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Lesson 16. DEBITTERING OF PROTEIN HYDROLYSATES
Module 2. Skim milk and its by-products
Lesson 16
DEBITTERING OF PROTEIN HYDROLYSATES
16.1 Introduction
Bitterness, which is a natural consequence of development of bitter-tasting components during enzymatic hydrolysis of protein, is the main defect that limits the use of protein hydrolysates for human consumption. Consequently, the reduction, prevention or removal of bitterness from protein hydrolysates has been investigated intensively. Debittering of the protein hydrolysates by different techniques like selective separation of bitter components, masking, enzymatic treatment and palstein reaction, are the key to the production of an acceptable protein hydrolysate, which can be used in the formulation of novel functional foods for geriatric, sports nutrition, weight control, anti milk allergic foods and for special medical diets like opiodic, anti hypersensitive and antimicrobial active foods.
16.2 Application of exopeptides
Free amino acids are much less bitter than the corresponding peptides, and the bitterness is highest when the hydrophobic amino acids are non-terminal. Bitter peptides in casein hydrolysates can be degraded by the application of carboxypeptidases into non-bitter peptides. Minagawa et al. (1989) reported that aminopeptidase T hydrolyses hydrophobic amino acid residues at N-terminal of peptides and proteins and hence remove bitter components from bitter peptides. Flavourzyme is the enzyme obtained from Aspergillus oryzae which has both exo- and endo- peptidase activity. This helps in debittering the protein hydrolysate.
Debittering methods using exopeptidases encounters certain limitations. The use of exopeptidases results in very high degree of hydrolyses. The hydrolysates will consist mainly of free amino acids and small peptides, resulting in high osmolality, which may limit its use.
16.3 Selective separation of bitter components
Selective separation of bitter components was first applied in 1952 for treating casein hydrolysate with activated carbon. This separation is based on the adsorption of hydrophobic group by means of the followings:
The activated carbon treatment method has been found safe, promising and most effective in the elimination of the bitter taste. Activated carbon functions as a hydrophobic adsorbent and as such binds peptides and amino acids that are mainly hydrophobic and bitter tasting. This however results, in about 26% loss of protein nitrogen. The treatment of activated carbon lowers the bitterness of casein hydrolysates.
Bitterness in the hydrolysates obtained from Rhozyme 62 was eliminated by employing 0.5 g of activated carbon per g protein and storing it for 60 min at 25°C. However, such treatment was accompanied by a selective loss of tryptophan (63%), phenylalanine (36%), arginine (30%) and 26±2% of protein nitrogen due to adsorption of peptides and amino acids on to the activated carbon. As remedial measure, supplementation of the treated hydrolysates with the proper amounts of tryptophan and phenylalanine was suggested for the production of casein hydrolysates of acceptable taste and high nutritive quality.
Soluble casein hydrolysates for use in acid beverages could be obtained by stirring 5% casein hydrolysates solutions with 10% carbon of various kinds and mesh sizes at 22°C for 2 h or at 90°C for 10 min. Khanna and Gupta (1996) observed that minimum 15% activated carbon treatment was necessary for debittering the casein hydrolysate, though this treatment resulted in 40.90% N loss through adsorption on activated carbon. The yield and recovery of liquid casein hydrolysate were 47.98% and 46.23%, respectively. The liquid product had 10.25% TS, 1.93% nitrogen, 1.19% ash and a low viscosity of 1.99 cP at 20°C.
16.3.2 Hydrophobic chromatography
During chromatography, binding forces occurring between the structurally similar phenolic resin and peptide amino acid residues (containing aromatic/heterocyclic side chains) delay the emergence of these bitter components, permitting selective preparation of a non-bitter peptide hydrolysate. However, this method is reported to be impractical and costly for application to casein hydrolysates. Debittering by hydrophobic chromatography on hexyl Sepharose 6B has also been described.
16.3.3 Azeotropic mixture of secondary butanol and water
Selective extraction of bitter peptides using an azeotropic mixture of secondary butanol and water has also been observed to be very effective in reducing bitterness.
