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Research review paper
Debittering of protein hydrolyzates
Badal C. Sahaa,*, Kiyoshi Hayashib
aFermentation Biochemistry Research Unit, National Center for Agricultural Utilization Research,
Agricultural Research Service, US Department of Agriculture1, 1815 North University Street,
Peoria, IL 61604, USAbEnzyme Applications Laboratory, National Food Research Institute, Ministry of Agriculture, Forestry and
Fisheries, Tsukuba, Ibaraki 305, Japan
Abstract
Enzymatic hydrolysis of proteins frequently results in bitter taste, which is due to the formation of
low molecular weight peptides composed of mainly hydrophobic amino acids. Methods for
debittering of protein hydrolyzates include selective separation such as treatment with activated
carbon, extraction with alcohol, isoelectric precipitation, chromatography on silica gel, hydrophobic
interaction chromatography, and masking of bitter taste. Bio-based methods include further hydrolysis
of bitter peptides with enzymes such as aminopeptidase, alkaline/neutral protease and carboxypepti-
dase, condensation reactions of bitter peptides using protease, and use of Lactobacillus as a
debittering starter adjunct. The causes for the production of bitter peptides in various food protein
hydrolyzates and the development of methods for the prevention, reduction, and elimination of
bitterness as well as masking of bitter taste in enzymatic protein hydrolyzates are presented. Published
by Elsevier Science Inc.
Keywords: Protein hydrolyzate; Bitter peptides; Debittering; Protease; Aminopeptidase; Carboxypeptidase;
Lactobacillus; Bitterness masking
0734-9750/01/$ – see front matter. Published by Elsevier Science Inc.
PII: S0734 -9750 (01 )00070 -2
* Corresponding author. Tel.: +1-309-681-6276; fax: +1-309-681-6427.
E-mail address: [email protected] (B.C. Saha).1 Names are necessary to report factually on available data; however, the USDA neither guarantees nor
warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the
exclusion of others that may also be suitable.
Biotechnology Advances 19 (2001) 355–370
1. Introduction
Proteins are modified by treatment with proteolytic enzymes for the purpose of improving
their solubility, heat stability, and resistance to precipitation in acidic environments. Protein
hydrolyzates can be used as emulsifying agents in a number of applications such as salad
dressings, spreads, ice cream, coffee whitener, and emulsified meat products like sausages or
luncheon meat. However, the enzymatic treatment of various food proteins results in a bitter
taste due to the formation of low molecular weight peptides composed mainly of hydrophobic
amino acids. Thus, the formation of bitter peptides is the most serious problem in the practical
use of food protein hydrolyzates. In this review article, the authors describe the causes of
production of bitter peptides in various food protein hydrolyzates and progress to date in the
development of methods for the prevention, reduction, and elimination of bitterness in
enzymatic food protein hydrolyzates.
2. Formation of bitter peptides in protein hydrolyzates
According to the hypothesis of Ney (1979), bitterness is related to the average hydro-
phobicity of the peptide. The average hydrophobicity of peptide (Q value) is defined as the
sum of the free energies of transfer of the amino acid side chains from ethanol to water,
divided by the number of amino acid residues in the peptide, i.e.
Q ¼X
Dg=n
where Dg is the transfer free energy and n is the number of amino acid residues. A peptide is
almost certainly bitter when its Q value exceeds 1400 cal/mol (Fukui et al., 1983; Otagiri et
al., 1983). Ney and Gisela (1986) developed a computer program to predict the bitterness of
peptides, especially in protein hydrolyzates, based on amino acid composition and chain
length. The bitterness of peptides is caused by the hydrophobic action of its amino acid
residues but is limited by its molecular weight. It could be predicted based on a calculation
(Q rule) taking into account only the amino acid composition (and the resultant chain length).
Application of this rule to proteins allows prediction of whether bitterness would occur in the
hydrolyzate and how to avoid this by controlling the degree of hydrolysis (DH). Proteins with
high Q value such as casein (1605 cal/mol), soybean protein (1540 cal/mol), and zein (1480
cal/mol) would give to bitter peptides. The bitterness seems to be related to a high DH (Adler-
Nissen et al., 1978). DH is defined as the percentage of cleaved peptide bonds and serves as
the controlling parameter for the hydrolysis process. Small peptides have been shown to be
bitter if they contain predominately hydrophobic amino acid residues. Gulgoz and Solms
(1976) detected about 206 bitter peptides with the majority being between 2 and 15 amino
acid residues and two having more than 20 amino acid residues. Endoproteases with a broad
specificity have a tendency of hydrolyzing at hydrophobic amino acid residues, leaving a
nonpolar amino acid residue at the C-terminus of the peptide formed. This leads to relatively
high bitterness (Adler-Nissen, 1986).
