uht milk rupesh

UHT Milk Processing and Effect of PlasminActivity on Shelf Life: A ReviewRupesh 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 UHTand aseptic packaging systems reflect customer preferences and the processes are designed to ensure commercial sterilityand acceptable sensory attributes throughout shelf life. Advantages of UHT processing include extended shelf life, lowerenergy costs, and the elimination of required refrigeration during storage and distribution. Desirable changes taking placeduring 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. Gelationof UHT milk during storage (age gelation) is a major factor limiting its shelf life. Significant factors that influence theonset 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 theeffect of plasmin activity on enzymatic gelation in UHT milk during a prolonged storage period. Measuring enzymeactivity is a major concern to commercial producers, and many techniques, such as enzyme-linked immunosorbentassay, spectrophotometery, high-performance liquid chromatography, and so on, are available. Extension of shelf life ofUHT 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) MilkConsumers demand foods that are as fresh as possible with good

sensory properties (especially taste), additionally being safe andhaving a substantial shelf life, yet without application of addi-tives. Because of its high nutritional value, milk is an excellentmedium for microbiological growth. Consequently, fresh milknecessitates a heat treatment in order to guarantee a safe and shelf-stable product. The most commonly applied technique to achievethis is heat treatment. The first system consisting of indirect heat-ing with continuous flow (125 ◦C for 6 min) was manufacturedin 1893. Patented in 1912, the continuous-flow, direct heatingmethod mixed steam with milk to achieve temperatures of 130 to140 ◦C. Development of the UHT process was hindered dueto possible contamination without commercial aseptic systems. In1953, UHT milk was filled aseptically into cans after heat treatmentwith an Uperiser® processor followed by tetrahedral paperboardcartons in 1961 (Datta and Deeth 2007). The development ofaseptic processing in the United States started through the effortsof C. Olin Ball, and hot-cool-fill process was commercialized in

MS 20110127 Submitted 1/28/2011, Accepted 5/5/2011. Author Chavan is withNational 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 withCollege of Dairy Technology, Warud, Pusad, Maharashtra 452004, India. AuthorJana is with S.M.C. College of Dairy Science, Anand, Gujarat 388110, India.Direct inquiries to author Chavan (E-mail: [email protected]).

1938 for a chocolate milk beverage. In 1942, the Avoset processwas used to package a cream product by utilizing a continuous hotair system and ultraviolet (UV) lamps in the filling and sealing area.In 1948, the Dole aseptic process developed by William McKinleyMartin was used for pea soup and sterilized milk. Real Fresh, Inc.became the 2nd dairy in the United States in 1952 to use UHTand aseptic packaging (AP), and in 1981, it was the forerunner inusing hydrogen peroxide (H2O2) to sterilize packaging material(David and others 1996).

Market Status of UHT MilkConsumption trends for aseptic dairy foods have shown in-

creased demand for aseptic dairy foods and the global market isforecast to climb steeply to 2013 both in terms of pack numbersand 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 buoyantgrowth, rising 31% from 86 billion L in 2008 to 113 billion L in2013. In 2008, cartons dominated the market, accounting for al-most 75% of the total volume but lost some of the market share topolyethylene terephthalate bottles and pouches. In terms of num-bers of white milk packs, the Asian market already accounts for56% of world use and would rise to nearly 70% by 2013. Volumeshave grown annually by over 6% since 2003, with Asia achievingthe fastest rise at over 13% a year. In 2008, white milk accountedfor around 45% of aseptic package use, with beverage volume

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doi: 10.1111/j.1541-4337.2011.00157.x Vol. 10, 2011 � Comprehensive Reviews in Food Science and Food Safety 251

UHT milk processing and effect . . .

reaching over 40% (Harrington 2009). The category is growing ata steady annual rate of 20% in India.

Products are manufactured with UHT and aseptic processing inover 60 countries (Burton 1988). The market share of UHT milkconsumed varies considerably by country: Australia 9%, France88%, 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, andlack of freshness as negative attributes with UHT milk. UHT milkquality depends more on the manufacturing process than countryof origin or fat content. Customer acceptability of UHT milk ispositively correlated to consumption habits that include UHT milk(Oupadissakoon 2007). Aseptic processing has great potential toincrease through dairy consumption in tropical countries, as thereis a low milk consumption trend due to high temperatures andlimited refrigerated distribution (Goff 2008). Hedrick and others(1981) predicted UHT milk with flavor attributes comparable topasteurized milk would reduce energy costs, since the shelf-stablemilk would not require refrigeration throughout distribution. Thegrowth of this industry is limited by government regulations, fillerspeeds, and packaging costs (David and others 1996).

