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β-Casomorphins: Analysis in Cheese and Susceptibility to Proteolytic Enzymes from Lactococcus lactis ssp. cremoris1
J3-Casomorphins: Analysis in Cheese and Susceptibility to Proteolytic Enzymes from Lactococcus lactis ssp. cremoris1 M. R. MUEHLENKAMP and J. J. WARTHESEN Minnesota-South Dakota Dairy Foods Research Center, Department of Food Science and Nutrition, University of Minnesota, SI. Paul 55108
ABSTRACT
INTRODUCTION
{3-Casomorphins are small peptides derived from {3casein ranging from three to seven amino acids. The biologically active sequence of seven amino acids (Tyr-Pro-Phe-Pro-Gly-Pro-Ile), residues 60 to 66, was first isolated from a tryptic digest of casein by Brantl et al. (4). This peptide and fragments of the amino acid sequence possessed opioid activity in receptorbinding assays (4, 6) and isolated organ preparations (4). A common N-terminal sequence, Tyr-Pro-Phe, is present in all the peptides, indicating a specific action for binding to opioid receptors. Currently, there is no confirmed evidence that these peptides influence physiological functions upon ingestion. However, intravenous administration indicated opioid activity. Analgesia resulted after intracerebroventricular injection of (3-casomorphin into rats (4). In 1989, Daniel et al. (8) used intragastric application of labeled (3casomorphin with kinetic monitoring of gastric emptying and gastrointestinal transit time; that transit time was increased by feeding casein suspensions. Some evidence (19) suggested that {3-casomorphins are created as a result of casein digestion and are naturally present in dairy products. Recently, {3casomorphin precursors have been identified in Parmesan cheese (1). However, it is not known whether {3-casomorphins are created or degraded in dairy products as a result of enzymatic hydrolysis by native milk enzymes or bacterial starter enzymes. In addition, the release of {3-casomorphin peptides from larger protein sequences may be influenced by product processing conditions (14). Creation of {3casomorphins upon ingestion of milk was investigated Abbreviation key: DP IV = dipeptidyl-dipeptidase in vitro and in vivo with adult humans (21). {3IV, Pep X = X-prolyl dipeptidyl aminopeptidase. Casomorphins were found in digests in vitro and in vivo; however, opioid activity could not be determined. Similarly, Meisel ( 18) found opioids in vivo in miniature pigs, but physiological significance could Received February 9, 1995. not be determined. Accepted September 5, 1995. lPublished as Paper Number 21,689 of the contribution series of According to Hazum (13), the high content of prothe Minnesota Agricultural Experiment Station. Support also line residues in {3-casomorphins confers resistance to provided by the Minnesota-South Dakota Dairy Foods Research many degradative enzymes-pepsin, trypsin, and Center.
Commercial cheese products were surveyed for (3casomorphin peptides. Two extraction methods were compared: 1) water and 2) chloroform and methanol. Peptide profiles were determined using reverse-phase HPLC and multiple wavelength detection. {3-Casomorphin standards were used for comparison with cheese peptide profiles. Results indicated that peptides were present in cheeses with HPLC elution times that were similar to those for {3-casomorphins. However, comparison of absorbancies of the peaks at multiple wavelengths did not indicate peptides similar to {3-casomorphins. Therefore, {3-casomorphins were absent, or concentrations were below the HPLC detection threshold for {3-casomorphin of 2 J.tglml of cheese extract. The susceptibility of {3-casomorphins to the proteolytic system of a commercial strain of Lactococcus lactis ssp. cremoris was investigated. {3-Casomorphin standards were incubated at 4°C with bacterial cell lysate at pH 5.0,5.2,5.4, and 5.7. Salt concentrations varied among 0, 1.5, and 5%. The concentration of added {3-casomorphins and the degradation products were monitored over 15 wk using HPLC. Enzymatic degradation of {3-casomorphins was influenced by the combination of pH and salt concentrations at cheese ripening temperatures. Therefore, if formed in cheese, {3-casomorphins may be degraded under conditions common for Cheddar cheese. (Key words: (3-casomorphins, cheese proteolysis, Xprolyl dipeptidyl aminopeptidase)
1996 J Dairy Sci 79:20-26
20
ll-CASOMORPHINS IN CHEESE
21
chymotrypsin. Nevertheless, l3-casomorphins would dard was prepared in a series of concentrations behave to be created and avoid degradation by starter tween 1 mg and 1 p.g/ml of water. These samples were enzymes in cheese and intestinal enzymes in humans analyzed by reverse-phase HPLC to determine retenif they were to reach opioid receptors and cause a tion times and detection thresholds. I3-Casomorphin standards -3, -5, and -7 also were physiological response. Tome et al. (26) observed that 13-casomorphins could be degraded on the prepared in water as solutions of 0.5 mg/ml for use in mucosal side of the intestinal lining by the action of the degradation study. dipeptidyl-dipeptidase IV (DP IV) if they were ingested. The action of DP IV is to cleave X-Pro (N- Reverse-Phase HPLC Conditions terminal) dipeptides from larger peptides (13, 16). The reverse-phase HPLC analyses of 13For 13-casomorphins, Tyr-Pro is released. Therefore, 13casomorphin standards and cheese peptide extracts casomorphin peptides would likely be destroyed bewere done on a CI8 (201TP54) analytical column (5 fore crossing the intestinal lining and reaching opioid p.m, 25 em x 4.6 mm; VYDAC, Hesperia, CA) with a receptors. 50-p.1 loop. Gradient solvent delivery was achieved Similarly, bacterial starter cultures contain many using a Spectra Physics SP8800 ternary pump at a proteolytic enzymes that are responsible for the rate of 1 mVmin. Solvent A was 0.1% triflouroaflow breakdown of protein into peptides and amino acids. and HPLC grade water. Solvent B was 0.1% cetic acid The peptidases that have been found and characterand 9.9% HPLC trifluoroacetic acid, 90% acetonitrile, ized in lactic acid bacteria used in cheese products grade water. A linear gradient increase of 1% solvent have been summarized (5, 22, 23, 27). Because was used for all peptide separations. After the B/min caseins have high proline contents, bacterial starter initial gradient to 35% B (l3-casomorphin standards) cultures produce proline-specific peptidases that recognize the pyrolidine ring of proline. These pepti- or 50% B (cheese extracts), an accelerated gradient dases also are present in dairy products as a result of was programmed to 80% B and then back to 100% A starter culture addition. The peptidases break down to condition the column prior to the next run. Multipeptides containing proline into free amino acids that ple wavelength detection at 220, 254, and 280 nm was can be utilized by the cells. X-Prolyl dipeptidyl used for all samples (diode array 1040A; Hewlett aminopeptidase (Pep X), aminopeptidase P, proline Packard, St. Paul, MN). aminopeptidase, aminodipeptidase, amidodipeptidase, and dipeptidyl-peptidase have been characterized and Cheese are important for the breakdown of peptides containCommercial cheeses were used for water and oring proline (23). In particular, Pep X can cleave Xganic peptide extractions. The varieties were chosen Pro dipeptides from larger peptides, which are similar based on a range of ages and mode of ripening: Extra in action to DP IV. Also, Pep X has been shown to Sharp Cheddar, Swiss (aged 60 d), Blue, Brie, and have enzyme activity in a cheese matrix (7) and Limburger. This cross-section of cheeses would result activity on l3-casomorphin-7 substrate at pH 7.0 (3, in a range of proteolysis products with potential for 1315). casomorphins. All cheeses were purchased commerThe objectives of this study were to evaluate comcially, stored at 4°C until extracted, and analyzed mercial cheese varieties for l3-casomorphin peptides twice. and to investigate whether enzymes found in the proteolytic system of Lactococcus lactis ssp. cremons would degrade 13-casomorphins, if they were present, Peptide Extractions in a cheese system. Two methods were used to extract peptides from cheese. A water extraction method developed by MATERIALS AND METHODS Kuchroo and Fox (1 7) was modified to use 20 g of cheese homogenized with 40 ml of water in a Sorvall Omni mixer (model 17105; Omni Corp, Waterbury, i3-Casomorphin Standards CT) for 1.5 min on speed 3.5 (25). The slurry was 13-Casomorphin standards, -3, -5, and -7 amino acid centrifuged using an lEC model HT benchtop censequences and morphiceptin, were obtained from trifuge (lEC, a division of Damon, Needham Heights, Sigma Chemical Company (St. Louis, MO) and 13- MA) at 18,000 x g for 40 min. Fat was removed, and casomorphin-4 and -6 were obtained from American the water extracts were retained. The solids were Peptide Company, Inc. (Sunnyvale, CA). Each stan- reextracted using 40 ml of water. The combined exJournal of Dairy Science Vol. 79, No.1, 1996