B.C. Saha, K. Hayashi / Biotechnology Advances 19 (2001) 355–370356
The bitterness of isolated peptides is usually evaluated by sensory evaluation using a
group of sensory test panels. This is done by comparing the protein hydrolyzate with a
standard bitter solution such as quinine sulfate solution. The degree of bitterness is expressed
in mole concentration of quinine sulfate solution (Matsuoka et al., 1991). The effect of
debittering agents on the a- and b-casein (CN) hydrolyzates can be detected and evaluated
qualitatively because the tryptic hydrolyzates of them have been well studied and some bitter
peptide sequences have been identified. Without sensory evaluation, one can compare the
chromatogram of the hydrolyzates before and after addition of the specific debittering agent
and then predict whether or not it possesses a powerful ability to debitterize (Habibi-Najafi
and Lee, 1996).
2.1. Milk protein
b-CN contains three Pro residues in a sequence of 209 amino acids (O’Cuinn et al., 1999).
Gulgoz and Solms (1974) isolated a bitter-tasting peptide, Leu–Trp, from Alpkaese, a Swiss
mountain cheese. The peptide corresponds to residues 198–199 of the carboxyl end of
as1-CN. A preparative procedure including water extraction, membrane ultrafiltration, and
reverse-phase HPLC separation was developed to isolate bitter peptides in cheddar cheese
(Lee and Warthesen, 1996). The most bitter fractions in cheese samples were in a molecular
weight range of either 500 and 3000 or > 3000. These isolated peptides contained a high level
of hydrophilic amino acid residues such as Glu/Gln and Ser. Hydrophobic amino acid
residues such as Leu, Ile, and Pro were high in some bitter peptide fractions, but Phe and Tyr
were absent in all isolated bitter peptides. The Bacillus subtilis protease action on bovine
casein resulted in the production of a variety of hydrophobic, low molecular weight
( < 10 000) peptides, due to more extensive proteinase activity (Gallagher et al., 1994a).
Bromelain action, however, resulted in a hydrophobic hydrolyzate containing a number of
high molecular weight (>10 000) peptides, which are not normally associated with flavor
fractions. Vreeman et al. (1994) developed a rapid procedure for isolating the carboxy-
terminal fragment 193–209 (the bitter C-peptide) of b-CN on a preparative scale. The
C-peptide was preferentially cleaved from b-CN by chymosin and subsequently purified by
acid precipitation of residual large b-CN fragments and ultrafiltration. Bumberger and Belitz
(1993) showed that hydrophobicity or size alone is not responsible for bitter potency in the
case of larger peptides obtained from the tryptic hydrolyzate of casein and conformational
parameters must be of great importance. Tchorbanov and Iliev (1993) reported that
proteinases caused limited hydrolysis of casein in the presence of 20–60% (v/v) ethanol.
Hydrolysis by subtilisin resulted in more bitter peptides with increased ethanol concentration,
whereas trypsin and a-chymotrypsin action led to products with reduced bitterness. Le Bars
and Gripon (1989) characterized the milk plasmin cleavage of as2-CN. Five as2-CN sites
were particularly sensitive to plasmin. Three peptides from the C-terminus of as2-CN were
hydrophobic (1540–1790 cal/residue) and similar to bitter peptides of cheese (Visser et al.,
1975). Hynek et al. (1999) measured the activity of acid phosphatase during the 120 days of
ripening of Gouda cheese made with five different mesophilic starters. Low activity of acid
phosphatase led to accumulation of hydrophobic, often bitter, peptides that are undesirable for
B.C. Saha, K. Hayashi / Biotechnology Advances 19 (2001) 355–370 357
final sensory properties of cheese. Renz and Puhan (1975) concluded that the major cause of
bitterness in some yogurt is the proteolytic activity of Lactobacillus bulgaricus during
storage, based on correlations between increases of lactic acid and bitterness and lactic acid
and whey nonprotein nitrogen. Huber and Klostermeyer (1974) isolated a bitter peptide with
the sequence Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser from the water soluble extract of the
bitter cheese Butterkaese. Clegg et al. (1974) reported that digestion of the unfractionated
bovine casein with papain produced a bitter-tasting peptide that was derived from the residues
53–79 of b-CN. Hamilton et al. (1974) isolated a bitter peptide (molecular weight: 2500)
from cheddar cheese whose N-terminus represented residue 46 (Gln) of that of casein.
