National Dairy Research Institute, NDRI, Karnal

Course Title: Advances in chemistry of milk processing (DC-721) Date: 05.02.2011

Course Teacher: Dr. Sumit Arora Place: Karnal
Name of the student: Ankit Goyal
Class: Ph.D, I year, II sem (D.C.)

Effects of high pressure processing on milk proteins

Introduction
The application of high pressure, rather than heat, to food enables destruction of microorganisms without causing
significant changes to the colour, flavour and nutritional attributes of the food. In this way, the food can be preserved
in a safe state but still have many of the attributes of a fresh product. In addition, high pressure can cause rheological
changes in food which result in beneficial sensory and structural effects. The potential of high-pressure treatment of
food was first recognized by Hite in 1899 who investigated it as an alternative method for pasteurization of milk.
However, it attracted little attention until it was `rediscovered' in the 1980s. The first foods produced commercially by
this technology appeared on the market in Japan in 1990. They included yoghurts, fruit jellies, salad dressings and
fruit sauces. Several other pressure-treated foods are now manufactured, including pressure- pasteurized' milk, but the
total amount remains relatively small.

High Hydrostatic Pressure (HHP)

According to Pascal’s law, pressure acts instantly, isostatically and homogenously, independently of the size and
shape of the material. In high pressure treatment of foods, pressures between 100 and 1,000 MPa are used. This is
higher than pressures present in deep sea. During HHP treatment the food packed in a flexible packaging material is
put in a high pressure cylindrical vessel where it is surrounded by a non-compressible pressure-transmitting medium,
usually water. The transmitting medium is pressurized up to the treatment pressure. This pressure is kept constant
from a few minutes to multiples of times 10 minutes.
It has been established for both solid and liquid foods with moderate or high water content that the pressure is equal at
each point of the treated product to the pressure of the transmitting medium.

Purpose of HHP
In the food industry the main field of application of HHP is food preservation. Food spoilage is very often caused
by microorganisms and biochemical processes catalyzed by enzymes. With HHP a great part of microorganisms can
be destroyed and most of the enzymes can be inactivated. Using HHP treatment, undesirable changes and thermal
degradation of heat-sensitive food components can be avoided, a major advantage. The treatment is effective at
ambient or moderate temperatures. Tests show that this treatment affects only the non-covalent bonds (i. e. hydrogen,
ionic and hydrophobic) bonds, and impacts taste, colour and nutritional value of foods to a negligible degree. Thermal
treatment, on the other hand, changes the covalent bonds and significant changes are also observed in food
components. Depending on the kind of food the effects can be beneficial or undesirable (colour, texture, structure
etc.). In general, components with low molecular weight remain intact while macromolecules (proteins, complex
carbohydrates) undergo changes (Datta & Deeth, 1999).
HHP also affects biochemical reactions. Pressure reduces the size of the molecules and promotes bond formation
between side-chains. Protein molecules are denatured under high pressure. This is a complex phenomenon: it depends
on the structure of the proteins, the extent of the pressure, the temperature and the pH. The effect of HHP on
microorganisms depends on the composition of the foodstuffs and the physiological condition of the microorganisms.
HHP also affects the morphology of microorganisms. Survival of the microorganisms depends on the extent of
pressure, holding time and temperature, composition of the food and the condition and growth phase of microbes
(Patterson et al., 1995). Pressures between 300-600 MPa inactivate yeasts, moulds and most of the vegetative bacteria.
Bacterial spores can be destroyed substantially only with pressures higher than 1000 MPa. Pressures between 50 and
300 MPa may even stimulate spore germination.
Schematic diagram of basic equipment design
for HHP of foods

It consists of four parts:

1. A high pressure vessel and its closure

2. A pressure generating system

3. A temperature control device

4. A material handling system

Hinrichs et al., (1996)

Product (liquid or solid) is packed in poly-ethylene bags. Milk can be packed in PE bags or poly-
propylene tubes. This packaged product is put in vessel. Vessel is filled with non-compressible fluid (water or
ethanol). And then pressure is applied for 2-60 min depending upon the product, desired safety and temperature.
About 3°C temp is increased for each 100 MPa; so to maintain the temperature, temperature controllers (water flow or
exchangers) are used.
 Principle of HHP

Under pressure biomolecules obey the “Le-Chatelier” principle i.e. whenever stress is applied to a system
in equilibrium, the system will react so as to counteract the applied stress; thus, reactions that result in reduced
volume will be triggered under HHP. Such reactions may result in inactivation of microorganisms or enzymes and in
textural changes in foods.

Effects on bacterial flora and keeping quality of milk under HHP

1. Studies show that beneficial effects of HPP of foods are evident only when applied pressure exceeds 400 MPa.

2. Vegetative bacterial cells are inactivated by pressures 400-600 MPa.

3. About >1000 MPa pressure is required to inactivate bacterial spores.

4. Gram positive are more resistant to HP than gram negatives- due to presence of teichoic acid in former. Teichoic
acid is polysaccharide of bacterial cell wall. It is present only in gram positive bacteria and absent in gram negative
bacteria. Teichoic acid provides rigidity to cell wall.