22
MUEHLENKAMP AND WARTHESEN
tracts were filtered through glass wool and ultrafiltered into two molecular weight fractions. Each extract was added to an Amicon ultrafiltration 8400 stirred cell unit (Beverly, MA) with a YM2 or YM5 membrane (1000 or 5000 molecular weight cutoff). Both retentate and permeate were frozen, freeze-dried, and stored at -20°C. The organic extraction procedure developed by Han'lalkar and Elliott ( 12) was modified and used to extract peptides from the cheeses. Approximately 4 g of shredded, freeze-dried cheese were added to 10 ml of chloroform-methanol (6.5 ml of chloroform and 3.5 ml of methanol) and homogenized in a Sorvall Omni mixer for 1 min on speed 3.5. The slurry was centrifuged in an IEC model HT benchtop centrifuge at 2300 x g for 10 min (4500 rpm). The supernatant for each of three extractions were made biphasic by addition of 5 ml of water. The lower chloroform layer and the precipitate were discarded. The upper methanol and water layer was diluted further with water and ultrafiltered as described. Methanol was removed from the samples using a roto evaporator (model 410-2000; Haake Buchler, Saddle Brook, NJ). The extracts were then frozen and freeze-dried. In both cases, freeze-dried extracts were reconstituted as 10 mg/ml of water, and 50 1-'1 were injected under the HPLC conditions described. Sample HPLC runs were 50 min.
TABLE 1. Mean retention times and detection thresholds of 13casomorphin standards. Standard
Retention time!
SD
I3-Casomorphin-3 I3-Casomorphin-4 (3-Casomorphin-5 I3-Casomorphin-6 I3-Casomorphin-7 Morphiceptin
28.12 30.16 27.72 29.81 35.19 25.39
0.17 0.21 0.19 0.17 019 0.40
Detection threshold 2
(min) 1.5 2.0 2.0 1.0 2.5 2.5
!Means are the result of five HPLC runs. 2Thresholds were determined as a peak area dOO.
phosphate (4 ruM) buffer, pH 6.4. The resulting paste was lysed with an Eaton press (9) and centrifuged (35,000 xg for 30 min at 4°C). The supernatant was retained and stored at -50°C. In Vitro Enzymatic Tests
Test tubes were prepared with 5 ml of sodium acetate buffer at 4°C, pH 5.0, 5.2, 5.4, or 5.7. Sodium chloride was added at 0, 1.5, or 5% (wt/vol) to the buffer followed by 100 1-'1 of the cell lysate preparation, which was thoroughly mixed before the addition of 0.5 ml of l3-casomorphin standard, -3, -5, or -7. Each tube was vortexed, and one drop of toluene was added Samples Augmented with JS-Casomorphin before storage at 4°C. Samples of each combination of Cheddar cheese samples, 20 and 15 g, were aug- pH, salt, and l3-casomorphin were prepared. mented with 5 mg of l3-casomorphin before homogeniSamples were prepared for HPLC analysis within 5 zation with water or chloroform and methanol. Ex- min and after 2, 4, 6, 8, 10 and 15 wk. At each tractions were performed on augmented and interval, 300 1-'1 of sample from each reaction tube unaugmented samples for comparison. The resulting were removed and added to 500 1-'1 of 12% trichloroextracts were not ultrafiltered, but freeze-dried and acetic acid. The resulting 800-1-'1 samples were stored reconstituted as 10 mg/ml in water before analysis by at -20°C until HPLC analysis. HPLC. Control samples were also prepared and analyzed throughout 15 wk under conditions common for Cell Lysate Preparation cheese. Controls consisted of acetate buffer alone (pH A bacterial culture cell lysate was used as an en- 5.0 and 5.7); acetate buffer and cell lysate; acetate zyme preparation for hydrolysis of l3-casomorphin. buffer, cell lysate, and salt; and buffer with each 13The bacterial strain was a commercial preparation of casomorphin standard (no cell lysate added). L. lactis ssp. cremoris from Systems Bio-Industries, Inc. (Waukesha, WI). Ten milliliters of culture from RESULTS AND DISCUSSION a 125-ml can of frozen concentrate prepared by Systems Bio-Industries, Inc. were inoculated into 1 L of milk medium as described by Thomas and Turner JS-Casomorphin Standards (23). This culture was allowed to grow for 16 hand Retention times of l3-casomorphin standards are then was neutralized with sodium hydroxide (ION) and 60 ml of sodium citrate 25% (wt/vol). Cells were listed in Table 1. These results defined the time frame recovered from centrifugation (10,000 x g for 5 min at between 26 and 36 min as the area to investigate in 4°C) and washed twice with sodium acetate (46 mM) peptide profiles for l3-casomorphins. These retention Journal of Dairy Science Vol. 79, No.1, 1996
~·CASOMORPHINS
times were reproducible and had standard deviations of 0.1 to 0.4 min. The concentrations of {3casomorphins were varied to determine the minimum concentration that was necessary for peak identification. Threshold values were determined as twice the height of baseline noise (40 to 50 mAU). If {3casomorphin concentrations were below these threshold values, they could not be distinguished from the baseline and were reported as less than the threshold concentration. Data collected for the threshold determination was used also to calculate {3-casomorphin calibration lines by plotting HPLC peak area versus known concentrations. Data were fitted to linear equations for each standard and were used in subsequent analyses to determine {3-casomorphin concentrations from peak area (data not shown). The r 2 values for each {3casomorphin standard calibration curve was 0.99. Detection at 220, 254, and 280 nm was chosen for several reasons. All peptide bonds absorb at 220 nm. Therefore, peaks would be expected at this wavelength for all peptides, including {3casomorphins. Tyrosine and Phe absorb at 280 nm because of their aromatic side chains, and Phe also absorbs at 254 nm. Therefore, {3-casomorphins, which contain Tyr and Phe, formed a peak at all three wavelengths. This property was used to differentiate the {3-casomorphins from other peptides. {3-Casomorphin Standards Recovery
{3-Casomorphin-7 was recovered from augmented cheese samples using both extraction methods. Controls were prepared along with the augmented samples so that direct comparisons could be made. Calculations of the potential {3-casomorphin produced from 15 to 20 g of cheese indicated that 2% of the potential {3-casomorphin would be necessary before a peak in the chromatogram could be observed. Figure 1 shows chromatograms comparing organic extracts of mild Cheddar cheese with {3-casomorphin-7 standard. A major peak occurred at 34.6 min, corresponding to {3-casomorphin-7 in the augmented sample. Recovery of {3-casomorphin in the organic extract, using the HPLC calculated peak area and the {3-casomorphin-7 calibration line, was 55 ± 12%. {3Casomorphin could be lost during extraction because the procedure required several transfer steps during which losses could occur. Also, during extraction, the proteolytic enzymes that were present in cheese might degrade the peptide. Finally, {3-casomorphins might be retained by cheese solids.
