Plasmin Activity in UHT Milk: Relationship between Proteolysis, Age

Jun 25, 2014 - Age gelation of UHT milk is previously suggested to be caused by ... The UHT milk was stored in the dark at 20 °C in a climate chamber...
0 downloads 0 Views 524KB Size
Download PDF
Article pubs.acs.org/JAFC

Plasmin Activity in UHT Milk: Relationship between Proteolysis, Age Gelation, and Bitterness Valentin M. Rauh,*,†,‡ Lene B. Johansen,† Richard Ipsen,§ Marie Paulsson,∥ Lotte B. Larsen,‡ and Marianne Hammershøj‡ †

Arla Foods Strategic Innovation Centre, Rørdrumvej 2, DK-8220 Brabrand, Denmark Faculty of Science and Technology, Department of Food Science, Aarhus University, Blichers Allé 20, DK-8830 Tjele, Denmark § Department of Food Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg, Denmark ∥ Department of Food Technology, Engineering and Nutrition, Lund University, SE-221 00 Lund, Sweden ‡

S Supporting Information *

ABSTRACT: Plasmin, the major indigenous protease in milk, is linked to quality defects in dairy products. The specificity of plasmin on caseins has previously been studied using purified caseins and in the indigenous peptide profile of milk. We investigated the specificity and proteolytic pathway of plasmin in directly heated UHT milk (>150 °C for 1400 Are Potentially Bitter and Are Shown in Bold storage time peptidea αS1-CN f(1−7) αS1-CN f(1−22) αS1-CN f(23−34) αS1-CN f(23−36) αS1-CN f(35−42) αS1-CN f(80−90) αS1-CN f(80−100) αS1-CN f(80−102) αS1-CN f(91−100) αS1-CN f(91−102) αS1-CN f(91−103) αS1-CN f(91−105) αS1-CN f(100−105) αS1-CN f(101−124) + αS1-CN f(103−124) + αS1-CN f(104−119) + αS1-CN f(104−124) + αS1-CN f(106−119) + αS1-CN f(106−124) + αS1-CN f(120−124) αS1-CN f(125−132) αS1-CN f(194−199)

mass

P P P P P P

874.55 2615.48 1383.72 1640.86 945.51 1336.67 2585.37 2826.54 1266.70 1507.88 1635.97 1927.14 678.45 2916.53 2675.35 1950.95 2547.26 1659.79 2256.10 614.32 909.47 747.36

day 1

x

4 weeks

8 weeks

14 weeks

Q valueb

x x x

x x x x x x

x x x x x x x

1777 1297 1833 1718 1030 1054 1342 1396 1660 1710 1694 1759 1753 1358 1304 1339 1294 1218 1201 1152 756 1703

x

x x x x x x

x

x

x x x x

x

x

x

x x x x x x x x x x x x x x

Phosphorylations are indicated after the peptide. bQ value was calculated according to Ney (1971) in cal mol−1. Presence of peptides at different storage times is indicated by ‘x’. a

this time more than 90% of α- and β-CNs were hydrolyzed. The relatively low degree of β-lg denaturation in the UHT milk of 36.7 ± 3.2% indicated that the age gelation may be caused by a different mechanism than complexation of κ-CN with denatured β-lg. At this level of β-lg denaturation, only a very limited fraction of β-lg is associated with κ-CN.22,23 The aim of the present study was to analyze the peptide formation and specificity of PL in this UHT milk and to relate the peptides found with the previously observed bitter off-flavor and age gelation of the UHT milk during storage.

■

of-flight mass spectrometer (Q-TOF MS, Agilent 6530 Accurate Mass Q-TOF LC/MS, Agilent Technologies) with a Jetstream interface. Depending on the peptide concentration, 10 or 20 μL of the sample was injected to the uHPLC-Q-TOF-MS system. The column was an Xbridge BEH 300 (rp-C18, 3.5 μm 2.1 mm × 250 mm) (Waters, Milford, MA, USA) operated at 45 °C. Buffer A was Milli-Q water with 0.05% trifluoroacetic acid (TFA) (v/v and buffer B acetonitrile with 0.1% (v/v) TFA. A linear gradient was applied with 100% buffer A from 3 min, 100% to 45% buffer A at 45 min at a flow rate of 0.3 mL min−1. Peptides were monitored at 214 nm. The Q-TOF MS was run in 4 GHz high resolution mode. The Jetstream interface added a sheath gas at 350 °C with a flow of 8 L min−1. A nozzle voltage of 100 V was applied to improve the ionization. The drying gas temperature was set to 325 °C at a flow rate of 10 L min−1. The capillary voltage was set to 2.5 kV in the first time segment (3.0−24.0 min) and to 4.0 kV in the second time segment (24.0−35.2 min) to achieve optimal ionization for both small and large peptides. Spectra in the mass range from 300 to 3200 m/z were recorded in positive mode with a resolution of 20000 in the Q-TOF. Tandem mass spectra of the two most abundant ions with a charge state of 1, 2, or 3 in each MS spectrum were obtained in collisioninduced dissociation (CID) mode (collision gas nitrogen, isolation width 1.3 m/z (narrow) at 5 different fixed collision energies and flexible collision energies optimized for larger peptides. The fixed collision energies were 10, 20, 30, 50, and 100 V, and the flexible energy settings were based on charge state and m/z (singly charged: offset 18 V, slope 3 V m/z−1; doubly charged: offset 2 V, slope 2.2 V m/z−1; triply charged: offset 3.5 V, slope 1.5 V m/z−1). Mass spectra and UV chromatograms were analyzed by MassHunter software (version B 6.01, Agilent Technologies).The MS spectra were investigated for potential peptides formed by plasmin, which were not identified by MS/MS, using the “Find by Formula” feature in MassHunter. Matching ions and isotopic patterns were further evaluated with regard to their retention time. Database Search. The obtained tandem mass spectra were analyzed with Mascot v2.4 (Matrix Science, London, UK). Tandem mass spectra were searched against a custom database containing