16.4 Masking
Masking of bitter compounds is advantageous over selective separation of bitter compounds as some of the bitter compounds are removed by separation technique. In masking, bitter peptides/components are retained for nutrition or medicinal property. A number of components have been shown to mask bitter taste of protein hydrolysates. These components are:
Masking of bitterness by adding acidic dipeptide and acidic amino acids like aspartic acid, glutamic acid and taurine have been reported. Glutamic acid or aspartic acid can be used to mask bitterness by weakening the bitter compound. Taurine in acid solution reduces bitterness without imparting sourness and is as effective as glutamic acid and aspartic acid.
16.4.2 Fatty substances
Fatty substances such as creaming powder, margarine and vegetable oil exert varying effect in their debittering effectiveness probably because of their affinity for hydrophobic compounds.
16.4.3 Cyclodextrin
Cyclodextrin can also mask bitterness because of its ability to wrap up the hydrophobic groups of bitter peptides, but its safety as a food additive still needs to be established. The bitterness from casein hydrolysates can be removed through treatment of 10% ß- cyclodextrin.
16.4.4 Glycine
Glycine, an amino acid, has been characterized as sweet and has sweetness equivalent to a 0.45 percent sucrose solution at 0.30 percent in aqueous solution. Addition of 0.5 percent glycine in a protein hydrolysate could mask the bitterness, resulting in a sweet taste, which, however, may not always be desirable in the product.
16.4.5 Gelatin
Gelatin is as effective as cyclodextrin in debittering, although not as good as glycine. When gelatin is added along with the protein for hydrolysis, the enzymes used for hydrolysis (trypsin, chemotrypsin/flavourze) also attack gelatin along with the desired protein. It seems that gelatin could donate hydrolysate substances including glycine, which are capable of masking bitterness.
16.4.6 Polyphosphates and starch
Addition of polyphosphates during the hydrolysis process could successfully mask the bitterness of casein hydrolysates; addition of hexametaphosphate @ 0.1% before hydrolysis is recommended.
Bitterness could be masked using gelatinized starch as the bitter peptides get hidden in the network structure of starch and thereby prevent them from reaching the bitter taste buds. To achieve this effect, it is necessary to heat the mixture of starch and bitter peptides.
The plastein reaction has been claimed to yield the following advantages:
Selected references
Jenny Ann John and Ghosh, B.C. 2008. Bitterness and its reduction in milk protein hydrolyzates. Indian Dairyman, 60 (1): 36-45.
Khanna, R.S. and Gupta, V.K. 1996. Process optimization for the production of buffalo milk casein hydrolysate. Indian J. Dairy Sci., 49: 386.
Minagawa, E, Kaminogawa, S., Tsukasaki, F, and Yamauchi, K. 1989. Debittering mechanism in bitter peptides of enzymic hydrolysates from milk casein by aminopeptidase T. J. Food Sci, 54: 1225-1231.
Bitterness, which is a natural consequence of development of bitter-tasting components during enzymatic hydrolysis of protein, is the main defect that limits the use of protein hydrolysates for human consumption. Consequently, the reduction, prevention or removal of bitterness from protein hydrolysates has been investigated intensively. Debittering of the protein hydrolysates by different techniques like selective separation of bitter components, masking, enzymatic treatment and palstein reaction, are the key to the production of an acceptable protein hydrolysate, which can be used in the formulation of novel functional foods for geriatric, sports nutrition, weight control, anti milk allergic foods and for special medical diets like opiodic, anti hypersensitive and antimicrobial active foods.
16.2 Application of exopeptides
Free amino acids are much less bitter than the corresponding peptides, and the bitterness is highest when the hydrophobic amino acids are non-terminal. Bitter peptides in casein hydrolysates can be degraded by the application of carboxypeptidases into non-bitter peptides. Minagawa et al. (1989) reported that aminopeptidase T hydrolyses hydrophobic amino acid residues at N-terminal of peptides and proteins and hence remove bitter components from bitter peptides. Flavourzyme is the enzyme obtained from Aspergillus oryzae which has both exo- and endo- peptidase activity. This helps in debittering the protein hydrolysate.