Matoba et al. (1970) isolated three bitter peptides from a tryptic hydrolyzate of casein. The
primary structures of these peptides were: Gly-Pro-Phe-Pro-Val-Ile, Phe-Phe-Val-Ala-Pro-
Phe-Pro-Glu-Val-Phe-Gly-Lys, and Phe-Ala-Leu-Pro-Gln-Tyr-Leu-Lys (Table 1). Minamiura
et al. (1972b) isolated a bitter peptide consisting of equimolar Trp and Leu from B. subtilis
alkaline proteinase-digested casein. Takahashi et al. (1995) converted bitterness of the
C-terminal octapeptide of bovine b-CN (Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val) into sweetness
(667-fold stronger than sucrose) by substituting both Arg and Phe by Glu residues. Bachmann
and Farah (1982) reported that the occurrence of bitter taste in mixtures of milk proteins and
kiwi fruit (Actinidia chinensis) was due to the presence of a very active caseinolytic protease
in kiwi fruit, which splits casein into bitter peptides.
2.2. Soybean protein
The bitterness of soybean protein hydrolyzates prepared by Alcalase (a commercial
preparation of subtilisin Carlsberg from B. licheniformis obtained from Novo Nordisk,
Table 1
Some bitter peptides isolated from protein hydrolyzates
Source Peptides Reference
Tryptic hydrolyzate
of casein
Gly-Pro-Phe-Pro-Val-Ile Matoba et al., 1970
Phe-Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys
Phe-Ala-Leu-Pro-Gln-Tyr-Leu-Lys
Bacterial proteinase
hydrolyzate of casein
Arg-Gly-Pro-Pro-Phe-Ile-Val Minamiura et al., 1972b
Beer yeast residue Trp-Phe, Trp-Pro, Leu-Pro-Trp Matsusita and Ozaki, 1993
Pepsin hydrolyzate of zein Ala-Ile-Ala, Ala-Ala, Leu, Gly-Ala-Leu, Wieser and Belitz, 1975
Leu-Gln-Leu, Leu-Glu-Leu, Leu-Val-Leu
Leu-Pro-Phe-Asn-Gln-Leu, Leu-Pro-Phe-Ser-Gln-Leu
Pepsin hydrolyzate
of hemoglobin
Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe Aubes-Dufau et al., 1995
Alkalase hydrolyzate
of hemoglobin
Val-Val-Tyr-Pro-Trp Aubes-Dufau and
Combes, 1997
Soy protein Arg–Leu, Arg-Leu-Leu, Ser-Lys-Gly-Leu Fujimaki et al., 1971
Bovine b-CN(C-terminal)
Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val Takahashi et al., 1995
B.C. Saha, K. Hayashi / Biotechnology Advances 19 (2001) 355–370358
Bagsvaerd, Denmark) treatment was mainly caused by hydrophobic bitter peptides of
molecular weight less than 1000 (Kukman et al., 1995, 1996). The formation of bitterness
during tryptic hydrolysis of soybean 11S glycinin was due to the formation of peptide
fractions of molecular weight 360–2100 in which case the acidic subunit of 11S glycinin was
mostly hydrolyzed (Kim et al., 1999). Yamasaki (1987) characterized a bitter peptide in natto
with the amino acid composition of Asp 1, Thr 1, Glu 1, Ala 1, Pro 2, Val 3, Ile 3, and Leu 5.
The N-terminus was Leu and the C-terminal sequence was -Ala-Val-Ile-Leu.
2.3. Corn protein zein
Wieser and Belitz (1975) isolated eight peptides with bitter taste from pepsin hydrolyzates
of zein using several chromatography procedures (Table 1). The threshold for bitter taste
decreased with increasing number of hydrophobic side chains (� 3) in the peptide. It
increased in the presence of hydrophilic side chains parallel to their polarity. Tanimoto et
al. (1991) hydrolyzed zein first with alkalophilic proteinase and then with actinase to liberate
aromatic amino acids. The hydrolyzate, however, tasted bitter.
2.4. Fish protein
Hevia and Olcott (1977) fractionated the soluble protein of fish protein concentrate,
obtained by treatment with bromelain, ficin, or pronase by size and charge. They found that
bitterness and Glu taste (acid-like) were the main contributors to off-flavors in the
hydrolyzates. Pronase hydrolyzate was less bitter than that from bromelain or ficin. A
basic bitter mixture isolated from the 800 molecular weight region of a ficin hydrolyzate
contained Gly, Ile, Phe, and Val as N-terminal residues. Further separation of this bitter
fraction indicated a basic bitter tripeptide or tripeptides with Asn as the middle amino acid
and Lys as the C-terminus.