MATERIALS AND METHODS

Milk Processing. Three batches of 1 day old pasteurized (72 °C for 15 s) and homogenized milk (1.5% fat) were obtained from Arla Foods Stockholm Dairy (Sweden) during three consecutive weeks. The milk was preheat treated at 74 °C for 180 s in a tubular heat exchanger before being subjected to a direct steam infusion heat treatment at >150 °C for 1400 Are Potentially Bitter and Are Shown in Bold storage time

a

peptidea

mass

day 1

4 weeks

8 weeks

14 weeks

Q valueb

αS2-CN f(1−21) + 4P αS2-CNf(1−24)c + 4P αS2-CN f(25−45)d αS2-CN f(71−80) αS2-CN f(115−125) αS2-CN f(115−136) + 2P αS2-CN f(115−137) + 2 P αS2-CN f(115−149) + 3 P αS2-CN f(115−150) + 3 Pc αS2-CN f(137−149) + P αS2-CN f(137−150) + P αS2-CNf(138−149) + P αS2-CN f(138−150) + P αS2-CN f(150−165) αS2-CN f(151−165) αS2-CN f(151−166) αS2-CN f(151−173) αS2-CN f(153−165) αS2-CN f(166−173) αS2-CN f(167−173) αS2-CN f(171−181) αS2-CN f(174−181) αS2-CN f(174−188) αS2-CN f(182−188) αS2-CN f(182−197) αS2-CN f(182−207) αS2-CN f(189−197) αS2-CN f(189−199) αS2-CNf(189−207) αS2-CNf(198−205) αS2-CNf(198−207) αS2-CNf(200−207)

2745.99 3131.19 2380.15 1245.64 1194.67 2587.16 2715.25 4162.85 4290.94 1593.70 1721.80 1465.61 1593.70 1990.12 1862.03 1990.12 2893.61 2632.88 1049.60 921.50 1397.77 978.55 1863.00 902.46 1982.06 3214.78 1097.61 1326.75 2330.33 974.59 1250.74 1021.60

x x

x x

x x

x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x

880 851 1019 1245 1331 939 963 971 986 1025 1059 986 1025 1215 1177 1197 1177 1208 1176 1130 1762 1846 1463 971 1434 1644 1793 1644 1892 1814 1980 2233

x x x x x x x x

x x x x x x

x

x

x x

x x x x x x x x x x x x x x x x x x x x x x x x

Phosphorylations are indicated after the peptide. bQ value was calculated according to Ney (1971) in cal mol−1. cPeptide was identified by MS only. Intramolecular disulfide bond. Presence of peptides at different storage times is indicated by ‘x’.

d

Lys150, and Lys150-Thr151. Earlier studies suggest that the cleavage sites Lys149-Lys150 and Lys150-Thr151 are preferred over Arg114-Asn115,16 but the simultaneous appearance of αS2-CN f(115−136), αS2-CN f(137−149), and αS2-CN f(138−150) indicate that PL readily hydrolyzed all of these cleavage sites. After 8 weeks of storage peptides originating from the region 151−173 in αS2-CN were identified. The late appearance of these peptides indicates a sequential hydrolysis of this region from an N- and C-terminal direction after the preferred cleavage sites Lys150-Thr151 and Lys173-Phe174 are hydrolyzed. The least preferred cleavage sites in αS2-CN seem to be Arg125Glu126 and Lys152-Leu153, which are either in close proximity to phosphoserine residues or to major cleavage sites (Figure 2). Although the region 25−114 contains eight potential cleavage sites for PL, only αS2-CN f(71−80) and αS2-CN f(25−45) could be identified. Masses corresponding to peptides including the cleavage site Lys91-Phe92, Lys32-Glu33, and Lys41-Glu42 were not present in the milk. The not-present cleavage at Lys91-Phe92 is located in a hydrophobic region of αS2-CN (Figure 4B), which may be inaccessible for enzymatic hydrolysis.35 Cleavage of this bond can be additionally hindered by Pro93 which induces a change in the secondary structure of αS2-CN.35 Lys32Glu33 and Lys41-Glu42 are located in the proximity of the two

Figure 2. Primary structure of bovine αS2-CN A-11P. Phosphoserine residues are indicated by circles. The dotted line between Cys36 and Cys40 indicates the intramolecular disulfide bridge. Identified pH 4.6 soluble peptides formed by plasmin during storage at 20 °C are indicated by arrows.

