UHT Milk Processing and Effect of Plasmin

Activity on Shelf Life: A Review

Rupesh S. Chavan, Shraddha Rupesh Chavan, Chandrashekar D. Khedkar, and Atanu H. Jana

Abstract: The demand for ultra-high-temperature (UHT) processed and aseptically packaged milk is increasing world-
wide. A rise of 47% from 187 billion in 2008 to 265 billon in 2013 in pack numbers is expected. Selection of UHT
and aseptic packaging systems reflect customer preferences and the processes are designed to ensure commercial sterility
and acceptable sensory attributes throughout shelf life. Advantages of UHT processing include extended shelf life, lower
energy costs, and the elimination of required refrigeration during storage and distribution. Desirable changes taking place
during UHT processing of milk such as destruction of microorganisms and inactivation of enzymes occur, while unde-
sirable effects such as browning, loss of nutrients, sedimentation, fat separation, cooked flavor also take place. Gelation
of UHT milk during storage (age gelation) is a major factor limiting its shelf life. Significant factors that influence the
onset of gelation include the nature of the heat treatment, proteolysis during storage, milk composition and quality,
seasonal milk production factors, and storage temperature. This review is focused on the types of age gelation and the
effect of plasmin activity on enzymatic gelation in UHT milk during a prolonged storage period. Measuring enzyme
activity is a major concern to commercial producers, and many techniques, such as enzyme-linked immunosorbent
assay, spectrophotometery, high-performance liquid chromatography, and so on, are available. Extension of shelf life of
UHT milk can be achieved by deactivation of enzymes, by deploying low-temperature inactivation at 55 ◦ C for 60 min,
innovative steam injection heating, membrane processing, and high-pressure treatments.

History of Ultra-High-Temperature (UHT) Milk
Consumers demand foods that are as fresh as possible with good
sensory properties (especially taste), additionally being safe and
having a substantial shelf life, yet without application of addi-
tives. Because of its high nutritional value, milk is an excellent
medium for microbiological growth. Consequently, fresh milk
necessitates a heat treatment in order to guarantee a safe and shelf-
stable product. The most commonly applied technique to achieve
this is heat treatment. The first system consisting of indirect heat-
ing with continuous flow (125 ◦ C for 6 min) was manufactured
in 1893. Patented in 1912, the continuous-flow, direct heating
method mixed steam with milk to achieve temperatures of 130 to
140 ◦ C. Development of the UHT process was hindered due
to possible contamination without commercial aseptic systems. In
1953, UHT milk was filled aseptically into cans after heat treatment
with an Uperiser® processor followed by tetrahedral paperboard
cartons in 1961 (Datta and Deeth 2007). The development of
aseptic processing in the United States started through the efforts
of C. Olin Ball, and hot-cool-fill process was commercialized in
MS 20110127 Submitted 1/28/2011, Accepted 5/5/2011. Author Chavan is with
National Institute of Food Technology Entrepreneurship and Management, Kundli-
131028, Haryana, India. Author S. R. Chavan is with Department of Microbiology,
BACA College, AAU, Anand, Gujarat 388110, India. Author Khedkar is with
College of Dairy Technology, Warud, Pusad, Maharashtra 452004, India. Author
Jana is with S.M.C. College of Dairy Science, Anand, Gujarat 388110, India.
Direct inquiries to author Chavan (E-mail: rschavanb_tech@rediffmail.com).
1938 for a chocolate milk beverage. In 1942, the Avoset process
was used to package a cream product by utilizing a continuous hot
air system and ultraviolet (UV) lamps in the filling and sealing area.
In 1948, the Dole aseptic process developed by William McKinley
Martin was used for pea soup and sterilized milk. Real Fresh, Inc.
became the 2nd dairy in the United States in 1952 to use UHT
and aseptic packaging (AP), and in 1981, it was the forerunner in
using hydrogen peroxide (H2 O2 ) to sterilize packaging material
(David and others 1996).
Market Status of UHT Milk
Consumption trends for aseptic dairy foods have shown in-
creased demand for aseptic dairy foods and the global market is
forecast to climb steeply to 2013 both in terms of pack numbers
and volume. An industry study entitled “Global Aseptic Packag-
ing” by Zenith Intl. and Warrick Research, estimated a rise of 47%
from 187 billion in 2008 to 265 billion by 2013 in pack numbers.
Similarly, volumes of aseptic packs are also likely to see buoyant
growth, rising 31% from 86 billion L in 2008 to 113 billion L in
2013. In 2008, cartons dominated the market, accounting for al-
most 75% of the total volume but lost some of the market share to
polyethylene terephthalate bottles and pouches. In terms of num-
bers of white milk packs, the Asian market already accounts for
56% of world use and would rise to nearly 70% by 2013. Volumes
have grown annually by over 6% since 2003, with Asia achieving
the fastest rise at over 13% a year. In 2008, white milk accounted
for around 45% of aseptic package use, with beverage volume


c 2011 Institute of Food Technologists®
doi: 10.1111/j.1541-4337.2011.00157.x Vol. 10, 2011 r Comprehensive Reviews in Food Science and Food Safety 251
UHT milk processing and effect . . .
reaching over 40% (Harrington 2009). The category is growing at
a steady annual rate of 20% in India.
Products are manufactured with UHT and aseptic processing in
over 60 countries (Burton 1988). The market share of UHT milk
consumed varies considerably by country: Australia 9%, France
88%, Spain 83%, Germany 63%, Italy 55%, and the United King-
dom 5% to 13% (Harrington 2009). Based on sensory work,
Oupadissakoon (2007) reported butyric acid, sour aromatics, and
lack of freshness as negative attributes with UHT milk. UHT milk
quality depends more on the manufacturing process than country
of origin or fat content. Customer acceptability of UHT milk is
positively correlated to consumption habits that include UHT milk
(Oupadissakoon 2007). Aseptic processing has great potential to
increase through dairy consumption in tropical countries, as there
is a low milk consumption trend due to high temperatures and
limited refrigerated distribution (Goff 2008). Hedrick and others
(1981) predicted UHT milk with flavor attributes comparable to
pasteurized milk would reduce energy costs, since the shelf-stable
milk would not require refrigeration throughout distribution. The
growth of this industry is limited by government regulations, filler
speeds, and packaging costs (David and others 1996).
UHT Milk Processing Principles
Heat treatment in the production of long life products is called
“sterilization.” In such processes, the treated product is exposed to
such powerful heat treatment that the relevant microorganisms and
most of the enzymes are inactivated, and the processed product
is given excellent keeping qualities and can be stored for several
months under ambient conditions.
UHT processing uses continuous flow of milk, which renders
less chemical change in comparison to retort processing (Datta
and Deeth 2007). Product characteristics, such as pH, water ac-
tivity, viscosity, composition, and dissolved oxygen, indicate the
processing conditions necessary to achieve commercial sterility.
The selection criteria of UHT and AP systems reflect customer
preferences. The production processes are designed to ensure com-
mercial sterility and acceptable sensory attributes throughout shelf
life.
To compare the various effects of heat treatments, different
values are calculated:
Q10 value
The Q10 value has been introduced as an expression of this
increase in speed of a reaction. It states how many times the speed
of a reaction increases if the temperature of the system is raised by
10 ◦ C. The Q10 value for flavor changes—and for most chemical
reactions—is around 2 to 3, which means if the temperature of a
system is raised by 10 ◦ C, the speed of chemical reactions doubles
or triples. Q10 values can also be determined for the killing of
bacterial spores and is normally found in the range of 8 to 30
(Kessler 1981). The variation is so wide because different kinds
of bacterial spores react differently as the temperature increases.
The changes in chemical properties and spore destruction by the
influence of increased temperature are shown in Figure 1.
F 0 value
For the microbiological effect, F 0 value is already used in clas-
sical canned sterilization technology and is defined as the number
of minutes at 121.1 ◦ C (250 ◦ F) to which the process is equivalent
and is calculated according the following formula:
◦
F0 = t /60 × 10(T−121.1 C)/Z
, vate bacterial endospores and limit chemical changes with minimal
252 Comprehensive Reviews in Food Science and Food Safety r Vol. 10, 2011
Figure 1–Curves representing the speed of changes in chemical
properties and of spore destruction with increasing temperature. (Source:
Gösta 2003.)
where
t = sterilization time in seconds at T ◦ C
T = sterilization temperature in ◦ C
z = a value expressing the increase in temperature to obtain the
same lethal effect in 1 of 10 of the time. The value varies with the
origin of the spores (10 to 10, 8 ◦ C) and can generally be set as
10 ◦ C.
F 0 = 1 after the product is heated at 121.1 ◦ C for 1 min. To
obtain commercially sterile milk from good quality raw milk, a F 0
value of minimum 5 to 6 is required.
B∗ and C∗ values
The effective working range of UHT treatments is also defined
in some countries by reference to 2 parameters: bacteriological
effect: B∗ (known as B star) and chemical effect: C∗ (known as
C star). B∗ is based on the assumption that commercial sterility
is achieved at 135 ◦ C for 10.1 s with a corresponding z-value
of 10.5 ◦ C. This reference process is given a B∗ value of 1.0,
representing a reduction of thermophilic spore count of 109 per
unit. The chemical effects can be assessed in similar ways to those
used for the sterilization performance (Figure 2). The same data
for the time-temperature performance are used. The C∗ value
is based on the conditions for 3% destruction of thiamine per
unit. This is equivalent to 135 ◦ C for 30.5 s with a z-value of
31.4 ◦ C (Horak 1980; Kessler 1981; Kessler and Horak 1981).
A UHT process operates satisfactorily with regard to the keep-
ing quality of the product when the conditions of B∗ > 1 and
C∗ < 1 are fulfilled.
The U.S. FDA accepts F 0 values for thermal processes calcu-
lated only from the time and temperature of the product in the
holding tube (David and others 1996). The D-value is defined
as the required time to decrease microorganism numbers 10-fold
at a given temperature (Singh 2007). The process filing and sup-
porting documentation (trial run data, critical factors, equipment
sterilization, quality control procedures, and operational proce-
dures) are submitted to FDA for approval of a scheduled process
(David and others 1996). Ideal time-temperature profiles inacti-