23
IN CHEESE
71111
6ell 5
g see ~ 4ell
~
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~
3ell
:il0 2e"
~
lee e
Ie
ze
TIME (MIN) 3 e
Figure 1. Comparison of chromatograms from organic extracts of augmented and control medium Cheddar cheese to the {3casomorphin-7 (SCM-7) standard. AU '" Absorbance units.
Similarly, recovery of {3-casomorphin-7 from the water extract of Cheddar cheese was 88 ± 12%. These results indicated that {3-casomorphins would be recovered using either extraction method. Even though {3-casomorphins tended to be hydrophobic peptides, the water extraction method was more efficient than the organic extraction. Cheese Extracts
Peptide profiles of each cheese variety were determined by HPLC. The HPLC elution time frame of interest, as defined by the {3-casomorphin standards, was 26 to 36 min. After this area in the chromatogram was enlarged, individual peaks could be seen. However, each peak might not represent one peptide. In most cases, there were probably several peptides that coeluted. Further clarification of peptides was sought by viewing the same peptide profiles at 254 and 280 nm. Using Brie cheese as an example, inspection of all three wavelengths indicated that the cheese did not contain detectable amounts of {3-casomorphins (Figure 2). All three wavelengths are shown in Figure 2, and there is a peak at 28 min and 220 nm that does correspond to {3-casomorphin. However, the peak has no detectable absorbance at either 280 or 254 nm. Therefore, it does not represent a {3casomorphin peptide. Elution time and absorbance at three wavelengths were the criteria used to detect (3casomorphins in all other cheese varieties. Similarly, peaks appeared with retention times that were similar to those of {3-casomorphins, but peaks failed to indicate absorbance at all three wavelengths. This evaluation was performed twice on each cheese variety with the same results. Journal of Dairy Science Vol. 79, No.1, 1996
24
'"
1
MUEHLENKAMP AND WARTHESEN
However, if no physiological benefits could be gained by trace amounts, further analysis to detect even lower concentrations or broader product surveys might be unnecessary. This product survey was limited to five varieties of cheese; therefore, the potential for other varieties to contain 13casomorphins is conceivable.
2BB
0'
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IBB
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Iil.j-----~--------~~....A---
26
28
39
In Vitro Enzymatic Tests
32
T1\1E ;\1r:\)
Figure 2. Comparison of peptide ptofiles of Bne cheese at elution of 220, 254, and 280 nm between 20 and 34 min. AU = Absorbance units.
The absence of l3-casomorphins from the cheese samples in this study could be the result of several factors. First, l3-casomorphins might not be released from the l3-casein sequence. Specific proteolytic enzymes would be necessary to release the 60 to 66 residues from l3-casein. To date, only larger fragments containing $-casomorphin in their sequence have been identified in Cheddar cheese (2). The sequence of cleavage leading to l3-casomorphin is not known. Second, i3-casomorphins might be degraded, even as they are created, by starter culture proteolytic enzymes. Finally, i3-casomorphins might be present in amounts smaller than the determined threshold values. The threshold value, expressed in micrograms per milliliter of cheese extract, was 2 ",g/ml, indicating that 2% of the theoretical i3-casomorphin potential could be detected if it were formed. A more sensitive analysis method, such as radioimmunoassays, might be appropriate to detect lower concentrations.
Control samples were prepared to characterize chromatographic profiles that would be expected during the study as a result of the buffer medium. Results of these samples indicated that the HPLC peaks between 4 and 8 min were the result of TCA and acetate buffers and that the peak at 19 to 20 min was from the acid only. As expected, these peaks were found in all samples and remained constant throughout 15 wk. Buffer and enzyme controls indicated that no peaks were introduced by the lysate preparation. i3-Casomorphins in solutions without enzymes were analyzed to determine whether they remained stable in the buffer medium throughout the study. The percentage of each remaining standard was determined at pH 5.0 and 5.7 only. At both pH conditions, no 13casomorphin degradation was observed. Any peak formation in samples would have been due to enzyme degradation of i3-casomorphins. Table 2 contains data for one complete set of experiments through 15 wk. These data are specific for this strain and would vary depending on preparation and strain selection. Figure 3 shows i3-casomorphin-3 after 2 wk at pH 5.0 and at 0, 1.5, and 5% of salt. Only with 5% salt did l3-casomorphin-3 remain after 15 wk. At 15 wk, 30% of the original l3-casomorphin concentration re-
TABLE 2. Concentrations of p-casomorphin standards at 0, 6, and 15 wk; pH of 5.0, 5.2, 5.4, and 5.7; and 0, 1.5, and 5% salt. p-Casornorphin-3 pH
Salt
o wk
6 wk
15 wk
o wk
33 32 33 48 42 46 50 58 32 31 21 32.4
<1.5 <1.5 20
<1.5 <1.5 10.9
<1.5 <1.5 4 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5
<1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5
36 39 31 30 33 32 22 38 36 31 32 32
(o/c)
5.0
5.2
5.4
5.7
0 1.5 5 0 1.5 5 0 1.5 5 0 1.5 5
Journal of Dairy Science Vol. 79, No. 1, 1996
p-Casomorphin-7
p-Casomorphin-5 6 wk
( "g/m!) <2.0 4 22 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <20 <2.0
15 wk
o wk
6 wk
15 wk
<2.0 <2.0 26 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0
39 38 34 47 46 46 38 39 46 31 26 31
<2.5 22 31 <2.5 17 42 <2.5 <2.5 29 <2.5 <2.5 <2.5
<2.5 20 31 <2.5 <2.5 40 <2.5 <2.5 25 <2.5 <2.5 <2.5
~-CASOMORPHINS
25
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HIll
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I
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,
S%SALT
l.l% SALT I
3all
1
0% SALT
A
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TIME (MIN)
3a
48
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9
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1.5% SALT
•
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S%SALT
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TIME (MIN)
3a
4a
Figure 3. Chromatograms of l3-casomorphin-3 stored for 2 wk at pH 5.0 in 0, 1.5, and 5% NaCl. I3-Casomorphin peak is at 28 minutes. AU = Absorbance units.
