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