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Preparation, Properties and Uses of Enzymatic Milk Protein Hydrolysates a
a
M.H. Abd El-Salam & S. El-Shibiny a
Dairy Department, National Research Centre, Dokki, Cairo, Egypt Accepted author version posted online: 16 Apr 2015.
Click for updates To cite this article: M.H. Abd El-Salam & S. El-Shibiny (2015): Preparation, Properties and Uses of Enzymatic Milk Protein Hydrolysates, Critical Reviews in Food Science and Nutrition, DOI: 10.1080/10408398.2014.899200 To link to this article: http://dx.doi.org/10.1080/10408398.2014.899200
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
ACCEPTED MANUSCRIPT Preparation, properties and uses of enzymatic milk protein hydrolysates M.H.Abd El-Salam1,* and S. El-Shibiny1 Dairy Department, National Research Centre, Dokki, Cairo, Egypt Corresponding author Email: [email protected] Abstract Enzymatic hydrolysis of milk proteins has been the subject of numerous research studies and
Downloaded by [Gazi University] at 07:13 05 May 2015
patents. The driving force for these studies has been the increased utilization of milk proteins. The industrial uses of milk proteins are based on their unique composition, functionality and nutritive value. The diversity of milk protein fraction, the large number of proteinases and controlled hydrolysis conditions used resulted in the preparation of hydrolysates suitable for several purposes. Enzymatic hydrolysis of milk proteins modifies the technofunctional and biofunctional properties of the hydrolysates depending on the enzyme(s) and hydrolysis conditions used. Milk protein hydrolysates (MPH) are used commonly in normal and clinical nutrition and as functional food ingredient. In the present review emphasis has been made to highlight methods applied for the preparation of MPH, and the functional properties and utilization of the obtained hydrolysates. Key words: hydrolysates, casein, whey proteins, functional properties, nutrition, bioactive peptides, sport drinks, phenylketonuria, hypoallergenic
1
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ACCEPTED MANUSCRIPT Introduction Protein hydrolysates are defined as “mixtures of polypeptides, oligopeptides and amino acids that are manufactured from protein sources using partial hydrolysis" (Schaafmsa, 2009). Hydrolysis is done by using food grade proteolytic enzymes, or suitable acids and alkalis. Enzyme hydrolysis is greatly preferred because acid and alkali hydrolysis are difficult to control. Also, acid hydrolysis oxidizes Cys and Met and partially destroys Ser and Thr and converts Gln
Downloaded by [Gazi University] at 07:13 05 May 2015
and Asn to Glu and Asp acids and thus lowering the quality and biological value of the protein. On the other hand alkali hydrolysis causes racemization in amino acids. In addition, enzymatic hydrolysis of proteins is an affordable technology to produce high value added products. Enzyme-catalyzed hydrolysis provides several advantages including fast reaction rate and high specificity. Also, enzyme hydrolysis is carried out under mild conditions resulting in minimum changes in proteins and generates food-grade protein hydrolysates. Proteolysis reduces the molecular weight of the hydrolyzed protein and changes its conformation and hydrophobicity. These changes depend on the specificity of the used protease, degree of hydrolysis (DH) and the protein subjected to hydrolysis. Enzymatic hydrolysis of proteins is carried for several purposes and are discussed here in briefly category wise: 1- Human nutrition. Consumption of protein hydrolysates has been shown to result in more rapid uptake of amino acids compared to the intact protein or the free amino acid mixtures. Also, protein hydrolysates may have biologically active peptides that exhibit several physiological functions. In addition, protein hydrolysates are characterized by hypoallergenic properties compared to the intact proteins. The high availability of the
2
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ACCEPTED MANUSCRIPT amino acids, the presence of the bioactive peptides, and the hypoallergenic characteristics have resulted in the incorporation of protein hydrolysates as a principal ingredient in medical foods such as geriatric food supplements, weight-control, sport nutrition, infant formulae and therapeutic diets or for clinical treatments in patients suffering from digestion or absorption disorders or malnutrition association with cancer, trauma and burns (Clemente, 2000). Modified protein hydrolysates have been used in the nutrition of
Downloaded by [Gazi University] at 07:13 05 May 2015
subjects with metabolic disorders such as phenylketonuria. 2- Biotechnology applications. Traditionally protein hydrolysates have been long used to provide the nitrogen requirements for microorganisms grown in laboratory. Today, protein hydrolysates are widely used in other areas of biotechnology such as (1) animal cell cultures for the production of monoclonal antibodies, therapeutic proteins and enzymes (2) recombinant cultures for the production of therapeutic drugs and vaccine (3) tissue cultures (4) plant cell cultures for a variety of end products (5) specialized media for growing and expressing genetically modified microorganisms (Pasupuleti and Demain, 2010). 3- Food industry. Limited hydrolysis of proteins changes their functional properties (Neklyudov et al., 2000). Due to their better emulsifying properties, the partially hydrolysed proteins have been used as emulsifiers in food products such as meat products. 4- Animal feeding. The productivity of livestock is largely dependent of the quality of feed particularly high quality protein. Protein hydrolysates from mixed sources can provide products of improved protein quality to be incorporated in animal feed. Protein
3