Debittering methods using exopeptidases encounters certain limitations. The use of exopeptidases results in very high degree of hydrolyses. The hydrolysates will consist mainly of free amino acids and small peptides, resulting in high osmolality, which may limit its use.
16.3 Selective separation of bitter components
Selective separation of bitter components was first applied in 1952 for treating casein hydrolysate with activated carbon. This separation is based on the adsorption of hydrophobic group by means of the followings:
- Activated carbon
- Hydrophobic chromatography
- Hexylepoxy cellulose, soft glass fibre, flint glass powder or micro fibre paper.
- Azeotropic mixture of secondary butanol and water.
- Phenolic formaldehyde resin.
- Aqueous ethanol
The activated carbon treatment method has been found safe, promising and most effective in the elimination of the bitter taste. Activated carbon functions as a hydrophobic adsorbent and as such binds peptides and amino acids that are mainly hydrophobic and bitter tasting. This however results, in about 26% loss of protein nitrogen. The treatment of activated carbon lowers the bitterness of casein hydrolysates.
Bitterness in the hydrolysates obtained from Rhozyme 62 was eliminated by employing 0.5 g of activated carbon per g protein and storing it for 60 min at 25°C. However, such treatment was accompanied by a selective loss of tryptophan (63%), phenylalanine (36%), arginine (30%) and 26±2% of protein nitrogen due to adsorption of peptides and amino acids on to the activated carbon. As remedial measure, supplementation of the treated hydrolysates with the proper amounts of tryptophan and phenylalanine was suggested for the production of casein hydrolysates of acceptable taste and high nutritive quality.
Soluble casein hydrolysates for use in acid beverages could be obtained by stirring 5% casein hydrolysates solutions with 10% carbon of various kinds and mesh sizes at 22°C for 2 h or at 90°C for 10 min. Khanna and Gupta (1996) observed that minimum 15% activated carbon treatment was necessary for debittering the casein hydrolysate, though this treatment resulted in 40.90% N loss through adsorption on activated carbon. The yield and recovery of liquid casein hydrolysate were 47.98% and 46.23%, respectively. The liquid product had 10.25% TS, 1.93% nitrogen, 1.19% ash and a low viscosity of 1.99 cP at 20°C.
16.3.2 Hydrophobic chromatography
During chromatography, binding forces occurring between the structurally similar phenolic resin and peptide amino acid residues (containing aromatic/heterocyclic side chains) delay the emergence of these bitter components, permitting selective preparation of a non-bitter peptide hydrolysate. However, this method is reported to be impractical and costly for application to casein hydrolysates. Debittering by hydrophobic chromatography on hexyl Sepharose 6B has also been described.
16.3.3 Azeotropic mixture of secondary butanol and water
Selective extraction of bitter peptides using an azeotropic mixture of secondary butanol and water has also been observed to be very effective in reducing bitterness.
16.4 Masking
Masking of bitter compounds is advantageous over selective separation of bitter compounds as some of the bitter compounds are removed by separation technique. In masking, bitter peptides/components are retained for nutrition or medicinal property. A number of components have been shown to mask bitter taste of protein hydrolysates. These components are:
- Amino acids ( aspartic acid, glutamic, taurine in acid solution and glycine)
- Fatty substances
- Cyclodextrin
- Gelatin
- Polyphosphates and starch
Masking of bitterness by adding acidic dipeptide and acidic amino acids like aspartic acid, glutamic acid and taurine have been reported. Glutamic acid or aspartic acid can be used to mask bitterness by weakening the bitter compound. Taurine in acid solution reduces bitterness without imparting sourness and is as effective as glutamic acid and aspartic acid.
16.4.2 Fatty substances
Fatty substances such as creaming powder, margarine and vegetable oil exert varying effect in their debittering effectiveness probably because of their affinity for hydrophobic compounds.