2.5. Other proteins
Seki et al. (1996) hydrolyzed 12 kinds of food proteins by B. licheniformis alkaline
proteases to peptides of average chain length (2.26–4.02). Hydrophobic amino acid residues
situated in the interior of protein molecules were exposed by fragmentation with proteases,
and the peptides containing hydrophobic amino acid residues were found in aqueous solution.
The peptides from casein showed the highest hydrophobicity and most bitter taste. Matsusita
and Ozaki (1993) isolated oligopeptides (Trp-Phe, Trp-Pro, and Leu-Pro-Trp) of bitter taste
from the hydrolyzate of beer yeast residue (Table 1). Aubes-Dufau and Combes (1997)
investigated the effect of different proteases on bitterness of hemoglobin hydrolyzates. All
bitter peptides (molecular weight: 500–5000) belonged to the same fragment of the b-chainof bovine hemoglobin. Henriksen and Stahnke (1997) performed sensory and chromato-
graphic evaluation of water soluble fractions from dried sausages. They found that bitterness
was dependent on the level of hydrophobic amino acids present in these fractions.
B.C. Saha, K. Hayashi / Biotechnology Advances 19 (2001) 355–370 359
3. Synthetic bitter peptides
To investigate the role of the Pro residue in the bitter taste of peptides, Ishibashi et al. (1988)
synthesized some oligopeptides containing Pro and evaluated their tastes. Pro peptides
exhibited bitterness. The most significant role of the Pro residue in peptide bitterness was
dependent on the conformational alternation of the peptide molecule by folding the peptide
skeleton due to the imino ring of the Pro molecule. They concluded that two bitter taste
determinant sites were essential for peptide bitterness and should be adjacent in the steric
conformation of the peptides. Ishibashi et al. (1987b) synthesized some oligopeptides contain-
ing Phe or Tyr and evaluated their taste. The hydrophobicity of Phe or Tyr molecule caused the
marked bitter taste in peptides. The bitterness was more intense when Phe was located at the
C-terminus and when the content of Phe or Tyr was increased in peptides. The hydrophobicity
of Leu residues markedly caused the bitterness of peptides. Bitterness was always found when a
Leu residue was located at the C-terminus of peptides (Ishibashi et al., 1987a). Shinoda et al.
(1986) examined the bitterness of diastereomers of a hexapeptide (Arg-Arg-Pro-Pro-Phe-Phe)
containing D-Phe in place of L-Phe. The analogs with L-Phe at the C-terminal exhibited stronger
bitterness than those with D-Phe at the C-terminal. Evidently, the configuration of the
C-terminal hydrophobic amino acid is important for the increase in bitterness. Hashimoto et
al. (1980) synthesized a bitter heptapeptide, Gly-Pro-Phe-Pro-Ile-Ile-Val, and found it to be
indistinguishable from a natural peptide isolated from a tryptic hydrolyzate of Hammersten
casein. Nakatani et al. (1994) synthesized Arg-Gly-Pro-X-Pro-Ile-Ile-Val (I, X-Phe) and analog
I (X = D-Phe, Lys, Gly, Glu, or L-Phe) in order to elucidate the relationship between the chemical
structure and bitter taste of the C-terminal portion of b-CN. The location of the hydrophobic
amino acid with the L-configuration between the two Pro residues was shown to be important
for this series of peptides in order to produce strong bitterness. Tamura et al. (1990) synthesized
and tasted several O-aminoacyl sugars, in which amino acids or peptides were attached to the
2- and 3-position of methyl-a-D-glucopyranoside in order to study the role of hydrophobicity inbitter peptides. The bitterness increased as the hydrophobicity increased, implying that the
bitterness receptor recognizes the hydrophobicity of bitter peptides.
4. Debittering of protein hydrolyzates
Various attempts have been made to prevent, remove, eliminate, or mask the bitterness of
peptides, which otherwise limits the sensory acceptability of food proteins.
4.1. Treatment with activated carbon, extraction with alcohol, and isoelectric precipitation
Murray and Baker (1952) demonstrated that treatment of enzymic casein hydrolyzate with
activated carbon resulted in a substantial improvement in the taste of the preparations.
However, this method was impractical due to simultaneous loss of a large proportion of Trp
during treatment. Cogan et al. (1981) reported that debittering of the commercially available
proteolytic enzyme hydrolyzate of casein with activated carbon was accompanied by a
B.C. Saha, K. Hayashi / Biotechnology Advances 19 (2001) 355–370360
selective loss of Trp (63%), Phe (36%), and Arg (30%). Supplementation of the activated
carbon-treated debittered hydrolyzate with the proper amounts of Trp and Phe improved their
nutritional quality, which permits the production of casein hydrolyzate of acceptable taste of
high nutritive value. Tossavainen et al. (1996) removed the residual b-lactoglobulin from an
extensively hydrolyzed bitter whey protein hydrolyzate with a polystyrene-based adsorption
resin. This resin treatment removed principally macropeptides including the bitter peptides
and caused only slight changes in the amino acid composition of the product.
