Specificity of Lactococcus lactis subsp. cremoris SK11 Proteinase, Lactocepin III, in Low-Water-Activity, High-Salt-Concentration Humectant Systems and Its Stability Compared with That of Lactocepin I

Abstract

Marked changes in the specificity of hydrolysis of α_s1-, β-, and κ-caseins by lactocepin III from Lactococcus lactis subsp. cremoris SK11 were found in humectant systems giving the equivalent water activity (a_w) and salt concentration of cheddar cheese. Correlations were noted between certain peptides produced by the activity of lactocepin III in the humectant systems and peptides found in cheddar cheese. The stability of lactocepin III was compared with that of lactocepin I from L. lactis subsp. cremoris HP in the humectant systems at different pHs. Significant differences between the stability of each of the lactocepin types were evident. The relationship between stability and humectant type, a_w, pH, and NaCl concentration was complex. Nevertheless, in those systems where a_w, pH, and NaCl concentration were equivalent to those in cheddar cheese, lactocepin I was generally more stable than lactocepin III. It was concluded that differences in the specificity and/or stability of various lactocepin types are likely to persist in cheese itself and therefore potentially contribute to differences in the peptide composition of ripened cheese.

It is generally accepted that proteolysis represents the most important series of biochemical events during cheese ripening, affecting both flavor and texture. Proteolytic enzymes in cheese can originate from various sources (9), and the relative stabilities of these enzymes in cheese will govern the time over which they are active in the ripening process. The ripening process is therefore dependent on both the specificity and the stability characteristics of the proteases in cheese. Determination of both of these parameters, as exhibited in the cheese environment, is prerequisite to gaining control over the ripening process.

Proteolysis by lactococcal starters plays an important role in cheese ripening (3) and is initiated by the cell envelope-associated proteinase (21), now named lactocepin (EC 3.4.21.96) (22). A considerable number of studies in simple aqueous buffers (10, 18–20, 24–26, 32, 34, 35) have identified that different lactocepin types produce different peptides from the hydrolysis of the caseins. Lactocepin I represents one extreme of the lactocepin specificity types. It has long been suspected that different lactocepin specificity types contribute differently to cheese flavor development through differences in the peptides they produce (6). It has been postulated that starters possessing lactocepin III activity (the other extreme of specificity types) tend not to produce bitter flavors in cheese.

It is now becoming clear, however, that studies in simple buffered systems may be misleading, since environmental parameters within cheese have been shown to affect the specificity of chymosin (8) and lactocepin I (23) on intact caseins. There is a clear requirement to be able to understand, control, and manipulate flavor development for an increasingly competitive and sophisticated marketplace. The potential role of proteolysis in cheese flavor development and the impact of both enzyme specificity and stability on the direction and extent of proteolysis is equally clear, but real understanding of these factors is actually quite limited.

We have previously shown that the low water activity (a_w) and high-salt-concentration characteristic of cheese imparts lactocepin III characteristics to lactocepin I (23). We now show the effects of these parameters on the specificity of lactocepin III and, together with pH, on the stability of both lactocepin I and lactocepin III. These studies form part of a concerted effort in our laboratory to elucidate the key factors in cheese flavor development through proteolysis.

Very little is known for certain, however, about the relationship between the proteolytic activities of enzymes in cheese and the generation of flavor peptides, including bitter peptides. Some starter strains have been designated “bitter” or “nonbitter” because they have been found to give bitter and nonbitter cheeses under similar conditions of cheese manufacture (14, 15). This is somewhat simplistic, because nonbitter starters give poor flavored cheese under different conditions of cheese manufacture (rennet levels, cook temperatures, etc.) (16). It is the ratios, catalytic rates, and stabilities of the proteinases and peptidases which determine the levels and turnover of peptides and the rates and relative levels of free amino acid production and therefore the levels of a host of derivative flavor compounds. It should be noted, however, that proteinase activity primes the entire proteolytic system. Differences in the specificities of the lactocepins will have a downstream effect on the identities and levels of flavor peptides and peptide precursors of flavor compounds. Where possible, conclusions have been drawn in this study between the specificity of lactocepin III in the different humectant systems and peptides found in cheese which have bitter flavor.