Figure 5. Chromatograms of l3-casomorphin-7 at pH 5.2 after 2 wk in 0, 1.5, and 5% NaC!. I3-Casomorphin peak is at 34 min. AU = Absorbance units.
mained. In addition to the loss of i3-casomorphins, we also observed an increase in the peak at 16 min. This peak was determined to be Tyr-Pro by amino acid analysis. The production of this peptide suggests the action of the Pep X enzyme. Results were similar for i3-casomorphin-5. The only conditions under which i3-casomorphin survived for 15 wk were in 5% salt and pH 5.0 (75% remaining). Figure 4 shows i3-casomorphin-5 after 2 wk in each of the buffers and 5% salt. Again, a peak at 16 min was formed at each pH. i3-Casomorphin-7 peptide was more resistant to proteolysis than was i3-casomorphin-5 or -3. No loss of i3-casomorphin-7 occurred throughout 15 wk at pH 5.0 in the presence of 5% salt. i3-Casomorphin-7 also remained after 15 wk at pH 5.2 and 5.4, but only in the presence of 5% salt. The only case in which i3casomorphin at 1.5% salt survived 15 wk was at pH
5.0. Small amounts of i3-casomorphin did remain after 6 wk at pH 5.2 and 1.5% salt but was degraded after 15 wk. Figure 5 shows i3-casomorphin after 2 wk at pH 5.2 in 0, 1.5, and 5% salt. When salt was absent, i3-casomorphin was degraded within 2 wk. The results of the stability tests on all i3casomorphin standards agreed with similar results in the literature that were related to pH and salt influence on proteolysis (10). In most cases, lower pH and higher salt content decreased proteolytic activity, which was in agreement with our observations of i3casomorphin substrates. Stepaniak et a1. (20) also found that i3-casomorphin-7 was susceptible to degradation by Pep X and further suggested that Pep X inhibited the action of other peptidases. The optimal pH for typical proteolytic enzymes in starter cultures is near neutrality (19). Therefore, less enzyme activity would be expected at the lower pH used in this study. However, the activity at the low pH and low temperature was still rapid in the absence of salt. Rate of proteolysis in cheese was influenced by the content of salt in moisture (10, 11). The combination in this study oflow pH and 1.5 to 5% salt created an environment that partially influenced activity of the starter enzymes.
499
S
1
399 pH
s.?
~
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2aa
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laa
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s.a
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TIME (MIN)
3a
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48
Figure 4. Chromatograms of l3-casomorphin-5 stored for 2 wk in 5% NaCl at pH 5.0, 5.2, 5.4, and 5.7. I3-Casomorphin peak is at 27.5 min. AU = Absorbance units.
CONCLUSIONS
Low concentrations of i3-casomorphins might be present in fermented dairy products, but they might also be degraded even as they are formed. The stability of i3-casomorphins under model conditions for cheese ripening was dependent on pH, salt, and type of enzymes present. The enzyme Pep X, a serine protease, might degrade i3-casomorphins that are produced in dairy products by the action of proteinases and peptidases. Journal of Dairy Science Vol. 79, No.1, 1996
26
MUEHLENKAMP AND WARTHESEN
We used proteolytic enzymes from a commercial strain of L. lactis ssp. cremons. Other strains may have similar proteolytic systems but variable enzymes and substrate specificities. Understanding the characteristics of the proteolytic system of cheese and, particularly, the action of the Pep X proteolytic enzyme would be important for estimating 13casomorphin formation. Because Pep X has been purified from several lactic acid bacteria, similar 13casomorphir. degradation characteristics might be expected in other products.
REFERENCES 1 Addeo, F., 1. Chianese, A. Salzano, R. Sacchi, U. Cappuccio, P. Ferranti, and A. Malomi. 1992. Characterization of the 12% trichloroacetic acid soluble oligopeptides of ParmigianoReggiano cheese. J. Dairy Res. 59:401. 2 Belitz, H. D., and K. P. Kaiser. 1993. Monitoring Cheddar cheese ripening by chemical indices of proteolysis. Z. Lebensm. Unters. Forsch. 197:118. 3 Booth, M., 1. N. Fhaolain, P. V. Jennings, and G. O'Cuinn. 1990. Purification and characterization of a post proline dipeptidyl aminopeptidase from Streptococcus cremoris AM2. J. Dairy Res. 57:89. 4 Brantl, V., H. Teschemacher, A. Henschen, and F. Lottspeich. 1979. Novel opioid peptides derived from casein ( (3casomorphins). Hoppe-Seyler's Z. Physiol. Chern. 360:1211. 5 Casey, M. G., and J. Meyer. 1985. Presence of X-prolyldipeptidyl-peptidase in lactic acid bacteria. J. Dairy Sci. 68: 3212. 6 Chang, K. J., A. Killian, E. Hazum, and P. Cuatrecasas. 1981. Morphiceptin (NH4-Tyr-Pro-Phe-Pro-CONH2) A potent and specific agonist for morphine (I') receptors. Science 212:75. 7 Chapot-Chartier, M.-P., C. Deniel, M. Rousseau, L. Vassal, and J.-C. Gripon. 1994. Autolysis of two strains of Lactococcus lactis during cheese ripening. Int. Dairy J. 4:251. 8 Daniel, H., M. Vohwinkel, and G. Rehner. 1989. Effect of casein and beta-casomorphins on gastrointestinal motility in rats. J. Nutr. 120:252. 9 Eaton, N. 1962. Kew press for disruption of microorganisms. J. Bacterial. 83:1359. 10 Fox, P. F. 1987. Cheese: Chemistry, Physics and Microbiology. Vol. 1. General Aspects. Elsevier Appl. Sci., New York, NY. 11 Fox, P. F., and J. Law. 1991. Enzymology of cheese ripening. Food Biotechnol. (NY) 5:239.