The bitter compounds of enzymatic protein hydrolyzates can be removed by the extraction
of azeotropic secondary butyl alcohol, aqueous ethanol, or aqueous isopropyl alcohol. The
bitter peptides are concentrated in the alcohol phase, which has an extremely bitter taste
(Lalasidis, 1978; Lalasidis and Sjoberg, 1978).
Hydrophobic peptides have very low solubility at around isoelectric pH. They can be
removed by isoelectric precipitation (pH adjustment) (Adler-Nissen, 1984).
4.2. Chromatographic separation
Visser et al. (1975) reported that chromatography on silica gel utilizing an organic solvent
such as propyl alcohol as a part of the eluent provides a suitable way for the purification of
bitter fractions from caseins and is also very promising for the fractionation of bitter peptides
from cheese.
Reduction in bitterness of protein hydrolyzates can be achieved by applying hydrophobic
interaction chromatography. Helbig et al. (1980) compared various adsorption methods for
debittering skim milk hydrolyzates in an attempt to develop acid-soluble milk solids for
fortification of beverages. They found that bitter peptides were mostly hydrophobic and could
be completely eliminated by hydrophobic chromatography on hexylepoxy Sepharose.
Activated carbon talc and b-cyclodextrin were effective in adsorbing bitter peptides. Two
bitter peptides were isolated from the butanol extract of the activated C used in debittering
pronase-hydrolyzed skim milk, which were derived from casein especially as1-CN. Rolandet al. (1978) developed a process for debittering casein and soybean protein hydrolyzates
based on the application of hydrophobic chromatography on phenolic formaldehyde resin.
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 and permit the selective preparation of a nonbitter
peptide hydrolyzate. van Leuwen (1978) developed a debittering method using an immune
specific adsorbent (antibody raised against tryptic hydrolyzate from casein) for selective
adsorption of hydrophobic peptides. However, this method was impractical because it was
difficult to dissociate the antigen–antibody complex again.
4.3. Masking of bitter taste
Noguchi et al. (1975) proposed a bitterness masking method by using monosodium
glutamate and several glutamyl oligopeptides. Matsuura et al. (1980) synthesized eight
structural and optical isomers of glutamylglutamic acid with similar bitter-masking effects
B.C. Saha, K. Hayashi / Biotechnology Advances 19 (2001) 355–370 361
against Leu (1%). Addition of polyphosphates (Tokita, 1969) and gelatin or glycine (Stanly,
1981) during hydrolysis decreased bitterness in protein hydrolyzates. Cyclodextrins can mask
bitterness by covering the hydrophobic parts of peptides inside their ring structures (Tamura et
al., 1990). Bitter taste of amino acids and peptides is controlled by adding acidic phospholipids
and lysophospholipids (0.01–50-fold by weight of amino acids and peptides) in health food
(Sugiura, 1996).
4.4. Enzymatic hydrolysis of bitter peptides
The purpose of enzymatic treatment is to hydrolyze bitter peptides further by exopeptidases.
4.4.1. Treatment with aminopeptidase
Aminopeptidases are exopeptidases that selectively release N-terminal amino acid residues
from polypeptides and proteins. These enzymes from various sources can be used successfully
to debitter protein hydrolyzates. Izawa et al. (1997) reported that aminopeptidase from
Aeromonas caviae T-64 hydrolyzed bitter peptides containing hydrophobic amino acids in
the N-terminal region of protease hydrolyzates of milk casein and soy protein. Bitterness of the
peptides were reduced by removal of these amino acids. Fernandez-Espia and Rul (1999)
reported that an aminopeptidase (PepS) from Streptococcus thermophilus exhibits a high
specificity towards peptides possessing Arg or aromatic amino acids at the N-terminus. PepS
may be involved in bacterial growth by supplying amino acids, and in the development of
flavor of dairy products, by hydrolyzing bitter peptides and liberating aromatic amino acids,
which are important precursors of aroma compounds. Donnell et al. (1997) purified and
characterized an aminopeptidase P from L. lactis subsp. cremoris. The purified enzyme
removed the N-terminal amino acid from peptides only where Pro (and in one case Ala) was
present in the penultimate position. As bitter casein-derived peptides are likely to contain
single Pro or pairs of Pro, aminopeptidase P appears to be an important enzyme for debittering.