6855

dx.doi.org/10.1021/jf502088u | J. Agric. Food Chem. 2014, 62, 6852−6860

Journal of Agricultural and Food Chemistry

Article

Table 3. Presence and Identity of pH 4.6 Soluble Peptides from β-CN Formed by Plasmin in Directly Heated UHT Milk; Peptides with Q Values >1400 Are Potentially Bitter and Are Shown in Bold storage time peptidea β-CN β-CN β-CN β-CN β-CN β-CN β-CN β-CN β-CN β-CN β-CN β-CN

f(1−25) + 4P f(1−28) + 4P f(1−29) + 4P f(29−48) + P f(33−48) + P f(98−105) f(98−107) f(100−105) f(100−107) f(106−113) f(108−113) f(170−176)

mass 3121.26 3476.48 3604.58 2559.13 2060.82 872.48 1137.63 645.32 910.47 1012.52 747.36 779.49

day 1

4 weeks

x x x

x x x x x x x x x x

x

8 weeks

14 weeks

Q valueb

x x x x x x x x x x x x

x x x x x x x x x x x x

1006 1057 1072 818 614 1328 1262 1238 1179 1655 1873 1777

a Phosphorylations are indicated after the peptide. bQ value was calculated according to Ney (1971) in cal mol−1. Presence of peptides at different storage times is indicated by ‘x’.

cysteine residues of αS2-CN. The majority of the αS2-CN molecules is present in a form with an intramolecular disulfide bond, which induces a tight loop in the protein34,36 which may limit the accessibility of Lys41-Glu42 and Lys32-Glu33 for PL. To our knowledge, the cleavage of Arg45-Asn46, Lys70-Ile71, Lys80Ile81, Arg125-Glu126, Lys166-Ile167, and Arg205-Tyr206 has not been reported earlier for PL in either model studies or as part of the indigenous peptide profile in milk. This could be attributed to the low concentration of αS2-CN in milk or a different conformation of αS2-CN in model systems.31,35 In comparison to αS2-CN, only a relatively small number of peptides was identified derived from β-CN (Table 3). The peptides β-CN f(1−28), β-CN f(1−29), β-CN f(29−48), and β-CN f(98−107) were present in the milk after 1 day of storage. After 4 weeks of storage, all the observed peptides except β-CN f(33−48) and β-CN f(1−25) were present. The identified peptides mainly derived from cleavage around the two major cleavage sites of PL in β-CN, Lys105-His106, and Lys107-Glu108 (Figure 3). The peptide β-CN f(170−176) was the only identified peptide originating from the hydrophobic Cterminal part of β-CN and in contrast to earlier studies using trypsin,31,37 no peptides were identified from hydrolysis of Arg183-Asp184 and Arg202-Gly203.

Peptides Formed by Other Proteases. Peptides that were identified in the UHT milk samples and which cannot be assigned to be a result of PL activity alone are summarized in Table 4. Most peptides deriving from αS1-CN or β-CN indicate activity of cathepsins alone or in combination with PL.28,38,39 Peptides formed by cathepsins alone were identified after 1 day of storage or after 4 weeks of storage in the case of αS1-CN f(1−14). Peptides formed by the combined activity of cathepsins and PL on the other hand appear after 4−8 weeks of storage. This indicates that cathepsins were active in the raw or pasteurized milk, but inactivated by the UHT treatment and that the polypeptides formed by cathepsins were further hydrolyzed by residual PL to smaller peptides. With the exception of cathepsin D, the heat stability of cathepsins is largely unknown. Although cathepsin D in its active form can partially survive HTST pasteurization,40 it is unlikely that sufficient active cathepsins are present after the UHT treatment to significantly contribute to the observed proteolysis. The peptides deriving from αS2-CN originate from the C-terminal of the protein. With the exception of αS2-CN f(201−206), the peptides contained the PL cleavage site Lys197-Thr198. The apparently unspecific C-terminal cleavage site of the peptides suggests activity of carboxypeptidases in the raw milk or heatinduced hydrolysis.41,42 Peptide Development during Storage. The presence of identified peptides after different storage periods gives a good indication of preferred and uncleaved cleavage sites of PL in the UHT milk system, but only gives limited information on the actual concentration and development of the peptides. Semiquantitative analysis of the peptides using MS spectra was not possible because their response factors are unknown and their intensity might be affected by ion suppression of coeluting components. The UV chromatograms at 214 nm were therefore used for a relative quantification and comparison of the peptides. Due to substantial overlap of large areas in the chromatogram, only a limited number of peaks could be used for such relative quantification. Quantified peptides included αS1-CN f(1−22), αS1-CN f(106−124), αS2-CN f(1−21), αS2CN f(1−24), αS2-CN f(115−150), as well as β-CN f(1−28) and β-CN f(29−48). A comparison of the UV peaks and the extracted ion chromatograms are shown in the Supporting Information together with MS spectra of the peaks.