“It has been observed that to achieve the shelf-life of thermally pasteurized milk of 10 days at 10°C, a pressure
treatment of at least 400MPa for 15 min or 500MPa for 3 min is required”.
Advantages of HPP

1. Thermal degradation of heat sensitive foods can be avoided.

2. High retention of colour, aroma and nutritional value.

3. No re-contamination as treatment is given after final packaging of product.

4. Environmental friendly as no chemicals added.

5. Positive consumer acceptance

6. Operates at room temperature

Disadvantages

1. Very expensive. Capital cost is very high.

2. Non-continuous
3. Additional heat treatment is required to kill spores.

Effects of High Hydrostatic Pressure (HHP) on Milk Proteins

 In their native state, proteins are stabilized by covalent bonds (including disulphide bridges) plus
electrostatic interactions (ion pairs, polar groups), hydrogen bridges and hydrophobic interactions. Covalent bonds
are almost unaffected by HHP, at least at relatively low temperatures (0–40°C), and so the primary structure of
proteins remains intact during HHP treatment. High pressure affects:
a.) the quaternary structure (e.g. through hydrophobic interactions)

b.) the tertiary structure (hydrophobic and hydrogen bonding)

c.) the secondary structure ( H- and electrostatic interactions)

Stabilising hydrogen bonds are enhanced at low pressures and ruptured only at very high pressures. Significant
changes to the tertiary structure of proteins, which is maintained chiefly by hydrophobic and ionic interactions, are
observed at >200 MPa. Multimeric proteins, held together by non-covalent bonds, dissociate at relatively low
pressures (~150 MPa), thereby disrupting quaternary structures.

Sensitivity of different bonds to HHP

Hydrobhobic > electrostatic bonds > H- bonds > covalent bonds

Effects on beta-lactglobulin under HHP

ß- Lg is the most sensitive to HHP. Beta- lg has only 2 disulphide linkages and 1 free –SH group. So it is
less rigid as compared to alpha-la which has 4 disulphide bonds. At lower pressure (>100 MPa), unfolding of beta-lg
starts and free –SH group is exposed; which may interact with k- casein or other unfolded beta-lg molecules. It results
in increase in size of casein micelles and a small extent of aggregation among beta-lg molecules.

During storage, re-naturation occurs within 1-2 days at 20-40°C. At lower temperature (5°C) re-
association does not take place, because at lower temperature, mobility (energy) of atoms is too low to form
hydrophobic as well as ionic bonds. And thus at low temperature, strength of hydrophobic interactions is very low.

 At 400 MPa, denaturation of beta-Lg reaches 70-80% at 20°C.

 A synergistic effect of temperature on denaturation of beta-Lg has been reported.

 About 100% denaturation was observed at 300 MPa at 60°C or at 400 MPa at 40°C.

0.1 MPa 100 MPa 450 MPa 800 MPa

Mainly native monomers non-native monomers polymers

 and dimmers
Considine et al., (2007)

 At less than 100 MPa, beta-lg remains in native monomer form.

 Between 100 to 450 MPa, it gets unfold and forms dimmers via di-sulphide linkages. This conversion is reversible
during storage.
 Between 450-800 MPa it forms polymers via di-sulphide bonds and this is irreversible.

Effects of HHP on α-lactalbumin

 α-la is more resistant to denaturation under pressure because it has 4 di-sulphide linkages (beta-lg has 2).
 Denaturation starts only at >400 MPa.
 Under HHP of 400-800 MPa, no transformation of monomers into disulphide-bonded aggregates is observed
(Patel et al., 2004), because it has no free –SH group.
 At ~1000 MPa small aggregates are formed due to bonding between Cys 6- Cys 120, which is more sensitive to
cleavage due to its environment (Huppertz et al., 2004a)

Effect of HHP on bovine serum albumin (BSA) & Immunoglobulins (Igs)

BSA

 BSA is single polypeptide of 582 amino acids, having 17 disulphide bridges and one free thiol group, Cys 34.
 Structure is composed of 76% helix, 10% turns and 23% extended chain and no beta (ß) sheets.
 BSA is very resistant to pressure up to 400 MPa probably due to large number of disulphide linkages.
 Very little denaturation occurs above 400 MPa.

Igs

 Immunoglobulins are more resistant to pressures up to 300 MPa.

Lectoferrin and lysozyme

 Lf and lysozyme in milk are resistant to HP. More reports are not available on it.

