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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 worldwide. 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 reect 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 undesirable effects such as browning, loss of nutrients, sedimentation, fat separation, cooked avor also take place. Gelation of UHT milk during storage (age gelation) is a major factor limiting its shelf life. Signicant factors that inuence 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 additives. 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 shelfstable product. The most commonly applied technique to achieve this is heat treatment. The rst system consisting of indirect heating with continuous ow (125 C for 6 min) was manufactured in 1893. Patented in 1912, the continuous-ow, 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 lled 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-ll 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, Kundli131028, 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 lling 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 increased 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 Packaging 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 almost 75% of the total volume but lost some of the market share to polyethylene terephthalate bottles and pouches. In terms of numbers 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

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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 Kingdom 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 avor 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, ller speeds, and packaging costs (David and others 1996). Figure 1Curves representing the speed of changes in chemical

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 ow of milk, which renders less chemical change in comparison to retort processing (Datta and Deeth 2007). Product characteristics, such as pH, water activity, viscosity, composition, and dissolved oxygen, indicate the processing conditions necessary to achieve commercial sterility. The selection criteria of UHT and AP systems reect customer preferences. The production processes are designed to ensure commercial sterility and acceptable sensory attributes throughout shelf life. To compare the various effects of heat treatments, different values are calculated:

properties and of spore destruction with increasing temperature. (Source: G sta 2003.) o

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.

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 avor changesand for most chemical reactionsis 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 inuence of increased temperature are shown in Figure 1. F 0 value For the microbiological effect, F 0 value is already used in classical canned sterilization technology and is dened 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(T121.1


B and C values The effective working range of UHT treatments is also dened 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 keeping quality of the product when the conditions of B > 1 and C < 1 are fullled. The U.S. FDA accepts F 0 values for thermal processes calculated only from the time and temperature of the product in the holding tube (David and others 1996). The D-value is dened as the required time to decrease microorganism numbers 10-fold at a given temperature (Singh 2007). The process ling and supporting documentation (trial run data, critical factors, equipment sterilization, quality control procedures, and operational procedures) are submitted to FDA for approval of a scheduled process (David and others 1996). Ideal time-temperature proles inactivate bacterial endospores and limit chemical changes with minimal

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Figure 2Bacteriological 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 sufcient heat treatment with minimal avor change. Direct heating imparts less avor change but requires more energy in comparison to indirect heating. Total microbial lethality at constant time and temperature varies between direct and indirect heating systems (Westhoff 1981). The residence time distribution is the time range for a uid 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, ow rate, and ow rate attributes (viscosity) of specic products. Positive reactions in the hold tube include destruction of bacteria, inactivation of enzymes, and hydration of thickeners. Negative reactions include development of off-avor, initiation of off-color, and destruction of vitamins (David and others 1996).

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. Solutions 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.

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 possible undesired changes of physicochemical and sensory properties, as well as, what is even more important, preservation of its nutritional value (Jovanka and others 2008). A study conducted by Korel and Balaban (2002) suggested that odor changes in milk samples inoculated with Pseudomonas uorescens or Bacillus coagulans 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 uorescence spectroscopy and hydroxy methyl furfural (HMF) analysis (Schamberger and Labuza 2006). Elliott and others (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

Chemical changes Direct heat processing imparts less adverse chemical changes compared to indirect heat processing (Elliott and others 2003). In an indirect continuous-ow coiled tube system, the process holding 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: avor, acidity (decreases following direct UHT process), enzyme inactivation, and vitamin decomposition. The heated avor after UHT processing is due to sulfhydryl (SH) groups, which oxidize 5 to 10 d after processing. The oxidation then gradually reduces the cooked avor (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 heating, 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 rancidity 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 partially in UHT processing. A signicant reduction in vitamin B1 (thiamin), B2 (riboavin), B3 (niacin), B6 , B12 , and folate has been reported under the inuence of different milk processing

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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 minimally by UHT processing (Holdsworth 1992) and is correlated to the storage temperatures, initial oxygen content, and packaging choice (Dunkley and Stevenson 1987). le 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 sedimentation.

