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Enzymatic debittering of food protein hydrolysates
Biotechnology Advances 24 (2006) 234 – 237 www.elsevier.com/locate/biotechadv
Research review paper
Enzymatic debittering of food protein hydrolysates R.J. FitzGerald a,*, G. O’Cuinn b a
b
Life Sciences Department, University of Limerick, Ireland Life Sciences Department, Galway Mayo Institute of Technology, Galway, Ireland Available online 4 January 2006
Abstract Protein hydrolysates have a range of applications in the food and allied healthcare sectors. Bitterness is a negative attribute associated with most food protein hydrolysates. The development of biotechnological solutions for hydrolysate debittering is ongoing. Specific enzymatic debittering strategies have focused on the application of proline specific exo- and endopeptidases given the contribution of proline residues to peptide/hydrolysate bitterness. Hydrolysate manufacturing conditions may also play an important role in bitterness development. Practical solutions to hydrolysate debittering are likely to involve judicious choice of enzymatic processing conditions in conjunction with the use of peptidase activities having targeted hydrolytic specificity. D 2005 Elsevier Inc. All rights reserved. Keywords: Protein hydrolysate; Debittering; Peptidases
Contents 1. Introduction . . . . . . . . . . . . . . . . . . 2. Hydrolysate functionality and applications . . . 3. Food protein hydrolysate bitterness . . . . . . 4. Quantification of hydrolysate/peptide bitterness 5. Protein hydrolysate debittering . . . . . . . . . 6. Peptidase mediated debittering . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Proteins from animal (e.g., milk, meat and fish) and plant (e.g., soy, legume, cereal) origin are subjected to hydrolysis during food processing. Hydrolysis occurs via two main routes, i.e., during food * Corresponding author. Tel.: +353 61 202598; fax: +353 61 331490. E-mail address: [email protected] (R.J. FitzGerald). 0734-9750/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2005.11.002
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fermentations as a result of the action of bacterial/ fungal proteinase and peptidases activities, and during the direct manufacture of protein hydrolysates per se. Hydrolysis significantly impacts the functional and sensory properties of the resultant hydrolysate. The release of bitter tasting peptides is a negative aspect associated with most food protein hydrolysates. Different enzyme-based strategies may be employed in reducing the bitterness associated with hydrolysis of food protein.
R.J. FitzGerald, G. O’Cuinn / Biotechnology Advances 24 (2006) 234–237
235
2. Hydrolysate functionality and applications
4. Quantification of hydrolysate/peptide bitterness
Food protein hydrolysates have a wide range of applications, e.g., as nitrogen fortification agents in specialist beverages, as pre-digested ingredients for enteral/parenteral nutrition for general/specific population segments, as enriched/isolated peptide preparations for beneficial physiological function, as ingredients in cell culture/bacteriological growth media and as ingredients in cosmetics and healthcare products. It is possible, largely depending on enzyme specificity and the degree of hydrolysis (DH) achieved, to generate hydrolysate products with either enhanced or reduced functionality, e.g., solubility, emulsification, foaming and gelation properties. Hydrolysis also results in a reduction in allergenic potential by the destruction of allergenic epitopes. Significant current interest exists in the hydrolytic release of bioactive peptide sequences encrypted within the primary structure of food proteins (FitzGerald and Meisel, 2003).
The ideal route for quantification of hydrolysate bitterness is to use sensory evaluation panels (Spellman et al., 2005). This is a very time consuming activity requiring appropriate numbers of panellist and training to detect bitterness in order to obtain statistically relevant data. However, instrumental methods which distinguish hydrophobic peptides within hydrolysates such as reversed-phase chromatography and Fourier transform infrared (FTIR) spectroscopy in combination with multivariate data analysis may find application in fingerprinting bitterness in hydrolysates.
