16
Research review paper Debittering of protein hydrolyzates Badal C. Saha a, *, Kiyoshi Hayashi b a Fermentation Biochemistry Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, US Department of Agriculture 1 , 1815 North University Street, Peoria, IL 61604, USA b Enzyme 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

Debittering of protein hydrolyzates

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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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