Figure 3. Primary structure of bovine β-CN A2-5P. Phosphoserine residues are indicated by circles. Identified pH 4.6 soluble peptides formed by plasmin during storage at 20 °C are indicated by arrows. 6856

dx.doi.org/10.1021/jf502088u | J. Agric. Food Chem. 2014, 62, 6852−6860

Journal of Agricultural and Food Chemistry

Article

Table 4. Presence and Identity of pH 4.6 Soluble Casein Derived Peptides Formed by Other Proteases in Directly Heated UHT Milk; Peptides with Q Values >1400 Are Potentially Bitter and Are Shown in Bold proteaseb

storage time peptide

mass

day 1

αS1-CN f(1−8) αS1-CN f(1−14) αS1-CN f(1−21) αS1-CN f(1−24) αS1-CN f(3−7) αS1-CN f(24−36) αS1-CN f(25−36) αS1-CN f(180−199) αS1-CN f(185−199) αS2-CN f(198−202) αS2-CN f(198−203) αS2-CN f(198−204) αS2-CN f(201−206) β-CN f(96−105) β-CN f(96−107) β-CN f(106−125) β-CN f(108−125) β-CN f(163−169)

1011.62 1663.93 2459.38 2909.61 621.40 1089.59 1236.66 2215.05 1688.81 556.36 719.42 818.49 809.44 1087.61 1352.76 2390.16 2125.00 761.43

x x

x

a

4 weeks

8 weeks

14 weeks

Q value

N-terminal

C-terminal

x x x

x x x x

x x x x x

x x x x x x x

x x x x x x x x x x x

1618 1309 1324 1410 1598 1646 1563 1232 1232 1844 2015 1969 2292 1180 1262 1482 1535 909

− − − − CB CB,CD CB,CD CG ? PL PL PL ? CD,CG CD,CG PL PL CB,CD

? CB CG CB,CD PL PL PL − − CP CP CP CP PL PL CD CD PL

x x

x x x x

x x x x x

a Q value was calculated according to Ney (1971) in cal mol−1. bPossible responsible proteases for N- and C-terminal cleavage are indicated: CB, cathepsin B; CD, cathepsin D; CG, cathepsin G; PL, plasmin; CP, carboxypeptidase; −, no cleavage; ?, unknown protease. Presence of peptides at different storage times is indicated by ‘x’.

f(115−150) originates from two of the most preferred cleavage sites in αS2-CN and showed the highest formation rate. The peptide αS1-CN f(106−124) developed almost in the same manner as αS1-CN f(1−22) within the first 6 weeks of storage but showed a higher rate of formation from 7 weeks of storage on. These findings further highlight the preference of PL for cleavage sites located in the hydrophilic regions of the caseins. Gagnaire et al. (1998) found that the proteose peptones β-CN f(1−105/7) were retained in the casein micelle, while β-CN f(29−105/7) was released into the serum phase.31 This release limits further hydrolysis of these proteose peptones by the casein-associated PL, which explains why β-CN f(29−48) was only formed in very small amounts. Destabilization of Casein Micelles by Proteolysis. PL enters the milk from the blood44 and associates itself with the casein micelle.45 Whether PL is able to enter the micellar structure or is located entirely on the surface of the casein micelle is unknown. It becomes obvious from Figure 3 that the majority of the major cleavage sites of PL within the caseins, similar to those of trypsin,31,32 are located at the most hydrophilic and serum-exposed regions of the proteins. β-CN is mainly present in the interior of the casein micelle46 and is proposed to stabilize water or serum channels within the casein micelle.47,48 αS2-CN is involved in the binding to calcium phosphate clusters, regions that are hypothesized to be surrounded by hydrophobic domains11 and likely to be inaccessible for PL. The rapid cleavage, within 4 weeks of storage, of the N-terminal regions of β-CN and αS2-CN implies that PL is able to penetrate the casein micelle. In relation to a destabilization of the casein micelle by proteolysis, it is notable that the minor cleavage sites, together with some of the major cleavage sites, are located at the boundary between hydrophilic, hydrophobic, or phosphorylated regions of the caseins (Figure 4A−C). Thus, PL hydrolyses around the regions essential for the internal integrity and stabilization of the casein micelle, i.e. hydrophobic interaction between caseins, interactions of