3. Food protein hydrolysate bitterness Intact food proteins do not display bitterness as their molecular size militates against their ability to interact extensively with bitterness receptors in the oral cavity. Bitterness in protein hydrolysates has been classically associated with the release of peptides containing hydrophobic amino acid residues. Ney (1971) quantified peptide bitterness on the basis of the hydrophobicity of the side-chains associated with the residues within a given peptide sequence. This was calculated from the free energy of transfer of an amino acid side chain from ethanol to water. From synthetic peptide studies, Ney reported that peptides with hydrophobicity ( Q) values N 1400 cal per mole and molecular masses b 6 kDa display bitterness. However, peptides with Q values b 1300 cal per mole along with peptides with Q values N 1400 1400 cal per mole and molecular masses N 6 kDa would not be bitter. Therefore, peptides b 6 kDa having a high content of Leu, Pro, Phe, Tyr, Ile and Trp residues are likely to be bitter. Matoba and Hata (1972) subsequently reported that the presence of internally sited hydrophobic amino acid residues led to greater bitterness than when the hydrophobic residues were located at either the N- or C-terminus in peptides. The presence of internally sited Pro residues was shown to be a major and distinct contributor to peptide bitterness due to the unique conformation associated with this imino acid (Ishibashi et al., 1988).
5. Protein hydrolysate debittering Numerous options have been investigated in the debittering of food protein hydrolysates. These include absorption of bitter peptides on activated carbon, chromatographic removal using different matrices and selective extraction with alcohols. These procedures, however, lead to the loss of some amino acid residues from hydrolysates. Bitterness has also been masked in hydrolysates via the addition of polyphosphates, specific amino acids such as Asp and Glu, a-cyclodextrins and by the admixture of hydrolysates with intact protein samples. The debittering of hydrolysates via transpeptidation reactions in the dso-calledT plastein reaction in addition to cross-linking using transglutaminase represent other potential routes for hydrolysate bitterness reduction. However, these latter protocols may not always be suitable when products having high solubility are required. 6. Peptidase mediated debittering Much effort has concentrated on the use of peptidases, specifically exopeptidases including amino- and carboxypeptidases, in food protein hydrolysate debittering (for review see Raksakulthai and Haard, 2003 and references therein). Significant reductions in bitterness have been observed in proteinase digests of food proteins during concomitant or subsequent incubation with exopeptidase-rich enzyme preparations, particularily peptidases which cleave adjacent to hydrophobic amino acid residues. Given the unique contribution of Pro residues to hydrolysate bitterness, much research has focused on the application of proline specific exopeptidases in hydrolysate debittering strategies due to the general inability of most general aminopeptidases to hydrolyse the imino bond. Highly significant reductions in casein hydrolysate bitterness could be achieved using
236
R.J. FitzGerald, G. O’Cuinn / Biotechnology Advances 24 (2006) 234–237
post-proline dipeptidyl aminopeptidase (PPDA), which releases amino acyl proline residues from the N-terminus, in conjunction with a general aminopeptidase activity. Fig. 1 outlines the mechanism by which combinations of proline specific aminopeptidases (i.e. PPDA and aminopeptidase P, which removes the Nterminal amino acid where proline is present in the second position) could mediate hydrolysis of proline rich substrates, i.e., containing single and consecutive prolines (Bouchier et al., 1999).
(a)
The application of proline-specific endopeptidase activites, which hydrolyse peptides releasing C-terminal prolines, is an interesting development in the generation of low/reduced bitterness food protein hydrolysates. This has led to development of procedures for the overexpression of proline-specific endopeptidase activities, particularily from Aspergillus niger, for the generation of protein hydrolysates enriched in C-terminal proline residues (Edens et al., 2004 and references therein).
PPDA KpNA-H
KpNA-H
PPDA
Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln
Pep P
αs1-casein f(1-9)
KpNA-H KpNA-H
(b) KpNA-H
His-Pro-Ile-Lys-His-Gln
αs1-casein f(4-9)
Pep P
(c)
Pep P Pep P
PPDA KpNA-H
Val-Pro-Pro-Phe-Leu-Gln
β-casein f(84-89)
PPDA Fig. 1. Schematic representation of the potential routes of hydrolysis for peptides containing non-consecutive proline residues, i.e. (a) as1-casein f(1–9), (b) as1-casein f(4–9) and a peptide containing consecutive proline residues, i.e. (c) h-casein f(84–89) using Lys-paranitroanilide hydrolase (KpNA-H; a general aminopeptidase), post-proline dipeptidyl aminopeptidase (PPDA) and aminopeptidase P (Pep P) from Lactococcus lactis subsp. cremoris AM2 (taken from Bouchier et al., 1999).