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ACCEPTED MANUSCRIPT hydrolysates have been incorporated in animal for high milk output and meat quality (Gilbert et al., 2008). 5- Fermentation industry. The use of protein hydrolysates provide microbial cultures used in the fermentation industries with their nitrogen needs in more readily accessible form to improve the performance of microbial cultures and maximize their fermentation products. 6- Cosmetic industry. Enzymatic protein hydrolysates from plant and animal sources
Downloaded by [Gazi University] at 07:13 05 May 2015
function primarily as skin and hair conditioning agents in personal care products. Preparation of hydrolysates from milk proteins has received much attention due to the diversity, unique functional properties and high nutritional value of milk proteins and as rich sources of bioactive peptides. Milk protein hydrolysates represent a significant segment of world protein hydrolysates. It has been estimated that the world annual market for hydrolysates derived from whey proteins, milk proteins and casein at 8000, 4000 and 3000 metric tons respectively (Schaafmsa, 2009). Milk protein hydrolysates have been used in almost all areas of protein hydrolysates applications (Fig 1) The aim of the present article is to compile and discuss different aspects concerning the preparation and utilization of milk protein hydrolysates with special emphasis on their use in human nutrition. 2-Preparation of milk protein hydrolysates. 1-2 Methods used. The manufacturing processes of protein hydrolysates have been dramatically improved due to the increased demand for protein hydrolysates in new and diversified areas such as animal cell
4
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ACCEPTED MANUSCRIPT cultures. Three methods (Fig 2) can be followed in the preparation of protein hydrolysates. These are: 1- The direct addition of the selected enzyme to the protein solution to induce hydrolysis under the optimum conditions for the enzyme activity until a desired degree of hydrolysis (DH) is attained. The degree of hydrolysis is the proportion of cleaved peptide bonds in a protein hydrolysate. The reaction is then terminated by heating the solution to inactivate
Downloaded by [Gazi University] at 07:13 05 May 2015
the enzyme. The method is the widely used process on industrial scale for the preparation of protein hydrolysates due to its simplicity. However, it has several disadvantages: -
The enzyme is used only for one time. Due to the high cost of many enzymes, the process economy may limit its industrial application.
-
Difficulty to control the hydrolytic products. The reaction time and condition influence greatly the type and concentration of the hydrolytic products.
-
The inactivated enzyme remains in the reaction product which may add to the allergenic properties of the hydrolysates.
-
There is a risk of causing undesirable changes in the hydrosylates by the high heat treatment subjected to inactivate the enzyme.
2- Membrane bioreactor which can be used for continuous hydrolysis and removal of selected hydrolytic products. The reaction vessel is connected to a membrane system in a closed circuit. The selected enzyme is added to the protein solution in the reaction vessel. After an initial incubation time, the vessel contents are pumped in the membrane system which allows the selective separation of the hydrolytic products depending on their molecular mass and the molecular cutoff of the membrane. The enzyme, the
5
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT unhydrolysed protein and large hydrolytic products are retained by the membrane to be returned back to the reaction vessel. This enables the process efficiency and productivity exceeding those obtained by the classical methods. Casein was continuously hydrolysed with Aspergillus oryzae protease in a pilot scale formed-in-place membrane reactor (Chiang et al., 1995). A high percentage (>99%) of trichloro acetic acid (TCA) soluble nitrogen in the hydrolysate (product) was achieved after 45 min at 37°C and pH 7. The
Downloaded by [Gazi University] at 07:13 05 May 2015
product was completely soluble over pH range 2-9. Water sorption increased 4 to 6.5 times in the range of water activity of 0.35-0.95 as compared to intact casein. The immunologically active casein and immunologically active whey proteins in the product were reduced 99% and 97%, respectively. Long term operation showed that the membrane reactor maintained steady production of casein hydrolysate longer than 17 hr. Lin et al.,(1997) prepared casein hydrolysate (DH 23.2%) with low free amino acid using alcalase in a two-stage membrane bioreactor. The hydrolysate retained 2.32 and 1.09% immunologically active casein and whey protein respectively. A cyclic batch membrane bioreactor was used for the production of whey protein hydolysate of 1000 times less antigenicity than the original proteins (Prieto et al., 2007). Hydrolysis was carried out using substilisin at 60ºC and pH 8.5. In order to achieve the target for reduced antigenicity, a DH of 15% was needed. The reuse of the enzyme up to five times was reported which allowed for 59% saving in the used enzyme. Casein was hydrolysed with thermolysin and substilisin in a membrane reactor (Trusek-Holownia, 2008). Thermolysin was selected as the most appropriate catalyst. At high substrate retention (about 50%) a high hydrolysis was obtained. The use of enzymatic membrane bioreactor
6
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ACCEPTED MANUSCRIPT for the preparation of whey protein hydrolysate has been the subject of many patents. Prieto et al. (2010) surveyed 20 patent issued between 1989 and 2009 for the use of membrane bioreactor to prepare whey protein hydrolysates for different purposes. The DH and the molecular cut off of the membrane used for the fractionation of the hydrolysate determine mainly the composition and properties of the obtained hydrolysates.