Tan et al. (1993) reported that trypic digest of b-CN exhibits a strong bitter taste, which
corresponds to the strong hydrophobicity of several peptides in the tryptic digest of b-CN. Thedegradation of the tryptic digest by aminopeptidase N from L. lactis subsp. cremoris WG2
resulted in the decrease of hydrophobic peptides and a drastic decrease of bitterness of the
reaction mixture. Watanabe et al. (1990) noticed that the bitter flavor of a tryptic hydrolyzate of
casein was decreased after it was incubated in the presence of Erwinia ananas, an ice
nucleation-active bacterium. This is due to the action of aminopeptidase on these peptides at
low temperature. Park et al. (1995) investigated the effects of crude enzyme extract of L. casei
subsp. casei LLG on the water-soluble peptides of enzyme-modified cheddar cheese (EMC).
The crude enzyme extract contained a wide range of peptidolytic activities (aminopeptidase
1616 U/mL, x-prolyldipeptidyl peptidase 67 U/mL, proline-iminopeptidase 39 U/mL). They
found that supplementation of cheddar cheese slurries with crude enzyme extract in addition to
Neutrase produced cheese without bitter taste. They concluded that both aminopeptidase and
Pro-specific peptidases present in the crude extract were responsible for degrading the
hydrophobic peptides in bitter EMC. Gallagher et al. (1994b) used two protease preparations,
papain and neutral Bacillus (B. subtilis) separately to produce bitter a-CN hydrolyzate. The
B.C. Saha, K. Hayashi / Biotechnology Advances 19 (2001) 355–370362
action of a food grade fungal peptidase (Aspergillus oryzae) was then studied by observing
changes in the peptide profiles of the bitter a-CN hydrolyzate. The production of novel savory
(hydrophilic) peptides in the a-CN hydrolyzate was evident from their study. Prost and
Chamba (1994) demonstrated that a high aminopeptidase activity is necessary when strains of
lactic acid bacteria are intended for manufacture of cheese. They prepared cheese with L.
helveticus strain L1, which has high aminopeptidase activity and two clones (L2, L3) selected
for their lack of aminopeptidase activity. Cheeses made with aminopeptidase-deficient
Lactobacilli (L2, L3) were more stiff and more bitter than those made with L1. Blanc et al.
(1993) reported that aminopeptidase (APII), which catalyzed L-lysine p-nitroanilide hydro-
lysis, was able to degrade several dipeptides and tripeptides with hydrophobic N-terminal
amino acid (Leu, Ala). It was inactive on peptides containing Pro or Gly. L. delbrueckii subsp.
bulgaricus cells treated at 67–68�C for 15.5–16 s had significantly less acidification capacity
and much lower endopeptidase activity than untreated cells, while they still retained high
aminopeptidase activities against amino acids commonly found in bitter peptides (Lopez-
Fandino and Ardoe, 1991). A stimulatory effect of heat treatment on the leakage of amino-
peptidase from the cells was observed at pH 5.4. These properties make them suitable as an
extra source of important ripening enzymes in cheese-making. The purified aminopeptidase II
from Penicillium caseicolum had a wide specificity for various substrates, and it was capable
of cleaving amino-terminal Leu and Phe residues of dipeptides and oligopeptides (Matsuoka
et al., 1991). The enzyme treatment for 3 h was effective in debittering the bitter peptide
fraction from peptic casein. Minagawa et al. (1989) treated the bitter peptide fraction present in
casein hydrolyzates obtained by using three proteases (subtilisin, papain, and trypsin) with
aminopeptidase T from Thermus aquaticus YT-1. The bitterness of the bitter peptide fraction
was decreased and sometimes disappeared completely, with an increase in free amino acids.
The percentage of total free amino acids released from each bitter peptide fraction (subtilisin,
papain, and trypsin) by aminopeptidase digestion for 20 h were approximately 11, 8.7, and 6.5,
respectively. Further, a bitter peptide [as1-CN(f91–100)] with a threshold value of bitterness
of 2.9 ppm (w/v) was isolated from a tryptic hydrolyzate of casein. The peptide [as1-CN(f 96–100)], obtained from the aminopeptidase digestion of this bitter peptide, was not bitter.
