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HPLC Separation of Bitter Peptides from Cheddar Cheese
Can. Inst. Food Sei. Technol. J. Vo/. 15, No. 4, pp. 283-288, 1982 Pergamon Press Ltd. Printed in Canada.
HPLC Separation of Bitter Peptides from Cheddar Cheese H.M. Champion and D.W. Stanley Department of Food Science University of Guelph Guelph, Ontario NIG 2WI
Abstract
soumises aux analyses sensorielles et chimiques. Les deux fractions ameres obtenues par chromatographie en phase Iiquide 11 haute resolution eurent des indices d'hydrophobicite moyens quelque peu plus eleves que ceux des fractions nOn ameres, mais ceci ne fut pas le cas pour la fraction amere obtenue par chromatographie sur gel. Pour les fractions ameres obtenues par chromatographie en phase Iiquide 11 haute resolution, les fractions ameres furent plus riches en valine et en leucine que les fractions nOn ameres. Chez les fractions separees par chromatographie sur gel, la fraction amere fut plus riche en lysine que la fraction non amere. Le temps de retention augmenta avec l'hydrophobicite moyenne des fractions ameres. Cette tendance ne fut pas observee avec les proteines pures et les peptides etudies. Une relation positive apparente fut toutefois observee entre le temps de retention et le poids moleculaire.
High performance liquid chromatography was used to separate bitter peptides extracted from Cheddar cheese coagulated with chicken pepsin. A reversed phase octadecylsilane type column coupled with linear gradient elution from water to 91 % methanol enabled the bitter extract to be separated into at least seventy-one compounds shown to be peptides. Gel filtration was also used but the one bitter fraction, as determined by sensory testings, obtained in this manner was separated into at least forty-four different components by high performance liquid chromatography. The chromatograms from both methods were divided into bitter and nOnbitter fractions, each containing numerous peaks, according to sensory analysis. The average molecular weight of the bitter fraction obtained by gel filtration was established to be approximately 190 daltons. Ultracentrifugation of the high performance liquid chromatography fractions supported this estimate. Fractions collected by both separation techniques were subjected to sensory and chemical analyses. The two bitter high performance liquid chromatography fractions had slightly higher average hydrophobicity values than the nonbitter fractions, but this did not hold for the bitter fraction obtained by gel chromatography. Valine and leucine occurred at a higher level in the high performance liquid chromatography separated bitter fractions than in the nonbitter fractions, while lysine values were elevated in the bitter fraction separated by gel chromatography. There was an increase in retention time with increasing average hydrophobicity of the bitter extract fractions, but nO such trend appeared for the pure proteins and peptides tested; there was, however, an apparent positive relationship between retention time and molecular weight.
Introduction Bitterness is a flavour defect sometimes encountered in cheeses. It has been traced to the formation of bitter peptides during curing as a result of enzymatic casein hydrolysis (Harwalkar and Elliot, 1965). In Canada there is a growing interest in rennet substitutes for Cheddar cheese making, but bitterness may result from some rennet replacers due to the formation of bitter peptides. Recently, for example, commercial chicken pepsin was found to produce an objectionable level of bitterness in Cheddar cheese (Stanley et al., 1980). The bitter fractions of various cheeses have been separated and bitter peptides isolated. Harwalkar and Elliot (1970) used gel filtration to fractionate a bitter extract from Cheddar cheese and achieved further separation through electrophoresis. Huber and Klostermeyer (1974) isolated a bitter nonapeptide from a non-Cheddar variety of cheese using successively gel filtration, ion exchange chromatography and thin layer chromatography. Ion exchange chromatography was also used by Novgorodova et al. (1975) to separate extracts of various cheeses but the degree of bitterness and composition were not identified. Only a few peptides from cheese have been sequenced. Hodges et al. (1972) identified three of these in Cheddar cheese but did not test them individually for bitterness, although subsequently Richardson
Resume La separation des peptides ameres extraits du fromage Cheddar coagule 11 la pepsine de poulet fut realisee 11 I' aide de la chromatographie en phase liquide 11 haute resolution. Une phase inversee d'une colonne de type octadecylsilane plus une elution par gradient lineaire allant de I' eau 11 du methanol 11 91 % ont perm is de separer les extraits amers au moins en soixante-et-onze constituants peptidiques. Une fraction amere, obtenue par filtration sur gel et qui a ete identifiee par evaluation sensorielle, a ete separee en au moins quarante-quatre constituants differents par chromatographie en phase Iiquide 11 haute resolution. Les chromatogrammes obtenus par I'une ou l'autre des deux methodes furent divises en fractions amere et nOn amere, tel que juge par analyse sensorielle. Le poids moleculaire moyen de la fraction amere obtenue par filtration sur gel a ete etabli 11 environ 190 daltons. Cette evaluation a ete corroboree par ultrafiltration des fractions obtenues 11 l'aide de la chromatographie en phase liquide 11 haute resolution. Les fractions recueillies par les deux techniques de separation ont ete
0315-5463/82/040283-06$3.00/0 Copyright
© 1982 Canadian Institute of Food Science and Technology
283
and Creamer (1973) demonstrated one of them to be bitter. Several reports, however, have indicated that peptides with high contents of hydrophobic side chains will develop a bitter taste (Ney, 1971, 1972; Matoba and Hata, 1972; Guigoz and Solms, 1976). Many of the techniques heretofore used for investigating bitter peptides are time consuming and complex. The scope and popularity of high performance liquid chromatography (HPLC) as a separation device for biological molecules has grown tremendously and has prompted the present investigation into its use as a means of separating bitter peptides derived from Cheddar cheese. Recently published reports on the separation of peptides and proteins (Molnar and Horvath, 1977; Monch and Dehnen, 1977, 1978; Hancock et al., 1978a,b; Rivier, 1978) have indicated the potential for such a separation in a system such as a bitter extract from cheese.