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3- Immobilization of enzymes is carried out on a solid matrix or a membrane by covalent binding and utilized in the preparation of protein hydrolysates. The immobilized enzyme is either stirred in the protein solution for a determined time and then removed by filtration or the protein solution is passed through the immobilized enzyme bed. The protein hydrolysate is obtained at the column outlet. In the case of enzymes immobilized on membranes, the hydrolytic products are selectively separated by the membrane filtration. Several proteolytic enzymes were immobilized on amino propyl-glass beads (Haque and Mozaffer, 1992a) and used in the hydrolysis of casein. Immobilized papain, trypsin and rhozyme 41 extensively hydrolyzed casein within 30 min whereas hydrolysis of casein with immobilized chymotrypsin was slow and gave few peptides. 2.2. Enzymes used and hydrolysis conditions Selection of the proteolytic enzyme(s) for the preparation of protein hydrolysates, hydrolysis condition and DH depends on the target uses of the hydrolysates. Hydrolysates are usually classified according to DH to partial hydrolysates and extensive hydrolysates. Partial (limited) hydrolysis of proteins aims to improve the functional properties of proteins, while extensively hydrolysed proteins are mainly used in normal and clinical nutrition. Proteolytic enzymes from
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ACCEPTED MANUSCRIPT animal (pepsin, trypsin, chymotrypsin and pancreatin), plant ( such as papain, bromelin and ficin) and microbial ( such as neutrase®, alcalase®, substilisin®, protamex®, flavourzyme® and cornolase®) sources have been singly or in combination or sequential in the preparation of protein hydrolysates. The hydrolysis conditions (pH, temperature, ionic strength, enzyme: substrate ratio, and time) are selected within the specified ranges for the enzyme action and the required DH. The use of microbial proteases is increasing due to their wide and diversified
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specificities and ranges of their actions (Table 1). The commercial preparation of the different proteolytic enzymes may contain traces of other proteolytic enzymes which may modify the obtained hydrolysate. Heat denatured whey protein isolate (HDWPI) was hydrolysed with trypsin, chymotrypsin, alcalase or neutrase to 2.8, 4.3, 6.0 or 8.0% DH. Analysis of freeze-dried fractions revealed a linear increase in primary amino groups, non-protein nitrogen and ash contents. Polyacrylamide gel electrophoresis showed that high and intermediate molecular weight peptides were converted to lower molecular weights with progress of hydrolysis. Differences in proteolysis patterns were observed with different enzymes. The time required to achieve equivalent hydrolysis at 1, 2, 3 or 4% enzyme/substrate (E: S) ratio varied with the type of enzyme and DH (Mutilangi et al.,1995). Whey protein components were hydrolysed with corolase 7092 (peptidases from Aspergillus strains), pepsin and corolase PP (a mixture of pancreatic enzymes), either individually or in combination, to eliminate protein allergenicity (Ena et al., 1995). Enzyme specificity rather than DH or molecular mass distribution of hydrolysates determined the residual antigenicity of the whey proteins in the hydrolysates. Ultrafiltration was performed to remove large peptides the
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ACCEPTED MANUSCRIPT permeate containing the small peptides was used to prepare the hypoallergenic whey protein hydrolysates. In order to obtain casein hydrolysates containing high dipeptides and tripeptides contents, substilisin and trypsin were used separately or in mixture or simultaneously (Morato et al., 2000). The hydrolysate with the highest small peptide was obtained when substilisin was only used at E:S 4%. Using substilisin first for 5 min and then trypsin gave hydolysate with the lowest
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large peptide content. Hydrolysis of native and heated whey protein concentrate (WPC) with pepsin followed by trypsin revealed greater hydrolysis of WPC (Kim et al., 2007a). The highest hydrolysis and the lowest antigenicity were obtained when heated WPC was incubated with 1% pepsin followed by 1% trypsin for 120 min. Heat treatment altered the rate of whey protein hydrolysis and the peptides released from the different whey proteins (O’Loghlin et al.,2013). Native and heat denatured WPI were subjected to hydrolysis (DH 5%) with corolase PP. Hydrolysis of denatured WPI favoured the generation of higher levels of free essential amino acids and increased the number of peptides derived from α-lactalbumin and glycomacropeptide. The use of combinations of different enzyme was more effective in reducing the antigenicity of whey proteins than the use of single enzyme (Nakamura et al., 1993). Hydrolysis of whey proteins with alcalase followed by treatment with papain was highly effective in reducing the immune reactivity and improving the sensory properties of the hydrolysate (Wröblewska et al.,2004). Two enzyme (1:1) combinations of trypsin (T), papain (P), nutrase (N) and protease S (S) were used for the hydrolysis of whey proteins (Shin et al.,2007). The lowest antigenicity was observed in hydrolysates prepared with the use of trypsin-containing combinations and that the
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ACCEPTED MANUSCRIPT TP and TN mixtures were the most effective in removing of ß-lactoglobulin from the hydrolysate. The newly acid proteinases MPiAP1 and MPiAP2 produced by Monascus pilosus were found effective in complete digestion of α-lactalbumin (Lakshman et al., 2011). Complete digestion of WPC proteins was achieved with the combination of Monascus pilosus proteinases and trypsin. The obtained hydrolysates were characterized by the lowest antigenicity compared to hydrolysates prepared by the use of trypsin and Monascus pilosus proteinases separately.