Arora and Lee (1990) evaluated six selected strains of L. casei (subspecies casei,
pseudoplantarum, and rhamnosus) for their amino, di-, tri-, and carboxypeptidase activities
using 30 different synthetic peptides and peptide derivatives. All six strains showed similar
substrate specificities toward various peptides, except for Phe-Pro, which was only hydro-
lyzed by L. casei subsp. casei strains. L. casei subsp. casei strains showed strong activities
against bitter peptides (Phe-Pro, Pro-Phe, Pro-Ile) and S-containing amino and peptide
derivatives (methionine p-nitroanilide, cysteine p-nitroanilide, and Ala-Met), which are of
great importance in removing bitterness during cheese ripening. None of the strains showed
any detectable carboxypeptidase activity when tested on seven carboxyl end-substituted
peptides. Liberation of the N-terminal Arg from the bitter peptide Arg-Gly-Pro-Pro-Phe-Ile-
Val obtained by digestion of casein with bacterial proteinase did not affect the bitterness
(Minamiura et al., 1972a). Also, the splitting of Val and Ile or Ile-Val at the C-terminus by
carboxypeptidase or bacterial neutral proteinase had no influence on the bitterness. The
liberation of Arg and Gly from the peptide with bacterial aminopeptidase gave rise to a
B.C. Saha, K. Hayashi / Biotechnology Advances 19 (2001) 355–370 363
nonbitter peptide, which indicates that the Gly-Pro-Pro-Phe peptide core was responsible for
bitterness. Asano et al. (1998) showed that purified aminopeptidase derived from germinated
soybean cotyledon efficiently decomposes low molecular weight peptide containing Glu or
Asp in its sequence. Gobbetti et al. (1995) demonstrated debittering activity of purified
aminopeptidase from Pseudomonas fluorescens on synthetic bitter peptides, bitter hydro-
lyzate of ultra-high-temperature treated milk proteins, and on the ripening of Italian caciotta-
type cheese. The enzyme specifically hydrolyzed peptide bonds involving Leu, Trp, and Val,
usually identified as major components of bitter peptides.
4.4.2. Treatment with alkaline/neutral protease
Kanekanian et al. (2000) studied the profile of bitter peptides of an a3-CN sample from
skim milk by treating with neutral Bacillus protease. After gel filtration, three bitter fractions
were analyzed by reverse-phase HPLC. The first fraction, which showed a high level of
bitterness, was debittered by an alkaline/neutral Aspergillus protease P194. The debittered
hydrolyzate showed a shift from the hydrophobic to the hydrophilic region (di- and
tripeptides, several free amino acids). Lee et al. (1996) reduced bitterness significantly in
the bitter peptides extracted from cheddar cheese with peptidases from Lactococcus lactis
subsp. cremoris SK11. They have isolated five peptides responsible for bitterness in cheddar
cheese. Their origins were identified as as1-CN(f1–7), as1-CN(f11–14), as2-CN(f191–197), and b-CN(f 8–16). These bitter peptides were rich in Pro, and Pro commonly occurred
in the penultimate position. The calculated hydrophobicities suggested that the isolated bitter
peptides were both hydrophobic and hydrophilic. Baankreis et al. (1995) reported that an
intracellular neutral thermolysin-like oligopeptidase purified from L. lactis plays a crucial role
in the degradation of an important bitter peptide in cheese, the b-CN 193–209 fragment.
4.4.3. Treatment with carboxypeptidase
Arai et al. (1970) showed that the bitterness of peptides from soybean protein hydrolyzates
was decreased by treatment of Aspergillus acid carboxypeptidase. This method for debittering
is based on the further hydrolysis of bitter peptides with exopeptidases. Umetsu and
Ichishima (1988) treated the bitter peptide fraction obtained from the peptic hydrolyzate of
casein with crystalline wheat carboxypeptidase. The bitterness of the bitter fraction decreased
with an increase in free amino acids. Kawabata et al. (1996) reported that serine carboxy-
peptidase isolated from squid liver can eliminate bitter peptides prepared from soy protein
and corn gluten by enzymatic hydrolysis with pepsin and trypsin. The enzyme reacts well at
the C-terminal position of peptides having hydrophobic amino acids. Ge and Zhang (1996)
immobilized porcine pancreatic exopeptidases on thin shrimp chitin film by cross-linking
with glutaraldehyde. The immobilized enzyme preparation was able to remove the bitterness
of the tryptic/chymotryptic casein hydrolyzates.
4.5. Condensation reactions of bitter peptides using protease
The plastein reaction is a protease-catalyzed process involving the formation of a plastic
gel-like product from an oligopeptide mixture or a partial protein hydrolyzate. Both trans-
B.C. Saha, K. Hayashi / Biotechnology Advances 19 (2001) 355–370364
peptidation and condensation are considered to contribute to the plastein product (Watanabe
and Arai, 1988). The plastein reaction can be successfully applied to the bitter hydrolyzate in
order to debitter it. Papain-catalyzed resynthesis reaction permitted covalent incorporation of
added amino acid esters into proteins. This led to improving the functional and nutritional
properties of food proteins, giving rise to peptide surfactants with considerable oil-emulsify-
ing capacity and modified peptides with balanced amino acid compounds (Watanabe, 1997).