Materials and Methods Cheese Cheddar cheese was made using a direct set culture and commercial chicken pepsin as a coagulant. Chemical analyses of the newly made cheese indicated 32.4% moisture, 37.9% fat and 23.5% protein. The cheese was judged to be bitter, relative to a control produced with commercial rennet, by a sensory panel 3 mo after manufacture, and enhanced bitterness was encountered at 10 and 17 mo (Stanley et al., 1980). HPLC Solvents For use as a solvent for HPLC, deionized, glass distilled water was filtered through a low extractable 0.45 j), HPLC grade filter (Millipore Corp., Bedford, Mass.) and sonicated for 30 min to remove dissolved gases. It was stored under refrigeration for no longer than 48 h prior to use. Methanol was of HPLC grade and degassed by sonication. Equipment HPLC was performed using two proportioning pumps, a solvent programmer, a sample injector and an absorbance detector with a 280 nm light filter (Waters Associates, Mississauga, Ont.). A j),Bondapak CI8 reverse phase column (3.9 mm ID x 30 cm) from the same supplier was employed. Preparation of Bitter Extract Cheese was sliced, frozen and then freeze-dried. The extraction procedure of Harwalkar and Elliot (1970) was followed, which involved grinding 55 g of freeze-dried cheese and 150 mL of chloroform methanol (2: I, v/v) and filtering the resulting mixture. The residue was reextracted three times, the filtrate combined and made biphasic with water. After being shaken and separated, the layer containing water and methanol was removed, evaporated under reduced pressure, redissolved in water and frozen for further use. The bitter extract was clear, pale yellow and did not show any precipitation during storage. Sensory Methods Fourteen individuals were screened for their ability to detect bitterness in quinine sulphate solutions. An 284/Champion and Stanley
unstructured rating scale was used in the form of a 10 cm line with the anchor point on the far left labelled "not bitter" and that on the far right labelled "extremely bitter." Individuals indicated perceived bitterness by placing a vertical mark on a separate line for each sample. Five of the fourteen individuals screened were selected to form the panel; their average threshold value for quinine sulphate was 9.3 x 10-- li M.
Standard Conditions for HPLC Separations A method for HPLC separation of the bitter extract was developed that consisted of applying j),L quantities of the extract to the column equilibrated with water at a flow rate of I. 5 mL/min. A water/91 % methanol linear gradient was employed using a rate of change of 1.14% methanol/min. Fractions selected for sensory analysis were evaporated to dryness, redissolved in water and presented, along with a standard, to the panel. Gel Filtration Gel filtration was also used as a method of separating the bitter extract. Sephadex G-50 was used in a 2.6 x 40 cm column with water as the eluent. A 2 mL sample of bitter extract was applied, the flow rate set at 5.0 mL/min and the eluent monitored continuously at 280 nm prior to the fractions being collected. A standard curve of elution volume vs. log molecular weight (Mw) was constructed using molecules of known Mw ranging from bovine serum albumin (67,000) to glycine (75). Fractions selected for sensory analysis were diluted with an equal volume of water and presented, along with a standard, to the panel. Similar fractions were analyzed for protein (Lowry et al., 1951), a-amino nitrogen (Paik and Kim, 1972) and constituent amino acids by means of a Technicon Auto Analyzer amino acid analyzer. Ultracentrifugation Samples were centrifuged in a Beckman Model 3 ultracentrifuge at 20cC at about 63,000 rpm with the actual rpm determined from the revolution counter. An AN-H rotor was used as were double sector, synthetic boundary cells. Sedimentation was followed using Schlieren optics and the sedimentation coefficient obtained by calculation from measurements of photographic plates.
Results and Discussion Gel Filtration A plot of absorbance (280 nm) vs. elution volume during gel filtration of the bitter extract is shown in Figure I. Sensory analysis of the fractions (Table 1) showed that only one fraction, that eluted at 150 mL, was bitter. The standard curve of molecules of known Mw indicated an estimated average Mw of 190 daltons for the bitter 150 mL fraction. Table 1 also gives results for ultraviolet absorbance (A tHO )' Lowry protein (A 7:;o) and a-amino nitrogen (A:;HO) of fractions collected at 120, 150, 180 and 210 mL (before and at the bitter peak, and on and after the shoulder in Figure 1). These data indicate that the first three fractions contain total concentrations of proteins, peptides and amino acids of a similar order of magnitude, while only the 150 mL fraction was bitter. J. Insl. Can. Sci. TecJmol. Alime·ll. Vol. 15. No. 4. 1982
Table I. Analysis of gel filtration fractions. Absorbance
Elution volume (mL)
Bitterness score I
280 nm
750 nm
580 nm
Hydrophobicity (cal/residue)
-2.1 6.7 -2.2 -2.5
0.39 1.65 1.18 0.20
0.52 0.68 0.56 0.08
0.56 0.64 0.64 0.19
1,460 1,460 2,000 1,580
120 150 180 210
;r)istance (cm) on 10 cm line relative to 11.6 x 10-'; M quinine sulphate. Table 2. Amino acid analysis of gel filtration fractions.
BITTER
j 1.5
Amino acid
NON· BITTER
j 1.0
\
0.5
NON· BITTER
j
vo 50
100
150
200
250
ELUENT VOLUME (ml)
Fig. I. Separation of bitter extract by gel filtration.
Also shown in Table 1 are the average hydrophobicities of the fractions, calculated according to the method of Bigelow (1967). Considering the theory (Ney, 1971) that proteins having average hydrophobicities of less than 1,300 cal/residue are not bitter while those above 1,400 cal/residue are, it might be expected that all the gel filtration fractions would be perceived as bitter. That this is not the case possibly reflects the concurrent assumptions made when treating fractions as molecular entities and using total amino acid values for calculating average hydrophobicities, even though each fraction contains many compounds. Apparently, low hydrophobicity, nonbitter peptides predominated in the 150 mL fraction despite the presence of sufficient bitter material to yield an overall sensory sensation of bitterness. The results for amino acid analysis of the fractions (Table 2) show the bitter fraction to have the highest level of total amino acids and to contain the greatest concentration of lysine, but low levels of most other amino acids including glycine. These data are consistent with the findings of Kirimura et al. (1969) who found that the most bitter peptides were those which contained lysine or arginine, and Stanley (1981) who observed that the addition of glycine to protein hydrolysates tended to mask bitterness.
