Comprehensive analysis of proteolysis in long-ripened hard cooked Old Saare cheese

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1 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

2 Outline 1. Backround 2. Experimental design 3. Results Distribution of proteolysis fractions Evolution of small peptides Release of free amino acids

3 Old Saare cheese Milk Cow Coagulation time (min) Cutting time (min) 11.5 Cooking/heating time (min) at 52 0 C 40 Pre-press 21,4, 1 bar Pressing (h) 8 Brining 72h, % salt in brine, 10⁰C Water content of cheese 38,4 ph of cheese 5,29 Ripening time at 12 0 C (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

4 Relative microbial counts, % Microbial counts in Old Saare detected by pyrosequencing of cdna library St.thermophilus Lb.casei Lb.Helveticus Lc.lactis Age, months Blank et al., Manuscript in preparation.

5 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

6 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

7 Experimental design GRATED CHEESE Citric dispersion (CD) Water soluble extract (WSE) Kjeldahl total protein in CD CE intact CN and long peptides Kjeldahl total protein in WSE LC-MS/MS small peptides 4-25 AA long UPLC FAA

8 Content, g/ 100 g Results Distribution of casein proteolysis products Free amino acids 20 Small peptides (striped) 15 Long peptides (spotted) α-s1-casein Ripening time, months α-s1-casein α-s2-casein β-casein Intact protein (solid) para-κ-casein Unknown long peptides Free amino acids

9 Content, g/100 g Results Accumulation of small peptides Ripening time, months Number of identified peptides α-s1-casein 1033 α-s2-casein 872 β-casein 1112 κ-casein 260 Total 3277 α-s1-casein α-s2-casein β-casein κ-casein

10 Results Allocation of small peptides on α-s1-casein sequence 0 months: 8 months:

11 Results Allocation of small peptides on α-s2-casein sequence 0 months: 8 months:

12 Results Allocation of small peptides on β-casein sequence 0 months: 8 months:

13 0 months: Results Allocation of small peptides on κ-casein sequence 8 months:

14 Results Analysis of cleavage sites I Chymosin Plasmin, cathepsin CEPs CEPs cathepsin Plasmin? Plasmin Chymosin

15 Results Analysis of cleavage sites II Plasmin CEPs, cathepsin Plasmin Chymosin, CEPs CEPs, cathepsin Chymosin

16 Results Release of free amino acids Thr Ser Trp Tyr Val Ala Arg Asn Asp GABA Gln 0 0, AA distribution in caseins: Ala Val 50 Arg Tyr 40 Asn Trp Thr Asp Cys Ser 0 Gln Pro Glu 4 Pro Glu 5 Phe Gly Phe Orn Met Lys μmol/g Leu Ile His Gly Met Lys Leu Ile His

17 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

18 Thank you!

19 Results Loss of intact caseins 100.0% 94.7% 91.2% 76.2% α-s1-casein 100.0% α-s2-casein 54.4% 38.3% 36.0% 33.4% 27.5% 35.1% 66.3% 69.3% 50.4% 28.7% 25.8% 21.1% 19.6% 18.5% 22.4% Ripening time, months Ripening time, months 100.0% 87.1% 87.5% β-casein 63.2% 42.6% 24.6% 27.0% 21.6% 19.7% 19.8% 100.0% 92.2% 81.9% 77.3% para-κ-casein 58.4% 61.5% 54.9% 51.0% 47.4% 62.5% Ripening time, months Ripening time, months

20 Content, nmol/g '1-23' '1-24' '7-13' '7-14' '7-23' '7-24' '7-30' '8-14' '8-16' '8-22' '8-23' '8-24' '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' '80-102' '80-103' '83-98' '83-99' '83-102' '83-103' '83-105' '85-93' '85-98' '85-102' '88-102' ' ' ' ' ' ' ' ' ' ' Results Proteolysis of α-s1-casein months 3 months 8 months

21 Results The progess of proteolysis 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Ripening time, months Concentrations, g/100 g Time, Intact Long peptides Small peptides FAA months 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) TN: ~28 g/100 g SN: 0.9 to 8.2 g/100 g FAA Small peptides Long peptides Intact