A comparison of peptides originating from the N-terminus of their parent proteins is shown in Figure 5. The peptide β-CN f(1−28) had the highest formation rate. During the first 4−5 weeks of storage, the relative concentration increased exponentially, reflecting the previously described increase in PL activity and rate of peptide formation. From 5 weeks of storage and further on, the relative concentration of β-CN f(1− 28) increased almost linearly and then leveled out after 12 weeks of storage. The decrease in the rate of formation could be linked to changes in the matrix induced by the starting gelation of the milk after 11 weeks of storage. Further degradation of β-CN f(1−28) to β-CN f(1−25) may also reduce the concentration. Since the proteose peptones β-CN f(1−105) and β-CN f(1−107) were still present after 14 weeks of storage (data not shown), substrate depletion can be excluded as a cause for the reduced rate of formation. The peptide αS2-CN f(1−24) showed the same initial rate of formation as β-CN f(1−28) and leveled off after 6 weeks of storage. The peptide αS2-CN f(1−21) exhibited a lower rate of formation than αS2-CN f(1−24), but in contrast to αS2-CN f(1−24), continued to increase after 6 weeks of storage. This indicates a hydrolysis of αS2-CN f(1−24) to αS2-CN f(1−21) that may be caused by substrate depletion of the cleavage site Lys24-Asn25. The development of αS1-CN f(1−22) differed from the other N-terminal peptides. The initial lag phase was not observable for α S1 -CN f(1−22), and the overall concentration was significantly lower compared to that of the N-terminal peptides derived from αS2- and β-CN. The cleavage sites for these peptides in αS2- and β-CN are in very hydrophilic domains of the proteins (Figure 4 A−C), while the cleavage site Arg22-Phe23 in αS1-CN is in a hydrophobic domain involved in casein−casein interactions.43 Peptides being present as the result of two proteolytic cleavages are shown in Figure 6. They exhibited an extended lag phase compared to the N-terminal peptides, due to the time needed to form their precursors. As discussed above, αS2-CN 6857

dx.doi.org/10.1021/jf502088u | J. Agric. Food Chem. 2014, 62, 6852−6860

Journal of Agricultural and Food Chemistry

Article

Figure 5. Development of casein-derived N-terminal peptides formed by plasmin in milk preheat treated at 72 °C for 180 s and subjected to direct steam infusion (>150 °C for 150 °C for 1400 cal mol−1 are highlighted in Table 1−4. Bitterness of peptides from β-CN was found in the γ-CN fraction after hydrolysis with PL,3 and analysis of tryptic digests of β-CN have shown that β-CN f(203−209) is extremely bitter.37 The potential bitter peptides found in the present study were β-CN f(108−113) and β-CN f(170−176), which have previously been described sensorially as tickling and burning.37 The bitter peptide β-CN f(203−209) was not identified in the UHT milk or was only present in a very low concentration. Previously described bitter peptides deriving from αS1-CN, that can be formed by PL, are αS1-CN f(23−34) and αS1-CN f(91−100).54,55 Besides αS1-CN f(91−100), all peptides from residue 91 to 100−105 found in the milk had Q values >1400, and were potentially bitter. The smaller peptides αS1-CN f(1−7) and αS1-CN f(194−199) were highly hydrophobic, and could have contributed to bitterness. The peptides αS2-CN f(182−207), αS2-CN f(189−207), and αS2-CN f(198− 207) have been correlated with bitterness and were also present early in the storage period in the UHT milk.16 Besides αS2-CN f(182−188), all peptides deriving from the C-terminal region of αS2-CN from residue 171 and onward are potentially bitter. Cleavage of Arg205-Tyr206 in αS2-CN gives rise to the bitter dipeptide Tyr-Leu.56 The identified potential bitter peptides indicate that the bitterness caused by PL might mainly be caused by hydrolysis of αS1- and αS2-CN, rather than β-CN as previously reported.3 Several peptides formed by the combined action of PL and cathepsins were potentially bitter as well. Most of these peptides derive from the N-terminal region of αS1-CN and the C-terminal region of αS2-CN. A bitter off-flavor was detected in the milk after 6 weeks of storage, which intensified rapidly within 1 week of storage.1 The concentration and sensory thresholds of the potential bitter peptides in the UHT milk are unknown, and the bitterness cannot be attributed to a specific peptide. We suggest that either the overall concentration of bitter peptides reached the sensory threshold after 7 weeks of storage or that one or several bitter peptides with a lower sensory threshold were formed very rapidly in the storage period between 5 and 7 weeks. Similar to trypsin, PL has the capability to form a range of potential bitter peptides, but further studies are necessary to confirm the bitterness of these peptides and their sensory threshold. In conclusion, the special UHT process used allowed the investigation of specificity and effect of PL in a sterile milk system, without causing substantial changes to the milk by the heat treatment. PL showed a different specificity in the milk system compared to previous model studies, and 6 new cleavage sites of PL in αS2-CN could be identified. The relative quantification of peptides and time of their appearance allowed a direct comparison of formation rates of peptides to assess the affinity of plasmin to certain cleavage sites. We could show that PL is able to produce potentially bitter peptides and bitterness was most likely caused by peptides arising from αS1- and αS2CN. The observed proteolytic pattern indicated that PLmediated proteolysis can destabilize the casein micelle and explain proteolysis-induced age gelation of milk.