MATERIALS AND METHODS

Organisms, growth, and harvesting.

Lactococcus lactis subsp. cremoris strains SK11 and HP were from the culture collection of the New Zealand Dairy Research Institute (NZDRI), Palmerston North, New Zealand. Cultures were grown, harvested, and washed as previously described (2).

Purification of lactocepin.

Proteinase was released from the surface of washed lactococcal cells by using calcium-free phosphate buffer and purified by using a combination of anion-exchange and gel exclusion chromatography (Mono-Q HR 10/10 and Superose 6 HR 10/30, respectively; Pharmacia Biotech, Uppsala, Sweden) as described by Coolbear et al. (2).

a_w measurements and humectants.

All a_w measurements were made by using a model CX-2 dewpoint electronic humidity meter (Decagon Devices, Inc., Pullman, Wash.). Humectants used were sorbitol (a relatively hydrophilic humectant), polyethylene glycol (PEG) 20,000 (both from BDH Ltd., Poole, England) and PEG 300 (Meck KGaA, Darmstadt, Germany). The a_w measurements of a series of solutions of each humectant covering a range of concentrations in the presence or absence of 5% (wt/vol) NaCl were used to construct standard curves relating humectant concentration to a_w. Stock solutions of each humectant in bis-Tris-propane (BTP), pH 6.4, were used for casein hydrolysis and lactocepin stability studies; for lactocepin stability studies at pH 5.2 stock solutions were made in 20 mM sodium acetate-acetic acid at this pH. The concentrations of humectants and NaCl used and the resulting a_w values are shown in Table 1.

TABLE 1.

Half-life at 40°C of lactocepin I and lactocepin III in humectant systems

Humectant systema	aw	Half-life (h)
		Lactocepin I at:		Lactocepin III at:
		pH 6.4	pH 5.2	pH 6.4	pH 5.2
1. Control (no humectant)	1	0.3	1.0	0.1	0.3
2. PEG 20,000 (20.5%, wt/vol) + NaCl	0.95	1.2	2.0/6.0b	0.3	0.3/0.9b
3. PEG 20,000 (20.5%, wt/vol)	0.99	0.3	0.4	0.3	0.1
4. PEG 300 (14%, wt/vol) + NaCl	0.95	0.6	1.9	0.1	0.7
5. PEG 300 (14%, wt/vol)	0.99	0.9	2.5	0.3	0.4
6. PEG 300 (37%, wt/vol)	0.95	1.9	0.2	0.6	0.2
7. Sorbitol (20%, wt/vol) + NaCl	0.95	17.5	>24/12.9b	6.1	15.0
8. Sorbitol (20%, wt/vol)	0.98	12.5	>24/6.9b	8.8	12.5
9. Sorbitol (40%, wt/vol)	0.95	24.0	14.0	10.3	10.5
10. NaCl	0.97	0.4	2.4	0.1	3.4

^aNaCl was 5% (wt/vol) where included. 

^bIndicates a biphasic curve; the notation n₁/n₂ indicates the half-life calculated from the first and second phases of the curve, respectively. 

Lactocepin stability.

Lactocepin (50 μg) was incubated at 40°C in the presence of either humectant (240 μl of stock solution at pH 5.2 or 6.4) or, in the case of controls, buffer alone (at pH 5.2 or 6.4). CaCl₂ (1 mM) was included in all incubations. Residual proteolytic activity in samples (40 μl) taken at various times during the incubation was determined at 25°C in 0.1 M sodium acetate-NaH₂PO₄, pH 6.4, with fluorescein isothiocyanate-labelled β-casein (FITCβ-CN), essentially according to the method of Twining (31). The log₁₀ of the percentage of residual activity was plotted against time, and the half-life of lactocepin was taken as the time required for a 50% reduction of initial activity.

CN substrates.

κ-CN (A variant) was a gift from K. Coolbear, Massey University, Palmerston North, New Zealand. β-CN was a gift from R. Burr, NZDRI, Palmerston North, New Zealand, and α_s1-CN was from Sigma Chemical Company (St. Louis, Mo.). Stock solutions of κ- and β-CNs (15 mg/ml) were made in 20 mM BTP, pH 6.4, while α_s1-CN was dissolved (15 mg/ml) in water.

