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Comprehensive analysis of proteolysis in long-ripened hard
cooked Old Saare cheese
Minna Varikmaa, Tiina Kriščiunaite, Natalja Kabanova, Irina Stulova, Viktoria Põžjanova, Raivo Vilu
Competence Centre of Food and Fermentation Technologies
Tallinn University of Technology
Outline
1. Backround
2. Experimental design
3. Results
Distribution of proteolysis fractions
Evolution of small peptides
Release of free amino acids
Old Saare cheese
Milk Cow
Coagulation time (min) 37-43
Cutting time (min) 11.5
Cooking/heating time (min) at 520C 40
Pre-press 21,4, 1 bar
Pressing (h) 8
Brining 72h, 18-19.5% salt in brine, 10⁰C
Water content of cheese 38,4
pH of cheese 5,29
Ripening time at 120C (months) 8
Starter cultures : Lc. lactis subsp. lactis, Lc. lactis subsp. cremoris Lc. lactis subsp. diacetylactis Ln. mesenteroides subsp. cremoris St.thermophilus Lb. Casei Lb. Acidophilus Lb. Helveticus
Microbial counts in Old Saare detected by pyrosequencing of cDNA library
Blank et al., 2014. Manuscript in preparation.
0
20
40
60
80
100
120
0 0.5 1 3 4
Re
lati
ve m
icro
bia
l co
un
ts, %
Age, months
St.thermophilus
Lb.casei
Lb.Helveticus
Lc.lactis
Objective
Aim of the study: Develop in silico tools to analyze casein proteolysis Obtain full casein proteolysis profile of Old Saare cheese
throughout 8 months of ripening
Samples: 2 industrial trials of Old Saare 10 time-points (months): 0 (after pressing, before salting),
0.5, 1, 2, 3, 4, 5, 6, 7, 8
Comprehensive description of casein hydrolysis
• Capillary electrophoresis (CE) – caseins and big peptides
• LC-MS/MS – water soluble peptides
• UPLC – free amino acids
Fox et al., 2004
Experimental design
GRATED CHEESE
Citric dispersion (CD) Water soluble extract (WSE)
Kjeldahl total protein in CD
CE intact CN and long peptides
LC-MS/MS small peptides 4-25 AA long
UPLC FAA
Kjeldahl total protein in
WSE
Results Distribution of casein proteolysis products
Small peptides (striped)
Long peptides (spotted)
Intact protein (solid)
Free amino acids
α-s1-casein
α-s2-casein
β-casein
para-κ-casein
Unknown long peptides
Free amino acids
0
5
10
15
20
25
30
0 0.5 1 2 3 4 5 6 7 8
Co
nte
nt,
g/
10
0 g
Ripening time, months
α-s1-casein
Results Accumulation of small peptides
Number of identified peptides
α-s1-casein 1033
α-s2-casein 872
β-casein 1112
κ-casein 260
Total 3277 0.0
0.5
1.0
1.5
2.0
2.5
0 0.5 1 2 3 4 5 6 7 8
Co
nte
nt,
g/1
00
g
Ripening time, months
α-s1-casein α-s2-casein β-casein κ-casein
Results Allocation of small peptides on α-s1-casein
sequence
8 months:
0 months:
Results Allocation of small peptides on α-s2-casein
sequence 0 months:
8 months:
Results Allocation of small peptides on β-casein
sequence 0 months:
8 months:
Results Allocation of small peptides on κ-casein
sequence 0 months:
8 months:
Results Analysis of cleavage sites I
Chymosin
CEPs
Plasmin, cathepsin
Plasmin
Chymosin Plasmin
CEPs
?
cathepsin
Results Analysis of cleavage sites II
CEPs, cathepsin
Plasmin
CEPs, cathepsin
Chymosin, CEPs
Plasmin
Chymosin
Results Release of free amino acids
0
10
20
30
40
50
60
70
Ala Arg
Asn
Asp
GABA
Gln
Glu
Gly
His
Ile Leu Lys
Met
Orn
Phe
Pro
Ser
Thr
Trp
Tyr
Val 0
0,5
1
2
3
4
5
6
7
8
μmol/g
0
10
20
30
40
50 Ala
Arg
Asn
Asp
Cys
Gln
Glu
Gly
His
Ile Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
AA distribution in caseins:
Summary Full proteolysis profile of Old Saare cheese during 8 months of ripening
was determined: High proteolysis extent and depth was observed (only 25% of intact left;
~21% of TN was FAA) with most pronounce changes taking place during first 4 months of ripening
β-casein was most extensively hydrolyzed (19,3% intact remained) followed by α-s2-casein>α-s1-casein>κ-casein
Extensive hydrolysis of β-casein suggest significant contribution of plasmin and CEPs (eg Lb.Helveticus) to Old Saare ripening
Parallel increase in free amino acid and small peptides and Lb.Casei counts at 3-4 months, suggest important impact of this NSLAB in promotion of proteolysis
Number of in silico tools were developed to analyze the formation of small peptides, that can be applied to improve the choice of starters and control the process of ripening
Further studies are needed to verify how particular starter type and inoculation number impacts the progress of proteolysis
Thank you!