HPLC The bitter extract was applied to the reversed phase column and eluted with a water/methanol gradient over 80 min. At the end of the gradient there were no compounds left on the column that could be eluted by increased concentrations of methanol. Figure 2 shows a typical chromatogram for the HPLC separation of the Can. Inst. Food Sci. Techflol. J. Vol. 15, No. 4, 1982
ASP THR SER GLU PRO GLY ALA VAL MET ILE LEU TYR PHE LYS HIS ARG
Mole percentages 120 mL 2.8 2.8 2.3 38.1 10.1 1.6 1.8 5.4 0.2 1.9 24.2 1.2 2.6 3.0 1.0 0.9
150 mL 1.6 1.4 0.8 28.1 5.2 1.0 1.8 3.4 0.2 2.2 23.1 1.1 1.9 24.8 2.1 1.4
180 mL 2.3 2.0 1.5 7.3 4.9 1.5 3.0 4.5 0.3 1.5 56.4 2.3 6.5 2.9 0.8 1.9
210 mL 5.6 3.3 4.0 8.9 0.0 4.3 10.3 4.0 11.6 2.0 13.2 1.0 27.5 3.0 0.0 1.3
bitter extract (A~xo) with the results for Lowry protein (A,:;o) and a-amino nitrogen (A.,xo) superimposed. The compounds eluted before 26 min appeared to be short chain peptides (low Lowry/high a-amino nitrogen values), while after this the ratio increased, indicating longer chain peptides. Compounds eluted after 59 min resembled longer chain proteins, in that the Lowry reaction was much higher than the ninhydrin reaction. These molecules are present in lower concentrations, judging from the lower A,:;o values. The chromatogram was divided into four fractions according to two schemes (Figure 3) for the primary reason of collecting sufficient material for sensory analysis. In the first (Fractions I-IV), the divisions were assigned as a result of HPLC analysis of the four gel chromatography fractions; the boundaries between the four fractions were selected arbitrarily according to the positions of peaks produced when the bitter fraction from gel filtration was separated by HPLC, and also according to the presence of certain large peaks. Fraction I roughly corresponded to a gel filtration elution volume of 180 mL, Fraction 11 to 210 mL, Fraction III to 120 mL and Fraction IV to 150 mL. The chromatogram was also divided into four (1-4) fractions of approximately equal total peak areas. These fractions were collected following repeated injections of bitter extract and pooled. Sensory analysis (Table 3) of the two sets of fractions showed that Fractions IV, 3 and 4 were the most bitter. Considering which of these were eluted in overlapping time intervals, it appears that the bitter compounds from the cheese extract eluted between 35 and 66 min by HPLC and could, therefore, be any of many compounds. When the fractions were rechromatographed, they eluted in the Champion and Stanley/285
1.5 -A2.0 ASIO " A7S0
I.'
1.3 1.2 1.1 1.0
91
0.9
...
0.8 0.7 0.6
3:
...:I:m
0.5
> z ~
0.' 0.3 0.2
~
0.1 0
,
0
10
20
30
50
'0
70
60
0 80
TIME (MIN)
INJECT
Fig. 2. Separation of bitter extract by HPLC.
same time periods in which they had been collected. Fraction IV, which corresponds to the bitter gel filtration fraction, was separated by HPLC into at least forty-four compounds. Results of analysis of the fractions taken by area (Table 4) indicate that the level of free amino acids is far higher in Fraction 1 and that the amount of peptides or proteins is greater as well. Fractions 3 and 4, which were both bitter, had slightly higher hydrophobicities than either 1 or 2. There was a correlation between elution time and average hydrophobicity of the fractions which implies that the elution of peptides from the bitter extract Table 3. Bitterness score l of HPLC fractions. Fractionated according to area
Fractionated according to gel chromatography separation
-1.7 1.7 1.9 6.7
I II III IV
I 2
3 4
'Distance (cm) on 10 cm line relative to 11.6
X
3.6 0.8 6.9 6.2
IO- li M quinine sulphate.
1.S,
1.4; 1.31
1.': ,L'.. ,.,., 1,1
I
J ::: ,..
'.3 ,.,
';ljJ,~~4~~~~~-=;:::::::::::::;;=::=:::±
,
TIME ,"'N.
INJECT
Fig. 3. Division of HPLC fractions according to gel filtration fractions (I-IV) and area (1-4); .... ·gel filtration, - - area.
under the present conditions of separation is affected by their average hydrophobicity. The least hydrophobic peptides elute first by the least hydrophobic eluent and vice versa, which is consistent with the hydrophobic nature of the column packing. Marked differences were seen in amino acid composition of the four fractions (Table 5). Fraction I was moderately bitter and it is possible that its high lysine and arginine content may be due to a bitter peptide high in these amino acids. Two hydrophobic amino acids, valine and leucine, occurred at substantially higher levels in the later, more bitter fractions while two less hydrophobic amino acids, lysine and arginine, appeared in greater quantities in the first fraction. Fractions 3 and 4 resembled the bitter fraction from gel filtration (Table 2) in having higher levels of glutamic acid, leucine, proline and valine, but unlike the bitter gel filtration fraction, did not exhibit elevated lysine levels. Synthetic boundary ultracentrifugation of the four fractions yielded the sedimentation coefficients given in Table 4. Since sedimentation coefficients are proportional to Mw, barring conformational differences, it appears that the bitter fractions, 3 and 4, had average Mw less than the 448 dalton value of the pure peptide pro-phe-gly-lys that gave a sedimentation coefficient of 2.04. This is consistent with the range of 158-218 estimated for the bitter compounds by gel filtration. The sedimentation values indicate that while higher Mw compounds are eluted earlier from the HPLC column, there is no obvious relationship between the Mw' of the compound in the bitter extract and their retention on the column when using a water/methanol gradient. In order to clarify the relationship between retention time, hydrophobicity and Mw, an attempt was made to chromatograph known peptides under standard separation
Table 4. Analysis of HPLC fractions according to area. % CH"OH at
Fraction I
2 3 4
midpoint of fraction
14.3 33.6 52.7 76.5
286/Champion and Stanley
Sedimentation coefficient
Absorbance
580nm 1.56 1.64
0.53 0.76
1.81 0.29 0.27 0.16
750 nm 0.52 0.35 0.48 0.39
Total amino acids'
H ydrophobicity (cal/residue)
170
1,460
200 230 180
1,500 1,530 1,610
J. Insl. Can. Sci. Technol. Aliment. Vol. 15, No. 4, 1982
Table 5. Amino acid analysis of HPLC fractions according to area. Amino acid ASP THR SER GLU PRO GLY ALA VAL MET ILE LEU TYR PHE LYS HIS ARG
Mole percentages I J.2 I.3 0.8 29.9 7.0 I.3 1.4 2.5 0.5 2.5 5.5 2.0 4.7 34.0 3.0 2.6
2
3
4
3.3 1.7 0.8 36.0 9.5 0.8 1.5 3.9 0.9 3.0 27.4 1.8 2.6 4.3 1.5 0.9
2.7 1.4 J.2 34.2 10.8 1.4 1.6 9.2 0.2 2.2 23.9 1.9 3.4 3.6 1.4 1.1
1.8 1.7 1.7 24.7 11.7 1.4 1.7 19.7 0.1 1.6 23.9 1.4 3.4 3.1 1.2 1.0
conditions. This work was hampered by the poor water solubility of many peptides and their low absorbance at 280 nm. The data (Table 6) indicate a possible relationship among the three variables. Considering (1) that molecules with a Mw higher than insulin did not elute, (2) the results where, in general, compounds of higher Mw eluted later, and (3) the positive relation between retention time and hydrophobicity, it seems that the retention of peptides and proteins from the bitter extract under the conditions of this work is influenced by a combination of Mw and hydrophobicity factors.