Hydrolysis of casein and analysis of hydrolysates.

CNs were hydrolyzed with purified SK11 lactocepin III, and samples were taken at 10 min, 1 h, 4 h, and 24 h as previously described (23). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was done as described by Laemmli (12) and as modified by Reid and Coolbear (23), with PEG 20,000 added to all samples. CN hydrolysates were also analyzed by reversed-phase high-performance liquid chromatography (HPLC) by the method of Reid et al. (25), modified by adding PEG 20,000 to all samples (23).

Peptide identification.

Peptides were identified as described previously (25) by performing N-terminal sequence analysis with a model 476A protein sequencer (Applied Biosystems, Foster City, Calif.) and by molar mass determination by mass spectrometry (triple quadrupole model API 300 [Perkin-Elmer-Sciex, Thornhill, Ontario, Canada] equipped with an ionspray source). Positively charged peptide ions were generated by spraying the sample solution through a 75-μm-internal-diameter fused-silica capillary within a stainless steel capillary held at a high potential of 4.5 kV. Nebulization was assisted by a coaxial air flow delivered at an inlet pressure of 0.4 MPa.

RESULTS

CN hydrolysis: SDS-PAGE analysis.

The high-M_r products formed in the hydrolysis of α_s1-, β-, and κ-CNs by lactocepin III in the humectant systems were investigated by using SDS-PAGE (Fig. 1). Since the hydrolysis rates varied between systems, digest samples taken at different times were compared to obtain relativity.

FIG. 1.

SDS-PAGE showing hydrolysis of α_s1-CN (A), β-CN (B), and κ-CN (C) by lactocepin III from L. lactis subsp. cremoris SK11 in humectant systems. Lane numbers correspond to the system numbers given in Table 1. Digest times were as follows: α_s1-CN samples were taken at 1 h (lanes 1 through 3) and at 4 h (lanes 4 through 10); β-CN samples were taken at 4 h (lanes 1 and 4 through 10) and at 10 min (lanes 2 and 3); κ-CN samples were taken at 24 h (lanes 1 and 4 through 10) and at 10 min (lanes 2 and 3). Bands corresponding to undigested substrate are indicated as are the hydrolysis products from α_s1- and κ-CNs (see text).

(i) α_s1-CN.

The rate and extent of hydrolysis varied greatly in the different humectant systems (Fig. 1A). With the notable exception of the system containing PEG 20,000 (Fig. 1A, lane 2), the presence of NaCl reduced the production of one lower-M_r product (marked with “‡” in Fig. 1A, lanes 4, 7, and 10). Some distinct differences were apparent between hydrolysis products obtained in the control and those obtained in the humectant systems; notably absent from or at very low levels in the majority of humectant systems were two products marked with a “†” in Fig. 1A. In addition, one product clearly visible in the majority of samples digested in the humectant systems (marked with an asterisk in Fig. 1A) was present in only minute quantities in the control digest.

(ii) β-CN.

The highest rate of β-CN hydrolysis was observed in the systems containing PEG 20,000 (Fig. 1B, lanes 2 and 3), where the amount of β-CN remaining undigested in the 10-min samples was approximately the same as the amount remaining undigested in the 4-h samples obtained from the control and the system containing 14% (wt/vol) PEG 300 alone (Fig. 1B, lanes 1 and 5, respectively). In all other digests the intensity of the band corresponding to β-CN at 4 h clearly indicated a lower rate of hydrolysis. Apart from quantitative differences, the major banding pattern given by samples from each of the humectant systems were similar to that obtained from the control, indicating that the major high-M_r products formed in each of the digests were similar.

(iii) κ-CN.