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Changing the hydrolysis conditions can be used to regulate the antigenicity of whey protein hydrolysate. Treatment of whey protein concentrate with alcalase at different pH, temperature and enzyme to substrate ration resulted in different rates of antigenicity reduction (Zheng et al., 2008). Combinations of these parameters can be used to regulate the antigenicity of whey protein hydrolysate. The anti-α-lactalbumin and IgG binding inhibition was negatively related to the DH, while the anti-ß-lactoglobulin binding inhibition was not related to DH. Combined microwave treatment and peptic hydrolysis reduced the immune reactive binding properties of whey proteins better than the combined heat treatment and enzymatic hydrolysis (El-Mecherfi et al., 2011). The proteolytic effects of pancreatin and proteases from Bacillus licheniformis, Aspergillus oryzae and Aspergillus sojae on whey protein concentrate (WPC) at enzyme: substrate ratios (E:S) of 0.5:100, 1:100, 2:100,3:100, 4:100 and 8:100 were evaluated (Morais et al., 2013). Protease from Bacillus licheniformis at E:S 8:100 and pancreatin at E:S 2:100,3:100, 4:100 and 8:100 respectively yielded WPC hydrolysates with the highest sums of dipeptides and tripeptides and free amino acid contents and the lowest amounts of large peptides compared to other proteases used (Morais et al.,2013).
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ACCEPTED MANUSCRIPT β-Casein (β-CN) and β-lactoglobulin (β-Lg), were hydrolysed with alcalase, neutrase and bromelain at different time points. Hydrolysates were analyzed by SDS-PAGE and RP-HPLC. Selected samples were tested against 6 patients’ IgE sera by inhibition ELISA respectively (Herranz et al., 2013). Higher values of IC50 (lower allergenicity) for β-CN and β-Lg hydrolysates were obtained at higher incubation times when alcalase or neutrase were employed as enzymes. β-CN was hydrolyzed more efficiently showing higher IC50 values than β-Lg, at
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similar incubation times. Between the two enzymes, neutrase produced hydrolysates with lower allergenicity. On the other hand, bromelain had no influence on β-Lg hydrolysis or allergenicity, but it showed a discrete effect on β-CN hydrolysis with a moderate influence on its allergenicity (Herranz et al., 2013). 2.3. Debittering of milk protein hydrolysates The release of bitter tasting properties is a negative aspect associated with most protein hydolysates. Bitterness has been attributed to the release of peptides containing hydrophobic amino acid residues (Ney, 1971). Hydrophobic peptides are usually characterized by bitter taste. Compared to casein, whey proteins develop less bitterness on hydrolysis due to their low contents of hydrophobic amino acids. Therefore, peptides found in whey protein hydrolysates are less hydrophobic than that found in casein hydrolysates. Removal of bitter peptides from protein hydrolysates using different matrices or extraction has been proposed but these methods suffer from losses in some amino acids from the hydrolysates. The reaction conditions during the preparation of the hydrolysates may play a highly significant role in the development of bitterness. Increasing the total solids during the preparation of whey protein hydrolysates (WPH) significantly decrease its bitterness (Spellman et al., 2005). The bitterness of WPC hydrolysates
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ACCEPTED MANUSCRIPT was affected by the type of enzyme used (Spellman et al., 2009). Comparing the bitterness of WPH generated by three commercially available Bacillus proteinases revealed that alcalase 2.4L hydrolysates were more bitter than those obtained from the action of prolyve 1000 and corolase 7089 on WPC. These differences were attributed to the presence of a glutamyl endopeptidase activity in the alcalase preparation but not found in the other two enzymes (Spellman et al., 2009). Debittering can be mediated using exopeptidases including amino and carboxypeptidases
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with special emphasis on proline exopeptidases (Rakasakulthai and Haard, 2003). Thus, bitterness of tryptic casein hydrolysate was removed by treatment with Procine exopeptidases immobilized on chitin film (Ge and Zhang, 1996). The bitter taste and hygroscopic property of WPH limit its direct utilization as food ingredient. Encapsulation of WPH by spray drying using maltodextrin/b-cyclodextrin mixture as wall materials attenuated the bitter taste and enhanced the stability of whey protein hydrolysate (Yang et al, 2012). Masking bitterness has been proposed as an alternative for its removal. FitzGerald and O’Cuinn (2006) reported that bitterness can be masked in hydrolysates by the addition of polyphosphate or specific amino acids such as aspartic and glutamic acids. Several bitter taste inhibitors have been found effective (P
Critical Reviews in Food Science and Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bfsn20
Preparation, Properties and Uses of Enzymatic Milk Protein Hydrolysates a
a
M.H. Abd El-Salam & S. El-Shibiny a
Dairy Department, National Research Centre, Dokki, Cairo, Egypt Accepted author version posted online: 16 Apr 2015.