Conclusion This research has shown that HPLC, using gradient elution from an octadecylsilane type column, is effective for separation of the components of an extract from bitter Cheddar cheese. The bitter extract contained at least seventy-one compounds of which only some were bitter. While gel filtration achieved limited fractionation of the bitter extract, HPLC enabled more extensive and reproducible separation. Elution order was related to the calculated average hydrophobicity of fractions collected; more hydrophobic peptides were eluted only by higher concentrations of methanol. There was no consistent relationship between Mw of the fractions and either retention time or methanol concentration, however, correlation was found between the Mw of pure peptides and retention time under standard chromatography conditions. Thus, both hydrophobicity and Mw seem to be factors in influencing separation.
Two bitter fractions collected by HPLC had higher average hydrophobicities than nonbitter fractions, but the bitter fraction from gel filtration did not show this relationship. Casein has a high average hydrophobicity (1,605 cal/residue; Guigoz and Solms, 1976) and it is to be expected that many of its peptides should exhibit high hydrophobicities. Since the fractions collected were mixtures, the compounds present in the greatest concentration could make large contributions to hydrophobicity but not contribute as much to bitterness as other less plentiful molecules.
Acknowledgements Dr. ER. van de Voort provided assistance with the ultracentrifugation. This work was funded in part by the Ontario Milk Marketing Board, Kraft Foods and the Ontario Ministry of Agriculture and Food.
References Bigelow, c.c. 1967. On the average hydrophobicity of proteins and the relationship between it and protein structure. J. Theor. BioI. 16:187. Guigoz, Y. and Solms, J. 1976. Bitter peptides, occurrence and structure. Chem. Senses Flavor 2:71. Hancock, W.S., Bishop, C.A., Prestidge, R.L., Harding, D.R.K. and Hearn, M.T. 1978a. High pressure liquid chromatography of peptides and proteins. 11. Use of phosphoric acid in the analysis of underivatised peptides by reversed phase high pressure liquid chromatography. J. Chromatogr. 153:391. Hancock, W.S., Bishop, C.A., Prestidge, R.L., Harding, D.R.K. and Hearn, M.T. 1978b. Reversed phase high pressure liquid chromatography of peptides and proteins with ion-pairing reagents. Science 200: 1168. Harwalkar, V.R. and Elliot, J.A. 1965. Isolation and partial purification of bitter components from Cheddar cheese. J. Dairy Sci. 48:784. Harwalkar, V.R. and Elliot, J.A. 1970. Isolation of bitter and astringent fractions from Cheddar cheese. J. Dairy Sci. 54:8. Hodges, R., Kent, S.B.H. and Richardson, B.C. 1972. The mass spectra of some permethylated acetylpeptides. Biochim. Biophys. Acta 257:54. Huber, L. and Klostermeyer, H. 1974. Isolierung und identifizierung eins bitterstoffes aus butterkase. Milchwissenschaft 29:449. Kirimura, J., Shimizu, A., Kimizuka, A., Ninomiya, T. and Katsuya, N. 1969. The contribution of peptides and amino acids to the taste of foodstuffs. J. Agric. Food Chem. 17:689. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, RJ. 1951. Protein measurement with the Folin phenol reagent. J. BioI. Chem. 193:265. Matoba, T. and Hata, T. 1972. Relationships between bitterness of peptides and their chemical structure. Agric. BioI. Chem. 36:1423. Molnar, I. and Horvath, C. 1977. Separation of amino acids and peptides on nonpolar stationary phases by high performance liquid chromatography. J. Chromatogr. 142:623.
Table 6. HPLC of pure peptides under standard conditions. Peptide gly-gly gly-gly-gly gly-gly-gly-gly leu-val gly-tyr glu-cys-gly pro-phe-gly-Iys Iys-phe-ile-gly-Ieu-met insulin, A chain
Can. Inst. Food Sci. Technol. J. Vol. 15. No. 4, 1982
Retention time (min) 2.1 2.1 3.6 2.5 5.0 3.9 47.0 37.0
Mw 132 189 246 230 238 307 448 708 2,697
Hydrophobicity (cal/residue)
o
o o
2,055 1,435 183 1,688 1,800 712
Champion and Stanley/287
Monch W. and Dehnen, W. 1977. High perfonnance liquid chromatography of peptides. J. Chromatogr. 140:260. Monch W. and Dehnen, W. 1978. High perfonnance liquid chromatography of polypeptides and proteins on a reversed phase suppOI1. J. Chromatogr. 147:415. Ney, K.H. 1971. Voraussage der bitterkeit von peptiden aus deren aminosaurezusammensetzung. Z. Lebensm. Vnters. Forsch. 147:64. Ney, K.H. 1972. Aminosaurezusammensetzung von proteinen und die bitterkeit ihrer peptide. Z. Lebensm. Vnters. Forsch. 149:321. Novgorodova, N .S., Buzov, I.P. and Nebel1, V.K. 1975. Trudy, Vsesoyuznyi Nauchnoissledovatel'skii Intitut Maslodel'noi i Syrodel'noi Promyshlennosti. No. 18:107. English abstract Food Sci. Technol. Abstr. 11 :5P767. Paik, W.K. and Kim, S. 1972. Effect of methylation on susceptibility of protein to proteolytic enzymes. Biochemistry 11:2589.
288/Champion and Stanley
Richardson, B.C. and Creamer, L.K. 1973. Casein proteolysis and bitter peptides in Cheddar cheese. N.Z. J. Dairy Sci. Technol.
8:46. Rivier, J. 1978. Use oftrialkyl ammonium phosphate buffers in reversed phase high performance liquid chromatography for high resolution and recovery of peptides and proteins. J. Liq. Chromatogr. 1:343. Stanley, D.W., Emmons, R.B., Modler, H.W. and Irvine, D.M. 1980. Cheddar cheese made with chicken pepsin. Can. Inst. Food Sci. Technol. J. 13:97. Stanley, D. W. 1981. Nonbitter protein hydrolysates. Can. Inst. Food Sci. Technol. J. 14:49.