As for β-CN hydrolysis, the rate of κ-CN hydrolysis by lactocepin III was highest in the systems containing PEG 20,000 (Fig. 1C, lanes 2 and 3). Under these conditions less κ-CN remained undigested at 10 min than at 24 h in all other systems. The band patterns obtained in the presence of NaCl alone (Fig. 1C, lane 10) and PEG 20,000 alone (Fig. 1C, lane 3) were very similar to that obtained in the control (Fig. 1C, lane 1). In each of the systems containing either PEG 300 or sorbitol as humectant with or without NaCl present (Fig. 1C, lanes 4 to 9), a band tentatively identified as fragment 1-151 (by comparison with the results of Visser et al. [35]) was a major product. Accumulation of this fragment appeared to be independent of a_w since it was present at similar levels in each of the six digests (a_w values of 0.99 to 0.95). In contrast, production of one unidentified fragment (indicated with an asterisk in Fig. 1C) did appear to be dependent on a_w. This fragment, which was completely absent from the control digest, was produced in significant amounts only in the five systems of a_w = 0.95 (Fig. 1C, lanes 2, 4, 6, 7, and 9).

CN hydrolysis: HPLC analysis.

HPLC chromatograms of the trifluoroacetic acid (TFA)-soluble peptides (i.e., low-M_r peptides) resulting from the hydrolysis of α_s1-, β-, and κ-CNs in the humectant systems are shown in Fig. 2, 3, and 4, respectively. Peptides recovered from the major peaks were identified by a combination of mass spectrometry and N-terminal sequencing and are shown in Tables 2, 3, and 4, respectively.

FIG. 2.

HPLC chromatograms of the 1% TFA-soluble peptides resulting from 24 h of hydrolysis of α_s1-CN in humectant systems by lactocepin III from L. lactis subsp. cremoris SK11. Chromatogram numbers correspond to the system numbers given in Table 1. Peptides identified within the numbered peaks are given in Table 2.

FIG. 3.

HPLC chromatograms of the 1% TFA-soluble peptides resulting from 24 h of hydrolysis of β-CN in humectant systems by lactocepin III from L. lactis subsp. cremoris SK11. Chromatogram numbers correspond to system numbers given in Table 1. Chromatograms obtained with samples from humectant systems 5, 6, and 8 were very similar to the chromatogram obtained with samples from system 4, and the chromatogram obtained with sample from system 9 was similar to that obtained with samples from system 7. The chromatograms of samples from systems 5, 6, 8, and 9 are therefore not shown. Peptides identified within the numbered peaks are given in Table 3.

FIG. 4.

HPLC chromatograms of the 1% TFA-soluble peptides resulting from hydrolysis of κ-CN in humectant systems by lactocepin III from L. lactis subsp. cremoris SK11. Chromatogram numbers correspond to system numbers given in Table 1. Digest times were 24 h (chromatograms 1 and 4 through 10) and 4 h (chromatograms 2 and 3). Peptides identified within the numbered peaks are given in Table 4.

TABLE 2.

Peptides produced from hydrolysis of α_s1-CN by lactocepin III in humectant systems and under control conditions

HPLC peaka	Peptide(s) identified	Monoisotopic mass (Mr)
		Predicted	Observed
1b	1-9	1,139.7	1,139.7
2	115-121	920.4	920.4
	122-130cd	1,015.8	1,015.8
	157-161d	551.2	551.2
3	122-130	999.5	999.6
	115-120	783.3	783.2
	33-41e	1,002.5	1,002.5
	100-105e	706.5	706.3
4f	55-60	762.4	762.2
5	143-148	788.4	788.4
6	10-16	754.4	754.4
	93-98b	721.4	721.2
	105-114b	1,135.7	1,135.5
7	17-23b	904.5	904.5
	18-23b	790.4	790.4
	25-35b	1,218.6	1,218.6
	191-199b	1,091.5	1,091.4
	193-199b	875.5	875.3
	99-120e	2,703.5	2,703.6
8	170-199c	3,299.5	3,300.3
9	162-169	967.5	967.6
10	170-199	3,283.5	3,284
11	75-121	5,792	5,795.1
12	74-114	4,888.6	4,889.0

^aSee Fig. 2. 

^bPresent in all samples excluding the control. 

^cContains methionine sulfoxide. 

^dPresent in all samples except those from systems containing PEG 20,000. 

^ePresent only in samples from the control. 