Susceptibility to high pressure of whey proteins

WHEY PROTEINS SUSUSCEPTIBILITY TO HP

SUSUSCEPTIBILITY TO
 ININDUCED DENATURATION THTHERMAL DENATURATION
ß- Lactglobulin +++++ ++
Immunoglobulins +++ +++++
α- lactalbumin ++ +
BSA ++ +++

Effects of HHP on caseins

 Casein micelles are generally unaffected at low HHP treatments, as casein has very little secondary and 3D
structures. Casein’s native form is primary structure having covalent bonds (peptide and di-sulphide bonds). Because
HHP does not affect the covalent bonds, so doesn’t affect the casein micelles under <300 MPa.
 But at higher pressures, defragmentation occurs, in which micelles are broken up into more soluble components
like αs-1, αs-2, ß- and k-caseins.
 Defragmentation occurs due to 2 following reasons:
1. Solublization of colloidal calcium phosphate (CCP), so stability of micelles are disrupted.
2. Disruption of hydrophobic and electrostatic interactions

Effect of HHP on casein micelle size

Pressure (MPa) Temperature Effects on casein Change

micelles
≤ 200 MPa 20-40°C No effect -
250 MPa 40°C ~30% increase in size of Reversible during storage
micelle
>300 MPa 40°C ~50% decrease in size of Irreversible during storage
micelle
Huppertz et al., (2004c)

 Increase in size at 250 MPa is due to interaction of denatured beta-lg with k-casein micelle.

 At >300 MPa reduction in size is due to fragmentation of casein micelles.

Effect of HHP on hydration of casein

Hydration of micelles is increased due to following reasons:

o Due to interaction of denatured beta- lactglobulin with k-casein, net negative charge on casein/ or casein
components increases; and thus hydration or solubility increases.
o By disruption of casein micelles into αs2, αs1, ß and k-caseins.

The order of dissociation of casein variants in bovine milk under HHP-

k > ß > αs-1 > αs-2

Order of dissociation depends upon the phosphate content of casein variants. More phosphate residues, more is the
stability or less is the dissociation. k = 1, ß = 5, αs1 = 8, αs2= 11 (phosphate residues)

Effect in pH of milk by HHP

o Slight change in pH of milk under HHP.

o Under 200-600 MPa , pH increased from 6.38 to 6.48; only by 1 unit.

Effect on turbidity, L value & transmittance of milk by HHP

• Turbidity (cloudiness) decreases with increase in pressure (Huppertz et al., 2004b). As pressure is increased
(>300MPa), casein micelles are disrupted and fragmented into small particles. Scattering of light depend upon the
size and is directly proportional to it. On fragmentation of micelles, scattering is reduced, thus turbidity is also
reduced.

• At lower pressure (200-300 MPa), turbidity changes are reversible during storage.

• Turbidity can be measured by nephlometer in terms of NTU (nephlometric turbidity units).

• Luminance (L) value of milk decreases with HP treatment. L=0 indicates black (toward darkness) and L=100
denotes white (lightness). Raw milk is more white than HP induced milk. At 600 MPa for 30 min, L value is
decreased from ~78 to ~42 (Huppertz et al., 2004b).

• Light transmittance of milk is increased under HP treatment. As turbidity is decreased, transmittance increases.

•
Raw milk Milk at >300 MPa for 5 min
Conclusion
 Under HHP, beta-lg is unfolded and forms dimers, polymers and aggregates with k-casein depending upon the
extent of pressure and temperature. Solublization of CCP occurs under HP resulting fragmentation of casein micelles.
Disruption of hydrophobic, ionic and H-bonds results the conversion of 2, 3 & 4° structures in to primary structure;
which could be reversible or irreversible depending upon the intensity of pressure.

References

Considine, T., Patel, H. A., Singh, H. & Creamer, L. K. (in press). Influence of binding of conjugated linoleic acid
and myristic acid on the heat-and pressureinduced unfolding and aggregation of β-lactoglobulin B. Food Chemistry.

Datta, N. and Deeth, H. C. (1999). High pressure processing of milk and dairy products. The Australian Journal of
Dairy Technolog. 54, 41-48.

Hinrichs, J., Rademacher, B. and Kessler, H. G. (1996) Food processing of milk products with ultra high pressure. In:
Heat Treatments and Alternative Methods, International Dairy Federation Document no. 9602, pp. 185- 201.
Brussels: International Dairy Federation.

Huppertz, T., Fox, P. F., & Kelly, A. L. (2004a). High pressure treatment of bovine milk: Effects on casein micelles
and whey proteins. Journal of Dairy Research, 71, 97−106.

Huppertz, T., Fox, P. F., & Kelly, A. L. (2004b). Properties of casein micelles in high-pressure treated bovine milk.
Food Chemistry, 87, 103−110.

Huppertz, T., Fox, P. F., & Kelly, A. L. (2004c). Dissociation of caseins in high pressure-treated bovine milk.
International Dairy Journal, 14, 675−680.

Jegouic, M., Grinberg, V. Y., Guingant, A., & Haertle, T. (1996). Thiol-induced oligomerization of α-lactalbumin at
high pressure. Journal of Protein Chemistry, 15, 501−509.