Click for updates To cite this article: M.H. Abd El-Salam & S. El-Shibiny (2015): Preparation, Properties and Uses of Enzymatic Milk Protein Hydrolysates, Critical Reviews in Food Science and Nutrition, DOI: 10.1080/10408398.2014.899200 To link to this article: http://dx.doi.org/10.1080/10408398.2014.899200
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
ACCEPTED MANUSCRIPT Preparation, properties and uses of enzymatic milk protein hydrolysates M.H.Abd El-Salam1,* and S. El-Shibiny1 Dairy Department, National Research Centre, Dokki, Cairo, Egypt Corresponding author Email: [email protected] Abstract Enzymatic hydrolysis of milk proteins has been the subject of numerous research studies and
Downloaded by [Gazi University] at 07:13 05 May 2015
patents. The driving force for these studies has been the increased utilization of milk proteins. The industrial uses of milk proteins are based on their unique composition, functionality and nutritive value. The diversity of milk protein fraction, the large number of proteinases and controlled hydrolysis conditions used resulted in the preparation of hydrolysates suitable for several purposes. Enzymatic hydrolysis of milk proteins modifies the technofunctional and biofunctional properties of the hydrolysates depending on the enzyme(s) and hydrolysis conditions used. Milk protein hydrolysates (MPH) are used commonly in normal and clinical nutrition and as functional food ingredient. In the present review emphasis has been made to highlight methods applied for the preparation of MPH, and the functional properties and utilization of the obtained hydrolysates. Key words: hydrolysates, casein, whey proteins, functional properties, nutrition, bioactive peptides, sport drinks, phenylketonuria, hypoallergenic
1
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Introduction Protein hydrolysates are defined as “mixtures of polypeptides, oligopeptides and amino acids that are manufactured from protein sources using partial hydrolysis" (Schaafmsa, 2009). Hydrolysis is done by using food grade proteolytic enzymes, or suitable acids and alkalis. Enzyme hydrolysis is greatly preferred because acid and alkali hydrolysis are difficult to control. Also, acid hydrolysis oxidizes Cys and Met and partially destroys Ser and Thr and converts Gln
Downloaded by [Gazi University] at 07:13 05 May 2015
and Asn to Glu and Asp acids and thus lowering the quality and biological value of the protein. On the other hand alkali hydrolysis causes racemization in amino acids. In addition, enzymatic hydrolysis of proteins is an affordable technology to produce high value added products. Enzyme-catalyzed hydrolysis provides several advantages including fast reaction rate and high specificity. Also, enzyme hydrolysis is carried out under mild conditions resulting in minimum changes in proteins and generates food-grade protein hydrolysates. Proteolysis reduces the molecular weight of the hydrolyzed protein and changes its conformation and hydrophobicity. These changes depend on the specificity of the used protease, degree of hydrolysis (DH) and the protein subjected to hydrolysis. Enzymatic hydrolysis of proteins is carried for several purposes and are discussed here in briefly category wise: 1- Human nutrition. Consumption of protein hydrolysates has been shown to result in more rapid uptake of amino acids compared to the intact protein or the free amino acid mixtures. Also, protein hydrolysates may have biologically active peptides that exhibit several physiological functions. In addition, protein hydrolysates are characterized by hypoallergenic properties compared to the intact proteins. The high availability of the
2
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT amino acids, the presence of the bioactive peptides, and the hypoallergenic characteristics have resulted in the incorporation of protein hydrolysates as a principal ingredient in medical foods such as geriatric food supplements, weight-control, sport nutrition, infant formulae and therapeutic diets or for clinical treatments in patients suffering from digestion or absorption disorders or malnutrition association with cancer, trauma and burns (Clemente, 2000). Modified protein hydrolysates have been used in the nutrition of
Downloaded by [Gazi University] at 07:13 05 May 2015
subjects with metabolic disorders such as phenylketonuria. 2- Biotechnology applications. Traditionally protein hydrolysates have been long used to provide the nitrogen requirements for microorganisms grown in laboratory. Today, protein hydrolysates are widely used in other areas of biotechnology such as (1) animal cell cultures for the production of monoclonal antibodies, therapeutic proteins and enzymes (2) recombinant cultures for the production of therapeutic drugs and vaccine (3) tissue cultures (4) plant cell cultures for a variety of end products (5) specialized media for growing and expressing genetically modified microorganisms (Pasupuleti and Demain, 2010). 3- Food industry. Limited hydrolysis of proteins changes their functional properties (Neklyudov et al., 2000). Due to their better emulsifying properties, the partially hydrolysed proteins have been used as emulsifiers in food products such as meat products. 4- Animal feeding. The productivity of livestock is largely dependent of the quality of feed particularly high quality protein. Protein hydrolysates from mixed sources can provide products of improved protein quality to be incorporated in animal feed. Protein
3