Factors Inuencing Shelf Life of UHT Milk

The factors that inuence the quality of milk that have an effect on the gelation behavior of UHT milk are discussed here:

Physical changes The unwanted physical attributes associated with UHT milk are brown color, sedimentation, protein destabilization, and fat separation (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, homogenization in direct UHT plants takes place in the downstream position (after the nal 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 direct 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, avor, 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 -lactoglobulin associated with the casein micelle increases with the heating time and the trend is similar for -lactalbumin but to a lesser extent (Elfagm and Wheelock 1978). -Lactalbumin can interact with -casein only in the presence of -lactoglobulin, possibly through the initial formation of -lactalbumin/-lactoglobulin aggregates, which then interact with -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 consequently the attachment to the casein micelles will be less efcient (Oldeld 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 inactivation of a transglutaminase (TG) inhibitor provides improved cross-linking of casein micelles, resulting in improved product texture (Bonisch and others 2004). The color of the UHT milk, that is its intensity, basically represents reection 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 lactuloselysine or fructose-lysine, as well as low molecular weight acids. Lactulose, a molecule derived from lactose isomerization, is a wellknown 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 unjustied overprocessing. When combined with furosine, lactulose also allows assessing authenticity of this type of uid 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-

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 increased proteolytic activity resulting from an elevated level of plasmin. 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 susceptible 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 increased growth of psychrotropic microorganisms and concomitant production of heat-stable enzymes, especially proteinases and lipases. 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 1Effect of psychrotrophic bacteria count in raw milk on gelation time of UHT milk. Bacterial count/CFU/mL <8.0 106 8.0 106 5.0 107
Source: Law and others 1977.

Gelation time, days >140 63 12

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UHT milk processing and effect . . . Storage temperature The temperature of storage markedly inuences 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, 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 if regions of proteins that could take part in proteinprotein interactions are blocked by caseinlactose interactions involving lysine residues. Such interactions precede browning in UHT milk stored at temperatures above 30 C. This hypothesis is supported by Hill and Cracker (1968) who observed that when lysine and arginine residues of -casein molecules were blocked, there was a resultant loss of sensitivity to rennet coagulation, indicating that Maillard browning may lead to an inhibition of -casein hydrolysis.

Standardized milk

Pasteurization 20 s 80 C

Homogenization 20 MPa

Steam Injection 2 s 150 C


Direct expansion cooling 80 C


Aseptic homogenization

15 s 142 C cool to 20 C 40 MPa Fat content UHT-processed skim milk is more susceptible to gelation than UHT whole milk. This can be attributed to an enhanced action Packaging material Cool to 20 C of plasmin and bacterial proteinases in skim milk over whole milk. 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 Aseptic Aseptic suggested that the higher proportion of denatured whey proteins Sterilization packaging packaging not attached to the micelle surface of skim milk may be a reason for its lower resistance to gelation. Gaucher and others (2008b) examined the effects of storage up to 6 mo at different temperFigure 3Examples 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 storage 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 lling 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 surof 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 heatstorage temperature of 5 C as measured by -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-avors 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 coefcient 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 barrier is minimized by maintaining a greater product pressure on Types of UHT Processing Systems for Milk UHT plants became commercially available in 1960 when asep- the sterile side compared to the raw side. The comparisons of timetic lling 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

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Table 2Commercial UHT systems and heating modes. Commercial UHT sterilizer Actijoule Gerbig, sterideal system High heat infusion, tetra therm aseptic plus 2 Languilharre system, thermovac, palarisator, steritwin UHT sterilizer, ultra therm, Da-Si Sterilizer Rotatherm Spiratherm Ultramatic, ahlborn process, sordi sterilizer, UHT steriplak-R, dual purpose sterilizer Votator scraped surface heater, thermutator heater VTIS, ARO-VAC process, uperiser, grindrod
Adapted from Datta and Deeth 2007.

Table 3Aseptic packaging systems. System Asepak ASTEC CKD Combibloc Dole Aseptic Canning System DuPont Canada ERCA Evergreen Gasti Gaulin Hamba manufacturing Hassia Ingko Inpaco International Paper Co. Lieffeld & Lemke Liqui-Box Corp. Manccini Mead Packaging Co. Metal-Box Freshll (Autoprod) Pure-Pak, Inc. Purity Packaging Co. Remy Remy Scholle Corp. Serac Tetra Pak, Inc. Wright Sel
Source: David and others 1996.

Heating mode Indirect electrically heated Indirect heat with tubes Combined heating modes Direct heat with steam infusion

Direct heat with scraped surface Indirect heat with spiral tubes Indirect heat with plates Indirect heat with scraped surface Direct heat with steam injection

Package Bags Bins and tanks Cups Cartons Steel/aluminum cans and lids Bags, pouches Cups Cartons Cups Bags Cups Cups Bags Pouches Rectangular packages Cups Bags Bags Cups Cups Cartons Cups Cups Bottles Bags Bottles Cartons Bags

Sterilant Heat Pressurized steam H 2 O2 H2 O2 + heat Superheated steam H 2 O2 H2 O2 +heat H2 O2 +heat High-pressure steam Ethylene oxide Ultraviolet rays H2 O2 + heat or pressurized steam Chlorine solution + heat H2 O2 + heat H2 O2 + heat H2 O2 + heat Gamma radiation Gamma radiation Citric acid + heat H2 O2 + heat H2 O2 + heat, oxonia H 2 O2 H2 O2 +heat H2 O2 or oxonia Gamma radiation or ethylene oxide H 2 O2 H 2 O2 Gamma radiation or ethylene oxide

point where sterilized packaging enters the sterile zone to where the sealed package is evacuated.

