What are casein micelles? Calcium phosphate Polar surface Non-Polar interior layer, rich in (rich in Alpha-s1, Kappa-casein Alpha -s2, Beta casein) Adapted from: Horn, D. 1998. International Dairy J. 8:171- 177
Kappa casein nonpolar + + polar AA 169
Calcium phosphate Polar surface Non-Polar interior layer, rich in Kappa-casein (rich in Alpha-s1, Alpha -s2, Beta casein) Adapted from: Horn, D. 1998. International Dairy J. 8:171- 177
Mechanisms of coagulation • Rennet (rapid: 30 – 60 min) • Acid (slow: 5 – 24 hr) • Acid-rennet (slow: 12 -24 hr) • Acid-heat
What is rennet? • General term for enzymes used to coagulate milk • Technically restricted to enzymes derived from ruminant stomachs • All are protein degrading enzymes (proteinase, protease, proteolytic enzyme) • All are members of the aspartic proteinase family
All aspartic proteinases have 2 characteristics in common aspartic aspartic – – Source: after Crabbe, M.J.C. 2004. Rennets: General and molecular aspects In Cheese: Chemistry, Physics and Microbiology, Elsevier Academic Press, London
Kappa casein is uniquely vulnerable to the action of aspartic proteases nonpolar AA 105-106 + (very vulnerable + to aspartic proteinases) polar AA 169
Rennet cleavage of kappa casein Kappa casein chymosin, etc. – + + – – – – – – – – –
Rennet coagulation occurs in two phases 1. Enzymatic 2. Non-enzymatic
1. Enzymatic phase: cleavage of k-casein Rennet enzymes shave off polar surface Adapted from: Horn, D. 1998. International Dairy J. 8:171- 177
1. Enzymatic phase: cleavage of k-casein casein micellecasein micelle Kappa-Casein with exposed polar region
2. Nonenzymatic phase: Ca++ induced aggregation casein micelle ++ Ca Ca++ Ca++ Ca++ VERY Nonpolar surface CMP/GMP (AA106-169)
2. Nonenzymatic phase: aggregation of casein micelles + Ca+ + Permanent bonding Nonpolar micelles (commences after 80-90% cleavage of k-casein)
2. Nonenzymatic phase continued: chain formation/flocculation visible flocs = rennet clotting time
continuous gel – coagulation
Matrix rearrangement Fine matrix – small coarse matrix – large pores, firm gel pores,weak gel
Repercussions of matrix rearrangement • Cheese moisture content • Acid development during cheese making • Cheese yield
Matrix rearrangement Coarse matrix contracts, Fine matrix – small syneresis pressure pores,weak gel
Repercussions of matrix rearrangement 1. Cheese moisture content • Cutting early (weak set) enables much rearrangement to occur after cutting; syneresis , cheese moisture content Ø – ex: alpine cheeses • Cutting late (firm set) limits rearrangement after cutting; syneresis Ø, cheese moisture content – ex: traditional Brie, Camembert • Cutting firmness should be consistent from vat-to-vat to reduce moisture variation
Repercussions of matrix rearrangement 2. Acid development • If the amount of time to the desired cutting firmness varies greatly from day-to-day, the subsequent rate of acidification during the rest of cheese making may be affected: – Extended cutting time, starter culture population, rate of acidification – Reduced cutting time, Ø starter culture population, Ø rate of acidification • Therefore, both cutting firmness and cutting time should be optimized and held constant from day-to- day
Bottom line • Cutting should be initiated at a consistent curd firmness that is optimized for the type of cheese being made • The time required to achieve the target cutting firmness should be consistent from vat-to-vat across season • In practice, this can be challenging because several factors may influence coagulation and cause curd firmness at cutting and/or cutting time to vary
Repercussions of matrix rearrangement • Cheese moisture content • Acid development during cheese making • Cheese yield