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT hydrolysates have been incorporated in animal for high milk output and meat quality (Gilbert et al., 2008). 5- Fermentation industry. The use of protein hydrolysates provide microbial cultures used in the fermentation industries with their nitrogen needs in more readily accessible form to improve the performance of microbial cultures and maximize their fermentation products. 6- Cosmetic industry. Enzymatic protein hydrolysates from plant and animal sources
Downloaded by [Gazi University] at 07:13 05 May 2015
function primarily as skin and hair conditioning agents in personal care products. Preparation of hydrolysates from milk proteins has received much attention due to the diversity, unique functional properties and high nutritional value of milk proteins and as rich sources of bioactive peptides. Milk protein hydrolysates represent a significant segment of world protein hydrolysates. It has been estimated that the world annual market for hydrolysates derived from whey proteins, milk proteins and casein at 8000, 4000 and 3000 metric tons respectively (Schaafmsa, 2009). Milk protein hydrolysates have been used in almost all areas of protein hydrolysates applications (Fig 1) The aim of the present article is to compile and discuss different aspects concerning the preparation and utilization of milk protein hydrolysates with special emphasis on their use in human nutrition. 2-Preparation of milk protein hydrolysates. 1-2 Methods used. The manufacturing processes of protein hydrolysates have been dramatically improved due to the increased demand for protein hydrolysates in new and diversified areas such as animal cell
4
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT cultures. Three methods (Fig 2) can be followed in the preparation of protein hydrolysates. These are: 1- The direct addition of the selected enzyme to the protein solution to induce hydrolysis under the optimum conditions for the enzyme activity until a desired degree of hydrolysis (DH) is attained. The degree of hydrolysis is the proportion of cleaved peptide bonds in a protein hydrolysate. The reaction is then terminated by heating the solution to inactivate
Downloaded by [Gazi University] at 07:13 05 May 2015
the enzyme. The method is the widely used process on industrial scale for the preparation of protein hydrolysates due to its simplicity. However, it has several disadvantages: -
The enzyme is used only for one time. Due to the high cost of many enzymes, the process economy may limit its industrial application.
-
Difficulty to control the hydrolytic products. The reaction time and condition influence greatly the type and concentration of the hydrolytic products.
-
The inactivated enzyme remains in the reaction product which may add to the allergenic properties of the hydrolysates.
-
There is a risk of causing undesirable changes in the hydrosylates by the high heat treatment subjected to inactivate the enzyme.
2- Membrane bioreactor which can be used for continuous hydrolysis and removal of selected hydrolytic products. The reaction vessel is connected to a membrane system in a closed circuit. The selected enzyme is added to the protein solution in the reaction vessel. After an initial incubation time, the vessel contents are pumped in the membrane system which allows the selective separation of the hydrolytic products depending on their molecular mass and the molecular cutoff of the membrane. The enzyme, the
5
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT unhydrolysed protein and large hydrolytic products are retained by the membrane to be returned back to the reaction vessel. This enables the process efficiency and productivity exceeding those obtained by the classical methods. Casein was continuously hydrolysed with Aspergillus oryzae protease in a pilot scale formed-in-place membrane reactor (Chiang et al., 1995). A high percentage (>99%) of trichloro acetic acid (TCA) soluble nitrogen in the hydrolysate (product) was achieved after 45 min at 37°C and pH 7. The
Downloaded by [Gazi University] at 07:13 05 May 2015
product was completely soluble over pH range 2-9. Water sorption increased 4 to 6.5 times in the range of water activity of 0.35-0.95 as compared to intact casein. The immunologically active casein and immunologically active whey proteins in the product were reduced 99% and 97%, respectively. Long term operation showed that the membrane reactor maintained steady production of casein hydrolysate longer than 17 hr. Lin et al.,(1997) prepared casein hydrolysate (DH 23.2%) with low free amino acid using alcalase in a two-stage membrane bioreactor. The hydrolysate retained 2.32 and 1.09% immunologically active casein and whey protein respectively. A cyclic batch membrane bioreactor was used for the production of whey protein hydolysate of 1000 times less antigenicity than the original proteins (Prieto et al., 2007). Hydrolysis was carried out using substilisin at 60ºC and pH 8.5. In order to achieve the target for reduced antigenicity, a DH of 15% was needed. The reuse of the enzyme up to five times was reported which allowed for 59% saving in the used enzyme. Casein was hydrolysed with thermolysin and substilisin in a membrane reactor (Trusek-Holownia, 2008). Thermolysin was selected as the most appropriate catalyst. At high substrate retention (about 50%) a high hydrolysis was obtained. The use of enzymatic membrane bioreactor
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ACCEPTED MANUSCRIPT for the preparation of whey protein hydrolysate has been the subject of many patents. Prieto et al. (2010) surveyed 20 patent issued between 1989 and 2009 for the use of membrane bioreactor to prepare whey protein hydrolysates for different purposes. The DH and the molecular cut off of the membrane used for the fractionation of the hydrolysate determine mainly the composition and properties of the obtained hydrolysates.