Accepted May 4, 1982
J. Inst. Can. Sei. Tecimol. A/imem. Vol. 15, No. 4, 1982
HPLC Separation of Bitter Peptides from Cheddar Cheese H.M. Champion and D.W. Stanley Department of Food Science University of Guelph Guelph, Ontario NIG 2WI
Abstract
soumises aux analyses sensorielles et chimiques. Les deux fractions ameres obtenues par chromatographie en phase Iiquide 11 haute resolution eurent des indices d'hydrophobicite moyens quelque peu plus eleves que ceux des fractions nOn ameres, mais ceci ne fut pas le cas pour la fraction amere obtenue par chromatographie sur gel. Pour les fractions ameres obtenues par chromatographie en phase Iiquide 11 haute resolution, les fractions ameres furent plus riches en valine et en leucine que les fractions nOn ameres. Chez les fractions separees par chromatographie sur gel, la fraction amere fut plus riche en lysine que la fraction non amere. Le temps de retention augmenta avec l'hydrophobicite moyenne des fractions ameres. Cette tendance ne fut pas observee avec les proteines pures et les peptides etudies. Une relation positive apparente fut toutefois observee entre le temps de retention et le poids moleculaire.
High performance liquid chromatography was used to separate bitter peptides extracted from Cheddar cheese coagulated with chicken pepsin. A reversed phase octadecylsilane type column coupled with linear gradient elution from water to 91 % methanol enabled the bitter extract to be separated into at least seventy-one compounds shown to be peptides. Gel filtration was also used but the one bitter fraction, as determined by sensory testings, obtained in this manner was separated into at least forty-four different components by high performance liquid chromatography. The chromatograms from both methods were divided into bitter and nOnbitter fractions, each containing numerous peaks, according to sensory analysis. The average molecular weight of the bitter fraction obtained by gel filtration was established to be approximately 190 daltons. Ultracentrifugation of the high performance liquid chromatography fractions supported this estimate. Fractions collected by both separation techniques were subjected to sensory and chemical analyses. The two bitter high performance liquid chromatography fractions had slightly higher average hydrophobicity values than the nonbitter fractions, but this did not hold for the bitter fraction obtained by gel chromatography. Valine and leucine occurred at a higher level in the high performance liquid chromatography separated bitter fractions than in the nonbitter fractions, while lysine values were elevated in the bitter fraction separated by gel chromatography. There was an increase in retention time with increasing average hydrophobicity of the bitter extract fractions, but nO such trend appeared for the pure proteins and peptides tested; there was, however, an apparent positive relationship between retention time and molecular weight.
Introduction Bitterness is a flavour defect sometimes encountered in cheeses. It has been traced to the formation of bitter peptides during curing as a result of enzymatic casein hydrolysis (Harwalkar and Elliot, 1965). In Canada there is a growing interest in rennet substitutes for Cheddar cheese making, but bitterness may result from some rennet replacers due to the formation of bitter peptides. Recently, for example, commercial chicken pepsin was found to produce an objectionable level of bitterness in Cheddar cheese (Stanley et al., 1980). The bitter fractions of various cheeses have been separated and bitter peptides isolated. Harwalkar and Elliot (1970) used gel filtration to fractionate a bitter extract from Cheddar cheese and achieved further separation through electrophoresis. Huber and Klostermeyer (1974) isolated a bitter nonapeptide from a non-Cheddar variety of cheese using successively gel filtration, ion exchange chromatography and thin layer chromatography. Ion exchange chromatography was also used by Novgorodova et al. (1975) to separate extracts of various cheeses but the degree of bitterness and composition were not identified. Only a few peptides from cheese have been sequenced. Hodges et al. (1972) identified three of these in Cheddar cheese but did not test them individually for bitterness, although subsequently Richardson
Resume La separation des peptides ameres extraits du fromage Cheddar coagule 11 la pepsine de poulet fut realisee 11 I' aide de la chromatographie en phase liquide 11 haute resolution. Une phase inversee d'une colonne de type octadecylsilane plus une elution par gradient lineaire allant de I' eau 11 du methanol 11 91 % ont perm is de separer les extraits amers au moins en soixante-et-onze constituants peptidiques. Une fraction amere, obtenue par filtration sur gel et qui a ete identifiee par evaluation sensorielle, a ete separee en au moins quarante-quatre constituants differents par chromatographie en phase Iiquide 11 haute resolution. Les chromatogrammes obtenus par I'une ou l'autre des deux methodes furent divises en fractions amere et nOn amere, tel que juge par analyse sensorielle. Le poids moleculaire moyen de la fraction amere obtenue par filtration sur gel a ete etabli 11 environ 190 daltons. Cette evaluation a ete corroboree par ultrafiltration des fractions obtenues 11 l'aide de la chromatographie en phase liquide 11 haute resolution. Les fractions recueillies par les deux techniques de separation ont ete
0315-5463/82/040283-06$3.00/0 Copyright
© 1982 Canadian Institute of Food Science and Technology
283
and Creamer (1973) demonstrated one of them to be bitter. Several reports, however, have indicated that peptides with high contents of hydrophobic side chains will develop a bitter taste (Ney, 1971, 1972; Matoba and Hata, 1972; Guigoz and Solms, 1976). Many of the techniques heretofore used for investigating bitter peptides are time consuming and complex. The scope and popularity of high performance liquid chromatography (HPLC) as a separation device for biological molecules has grown tremendously and has prompted the present investigation into its use as a means of separating bitter peptides derived from Cheddar cheese. Recently published reports on the separation of peptides and proteins (Molnar and Horvath, 1977; Monch and Dehnen, 1977, 1978; Hancock et al., 1978a,b; Rivier, 1978) have indicated the potential for such a separation in a system such as a bitter extract from cheese.