Figure 4Time-temperature curve for processing of milk in a direct system (A) and indirect system (B). (Source: G sta 2003.) o

is dependent upon factors such as, product (pH, water activity, viscosity, specic gravity); microbial prole (number, type, heat resistance); equipment design, and package.

AP Systems for Milk

In AP, raw or unprocessed product is heated, sterilized by holding at high temperature for a predetermined period, then cooled and delivered to a packaging unit for packaging. Packaging material 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 ll the sterile product into sterile packages within the connes of the sterile zone of the ller. The aseptic zone/sterile zone extends from the

Types of milk AP lines There are 5 basic types of AP lines: (1) Fill and seal: preformed containers made of thermoformed plastic, glass, or metal are sterilized, lled in aseptic environment, and sealed. (2) Form, ll, and seal: roll of material is sterilized, formed in sterile environment, lled, sealed, for example, tetrapak. (3) Erect, ll, and seal: using knocked-down blanks, erected, sterilized, lled, sealed, for example, gable-top cartons, cambi-bloc. (4) Thermoform, ll, sealed roll stock sterilized thermoformed, lled, sealed aseptically, for example, creamers, plastic soup cans. (5) Blow mold, ll, 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 include 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 llers 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 llers and associated pipes are sterilized typically

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with heat in the form of steam. Wet heat sterilization using saturated steam is the most dependable sterilant, as microorganisms are more resistant to dry heat, which necessitates higher temperatures (Burton 1988). Sterilants are applied uniformly to the aseptic zone by misting equipment, whereas packaging typically is sterilized by misting or passing through sterilant bath. Sterilization of packaging material is a critical step in the AP system. Therefore, the sterilization process should meet the following requirements for sterilization of packaging materials: (1) Rapid microbiocidal activity; (2) compatibility with surfaces treated, especially packaging material and equipment; (3) easily removed from surface, minimum residue; (4) present no health hazard to the consumer; (5) no adverse effect on product quality in the case of unavoidable residue or erroneous high concentration; (6) present no health hazard to operation personnel around the packaging equipment; (7) compatibility with environment; (8) noncorrosive to surfaces treated; (9) reliable and economical (Ansari and Datta 2003). Sterilants that are commonly used at industrial level include chlorine, iodine, oxonia, food acids, ozone, H2 O2 , and UV light (David and others 1996). Some of these methods are listed in Table 4. The H2 O2 is now the only chemical sterilant for sterilization of packaging materials that has been proved to be acceptable in the United States. The FDA regulations specify that a maximum concentration of 35% H2 O2 may be used for sterilizing
Table 4Methods for sterilizing aseptic packages. Methods Superheated steam Application Metal containers Advantages/ disadvantages High temperature at atmospheric pressure. Microorganisms are more resistant than in saturated steam High temperature at atmospheric pressure. Microorganisms are more resistant than in saturated steam Fast and efcient method UV increases effectiveness of hydrogen peroxide Cannot be used where chlorides are present or where residuals would remain No chemicals used Can be used to sterilize heat-sensitive packaging materials. Expensive. Problems with location of radiation source Reference Collier and Townsend (1956)

food contact surfaces. In a properly designed APs system, a good microbiocidal effect using H2 O2 can be achieved and the level of residue can be effectively controlled to within permissible limits. The residual level of H2 O2 is regulated with a maximum level of 0.5 ppm. Infrared radiation and vaporized H2 O2 have been studied as sterilants for packaging materials (Kulozik and Guilmineau 2003). There are many other chemicals such as peracetic acid, beta propiolactone, alcohol, chlorine, and its oxide, and ozone that have been suggested as having potential for use in sterilizing AP materials (Ansari and Datta 2003).