c 2011 Institute of Food Technologists®
UHT milk processing and effect . . .
Figure 2–Bacteriological killing effects and chemical changes in heat-treated milk. (Source: Kessler 1981.)
decrease in nutritional and sensory quality (Datta and others 2002).
The major challenge in UHT milk production is sufficient heat
treatment with minimal flavor change. Direct heating imparts less
flavor change but requires more energy in comparison to indirect
heating. Total microbial lethality at constant time and tempera-
ture varies between direct and indirect heating systems (Westhoff
1981). The residence time distribution is the time range for a fluid
product such as milk to enter and exit the holding system (Singh
2007). Flow through the heating system is controlled by timing or
metering pumps. The residence time is determined by hold tube
volume, flow rate, and flow rate attributes (viscosity) of specific
products. Positive reactions in the hold tube include destruction
of bacteria, inactivation of enzymes, and hydration of thickeners.
Negative reactions include development of off-flavor, initiation of
off-color, and destruction of vitamins (David and others 1996).
Physicochemical Changes Occurring in UHT Milk
One of the principal goals of milk preservation methods by
its short time treatment at increased temperatures is to obtain a
desired degree of destruction of microorganisms and inactivation
of enzymes, with, at the same time, introducing the least possi-
ble undesired changes of physicochemical and sensory properties,
as well as, what is even more important, preservation of its nu-
tritional value (Jovanka and others 2008). A study conducted by
Korel and Balaban (2002) suggested that odor changes in milk
samples inoculated with Pseudomonas fluorescens or Bacillus coagu-
lans could be detected by an electronic nose. The odor changes
correlated with microbial and sensory data. Maillard browning, as
a function of heat treatment given to milk, can be detected by
front-face fluorescence spectroscopy and hydroxy methyl furfural
(HMF) analysis (Schamberger and Labuza 2006). Elliott and oth-
ers (2003) concluded that lactulose is the most reliable index of
heat treatment, since it is not affected by milk storage before or
after UHT processing. Heat treatment involves 2 reactions: type 1
reactions involve the denaturation, degradation, and inactivation
of whey proteins, enzymes, and vitamins. Type 2 reactions involve

c 2011 Institute of Food Technologists®
the formation of lactulose, HMF, furosine, and others, which are
not detected in the raw milk (Morales and others 2000). Singh
(2004) stated that the heat stability of the milk is its ability to
undergo high heat treatment without coagulating or gelling. So-
lutions to improve heat stability include preheating the product
in the UHT processor, adjusting pH to the ideal heat stability
maximum, and adding phosphate, buttermilk, or phospholipids.
Chemical changes
Direct heat processing imparts less adverse chemical changes
compared to indirect heat processing (Elliott and others 2003). In
an indirect continuous-flow coiled tube system, the process hold-
ing time accounted for >80%, the process heating time <10%,
and the cooling phase <2% of the accumulated chemical changes
(Labropoulos and Varzakas 2008). Hsu (1970) reported that dairy
foods undergo the following chemical changes to varying degrees:
flavor, acidity (decreases following direct UHT process), enzyme
inactivation, and vitamin decomposition. The heated flavor after
UHT processing is due to sulfhydryl (S–H) groups, which oxidize
5 to 10 d after processing. The oxidation then gradually reduces
the cooked flavor (Hsu 1970). Heating has little effect on milk
salts with 2 exceptions, carbonates and calcium phosphates. Most
of the potential carbonate occurs as CO2 , which is lost on heat-
ing, with a consequent increase in pH. Among the salts of milk,
calcium phosphate is unique in that its solubility decreases with
increasing temperature. On heating, soluble calcium phosphate
precipitates onto the casein micelles, with a concomitant decrease
in the concentration of calcium ions and pH. Milk oxidative ran-
cidity is the reaction of oxygen on milkfat components resulting
in short-chain aldehyde and ketone volatiles (Solano-Lopez and
others 2005). Enzyme inactivation is a positive chemical change
of UHT processing. Fat-soluble vitamins are affected minimally
by heat, whereas water-soluble vitamins can be destroyed par-
tially in UHT processing. A significant reduction in vitamin B1
(thiamin), B2 (riboflavin), B3 (niacin), B6 , B12 , and folate has
been reported under the influence of different milk processing
Vol. 10, 2011 r Comprehensive Reviews in Food Science and Food Safety 253
UHT milk processing and effect . . .
treatments (Asadullah and others 2010). UHT processing reduces
B vitamins by 10%, folic acid by 15%, and vitamin C by 25%. The
nutritional value of proteins, minerals, and fats is affected mini-
mally by UHT processing (Holdsworth 1992) and is correlated to
the storage temperatures, initial oxygen content, and packaging
choice (Dunkley and Stevenson 1987).
Physical changes
The unwanted physical attributes associated with UHT milk are
brown color, sedimentation, protein destabilization, and fat sepa-
ration (Hsu 1970). Direct heating after homogenization appears to
cause reagglomeration of the small fat globules with the formation
of a solid fat layer during storage. To prevent this fat separation, ho-
mogenization in direct UHT plants takes place in the downstream
position (after the final heating step and vacuum cooling). The
production of free fatty acids during storage is more noticeable in
milk with higher fat content and is greater in milk produced in di-
rect rather than indirect systems (Schmidt and Renner 1978). Milk
proteins change more than any other milk constituent due to UHT
processing that contributes to loss of color, flavor, and nutrition,
as well as gelation and sedimentation. Denatured whey proteins
form complexes with other whey proteins, caseins, and fat globules
(Dunkley and Stevenson 1987). The amount of β-lactoglobulin
associated with the casein micelle increases with the heating time
and the trend is similar for α-lactalbumin but to a lesser extent
(Elfagm and Wheelock 1978). α-Lactalbumin can interact with
κ-casein only in the presence of β-lactoglobulin, possibly through
the initial formation of α-lactalbumin/β-lactoglobulin aggregates,
which then interact with κ-casein (Elfagm and Wheelock 1978).
If milk is heat-treated instantaneously (by direct heating), all whey
protein begins unfolding at the same time and this gives a greater
opportunity for the unfolded of monomers to aggregate and con-
sequently the attachment to the casein micelles will be less efficient
(Oldfield and others 1998). The association with casein micelles
is pH-dependent and decreases as the pH increases in the range
of pH 6.3 to 7.3. The micellar size shows a similar dependence in
range of pH 6.5 to 6.7 (Skelte and Yuming 2003). Thermal in-
activation of a transglutaminase (TG) inhibitor provides improved
cross-linking of casein micelles, resulting in improved product tex-
ture (Bonisch and others 2004).
The color of the UHT milk, that is its intensity, basically repre-
sents reflection of physicochemical changes in the product. Dairy
foods with greater quantities of reducing sugars have more issues
with browning and it increases with process severity and storage
temperature (Dunkley and Stevenson 1987). These reactions are
known as Maillards reactions, and consist of a series of changes
whose consequence is the formation of brown-colored pigments,
such as pyralysins and melanoidins, polymers such as lactulose-
lysine or fructose-lysine, as well as low molecular weight acids.
Lactulose, a molecule derived from lactose isomerization, is a well-
known indicator for assessing the severity of the thermal treatment
applied to milk, as it is minimally affected by storage conditions.
Actually, the proposed limit (600 mg/L) seems to be effective for
indirect UHT milk, while in the case of direct UHT milk, it
does not give clear evidence of unjustified overprocessing. When
combined with furosine, lactulose also allows assessing authenticity
of this type of fluid milk (Cattaneo and others 2008). Sediment
is more prevalent in products that are more severely processed,
that have a targeted pH of <6.6, and that have undergone direct
instead for indirect UHT processing (Holdsworth 1992). Other
factors affecting sedimentation include homogenization pressure
that is used to control fat separation, time, and temperature pro-
254 Comprehensive Reviews in Food Science and Food Safety r Vol. 10, 2011
file that is used to ensure product sterility, and formulations that
can increase product variability (Hsu 1970). For example, sodium
citrate inhibits sedimentation, whereas calcium salts increase sedi-
mentation.
Factors Influencing Shelf Life of UHT Milk
The factors that influence the quality of milk that have an effect
on the gelation behavior of UHT milk are discussed here:
Age of cow
Milk from older cows gels faster than that of young cows (Datta
and Deeth 2003).
Stage of lactation
Auldist and others (1996) reported that early-lactation UHT
milk gelled in 5 to 6 mo, while late-lactation milk did not gel
during the 9 mo after their experiment. The reason behind the
phenomenon is that greater amount of denatured whey protein
is complexed with casein in late-lactation milks as compared to
early-lactation counterparts.
Mastitis
Mastitic milk (that is, milk with high somatic cell count, SCC)
subjected to UHT treatment is more susceptible to gelation than
normal milk (Swartling 1968). This has been attributed to in-
creased proteolytic activity resulting from an elevated level of plas-
min.
Season
Seasonal variations in the composition of milk may indirectly
affect the gelation behavior of UHT-sterilized milk. Spring and
late autumn milk shows more age gelation problems than milk
produced in other seasons; this can be attributed to the mineral
composition of milk (Hardham and Auldist 1996). Zadow and
Chituta (1975) observed that milk produced between August and
October was more prone to gel during storage than that produced
during the remainder of the year. Gel time was ranging from 77
to 140 d as compared with 120 to 180 d. Spring milk has higher
values of pH, lactose, soluble phosphate, and micellar hydration
than milk collected in autumn, while spring milk has low fat and
heat stability (Gaucher and others 2008a).
Microbiological quality of raw milk
Milk with a high preprocessing microbial count is more suscep-
tible to gel formation than milk with a low count. Microorganisms
that produce heat-stable enzymes cause the most serious gelation
problems. Longer refrigeration times prior to sterilization allow in-
creased growth of psychrotropic microorganisms and concomitant
production of heat-stable enzymes, especially proteinases and li-
pases. In work by Law and others (1977), when the psychrotrophic
bacterial count was less than 8 × 106 CFU/mL, shelf life was of
the order of 6 mo, whereas at higher counts, a marked reduction
in the time to onset of gelation was observed (see Table 1).
Table 1–Effect of psychrotrophic bacteria count in raw milk on gelation
time of UHT milk.
Bacterial count/CFU/mL Gelation time, days
<8.0 × 106 >140
8.0 × 106 ∼63
5.0 × 107 ∼12
Source: Law and others 1977.

c 2011 Institute of Food Technologists®
UHT milk processing and effect . . .