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3- Immobilization of enzymes is carried out on a solid matrix or a membrane by covalent binding and utilized in the preparation of protein hydrolysates. The immobilized enzyme is either stirred in the protein solution for a determined time and then removed by filtration or the protein solution is passed through the immobilized enzyme bed. The protein hydrolysate is obtained at the column outlet. In the case of enzymes immobilized on membranes, the hydrolytic products are selectively separated by the membrane filtration. Several proteolytic enzymes were immobilized on amino propyl-glass beads (Haque and Mozaffer, 1992a) and used in the hydrolysis of casein. Immobilized papain, trypsin and rhozyme 41 extensively hydrolyzed casein within 30 min whereas hydrolysis of casein with immobilized chymotrypsin was slow and gave few peptides. 2.2. Enzymes used and hydrolysis conditions Selection of the proteolytic enzyme(s) for the preparation of protein hydrolysates, hydrolysis condition and DH depends on the target uses of the hydrolysates. Hydrolysates are usually classified according to DH to partial hydrolysates and extensive hydrolysates. Partial (limited) hydrolysis of proteins aims to improve the functional properties of proteins, while extensively hydrolysed proteins are mainly used in normal and clinical nutrition. Proteolytic enzymes from
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ACCEPTED MANUSCRIPT animal (pepsin, trypsin, chymotrypsin and pancreatin), plant ( such as papain, bromelin and ficin) and microbial ( such as neutrase®, alcalase®, substilisin®, protamex®, flavourzyme® and cornolase®) sources have been singly or in combination or sequential in the preparation of protein hydrolysates. The hydrolysis conditions (pH, temperature, ionic strength, enzyme: substrate ratio, and time) are selected within the specified ranges for the enzyme action and the required DH. The use of microbial proteases is increasing due to their wide and diversified
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specificities and ranges of their actions (Table 1). The commercial preparation of the different proteolytic enzymes may contain traces of other proteolytic enzymes which may modify the obtained hydrolysate. Heat denatured whey protein isolate (HDWPI) was hydrolysed with trypsin, chymotrypsin, alcalase or neutrase to 2.8, 4.3, 6.0 or 8.0% DH. Analysis of freeze-dried fractions revealed a linear increase in primary amino groups, non-protein nitrogen and ash contents. Polyacrylamide gel electrophoresis showed that high and intermediate molecular weight peptides were converted to lower molecular weights with progress of hydrolysis. Differences in proteolysis patterns were observed with different enzymes. The time required to achieve equivalent hydrolysis at 1, 2, 3 or 4% enzyme/substrate (E: S) ratio varied with the type of enzyme and DH (Mutilangi et al.,1995). Whey protein components were hydrolysed with corolase 7092 (peptidases from Aspergillus strains), pepsin and corolase PP (a mixture of pancreatic enzymes), either individually or in combination, to eliminate protein allergenicity (Ena et al., 1995). Enzyme specificity rather than DH or molecular mass distribution of hydrolysates determined the residual antigenicity of the whey proteins in the hydrolysates. Ultrafiltration was performed to remove large peptides the
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ACCEPTED MANUSCRIPT permeate containing the small peptides was used to prepare the hypoallergenic whey protein hydrolysates. In order to obtain casein hydrolysates containing high dipeptides and tripeptides contents, substilisin and trypsin were used separately or in mixture or simultaneously (Morato et al., 2000). The hydrolysate with the highest small peptide was obtained when substilisin was only used at E:S 4%. Using substilisin first for 5 min and then trypsin gave hydolysate with the lowest
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large peptide content. Hydrolysis of native and heated whey protein concentrate (WPC) with pepsin followed by trypsin revealed greater hydrolysis of WPC (Kim et al., 2007a). The highest hydrolysis and the lowest antigenicity were obtained when heated WPC was incubated with 1% pepsin followed by 1% trypsin for 120 min. Heat treatment altered the rate of whey protein hydrolysis and the peptides released from the different whey proteins (O’Loghlin et al.,2013). Native and heat denatured WPI were subjected to hydrolysis (DH 5%) with corolase PP. Hydrolysis of denatured WPI favoured the generation of higher levels of free essential amino acids and increased the number of peptides derived from α-lactalbumin and glycomacropeptide. The use of combinations of different enzyme was more effective in reducing the antigenicity of whey proteins than the use of single enzyme (Nakamura et al., 1993). Hydrolysis of whey proteins with alcalase followed by treatment with papain was highly effective in reducing the immune reactivity and improving the sensory properties of the hydrolysate (Wröblewska et al.,2004). Two enzyme (1:1) combinations of trypsin (T), papain (P), nutrase (N) and protease S (S) were used for the hydrolysis of whey proteins (Shin et al.,2007). The lowest antigenicity was observed in hydrolysates prepared with the use of trypsin-containing combinations and that the
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ACCEPTED MANUSCRIPT TP and TN mixtures were the most effective in removing of ß-lactoglobulin from the hydrolysate. The newly acid proteinases MPiAP1 and MPiAP2 produced by Monascus pilosus were found effective in complete digestion of α-lactalbumin (Lakshman et al., 2011). Complete digestion of WPC proteins was achieved with the combination of Monascus pilosus proteinases and trypsin. The obtained hydrolysates were characterized by the lowest antigenicity compared to hydrolysates prepared by the use of trypsin and Monascus pilosus proteinases separately.