Materials and Methods Cheese Cheddar cheese was made using a direct set culture and commercial chicken pepsin as a coagulant. Chemical analyses of the newly made cheese indicated 32.4% moisture, 37.9% fat and 23.5% protein. The cheese was judged to be bitter, relative to a control produced with commercial rennet, by a sensory panel 3 mo after manufacture, and enhanced bitterness was encountered at 10 and 17 mo (Stanley et al., 1980). HPLC Solvents For use as a solvent for HPLC, deionized, glass distilled water was filtered through a low extractable 0.45 j), HPLC grade filter (Millipore Corp., Bedford, Mass.) and sonicated for 30 min to remove dissolved gases. It was stored under refrigeration for no longer than 48 h prior to use. Methanol was of HPLC grade and degassed by sonication. Equipment HPLC was performed using two proportioning pumps, a solvent programmer, a sample injector and an absorbance detector with a 280 nm light filter (Waters Associates, Mississauga, Ont.). A j),Bondapak CI8 reverse phase column (3.9 mm ID x 30 cm) from the same supplier was employed. Preparation of Bitter Extract Cheese was sliced, frozen and then freeze-dried. The extraction procedure of Harwalkar and Elliot (1970) was followed, which involved grinding 55 g of freeze-dried cheese and 150 mL of chloroform methanol (2: I, v/v) and filtering the resulting mixture. The residue was reextracted three times, the filtrate combined and made biphasic with water. After being shaken and separated, the layer containing water and methanol was removed, evaporated under reduced pressure, redissolved in water and frozen for further use. The bitter extract was clear, pale yellow and did not show any precipitation during storage. Sensory Methods Fourteen individuals were screened for their ability to detect bitterness in quinine sulphate solutions. An 284/Champion and Stanley
unstructured rating scale was used in the form of a 10 cm line with the anchor point on the far left labelled "not bitter" and that on the far right labelled "extremely bitter." Individuals indicated perceived bitterness by placing a vertical mark on a separate line for each sample. Five of the fourteen individuals screened were selected to form the panel; their average threshold value for quinine sulphate was 9.3 x 10-- li M.
Standard Conditions for HPLC Separations A method for HPLC separation of the bitter extract was developed that consisted of applying j),L quantities of the extract to the column equilibrated with water at a flow rate of I. 5 mL/min. A water/91 % methanol linear gradient was employed using a rate of change of 1.14% methanol/min. Fractions selected for sensory analysis were evaporated to dryness, redissolved in water and presented, along with a standard, to the panel. Gel Filtration Gel filtration was also used as a method of separating the bitter extract. Sephadex G-50 was used in a 2.6 x 40 cm column with water as the eluent. A 2 mL sample of bitter extract was applied, the flow rate set at 5.0 mL/min and the eluent monitored continuously at 280 nm prior to the fractions being collected. A standard curve of elution volume vs. log molecular weight (Mw) was constructed using molecules of known Mw ranging from bovine serum albumin (67,000) to glycine (75). Fractions selected for sensory analysis were diluted with an equal volume of water and presented, along with a standard, to the panel. Similar fractions were analyzed for protein (Lowry et al., 1951), a-amino nitrogen (Paik and Kim, 1972) and constituent amino acids by means of a Technicon Auto Analyzer amino acid analyzer. Ultracentrifugation Samples were centrifuged in a Beckman Model 3 ultracentrifuge at 20cC at about 63,000 rpm with the actual rpm determined from the revolution counter. An AN-H rotor was used as were double sector, synthetic boundary cells. Sedimentation was followed using Schlieren optics and the sedimentation coefficient obtained by calculation from measurements of photographic plates.
Results and Discussion Gel Filtration A plot of absorbance (280 nm) vs. elution volume during gel filtration of the bitter extract is shown in Figure I. Sensory analysis of the fractions (Table 1) showed that only one fraction, that eluted at 150 mL, was bitter. The standard curve of molecules of known Mw indicated an estimated average Mw of 190 daltons for the bitter 150 mL fraction. Table 1 also gives results for ultraviolet absorbance (A tHO )' Lowry protein (A 7:;o) and a-amino nitrogen (A:;HO) of fractions collected at 120, 150, 180 and 210 mL (before and at the bitter peak, and on and after the shoulder in Figure 1). These data indicate that the first three fractions contain total concentrations of proteins, peptides and amino acids of a similar order of magnitude, while only the 150 mL fraction was bitter. J. Insl. Can. Sci. TecJmol. Alime·ll. Vol. 15. No. 4. 1982
Table I. Analysis of gel filtration fractions. Absorbance
Elution volume (mL)
Bitterness score I
280 nm
750 nm
580 nm
Hydrophobicity (cal/residue)
-2.1 6.7 -2.2 -2.5
0.39 1.65 1.18 0.20
0.52 0.68 0.56 0.08
0.56 0.64 0.64 0.19
1,460 1,460 2,000 1,580
120 150 180 210
;r)istance (cm) on 10 cm line relative to 11.6 x 10-'; M quinine sulphate. Table 2. Amino acid analysis of gel filtration fractions.
BITTER
j 1.5
Amino acid
NON· BITTER
j 1.0
\
0.5
NON· BITTER
j
vo 50
100
150
200
250
ELUENT VOLUME (ml)
Fig. I. Separation of bitter extract by gel filtration.
Also shown in Table 1 are the average hydrophobicities of the fractions, calculated according to the method of Bigelow (1967). Considering the theory (Ney, 1971) that proteins having average hydrophobicities of less than 1,300 cal/residue are not bitter while those above 1,400 cal/residue are, it might be expected that all the gel filtration fractions would be perceived as bitter. That this is not the case possibly reflects the concurrent assumptions made when treating fractions as molecular entities and using total amino acid values for calculating average hydrophobicities, even though each fraction contains many compounds. Apparently, low hydrophobicity, nonbitter peptides predominated in the 150 mL fraction despite the presence of sufficient bitter material to yield an overall sensory sensation of bitterness. The results for amino acid analysis of the fractions (Table 2) show the bitter fraction to have the highest level of total amino acids and to contain the greatest concentration of lysine, but low levels of most other amino acids including glycine. These data are consistent with the findings of Kirimura et al. (1969) who found that the most bitter peptides were those which contained lysine or arginine, and Stanley (1981) who observed that the addition of glycine to protein hydrolysates tended to mask bitterness.