Repercussions of matrix rearrangement 3. Cheese yield • Weak curds are fragile and tend to shatter during cutting – fat and casein losses , cheese yield Ø – however, matrix rearrangement after cutting occurs rapidly, curd particles firm up quickly • Firm curds are more forgiving with respect to shattering during cutting – however, matrix rearrangement after cutting occurs slowly – curd particles firm up slowly and remain vulnerable to shattering for longer time after cutting
Mechanisms of coagulation • Rennet (rapid: 30 – 60 min) • Acid (slow: 5 – 24 hr) • Acid-rennet (slow: 12 -24 hr) • Acid-heat
Acid coagulation 1. Starter culture 3. H+ ions produces lactic acid, neutralize the H+ ions accumulate negative charges on k-casein H+ H+ H+ H+ H+ H+ H+ H+ 2. Calcium phosphate 4. Neuralized dissolves into k-casein collapses water phase
Acid coagulation H+ pH 4.6 Collapsed neutralized Polar k-cn nonpolar surface Surface Micelle rich in Micelle depleted of calcium phosphate calcium phosphate
Aggregation of casein micelles continuous gel – coagulation
Acid coagulation • Acid gels lack the capacity to contract and synerese • Therefore, final cheese moisture content is very high (around 70-80%, depending of the fat content) • In general, acid coagulated cheeses are eaten fresh, not ripened
Mechanisms of coagulation • Rennet (rapid: 30 – 60 min) • Acid (slow: 5 – 24 hr) • Acid-rennet (slow: 12 -24 hr) • Acid-heat
Key parameters of acid-rennet coagulation 1. Amount of rennet added to the milk: – Anywhere from 1 – 30% of the level used in rennet coagulation 2. Coagulation temperature: – Anywhere from 18 – 32°C
Example 1: Quark • Lactic fermentation at ca. 30°C for 16 hr • A small amount of rennet (e.g., 1 – 10% of level used in rennet coagulation) added at around pH 6.3 • Rennet proceeds through enzymatic and non- enyzmatic phases as milk pH Ø • Coagulation occurs at pH 4.8 or 4.9 instead of ph 4.6 • The resulting curd develops hybrid characteristics that fall somewhere between those of rennet curd and acid curd
Advantages of acid-rennet coagulated Quark –Better draining results in a lower moisture content – Lower moisture content along with a firmer coagulation result in a firmer cheese body, improved texture –Higher cutting pH (e.g. from pH 4.6 to 4.8 or 4.9) results in a less acidic flavor
Example 2: soft ripened goat ’s milk cheese • The milk undergoes lactic acidification at around 20°C for 24 hr • Rennet (about 1/3 the level used in rennet coagulation) is added, often around pH 6.3. • The rennet proceeds through enzymatic phase but the non-enyzmatic phase is strongly impeded at 20°C, • Coagulation occurs in 24 hr when the pH reaches around pH 5.3 • The resulting curd develops hybrid characteristics that fall somewhere between those of rennet curd and acid curd
Advantages of acid-rennet coagulation for goat’s cheeses • Syneresis is improved, resulting in a final cheese with ca. 60-70% moisture. • This moisture range is low enough to support controlled ripening • The end result is a group of soft ripened goat’s milk cheeses with a unique lactic acid dominated texture
Factors that affect rennet coagulation • Temperature history of milk • pH of milk during coagulation • Temperature of milk during coagulation • Ca++ ion content of milk • Casein content of milk
Temperature history of milk: 1. Cooling (< 10ºC) H+ Ca++ ß- casein HPO4 = 0.2 – 0.3 pH
Repercussions of cooling milk to 4ºC • milk pH – Slower enzymatic phase – Ø attraction between rennet enzymes and casein micelles, cleavage of k-casein needed to induce coagulation – Therefore, longer time needed to attain target cutting firmness • Altered casein micelle structure – Curd matrix less able to undergo structural rearrangement, contraction – Therefore, weaker set, slower syneresis, higher moisture content in cheese (especially in high moisture types, e.g., bloomy rind) • May cause problems when switching from warm milk fresh from the animal to cold stored milk