Storage temperature
Standardized milk
The temperature of storage markedly influences the time of
gelation of UHT-sterilized milk. In general, gelation occurs more
readily at room temperatures (20 to 25 ◦ C) than at low (4 ◦ C)
and high (35 to 40 ◦ C) temperatures. Kocak and Zadow (1985a, Pasteurization
20 s 80 °C
1985b) reported the order of gelation at different temperatures
to be 30 > 25 > 20 > 15 > 10 > 2 > 40, 50 ◦ C. Samel and
others (1971) suggested that, at 37 ◦ C, gelation may be inhibited
Homogenization Steam Injection
if regions of proteins that could take part in protein–protein inter-
20 MPa 2 s 150 °C
actions are blocked by casein–lactose interactions involving lysine
residues. Such interactions precede browning in UHT milk stored
at temperatures above 30 ◦ C. This hypothesis is supported by Hill
Direct expansion cooling
and Cracker (1968) who observed that when lysine and arginine De-aeration
80 °C
residues of κ-casein molecules were blocked, there was a resultant
loss of sensitivity to rennet coagulation, indicating that Maillard
browning may lead to an inhibition of κ-casein hydrolysis.
Sterilization Aseptic homogenization

Fat content 15 s 142 °C cool to 20 °C 40 MPa

UHT-processed skim milk is more susceptible to gelation than

UHT whole milk. This can be attributed to an enhanced action
of plasmin and bacterial proteinases in skim milk over whole milk. Packaging material Cool to 20 °C
An explanation for the effect is that the fat in whole milk hinders
access of the enzymes to their casein substrates. It has also been
suggested that the higher proportion of denatured whey proteins Aseptic
Sterilization
Aseptic
not attached to the micelle surface of skim milk may be a reason packaging packaging
for its lower resistance to gelation. Gaucher and others (2008b)
examined the effects of storage up to 6 mo at different temper-
Figure 3–Examples of the manufacture of UHT-sterilized milk (indirect or
atures (4, 20, and 40 ◦ C) of partially defatted UHT milk on its direct heating) with aseptic packaging.
particular physicochemical characteristics, and an increase of stor-
age temperature essentially affects the rate and degree of individual
changes. purpose of a UHT processing plant is to heat the product to the
sterilization temperature (in the range 135 to 150 ◦ C), hold it there
Hydrolysis of lactose for a few seconds, and then cool it to a suitable filling temperature.
Tossavainen and Kallioinen (2007) studied proteolytic changes There are 2 main technologies distinguished by the medium used
in lactose-unhydrolyzed and lactose-hydrolyzed direct UHT- for heating to the UHT, direct and indirect systems (Figure 3).
treated milks for a storage period of 12 wk. Enzymatic Steam, hot water, and electricity are heating methods for UHT
hydrolysis was performed either before (prehydrolyzed) or after equipment. The sterilizers utilizing steam or hot water can be sub-
(posthydrolyzed) UHT treatment. The enzymatic hydrolysis of categorized as direct or indirect heating systems. In the indirect
lactose resulted in an increase in proteolysis, compared to unhy- system, the product and heating medium do not have contact, as
drolyzed milk, during the storage regardless whether hydrolysis was a barrier (stainless steel) is present (Burton 1988). Direct heating
performed before or after the UHT treatment. The highest degree modes include steam injection, steam infusion, and scraped sur-
of proteolysis was found at the highest storage temperature tested face. Indirect heating modes include indirect spiral tubes, indirect
(45 ◦ C), while proteolysis was almost nonexistent at the lowest tubes, indirect plate, scraped surface, and electricity. Indirect heat-
storage temperature of 5 ◦ C as measured by α-amino nitrogen/ ing with electricity includes electric elements, conductive heating,
total nitrogen or as changes in sodium dodecyl sulfate- and friction (Burton 1988). Table 2 lists commercial UHT systems
polyacrylamide gel electrophoresis analyses. Proteolysis was also and their respective heating modes.
noticed in unhydrolyzed milk where it was caused by the plasmin Direct heating systems include steam injection (steam into milk)
enzyme system and possibly by the microbial contaminants in milk. and steam infusion (milk into steam). The culinary steam must be
The lactase dosage in prehydrolyzed milk was 30 times higher than of high quality and must not impart any off-flavors to the milk
in posthydrolyzed milk but proteolysis was only slightly stronger product. The product temperature increases almost instantly due to
than in posthydrolyzed milk. This means that most of the prote- the latent heat of vaporization. The condensed steam that dilutes
olytic side activity of the lactase was destroyed during the UHT the milk is removed later as the heated milk is cooled in a vacuum
treatment of prehydrolyzed milk. Thus, increasing the heat treat- chamber. Plate or tubular heat exchangers are 2 heating modes for
ment during the UHT process could destroy more of the harmful indirect heating. Heating in the indirect system occurs at a slower
proteolytic activities in milk. However, this may lead also to in- rate; therefore, the milk is subjected to the overall heat treatment
creased protein damage due to enhanced Maillard reaction and for a longer time. The heat transfer coefficient is greater with plate
browning of the product. heat exchangers due to turbulence (Datta and others 2002). The
potential for contamination due to pinholes in the stainless steel
Types of UHT Processing Systems for Milk barrier is minimized by maintaining a greater product pressure on
UHT plants became commercially available in 1960 when asep- the sterile side compared to the raw side. The comparisons of time-
tic filling technology, which is a necessity to maintain the com- temperature curves characteristic for treatment of milk in direct
mercial sterility of the UHT-treated product, was developed. The and indirect systems are shown in Figure 4. The thermal process


c 2011 Institute of Food Technologists® Vol. 10, 2011 r Comprehensive Reviews in Food Science and Food Safety 255
UHT milk processing and effect . . .

Table 2–Commercial UHT systems and heating modes. Table 3–Aseptic packaging systems.

Commercial UHT sterilizer Heating mode System Package Sterilant

Actijoule Indirect electrically heated Asepak Bags Heat
Gerbig, sterideal system Indirect heat with tubes ASTEC Bins and tanks Pressurized steam
High heat infusion, tetra therm Combined heating modes CKD Cups H 2 O2
aseptic plus 2 Combibloc Cartons H2 O2 + heat
Languilharre system, thermovac, Direct heat with steam infusion Dole Aseptic Canning Steel/aluminum Superheated steam
palarisator, steritwin UHT System cans and lids
sterilizer, ultra therm, Da-Si DuPont Canada Bags, pouches H 2 O2
Sterilizer ERCA Cups H2 O2 +heat
Rotatherm Direct heat with scraped surface Evergreen Cartons H2 O2 +heat
Spiratherm Indirect heat with spiral tubes Gasti Cups High-pressure steam
Ultramatic, ahlborn process, sordi Indirect heat with plates Gaulin Bags Ethylene oxide
sterilizer, UHT steriplak-R, dual Hamba manufacturing Cups Ultraviolet rays
purpose sterilizer Hassia Cups H2 O2 + heat or
Votator scraped surface heater, Indirect heat with scraped surface pressurized steam
thermutator heater Ingko Bags Chlorine solution + heat
VTIS, ARO-VAC process, uperiser, Direct heat with steam injection Inpaco Pouches H2 O2 + heat
grindrod International Paper Co. Rectangular H2 O2 + heat
Adapted from Datta and Deeth 2007.
packages
Lieffeld & Lemke Cups H2 O2 + heat
Liqui-Box Corp. Bags Gamma radiation
Manccini Bags Gamma radiation
Mead Packaging Co. Cups Citric acid + heat
Metal-Box Freshfill Cups H2 O2 + heat
(Autoprod)
Pure-Pak, Inc. Cartons H2 O2 + heat, oxonia
Purity Packaging Co. Cups H 2 O2
Remy Cups H2 O2 +heat
Remy Bottles H2 O2 or oxonia
Scholle Corp. Bags Gamma radiation or
ethylene oxide
Serac Bottles H 2 O2
Tetra Pak, Inc. Cartons H 2 O2
Wright Sel Bags Gamma radiation or
ethylene oxide
Source: David and others 1996.

point where sterilized packaging enters the sterile zone to where

the sealed package is evacuated.

Types of milk AP lines

Figure 4–Time-temperature curve for processing of milk in a direct system
(A) and indirect system (B). (Source: Gösta 2003.)
is dependent upon factors such as, product (pH, water activity,
viscosity, specific gravity); microbial profile (number, type, heat
resistance); equipment design, and package.
AP Systems for Milk
In AP, raw or unprocessed product is heated, sterilized by hold-
ing at high temperature for a predetermined period, then cooled
and delivered to a packaging unit for packaging. Packaging mate-
rial and equipment surface may be sterilized by various methods
such as heat, H2 O2 , irradiation, infrared light, and combinations
of methods (Ansari and Datta 2003). AP systems fill the sterile
product into sterile packages within the confines of the sterile
zone of the filler. The aseptic zone/sterile zone extends from the
There are 5 basic types of AP lines:
(1) Fill and seal: preformed containers made of thermoformed
plastic, glass, or metal are sterilized, filled in aseptic environ-
ment, and sealed.
(2) Form, fill, and seal: roll of material is sterilized, formed in
sterile environment, filled, sealed, for example, tetrapak.
(3) Erect, fill, and seal: using knocked-down blanks, erected,
sterilized, filled, sealed, for example, gable-top cartons,
cambi-bloc.
(4) Thermoform, fill, sealed roll stock sterilized thermoformed,
filled, sealed aseptically, for example, creamers, plastic soup
cans.
(5) Blow mold, fill, seal (Gedam and others 2007).
Commercial manufacturers include Tetra-Pak, Scholle, and the
Dole Aseptic Canning System® . Table 3 lists several manufacturers
of aseptic equipment. AP systems available for dairy foods in-
clude drum and bin systems, heat during blow-molding, carton
packaging machines, bag-in-box packaging systems, bulk tanks
and containers, plastic cups/pots/cartons, and pouches/sachets
(Holdsworth 1992).
Filler and container sterilization
Aseptic fillers have sections containing sterile contact pipes and
valves along with noncontact sections (sterile chambers). Both
sections must be sterilized prior to production and must maintain
sterility throughout production (Burton 1988). Rippen (1969)
stated aseptic fillers and associated pipes are sterilized typically

256 Comprehensive Reviews in Food Science and Food Safety r Vol. 10, 2011 
c 2011 Institute of Food Technologists®
UHT milk processing and effect . . .