HPLC The bitter extract was applied to the reversed phase column and eluted with a water/methanol gradient over 80 min. At the end of the gradient there were no compounds left on the column that could be eluted by increased concentrations of methanol. Figure 2 shows a typical chromatogram for the HPLC separation of the Can. Inst. Food Sci. Techflol. J. Vol. 15, No. 4, 1982
ASP THR SER GLU PRO GLY ALA VAL MET ILE LEU TYR PHE LYS HIS ARG
Mole percentages 120 mL 2.8 2.8 2.3 38.1 10.1 1.6 1.8 5.4 0.2 1.9 24.2 1.2 2.6 3.0 1.0 0.9
150 mL 1.6 1.4 0.8 28.1 5.2 1.0 1.8 3.4 0.2 2.2 23.1 1.1 1.9 24.8 2.1 1.4
180 mL 2.3 2.0 1.5 7.3 4.9 1.5 3.0 4.5 0.3 1.5 56.4 2.3 6.5 2.9 0.8 1.9
210 mL 5.6 3.3 4.0 8.9 0.0 4.3 10.3 4.0 11.6 2.0 13.2 1.0 27.5 3.0 0.0 1.3
bitter extract (A~xo) with the results for Lowry protein (A,:;o) and a-amino nitrogen (A.,xo) superimposed. The compounds eluted before 26 min appeared to be short chain peptides (low Lowry/high a-amino nitrogen values), while after this the ratio increased, indicating longer chain peptides. Compounds eluted after 59 min resembled longer chain proteins, in that the Lowry reaction was much higher than the ninhydrin reaction. These molecules are present in lower concentrations, judging from the lower A,:;o values. The chromatogram was divided into four fractions according to two schemes (Figure 3) for the primary reason of collecting sufficient material for sensory analysis. In the first (Fractions I-IV), the divisions were assigned as a result of HPLC analysis of the four gel chromatography fractions; the boundaries between the four fractions were selected arbitrarily according to the positions of peaks produced when the bitter fraction from gel filtration was separated by HPLC, and also according to the presence of certain large peaks. Fraction I roughly corresponded to a gel filtration elution volume of 180 mL, Fraction 11 to 210 mL, Fraction III to 120 mL and Fraction IV to 150 mL. The chromatogram was also divided into four (1-4) fractions of approximately equal total peak areas. These fractions were collected following repeated injections of bitter extract and pooled. Sensory analysis (Table 3) of the two sets of fractions showed that Fractions IV, 3 and 4 were the most bitter. Considering which of these were eluted in overlapping time intervals, it appears that the bitter compounds from the cheese extract eluted between 35 and 66 min by HPLC and could, therefore, be any of many compounds. When the fractions were rechromatographed, they eluted in the Champion and Stanley/285
1.5 -A2.0 ASIO " A7S0
I.'
1.3 1.2 1.1 1.0
91
0.9
...
0.8 0.7 0.6
3:
...:I:m
0.5
> z ~
0.' 0.3 0.2
~
0.1 0
,
0
10
20
30
50
'0
70
60
0 80
TIME (MIN)
INJECT
Fig. 2. Separation of bitter extract by HPLC.
same time periods in which they had been collected. Fraction IV, which corresponds to the bitter gel filtration fraction, was separated by HPLC into at least forty-four compounds. Results of analysis of the fractions taken by area (Table 4) indicate that the level of free amino acids is far higher in Fraction 1 and that the amount of peptides or proteins is greater as well. Fractions 3 and 4, which were both bitter, had slightly higher hydrophobicities than either 1 or 2. There was a correlation between elution time and average hydrophobicity of the fractions which implies that the elution of peptides from the bitter extract Table 3. Bitterness score l of HPLC fractions. Fractionated according to area
Fractionated according to gel chromatography separation
-1.7 1.7 1.9 6.7
I II III IV
I 2
3 4
'Distance (cm) on 10 cm line relative to 11.6
X
3.6 0.8 6.9 6.2
IO- li M quinine sulphate.
1.S,
1.4; 1.31
1.': ,L'.. ,.,., 1,1
I
J ::: ,..
'.3 ,.,
';ljJ,~~4~~~~~-=;:::::::::::::;;=::=:::±
,
TIME ,"'N.
INJECT
Fig. 3. Division of HPLC fractions according to gel filtration fractions (I-IV) and area (1-4); .... ·gel filtration, - - area.
under the present conditions of separation is affected by their average hydrophobicity. The least hydrophobic peptides elute first by the least hydrophobic eluent and vice versa, which is consistent with the hydrophobic nature of the column packing. Marked differences were seen in amino acid composition of the four fractions (Table 5). Fraction I was moderately bitter and it is possible that its high lysine and arginine content may be due to a bitter peptide high in these amino acids. Two hydrophobic amino acids, valine and leucine, occurred at substantially higher levels in the later, more bitter fractions while two less hydrophobic amino acids, lysine and arginine, appeared in greater quantities in the first fraction. Fractions 3 and 4 resembled the bitter fraction from gel filtration (Table 2) in having higher levels of glutamic acid, leucine, proline and valine, but unlike the bitter gel filtration fraction, did not exhibit elevated lysine levels. Synthetic boundary ultracentrifugation of the four fractions yielded the sedimentation coefficients given in Table 4. Since sedimentation coefficients are proportional to Mw, barring conformational differences, it appears that the bitter fractions, 3 and 4, had average Mw less than the 448 dalton value of the pure peptide pro-phe-gly-lys that gave a sedimentation coefficient of 2.04. This is consistent with the range of 158-218 estimated for the bitter compounds by gel filtration. The sedimentation values indicate that while higher Mw compounds are eluted earlier from the HPLC column, there is no obvious relationship between the Mw' of the compound in the bitter extract and their retention on the column when using a water/methanol gradient. In order to clarify the relationship between retention time, hydrophobicity and Mw, an attempt was made to chromatograph known peptides under standard separation
Table 4. Analysis of HPLC fractions according to area. % CH"OH at
Fraction I
2 3 4
midpoint of fraction
14.3 33.6 52.7 76.5
286/Champion and Stanley
Sedimentation coefficient
Absorbance
580nm 1.56 1.64
0.53 0.76
1.81 0.29 0.27 0.16
750 nm 0.52 0.35 0.48 0.39
Total amino acids'
H ydrophobicity (cal/residue)
170
1,460
200 230 180
1,500 1,530 1,610
J. Insl. Can. Sci. Technol. Aliment. Vol. 15, No. 4, 1982
Table 5. Amino acid analysis of HPLC fractions according to area. Amino acid ASP THR SER GLU PRO GLY ALA VAL MET ILE LEU TYR PHE LYS HIS ARG
Mole percentages I J.2 I.3 0.8 29.9 7.0 I.3 1.4 2.5 0.5 2.5 5.5 2.0 4.7 34.0 3.0 2.6
2
3
4
3.3 1.7 0.8 36.0 9.5 0.8 1.5 3.9 0.9 3.0 27.4 1.8 2.6 4.3 1.5 0.9
2.7 1.4 J.2 34.2 10.8 1.4 1.6 9.2 0.2 2.2 23.9 1.9 3.4 3.6 1.4 1.1
1.8 1.7 1.7 24.7 11.7 1.4 1.7 19.7 0.1 1.6 23.9 1.4 3.4 3.1 1.2 1.0
conditions. This work was hampered by the poor water solubility of many peptides and their low absorbance at 280 nm. The data (Table 6) indicate a possible relationship among the three variables. Considering (1) that molecules with a Mw higher than insulin did not elute, (2) the results where, in general, compounds of higher Mw eluted later, and (3) the positive relation between retention time and hydrophobicity, it seems that the retention of peptides and proteins from the bitter extract under the conditions of this work is influenced by a combination of Mw and hydrophobicity factors.