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Changing the hydrolysis conditions can be used to regulate the antigenicity of whey protein hydrolysate. Treatment of whey protein concentrate with alcalase at different pH, temperature and enzyme to substrate ration resulted in different rates of antigenicity reduction (Zheng et al., 2008). Combinations of these parameters can be used to regulate the antigenicity of whey protein hydrolysate. The anti-α-lactalbumin and IgG binding inhibition was negatively related to the DH, while the anti-ß-lactoglobulin binding inhibition was not related to DH. Combined microwave treatment and peptic hydrolysis reduced the immune reactive binding properties of whey proteins better than the combined heat treatment and enzymatic hydrolysis (El-Mecherfi et al., 2011). The proteolytic effects of pancreatin and proteases from Bacillus licheniformis, Aspergillus oryzae and Aspergillus sojae on whey protein concentrate (WPC) at enzyme: substrate ratios (E:S) of 0.5:100, 1:100, 2:100,3:100, 4:100 and 8:100 were evaluated (Morais et al., 2013). Protease from Bacillus licheniformis at E:S 8:100 and pancreatin at E:S 2:100,3:100, 4:100 and 8:100 respectively yielded WPC hydrolysates with the highest sums of dipeptides and tripeptides and free amino acid contents and the lowest amounts of large peptides compared to other proteases used (Morais et al.,2013).
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ACCEPTED MANUSCRIPT β-Casein (β-CN) and β-lactoglobulin (β-Lg), were hydrolysed with alcalase, neutrase and bromelain at different time points. Hydrolysates were analyzed by SDS-PAGE and RP-HPLC. Selected samples were tested against 6 patients’ IgE sera by inhibition ELISA respectively (Herranz et al., 2013). Higher values of IC50 (lower allergenicity) for β-CN and β-Lg hydrolysates were obtained at higher incubation times when alcalase or neutrase were employed as enzymes. β-CN was hydrolyzed more efficiently showing higher IC50 values than β-Lg, at
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similar incubation times. Between the two enzymes, neutrase produced hydrolysates with lower allergenicity. On the other hand, bromelain had no influence on β-Lg hydrolysis or allergenicity, but it showed a discrete effect on β-CN hydrolysis with a moderate influence on its allergenicity (Herranz et al., 2013). 2.3. Debittering of milk protein hydrolysates The release of bitter tasting properties is a negative aspect associated with most protein hydolysates. Bitterness has been attributed to the release of peptides containing hydrophobic amino acid residues (Ney, 1971). Hydrophobic peptides are usually characterized by bitter taste. Compared to casein, whey proteins develop less bitterness on hydrolysis due to their low contents of hydrophobic amino acids. Therefore, peptides found in whey protein hydrolysates are less hydrophobic than that found in casein hydrolysates. Removal of bitter peptides from protein hydrolysates using different matrices or extraction has been proposed but these methods suffer from losses in some amino acids from the hydrolysates. The reaction conditions during the preparation of the hydrolysates may play a highly significant role in the development of bitterness. Increasing the total solids during the preparation of whey protein hydrolysates (WPH) significantly decrease its bitterness (Spellman et al., 2005). The bitterness of WPC hydrolysates
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ACCEPTED MANUSCRIPT was affected by the type of enzyme used (Spellman et al., 2009). Comparing the bitterness of WPH generated by three commercially available Bacillus proteinases revealed that alcalase 2.4L hydrolysates were more bitter than those obtained from the action of prolyve 1000 and corolase 7089 on WPC. These differences were attributed to the presence of a glutamyl endopeptidase activity in the alcalase preparation but not found in the other two enzymes (Spellman et al., 2009). Debittering can be mediated using exopeptidases including amino and carboxypeptidases
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with special emphasis on proline exopeptidases (Rakasakulthai and Haard, 2003). Thus, bitterness of tryptic casein hydrolysate was removed by treatment with Procine exopeptidases immobilized on chitin film (Ge and Zhang, 1996). The bitter taste and hygroscopic property of WPH limit its direct utilization as food ingredient. Encapsulation of WPH by spray drying using maltodextrin/b-cyclodextrin mixture as wall materials attenuated the bitter taste and enhanced the stability of whey protein hydrolysate (Yang et al, 2012). Masking bitterness has been proposed as an alternative for its removal. FitzGerald and O’Cuinn (2006) reported that bitterness can be masked in hydrolysates by the addition of polyphosphate or specific amino acids such as aspartic and glutamic acids. Several bitter taste inhibitors have been found effective (P