Results Loss of intact caseins
100.0% 94.7% 91.2% 76.2%
54.4%
38.3% 36.0% 33.4% 27.5% 35.1%
0 0.5 1 2 3 4 5 6 7 8
Ripening time, months
α-s1-casein
100.0%
66.3% 69.3% 50.4%
28.7% 25.8% 21.1% 19.6% 18.5% 22.4%
0 0.5 1 2 3 4 5 6 7 8
Ripening time, months
α-s2-casein
100.0% 92.2%
81.9% 77.3% 58.4% 61.5% 54.9% 51.0% 47.4%
62.5%
0 0.5 1 2 3 4 5 6 7 8
Ripening time, months
para-κ-casein 100.0% 87.1% 87.5%
63.2%
42.6%
24.6% 27.0% 21.6% 19.7% 19.8%
0 0.5 1 2 3 4 5 6 7 8
Ripening time, months
β-casein
Results Proteolysis of α-s1-casein
0
50
100
150
200
250
300
350
400
'1-2
3'
'1-2
4'
'7-1
3'
'7-1
4'
'7-2
3'
'7-2
4'
'7-3
0'
'8-1
4'
'8-1
6'
'8-2
2'
'8-2
3'
'8-2
4'
'10
-22
'
'10
-23
'
'10
-24
'
'10
-30
'
'10
-34
'
'14
-22
'
'14
-23
'
'15
-22
'
'15
-23
'
'16
-23
'
'17
-22
'
'17
-23
'
'24
-30
'
'24
-34
'
'24
-36
'
'24
-39
'
'24
-42
'
'25
-30
'
'25
-34
'
'25
-36
'
'33
-40
'
'34
-40
'
'80
-90
'
'80
-97
'
'80
-98
'
'80
-99
'
'80
-10
2'
'80
-10
3'
'83
-98
'
'83
-99
'
'83
-10
2'
'83
-10
3'
'83
-10
5'
'85
-93
'
'85
-98
'
'85
-10
2'
'88
-10
2'
'10
5-1
14
'
'10
9-1
14
'
'17
8-1
99
'
'18
0-1
99
'
'18
1-1
99
'
Co
nte
nt,
nm
ol/
g
0 months 3 months 8 months
Results The progess of proteolysis
Concentrations, g/100 g
Time,
months
Intact Long peptides Small peptides FAA
0 21,88 (±0,69) 5,40 (±0,13) 0,74(±0,01) 0,12(±0,00)
0,5 19,55 (±0,97) 7,24(±0,35) 1,05(±0,02) 0,31(±0,01)
1 19,03 (±0,68) 6,91(±0,2) 1,37(±0,05) 0,84(±0,03)
2 15,17 (±0,89) 9,32(±0,22) 1,61(±0,06) 2,05(±0,04)
3 10,59 (±0,36) 11,82(±0,37) 2,34(±0,13) 3,40(±0,07)
4 7,66(±0,26) 13,07(±0,31) 2,28(±0,17) 5,14(±0,12)
5 7,42(±0,33) 13,39(±0,33) 1,75(±0,12) 5,59(±0,05)
6 6,57(±0,43) 13,34(±0,39) 1,98(±0,16) 6,26(±0,08)
7 5,78(±0,29) 13,88(±0,34) 1,84(±0,22) 6,65(±0,14)
8 6,94(±0,47) 13,03(±0,41) 2,19(±0,16) 5,99(±0,05)
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 0.5 1 2 3 4 5 6 7 8
Ripening time, months
FAA
Small peptides
Long peptides
Intact
TN: ~28 g/100 g SN: 0.9 to 8.2 g/100 g