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Enzymes for Flavour Development in
Dairy Substrates
Presented by:
Blanca Camarasa
Senior Business Manager
Cheese composition
Cheese consists of proteins and fat from milk, usually the milk of
cows, goat, sheep or buffalo
MILK :
Protein
Fat
Lactose
Water
Cow’s milk
3.4%
3.7%
4.8%
88%
Cheese
making
ENDOPEPTIDASE /
EXOPEPTIDASE
LIPASE / ESTERASE
CHEESE :
Protein
Fat
Mineral matters
Water
Cheddar
25%
33%
(Min 50% solids)
Max 39% water
EMC Process
Benefits of Using Enzymes in Dairy
Flavour
Texture
Cost
Savings
Add
Value
Benefits of EMC as an Ingredient
•
Can give high value to out cuts of cheese and out of the spec batches.
•
Used in processed & analogue cheese, snacks, sauces or toppings.
•
EMC used at 1-3%
•
1% EMC can replace 20% natural cheese
•
Gives consistent and flavour
•
Considerable cost savings when EMC
is used instead of mature cheese.
Hydrolysis of Fat with Lipase / Esterase
Triglycerides are the major type of lipid in milk fat.
Approximately 15 to 20 different fatty acids make up 90% of the milk fat. The
major fatty acids in milk fat are straight chain fatty acids that are saturated and have 4
to 18 carbons (4:0, 6:0, 8:0, 10:0, 12:0, 14:0, 16:0, 18:0), monounsaturated fatty acids
(16:1, 18:1), and polyunsaturated fatty acids (18:2, 18:3).
Butyric acid (C4:0)
Triglyceride
(Triacylgycerol)
Oleic acid (C16:1)
Most of the short chain fatty acids are at the position 3 of the triglyceride molecule, and
the longer fatty acids tend to be in the positions 1 and 2.
Hydrolysis of Fat with Lipase / Esterase
Esterase or
Lipase
Diacylgycerol
Triglyceride
(Triacylgycerol)
Free fatty
Acid
(FFA)
Lipases act at the water-oil
interface (high activity toward
water insoluble substrates) and
esterases show highest
activity toward water soluble
substrates.
Commercial lipases / esterases for EMC are generally produced from:
- Animal sources : from bovine and porcine pancreatic tissues (pancreatin and
pancreatic lipase) and the pregastric tissues of calf, lamb and goat kid (PGE).
Animal lipases are highly specific for the liberation of free fatty acids (FFA) from the
sn-1 and sn-3 positions. Butanoic acid (C4:0) is located mainly at the sn-3 position
and is preferentially released by PGE enzymes.
-
Fungal sources (Penicillium spp., Aspergillus spp., Rhizopus spp., Rhizomucor
spp., Mucor spp. or Candida spp.). Lipases derived from microbial sources tend to
have a much wider range of activity and specificity.
Hydrolysis of Fat with Lipase / Esterase:
Impact on Flavour
Impact on flavour in cheese:
-
FFA released upon lipolysis contribute directly to cheese flavour, especially short and
medium-chain FFA. Long-chain FFA (> C12:0) are considered to play a minor role in
cheese flavour due to their high perception thresholds.
-
Short and intermediate-chain FFAs have a considerably lower perception thresholds and
each gives a characteristic flavour note:
Impact on flavour in EMC:
- Short chain FFA (C4:0-C6:0) : rancid, pungent, cheesy, sharp, piquant (Blue cheese /
Italian-type EMC)
- Medium chain FFA (C8:0-C14:0) : Buttery
- Long chain FFA (>C14:0): Soapy
It is important to find the right balance between short / medium / long
chain FFAs to produce the desired flavour.
Hydrolysis of Fat with
Lipase/Esterase: Analysis of FFA
FFAs in EMC or cheese can be analysed by Gas Chromatography (GC)
to generate free fatty acid profiles (FFAP)
1/ Extraction lipids
from EMC or cheese
2/ Purification / Isolation
of the FFA
3/ Gas Chromatography
(GC)
4/ Free Fatty Acid
Profile (FFAP)
L187P
Hydrolysis of Fat with Lipase: FFAPs
L187P “Cheddar”
L338P “Blue notes”
L801MDP - Microbial
Pancreatin lipase
FlavorproTM C (Microbial
calf PGE ) - very high level
of C4:0 and C6:0,
“Piccante” flavour
Catabolism of FFA : generation of other
flavour compounds
Lactones identified in blue
cheese, Cheddar, ParmagianoReggiano
Methyl ketones (due to the action
of mould lipase) - blue cheese
38 esters were identified in
Parmagiano-Reggiano cheese
Ethyl ester – fruity notes
Hydrolysis of Proteins by Proteases
Proteins (polypeptides) are made of amino acids arranged in a linear
chain and folded into a globular form. The amino acids are joined
together by the peptides bond between the carboxyl and amino groups
of adjacent amino acid residues.