Postprocess Contamination Concerns in UHT Milk

The problem of posttreatment contamination of in container sterilized product is well known. The contamination can either through poor seal or through pinhole in the container. Post treatment contaminants in UHT milk may be either spores, which would not be expected to be heat resistant enough to survive the heat treatment or nonheat-resistant vegetative organisms. Organisms of the 1st type will probably have entered from the ineffectively sterilized plant downstream from the heat treatment stage of the process, which includes spores of Bacillus cereus (Wilson and others 1960; Davies 1975) and Bacillus licheniformis (Wilson and others 1960). Organisms of the 2nd type will probably have entered through a poorly sealed container after aseptic lling (Hassan and others 2009). Postprocess contamination of the aseptic zone can be attributed to several variables: environmental bioburden, positive air pressure, processing equipment or line turbulence, system gasping, indexing operations, condensate accumulation, unsterile product entry, or bacteriological seeding (David and others 1996). Postprocess contamination occurs in individual cartons if package integrity is compromised. Contamination from isolated package integrity issues occurs more frequently than processing contamination. Rippen (1969) cited typical spoilage in UHT-AP production at a defect rate of 1 of 1000. Manufacturers of aseptic llers target a defect rate of 1/1000 or 1/3000, whereas 1/10000 is an industry standard for aseptically packaged low-acid foods in rigid, semi-rigid, and exible containers (David and others 1996). The following 7 potential failure modes exist for aseptic processing and packaging of foods: (1) Type 1 failure results from raw ingredient, handling, storage, or batching issues. (2) Type 2 failure results from processor and ller cleaning in place, sanitation, preventive maintenance, and presterilization issues. (3) Type 3 failure results from the thermal process heating cycle including regeneration. (4) Type 4 failure results from the cooling cycle including surge tanks. (5) Type 5 failure results from sterilization issues with the package. (6) Type 6 failure results from sterility loss in the aseptic zone or from environmental load. (7) Type 7 failure results from loss of package integrity (David and others 1996).

Dry hot air

Metal or composite juice and beverage containers Plastic containers, laminated foil Plastic containers (preformed cartons) Glass and plastic containers

Denny and Mathys (1975)

Hot hydrogen peroxide Hydrogen peroxide/UV light combination Ethylene oxide

Denny and others (1974) Bayliss and Waites (1982) Blake and Stumbo (1970)

Heat from coextrusion process Radiation

Plastic containers Heat-sensitive plastic containers

Commercial Sterility Testing of UHT Milk Process

Scheduled processes in retort operations and UHT processes inactivate vegetative cells and spores of pathogenic bacteria. The genera Bacillus and Clostridium are the primary sporeforming spoilage microbes (Ravishankar and Maks 2007). Spoiled packages are
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identied as at sours or swells. Spoilage organism identication 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 contamination is indicated by mixed ora containing heat-sensitive organisms (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 identied 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 ller 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, ller restarts, and production end. An ideal sampling plan provides sterility assurance within a reasonable cost structure (Farahnik 1982). Microbial testing is viewed as an additional verication 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 enumeration and viable enumeration. Viable cells are counted using standard plate counts, most probable number, membrane ltration, 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). treatments. Proteolysis in UHT milk can cause the development of bitter avor and lead to an increase in viscosity, with eventual formation of a gel during storage, which is a major factor limiting its shelf life and market potential. The enzymes responsible for the proteolysis are the native milk alkaline proteinase, plasmin, and heat-stable extracellular bacterial proteinases produced by psychrotrophic bacterial contaminants in raw milk. Proteolysis of casein caused by plasmin is responsible for gelation and bitterness of UHT milk during storage.

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, distribution, 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 inspections for exible containers include visual observation, dye test, squeeze test, seal teardown, and conductivity (Grow 2000). Additional tests identied by Holdsworth (1992) include the ination test, compression test, decompression test, biotesting, ultrasound imaging, mechanical tests, and headspace indicators. The mechanical tests on lled packages include stress testing, stack testing, load vibration, and impact resistance. Secretion and biochemical properties of proteinases and

Psychrotrophic bacteria in milk Psychrotrophic microorganisms that are capable of growing in milk at temperatures close to 0 C are represented by both Gramnegative bacteria (such as Pseudomonas, Achromobacter, Aeromonas, Serratia, Alcaligenes, Chromobacterium, and Flavobacterium spp.) and Gram-positive bacteria (such as Bacillus, Clostridium, Corynebacterium, Streptococcus, Lactobacillus, and Microbacterium spp.). Furthermore, refrigeration conditions under which raw milk is stored selects for growth of psychrotrophs, many of which produce heatstable enzymes while milk awaits processing. Following heat treatment, these enzymes can continue to degrade milk in the absence of viable bacterial cells. A variety of psychrotrophic organisms, including P. uorescens, 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. Pseudomonas spp. usually represents no more than 10% of the microora of freshly drawn milk; however, they are the most important of the psychrotrophs that dominate the microora of raw or pasteurized milk at the time of spoilage. This genus is also known to be strongly lipolytic (Srhaug and Stepaniak 1997). Thermoresistant psychrotrophs. Among the microorganisms that survive pasteurization (the thermoduric microorganisms), sporeforming Bacillus spp. dominate those that are also psychrotrophs. Other thermoduric psychrotrophs are represented in the genera Arthrobacter, Microbacterium, Streptococcus, Corynebacterium, and Clostridium. Clostridium is also sporeformer and anaerobic. 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 rst detected in UHT milk in 1985 in southern Europe and in UHT milk in Germany in 1990 (Pettersson 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). lipases from psychrotrophs Among the hydrolases from psychrotrophic bacteria in milk, those from Pseudomonas spp. have been the most frequently studied and are secreted mainly at the end of the stationary phase of growth. The maximum concentrations of proteinases get accumulated 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 containing 1 zinc atom and up to 8 calcium atoms per molecule. Most proteinases from psychrotrophs have milk-clotting activity, are readily able to degrade -, s1 - and -casein, and have low activity on nondenaturated whey proteins (Srhaug and Stepaniak 1997).