Article

ASSOCIATED CONTENT

S Supporting Information *

Spectra of peptides used for relative quantification and proteins in Mascot database used for identification. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*(V.M.R.) Phone: +45 87 33 2783. Fax: +45 87 46 6688. Email: [email protected]. Funding

The Danish Agency for Science, Technology and Innovation (DASTI) is thanked for financial support of the study. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank laboratory technicians Vibeke Mortensen and Betina Hansen at the Arla Strategic Innovation Center for assistance with the analysis and helpful discussions.

■

REFERENCES

(1) Rauh, V. M.; Sundgren, A.; Bakman, M.; Ipsen, R.; Paulsson, M.; Larsen, L. B.; Hammershøj, M. Plasmin activity as a possible cause for age gelation in UHT milk produced by direct steam infusion. Int. Dairy J. 2014, DOI: 10.1016/j.idairyj.2013.12.007. (2) Kelly, A. L.; Foley, J. Proteolysis and storage stability of UHT milk as influenced by milk plasmin activity, plasmin/β-lactoglobulin complexation, plasminogen activation and somatic cell count. Int. Dairy J. 1997, 7, 411−420. (3) Harwalkar, V. R.; Cholette, H.; McKellar, R. C.; Emmons, D. B. Relation Between Proteolysis and Astringent Off-Flavor in Milk. J. Dairy Sci. 1993, 76, 2521−2527. (4) Bastian, E. D.; Lo, C. G.; David, K. M. M. Plasminogen Activation in Cheese Milk: Influence on Swiss Cheese Ripening. J. Dairy Sci. 1997, 80, 245−251. (5) Crudden, A.; Patrick; Fox, F.; Kelly, A. L. Factors affecting the hydrolytic action of plasmin in milk. Int. Dairy J. 2005, 15, 305−313. (6) Mara, O.; Roupie, C.; Duffy, A.; Kelly, A. L. The Curd-forming Properties of Milk as Affected by the Action of Plasmin. Int. Dairy J. 1998, 8, 807−812. (7) Ismail, B.; Nielsen, S. S. Invited review: Plasmin protease in milk: current knowledge and relevance to dairy industry. J. Dairy Sci. 2010, 93, 4999−5009. (8) Prado, B. M.; Sombers, S. E.; Ismail, B.; Hayes, K. D. Effect of heat treatment on the activity of inhibitors of plasmin and plasminogen activators in milk. Int. Dairy J. 2006, 16, 593−599. (9) Prado, B. M.; Ismail, B.; Ramos, O.; Hayes, K. D. Thermal stability of plasminogen activators and plasminogen activation in heated milk. Int. Dairy J. 2007, 17, 1028−1033. (10) Saint-Denis, T.; Humbert, G.; Gaillard, J. L. Heat inactivation of native plasmin, plasminogen and plasminogen activators in bovine milk: a revisited study. Lait 2001, 81, 715−729. (11) McMahon, D. J.; Oommen, B. S. Casein Micelle Structure, Functions, and Interactions. In Advanced Dairy Chemistry; McSweeney, P. L. H., Fox, P. F., Eds.; Springer: New York, 2013; pp 185−209. (12) White, J. H.; Zavizion, B.; O’Hare, K.; Gilmore, J.; Guo, M. R.; Kindstedt, P.; Politis, I. Distribution of plasminogen activator in different fractions of bovine milk. J. Dairy Res. 1995, 62, 115−122. (13) Ismail, B.; Choi, L. H.; Were, L. M.; Nielsen, S. S. Activity and nature of plasminogen activators associated with the casein micelle. J. Dairy Sci. 2006, 89, 3285−3295. (14) Eigel, W. N.; Butler, J. E.; Ernstrom, C. A.; Farrell, H. M.; Harwalkar, V. R.; Jenness, R.; Whitney, R. M. Nomenclature of Proteins of Cow’s Milk: Fifth Revision. J. Dairy Sci. 1984, 67, 1599− 1631.