Peptide bond
N-terminus
Amino acid
C-terminus
Protein (>100 amino acids)
Proteases (endo- or exopeptidases) are proteolytic enzymes that catalyze the
hydrolysis of peptide linkages:
Protease
Hydrolysis of Proteins by Proteases
Endopeptidase (also called endo-protease) catalyzes the cleavage of peptide
bonds within a protein.
●
Exopeptidase catalyzes the cleavage of the terminal (last) or next to last peptide
bond from a protein. There are 2 types of exopeptidases:
●
- Carboxypeptidase catalyses the removal of one or two amino acid residues
from the C-terminus of the protein.
- Aminopeptidase catalyses the removal of one or two single amino acid residue
from the N-terminus of the protein.
Aminopeptidase
Endopeptidase
Carboxypeptidase
Hydrolysis of Proteins by Proteases : Effect
on Flavour
Commercial proteases are generally derived from lactic acid bacteria (LAB) or from
Aspergillus oryzae. Acid, neutral or basic (alkaline) proteases are available.
In cheese, small peptides and amino acids are hypothesised to be mainly responsible
for the basic taste of cheese (contributing to the background flavour of cheese).
Amino acids and hydrophilic peptides contribute to savoury flavour in
cheese.
In EMC production, the action of proteases will:
- Develop a “savoury” flavour.
- Create some bitterness (that can be controlled).
- Decrease the viscosity (change of texture by breakdown of the
casein network).
Hydrolysis of Proteins by Proteases : Effect
on Flavour
Savoury flavour comes from:
- Hydrophilic peptides (increase of flavour due to increase of solubility): medium
and small peptides contribute to a brothy background flavour in many cheese
varieties.
- Amino acids which contribute directly to cheese flavour; as some amino acids
taste sweet (e.g. Gly, Ser, Thr, Ala, Pro), sour (e.g. His, Glu, Asp), or bitter (e.g.
Arg, Met, Val, Leu, Phe).
Example: The principal amino acids in Cheddar cheese are Glu, Leu, Arg, Lys, Phe,
and Ser.
Bitterness is caused by the accumulation of short hydrophobic peptides.
Although the bitter taste is considered a normal component of cheese taste, excessive
bitterness is not desirable for cheese or EMC.
Bitterness can be controlled by the use of debittering proteases : exopeptidases
Hydrolysis of Proteins by Proteases:
Glutamic Acid Release
Free glutamic acid is responsible
for umami flavour in cheese
(Parmesan contains 1680 mg of
glutamic acid for 100g of cheese).
Flavorpro™ Umami is an
exopeptidase with endopeptidase and
glutaminase side activities releasing
high level of glutamic acid (Glu) in
EMC applications.
Umami heightens the flavour
profile which helps increase the
apparent saltiness in EMC, as
confirmed by external sensory
analysis. EMC made with Flavorpro™
Umami can therefore be used for salt
reduction in processed cheese
applications.
Hydrolysis of Proteins by Proteases:
Catabolism of amino acids
Aromatic amino acids (phenylalanine, tyrosine, tryptophan), branched-chain
amino acids (leucine, isoleucine and valine) and methionine are the major
precursors of other important aroma and flavour compounds.
Branched-chain amino acids (Leu, Ile,
Val) are converted into:
-Specific aldehydes with malty flavours,
- Alcohols with fruity and alcoholic
flavours
- Acids with sweaty and also sour,
rancid, rotten, fruity and buttery flavours
(depending on which amino acid, Leu, Ile
or Val, is catabolised).
Sulfur compounds (characteristic
components of surface ripened
cheeses – ie camembert, brie)
Conclusion
EMC cheese
LIPASES
Fat
(triglycerides)
Protein
(caseins)
Free fatty acids
CHEESE FLAVOUR
Can be varied depending on the lipase(s) used
(specificity of FFA carbon chain length)
- Short chain FFAs (C4:0-C6:0) : cheesy, sharp, piquant
- Medium chain FFAs (C8:0-C14:0) : Buttery
- Long chain FFAs (>C14:0): Soapy
PROTEASES
- Peptides
- Amino acids
SAVOURY NOTES
Protease(s) specificity (endoand/or exopeptidase) and
dosage will influence savoury
taste, bitterness and EMC
texture.
Lipase(s) and/or protease(s) to be used will depend on the flavour profile needed
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