Compensating for cooling milk to 4ºC • The changes can be largely reversed by normal pasteurization before cheese making • These changes can be partly overcome by adding calcium chloride. • If necessary, increase cutting time to restore the target curd firmness • If necessary, adjust starter usage to restore target acifidification schedule • If necessary, take action during cheese making to enhance syneresis/draining to reduce cheese moisture content
Factors that affect rennet coagulation • Temperature history of milk • pH of milk during coagulation • Temperature of milk during coagulation • Ca++ ion content of milk • Casein content of milk
pH of milk during coagulation pH Ø from 6.7 – 6.0 Ca++ H PO – ß-casein 2 4
Repercussions of milk pH • As milk pH Ø • Rennet enzyme activity increases • Rennet enzymes are more strongly attracted to casein micelles • Therefore, the enzymatic phase occurs more rapidly • Furthermore: • Rennet enzymes adsorb more tightly onto the casein micelle surface, resulting in “patches” of k-casein cleavage • Therefore, the amount of k-casein cleavage needed to induce micelle aggregation Ø • Also, higher Ca++ ion concentration speeds up the aggregation of casein micelles • Therefore, Non-enzymatic phase occurs more rapidly • Consequently, rennet clotting time Ø, cutting time Ø, and curd firmness
• Temperature history of milk • pH of milk during coagulation • Temperature of milk during coagulation • Ca++ ion content of milk • Casein content of milk
As coagulation temperature from ca. 25 ° – 40 °C • Enzymatic phase occurs more rapidly: • Rennet enzyme activity increases with temperature • Non-enzymatic phase occurs more rapidly • Rennet enzymes adsorb more tightly onto the casein micelle surface • Therefore, the amount of k-casein cleavage needed to induce micelle aggregation Ø • Consequently, rennet clotting time and cutting time Ø, and curd firmness
As coagulation temperature Ø from ca. 25 ° – 15°C • Enzymatic phase gradually slows down but the non-enzymatic phase fails catastrophically – At 20°C, the non-enzymatic phase is severely impeded and casein aggregation/curd formation occurs very slowly (many hours) – This principle is exploited in the production of acid-rennet coagulated cheeses – At 15°C, the nonenzymatic phase is completely prevented; casein aggregation cannot occur even when k-casein has been completely cleaved from the micelle surface • Bottom line: coagulation temperature should be tightly controlled
Factors that affect rennet coagulation • Temperature history of milk • pH of milk during coagulation • Temperature of milk during coagulation • Ca++ ion content of milk • Casein content of milk
Ca++ ion content of the milk casein micelle ++ Ca Ca++ Ca++ Ca++ VERY Nonpolar surface CMP/GMP (AA106-169)
Calcium chloride addition • Supplies Ca++ ions, enables nonezymatic phase (aggregation of casein micelles) to proceed • Decreases the milk pH, thereby stimulating the enzymatic phase • End result: rennet clotting time Ø, cutting time Ø, and curd firmness
Factors that affect rennet coagulation • Temperature history of milk • pH of milk during coagulation • Temperature of milk during coagulation • Ca++ ion content of milk • Casein content of milk
As casein content : • The frequency of collisions between casein micelles increases dramatically • This causes micelles to aggregate at much lower levels of k-casein cleavage • End result: the nonezymatic phase is greatly accelerated: rennet clotting time Ø, cutting time Ø, and curd firmness
General rules of thumb • Cut curd at a consistent firmness (for moisture control) • Cut curd at a consistent time from rennet addition (for maintaining acidification schedule) • If necessary: – Add calcium chloride (up to 0.02% maximum) – adjust cutting time to hold cutting firmness constant – adjust starter usage to maintain target acidification schedule
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