Journal of Dairy Science Vol. 79, No.1, 1996
12 Harwalkar, V. R., and J. A. Elliott. 1971. Isolation of bitter and astringent fractions from Cheddar cheese. J. Dairy Sci. 54:8. 13 Hazum, E. 1991. Neuroendocrine peptides in milk. Trends Endocrinol. Metab. (Jan-Feb.):25. 14 Kaufmann, W. 1984. Influences of different technological treatments of milk on the digestion in the stomach. VI. Estimation of amino acids and urea concentrations in blood; conclusions regarding nutritional value. Milchwissenschaft 39:284 15 Kiefer-Partsch, 8., W. Bockelmann, A. Geis, and M. Teuber. 1989. Purification of an X-prolyl-dipeptidyl aminopeptidase from the cell wall proteolytic system of Lactococcus lactis subsp. cremoris. Appl. Microbiol. Biotechnol. 31:75. 16 Kriel, G., M. Umbach, V. Brant!, and H. Teschemacher. 1983. Studies on the enzymatic degradation of {3-casomorphins. Life Sci. 33: 137. 17 Kuchroo, C. N., and P. F. Fox. 1982. Soluble nitrogen in Cheddar cheese: comparison of extraction procedures. Milchwissenschaft 37:331. 18 Meisel, H. 1986. Chemical characterization and opioid activity of an exorphin isolated from in vivo digests of casem. FEBS (Fed. Eur. Biochem. Soc.) Lett. 196:223. 19 Meisel, H., and E. Schlirnrne. 1990. Milk proteins: precursors of bioactive peptides. Trends Food Sci. Technol. (Aug):41. 20 Stepaniak, L., P. F. Fox, T. Sorhaug, and J. Grabska. 1995. Effect of peptides from the sequence 58-72 of {3-casein on the activity of endopeptidase, aminopeptidase, and X-prolyldipeptidyl aminopeptidase from Lactococcus lactis ssp. lac tis MG1363. J. Agric. Food Chern. 43:849. 21 Svedberg, J., J. Haas, G. Leimenstoll, F. Paul, and H. Teschemacher. 1985. Demonstration of {3-casomorphin immunoreactive materials in in vitro digests of bovine milk and in small intestine contents after bovine milk ingestion in adult humans. Peptides 6:825. 22 Tan, T.P.S., B. Pollman, and W. N. Konings. 1993. Proteolytic enzymes of Lactococcus lactis. J. Dairy Res. 60:269. 23 Thomas, T. D., and G. G. Pritchard. 1987. Proteolytic enzymes of dairy starter cultures. FEMS (Fed. Eur. Microbial. Soc.) Microbiol. Rev. 46:245. 24 Thomas, T. D., and K. W. Turner. 1977. Preparation of skim milk to allow harvesting of starter cells from milk cultures. N.Z. J. Dairy Sci. Technol. 12:15. 25 Tieleman, A. E., and J. J. Warthesen. 1991. Comparison of three extraction procedures to characterize Cheddar cheese proteolysis. J. Dairy Sci. 74:3686. 26 Tome, D., A. M. Dumontier, M. Hautefeuiller, and J. F. Desjeux. 1987. Opiate activity and transepithelial passage of intact {3casomorphins in rabbit ileum. Am. J. Physiol. G737. 27 Visser, S. 1993. Proteolytic enzymes and their relation to cheese ripening and flavor: an overview. J. Dairy Sci. 76:329.
ABSTRACT
INTRODUCTION
{3-Casomorphins are small peptides derived from {3casein ranging from three to seven amino acids. The biologically active sequence of seven amino acids (Tyr-Pro-Phe-Pro-Gly-Pro-Ile), residues 60 to 66, was first isolated from a tryptic digest of casein by Brantl et al. (4). This peptide and fragments of the amino acid sequence possessed opioid activity in receptorbinding assays (4, 6) and isolated organ preparations (4). A common N-terminal sequence, Tyr-Pro-Phe, is present in all the peptides, indicating a specific action for binding to opioid receptors. Currently, there is no confirmed evidence that these peptides influence physiological functions upon ingestion. However, intravenous administration indicated opioid activity. Analgesia resulted after intracerebroventricular injection of (3-casomorphin into rats (4). In 1989, Daniel et al. (8) used intragastric application of labeled (3casomorphin with kinetic monitoring of gastric emptying and gastrointestinal transit time; that transit time was increased by feeding casein suspensions. Some evidence (19) suggested that {3-casomorphins are created as a result of casein digestion and are naturally present in dairy products. Recently, {3casomorphin precursors have been identified in Parmesan cheese (1). However, it is not known whether {3-casomorphins are created or degraded in dairy products as a result of enzymatic hydrolysis by native milk enzymes or bacterial starter enzymes. In addition, the release of {3-casomorphin peptides from larger protein sequences may be influenced by product processing conditions (14). Creation of {3casomorphins upon ingestion of milk was investigated Abbreviation key: DP IV = dipeptidyl-dipeptidase in vitro and in vivo with adult humans (21). {3IV, Pep X = X-prolyl dipeptidyl aminopeptidase. Casomorphins were found in digests in vitro and in vivo; however, opioid activity could not be determined. Similarly, Meisel ( 18) found opioids in vivo in miniature pigs, but physiological significance could Received February 9, 1995. not be determined. Accepted September 5, 1995. lPublished as Paper Number 21,689 of the contribution series of According to Hazum (13), the high content of prothe Minnesota Agricultural Experiment Station. Support also line residues in {3-casomorphins confers resistance to provided by the Minnesota-South Dakota Dairy Foods Research many degradative enzymes-pepsin, trypsin, and Center.