6859

dx.doi.org/10.1021/jf502088u | J. Agric. Food Chem. 2014, 62, 6852−6860

Journal of Agricultural and Food Chemistry

Article

(15) Eigel, W. N. Formation of γ1-A2, γ2-A2 and γ3-A caseins by in vitro proteolysis of β-CASEIN A2 with bovine plasmin. Int. J. Biochem 1977, 8, 187−192. (16) Le Bars, D.; Gripon, J.-C. Specificity of plasmin towards bovine αs2-casein. J. Dairy Res. 1989, 56, 817−821. (17) McSweeney, P. L. H.; Olson, N. F.; Fox, P. F.; Healy, A.; Højrup, P. Proteolytic specificity of plasmin on bovine αs1-casein. Food Biotechnol 1993, 7, 143−158. (18) Baum, F.; Fedorova, M.; Ebner, J.; Hoffmann, R.; Pischetsrieder, M. Analysis of the endogenous peptide profile of milk: identification of 248 mainly casein-derived peptides. J. Proteome Res. 2013, 12, 5447− 5462. (19) Dallas, D. C.; Guerrero, A.; Parker, E. A.; Garay, L. A.; Bhandari, A.; Lebrilla, C. B.; Barile, D.; German, J. B. Peptidomic profile of milk of holstein cows at peak lactation. J. Agric. Food Chem. 2013, 62, 58− 65. (20) McMahon, D. J., Age-gelation of UHT milk: Changes that occur during storage, their effect on shelf life and the mechanism by which age-gelation occurs. In Heat Treatments and Alternative Methods; International Dairy Federation: Brussels, Belgium, 1996; pp 315−325. (21) Datta, N.; Deeth, H. C. Age Gelation of UHT MilkA Review. Food Bioprod. Process. 2001, 79, 197−210. (22) Crudden, A.; Oliveira, J. C.; Kelly, A. L. Kinetics of changes in plasmin activity and proteolysis on heating milk. J. Dairy Res. 2005, 72, 493−504. (23) Anema, S. G.; Li, Y. Effect of pH on the Association of Denatured Whey Proteins with Casein Micelles in Heated Reconstituted Skim Milk. J. Agric. Food Chem. 2003, 51, 1640−1646. (24) ISO, Microbiology of food and animal feeding stuffs - horizontal method for the enumeration of microorganisms - Colony-count technique at 30 °C. ISO 4833. International Standardisation Organisation: Geneva, Switzerland, 2003. (25) Kuipers, B. J.; Gruppen, H. Prediction of molar extinction coefficients of proteins and peptides using UV absorption of the constituent amino acids at 214 nm to enable quantitative reverse phase high-performance liquid chromatography-mass spectrometry analysis. J. Agric. Food Chem. 2007, 55, 5445−5451. (26) Ney, K. H. Voraussage der Bitterkeit von Peptiden aus deren Aminosäurezu-sammensetzung. Z. Lebensm. Unters. Forsch. 1971, 147, 64−68. (27) Hachana, Y.; Kraiem, K.; Paape, M. J. Effect of plasmin, milk somatic cells and psychrotrophic bacteria on casein fractions of ultra high temperature treated milk. Food Sci. Technol. Res. 2010, 16, 79−86. (28) Larsen, L. B.; Benfeldt, C.; Rasmussen, L. K.; Petersen, T. E. Bovine milk procathepsin D and cathepsin D: coagulation and milk protein degradation. J. Dairy Res. 1996, 63, 119−130. (29) Le Bars, D.; Gripon, J. C. Hydrolysis of αs1-casein by bovine plasmin. Lait 1993, 73, 337−344. (30) Malin, E. L.; Brown, E. M.; Wickham, E. D.; Farrell, H. M. Contributions of terminal peptides to the associative behavior of αs1casein. J. Dairy Sci. 2005, 88, 2318−2328. (31) Gagnaire, V.; Léonil, J. Preferential sites of tryptic cleavage on the major bovine caseins within the micelle. Lait 1998, 78, 471−489. (32) Diaz, O.; Gouldsworthy, A. M.; Leaver, J. Identification of peptides released from casein micelles by limited trypsinolysis. J. Agric. Food Chem. 1996, 44, 2517−2522. (33) Holt, C.; de Kruif, C. G.; Tuinier, R.; Timmins, P. A. Substructure of bovine casein micelles by small-angle X-ray and neutron scattering. Colloids Surf., A 2003, 213, 275−284. (34) Farrell, H. M., Jr.; Malin, E. L.; Brown, E. M.; Mora-Gutierrez, A. Review of the chemistry of αS2-casein and the generation of a homologous molecular model to explain its properties. J. Dairy Sci. 2009, 92, 1338−1353. (35) Tauzin, J.; Miclo, L.; Roth, S.; Mollé, D.; Gaillard, J. L. Tryptic hydrolysis of bovine αS2-casein: identification and release kinetics of peptides. Int. Dairy J. 2003, 13, 15−27. (36) Rasmussen, L. K.; Højrup, P.; Petersen, T. E. Disulphide arrangement in bovine caseins: localization of intrachain disulphide