R.J. FitzGerald, G. O’Cuinn / Biotechnology Advances 24 (2006) 234–237
All these results indicate that specificity has a major role to play in choosing an enzymatic strategy for hydrolysate debittering. However, reaction conditions during hydrolysate manufacture may also play a highly significant role in the bitterness associated with the resultant hydrolysate. It has recently been demonstrated that the total solids (TS) content in the hydrolysis reaction can significantly affect bitterness generation. Hydrolysates of whey protein concentrate at equivalent DH values (15%) were significantly less bitter when generated at 300 g TS/L compared to hydrolysates generated at 50 g TS/ L (Spellman et al., 2005). The explanation for this difference was attributed, in part, to the presence of a specific hydrophobic peptide at higher concentration in the hydrolysate generated at lower TS. In conclusion, these results indicate the importance of carefully choosing reaction conditions, in addition to the specificity of the enzyme preparation, in the generation of reduced bitterness food protein hydrolysates. References Bouchier P, FitzGerald RJ, O’Cuinn G. Hydrolysis of as1-and hcasein-derived peptides with a broad specificity aminopeptidase and proline specific aminopeptidases from Lactococcus lactis supsp cremoris AM2. FEBs Lett 1999;445:321 – 4.
237
Edens L, Hoevan RAM, vander Delest V. Protein hydrolysates enriched in peptides having a carboxy terminal proline residues. United States Patent Application 2004 0241791. FitzGerald RJ, Meisel HM. Milk protein hydrolysis and bioactive peptides. In: Fox PF, McSweeney P, editors. Advanced dairy chemistry, Third Edition, Part B. New York7 Kluwer Academic/ Plenum Publishers; 2003. p. 675 – 98. Ishibashi N, Ono I, Kato K, Shigenaga T, Shinoda H, Okai H, et al. Role of the hydrophobic amino acid residue in the bitterness of peptides. Agric Biol Chem 1988;52(1):91 – 4. Matoba T, Hata T. Relationship between bitterness of peptides and their chemical structure. Agric Biol Chem 1972;36(8):1423 – 31. Ney KH. Prediction of bitterness of peptides from their amino acid composition. Z Lebensm-Unters Forsch 1971;147:64 – 8. Raksakulthai R, Haard NF. Exopeptidases and their application to reduce bitterness in food: a review. Crit Rev Food Sci Nutr 2003;43(4):401 – 45. Spellman D, O’Cuinn G, FitzGerald RJ. Physicochemical and sensory characteristics of whey protein hydrolysates generated at different total solids levels. J Dairy Res 2005;72:138 – 43.
Research review paper
Enzymatic debittering of food protein hydrolysates R.J. FitzGerald a,*, G. O’Cuinn b a
b
Life Sciences Department, University of Limerick, Ireland Life Sciences Department, Galway Mayo Institute of Technology, Galway, Ireland Available online 4 January 2006
Abstract Protein hydrolysates have a range of applications in the food and allied healthcare sectors. Bitterness is a negative attribute associated with most food protein hydrolysates. The development of biotechnological solutions for hydrolysate debittering is ongoing. Specific enzymatic debittering strategies have focused on the application of proline specific exo- and endopeptidases given the contribution of proline residues to peptide/hydrolysate bitterness. Hydrolysate manufacturing conditions may also play an important role in bitterness development. Practical solutions to hydrolysate debittering are likely to involve judicious choice of enzymatic processing conditions in conjunction with the use of peptidase activities having targeted hydrolytic specificity. D 2005 Elsevier Inc. All rights reserved. Keywords: Protein hydrolysate; Debittering; Peptidases
Contents 1. Introduction . . . . . . . . . . . . . . . . . . 2. Hydrolysate functionality and applications . . . 3. Food protein hydrolysate bitterness . . . . . . 4. Quantification of hydrolysate/peptide bitterness 5. Protein hydrolysate debittering . . . . . . . . . 6. Peptidase mediated debittering . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
1. Introduction Proteins from animal (e.g., milk, meat and fish) and plant (e.g., soy, legume, cereal) origin are subjected to hydrolysis during food processing. Hydrolysis occurs via two main routes, i.e., during food * Corresponding author. Tel.: +353 61 202598; fax: +353 61 331490. E-mail address: [email protected] (R.J. FitzGerald). 0734-9750/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2005.11.002
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234 235 235 235 235 235 237
fermentations as a result of the action of bacterial/ fungal proteinase and peptidases activities, and during the direct manufacture of protein hydrolysates per se. Hydrolysis significantly impacts the functional and sensory properties of the resultant hydrolysate. The release of bitter tasting peptides is a negative aspect associated with most food protein hydrolysates. Different enzyme-based strategies may be employed in reducing the bitterness associated with hydrolysis of food protein.
