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rennet types and uses
RENNET TYPES AND USES Keith Glewis FoodPlus Technical Consultants PL 9th October, 2013 [email protected] PRESENTATION OVERVIEW Rennet – what it is / types / preparation. Advantages Primary Role of Rennet – Clotting milk. Secondary Cheese & Disadvantages of each type. Role of Rennet – Cheese maturation. Peptide Profiles with different rennet types. 2 THE CHEESE MAKING PROCESS 3 RENNET – WHAT IT IS ? Enzyme preparation used to clot milk. Historically Plays Calf Rennet. two roles: Primarily – clot milk curd. Secondary – ongoing proteolysis in cheese. 4 RENNET – TYPES Type Source Active enzymes Examples Animal Calf Chymosin (88-94%) Naturen Bovine pepsin (6-12%) Animal • Ovine • Porcine • (Chicken) Chymosin Secondary proteases - Plant dried flowers Plant proteases - Microbial proteases Hannilase Fromase Kesozym. Chymosin (100%) Chy-max Maxiren Cynara cardunculus Microbial Fungal species e.g. Rhizomucor miehei, Fermentation Produced Chymosin Fungus (bovine chymosin gene cloned into fungus.) 5 RENNET PREPARATION - CALF RENNET 4th stomach of unweaned calves. Frozen stomach milled & placed in enzyme-extracting solution. Acid added to activate the enzyme. Neutralised. Filtered. Concentrated. 6 RENNET PREPARATION - Calf Gene Culture Aspergillus niger Kluyveromyces lactis Standardisation Filtration & Purification FPC Fermentation Culture inactivation 7 RENNET PREPARATION - MICROBIAL Culture Rhizomucor miehei Fermentation Standardisation Filtration & Purification Culture inactivation 8 RENNET – COMPARING THE DIFFERENT TYPES Rennet Type Advantages Disadvantages Animal (Calf) • Traditional use. • Historical acceptance. • Flavour can vary slightly (Farmhouse). • Can have slight variations in manufacture. • Animal origin. • Not suitable for vegetarians. FermentationProduced Chymosin • Very consistent in • Not GMO but GE manufacture. implications. • Suitable for vegetarians. Microbial • Non-animal, non GE. • Perception may cause • Suitable for vegetarians. bitterness. • More Thermoliable – • Curd sets slightly softer. Mozzarella. 9 RENNET – MODE OF ACTION 1 – CLOTTING MILK How does rennet clot milk ? 10 RENNET MODE OF ACTION – CLOTTING MILK - MILK PROTEINS 11 RENNET MODE OF ACTION – CLOTTING MILK - CASEIN MICELLE MODEL - HORNE (1998) 12 RENNET MODE OF ACTION – CLOTTING MILK - CASEIN MICELLE MODEL - HORNE (1998) Colloidal Calcium Phosphate 13 RENNET MODE OF ACTION – CLOTTING MILK - RENNET CLEAVAGE OF KAPPA CASEIN 1 105 106 Glu Phe Met Kappa 169 169 Val casein. amino acids long. Amino acids 105 & 106 critical. 14 RENNET MODE OF ACTION – CLOTTING MILK - RENNET CLEAVAGE OF KAPPA CASEIN 105 N-terminal Phe 106 Met C-terminal kappa casein (κ-CN) Rennet 15 RENNET MODE OF ACTION – CLOTTING MILK - RENNET CLEAVAGE OF KAPPA CASEIN 105 N-terminal Phe 106 C-terminal Met kappa casein (κ-CN) Rennet 105 Phe para-kappa casein (ρ- κ-CN) 106 Met caseinmacropeptide (CMP) 16 RENNET MODE OF ACTION – CLOTTING MILK 17 RENNET MODE OF ACTION – CLOTTING MILK Critical factors: pH – optimum 6.0. Temp: optimum 30-32oC. Coagulation does not occur <18oC. Calcium – 20% reduction in colloidal calcium phosphate prevents coagulation. 18 SECONDARY ROLE OF RENNET Cheese Maturation 19 SECONDARY ROLE OF RENNET - CHEESE MATURATION How does Rennet affect Cheese Maturation ? 20 SECONDARY ROLE OF RENNET – CHEESE MATURATION - RENNET RETENTION IN CURD Rennet Type Retention in Curd pH Effect Calf 5 - 8% Dependent pH (esp. drain) Rennet retention Microbial 3- 5% Independent Other Factors Amount of rennet used. Cook Temperature. Salt. 21 CHEESE MATURATION PROCESS OCCURRING IN CHEESE Maturation Lipolysis Glycolysis Proteolysis Fats breakdown Sugars breakdown Caseins breakdown 22 CHEESE MATURATION PROCESS OCCURRING IN CHEESE 23 CHEESE MATURATION - PROTEOLYSIS Casein Large Peptides Small Peptides Free Amino acids 24 CHEESE MATURATION - PROTEOLYSIS Residual coagulant Casein