p-Î³3-CN - IDF Cheese Ripening

University of Milan 

Pyroglutamyl- 3 

-Casein, a Proteolysis Resistant 

Peptide Present in Long Ripened Cheeses 

S. Cattaneo 1 , F. Masotti 1 , I. De Noni 1 , V. Rosi 1 , 1 , M. Brasca 2 , L. Pellegrino 1 

1 

Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente (DeFENS), 

Università degli Studi di Milano, Italia; 

2 

ISPA Istituto di Scienze delle Produzioni Alimentari, Italia 

IDF Cheese Ripening & Technology Symposium - Madison, 21-24 May 2012

Formation of pyro-glutamic acid 

‣ Cyclization of free GLU to pyro-GLU has been highlighted in several ripened cheeses 

‣ When N-terminal GLU or GLN cyclize, N-pyroglutamyl peptides form 

N-pyro-GLU 

N-GLN 

N-GLU 

Significance of the presence of N-pyroglutamyl peptides in food 

resistance to (gastro-intestinal) proteinases 

taste enhancers (umami taste) 


mV 

Background: identification of pyroglutamyl-γ 3 -casein (p-γ3-CN) in 

Grana Padano and Parmigiano Reggiano cheeses 

in cheese, plasmin plays a major role in primary hydrolysis of β-casein (CN) leading to 

formation of γ1-CN f(29-209), γ2-CN f(106-209) and γ3-CN f(108-209) 

cyclization of N-terminal GLU of γ3-CN and formation of p-γ3-CN was observed during 

ripening of Grana Padano and Parmigiano Reggiano cheeses (Masotti et al., 2010) 

128 

126 

124 

122 

120 

118 

116 

114 

112 

110 

108 

Parmigiano 

Reggiano 

Grana 

Padano 

3-CN 

p-3-CN 

bA 1 bA 2 

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 

Minutes 

CZE of Grana Padano (14-month ripened) 

and Parmigiano Reggiano (15-year ripened ) 

100 

95 

90 

85 

80 

75 

70 

65 

60 

55 

50 

45 

40 

35 

30 

25 

20 

15 

10 

5 


11539.4 

10000 10500 11000 11500 12000 

Mass 

HPLC/ESI-MS 

deconvoluted spectrum 


Cheese age (months) 

An application: Grana Padano and Parmigiano Reggiano age 

estimation (Masotti et al., 2010) 

45 

40 

35 

30 

: Grana Padano (n=149) 

: Parmigiano-Reggiano (n=27) 

R 2 = 0.93 

25 

20 

15 

10 

5 

0 

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 

Peak area ratio (100 x pγ 3 -CN/(p-γ 3 -CN + γ 3 -CN) 


Survey of commercial cheeses 

Bitto 

Gouda 

Grana Padano 

Tomme de Savoie 

Fontina 

Cheddar 

Parmigiano 

Reggiano 

Gruyère 

Beaufort 

Comté 

Provolone 

Sbrinz 

Emmentaler 

p-3-CN not detected 

p-3-CN detected 


Studied cheeses: 

Parmigiano Reggiano PDO and Fontina PDO cheeses 

p-γ3-CN detectable 

since 2 months of ripening 

p-γ3-CN not detectable 

in 6-month ripened cheese 

• Raw milk 

• Natural whey starter 

(Lactobacillus helveticus…) 

• Curd cooking up to 54 °C 

• Curd moulding for 48 h 

• Ripening ≥ 12 months 

• pH during ripening: 5.1-5.4 

• Raw milk 

• No starter (Streptococcus thermophilus, 

Enterococcus faecium, E. faecalis…) 

• Curd cooking up to 48 °C 

• Curd pressing 

• Ripening ≥ 3 months 

• pH during ripening: 6.0-6.5 


Aim of the research 

Elucidate some of the (bio)chemical phenomena 

involved in p-γ3-CN occurrence in cheese 

formation of p-γ3-CN in: 

degradation of p-γ3-CN by: 

model systems 

model cheeses 

real cheese 

starter bacteria of Parmigiano 

Reggiano cheese 

bacteria of Fontina cheese 


Peak area of p- 3 -CN (µV*s) 

