4, lane 3). Most likely, these bands correspond to a-lactalbumin (14.0kDa), -lactoglobulin (18.0 kDa) and proteins belonging to MFGM group (53.0 kDa and 80.0 kDa) as described in Britten et al. (2008) and Mather (2000). The treatment of the cream with actinidin (Fig. 4, lane 2) shows that the two bands likely corresponding to a-lactalbumin and -lactoglobulin (14.0 kDa and 18.0 kDa, respectively) are unaffected by the actinidin hydrolytic action, confirming the data previously obtained by Lo Piero et al. (2011), whereas an extensive degradation of the three main bands corresponding to a-casein, -casein and K-casein is produced (Fig. 4, lane 2). Regardless it has been previously shown that the hydrolysis of a-casein supplied either as total casein or as single fraction or in the milk mixture is only partial (Lo Piero et al., 2011). A higher degree of hydrolysis is observed in the case that this casein fraction is supplied as a mixture in the cream milk proteins (Fig. 4, lane 2). The analysis of these results emphasizes the crucial role of the cream fat in ameliorating the enzyme performance also against a-casein as shown for -casein (Figs. 2 and 4, lane 2). Moreover, the complete digestion of the MFGM proteins is also achieved (Fig. 4, lane 2) thus suggesting that cream milk might be subjected to actinidin treatment at the early stages of its processing to efficiently reduce the size of the protein components, in particular of the MFGM proteins. This kind of treatment upon the cream milk, efficiently reducing all the cream proteins in smaller than 29-30 kDa fragments (Fig. 4, lane 2), might be useful either before the addition of the cream to milk or curd in the manufacture of high fat cheeses or before the churning process. In the latter case, this novel enzymatic approach upon the cream milk might yield favourable implications during the downstream mierofiltration practise which buttermilk

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routinely undergoes to gain the fractionation of its components (Corredig et al, 2003; Morin et al, 2004).

4.

Conclusion

In the present study we evaluated the effect of the cream fat upon the proteolytic activity of actinidin, which has been recently characterized as milk coagulant enzyme (Lo Piero et al, 2011). The results indicate that actinidin totally retains the ability to degrade the casein fractions also in the presence of cream fat up to 5% thus suggesting a potential application in the manufacture of cheese with optimized fat content. The cream proteins are also degraded by actinidin as the SDS-PACE profile of the cream milk treated with actinidin reveals that both caseins, whey proteins and MFGF proteins are sharply digested by the enzyme thus suggesting a possible role of actinidin as preparatory treatment in view of the production of milk derived products characterized by a reduced content of undesirable proteins.

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