Plasmin Activity
Since the adoption of refrigerated bulk tanks for the collection 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 extremely 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

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UHT milk processing and effect . . . Plasmin, plasminogen, plasminogen activators (PAs), PA inhibitors (PAIs), and plasmin inhibitors (PIs) Plasmin (EC, 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 potential 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 -casein, s2 casein, and (more slowly) 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 plasminogen can vary considerably with the stage of lactation, breed, age, and presence of mastitis (Grufferty and Fox 1986). The nal plasmin activity of milk and, subsequently, milk casein hydrolysis, 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, -lactoglobulin also could inhibit these enzymes (Korycka-dahl and others 1983). Many interactions between plasminogen, plasmin, 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 microorganisms (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). 1 -Antitrypsin, 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 unstable. 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 plasmin 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 (Heat-labile) PAs (Heat-stable) PIs (Heat-labile)

75 C for 15 s. The PI in the isolated fractions was suggested to be 2 -antiplasmin, since it reacted immunochemically with polyclonal goat antihuman 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 claried samples indicated extensive breakdown 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 proteinase, preferentially hydrolyzes the hydrophilic glycomacropeptide from -casein on the outside of the micelle. This leaves the casein micelle largely intact and also reduces steric repulsion between micelles, allowing the formation of a more compact gel. By contrast, plasmin attacks -casein located inside the micelle, thereby disrupting the micelle and inhibiting the formation of a strong gel. Extensive proteolysis of skim milk by plasmin completely claries skim milk (Datta and Deeth 2003). Plasmin itself is a heat-stable enzyme that survives pasteurization and many UHT processes (D140 C is 32 s) and the initial (1 d) plasmin activity of UHT milk containing KIO3 was significantly 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 (oating 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 (Srhaug 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 modication (such as destruction of cystine cross-links and deamination of asparagine and glutamine residues). Proteinases, lipases, and phospholipase (from psychrotrophic bacteria, mostly Pseudomonads), which are stable at high temperatures and survive pasteurization 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 expanded hydrophobic core. Many of these features are apparently also stabilizing factors in heat-resistant proteinases, lipases, and phospholipase C from all psychrotrophic microorganisms. Lipases

Plasminogen (Heat-stable)

Plasmin (Heat-stable) Casein

Peptides (-Cn) and proteose peptone

Figure 5The plasmin-plasminogen system (adapted from Richardson 1983).

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Table 5Heat resistance of enzymes from Pseudomonads. Enzyme Protease (s) Protease (s) Protease Protease Protease Lipase Source Pseudomomas Total psychrotrophic ora of milk Pseudomomas uorescens P26 Pseudomomas Pseudomomas P. uorescens 22F Heat resistance D150 C = 90 s Survived 149 C/10 s D149 C = 90 s Survived unspecied UHT sterilization temperature Survived 135 C/3.5 min 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 (Srhaug and Stepaniak 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. Putrefactive 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 uid 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., Alcaligenes 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 recommends 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.

the highly reactive SH groups cause irreversible denaturation of plasmin (Crudden and others 2005). The above implies that a certain degree of denaturation of -lactoglobulin is necessary to increase the inactivation of plasmin and plasminogen. On the other hand, denaturation of -lactoglobulin induces the formation 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 -lactoglobulin is high enough to inactivate plasmin to avoid bitterness but low enough to minimize the formation of deposit and product degradation. Previous experiments showed that milk heated with the innovative steam injection (ISI) system had a degree of denaturation of -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 -lactoglobulin is usually not taken into account 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 -lactoglobulin, interacting with casein, chiey -casein, of the casein micelle. The major proteinaceous linkages that develop during the heat treatment result in formation of -lactoglobulin--casein complexes (-complexes). -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 SH groups became reactive after UHT treatment with an indirect heating system. The reactive SH can react intramolecularly to form -lactoglobulin aggregates, or intermolecularly to form disulfhide bonds between -lactoglobulin and other SH-containing molecules such as casein and proteins of the milk fat globule membrane. In the casein micelle, -casein exists at the surface and is available for such interaction with -lactoglobulin. Following these initial heat-induced changes, other changes occur slowly in UHT milk during storage and result in the formation of a 3D protein network that causes the milk to thicken and then gel. The ease with which a milk will gel is determined by the extent of 3 processes leading to gelation: the interaction between -lactoglobulin and -casein (as opposed to the self-association of denatured -lactoglobulin); the release of the -complex from the casein particle; and the cross-linking of the -complexes and associated proteins. When these associations (-complexes) are disrupted, -casein is released along with its attached -lactoglobulin. The released -complexes are observed as protuberances and tendrils on the micelle surface. The disruption of the -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 > > s1 and = s2 > s1 > , respectively. Overall, the relationship between added puried proteinase activity and gelation time can be obtained from the following equation:

Inactivation Kinetics of Enzymes Present in Milk

UHT treatments can be sufcient to inactivate microorganisms and obtain a sterile product but provide insufcient heat load to reduce plasmin activity and obtain a stable product. Insufcient inactivation causes bitter-tasting milk, resulting in product loss for producers and consumers of milk. To avoid bitterness, an inactivation 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 specic 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 -lactoglobulin is found to affects the rate of plasmin inactivation (Crudden and Gelation time (months) = 2.3916 X0.6449 , others 2005). Due to thermal processing free SH groups will become available when -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 -lactoglobulin, test kit using dehydrogenase as the substrate and relating the
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Figure 6Model of age gelation of UHT milk showing (1) formation of the -complex, (2) its dissociation from micelles during storage, and (3) subsequent gelation of the milk through cross-linking of the -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 directly on the -complex but cleave the peptide bonds that anchor the -casein to the casein micelle, facilitating release of the -complex. This dissociation of -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 -complexes and formation of a 3D network of cross-linked proteins (Figure 6) and effect of proteolytic enzymes 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 proteolysis, and led to the early formation of sediments. The results of this study suggest strongly that plasmin activity is a major inuence on the storage stability of UHT milk. Kelly and Foley (1997) concluded that KIO3 protected plasmin from inactivation by complexation with -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 Maillard 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 suggestion. Samel and others (1971) reported that blockage of -NH2 groups of lysine residues in casein micelles of UHT milk prevents micelles from interacting with each other and may retard age gelation due to modication 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

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plasmin, bacterial proteinase, or both. If it is caused by plasmin, it is likely that the UHT processing conditions are too mild that causes less denaturation of plasmin and whey proteins. This results in less whey proteincasein interaction and less inhibition of plasmin action on the casein that is most commonly encountered in the direct UHT processes, steam infusion, or injection (Manji and others 1986; Manji and Kakuda 1988). If the proteolysis is caused by bacterial proteinases, the quality of the raw milk is implicated. The most common cause is high levels of psychrotrophic bacteria in the raw milk. Inadequately cleaned equipment that supports bacterial growth and production of proteinases can also be a cause (Driessen 1983). Protease activity in the sterile skim milk was determined by measuring proteolysis at weekly intervals. Single samples from 3 bottles were assayed by the Hull method (1947) with FolinCiocalteau reagent. Measurements were continued until the increase in absorbance exceeded 0.6 or until whey separation and gelation. The rates of proteolysis were determined by calculating the regression of proteolysis on time. From the slopes of the regression lines, the percentage inactivation of proteolysis caused by low-temperature-inactivation (LTI) was calculated as: Percent of inactivation = 100 (slope of control slope of LTI sample)/slope of control. A sensitive assay for protease activity based on the reaction of primary amino groups of trichloroacetic acid (TCA) soluble peptides and amino acids with uorescamine (4-phenylspiro[furan-2(3H), 1 -phthalan]-3,3 dione) was applied to sterile milk (Chism and others 1979). The assay was linear in the range of 2 to 50 nmol of amino groups per aliquot. The assay is suitable for determining proteolytic activity in sterile milk products and for determining protease activity in general. Plasmin and plasminogen can also be determined spectrophotometrically using the chromogenic substrate H-D-valyl-L-leucylL-lysyl-4-nitroanilide (VLLN) as described by Korycka-dahl and others (1983) with some modications. Milk samples must be defatted at 10 C by centrifugation (5000 g for 20 min) and skimmed milk obtained must be incubated with 50-mM -aminon-caproic acid (EACA) for 2 h at room temperature to dissociate plasmin and plasminogen from casein micelles. A plasmin activity was measured without adding urokinase- and plasminogenderived activity and calculated as total activity minus plasmin activity and was expressed in the same units. One unit of activity was dened as the amount of enzyme that produces a change in absorbance (path length 1 cm) at 405 nm of 0.0001 in 1 min at pH 7.4 and 37 C in the dened reaction mixture (Manji and others 1986). Plasmin activity in dairy products can also be measured with synthetic chromogenic or uorogenic substrates. Synthetic substrates can be made by linking Lys peptides to chromogenic or uorogenic tags. VLLN is a synthetic substrate that can be used to determine plasmin activity as it hydrolyzes this peptide to release 4-nitroaniline, a compound that absorbs light at 405 nm (Bastian and others 1993). The most sensitive methods reported, including enzyme-linked immunosorbent assays (ELISAs), were able to detect enzyme activity or the presence of enzyme when the number of Pseudomonas cells present was about 106 CFU/mL. ELISAs that can detect 0.25 ng/mL of proteinases or 20 ng/mL of lipase from Pseudomonas spp. have been reported. An ELISA that detects proteolysis by measuring the accumulation of glycomacropeptide released from -casein by proteinases from Pseudomonas spp. has also been developed (Srhaug and Stepaniak 1997). Datta and Deeth (2003) differentiated the peptides produced by enzymes responsible for the proteolysis from the native milk alkaline proteinase, plasmin, and heat-stable, extracellular bacterial proteinases produced by psychrotrophic bacterial contaminants in the milk prior to heat processing. In order to differentiate, these peptide products, reversed-phase high-performance liquid chromatography (HPLC), and the uorescamine method were used to analyze the peptides soluble in 12% TCA and those soluble at pH 4.6. The TCA ltrate showed substantial peptide peaks only if the milk was contaminated by bacterial proteinase, while the pH 4.6 ltrate showed peptide peaks when either or both bacterial and native milk proteinases caused the proteolysis. Results from the uorescamine test were in accordance with the HPLC results whereby the TCA ltrate exhibited signicant proteolysis values only when bacterial proteinases were present, but the pH 4.6 ltrates showed signicant values when the milk contained either or both types of proteinase. A procedure based on these analyses can be proposed as a diagnostic test for determining which type of proteinase-milk plasmin, bacterial proteinase, or both are responsible for proteolysis in UHT milk.