^fPresent only in samples from the systems containing PEG 20,000. 

TABLE 3.

Peptides produced from hydrolysis of β-CN by lactocepin III in humectant systems and under control conditions

HPLC peaka	Peptide(s) identified	Monoisotopic mass (Mr)
		Predicted	Observed
1b	28-39	1,600.8	1,600.9
2b	28-37	1,344.6	1,344.6
3	176-182	801.4	801.7
	22-27	702.4	702.8
4	169-175	779.5	779.6
	166-175	1,081.6	1,081.6
5	47-52	755.4	755.8
	1-6	787.4	787.9
6c	73-82	1,078.6	1,078.8
7	44-52	1,125.6	1,126.1
8	94-123	3,417.7	3,417.6
9	94-119	2,971.6	2,971.9
	142-160d	2,255.1	2,255.4
	53-68(A2)de	1,728.9	1,728.6
	53-72(A1)	2,194.1	2,194.4
	106-119f	1,745.9	1,746.2
10	57-72(A1)	1,765.9	1,765.8
11	57-68(A2)	1,299.7	1,300.4
	53-72(A2)	2,154.1	2,154
12	83-93	1,254.8	1,255.2
13	199-209	1,150.7	1,151.0
14	194-209	1,718.0	1,718.0
	183-192	1,202.7	1,203
15	193-209	1,881.1	1,881.0
	183-193	1,365.7	1,366.0
16	134-143	1,202.7	1,203.1
	194-209g	1,701.0	1,700.5
17	53-93(A1)	4,492.4	4,492.8
	191-209	2,107.2	2,107.0
	133-160	3,280.7	3,280.4
18	57-93(A1)	4,064.2	4,064.1
	53-93(A2)	4,452.4	4,452.4
19	57-93(A2)	4,024.1	4,024.2
	133-163	3,579.9	3,580.0

^aSee Fig. 3. 

^bPresent in all samples excluding those from the control. 

^cPresent only in samples from systems containing NaCl. 

^dPresent in samples from all systems except those containing PEG 20,000. 

^eA1 and A2 are β-CN variants. 

^fPresent only in samples from systems containing PEG 20,000. 

^gN-terminal pyroglutamate. 

TABLE 4.

Peptides produced from hydrolysis of κ-CN by lactocepin III in humectant systems and under control conditions

HPLC peaka	Peptide(s) identified	Monoisotopic mass (Mr)
		Predicted	Observed
1	80-85	588.3	587.2
2	161-165	546.3	546.1
	161-167	734.2	734.2
3	86-95b	1,093.4	1,093.1
	132-142c	1,039.4	1,039.2
	143-151c	1,121.5	1,121.4
4	161-169	904.5	904.9
5	152-160	996.5	997.1
6b	33-41	1,055.5	1,055.5
7d	96-106e	1,344.7	1,344.5
8b	33-44	1,509.7	1,510.2
9	107-131	2,663.4	2,663.1
10	96-106	1,328.7	1,328.8
11	24-32	1,135.7	1,135.7
12	21-32	1,447.9	1,448.3
	107-160	5,768.8	5,768.3
13	66-79	1,607.9	1,607.9

^aSee Fig. 4. 

^bPresent in samples from control only. 

^cPresent in samples from all systems excluding the control. 

^dPresent only in samples from some humectant systems. 

^eContains methionine sulfoxide. 

(i) α_s1-CN.

Comparison of the α_s1-CN peptides present at 24 h in samples from the control and from the humectant systems revealed both quantitative and qualitative differences (Fig. 2). In the control the major peptide products were fragments 143-148, 162-169, and 170-199 (peaks 5, 9, and 10, respectively). These peptides were also the major products in the systems containing 14% (wt/vol) PEG 300 alone, 20% (wt/vol) sorbitol alone, 14% (wt/vol) PEG 300-NaCl, and NaCl alone (in the latter two systems, α_s1-CN peptide 170-199 contained a methionine sulfoxide residue and was mainly present in peak 8), but they were present at much lower levels in the remaining humectant systems. Peptide 1-9, although completely absent from the control sample, was detected in all of the humectant systems (peak 1) and was one of the major products in the PEG 20,000 systems. Peptide 55-60 (peak 4) was detected almost exclusively in the PEG 20,000 systems. The peptide composition of a number of peaks common both to samples from the control and from the humectant systems was variable (Table 2).