Commercial cheese products were surveyed for (3casomorphin peptides. Two extraction methods were compared: 1) water and 2) chloroform and methanol. Peptide profiles were determined using reverse-phase HPLC and multiple wavelength detection. {3-Casomorphin standards were used for comparison with cheese peptide profiles. Results indicated that peptides were present in cheeses with HPLC elution times that were similar to those for {3-casomorphins. However, comparison of absorbancies of the peaks at multiple wavelengths did not indicate peptides similar to {3-casomorphins. Therefore, {3-casomorphins were absent, or concentrations were below the HPLC detection threshold for {3-casomorphin of 2 J.tglml of cheese extract. The susceptibility of {3-casomorphins to the proteolytic system of a commercial strain of Lactococcus lactis ssp. cremoris was investigated. {3-Casomorphin standards were incubated at 4°C with bacterial cell lysate at pH 5.0,5.2,5.4, and 5.7. Salt concentrations varied among 0, 1.5, and 5%. The concentration of added {3-casomorphins and the degradation products were monitored over 15 wk using HPLC. Enzymatic degradation of {3-casomorphins was influenced by the combination of pH and salt concentrations at cheese ripening temperatures. Therefore, if formed in cheese, {3-casomorphins may be degraded under conditions common for Cheddar cheese. (Key words: (3-casomorphins, cheese proteolysis, Xprolyl dipeptidyl aminopeptidase)
1996 J Dairy Sci 79:20-26
20
ll-CASOMORPHINS IN CHEESE
21
chymotrypsin. Nevertheless, l3-casomorphins would dard was prepared in a series of concentrations behave to be created and avoid degradation by starter tween 1 mg and 1 p.g/ml of water. These samples were enzymes in cheese and intestinal enzymes in humans analyzed by reverse-phase HPLC to determine retenif they were to reach opioid receptors and cause a tion times and detection thresholds. I3-Casomorphin standards -3, -5, and -7 also were physiological response. Tome et al. (26) observed that 13-casomorphins could be degraded on the prepared in water as solutions of 0.5 mg/ml for use in mucosal side of the intestinal lining by the action of the degradation study. dipeptidyl-dipeptidase IV (DP IV) if they were ingested. The action of DP IV is to cleave X-Pro (N- Reverse-Phase HPLC Conditions terminal) dipeptides from larger peptides (13, 16). The reverse-phase HPLC analyses of 13For 13-casomorphins, Tyr-Pro is released. Therefore, 13casomorphin standards and cheese peptide extracts casomorphin peptides would likely be destroyed bewere done on a CI8 (201TP54) analytical column (5 fore crossing the intestinal lining and reaching opioid p.m, 25 em x 4.6 mm; VYDAC, Hesperia, CA) with a receptors. 50-p.1 loop. Gradient solvent delivery was achieved Similarly, bacterial starter cultures contain many using a Spectra Physics SP8800 ternary pump at a proteolytic enzymes that are responsible for the rate of 1 mVmin. Solvent A was 0.1% triflouroaflow breakdown of protein into peptides and amino acids. and HPLC grade water. Solvent B was 0.1% cetic acid The peptidases that have been found and characterand 9.9% HPLC trifluoroacetic acid, 90% acetonitrile, ized in lactic acid bacteria used in cheese products grade water. A linear gradient increase of 1% solvent have been summarized (5, 22, 23, 27). Because was used for all peptide separations. After the B/min caseins have high proline contents, bacterial starter initial gradient to 35% B (l3-casomorphin standards) cultures produce proline-specific peptidases that recognize the pyrolidine ring of proline. These pepti- or 50% B (cheese extracts), an accelerated gradient dases also are present in dairy products as a result of was programmed to 80% B and then back to 100% A starter culture addition. The peptidases break down to condition the column prior to the next run. Multipeptides containing proline into free amino acids that ple wavelength detection at 220, 254, and 280 nm was can be utilized by the cells. X-Prolyl dipeptidyl used for all samples (diode array 1040A; Hewlett aminopeptidase (Pep X), aminopeptidase P, proline Packard, St. Paul, MN). aminopeptidase, aminodipeptidase, amidodipeptidase, and dipeptidyl-peptidase have been characterized and Cheese are important for the breakdown of peptides containCommercial cheeses were used for water and oring proline (23). In particular, Pep X can cleave Xganic peptide extractions. The varieties were chosen Pro dipeptides from larger peptides, which are similar based on a range of ages and mode of ripening: Extra in action to DP IV. Also, Pep X has been shown to Sharp Cheddar, Swiss (aged 60 d), Blue, Brie, and have enzyme activity in a cheese matrix (7) and Limburger. This cross-section of cheeses would result activity on l3-casomorphin-7 substrate at pH 7.0 (3, in a range of proteolysis products with potential for 1315). casomorphins. All cheeses were purchased commerThe objectives of this study were to evaluate comcially, stored at 4°C until extracted, and analyzed mercial cheese varieties for l3-casomorphin peptides twice. and to investigate whether enzymes found in the proteolytic system of Lactococcus lactis ssp. cremons would degrade 13-casomorphins, if they were present, Peptide Extractions in a cheese system. Two methods were used to extract peptides from cheese. A water extraction method developed by MATERIALS AND METHODS Kuchroo and Fox (1 7) was modified to use 20 g of cheese homogenized with 40 ml of water in a Sorvall Omni mixer (model 17105; Omni Corp, Waterbury, i3-Casomorphin Standards CT) for 1.5 min on speed 3.5 (25). The slurry was 13-Casomorphin standards, -3, -5, and -7 amino acid centrifuged using an lEC model HT benchtop censequences and morphiceptin, were obtained from trifuge (lEC, a division of Damon, Needham Heights, Sigma Chemical Company (St. Louis, MO) and 13- MA) at 18,000 x g for 40 min. Fat was removed, and casomorphin-4 and -6 were obtained from American the water extracts were retained. The solids were Peptide Company, Inc. (Sunnyvale, CA). Each stan- reextracted using 40 ml of water. The combined exJournal of Dairy Science Vol. 79, No.1, 1996
22
MUEHLENKAMP AND WARTHESEN
tracts were filtered through glass wool and ultrafiltered into two molecular weight fractions. Each extract was added to an Amicon ultrafiltration 8400 stirred cell unit (Beverly, MA) with a YM2 or YM5 membrane (1000 or 5000 molecular weight cutoff). Both retentate and permeate were frozen, freeze-dried, and stored at -20°C. The organic extraction procedure developed by Han'lalkar and Elliott ( 12) was modified and used to extract peptides from the cheeses. Approximately 4 g of shredded, freeze-dried cheese were added to 10 ml of chloroform-methanol (6.5 ml of chloroform and 3.5 ml of methanol) and homogenized in a Sorvall Omni mixer for 1 min on speed 3.5. The slurry was centrifuged in an IEC model HT benchtop centrifuge at 2300 x g for 10 min (4500 rpm). The supernatant for each of three extractions were made biphasic by addition of 5 ml of water. The lower chloroform layer and the precipitate were discarded. The upper methanol and water layer was diluted further with water and ultrafiltered as described. Methanol was removed from the samples using a roto evaporator (model 410-2000; Haake Buchler, Saddle Brook, NJ). The extracts were then frozen and freeze-dried. In both cases, freeze-dried extracts were reconstituted as 10 mg/ml of water, and 50 1-'1 were injected under the HPLC conditions described. Sample HPLC runs were 50 min.
TABLE 1. Mean retention times and detection thresholds of 13casomorphin standards. Standard
Retention time!
SD
I3-Casomorphin-3 I3-Casomorphin-4 (3-Casomorphin-5 I3-Casomorphin-6 I3-Casomorphin-7 Morphiceptin
28.12 30.16 27.72 29.81 35.19 25.39
0.17 0.21 0.19 0.17 019 0.40
Detection threshold 2
(min) 1.5 2.0 2.0 1.0 2.5 2.5
!Means are the result of five HPLC runs. 2Thresholds were determined as a peak area dOO.