with heat in the form of steam. Wet heat sterilization using satu- food contact surfaces. In a properly designed APs system, a good
rated steam is the most dependable sterilant, as microorganisms are microbiocidal effect using H2 O2 can be achieved and the level of
more resistant to dry heat, which necessitates higher temperatures residue can be effectively controlled to within permissible limits.
(Burton 1988). Sterilants are applied uniformly to the aseptic zone The residual level of H2 O2 is regulated with a maximum level of
by misting equipment, whereas packaging typically is sterilized by 0.5 ppm. Infrared radiation and vaporized H2 O2 have been stud-
misting or passing through sterilant bath. ied as sterilants for packaging materials (Kulozik and Guilmineau
Sterilization of packaging material is a critical step in the AP sys- 2003). There are many other chemicals such as peracetic acid,
tem. Therefore, the sterilization process should meet the following beta propiolactone, alcohol, chlorine, and its oxide, and ozone
requirements for sterilization of packaging materials: that have been suggested as having potential for use in sterilizing
(1) Rapid microbiocidal activity; AP materials (Ansari and Datta 2003).
(2) compatibility with surfaces treated, especially packaging ma-
terial and equipment;
Postprocess Contamination Concerns in UHT Milk
(3) easily removed from surface, minimum residue;
The problem of posttreatment contamination of in container
(4) present no health hazard to the consumer;
sterilized product is well known. The contamination can either
(5) no adverse effect on product quality in the case of unavoid-
through poor seal or through pinhole in the container. Post treat-
able residue or erroneous high concentration;
ment contaminants in UHT milk may be either spores, which
(6) present no health hazard to operation personnel around the
would not be expected to be heat resistant enough to survive the
packaging equipment;
heat treatment or nonheat-resistant vegetative organisms. Organ-
(7) compatibility with environment;
isms of the 1st type will probably have entered from the ineffec-
(8) noncorrosive to surfaces treated;
tively sterilized plant downstream from the heat treatment stage
(9) reliable and economical (Ansari and Datta 2003).
of the process, which includes spores of Bacillus cereus (Wilson and
Sterilants that are commonly used at industrial level include
others 1960; Davies 1975) and Bacillus licheniformis (Wilson and
chlorine, iodine, oxonia, food acids, ozone, H2 O2 , and UV light
others 1960). Organisms of the 2nd type will probably have en-
(David and others 1996). Some of these methods are listed in
tered through a poorly sealed container after aseptic filling (Hassan
Table 4. The H2 O2 is now the only chemical sterilant for ster-
and others 2009). Postprocess contamination of the aseptic zone
ilization of packaging materials that has been proved to be ac-
can be attributed to several variables: environmental bioburden,
ceptable in the United States. The FDA regulations specify that a
positive air pressure, processing equipment or line turbulence, sys-
maximum concentration of 35% H2 O2 may be used for sterilizing
tem gasping, indexing operations, condensate accumulation, un-
sterile product entry, or bacteriological seeding (David and others
Table 4–Methods for sterilizing aseptic packages. 1996). Postprocess contamination occurs in individual cartons if
package integrity is compromised. Contamination from isolated
Advantages/
Methods Application disadvantages Reference package integrity issues occurs more frequently than processing
Superheated Metal containers High temperature Collier and contamination.
steam at atmospheric Townsend Rippen (1969) cited typical spoilage in UHT-AP production at
pressure. (1956) a defect rate of 1 of 1000. Manufacturers of aseptic fillers target
Microorganisms
are more a defect rate of ≤1/1000 or ≤1/3000, whereas ≤1/10000 is an
resistant than in industry standard for aseptically packaged low-acid foods in rigid,
saturated steam semi-rigid, and flexible containers (David and others 1996). The
Dry hot air Metal or High temperature Denny and
composite at atmospheric Mathys following 7 potential failure modes exist for aseptic processing and
juice and pressure. (1975) packaging of foods:
beverage Microorganisms (1) Type 1 failure results from raw ingredient, handling, storage,
containers are more
resistant than in or batching issues.
saturated steam (2) Type 2 failure results from processor and filler cleaning in
Hot hydrogen Plastic Fast and efficient Denny and
peroxide containers, method others place, sanitation, preventive maintenance, and presteriliza-
laminated foil (1974) tion issues.
Hydrogen Plastic UV increases Bayliss and (3) Type 3 failure results from the thermal process heating cycle
peroxide/UV containers effectiveness of Waites
light (preformed hydrogen (1982) including regeneration.
combination cartons) peroxide (4) Type 4 failure results from the cooling cycle including surge
Ethylene oxide Glass and plastic Cannot be used Blake and tanks.
containers where chlorides Stumbo
are present or (1970) (5) Type 5 failure results from sterilization issues with the pack-
where residuals age.
would remain
Heat from Plastic No chemicals used –
(6) Type 6 failure results from sterility loss in the aseptic zone or
coextrusion containers from environmental load.
process (7) Type 7 failure results from loss of package integrity (David
Radiation Heat-sensitive Can be used to –
plastic sterilize and others 1996).
containers heat-sensitive
packaging
materials. Commercial Sterility Testing of UHT Milk Process
Expensive. Scheduled processes in retort operations and UHT processes in-
Problems with
location of activate vegetative cells and spores of pathogenic bacteria. The gen-
radiation source era Bacillus and Clostridium are the primary sporeforming spoilage
Source: Ansari and Datta 2003. microbes (Ravishankar and Maks 2007). Spoiled packages are


c 2011 Institute of Food Technologists® Vol. 10, 2011 r Comprehensive Reviews in Food Science and Food Safety 257
UHT milk processing and effect . . .
identified as “flat sours” or swells. Spoilage organism identification
is useful in troubleshooting the cause of spoilage and the origin
of contamination (Burton 1988). Underprocessing is indicated by
spoilage due to spore-forming rods, whereas postprocess contam-
ination is indicated by mixed flora containing heat-sensitive or-
ganisms (Dunkley and Stevenson 1987). Lewis (1999) stated UHT
milk microbial counts should be <100 CFU/g following 15 d at
30 ◦ C. Hsu (1970) stated that souring and/or coagulation would
be identified after incubating UHT-AP products 7 to 10 d at 37 ◦ C.
An incubation and inspection program is recommended by FDA
to verify sterility of aseptically packaged products. Sampling plans
are more extensive when commissioning aseptic filler than during
routine production (Burton 1988). Sampling between 0.1% and
1.0% for routine production is recommended with samples taken
at the beginning of production, filler restarts, and production end.
An ideal sampling plan provides sterility assurance within a rea-
sonable cost structure (Farahnik 1982). Microbial testing is viewed
as an additional verification quality program and is completed
through traditional and rapid methods (Dunkley and Stevenson
1987). Visual inspections, sensory analysis, and pH measurements
are done in conjunction with rapid methods to verify product
quality (Grow 2000). Quantitative methods include direct enu-
meration and viable enumeration. Viable cells are counted using
standard plate counts, most probable number, membrane filtration,
plate loop methods, or spiral plating. Qualitative methods include
measuring metabolic activity or cellular constituents (Goff 2008).
The Cellscan Innovate System by Celsis uses bioluminescence to
measure adenosine triphosphate found in living microorganisms
(Grow 2000).
UHT Milk Container Integrity
Packaging plays an important role in the food manufacturing
process as it makes food more convenient. It gives the food greater
safety assurance from microorganisms and biological and chemical
changes such that the packed foods can have longer shelf life. Food
packaging for shelf-stable products must provide barrier properties
and physical strength so that they can withstand handling, distri-
bution, and storage. It is intriguing, however, that the number of
failures (nonsterile product) associated with UHT plants around
the world is an almost constant number, namely 1 to 4 in 104 .
The reasoning widely accepted for this is that nonsterility arises
through failure of packaging material or package seals or a plant
being “leaky” (Cerf and Davey 2001). Package integrity inspec-
tions for flexible containers include visual observation, dye test,
squeeze test, seal teardown, and conductivity (Grow 2000). Addi-
tional tests identified by Holdsworth (1992) include the inflation
test, compression test, decompression test, biotesting, ultrasound
imaging, mechanical tests, and headspace indicators. The mechan-
ical tests on filled packages include stress testing, stack testing, load
vibration, and impact resistance.
Plasmin Activity
Since the adoption of refrigerated bulk tanks for the collec-
tion and storage of milk, the predominant organisms in milk are
now psychrotrophic bacteria. These are organisms that are able to
grow at temperatures below 7 ◦ C, although their optimum growth
temperature may lie between 20 and 30 ◦ C. The majority of the
psychrotrophic bacteria (excluding Bacillus spp.) are destroyed by
pasteurization, but they produce extracellular enzymes that are ex-
tremely thermostable. The most important of these enzymes from
the commercial viewpoint are the proteases and lipases, both of
which are able to withstand high temperature short time and UHT
258 Comprehensive Reviews in Food Science and Food Safety r Vol. 10, 2011
treatments. Proteolysis in UHT milk can cause the development
of bitter flavor and lead to an increase in viscosity, with eventual
formation of a gel during storage, which is a major factor lim-
iting its shelf life and market potential. The enzymes responsible
for the proteolysis are the native milk alkaline proteinase, plas-
min, and heat-stable extracellular bacterial proteinases produced
by psychrotrophic bacterial contaminants in raw milk. Proteol-
ysis of casein caused by plasmin is responsible for gelation and
bitterness of UHT milk during storage.
Psychrotrophic bacteria in milk
Psychrotrophic microorganisms that are capable of growing in
milk at temperatures close to 0 ◦ C are represented by both Gram-
negative bacteria (such as Pseudomonas, Achromobacter, Aeromonas,
Serratia, Alcaligenes, Chromobacterium, and Flavobacterium spp.) and
Gram-positive bacteria (such as Bacillus, Clostridium, Corynebac-
terium, Streptococcus, Lactobacillus, and Microbacterium spp.). Further-
more, refrigeration conditions under which raw milk is stored
selects for growth of psychrotrophs, many of which produce heat-
stable enzymes while milk awaits processing. Following heat treat-
ment, these enzymes can continue to degrade milk in the absence
of viable bacterial cells. A variety of psychrotrophic organisms,
including P. fluorescens, P. putida, P. fragi, P. putrefaciens, Acinetobacter
spp., Achromobacter spp., Flavobacterium spp., Aeromonas spp., and
Serratia marcescens, produce heat-stable extracellular proteases and
some of them also produce heat-stable extracellular lipases. Pseu-
domonas spp. usually represents no more than 10% of the microflora
of freshly drawn milk; however, they are the most important of
the psychrotrophs that dominate the microflora of raw or pasteur-
ized milk at the time of spoilage. This genus is also known to be
strongly lipolytic (Sørhaug and Stepaniak 1997).
Thermoresistant psychrotrophs. Among the microorganisms
that survive pasteurization (the thermoduric microorganisms),
sporeforming Bacillus spp. dominate those that are also psy-
chrotrophs. Other thermoduric psychrotrophs are represented
in the genera Arthrobacter, Microbacterium, Streptococcus, Corynebac-
terium, and Clostridium. Clostridium is also sporeformer and anaero-
bic. Psychrotrophic Bacillus spp. secrete heat-resistant extracellular
proteinases, lipases, and phospholipase (lecithinase) that are of
comparable heat resistance to those of Pseudomonads. Bacillus
sporothermodurans is a mesophilic spore-former that produces highly
heat-resistant spores and was first detected in UHT milk in 1985
in southern Europe and in UHT milk in Germany in 1990 (Pet-
tersson and others 1996). The spores survive the heat process and
then multiply to a maximum of about 105/mL of milk during
incubation at 30 ◦ C for 5 d, but cause no noticeable spoilage. The
spores of B. sporothermodurans are more resistant than the spores of
many thermophiles (Brown 2000).
Secretion and biochemical properties of proteinases and
lipases from psychrotrophs
Among the hydrolases from psychrotrophic bacteria in milk,
those from Pseudomonas spp. have been the most frequently stud-
ied and are secreted mainly at the end of the stationary phase of
growth. The maximum concentrations of proteinases get accumu-
lated in shaken milk than those in static culture, but variable effects
of aeration on the production of lipases have been reported. Most
of the proteinases from Pseudomonas are metalloenzymes contain-
ing 1 zinc atom and up to 8 calcium atoms per molecule. Most pro-
teinases from psychrotrophs have milk-clotting activity, are readily
able to degrade κ-, α s1 - and β-casein, and have low activity on
nondenaturated whey proteins (Sørhaug and Stepaniak 1997).