Conclusion This research has shown that HPLC, using gradient elution from an octadecylsilane type column, is effective for separation of the components of an extract from bitter Cheddar cheese. The bitter extract contained at least seventy-one compounds of which only some were bitter. While gel filtration achieved limited fractionation of the bitter extract, HPLC enabled more extensive and reproducible separation. Elution order was related to the calculated average hydrophobicity of fractions collected; more hydrophobic peptides were eluted only by higher concentrations of methanol. There was no consistent relationship between Mw of the fractions and either retention time or methanol concentration, however, correlation was found between the Mw of pure peptides and retention time under standard chromatography conditions. Thus, both hydrophobicity and Mw seem to be factors in influencing separation.
Two bitter fractions collected by HPLC had higher average hydrophobicities than nonbitter fractions, but the bitter fraction from gel filtration did not show this relationship. Casein has a high average hydrophobicity (1,605 cal/residue; Guigoz and Solms, 1976) and it is to be expected that many of its peptides should exhibit high hydrophobicities. Since the fractions collected were mixtures, the compounds present in the greatest concentration could make large contributions to hydrophobicity but not contribute as much to bitterness as other less plentiful molecules.
Acknowledgements Dr. ER. van de Voort provided assistance with the ultracentrifugation. This work was funded in part by the Ontario Milk Marketing Board, Kraft Foods and the Ontario Ministry of Agriculture and Food.
References Bigelow, c.c. 1967. On the average hydrophobicity of proteins and the relationship between it and protein structure. J. Theor. BioI. 16:187. Guigoz, Y. and Solms, J. 1976. Bitter peptides, occurrence and structure. Chem. Senses Flavor 2:71. Hancock, W.S., Bishop, C.A., Prestidge, R.L., Harding, D.R.K. and Hearn, M.T. 1978a. High pressure liquid chromatography of peptides and proteins. 11. Use of phosphoric acid in the analysis of underivatised peptides by reversed phase high pressure liquid chromatography. J. Chromatogr. 153:391. Hancock, W.S., Bishop, C.A., Prestidge, R.L., Harding, D.R.K. and Hearn, M.T. 1978b. Reversed phase high pressure liquid chromatography of peptides and proteins with ion-pairing reagents. Science 200: 1168. Harwalkar, V.R. and Elliot, J.A. 1965. Isolation and partial purification of bitter components from Cheddar cheese. J. Dairy Sci. 48:784. Harwalkar, V.R. and Elliot, J.A. 1970. Isolation of bitter and astringent fractions from Cheddar cheese. J. Dairy Sci. 54:8. Hodges, R., Kent, S.B.H. and Richardson, B.C. 1972. The mass spectra of some permethylated acetylpeptides. Biochim. Biophys. Acta 257:54. Huber, L. and Klostermeyer, H. 1974. Isolierung und identifizierung eins bitterstoffes aus butterkase. Milchwissenschaft 29:449. Kirimura, J., Shimizu, A., Kimizuka, A., Ninomiya, T. and Katsuya, N. 1969. The contribution of peptides and amino acids to the taste of foodstuffs. J. Agric. Food Chem. 17:689. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, RJ. 1951. Protein measurement with the Folin phenol reagent. J. BioI. Chem. 193:265. Matoba, T. and Hata, T. 1972. Relationships between bitterness of peptides and their chemical structure. Agric. BioI. Chem. 36:1423. Molnar, I. and Horvath, C. 1977. Separation of amino acids and peptides on nonpolar stationary phases by high performance liquid chromatography. J. Chromatogr. 142:623.
Table 6. HPLC of pure peptides under standard conditions. Peptide gly-gly gly-gly-gly gly-gly-gly-gly leu-val gly-tyr glu-cys-gly pro-phe-gly-Iys Iys-phe-ile-gly-Ieu-met insulin, A chain
Can. Inst. Food Sci. Technol. J. Vol. 15. No. 4, 1982
Retention time (min) 2.1 2.1 3.6 2.5 5.0 3.9 47.0 37.0
Mw 132 189 246 230 238 307 448 708 2,697
Hydrophobicity (cal/residue)
o
o o
2,055 1,435 183 1,688 1,800 712
Champion and Stanley/287
Monch W. and Dehnen, W. 1977. High perfonnance liquid chromatography of peptides. J. Chromatogr. 140:260. Monch W. and Dehnen, W. 1978. High perfonnance liquid chromatography of polypeptides and proteins on a reversed phase suppOI1. J. Chromatogr. 147:415. Ney, K.H. 1971. Voraussage der bitterkeit von peptiden aus deren aminosaurezusammensetzung. Z. Lebensm. Vnters. Forsch. 147:64. Ney, K.H. 1972. Aminosaurezusammensetzung von proteinen und die bitterkeit ihrer peptide. Z. Lebensm. Vnters. Forsch. 149:321. Novgorodova, N .S., Buzov, I.P. and Nebel1, V.K. 1975. Trudy, Vsesoyuznyi Nauchnoissledovatel'skii Intitut Maslodel'noi i Syrodel'noi Promyshlennosti. No. 18:107. English abstract Food Sci. Technol. Abstr. 11 :5P767. Paik, W.K. and Kim, S. 1972. Effect of methylation on susceptibility of protein to proteolytic enzymes. Biochemistry 11:2589.
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Richardson, B.C. and Creamer, L.K. 1973. Casein proteolysis and bitter peptides in Cheddar cheese. N.Z. J. Dairy Sci. Technol.
8:46. Rivier, J. 1978. Use oftrialkyl ammonium phosphate buffers in reversed phase high performance liquid chromatography for high resolution and recovery of peptides and proteins. J. Liq. Chromatogr. 1:343. Stanley, D.W., Emmons, R.B., Modler, H.W. and Irvine, D.M. 1980. Cheddar cheese made with chicken pepsin. Can. Inst. Food Sci. Technol. J. 13:97. Stanley, D. W. 1981. Nonbitter protein hydrolysates. Can. Inst. Food Sci. Technol. J. 14:49.
Accepted May 4, 1982
J. Inst. Can. Sei. Tecimol. A/imem. Vol. 15, No. 4, 1982