Understanding formation of p-γ3-CN: model systems 

β-CN + bovine plasmin 

incubation at 40 °C for 20 h 

Incubation at 40 °C up to 

300 h at different pH values 

evaluation of 

p-γ3-CN by CZE 

formation of γ3-CN 

2000 

1800 

1600 

1400 

1200 

1000 

800 

600 

400 

200 

0 

pH=4.6 

pH=5.2 

pH=6.3 

pH=7.0 

0 50 100 150 200 250 300 

Incubation time (h) 


Peak area of p- 3 -CN (µV*s) 

Understanding formation of p-γ3-CN: 

model cheeses 

renneting 

microfiltered milk + 

plasmin at 40 °C for 20 h 

acidification 

800 

700 

600 

500 

400 

300 

200 

100 

0 

curd (pH=6.0) 

curd (pH=4.6) 

incubation at 40 °C 

up to 300 h 

evaluation of 

p-γ3-CN by CZE 

pH=4.6 

pH=6.0 

cheese without added plasmin 

0 50 100 150 200 250 300 



Peak area of p-γ3-CN (V*s) 

Understanding formation of p-γ3-CN: real cheese 

Fontina cheese 

(no detectable p-γ3-CN) 

adjusted to different pH 

values 

1400 

1200 

1000 

pH=4.6 

incubation in 

presence of NaN 3 

at 40 °C up to 300 h 

evaluation of 

p-γ3 –CN by CZE 

800 

600 

400 

200 

0 

0 50 100 150 200 250 300 


pH=5.2 

pH=6.3 

pH=7.0 


Understanding formation of p-γ3-CN: main outcomes 

protein 

protein 

N-terminal 

N-terminal 

1. Cyclization of 3-CN in cheese is a chemical phenomenon 

2. Formation of p-3-CN is pH dependent 


Absorbance at 210 nm (mV) 

Microbial degradation of p-γ3-CN solution 

Solution 

of p-3-CN 

Enterococcus faecium 

Enterococcus faecalis 

(from Fontina cheese) 

Lactobacillus helveticus 

(from natural whey starter of 

Parmigiano Reggiano cheese) 

pH=6.3 

Incubation at 40 °C 

up to 72 h 

pH=5.2 

evaluation of p-3-CN 

degradation by 

HPLC/ESI-MS 

1.8 

1.6 

1.4 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 


0 h 

5 h 

24 h 

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 

Time (min) 



Degradation of p-γ3-CN in cheese by 

bacteria from Fontina cheese 

Parmigiano Reggiano 

cheese (15-year ripened) 

Enterococcus faecium 

Enterococcus faecalis 

( from Fontina cheese) 


up to 72 h at pH=6.3 



HPLC/ESI-MS 


0 h 

24 h 

72 h 

Time (min) 



Degradation of p-γ3-CN in cheese by 

starter bacteria from Parmigiano Reggiano cheese 

Parmigiano Reggiano 

cheese (15-year ripened) 

Lactobacillus helveticus 

(from natural whey starter of 

Parmigiano Reggiano cheese) 


up to 72 h at pH=5.2 



HPLC/ESI-MS 


0 h 

24 h 

72 h 

Time (min) 


Conclusions (1) 

Parmigiano Reggiano cheese 

Fontina cheese 

pH value (5.2-5.4) during 

ripening promotes 3-CN 

cyclization 

whey starter bacteria do not 

proteolyze p-3-CN 

low rate of cyclization of 3- 

CN during ripening (pH value 

6.0-6.5) 

proteolysis of p-3-CN casein 

by Enterococcus spp. 

Presence of p-3-CN 

since 2 months of ripening 

No detectable p-3-CN 

up to 6 months of ripening 


Conclusions (2) 

The level of p-γ3-CN in cheese is the result of two 

conflicting phenomena: 

cyclization of N-terminal GLU of 3-CN 

proteolysis of p-3-CN by bacterial proteinases 

In very long ripened cheeses, cyclization prevails 

over proteolysis and occurrence of p-3-CN could 

be expected also in high pH cheeses 


Thank you!

p-Î³3-CN - IDF Cheese Ripening

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