c 2011 Institute of Food Technologists®
UHT milk processing and effect . . .
Plasmin, plasminogen, plasminogen activators (PAs), PA
inhibitors (PAIs), and plasmin inhibitors (PIs)
Plasmin (EC 3.4.21.7), the main native protease in milk, is
part of a complex system consisting of plasminogen, PA, PAI,
and PI (Crudden and Kelly 2003). An important part of the po-
tential plasmin activity is present as plasminogen. In fresh milk,
the concentration of plasminogen is much higher than that of
plasmin (Nielsen 2003). Plasmin is an alkaline serine proteinase
with pH optimum of 7.5, which readily hydrolyzes β-casein, α s2 -
casein, and (more slowly) α s1 -casein (Fox and McSweeney 1996).
Its enzymatic reactions result in desired and undesired effects on
dairy products. Plasmin plays a positive role in cheese ripening for
many varieties of cheeses (such as Emmental, Romano, Swiss, and
Gouda); however, its enzymatic action during milk clotting and
storage of UHT milk can affect the products adversely.
Plasmin activity is higher in mastitis and late-lactation milk due
to the increased level of PAs. The levels of plasmin and plas-
minogen can vary considerably with the stage of lactation, breed,
age, and presence of mastitis (Grufferty and Fox 1986). The final
plasmin activity of milk and, subsequently, milk casein hydroly-
sis, would depend not only on the amount of plasminogen and
PA but also on the quantity of PAI. The occurrence in milk of
blood serum trypsin inhibitors with both high and low molecular
weight has been documented. They presumably would interfere
with the function of serine proteinases and therefore, with plasmin
and PA activity. However, other milk constituents, for example,
β-lactoglobulin also could inhibit these enzymes (Korycka-dahl
and others 1983). Many interactions between plasminogen, plas-
min, PA, PAI, and PI characterize the plasmin system (Figure 5A).
The role of PA in the system is to mediate plasminogen conversion
into plasmin, whereas PAI and PI inhibit PA and plasmin activities,
respectively. PAs are likely native to milk or produced by microor-
ganisms (Deharveng and Nielsen 1991). Two major types of PA
are known to be present in fresh milk: a tissue type associated with
casein and a urokinase type associated with the somatic cells. PIs
and PAI are located mainly in milk serum; however, the inhibitors
might appear in several different forms, possibly due to formation
of complexes with other milk proteins (Precetti and others 1997).
α 1 -Antitrypsin, α 2 -antiplasmin, and PAI have been isolated from
milk serum and partially characterized (Weber and Nielsen 1991).
Little is known about the heat stability of PAI and PI, and it has
been generally proposed that the inhibitors are thermally unsta-
ble. Richardson (1983) suggested that PAI is inactivated by mild
thermal treatments. An increase in activity of PL and a subsequent
decrease in concentration of PG were observed in pasteurized
milk compared to raw milk after incubation at 37 ◦ C for up to
80 h. Effect of heat treatment on inhibitors of both PA and plas-
min was studied by (Prado and others 2006) and fractions of milk
with inhibitory activities against PA and plasmin were isolated.
Thermal inactivation of PA inhibitor (81.1%) was considerably
different from that of PI (35.8%) in milk after heat treatment at
PAIs PAs PIs
(Heat-labile) (Heat-stable) (Heat-labile)
Plasminogen Plasmin Peptides (γ-Cn) and
(Heat-stable) (Heat-stable) Casein proteose peptone
Figure 5–The plasmin-plasminogen system (adapted from Richardson
1983).

c 2011 Institute of Food Technologists®
75 ◦ C for 15 s. The PI in the isolated fractions was suggested to
be α 2 -antiplasmin, since it reacted immunochemically with poly-
clonal goat antihuman α 2 -antiplasmin and competitively inhibited
plasmin. Results showed that PA inhibitor is less heat stable than
PI, indicating that plasminogen activation could overcome any
inhibition of plasmin resulting after milk pasteurization.
Hard gel formation was caused by Pseudomonas proteinase and
partial digestion of the casein by plasmin in the samples incubated
at 40 ◦ C for 3 h. The clarified samples indicated extensive break-
down of casein as evident by turbidity, while the gelled samples
indicated limited proteolysis. The characteristics of hard gel are
similar to that caused by rennet, which, such as Pseudomonas pro-
teinase, preferentially hydrolyzes the hydrophilic glycomacropep-
tide from κ-casein on the outside of the micelle. This leaves the
casein micelle largely intact and also reduces steric repulsion be-
tween micelles, allowing the formation of a more compact gel.
By contrast, plasmin attacks κ-casein located inside the micelle,
thereby disrupting the micelle and inhibiting the formation of a
strong gel. Extensive proteolysis of skim milk by plasmin com-
pletely clarifies skim milk (Datta and Deeth 2003).
Plasmin itself is a heat-stable enzyme that survives pasteuriza-
tion and many UHT processes (D140◦ C is 32 s) and the initial
(1 d) plasmin activity of UHT milk containing KIO3 was signif-
icantly higher than that in raw milk (Kennedy and Kelly 1997).
The inhibitors present in fresh milk are heat labile, whereas the
activators are known to be heat stable (Richardson 1983; Lu and
Nielsen 1993). Consequently, heat treatment of milk alters the
natural balance between the activators and inhibitors in favor of
the activators. This can lead to enhanced proteolysis in heated
milk (Deharveng and Neilsen 1991). Driessen and van der Waals
(1978) reported the D142◦ C for milk proteinase (plasmin) to be
18 s. Rollema and Poll (1986) reported 28%, 6%, 4%, and 1.3%
plasmin/plasminogen remaining after indirect heating for 5 s at
110, 120, 140, and 147 ◦ C, respectively, but none after heating at
147 ◦ C for 10 s.
If proteases survive UHT sterilization, milk containing them
and stored without refrigeration would be a good environment
for their activity. The optimum pH for one protease ranges from
pH 7 to 8 with 85% to 90% of maximum activity at pH 6.5,
the pH of milk (Speck and Adams 1976). The production of
different phospholipases has been reported for Gram-negative and
Gram-positive psychrotrophs. The “bitty cream” defect (floating
clumps of fat) occurs in products that contain high numbers of
Bacillus cells, suggesting a unique ability of phospholipases from
this microorganism to damage the fat globule membrane (Sørhaug
and Stepaniak 1997).
Heat Resistance of Enzymes Present in Milk
Generally, heat-labile enzymes are inactivated by unfolding
followed by molecular scrambling (collapse to inactive forms),
whereas heat-resistant enzymes are inactivated by covalent mod-
ification (such as destruction of cystine cross-links and deamina-
tion of asparagine and glutamine residues). Proteinases, lipases, and
phospholipase (from psychrotrophic bacteria, mostly Pseudomon-
ads), which are stable at high temperatures and survive pasteuriza-
tion and UHT treatment, but are not active above 50 to 60 ◦ C
(Table 5). Among the features that stabilize thermoenzymes are
additional salt bridges, additional hydrogen bonds, tighter Ca2+ -
binding sites, maximized packing, shorter loops, and an ex-
panded hydrophobic core. Many of these features are apparently
also stabilizing factors in heat-resistant proteinases, lipases, and
phospholipase C from all psychrotrophic microorganisms. Lipases
Vol. 10, 2011 r Comprehensive Reviews in Food Science and Food Safety 259
UHT milk processing and effect . . .
Table 5–Heat resistance of enzymes from Pseudomonads.
Enzyme Source Heat resistance
Protease (s) Pseudomomas D150◦ C = 90 s
Protease (s) Total psychrotrophic Survived 149 ◦ C/10 s
flora of milk
Protease Pseudomomas D149◦ C = 90 s
fluorescens P26
Protease Pseudomomas Survived unspecified UHT
sterilization temperature
Protease Pseudomomas Survived 135 ◦ C/3.5 min
Lipase P. fluorescens 22F D150◦ C = 4.8 min
and proteinases are more sensitive to low-temperature treatment
at 50 to 60 ◦ C than to heat treatment at >100 ◦ C. Proteinases
are partially renatured after heat treatment (Sørhaug and Stepa-
niak 1997). The time required to reduce protease activity by 90%
was 90 s compared to 0.25 and 0.02 s for a 90% reduction in
PA3679 spores and B. stearothermophilus spores, respectively. Pu-
trefactive Anaerobe 3679 are the spores commonly are used for
the development of sterilization processes, and a heat treatment
of 149 ◦ C for 4 s should sterilize fluid milk products effectively.
These proteases showed less than 10% destruction during UHT
sterilization of milk at 149 ◦ C for 4 s (Speck and Adams 1976).
Kishonti (1975) showed that 24 of 60 strains of psychrotrophic
bacteria isolated from milk and including Pseudomonas spp., Al-
caligenes spp., and Aerobacter spp. produced extracellular enzymes
capable of retaining at least 75% of their activity after exposure to
63 ◦ C for 30 min. Stadhouders and Mulder (1960) showed that
strains of Achromobacter spp. and Serratia spp. produced lipases that
could withstand 74 ◦ C for 4 s, but certain strains of Alcaligenes spp.
and Flavobacterium spp. were not able to withstand this treatment.
Similarly, The Merck-BIOQUANT® Proteinase assay Kit recom-
mends a bacterial proteinase level <1 ng/mL (standard alcalase R
equivalent) for UHT milk to have a shelf life of at least 3 mo at
25 ◦ C. Mitchell and Ewings (1985) determined the threshold value
for proteinase to be about 0.3 ng/mL for a shelf life of at least
4 mo at 23 ◦ C.
Inactivation Kinetics of Enzymes Present in Milk
UHT treatments can be sufficient to inactivate microorganisms
and obtain a sterile product but provide insufficient heat load to
reduce plasmin activity and obtain a stable product. Insufficient
inactivation causes bitter-tasting milk, resulting in product loss
for producers and consumers of milk. To avoid bitterness, an in-
activation of 99% of the plasmin present is generally applied in
industries. Metwalli and others (1998) observed that irreversible
inactivation can be achieved at around 65 ◦ C, but above 92 ◦ C the
inactivation rate increases only slightly with temperature due to a
lower activation energy in that specific temperature range.
Thermal inactivation, at temperatures between 60 and 140 ◦ C,
of native plasmin, plasminogen, and PAs were studied in bovine
milk using improved enzymatic assays. Activation energies (Ea)
for the heat denaturation of plasmin, plasminogen, and PAs were
29, 35, and 24 kJ/mol, respectively, in the temperature range 95
to 140 ◦ C, and 244, 230, and 241 kJ/mol, respectively, in the
temperature range 70 to 90 ◦ C (Saint Denis and others 2001).
The inactivation kinetics of plasmin in milk has been described
by Rollema and Poll (1986) and (partly) denatured β-lactoglobulin
is found to affects the rate of plasmin inactivation (Crudden and
others 2005). Due to thermal processing free S–H groups will
become available when β-lactoglobulin unfolds, which is the 1st where X is proteinase activity measured by the Merck proteinase
stage of denaturation. As a result of unfolding of β-lactoglobulin, test kit using dehydrogenase as the substrate and relating the
260 Comprehensive Reviews in Food Science and Food Safety r Vol. 10, 2011
the highly reactive S–H groups cause irreversible denaturation
of plasmin (Crudden and others 2005). The above implies that
a certain degree of denaturation of β-lactoglobulin is necessary
to increase the inactivation of plasmin and plasminogen. On the
other hand, denaturation of β-lactoglobulin induces the forma-
tion of deposit on the wall of heat treatment equipment and is
also an indicator for product degradation. This implies that an
optimized heat treatment needs to be designed where the degree
of denaturation of β-lactoglobulin is high enough to inactivate
plasmin to avoid bitterness but low enough to minimize the for-
mation of deposit and product degradation. Previous experiments
showed that milk heated with the innovative steam injection (ISI)
system had a degree of denaturation of β-lactoglobulin of <30%,
whereas standard UHT treatments of milk resulted in a degree
of denaturation of >50% (Huijs and others 2004). However, the
effect of denatured β-lactoglobulin is usually not taken into ac-
count when predicting/designing UHT processes in relation to
the inactivation of plasmin (Crudden and others 2005).
Gelation of UHT Milk
The gel that forms in UHT milk is a 3-dimensional (3D) protein
matrix formed by the whey proteins, particularly β-lactoglobulin,
interacting with casein, chiefly κ-casein, of the casein micelle. The
major proteinaceous linkages that develop during the heat treat-
ment result in formation of β-lactoglobulin-κ-casein complexes
(βκ-complexes). β-Lactoglobulin begins to unfold and lose its
globular structure above 55 ◦ C (Gough and Jenness 1962; Sawer
1969); this causes rupture of cystine disulfhide bonds and a marked
increase in thiol group reactivity. Prolonged heat treatment above
80 ◦ C leads to degradation of all of the cystine residues. Lyster
(1964) observed that all of the S–H groups became “reactive”
after UHT treatment with an indirect heating system. The re-
active S–H can react intramolecularly to form β-lactoglobulin
aggregates, or intermolecularly to form disulfhide bonds between
β-lactoglobulin and other S–H-containing molecules such as κ-
casein and proteins of the milk fat globule membrane. In the
casein micelle, κ-casein exists at the surface and is available for
such interaction with β-lactoglobulin.
Following these initial heat-induced changes, other changes oc-
cur slowly in UHT milk during storage and result in the forma-
tion of a 3D protein network that causes the milk to thicken
and then gel. The ease with which a milk will gel is deter-
mined by the extent of 3 processes leading to gelation: the in-
teraction between β-lactoglobulin and κ-casein (as opposed to
the self-association of denatured β-lactoglobulin); the release of
the βκ-complex from the casein particle; and the cross-linking
of the βκ-complexes and associated proteins. When these asso-
ciations (βκ-complexes) are disrupted, κ-casein is released along
with its attached β-lactoglobulin. The released βκ-complexes are
observed as protuberances and tendrils on the micelle surface.
The disruption of the κ-casein anchors to the other caseins can
occur through enzymic (proteinase) action or nonenzymic means
(Harwalkar 1982). The order of susceptibility of the caseins to
hydrolysis by bacterial proteinases and milk plasmin are κ > β >
α s1 and β = α s2 > α s1 > κ, respectively. Overall, the relationship
between added purified proteinase activity and gelation time can
be obtained from the following equation:
Gelation time (months) = 2.3916 × X−0.6449 ,