phosphate (4 ruM) buffer, pH 6.4. The resulting paste was lysed with an Eaton press (9) and centrifuged (35,000 xg for 30 min at 4°C). The supernatant was retained and stored at -50°C. In Vitro Enzymatic Tests
Test tubes were prepared with 5 ml of sodium acetate buffer at 4°C, pH 5.0, 5.2, 5.4, or 5.7. Sodium chloride was added at 0, 1.5, or 5% (wt/vol) to the buffer followed by 100 1-'1 of the cell lysate preparation, which was thoroughly mixed before the addition of 0.5 ml of l3-casomorphin standard, -3, -5, or -7. Each tube was vortexed, and one drop of toluene was added Samples Augmented with JS-Casomorphin before storage at 4°C. Samples of each combination of Cheddar cheese samples, 20 and 15 g, were aug- pH, salt, and l3-casomorphin were prepared. mented with 5 mg of l3-casomorphin before homogeniSamples were prepared for HPLC analysis within 5 zation with water or chloroform and methanol. Ex- min and after 2, 4, 6, 8, 10 and 15 wk. At each tractions were performed on augmented and interval, 300 1-'1 of sample from each reaction tube unaugmented samples for comparison. The resulting were removed and added to 500 1-'1 of 12% trichloroextracts were not ultrafiltered, but freeze-dried and acetic acid. The resulting 800-1-'1 samples were stored reconstituted as 10 mg/ml in water before analysis by at -20°C until HPLC analysis. HPLC. Control samples were also prepared and analyzed throughout 15 wk under conditions common for Cell Lysate Preparation cheese. Controls consisted of acetate buffer alone (pH A bacterial culture cell lysate was used as an en- 5.0 and 5.7); acetate buffer and cell lysate; acetate zyme preparation for hydrolysis of l3-casomorphin. buffer, cell lysate, and salt; and buffer with each 13The bacterial strain was a commercial preparation of casomorphin standard (no cell lysate added). L. lactis ssp. cremoris from Systems Bio-Industries, Inc. (Waukesha, WI). Ten milliliters of culture from RESULTS AND DISCUSSION a 125-ml can of frozen concentrate prepared by Systems Bio-Industries, Inc. were inoculated into 1 L of milk medium as described by Thomas and Turner JS-Casomorphin Standards (23). This culture was allowed to grow for 16 hand Retention times of l3-casomorphin standards are then was neutralized with sodium hydroxide (ION) and 60 ml of sodium citrate 25% (wt/vol). Cells were listed in Table 1. These results defined the time frame recovered from centrifugation (10,000 x g for 5 min at between 26 and 36 min as the area to investigate in 4°C) and washed twice with sodium acetate (46 mM) peptide profiles for l3-casomorphins. These retention Journal of Dairy Science Vol. 79, No.1, 1996
~·CASOMORPHINS
times were reproducible and had standard deviations of 0.1 to 0.4 min. The concentrations of {3casomorphins were varied to determine the minimum concentration that was necessary for peak identification. Threshold values were determined as twice the height of baseline noise (40 to 50 mAU). If {3casomorphin concentrations were below these threshold values, they could not be distinguished from the baseline and were reported as less than the threshold concentration. Data collected for the threshold determination was used also to calculate {3-casomorphin calibration lines by plotting HPLC peak area versus known concentrations. Data were fitted to linear equations for each standard and were used in subsequent analyses to determine {3-casomorphin concentrations from peak area (data not shown). The r 2 values for each {3casomorphin standard calibration curve was 0.99. Detection at 220, 254, and 280 nm was chosen for several reasons. All peptide bonds absorb at 220 nm. Therefore, peaks would be expected at this wavelength for all peptides, including {3casomorphins. Tyrosine and Phe absorb at 280 nm because of their aromatic side chains, and Phe also absorbs at 254 nm. Therefore, {3-casomorphins, which contain Tyr and Phe, formed a peak at all three wavelengths. This property was used to differentiate the {3-casomorphins from other peptides. {3-Casomorphin Standards Recovery
{3-Casomorphin-7 was recovered from augmented cheese samples using both extraction methods. Controls were prepared along with the augmented samples so that direct comparisons could be made. Calculations of the potential {3-casomorphin produced from 15 to 20 g of cheese indicated that 2% of the potential {3-casomorphin would be necessary before a peak in the chromatogram could be observed. Figure 1 shows chromatograms comparing organic extracts of mild Cheddar cheese with {3-casomorphin-7 standard. A major peak occurred at 34.6 min, corresponding to {3-casomorphin-7 in the augmented sample. Recovery of {3-casomorphin in the organic extract, using the HPLC calculated peak area and the {3-casomorphin-7 calibration line, was 55 ± 12%. {3Casomorphin could be lost during extraction because the procedure required several transfer steps during which losses could occur. Also, during extraction, the proteolytic enzymes that were present in cheese might degrade the peptide. Finally, {3-casomorphins might be retained by cheese solids.
23
IN CHEESE
71111
6ell 5
g see ~ 4ell
~
UJ
~
3ell
:il0 2e"
~
lee e
Ie
ze
TIME (MIN) 3 e
Figure 1. Comparison of chromatograms from organic extracts of augmented and control medium Cheddar cheese to the {3casomorphin-7 (SCM-7) standard. AU '" Absorbance units.
Similarly, recovery of {3-casomorphin-7 from the water extract of Cheddar cheese was 88 ± 12%. These results indicated that {3-casomorphins would be recovered using either extraction method. Even though {3-casomorphins tended to be hydrophobic peptides, the water extraction method was more efficient than the organic extraction. Cheese Extracts
Peptide profiles of each cheese variety were determined by HPLC. The HPLC elution time frame of interest, as defined by the {3-casomorphin standards, was 26 to 36 min. After this area in the chromatogram was enlarged, individual peaks could be seen. However, each peak might not represent one peptide. In most cases, there were probably several peptides that coeluted. Further clarification of peptides was sought by viewing the same peptide profiles at 254 and 280 nm. Using Brie cheese as an example, inspection of all three wavelengths indicated that the cheese did not contain detectable amounts of {3-casomorphins (Figure 2). All three wavelengths are shown in Figure 2, and there is a peak at 28 min and 220 nm that does correspond to {3-casomorphin. However, the peak has no detectable absorbance at either 280 or 254 nm. Therefore, it does not represent a {3casomorphin peptide. Elution time and absorbance at three wavelengths were the criteria used to detect (3casomorphins in all other cheese varieties. Similarly, peaks appeared with retention times that were similar to those of {3-casomorphins, but peaks failed to indicate absorbance at all three wavelengths. This evaluation was performed twice on each cheese variety with the same results. Journal of Dairy Science Vol. 79, No.1, 1996
24
'"
1
MUEHLENKAMP AND WARTHESEN
However, if no physiological benefits could be gained by trace amounts, further analysis to detect even lower concentrations or broader product surveys might be unnecessary. This product survey was limited to five varieties of cheese; therefore, the potential for other varieties to contain 13casomorphins is conceivable.
2BB
0'
'"~
IBB
~
'"a:
'"
Iil.j-----~--------~~....A---
26
28
39
In Vitro Enzymatic Tests
32
T1\1E ;\1r:\)
Figure 2. Comparison of peptide ptofiles of Bne cheese at elution of 220, 254, and 280 nm between 20 and 34 min. AU = Absorbance units.