In addition, some differences were noted in certain transitory intermediate peptides. For example, in digest samples taken at either 1 or 4 h from the PEG 20,000 systems and those containing PEG 300 alone, the α_s1-CN peptides 75-122 and 75-114 (peaks 11 and 12, respectively) were major products and were detected at levels well above those in the corresponding 24 h samples (data not shown). However, in all other samples the levels of these two peptides remained low throughout the course of the digestion.

(ii) β-CN.

HPLC chromatograms showing the β-CN peptides detected in samples taken at 24 h from the control and from selected humectant systems are shown in Fig. 3. Several peptides detected in small to moderate quantities in the humectant systems were completely absent from the control sample, including β-CN fragments 28-39 and 28-37 (peaks 1 and 2, respectively). Production of β-CN peptide 28-37 in particular appeared to be sensitive to a_w, since the amount of this peptide relative to other peptides was higher in samples from the five systems where the a_w = 0.95. Production of fragment 73-82 (peak 6) appeared to be dependent on the presence of NaCl since it was only detected (albeit as a minor component) in the systems containing NaCl either alone or in the presence of a humectant.

Variations were also detected in the peptide composition of peaks common to samples from the humectant systems and from the control (Table 3). The relative proportions of β-CN peptides 47-52 and 1-6 in peak 5 appeared to be dependent on a_w; peptide 1-6 was produced in significant quantities only in those systems where the a_w = 0.95 (data not shown).

Further differences between the control and humectant systems included the relative quantities of β-CN peptide 94-123 (peak 8), a major product under control conditions but almost undetectable in the majority of humectant systems. Also, the peptides detected in peaks 14-19 (see Table 3) were major products in the control sample and in the sample from the digest containing PEG 20,000 alone, but were generally detected at greatly reduced levels in all other systems.

(iii) κ-CN.

HPLC chromatograms of κ-CN peptides are shown in Fig. 4. A major peptide present in all of the humectant systems was fragment 152-160 (peak 5), a finding consistent with elevated levels of the complementary fragment 1-151 detected by SDS-PAGE in all but the systems containing NaCl alone or PEG 20,000 (Fig. 1C). In the PEG 20,000 systems very high levels of κ-CN peptide 96-106 (peak 10) were consistent with low levels of the fragment 1-151 detected by SDS-PAGE (Fig. 1C), since peptide 96-106 is produced by secondary hydrolysis of fragment 1-151 as well as primary hydrolysis of κ-CN. High levels of peptide 96-106 coupled with lower levels of peptide 152-160 under control conditions were also consistent with the very low levels of the fragment 1-151 in the control detected by SDS-PAGE.

The levels of κ-CN peptide 161-169 (peak 4) varied significantly in samples from the different systems. Analysis of samples taken at earlier sampling times showed moderate levels of peptide 161-169 in all systems (data not shown). Therefore, the very low levels of this peptide at 24 h, particularly in systems where a_w = 0.95, resulted from secondary hydrolysis to give the peptides 161-165 and 161-167 (both in peak 2).

Additional differences were found in the composition of peak 3. This material from control samples contained κ-CN peptide 86-95, while that from the humectant systems contained peptides 132-142 and 143-151. Also, peptide 33-41 (peak 6) was only produced in significant quantity in the control. In samples from several of the humectant systems, peptide 96-106 was either completely or partially oxidized (methionine sulfoxide); the oxidized species eluted in peak 7. Production of κ-CN peptides 24-32, 21-32 and 107-160, and 66-79 (peaks 11, 12, and 13, respectively) was largely suppressed in the five systems possessing an a_w = 0.95.

Lactocepin stability.