Large Peptides Milk proteinases Small Peptides peptides Free Amino acids 25 CHEESE MATURATION - PROTEOLYSIS Residual coagulant Casein S tarter / NS LAB Large P roteinases Peptides Small Peptides Milk proteinases S tarter / NS LAB Peptidases Free Amino acids 26 CHEESE MATURATION – PROTEOLYSIS PROTEINASE & PEPTIDASES Casein Residual coagulant Milk proteinases Starter / LAB NS Proteinases Small Peptides Peptides Large Starter / LAB NS Peptidases Free Amino acids 27 CHEESE MATURATION - PROTEOLYSIS HPLC Profile (High Performance Liquid Chromatography) 28 CHEESE MATURATION : PROTEOLYSIS (HPLC PROFILES) mAU AN 4000 Tyr MR B H Trp G 3500 C A Phe B-Lg AB F 3000 12m 2500 E D 2000 6m 1500 1000 1m 500 10 20 30 40 50 60 min 29 CHEESE MATURATION - PROTEOLYSIS Alpha S1 casein sequence Rhizomucor meihei Rhizomucor meihei Cut site Cut site 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Arg Pro Lys His Pro Ile Lys His Gln Gly Leu Pro Gln [Glu Val Leu Asn Glu Asn Leu Leu Arg Phe Calf Rennet 1m 8 7 6 Chymosin Cut site Chymosin, Chymosin, 24 25 26 27 28 29 30 31 32 33 34 13 10 9 5 Microbial Rennet 1m 8 7 35 Phe Val Ala] Pro Phe Pro Glu Val Phe Gly Lys Glu 14 10 9 17 30 CHEESE MATURATION - PROTEOLYSIS Alpha S1 casein sequence Rhizomucor meihei Rhizomucor meihei Cut site Cut site 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Arg Pro Lys His Pro Ile Lys His Gln Gly Leu Pro Gln [Glu Val Leu Asn Glu Asn Leu Leu Arg Phe Calf Rennet 1m Chymosin Cut site Chymosin, Chymosin, 8 24 25 26 27 28 29 30 31 32 33 34 35 Phe Val Ala] Pro Phe Pro Glu Val Phe Gly Lys Glu 13 10 9 7 6 5 Microbial Rennet 1m 8 14 7 10 9 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Arg Pro Lys His Pro Ile Lys His Gln Gly Leu Pro Gln [Glu Val Leu Asn Glu Asn Leu Leu Arg Phe 1 24 25 26 27 28 29 30 31 32 33 34 10 9 Calf Rennet 12m 16 18 17 12 1 18 2 Microbial Rennet 35 Phe Val Ala] Pro Phe Pro Glu Val Phe Gly Lys Glu 31 10 9 12m 17 14 CHEESE MATURATION - PROTEOLYSIS Chymosin cut site, Chymosin Cut site Rhizomucor meihei Cut site Lactocepin cut site PIII type Beta Casein 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 Lactocepin cut site - PI and PIII-type 199 200 201 202 203 204 205 206 207 Pro Gln Arg Asp Met Pro Ile Gln Ala Phe Leu Leu Tyr Gln Gln Pro Val Leu Gly Pro Val Arg Gly Pro Phe Pro Ile Calf Rennet 44 1m 208 209 Ile Val OH 41 45 Microbial Rennet 181 182 183 184 1m 185 186 44 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 Pro Gln Arg Asp Met Pro Ile Gln Ala Phe Leu Leu Tyr Gln Gln Pro Val Leu Gly Pro Val Arg Gly Pro Phe Pro Ile Calf Rennet 44 12m 208 209 Ile Val OH 43 42 44 Microbial Rennet 12m 42 32 CHEESE MATURATION – PROTEOLYSIS….BITTERNESS Alpha S1 casein sequence Chymosin, Chymosin, Cut site Cut site 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Chymosin Cut site Rhizomucor meihei Rhizomucor meihei 22 23 Arg Pro Lys His Pro Ile Lys His Gln Gly Leu Pro Gln [Glu Val Leu Asn Glu Asn Leu Leu Arg Phe 1 24 25 26 27 28 29 30 31 32 33 34 35 Phe Val Ala] Pro Phe Pro Glu Val Phe Gly Lys Glu 10 9 Calf Rennet 12m 16 18 17 12 1 18 2 10 9 Microbial rennet 12m 17 14 17 18 Broadbent et al, 2002 1 Lemieux & Simard, 1992 Lemieux & Simard, 1992 Lee et al, 1996 Lee et al, 1996 Lee et al, 1996 Richardson & Creamer, 1973 Matoba et al, 1970 Lemieux & Simard, 1992 Lemieux & Simard, 1992 3 4 Pelissier et al, 1974 Broadbent et al, 2002 Broadbent et al, 2002 Pelissier et al, 1974 33 Hodges & Kent, 1972 Hodges & Kent, 1972 18 Hodges & Kent, 1972 CHEESE MATURATION – PROTEOLYSIS….BITTERNESS Beta Casein 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 Pro Gln Arg Asp Met Pro Ile Gln Ala Phe Leu Leu Tyr Gln Gln Pro Val Leu Gly Pro Val Arg Gly Pro Phe Pro Ile Calf Rennet 44 12m 208 209 Ile Val OH 43 42 44 Microbial Rennet 12m 42 KNOWN BITTER PEPTIDES IN CHEESE 44 43 Visser et al, 1983 Visser et al, 1983 Visser et al, 1983 Pelissier et al, 1974 Gordon, 1975 Matoba et al, 1970 34 UNDERSTANDING CHEESE MATURATION ??? 35