Table 6Effect of proteolytic enzymes on gelation. Enzyme/enzyme Plasmin/plasminogen (Manji and others 1986) Plasmin (Grufferty and Fox 1986). Bacterial proteinases (Speck and Adams 1976) Psychrotroph proteinases (Cogan 1977) Bacterial, P. uorescens (Law and others 1977) Bacterial, plasmin Plasmin (Snoeren and others 1979) Bacterial (Fox 1981; McMahon 1996) Proteinases (Harwalkar 1982) Proteinases (Samel and others 1971). Plasmin isolated from raw milk (Manji and Kakuda 1988). Proteinase inhibitors (de Koning and others 1985) Experiment Direct, indirect heating pH opt, 6.5; temp opt, 45 C Results and conclusions Indirect inactivated plasmin; no gelation to 182 d in indirect inactivated plasmin Caused degradation of casein and gelation in 60 d Caused bitter avor and gelation Responsible for gelation Responsible for gelation; time to gel dependent on extent of growth in raw milk Both caused gelation Plasmin caused gelation in 90 d Bacterial proteinases cause gelation in 21 d More proteolysis and gelation in directly heated milk More proteolysis but less gelation at 37 C; gelation not due to proteolysis No correlation between amount of proteolysis and gelation time but some proteolysis necessary for gelation Direct heating Inhibited plasmin action; retarded gelation, no gelation nor proteolysis after storage of 9 mo at 20 C

Good quality milk <2700 CFU/mL Poor quality milk, 2 106 CFU/mL Direct compared with indirect Storage at 4, 20, 30, 37 C Unconcentrated milk

Plasmin Deactivation in UHT Milk

Heat-stable proteinases produced by psychrotrophic bacterial contaminants of raw milk are also capable of causing gelation

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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 sterilization 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 sterilization 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) conrmed 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 psychrotrophs of dairy origin are most active at 45 C, but their activity 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 signicantly (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 proteolyticpsychrotroph counts, and protease concentration was 0.35, 0.41, and 0.54, respectively (West and others 1978). Topcu and others (2006) observed the effect of raw milk quality (total and psychrotrophic 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--casein, while the plasmin produces an increase of noncasein N and the formation of -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 specic protein breakdown components during refrigerated storage of raw milk show a signicant correlation with the bacterial 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 exhibited a bitter taste before gelation occurred showed increases in nonprotein nitrogen (NPN) content from 0.03% to 0.06%. Zalazar and others (1996) showed an increase of up to 21% in free (noncasein-bound) sialic acid (N-acetyl neuraminic acid), a carbohydrate present in -casein, in UHT milk during storage. This was attributable to the action of proteinases from psychrotropic bacteria on -casein, releasing the sialic acid-containing glycomacropeptide. 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 plasmin and plasminogen activities, found no correlation between the shelf life of the directly sterilized milk and the extent of proteolysis. 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