A process for the improvement of food proteins is suggested in which there occurs a
combination of enzymatic protein hydrolysis and plastein synthesis (Fig. 1). Stevenson et al.
(1998) used synthetic dipeptides of varying hydrophobicity as a model system to investigate
the feasibility of reversed reaction of protease as a means of improving the flavor of bitter
peptides found in protein hydrolyzates. They found that the condensation reaction was driven
by precipitation of insoluble reaction products, and this process is a potential means to
improve the flavor of bitter peptides extracted from protein hydrolyzates. Muro et al. (1992)
reported that treatment of bitter peptide (Phe-Val or Val-Phe) solution with protease from
Fig. 1. A combined process of enzymatic protein hydrolysis and resynthesis for producing a plastein with
improved acceptability and amino acid composition. (Reprinted from Fujimaki et al., 1977, with permission from
the American Chemical Society.)
B.C. Saha, K. Hayashi / Biotechnology Advances 19 (2001) 355–370 365
Streptomyces cellulosae produced insoluble condensation compounds. Treatment of solutions
containing the peptide and milk casein, soybean protein solutions, or their hydrolyzates with
the protease also caused precipitations, and the bitterness was largely removed. The
precipitates were mainly composed of 1:1 Phe and Val. Plastein formation generally takes
place at a pH value different from the optimum pH for hydrolysis.
4.6. Use of Lactobacilli as a debittering starter adjunct
The diversity of proteolytic activities among Lactobacilli may reflect different capacities of
starters to produce and degrade bitter peptides derived from milk proteins (Bruinenberg and
Limsowtin, 1995). Gomez et al. (1996a) studied the debittering activity of peptidases from
selected Lactobacillus strains in model cheeses. Reduction of the hydrophobic peptide level
in the water-soluble fraction of Neutrase cheeses with L. plantarum ESI144 was higher than
that obtained with L. paracasei subsp. paracasei ESi 207, pointing to its possible use as a
debittering starter adjunct in cheese manufacture. The use of L. plantarum ESI144 as adjunct
starter resulted in lower levels of hydrophobic peptides from Day 1 and lower bitterness
scores throughout ripening of a semihard cheese made from pasteurized milk to which B.
subtilis proteinase was added to induce bitter flavor formation (Gomez et al., 1996b).
Lemieux and Simard (1992) added Lactobacillus strains as an adjunct to the regular lactic
starter in cheddar cheese manufacture in order to accelerate ripening. Microbial cheese
proteolysis resulted in the release of free amino acids, which were extracted with the
astringent and bitter fractions. Lactobacillus strains generally increased the degree of
proteolysis. L. plantarum and L. brevis produced off-flavors due to an accumulation of
medium size peptides. The control cheese (without Lactobacilli) had the most peptides with a
mean molecular weight of < 1000 and had a flavor described as slightly bitter. Addition of L.
casei subsp. casei L2A accelerated ripening and yielded a well-aged cheddar cheese without
any bitterness even after 7 months at 6�C. Crow et al. (1995) suggested that a balance of lysed
and intact cells of L. lactis subsp. lactis is important for control of cheese ripening. Enzymes
released on cell lysis accelerated the rate-limiting peptidolytic steps and removal of some
bitter peptides, while intact cells are required for lactose removal.
5. Concluding remarks
Removing bitterness in enzymatic protein hydrolyzates is a major problem. Various
selective separation such as treatment with activated carbon, extraction with alcohol,
chromatography, masking with various agents, and further enzymatic treatment have proven
effective in removing the bitterness of protein hydrolyzates. Aminopeptidases show great
promise in removing the bitterness and will play an important role in enzymatic protein
hydrolysis. Research is in progress at the National Food Research Institute, Tsukuba, to
develop an efficient, cost-effective, and environmentally compatible (with respect to pH and
temperature) aminopeptidase that can be used together with protease to produce bitterless
protein hydrolyzate in a single step enzymatic reaction.
B.C. Saha, K. Hayashi / Biotechnology Advances 19 (2001) 355–370366
Acknowledgments
The work was prepared at the National Food Research Institute, Tsukuba, Japan, under a
Reimbursable Cooperative Agreement (58-3620-0-F108) between USDA-ARS and MAFF-
NFRI, Japan.
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