bridges in monomers of κ- and αs2-casein from bovine milk. J. Dairy Res. 1994, 61, 485−493. (37) Bumberger, E.; Belitz, H. D. Bitter taste of enzymic hydrolysates of casein. I. Isolation, structural and sensorial analysis of peptides from tryptic hydrolysates of beta-casein. Z. Lebensm. Unters. Forsch. 1993, 197, 14−19. (38) Considine, T.; Geary, S.; Kelly, A. L.; McSweeney, P. L. H. Proteolytic specificity of cathepsin G on bovine αs1- and β-caseins. Food Chem. 2002, 76, 59−67. (39) Considine, T.; Healy, Á .; Kelly, A. L.; McSweeney, P. L. H. Hydrolysis of bovine caseins by cathepsin B, a cysteine proteinase indigenous to milk. Int. Dairy J. 2004, 14, 117−124. (40) Moatsou, G.; Bakopanos, C.; Katharios, D.; Katsaros, G.; Kandarakis, I.; Taoukis, P.; Politis, I. Effect of high-pressure treatment at various temperatures on indigenous proteolytic enzymes and whey protein denaturation in bovine milk. J. Dairy Res. 2008, 75, 262−269. (41) Larsen, L. B.; Hinz, K.; Jorgensen, A. L.; Moller, H. S.; Wellnitz, O.; Bruckmaier, R. M.; Kelly, A. L. Proteomic and peptidomic study of proteolysis in quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus. J. Dairy Sci. 2010, 93, 5613−5626. (42) Gaucher, I.; Mollé, D.; Gagnaire, V.; Gaucheron, F. Effects of storage temperature on physico-chemical characteristics of semiskimmed UHT milk. Food Hydrocolloids 2008, 22, 130−143. (43) Farrell, H. M., Jr.; Brown, E. M.; Hoagland, P. D.; Malin, E. L. Higher Order Structures of the Caseins: A Paradox? In Advanced Dairy Chemistry1 Proteins; Fox, P. F., McSweeney, P. L. H., Eds.; Springer: New York, 2003; pp 203−231. (44) Berglund, L.; Andersen, M. D.; Petersen, T. E. Cloning and characterization of the bovine plasminogen cDNA. Int. Dairy J. 1995, 5, 593−603. (45) Rollema, H. S.; Visser, S.; Poll, J. K. Spectrophotometric assay of plasmin and plasminogen in bovine milk. Milchwissenschaft 1983, 38, 214−217. (46) Marchin, S.; Putaux, J. L.; Pignon, F.; Leonil, J. Effects of the environmental factors on the casein micelle structure studied by cryo transmission electron microscopy and small-angle X-ray scattering/ ultrasmall-angle X-ray scattering. J. Chem. Phys. 2007, 126, 045101. (47) Dalgleish, D. G. On the structural models of bovine casein micelles-review and possible improvements. Soft Matter 2011, 7, 2265−2272. (48) Trejo, R.; Dokland, T.; Jurat-Fuentes, J.; Harte, F. Cryotransmission electron tomography of native casein micelles from bovine milk. J. Dairy Sci. 2011, 94, 5770−5775. (49) Crudden, A.; Afoufa-Bastien, D.; Fox, P. F.; Brisson, G.; Kelly, A. L. Effect of hydrolysis of casein by plasmin on the heat stability of milk. Int. Dairy J. 2005, 15, 1017−1025. (50) Manji, B.; Kakuda, Y. The Role of Protein Denaturation, Extent of Proteolysis, and Storage Temperature on the Mechanism of Age Gelation in a Model System. J. Dairy Sci. 1988, 71, 1455−1463. (51) Kohlmann, K. L.; Nielsen, S. S.; Ladisch, M. R. Effects of a Low Concentration of Added Plasmin on Ultra-High Temperature Processed Milk. J. Dairy Sci. 1991, 74, 1151−1156. (52) Aroonkamonsri, J. The Role of Plasmin and Bacterial Proteinases on Age Gelation of UHT Milk (Pseudomonas f luorescens, Pseudomonas f ragi, Pseudomonas luteola, Vibrio fluvialis). Ph.D. Thesis, University of Guelph, Canada, 1996. (53) Newstead, D. F.; Paterson, G.; Anema, S. G.; Coker, C. J.; Wewala, A. R. Plasmin activity in direct-steam-injection UHTprocessed reconstituted milk: Effects of preheat treatment. Int. Dairy J. 2006, 16, 573−579. (54) Matoba, T.; Hayashi, R.; Hata, T. Isolation of bitter peptides from tryptic hydrolysate of casein and their chemical structure. Agric. Biol. Chem. 1970, 34, 1235−1243. (55) Hill, R.; Van Leeuwen, H. Bitter peptides from hydrolysed casein coprecipitate. Aust. J. Dairy Technol. 1974, 29, 32−34. (56) Kim, H. O.; Li-Chan, E. C. Quantitative structure-activity relationship study of bitter peptides. J. Agric. Food Chem. 2006, 54, 10102−10111.

6860

dx.doi.org/10.1021/jf502088u | J. Agric. Food Chem. 2014, 62, 6852−6860