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 Coagulation time (min) Cutting time (min) Cooking/heating time (min) at 520C Pre-press Pressing (h) Brining Water content of cheese pH of cheese Ripening time at 120C (months) Cow 37-43 11.5 40 21,4, 1 bar 8 72h, 18-19.5% salt in brine, 10⁰C 38,4 5,29 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 120 Relative microbial counts, % 100 80 St.thermophilus 60 Lb.casei Lb.Helveticus 40 Lc.lactis 20 0 0 0.5 1 3 4 Age, months Blank et al., 2014. Manuscript in preparation. 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) Kjeldahl total protein in CD CE intact CN and long peptides Water soluble extract (WSE) Kjeldahl total protein in WSE UPLC LC-MS/MS small peptides 4-25 AA long FAA Results Distribution of casein proteolysis products Content, g/ 100 g 30 25 Free amino acids 20 Small peptides (striped) 15 Long peptides (spotted) 10 5 α-s1-casein Intact protein (solid) 0 0 0.5 1 2 3 4 5 6 7 8 Ripening time, months α-s1-casein para-κ-casein α-s2-casein Unknown long peptides β-casein Free amino acids Results Accumulation of small peptides Content, g/100 g 2.5 Number of identified peptides α-s1-casein 1033 2.0 1.5 α-s2-casein 872 1.0 β-casein 1112 0.5 κ-casein 260 Total 3277 0.0 0 0.5 1 2 3 4 5 6 7 Ripening time, months α-s1-casein α-s2-casein β-casein κ-casein 8 Results Allocation of small peptides on α-s1-casein sequence 0 months: 8 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 Plasmin, cathepsin CEPs CEPs Plasmin cathepsin Plasmin ? Chymosin Results Analysis of cleavage sites II Plasmin Plasmin Chymosin, CEPs CEPs, cathepsin CEPs, cathepsin Chymosin Results Release of free amino acids AA distribution in caseins: Ala Val Tyr Ala Arg 70 50 Trp 0 Asn 60 Val Tyr 0,5 Asp 1 30 Thr GABA 20 Gln 0 Pro Glu 4 6 His Met 7 Ile Lys μmol/g 20 Thr Leu Cys 10 Ser Gly Orn Asp Gln 0 3 5 Phe Asn 30 2 10 Ser Arg 40 Trp 40 50 8 Pro Glu Phe Gly Met His Lys Ile Leu 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 α-s1-casein α-s2-casein 100.0% 94.7% 91.2% 100.0% 76.2% 66.3% 69.3% 54.4% 50.4% 38.3% 36.0% 33.4% 35.1% 27.5% 0 0.5 1 2 3 4 5 6 7 8 28.7% 25.8% 21.1% 19.6% 18.5% 22.4% 0 0.5 1 2 β-casein 87.1% 87.5% 100.0% 63.2% 92.2% 42.6% 81.9% 77.3% 24.6% 27.0% 21.6% 19.7% 19.8% 1 2 3 4 5 6 7 8 para-κ-casein 100.0% 0.5 4 Ripening time, months Ripening time, months 0 3 5 Ripening time, months 6 7 8 0 0.5 1 2 62.5% 58.4% 61.5% 54.9% 51.0% 47.4% 3 4 5 Ripening time, months 6 7 8 350 300 '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' '105-114' '109-114' '178-199' '180-199' '181-199' Content, nmol/g Results Proteolysis of α-s1-casein 400 0 months 3 months 8 months 250 200 150 100 50 0 Results The progess of proteolysis 100% TN: ~28 g/100 g SN: 0.9 to 8.2 g/100 g 90% 80% 70% 60% FAA 50% Small peptides 40% 30% Long peptides 20% Intact 10% 0% 0 0.5 1 2 3 4 5 6 7 8 Ripening time, months Time, months 0 0,5 1 2 3 4 5 6 7 8 Intact Long peptides 21,88 (±0,69) 19,55 (±0,97) 19,03 (±0,68) 15,17 (±0,89) 10,59 (±0,36) 7,66(±0,26) 7,42(±0,33) 6,57(±0,43) 5,78(±0,29) 6,94(±0,47) 5,40 (±0,13) 7,24(±0,35) 6,91(±0,2) 9,32(±0,22) 11,82(±0,37) 13,07(±0,31) 13,39(±0,33) 13,34(±0,39) 13,88(±0,34) 13,03(±0,41) Concentrations, g/100 g Small peptides 0,74(±0,01) 1,05(±0,02) 1,37(±0,05) 1,61(±0,06) 2,34(±0,13) 2,28(±0,17) 1,75(±0,12) 1,98(±0,16) 1,84(±0,22) 2,19(±0,16) FAA 0,12(±0,00) 0,31(±0,01) 0,84(±0,03) 2,05(±0,04) 3,40(±0,07) 5,14(±0,12) 5,59(±0,05) 6,26(±0,08) 6,65(±0,14) 5,99(±0,05)
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