c 2011 Institute of Food Technologists®
UHT milk processing and effect . . .

Figure 6–Model of age gelation of UHT milk showing (1) formation of the βκ-complex, (2) its dissociation from micelles during storage, and (3)
subsequent gelation of the milk through cross-linking of the βκ-complex. (Source: McMahon 1996.)

activity to the equivalent concentration of alcalase R (Novo Indus- plasmin activity, which increase on storage due to activation of
trials, Denmark) in nanogram per milliliter (Mitchell and Ewings plasminogen.
1985).
Enzymatic mechanism of gelation
According to McMahon (1996), the proteinases do not act di-
rectly on the βκ-complex but cleave the peptide bonds that an-
chor the κ-casein to the casein micelle, facilitating release of the
βκ-complex. This dissociation of βκ-complexes from the casein
micelles by proteinases is considered to be the 1st stage in a 2-stage
mechanism of age gelation. The 2nd stage involves the subsequent
aggregation of the βκ-complexes and formation of a 3D network
of cross-linked proteins (Figure 6) and effect of proteolytic en-
zymes on gelation in Table 6. Enright and others (1999) observed
that UHT milk with added KIO3 (0.23 M) at the rate of 13 mL/
30 L of milk behaved somewhat like raw milk during storage,
showing extensive plasminogen activation, rapid proteolysis, and
formation of sediments at a similar time, and of similar appearance,
to those seen in raw milk. The addition of plasmin to UHT milk
after heating reduced the stability of the milk, increased proteol-
ysis, and led to the early formation of sediments. The results of
this study suggest strongly that plasmin activity is a major influ-
ence on the storage stability of UHT milk. Kelly and Foley (1997)
concluded that KIO3 protected plasmin from inactivation by com-
plexation with β-lactoglobulin, leading to high residual levels of
Nonenzymatic mechanism of gelation
Andrews and Cheeseman (1972) suggested that gelation is
caused by polymerization of casein and whey proteins by Mail-
lard reactions that are promoted by higher storage temperatures.
However, the lack of gel formation during storage of UHT milk
at temperatures above 35 ◦ C does not corroborate their sugges-
tion. Samel and others (1971) reported that blockage of ε-NH2
groups of lysine residues in casein micelles of UHT milk prevents
micelles from interacting with each other and may retard age gela-
tion due to modification of the charge on the casein micelles.
According to another hypothesis, gelation of UHT milk results
from changes in the free energy of casein micelles. Differences in
potential energy promote aggregation of the casein micelles, the
extent of this depends upon the probability of contact and the
number of low potential micelles, both of which increase with
storage time. Micelle aggregation leads to increased viscosity of
the UHT milk.
Measuring Enzyme Activity in UHT Milk
Analysis of milk in the manner proposed can enable the UHT
milk manufacturer to determine if proteolysis is occurring, or
has occurred in the milk and, if so, whether it is caused by milk


c 2011 Institute of Food Technologists® Vol. 10, 2011 r Comprehensive Reviews in Food Science and Food Safety 261
UHT milk processing and effect . . .

plasmin, bacterial proteinase, or both. If it is caused by plasmin, it is A sensitive assay for protease activity based on the reaction of pri-
likely that the UHT processing conditions are too mild that causes mary amino groups of trichloroacetic acid (TCA) soluble peptides
less denaturation of plasmin and whey proteins. This results in and amino acids with fluorescamine (4-phenylspiro[furan-2(3H),
less whey protein–casein interaction and less inhibition of plasmin 1 -phthalan]-3,3 dione) was applied to sterile milk (Chism and
action on the casein that is most commonly encountered in the others 1979). The assay was linear in the range of 2 to 50 nmol
direct UHT processes, steam infusion, or injection (Manji and of amino groups per aliquot. The assay is suitable for determining
others 1986; Manji and Kakuda 1988). If the proteolysis is caused proteolytic activity in sterile milk products and for determining
by bacterial proteinases, the quality of the raw milk is implicated. protease activity in general.
The most common cause is high levels of psychrotrophic bacteria Plasmin and plasminogen can also be determined spectrophoto-
in the raw milk. Inadequately cleaned equipment that supports metrically using the chromogenic substrate H-D-valyl-L-leucyl-
bacterial growth and production of proteinases can also be a cause L-lysyl-4-nitroanilide (VLLN) as described by Korycka-dahl and
(Driessen 1983). others (1983) with some modifications. Milk samples must be
Protease activity in the sterile skim milk was determined defatted at 10 ◦ C by centrifugation (5000 × g for 20 min) and
by measuring proteolysis at weekly intervals. Single samples skimmed milk obtained must be incubated with 50-mM ε-amino-
from 3 bottles were assayed by the Hull method (1947) with n-caproic acid (EACA) for 2 h at room temperature to dissociate
Folin–Ciocalteau reagent. Measurements were continued until the plasmin and plasminogen from casein micelles. A plasmin activ-
increase in absorbance exceeded 0.6 or until whey separation and ity was measured without adding urokinase- and plasminogen-
gelation. The rates of proteolysis were determined by calculating derived activity and calculated as total activity minus plasmin
the regression of proteolysis on time. From the slopes of the re- activity and was expressed in the same units. One unit of activity
gression lines, the percentage inactivation of proteolysis caused by was defined as the amount of enzyme that produces a change in
low-temperature-inactivation (LTI) was calculated as: absorbance (path length 1 cm) at 405 nm of 0.0001 in 1 min at pH
7.4 and 37 ◦ C in the defined reaction mixture (Manji and others
Percent of inactivation = 100 (slope of control 1986). Plasmin activity in dairy products can also be measured
− slope of LTI sample)/slope of control. with synthetic chromogenic or fluorogenic substrates. Synthetic
substrates can be made by linking Lys peptides to chromogenic or
fluorogenic tags. VLLN is a synthetic substrate that can be used to
determine plasmin activity as it hydrolyzes this peptide to release
Table 6–Effect of proteolytic enzymes on gelation. 4-nitroaniline, a compound that absorbs light at 405 nm (Bastian
and others 1993).
Results and The most sensitive methods reported, including enzyme-linked
Enzyme/enzyme Experiment conclusions immunosorbent assays (ELISAs), were able to detect enzyme ac-
Plasmin/plasminogen Direct, indirect Indirect inactivated tivity or the presence of enzyme when the number of Pseudomonas
(Manji and others heating plasmin; no gelation
1986) to 182 d in indirect cells present was about 106 CFU/mL. ELISAs that can detect 0.25
inactivated plasmin ng/mL of proteinases or 20 ng/mL of lipase from Pseudomonas
Plasmin (Grufferty – Caused degradation of spp. have been reported. An ELISA that detects proteolysis by
and Fox 1986). casein and gelation in
60 d measuring the accumulation of glycomacropeptide released from
Bacterial pH opt, 6.5; temp Caused bitter flavor and κ-casein by proteinases from Pseudomonas spp. has also been
proteinases opt, 45 ◦ C gelation
(Speck and developed (Sørhaug and Stepaniak 1997). Datta and Deeth (2003)
Adams 1976) differentiated the peptides produced by enzymes responsible for
Psychrotroph – Responsible for gelation the proteolysis from the native milk alkaline proteinase, plasmin,
proteinases
(Cogan 1977) and heat-stable, extracellular bacterial proteinases produced by
Bacterial, P. – Responsible for psychrotrophic bacterial contaminants in the milk prior to heat
fluorescens (Law gelation; time to gel processing. In order to differentiate, these peptide products,
and others 1977) dependent on extent
of growth in raw milk reversed-phase high-performance liquid chromatography
Bacterial, plasmin – Both caused gelation (HPLC), and the fluorescamine method were used to analyze
Plasmin (Snoeren Good quality milk Plasmin caused gelation the peptides soluble in 12% TCA and those soluble at pH 4.6.
and others 1979) <2700 CFU/mL in 90 d
Bacterial (Fox 1981; Poor quality milk, 2 Bacterial proteinases The TCA filtrate showed substantial peptide peaks only if the
McMahon 1996) × 106 CFU/mL cause gelation in 21 d milk was contaminated by bacterial proteinase, while the pH 4.6
Proteinases Direct compared More proteolysis and filtrate showed peptide peaks when either or both bacterial and
(Harwalkar 1982) with indirect gelation in directly
heated milk native milk proteinases caused the proteolysis. Results from the
Proteinases (Samel Storage at 4, 20, 30, More proteolysis but fluorescamine test were in accordance with the HPLC results
and others 1971). 37 ◦ C less gelation at 37 ◦ C; whereby the TCA filtrate exhibited significant proteolysis values
gelation not due to
proteolysis only when bacterial proteinases were present, but the pH 4.6
Plasmin isolated Unconcentrated No correlation between filtrates showed significant values when the milk contained either
from raw milk milk amount of proteolysis
(Manji and and gelation time but or both types of proteinase. A procedure based on these analyses
Kakuda 1988). some proteolysis can be proposed as a diagnostic test for determining which type
necessary for gelation of proteinase-milk plasmin, bacterial proteinase, or both are
Proteinase inhibitors – Direct heating Inhibited
(de Koning and plasmin action; responsible for proteolysis in UHT milk.
others 1985) retarded gelation, no
gelation nor Plasmin Deactivation in UHT Milk
proteolysis after
storage of 9 mo at Heat-stable proteinases produced by psychrotrophic bacterial
20 ◦ C contaminants of raw milk are also capable of causing gelation