R.J. FitzGerald, G. O’Cuinn / Biotechnology Advances 24 (2006) 234–237
235
2. Hydrolysate functionality and applications
4. Quantification of hydrolysate/peptide bitterness
Food protein hydrolysates have a wide range of applications, e.g., as nitrogen fortification agents in specialist beverages, as pre-digested ingredients for enteral/parenteral nutrition for general/specific population segments, as enriched/isolated peptide preparations for beneficial physiological function, as ingredients in cell culture/bacteriological growth media and as ingredients in cosmetics and healthcare products. It is possible, largely depending on enzyme specificity and the degree of hydrolysis (DH) achieved, to generate hydrolysate products with either enhanced or reduced functionality, e.g., solubility, emulsification, foaming and gelation properties. Hydrolysis also results in a reduction in allergenic potential by the destruction of allergenic epitopes. Significant current interest exists in the hydrolytic release of bioactive peptide sequences encrypted within the primary structure of food proteins (FitzGerald and Meisel, 2003).
The ideal route for quantification of hydrolysate bitterness is to use sensory evaluation panels (Spellman et al., 2005). This is a very time consuming activity requiring appropriate numbers of panellist and training to detect bitterness in order to obtain statistically relevant data. However, instrumental methods which distinguish hydrophobic peptides within hydrolysates such as reversed-phase chromatography and Fourier transform infrared (FTIR) spectroscopy in combination with multivariate data analysis may find application in fingerprinting bitterness in hydrolysates.
3. Food protein hydrolysate bitterness Intact food proteins do not display bitterness as their molecular size militates against their ability to interact extensively with bitterness receptors in the oral cavity. Bitterness in protein hydrolysates has been classically associated with the release of peptides containing hydrophobic amino acid residues. Ney (1971) quantified peptide bitterness on the basis of the hydrophobicity of the side-chains associated with the residues within a given peptide sequence. This was calculated from the free energy of transfer of an amino acid side chain from ethanol to water. From synthetic peptide studies, Ney reported that peptides with hydrophobicity ( Q) values N 1400 cal per mole and molecular masses b 6 kDa display bitterness. However, peptides with Q values b 1300 cal per mole along with peptides with Q values N 1400 1400 cal per mole and molecular masses N 6 kDa would not be bitter. Therefore, peptides b 6 kDa having a high content of Leu, Pro, Phe, Tyr, Ile and Trp residues are likely to be bitter. Matoba and Hata (1972) subsequently reported that the presence of internally sited hydrophobic amino acid residues led to greater bitterness than when the hydrophobic residues were located at either the N- or C-terminus in peptides. The presence of internally sited Pro residues was shown to be a major and distinct contributor to peptide bitterness due to the unique conformation associated with this imino acid (Ishibashi et al., 1988).
5. Protein hydrolysate debittering Numerous options have been investigated in the debittering of food protein hydrolysates. These include absorption of bitter peptides on activated carbon, chromatographic removal using different matrices and selective extraction with alcohols. These procedures, however, lead to the loss of some amino acid residues from hydrolysates. Bitterness has also been masked in hydrolysates via the addition of polyphosphates, specific amino acids such as Asp and Glu, a-cyclodextrins and by the admixture of hydrolysates with intact protein samples. The debittering of hydrolysates via transpeptidation reactions in the dso-calledT plastein reaction in addition to cross-linking using transglutaminase represent other potential routes for hydrolysate bitterness reduction. However, these latter protocols may not always be suitable when products having high solubility are required. 6. Peptidase mediated debittering Much effort has concentrated on the use of peptidases, specifically exopeptidases including amino- and carboxypeptidases, in food protein hydrolysate debittering (for review see Raksakulthai and Haard, 2003 and references therein). Significant reductions in bitterness have been observed in proteinase digests of food proteins during concomitant or subsequent incubation with exopeptidase-rich enzyme preparations, particularily peptidases which cleave adjacent to hydrophobic amino acid residues. Given the unique contribution of Pro residues to hydrolysate bitterness, much research has focused on the application of proline specific exopeptidases in hydrolysate debittering strategies due to the general inability of most general aminopeptidases to hydrolyse the imino bond. Highly significant reductions in casein hydrolysate bitterness could be achieved using
236
R.J. FitzGerald, G. O’Cuinn / Biotechnology Advances 24 (2006) 234–237
post-proline dipeptidyl aminopeptidase (PPDA), which releases amino acyl proline residues from the N-terminus, in conjunction with a general aminopeptidase activity. Fig. 1 outlines the mechanism by which combinations of proline specific aminopeptidases (i.e. PPDA and aminopeptidase P, which removes the Nterminal amino acid where proline is present in the second position) could mediate hydrolysis of proline rich substrates, i.e., containing single and consecutive prolines (Bouchier et al., 1999).