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 samples stored at 37 C and 22 to 25 C but signicantly 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 caseins, 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 -lactoglobulin inhibited phosphoprotein phosphatases, including mammary alkaline phosphatase. The ability of -lactoglobulin to inhibit phosphatase activity is inuenced by acetate, calcium ions, and genetic variants of -lactoglobulin. They also hypothesized that phosphatase activity in milk regulated phosphate content and that phosphatase, in turn, is regulated by -lactoglobulin, calcium, potassium, and sodium. Thus, -lactoglobulin could have a physiological role in enzyme regulation. Garca-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, plasminderived activity after plasminogen activation. The pressure conditions assayed did not lead to plasmin inactivation and only decreased around 20% to 30% total plasmin activity after plasminogen activation. In milk, plasmin activity was shown to resist at least 400 MPa applied for 30 min at 25 C (L pez- Fandi o and o n others 1996), while these conditions reduced the total plasmin activity after plasminogen activation by 25% (Garca-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 significantly inactivated at 400 MPa in the presence of -lactoglobulin (Scollard and others 2000).

Ways to Improve Shelf Life of UHT Milk

To date, uid 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 denite 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, gelation, sedimentation, separation, and viscosity. These attributes can be affected by raw product quality, pretreatment process, process type, homogenization pressure, deaeration, postprocess contamination, 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).

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Storage of raw milk at low temperature (<4 C) for a minimum 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 difcult and expensive. 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 development of off-avors 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 signicantly alter the avor 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 treatment alone. The effect may be due to rapid autodigestion at 55 C, but this does not fully explain the change of inactivation characteristics after 60 min at this temperature. At 55 C, the proteinase undergoes a unique conformational change followed by aggregation 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 proteinase. 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 avor or protein content of the milk and -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 lowtemperature 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 protease 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 -lactoglobulin and complexation with casein. Such high heat treatment also inactivates plasmin. For the same bactericidal effect, 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 diisopropyluorophosphate, to UHT milk to inhibit the plasmin. 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 inhibitor1 at a concentration of 125 mg/mL; at this level, no PA could be detected. Plasmin does not hydrolyze whey proteins and they

Figure 7ISI heater-principle. (Source: van Asselt and others 2008.)

have some inhibitory effects on plasmin activity. -Lactoglobulin A, -lactalbumin, and bovine serum albumin at concentrations of 0.2 and 1 mg/mL inhibited plasmin plus plasminogen activity by 18% and 54%, 19% and 20%, 25% and 63%, respectively, while -lactoglobulin B had no inhibitory effect (Politis and others 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 avor 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 ethylenediaminetetraacetic 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 ionized groups of casein micelles that would not otherwise form an ionic bond. This would hold the -casein more tightly to the micelle and delay release of the -complex, thus retarding gelation of UHT milk during storage. The results suggest that polyphosphates, 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 several small openings through which high-pressure steam is injected,

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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 ash cooling (van Asselt and others 2008). The heat treatment in industrial applications is much shorter, but a higher temperature is applied. For example, Tetra Paks 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%) denatured -lactoglobulin was included. The level of 30% denatured -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 sufcient 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 sufcient inactivation of plasmin (that is, <1%) and to achieve a 6 decimal reduction of B. sporothermodurans. This opens the way for the production of extended shelf life milk with even less product degradation (that is, <50% denaturation of -lactoglobulin) compared with currently available UHT products (that is, >50% denaturation of -lactoglobulin) and improved taste characteristics (van Asselt and others 2008). exceeded 0.5. The higher the fat content with the same whole volume fraction, the lower the heat stability was because visible ocs were formed earlier. Increased heat stability was detected for UF > Nanoltration (NF) > Reverse Osmosisi (RO) concentrate because of the reduced ash content (UF < NF < RO). The storage stability can be improved if the ash content is reduced, which can be achieved by using electrodialysis, or nano or UF.

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 additive (at 18 wk); this was in contrast to the corresponding directand 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, disulde-reducing agents, such as mercaptoethanol and cysteine, markedly accelerated gelation (gelation time <1 mo). Lysine inhibits 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 (Srhaug 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 additives can be produced with long-life stability by means of UHT heating. Hirichs (2000) used ultraltration (UF) and reverse osmosis concentrates made from milk with differing fat and protein contents that were sheared in dened ow conditions to establish the critical concentration of the constituents beyond which ow properties and heat stability change. The heat coagulation time at 140 C of milk concentrates was dramatically inuenced by steric interactions if the whole volume fraction of fat and protein

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 stabilization 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 highpressure 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 thermal 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 microbial 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).

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-ll-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 -lactoglobulin-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, accelerates gelation by facilitating release of the complex from the micelle. Factors that inuence 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 hydrolysis of lactose. The shelf life of UHT milk can be increased by considering the above-mentioned factors and along with new improved manufacturing methods. Thus, by increasing the shelf life of UHT milk by using techniques such as ISI-heating, LTI, membrane processing, UHPH, consumers will have more choices in the marketplace.

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