262 Comprehensive Reviews in Food Science and Food Safety r Vol. 10, 2011 
c 2011 Institute of Food Technologists®
UHT milk processing and effect . . .
of UHT milk. Several authors have attempted to correlate the
level of bacterial proteinase with the time to gelation during
storage of UHT milk and then to make recommendations on
the maximum advisory level of proteinase to ensure a long shelf
life. The severity of heat treatments during preheating and steril-
ization is a very important consideration in retarding gelation in
UHT milk, especially where this is initiated by plasmin-catalyzed
proteolysis.
Samuelson and Holm (1966) observed that increasing the ster-
ilization temperature from 142 to 152 ◦ C and time from 6 to
12 s, allowed milk to be stored longer without gelation. Zadow
and Chituta (1975) confirmed that an increase in gelation time
is observed when the sterilization temperature is increased from
135 to 140 ◦ C along with holding time from 3 to 5 s. Adams
and others (1975) reported that the protease produced by psy-
chrotrophs of dairy origin are most active at 45 ◦ C, but their ac-
tivity is reduced to 25% of maximal at normal room temperature.
Psychrotrophic populations under 10000/mL are able to produce
about 10 or more units of heat-stable protease that would shorten
the shelf life of sterile milk significantly (Speck and Adams 1976).
In contrast, heat-resistant protease activity at 40 ◦ C did not appear
correlated with the bacterial populations in raw milk. Correlation
between Standard Plate Count, psychrotrophic, and proteolytic-
psychrotroph counts, and protease concentration was 0.35, 0.41,
and 0.54, respectively (West and others 1978). Topçu and others
(2006) observed the effect of raw milk quality (total and psy-
chrotrophic bacterial and SCCs, proteinase, and plasmin activity)
and UHT temperature (145 or 150 ◦ C for 4 s) on proteolysis in
UHT milk processed by a direct (steam injection) system during
storage at 25 ◦ C for 180 d. High proteinase activity was measured
in low-quality raw milk, which had high SCC, bacterial count,
and plasmin activity. Sterilization at 150 ◦ C extended the shelf life
of the UHT milk by reducing proteolysis, gelation, and bitterness.
Driessen (1983) suggested that proteolysis by bacterial enzymes
is accompanied by an increase of nonprotein N and the formation
of para-κ-casein, while the plasmin produces an increase of non-
casein N and the formation of γ -caseins. This also has diagnostic
value for the detection of the cause of gelation and bitterness.
Mottar and others (1985) reported that changes in the quantity
of 2 specific protein breakdown components during refrigerated
storage of raw milk show a significant correlation with the bacte-
rial count. Raw milk containing these compounds at higher than
a certain level should not be used for UHT processing.
Mitchell and Ewings (1985) reported that UHT milk that ex-
hibited a bitter taste before gelation occurred showed increases in
nonprotein nitrogen (NPN) content from 0.03% to 0.06%. Za-
lazar and others (1996) showed an increase of up to 21% in free
(noncasein-bound) sialic acid (N-acetyl neuraminic acid), a carbo-
hydrate present in κ-casein, in UHT milk during storage. This was
attributable to the action of proteinases from psychrotropic bacteria
on κ-casein, releasing the sialic acid-containing glycomacropep-
tide. They concluded that the determination of free sialic acid is a
useful method for monitoring proteolysis by bacterial proteinases
in UHT milk during storage and to provide an early warning of
the onset of gelation. Manji and others (1986), while attributing
the greater propensity to gelation of milk sterilized by direct UHT
processes (compared with indirectly sterilized milk) to higher plas-
min and plasminogen activities, found no correlation between the
shelf life of the directly sterilized milk and the extent of proteoly-
sis. It should also be noted that the difference in gelation times of
UHT milks stored at different temperatures cannot be explained
by the level of proteolysis; more proteolysis occurs at 40 ◦ C than

c 2011 Institute of Food Technologists® Vol. 10, 2011 r Comprehensive Reviews in Food Science and Food Safety 263
at 30 or 20 ◦ C (Renner 1988), but gelation is retarded in milks
stored at 40 ◦ C (Kocak and Zadow 1985b).
Manji and others (1986) observed that there is a correlation
between the extent of proteolysis and plasmin activity. Rate of
transformation of plasminogen to plasmin was similar for sam-
ples stored at 37 ◦ C and 22 to 25 ◦ C but significantly slower at
4 ◦ C. They also found that the extensively degraded proteins were
unable to form a gel matrix and if any gel structure that may
have formed may have been degraded by continued proteolytic
activity, thus reducing the apparent viscosity. Manji and Kakuda
(1988) observed that milks with 28% whey protein denaturation
gelled after 115 d, while more severely heat-treated milk with 66%
denatured whey protein gelled after 150 d. These results indicate
that the formation of complexes between whey proteins and ca-
seins, which accompanies denaturation, plays an important role in
determining the onset of gelation. McMahon (1996) concluded
that milk treated by indirect UHT processes is more stable than
milk treated by direct UHT processes.
Farrell and Thompson (1990) reported that β-lactoglobulin
inhibited phosphoprotein phosphatases, including mammary
alkaline phosphatase. The ability of β-lactoglobulin to inhibit
phosphatase activity is influenced by acetate, calcium ions, and
genetic variants of β-lactoglobulin. They also hypothesized that
phosphatase activity in milk regulated phosphate content and that
phosphatase, in turn, is regulated by β-lactoglobulin, calcium,
potassium, and sodium. Thus, β-lactoglobulin could have a phys-
iological role in enzyme regulation.
Garcı́a-Risco and others (2003) observed the effects of high
pressure (up to 400 MPa), applied at room temperature, on native
proteinase activity of milk by means of plasmin activity, plasmin-
derived activity after plasminogen activation. The pressure con-
ditions assayed did not lead to plasmin inactivation and only
decreased around 20% to 30% total plasmin activity after plas-
minogen activation. In milk, plasmin activity was shown to resist
at least 400 MPa applied for 30 min at 25 ◦ C (López- Fandiňo and
others 1996), while these conditions reduced the total plasmin
activity after plasminogen activation by 25% (Garcı́a-Risco and
others 1998). Pressurization at higher temperatures considerably
increased plasmin inactivation in milk, which reached 86.5% after
treatments at 60 ◦ C. Plasmin was also baro stable in buffer at room
temperature, resisting up to 600 MPa for 20 min, but it was signif-
icantly inactivated at 400 MPa in the presence of β-lactoglobulin
(Scollard and others 2000).
Ways to Improve Shelf Life of UHT Milk
To date, fluid product shelf life extension has focused primarily
on reducing and controlling the presence of bacterial contaminants
to achieve better product quality for longer periods. The extended
shelf life and shelf stability are definite advantages of UHT milk.
Shelf life is the storage time before quality drops to an unacceptable
level with subjective attributes that include taste, color, odor, gela-
tion, sedimentation, separation, and viscosity. These attributes can
be affected by raw product quality, pretreatment process, process
type, homogenization pressure, deaeration, postprocess contami-
nation, AP, and package barriers. Spoilage of raw milk prior to
processing can occur from poor sanitation and inadequate storage
temperatures. Some of the ways to improve shelf life of UHT milk
are described as follows:
Quality of raw milk
The use of high-quality raw milk is of utmost importance for
achieving a long shelf life of UHT milk (Law and others 1977).
UHT milk processing and effect . . .

Storage of raw milk at low temperature (<4 ◦ C) for a mini-

mum period of time (≤48 h) minimizes growth of psychrotrophic
bacteria and, consequently, the amount of extracellular bacterial
proteinases produced in the milk before heat treatment. Speck and
Adams (1976) suggested that preventing contamination of the raw
milk with psychrotrophic bacteria would be difficult and expen-
sive. The heat resistance of the enzymes precludes their destruction
at UHT. If the raw milk bacterial count is <25000 CFU/mL, then
the raw milk SCC will be the most important determinant of shelf
life in pasteurized extended shelf life milk with respect to devel-
opment of off-flavors when postpasteurization bacterial growth is
controlled (such as below 500000 CFU/mL) (Barbano and others
2006).