The effects of a_w, NaCl (5%, wt/vol), and pH on the stability of lactocepin I and lactocepin III were compared by measuring the half-life (at 40°C) of the proteinases in each of the humectant systems (Table 1). No clear relationship between a_w and proteinase stability was apparent; stabilization was humectant dependent and varied greatly between the different systems. Increased stability of both proteinases (relative to that in buffer alone) was found with the majority of systems. Stabilization by the PEG systems tended to be small compared with that given by the sorbitol systems. The stability of both lactocepins tended to be greater at pH 5.2 than at pH 6.4 under the majority of conditions. Further, lactocepin I was generally more stable than lactocepin III, except in NaCl alone at pH 5.2.

At pH 5.2 the addition of NaCl to the control system increased the stability of lactocepin I and lactocepin III by 2.4- and 11-fold, respectively. This was the only system in which lactocepin III was more stable than lactocepin I. NaCl also affected stability when added to the humectant systems, although this effect was pH, enzyme, and humectant dependent.

Stability data obtained at 40°C with the majority of humectant systems gave linear plots of log₁₀ percent residual lactocepin activity against time (data not shown). Exceptions to this were the data obtained with PEG 20,000-NaCl at pH 5.2 for both lactocepins and with both sorbitol-NaCl and sorbitol alone (20%, wt/vol) at pH 5.2 for lactocepin I, which gave biphasic plots.

DISCUSSION

Proteolysis in cheese is influenced by physicochemical parameters such as a_w, NaCl concentration, pH, and storage temperature (13). In the present study, the effects of a_w and NaCl on the specificity of lactocepin III from L. lactis subsp. cremoris SK11 were determined, complementing our previous work on lactocepin I (23), and the stabilities of both lactocepin I and lactocepin III were compared.

Many of the specificity changes observed in the humectant systems could be attributed directly to changes in a_w, e.g., significantly higher levels of α_s1-CN peptide 1-9, β-CN peptides 28-37 and 1-6, and two unidentified high-M_r hydrolysis products of α_s1-CN and κ-CN; reduced levels of κ-CN peptides 21-32, 24-32, 66-79, and 107-160; and secondary hydrolysis of κ-CN peptide 161-169. However, some specificity changes occurred only with a particular humectant, indicating that a humectant-specific side effect was responsible rather than reduced a_w. For example, the α_s1-CN peptides 122-130 and 157-161 were detected in all systems except those containing PEG 20,000, while α_s1-CN peptides 33-41, 55-60, and 100-105 and β-CN peptide 106-119 were detected only in such systems. These changes in specificity probably resulted from increased hydrophobicity generated by PEG 20,000, parallelling the case for lactocepin I (23).

Other changes could not be attributed to a particular effect. For example, significantly elevated levels of κ-CN peptide 1-151 in systems containing either PEG 300 or sorbitol or increased production of α_s1-CN peptides 93-98 and 105-114 and β-CN peptide 28-39 and decreased production of β-CN peptide 94-123 in all humectant systems did not correlate with a_w, NaCl concentration, or hydrophobicity. These changes appeared to result from nonspecific humectant effects, which is a finding still potentially relevant to the cheese environment given the variety of humectant-like components present therein.

Proteolysis in cheddar cheese has previously been investigated by identifying the peptides present in cheese and using the specificities of the proteinases and/or peptidases found in buffer systems, and attempts have been made to determine which enzymes are responsible for their generation (9, 17, 27, 28). Some of the peptides or CN cleavage sites identified in such studies can be matched with α_s1- or β-CN peptides identified in the present study as formed either exclusively in the humectant systems or at significantly higher levels than in the control (Table 5). Therefore, it would appear that the peptides formed in the humectant systems more accurately reflect the peptides formed by lactocepin within cheese itself. Remarkably, no peptides originating from κ-CN have so far been identified in cheddar cheese. During milk clotting, κ-CN is specifically hydrolyzed at the Phe-105–Met-106 bond by chymosin; the soluble macropeptide portion (residues 106 to 169) is lost in the whey, while the insoluble para-κ-CN portion (residues 1 to 105) is retained in the curd. Therefore, although the para-κ-CN region is hydrolyzed in the model systems, it may be that para-κ-CN is inaccessible or remains resistant to hydrolysis by lactocepin in cheese itself. The effect of combinations of the different caseins on overall specificity was not investigated.