16.4.3 Cyclodextrin
Cyclodextrin can also mask bitterness because of its ability to wrap up the hydrophobic groups of bitter peptides, but its safety as a food additive still needs to be established. The bitterness from casein hydrolysates can be removed through treatment of 10% ß- cyclodextrin.
16.4.4 Glycine
Glycine, an amino acid, has been characterized as sweet and has sweetness equivalent to a 0.45 percent sucrose solution at 0.30 percent in aqueous solution. Addition of 0.5 percent glycine in a protein hydrolysate could mask the bitterness, resulting in a sweet taste, which, however, may not always be desirable in the product.
16.4.5 Gelatin
Gelatin is as effective as cyclodextrin in debittering, although not as good as glycine. When gelatin is added along with the protein for hydrolysis, the enzymes used for hydrolysis (trypsin, chemotrypsin/flavourze) also attack gelatin along with the desired protein. It seems that gelatin could donate hydrolysate substances including glycine, which are capable of masking bitterness.
16.4.6 Polyphosphates and starch
Addition of polyphosphates during the hydrolysis process could successfully mask the bitterness of casein hydrolysates; addition of hexametaphosphate @ 0.1% before hydrolysis is recommended.
Bitterness could be masked using gelatinized starch as the bitter peptides get hidden in the network structure of starch and thereby prevent them from reaching the bitter taste buds. To achieve this effect, it is necessary to heat the mixture of starch and bitter peptides.
16.5 Plastein reaction
The plastein reaction is a process in which proteins are broken down by proteolytic enzymes into a mixture of peptides and amino acids and are then resynthesized enzymatically into products which consists of insoluble, highly aggregated materials having different structural, compositional and functional properties from that of initial protein. The synthesis of plastein involves following steps:
i. The protein is enzymatically hydrolysed to a low molecular weight mixture of peptides.
ii. After hydrolysis, the impurities which cause undesirable odours, colours and flavours are removed. Hydrolysis, however, results in a characteristic bitterness which has been attributed to peptides with more than three amino-acids. This material serves as substrate for the third step of plastein synthesis.
iii. During plastein synthesis, a high concentration of hydrolysate (30-50%) is incubated with an enzyme or heated to form viscous gel like material. (Fig. 16.1: Plastein reaction for debittering of casein hydrolysates)
ii. After hydrolysis, the impurities which cause undesirable odours, colours and flavours are removed. Hydrolysis, however, results in a characteristic bitterness which has been attributed to peptides with more than three amino-acids. This material serves as substrate for the third step of plastein synthesis.
iii. During plastein synthesis, a high concentration of hydrolysate (30-50%) is incubated with an enzyme or heated to form viscous gel like material. (Fig. 16.1: Plastein reaction for debittering of casein hydrolysates)
i. Elimination of bitterness present in the hydrolysate.
ii. Possibility of incorporating limiting essential amino acids into the plastein to form a protein like material with increased nutritional quality.
iii. Improvement in the acceptability and nutritional quality of a protein, as well as, alternation in physical properties.
iv. Facilitating the amino acids fortification in food by reducing loss during processing due to less solubility and thus helping in improving the amino-acid profile.
ii. Possibility of incorporating limiting essential amino acids into the plastein to form a protein like material with increased nutritional quality.
iii. Improvement in the acceptability and nutritional quality of a protein, as well as, alternation in physical properties.
iv. Facilitating the amino acids fortification in food by reducing loss during processing due to less solubility and thus helping in improving the amino-acid profile.
Selected references
Jenny Ann John and Ghosh, B.C. 2008. Bitterness and its reduction in milk protein hydrolyzates. Indian Dairyman, 60 (1): 36-45.
Khanna, R.S. and Gupta, V.K. 1996. Process optimization for the production of buffalo milk casein hydrolysate. Indian J. Dairy Sci., 49: 386.
Minagawa, E, Kaminogawa, S., Tsukasaki, F, and Yamauchi, K. 1989. Debittering mechanism in bitter peptides of enzymic hydrolysates from milk casein by aminopeptidase T. J. Food Sci, 54: 1225-1231.
Last modified: Tuesday, 16 October 2012, 8:51 AM