Low-temperature inactivation of proteinases

Barach and others (1976) reported that heat-resistant enzymes in
milk could be inactivated by treatment at low temperature (about
55 ◦ C) for a prolonged period of holding (30 to 60 min). The
effectiveness of such “low-temperature-inactivation” treatment is
independent of proteinase concentration and does not significantly
alter the flavor of the milk. The method can be applied before or
after sterilization or to aged sterile milk and is most effective when
used in milk at least 1 d after UHT treatment. LTI at 55 ◦ C is only
effective up to 60 min; thereafter, the inactivation rate of LTI when
combined with UHT treatment is similar to that for UHT treat-
ment alone. The effect may be due to rapid autodigestion at 55 ◦ C,
but this does not fully explain the change of inactivation charac-
teristics after 60 min at this temperature. At 55 ◦ C, the proteinase
undergoes a unique conformational change followed by aggrega-
tion of altered proteinase with casein to form an enzyme-casein
complex that causes inactivation of the enzyme. The combination
of LTI and UHT sterilization can prolong the shelf life (up to 3
times) of sterile skimmilk containing psychrotrophic bacterial pro-
teinase. Furthermore, some proteinases are quite resistant to heat
treatment at 55 ◦ C for an hour, so its usefulness for age gelation
may be limited. LTI did not alter the flavor or protein content of
the milk and κ-casein was affected by the protease. Such a process,
if feasible on a commercial scale, could offer the best solution to
the problem presented by heat-stable proteases. Maximum low-
temperature inactivation occurred at 55 ◦ C and only about 30%
loss in activity was expected to occur by heating for 60 min. The
extent of protease inactivation appeared to be independent of pro-
tease concentration and, therefore, could occur at the low protease
that might be in raw milk.
Heat treatment during preheating and sterilization
Adequate heating is required for denaturation of most of the
β-lactoglobulin and complexation with casein. Such high heat
treatment also inactivates plasmin. For the same bactericidal ef-
fect, indirect heating produces milk that is more stable to gelation
than that produced by direct heating. Lu and Nielsen (1993) added
serine proteinase inhibitors, namely, trypsin inhibitor, aprotinin,
and diisopropylfluorophosphate, to UHT milk to inhibit the plas-
min. In these cases, no proteolysis occurred and gelation was not
observed after 9 mo of storage at 20 ◦ C. Recently, 2 proteinase
inhibitors, PA inhibitor-1 and alpha 2-antiplasmin, were isolated
from bovine milk. Since these proteinase inhibitors are heat labile,
their use in control of the plasmin system would only be possible
if they were added (aseptically) after heat processing. The shelf life
of UHT milk could be enhanced by addition of PA inhibitor-
1 at a concentration of 125 mg/mL; at this level, no PA could
be detected. Plasmin does not hydrolyze whey proteins and they
Figure 7–ISI heater-principle. (Source: van Asselt and others 2008.)
have some inhibitory effects on plasmin activity. β-Lactoglobulin
A, α-lactalbumin, and bovine serum albumin at concentrations
of 0.2 and 1 mg/mL inhibited plasmin plus plasminogen activ-
ity by 18% and 54%, 19% and 20%, 25% and 63%, respectively,
while β-lactoglobulin B had no inhibitory effect (Politis and oth-
ers 1993). Indirect heating is a very practical means of retarding
age gelation. However, it is unfortunate that heating conditions
that minimize age gelation cause the most cooked flavor in UHT
milk, a characteristic that many consumers dislike.
Addition of sodium hexametaphosphate (SHMP)
Kocak and Zadow (1985b) added calcium chloride, SHMP
(0.05%, 0.1% w/w), sodium citrate, and ethylenediaminete-
traacetic acid (0.1%, 0.3% w/w) to raw milk that had been stored
at 2 ◦ C for 120 to 168 h and processed the milk at 140 ◦ C for
4 s. The addition of 0.05% calcium chloride or 0.1% SHMP to
milk before UHT processing resulted in a considerable increase in
stability, with no gelation evident after 500 d at 25 ◦ C. Addition
of a low level of SHMP would facilitate bridging between ion-
ized groups of casein micelles that would not otherwise form an
ionic bond. This would hold the κ-casein more tightly to the mi-
celle and delay release of the βκ-complex, thus retarding gelation
of UHT milk during storage. The results suggest that polyphos-
phates, such as SHMP, inhibit the 2nd stage of gelation involving
protein coagulation. Addition of sodium phosphate and sodium
citrate accelerate gelation in UHT milk, while polyphosphates,
such as SHMP, delay gelation (Samuelson and Holm 1966).
ISI heating
The ISI heater is a new type of steam injection that enables fast
heating (shorter than 0.2 s holding time) and high temperatures
(150 to 180 ◦ C). A schematic overview of the ISI heater is shown
in Figure 7. In the ISI, the product is pumped through a pipe with a
narrow end (nozzle, 1 to 2 mm). The wall of this pipe contains sev-
eral small openings through which high-pressure steam is injected,

264 Comprehensive Reviews in Food Science and Food Safety r Vol. 10, 2011 
c 2011 Institute of Food Technologists®
UHT milk processing and effect . . .
enabling very fast heating of the product. The milk can be heated
at 80 ◦ C (during different residence times) before (preheated) or
after (postheated) the heat treatment with the ISI (0.2 s, 180 ◦ C).
After heating, the product can be instantaneously cooled using
flash cooling (van Asselt and others 2008). The heat treatment in
industrial applications is much shorter, but a higher temperature
is applied. For example, Tetra Pak’s Tetra Therm Aseptic Plus 2
(Deeth and Datta 2003) includes a preheating of approximately
45 s at 90 ◦ C. With respect to plasmin inactivation, this heat load
is equivalent to a heat load of a treatment of 80 ◦ C during 300 s.
In order to optimize the process, the effect of partly (30%) dena-
tured β-lactoglobulin was included. The level of 30% denatured
β-lactoglobulin was chosen as previous experiments with the ISI
showed that application of ISI-heating resulted in that level of
denaturation (Huijs and others 2004). The results showed that a
postheat treatment is sufficient to reduce the amount of plasmin
below 1% of its initial level. By applying these new kinetics, the
heat load for currently applied UHT treatments of milk can be
reduced while obtaining a sufficient inactivation of plasmin (that
is, <1%) and to achieve a 6 decimal reduction of B. sporothermod-
urans. This opens the way for the production of extended shelf
life milk with even less product degradation (that is, <50% de-
naturation of β-lactoglobulin) compared with currently available
UHT products (that is, >50% denaturation of β-lactoglobulin)
and improved taste characteristics (van Asselt and others 2008).
Addition of the sulfhydryl (SH) group-blocking agent
N-ethylmaleimide (NEM) added to milk before heating inhibits
denaturation of whey proteins and interaction of these proteins
with caseins. Hong and others (1984) showed that UHT milk,
containing 0.5 g/L NEM, and processed by direct heating, gelled
later (at 52 wk) than indirectly heated milk with the same addi-
tive (at 18 wk); this was in contrast to the corresponding direct-
and indirect-processed control milks that gelled at 18 and 40 wk,
respectively. The reason for the opposite effects of NEM in the 2
milks is unexplained. In concentrated milks, NEM had little effect
on gelation time (7 mo compared with 6 mo for control); however,
disulfide-reducing agents, such as mercaptoethanol and cysteine,
markedly accelerated gelation (gelation time <1 mo). Lysine in-
hibits plasminogen activation by competing for the lysine-binding
site present in the kringles of plasmin/plasminogen. It also causes
dissociation of plasmin and plasminogen from casein micelles;
0.2 M lysine released 92% of the plasminogen 97% of the plasmin.
However, the high concentrations of lysine (0.2 M or 29.2 g/L)
necessary to completely inhibit plasminogen activation prohibit its
use as a practical measure for controlling plasmin activity in milk
(Bramley 1998).
Treatment of milk with carbon dioxide or nitrogen
This treatment can effectively inhibit the growth of different
psychrotrophs (Sørhaug and Stepaniak 1997).
Membrane processing of UHT milk
Milk can be treated by applying modern membrane technology
in such a way that high-quality milk concentrates without addi-
tives can be produced with long-life stability by means of UHT
heating. Hirichs (2000) used ultrafiltration (UF) and reverse os-
mosis concentrates made from milk with differing fat and protein
contents that were sheared in defined flow conditions to establish
the critical concentration of the constituents beyond which flow
properties and heat stability change. The heat coagulation time
at 140 ◦ C of milk concentrates was dramatically influenced by
steric interactions if the whole volume fraction of fat and protein

c 2011 Institute of Food Technologists® Vol. 10, 2011 r Comprehensive Reviews in Food Science and Food Safety 265
exceeded 0.5. The higher the fat content with the same whole
volume fraction, the lower the heat stability was because visible
flocs were formed earlier. Increased heat stability was detected for
UF > Nanofiltration (NF) > Reverse Osmosisi (RO) concentrate
because of the reduced ash content (UF < NF < RO). The stor-
age stability can be improved if the ash content is reduced, which
can be achieved by using electrodialysis, or nano or UF.
High-pressure treatments
The plasmin system is very pressure stable at room temperature.
A synergistic effect of high pressure and temperature is observed
in the 300 to 600 MPa and 35 to 65 ◦ C ranges, and a stabiliza-
tion effect could be observed for pressures above 600 MPa. Borda
and others (2004) concluded that particular attention could be
given to the stability of the plasmin system at pressures above 600
MPa and the possibilities of high-pressure thermal inactivation of
plasmin in the 300 to 600 MPa and 36 to 65 ◦ C range. Another
type of high-pressure processing that has been developed is high-
pressure homogenization (HPH). The principle of the operation
is similar to that of conventional homogenizers used in the dairy
industry except that it works at higher pressures (up to 400 MPa).
This technology is also called ultra-HPH (UHPH) depending on
the pressure achieved. Milk treated at 200 MPa at 30 ◦ C had the
longest microbial shelf life (about 21 d) and achieved an outlet
temperature of about 80 ◦ C for 0.7 s, which means that the ther-
mal effect on milk was less than that of the high-pasteurization
treatment. However, at 200 MPa and 30 ◦ C, a marked decrease
of milk pH was observed. With the other UHPH treatments, a
microbial shelf life between 14 and 18 d, similar to that observed
for high-pasteurized milk, was obtained. Therefore, the micro-
bial data indicate the possibility of obtaining UHPH-treated milk
with equal or better microbial shelf life than high-pasteurized milk.
The UHPH treatment, besides achieving a reduction in microbial
counts, generated changes in the physicochemical properties such
as color, viscosity, pH, and acidity. Color, texture, and mouthfeel
are important signals that determine consumer perception of the
freshness of milk (Pereda and others 2007).
Conclusion
UHT and AP of milk is a well-established technology in many
countries. Direct and indirect heating systems are used along with
sterile packages and form-fill-seal systems. Advantages of UHT
milk include reduced energy consumption, extended shelf life, and
ambient storage and distribution conditions. Commercial success
of UHT is affected by factors such as, postprocess contamination,
customer acceptance, chemical/physical changes resulting from
heat treatment, and extended storage. Age gelation is a major
factor limiting the shelf life of UHT milk. It can be explained
by a 2-stage process involving formation, during heating, of a
β-lactoglobulin–κ-casein complex that cross-links after partial or
complete release from the casein micelle to form a protein network
gel. Proteolysis, by native milk plasmin or bacterial proteinases, ac-
celerates gelation by facilitating release of the complex from the
micelle. Factors that influence the shelf life of UHT milk are age
of cow, stage of lactation, seasonal changes, microbial quality of
raw milk, storage temperature, fat content of milk, and hydrol-
ysis of lactose. The shelf life of UHT milk can be increased by
considering the above-mentioned factors and along with new im-
proved manufacturing methods. Thus, by increasing the shelf life
of UHT milk by using techniques such as ISI-heating, LTI, mem-
brane processing, UHPH, consumers will have more choices in
the marketplace.
UHT milk processing and effect . . .
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