TABLE 5.

Comparison of peptides produced by lactocepin III in humectant systems with those previously isolated from cheddar cheese

Peptides produced in humectant systemsa	Peptides isolated from cheddar cheese	Reference
αs1-CN 1-9	αs1-CN 1-9	29
αs1-CN 105-114	αs1-CN 105-?b	9
αs1-CN 25-35	αs1-CN 25-35	27
αs1-CN 75-114/122	αs1-CN 75-?b	28
αs1-CN 93-98	αs1-CN 93-?b	28
β-CN 57-72c	β-CN 58-72d	30
β-CN 28-39	β-CN 29-39d	28
β-CN 28-37	β-CN 29-37d	28
β-CN 1-6c	β-CN 1-6	28

^aSee Fig. 2 to 4 and Tables 2 through 4. 

^bOnly the N-terminal region of the peptide isolated from cheddar cheese was identified. 

^cPresent at significantly elevated levels in samples from the humectant systems. 

^dPossible modification by aminopeptidase. 

Recently, Broadbent et al. (1) have studied the relationship between peptide accumulation and bitterness in cheddar cheese by using starters with distinct proteinase specificities and have linked the α_s1-casein fragment 1-9 with bitterness. It has also been reported that the α_s1-CN peptide 1-9 has microbicidal activity (4). This peptide has been identified in Gouda and cheddar cheeses (11, 29) and is believed to be formed by the concerted action of chymosin and lactocepin I but not lactocepin III (29). This is based in part on the finding that bond Gln-9–Gly-10 of purified α_s1-CN peptide 1-23 (formed by chymosin action) is hydrolyzed in buffer systems by lactocepin I but not by lactocepin III (7). However, production of α_s1-CN peptide 1-9 by lactocepin III in the humectant systems suggests that lactocepin III may also contribute to the formation of this peptide in cheese directly from α_s1-CN (and probably from α_s1-CN peptide 1-23).

Overall, it is clear from this study and our previous study on lactocepin I (23), that significant specificity differences remain between the two enzyme types in humectant systems and therefore in cheese itself.

The relative stability of different lactocepin types is a factor in overall proteolysis and the ability of each enzyme to affect the peptide composition of cheese. Exterkate (5) has demonstrated that in the absence of Ca²⁺ the stabilities of the lactocepins from L. lactis subsp. cremoris strains SK11 and Wg2 are quite different. In the present study, significant differences were demonstrated between the stabilities of lactocepin I and lactocepin III in the humectant systems. Since the incubation temperature was 40°C, it is most likely that lactocepin instability resulted from a combination of thermally induced denaturation and autoproteolysis. Both factors would be reduced in cheddar manufacture and ripening due to the temperatures typical of ripening (e.g., 13°C) and the protein content of cheese. However, the relative stabilities of the different lactocepin types determined in the humectant systems are likely to be indicative of the relative stabilities of the lactocepins during cheese manufacture and ripening. The relationship between enzyme stability and humectant type, a_w, pH, and NaCl concentration was complex. The biphasic decay curve obtained for lactocepin in some systems indicated that the enzyme may adopt alternative conformations either more or less stable than the original. It was therefore difficult to draw firm conclusions regarding the effect of individual parameters on stability. Nevertheless, in the three humectant systems where pH, NaCl concentration, and a_w were all matched to cheese conditions, lactocepin I was generally more stable than lactocepin III.

Interestingly, lactocepin I used in the present study was from a strain known to consistently produce bitter Gouda cheese (33). An examination of the peptides in Gouda cheese made with different starter strains revealed that bitter peptides were present in cheese made either with the bitter strain or with nonbitter strains (33). It was concluded that the level of bitter peptides in cheese made with the bitter strain was simply higher than in nonbitter cheese and therefore exceeded the threshold value for bitter taste perception. Based on results from the present study, it is possible that these observations may be partly explained by an increased stability of lactocepin I under cheese conditions leading to the production of a greater quantity of peptides in total, including bitter peptides. This would serve to accentuate the differences between the specificity of lactocepin I and the specificity of other lactocepin types from nonbitter strains.