The absence of l3-casomorphins from the cheese samples in this study could be the result of several factors. First, l3-casomorphins might not be released from the l3-casein sequence. Specific proteolytic enzymes would be necessary to release the 60 to 66 residues from l3-casein. To date, only larger fragments containing $-casomorphin in their sequence have been identified in Cheddar cheese (2). The sequence of cleavage leading to l3-casomorphin is not known. Second, i3-casomorphins might be degraded, even as they are created, by starter culture proteolytic enzymes. Finally, i3-casomorphins might be present in amounts smaller than the determined threshold values. The threshold value, expressed in micrograms per milliliter of cheese extract, was 2 ",g/ml, indicating that 2% of the theoretical i3-casomorphin potential could be detected if it were formed. A more sensitive analysis method, such as radioimmunoassays, might be appropriate to detect lower concentrations.
Control samples were prepared to characterize chromatographic profiles that would be expected during the study as a result of the buffer medium. Results of these samples indicated that the HPLC peaks between 4 and 8 min were the result of TCA and acetate buffers and that the peak at 19 to 20 min was from the acid only. As expected, these peaks were found in all samples and remained constant throughout 15 wk. Buffer and enzyme controls indicated that no peaks were introduced by the lysate preparation. i3-Casomorphins in solutions without enzymes were analyzed to determine whether they remained stable in the buffer medium throughout the study. The percentage of each remaining standard was determined at pH 5.0 and 5.7 only. At both pH conditions, no 13casomorphin degradation was observed. Any peak formation in samples would have been due to enzyme degradation of i3-casomorphins. Table 2 contains data for one complete set of experiments through 15 wk. These data are specific for this strain and would vary depending on preparation and strain selection. Figure 3 shows i3-casomorphin-3 after 2 wk at pH 5.0 and at 0, 1.5, and 5% of salt. Only with 5% salt did l3-casomorphin-3 remain after 15 wk. At 15 wk, 30% of the original l3-casomorphin concentration re-
TABLE 2. Concentrations of p-casomorphin standards at 0, 6, and 15 wk; pH of 5.0, 5.2, 5.4, and 5.7; and 0, 1.5, and 5% salt. p-Casornorphin-3 pH
Salt
o wk
6 wk
15 wk
o wk
33 32 33 48 42 46 50 58 32 31 21 32.4
<1.5 <1.5 20
<1.5 <1.5 10.9
<1.5 <1.5 4 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5
<1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5 <1.5
36 39 31 30 33 32 22 38 36 31 32 32
(o/c)
5.0
5.2
5.4
5.7
0 1.5 5 0 1.5 5 0 1.5 5 0 1.5 5
Journal of Dairy Science Vol. 79, No. 1, 1996
p-Casomorphin-7
p-Casomorphin-5 6 wk
( "g/m!) <2.0 4 22 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <20 <2.0
15 wk
o wk
6 wk
15 wk
<2.0 <2.0 26 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0
39 38 34 47 46 46 38 39 46 31 26 31
<2.5 22 31 <2.5 17 42 <2.5 <2.5 29 <2.5 <2.5 <2.5
<2.5 20 31 <2.5 <2.5 40 <2.5 <2.5 25 <2.5 <2.5 <2.5
~-CASOMORPHINS
25
IN CHEESE
HIll
S
I
~
,
S%SALT
l.l% SALT I
3all
1
0% SALT
A
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on
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TIME (MIN)
3a
48
H
9
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a 19
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1.5% SALT
•
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TIME (MIN)
3a
4a
Figure 3. Chromatograms of l3-casomorphin-3 stored for 2 wk at pH 5.0 in 0, 1.5, and 5% NaCl. I3-Casomorphin peak is at 28 minutes. AU = Absorbance units.
Figure 5. Chromatograms of l3-casomorphin-7 at pH 5.2 after 2 wk in 0, 1.5, and 5% NaC!. I3-Casomorphin peak is at 34 min. AU = Absorbance units.
mained. In addition to the loss of i3-casomorphins, we also observed an increase in the peak at 16 min. This peak was determined to be Tyr-Pro by amino acid analysis. The production of this peptide suggests the action of the Pep X enzyme. Results were similar for i3-casomorphin-5. The only conditions under which i3-casomorphin survived for 15 wk were in 5% salt and pH 5.0 (75% remaining). Figure 4 shows i3-casomorphin-5 after 2 wk in each of the buffers and 5% salt. Again, a peak at 16 min was formed at each pH. i3-Casomorphin-7 peptide was more resistant to proteolysis than was i3-casomorphin-5 or -3. No loss of i3-casomorphin-7 occurred throughout 15 wk at pH 5.0 in the presence of 5% salt. i3-Casomorphin-7 also remained after 15 wk at pH 5.2 and 5.4, but only in the presence of 5% salt. The only case in which i3casomorphin at 1.5% salt survived 15 wk was at pH
5.0. Small amounts of i3-casomorphin did remain after 6 wk at pH 5.2 and 1.5% salt but was degraded after 15 wk. Figure 5 shows i3-casomorphin after 2 wk at pH 5.2 in 0, 1.5, and 5% salt. When salt was absent, i3-casomorphin was degraded within 2 wk. The results of the stability tests on all i3casomorphin standards agreed with similar results in the literature that were related to pH and salt influence on proteolysis (10). In most cases, lower pH and higher salt content decreased proteolytic activity, which was in agreement with our observations of i3casomorphin substrates. Stepaniak et a1. (20) also found that i3-casomorphin-7 was susceptible to degradation by Pep X and further suggested that Pep X inhibited the action of other peptidases. The optimal pH for typical proteolytic enzymes in starter cultures is near neutrality (19). Therefore, less enzyme activity would be expected at the lower pH used in this study. However, the activity at the low pH and low temperature was still rapid in the absence of salt. Rate of proteolysis in cheese was influenced by the content of salt in moisture (10, 11). The combination in this study oflow pH and 1.5 to 5% salt created an environment that partially influenced activity of the starter enzymes.
499
S
1
399 pH
s.?
~
~ N
2aa
'~'""
laa
~
'"
"H 5." pH
s.a
<
B~--'-----r----
9
la
~
29
TIME (MIN)
3a
~
48
Figure 4. Chromatograms of l3-casomorphin-5 stored for 2 wk in 5% NaCl at pH 5.0, 5.2, 5.4, and 5.7. I3-Casomorphin peak is at 27.5 min. AU = Absorbance units.
CONCLUSIONS
Low concentrations of i3-casomorphins might be present in fermented dairy products, but they might also be degraded even as they are formed. The stability of i3-casomorphins under model conditions for cheese ripening was dependent on pH, salt, and type of enzymes present. The enzyme Pep X, a serine protease, might degrade i3-casomorphins that are produced in dairy products by the action of proteinases and peptidases. Journal of Dairy Science Vol. 79, No.1, 1996
26
MUEHLENKAMP AND WARTHESEN
We used proteolytic enzymes from a commercial strain of L. lactis ssp. cremons. Other strains may have similar proteolytic systems but variable enzymes and substrate specificities. Understanding the characteristics of the proteolytic system of cheese and, particularly, the action of the Pep X proteolytic enzyme would be important for estimating 13casomorphin formation. Because Pep X has been purified from several lactic acid bacteria, similar 13casomorphir. degradation characteristics might be expected in other products.
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