(a)
The application of proline-specific endopeptidase activites, which hydrolyse peptides releasing C-terminal prolines, is an interesting development in the generation of low/reduced bitterness food protein hydrolysates. This has led to development of procedures for the overexpression of proline-specific endopeptidase activities, particularily from Aspergillus niger, for the generation of protein hydrolysates enriched in C-terminal proline residues (Edens et al., 2004 and references therein).
PPDA KpNA-H
KpNA-H
PPDA
Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln
Pep P
αs1-casein f(1-9)
KpNA-H KpNA-H
(b) KpNA-H
His-Pro-Ile-Lys-His-Gln
αs1-casein f(4-9)
Pep P
(c)
Pep P Pep P
PPDA KpNA-H
Val-Pro-Pro-Phe-Leu-Gln
β-casein f(84-89)
PPDA Fig. 1. Schematic representation of the potential routes of hydrolysis for peptides containing non-consecutive proline residues, i.e. (a) as1-casein f(1–9), (b) as1-casein f(4–9) and a peptide containing consecutive proline residues, i.e. (c) h-casein f(84–89) using Lys-paranitroanilide hydrolase (KpNA-H; a general aminopeptidase), post-proline dipeptidyl aminopeptidase (PPDA) and aminopeptidase P (Pep P) from Lactococcus lactis subsp. cremoris AM2 (taken from Bouchier et al., 1999).
R.J. FitzGerald, G. O’Cuinn / Biotechnology Advances 24 (2006) 234–237
All these results indicate that specificity has a major role to play in choosing an enzymatic strategy for hydrolysate debittering. However, reaction conditions during hydrolysate manufacture may also play a highly significant role in the bitterness associated with the resultant hydrolysate. It has recently been demonstrated that the total solids (TS) content in the hydrolysis reaction can significantly affect bitterness generation. Hydrolysates of whey protein concentrate at equivalent DH values (15%) were significantly less bitter when generated at 300 g TS/L compared to hydrolysates generated at 50 g TS/ L (Spellman et al., 2005). The explanation for this difference was attributed, in part, to the presence of a specific hydrophobic peptide at higher concentration in the hydrolysate generated at lower TS. In conclusion, these results indicate the importance of carefully choosing reaction conditions, in addition to the specificity of the enzyme preparation, in the generation of reduced bitterness food protein hydrolysates. References Bouchier P, FitzGerald RJ, O’Cuinn G. Hydrolysis of as1-and hcasein-derived peptides with a broad specificity aminopeptidase and proline specific aminopeptidases from Lactococcus lactis supsp cremoris AM2. FEBs Lett 1999;445:321 – 4.
237
Edens L, Hoevan RAM, vander Delest V. Protein hydrolysates enriched in peptides having a carboxy terminal proline residues. United States Patent Application 2004 0241791. FitzGerald RJ, Meisel HM. Milk protein hydrolysis and bioactive peptides. In: Fox PF, McSweeney P, editors. Advanced dairy chemistry, Third Edition, Part B. New York7 Kluwer Academic/ Plenum Publishers; 2003. p. 675 – 98. Ishibashi N, Ono I, Kato K, Shigenaga T, Shinoda H, Okai H, et al. Role of the hydrophobic amino acid residue in the bitterness of peptides. Agric Biol Chem 1988;52(1):91 – 4. Matoba T, Hata T. Relationship between bitterness of peptides and their chemical structure. Agric Biol Chem 1972;36(8):1423 – 31. Ney KH. Prediction of bitterness of peptides from their amino acid composition. Z Lebensm-Unters Forsch 1971;147:64 – 8. Raksakulthai R, Haard NF. Exopeptidases and their application to reduce bitterness in food: a review. Crit Rev Food Sci Nutr 2003;43(4):401 – 45. Spellman D, O’Cuinn G, FitzGerald RJ. Physicochemical and sensory characteristics of whey protein hydrolysates generated at different total solids levels. J Dairy Res 2005;72:138 – 43.