UHT Milk Processing PrinciplesHeat treatment in the production of long life products is called

“sterilization.” In such processes, the treated product is exposed tosuch powerful heat treatment that the relevant microorganisms andmost of the enzymes are inactivated, and the processed productis given excellent keeping qualities and can be stored for severalmonths under ambient conditions.

UHT processing uses continuous flow of milk, which rendersless chemical change in comparison to retort processing (Dattaand Deeth 2007). Product characteristics, such as pH, water ac-tivity, viscosity, composition, and dissolved oxygen, indicate theprocessing conditions necessary to achieve commercial sterility.The selection criteria of UHT and AP systems reflect customerpreferences. The production processes are designed to ensure com-mercial sterility and acceptable sensory attributes throughout shelflife.

To compare the various effects of heat treatments, differentvalues are calculated:

Q10 valueThe Q10 value has been introduced as an expression of this

increase in speed of a reaction. It states how many times the speedof a reaction increases if the temperature of the system is raised by10 ◦C. The Q10 value for flavor changes—and for most chemicalreactions—is around 2 to 3, which means if the temperature of asystem is raised by 10 ◦C, the speed of chemical reactions doublesor triples. Q10 values can also be determined for the killing ofbacterial spores and is normally found in the range of 8 to 30(Kessler 1981). The variation is so wide because different kindsof bacterial spores react differently as the temperature increases.The changes in chemical properties and spore destruction by theinfluence of increased temperature are shown in Figure 1.

F0 valueFor the microbiological effect, F0 value is already used in clas-

sical canned sterilization technology and is defined as the numberof minutes at 121.1 ◦C (250 ◦F) to which the process is equivalentand is calculated according the following formula:

F0 = t/60 × 10(T−121.1◦C)/Z,

Figure 1–Curves representing the speed of changes in chemicalproperties and of spore destruction with increasing temperature. (Source:Gosta 2003.)

wheret = sterilization time in seconds at T ◦CT = sterilization temperature in ◦Cz = a value expressing the increase in temperature to obtain the

same lethal effect in 1 of 10 of the time. The value varies with theorigin of the spores (10 to 10, 8 ◦C) and can generally be set as10 ◦C.

F0 = 1 after the product is heated at 121.1 ◦C for 1 min. Toobtain commercially sterile milk from good quality raw milk, a F0

value of minimum 5 to 6 is required.

B∗ and C∗ valuesThe effective working range of UHT treatments is also defined

in some countries by reference to 2 parameters: bacteriologicaleffect: B∗ (known as B star) and chemical effect: C∗ (known asC star). B∗ is based on the assumption that commercial sterilityis achieved at 135 ◦C for 10.1 s with a corresponding z-valueof 10.5 ◦C. This reference process is given a B∗ value of 1.0,representing a reduction of thermophilic spore count of 109 perunit. The chemical effects can be assessed in similar ways to thoseused for the sterilization performance (Figure 2). The same datafor the time-temperature performance are used. The C∗ valueis based on the conditions for 3% destruction of thiamine perunit. This is equivalent to 135 ◦C for 30.5 s with a z-value of31.4 ◦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 andC∗ < 1 are fulfilled.

The U.S. FDA accepts F0 values for thermal processes calcu-lated only from the time and temperature of the product in theholding tube (David and others 1996). The D-value is definedas the required time to decrease microorganism numbers 10-foldat a given temperature (Singh 2007). The process filing and sup-porting documentation (trial run data, critical factors, equipmentsterilization, 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-vate bacterial endospores and limit chemical changes with minimal

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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 heattreatment with minimal flavor change. Direct heating imparts lessflavor change but requires more energy in comparison to indirectheating. Total microbial lethality at constant time and tempera-ture varies between direct and indirect heating systems (Westhoff1981). The residence time distribution is the time range for a fluidproduct such as milk to enter and exit the holding system (Singh2007). Flow through the heating system is controlled by timing ormetering pumps. The residence time is determined by hold tubevolume, flow rate, and flow rate attributes (viscosity) of specificproducts. Positive reactions in the hold tube include destructionof bacteria, inactivation of enzymes, and hydration of thickeners.Negative reactions include development of off-flavor, initiation ofoff-color, and destruction of vitamins (David and others 1996).

Physicochemical Changes Occurring in UHT MilkOne of the principal goals of milk preservation methods by

its short time treatment at increased temperatures is to obtain adesired degree of destruction of microorganisms and inactivationof 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 byKorel and Balaban (2002) suggested that odor changes in milksamples inoculated with Pseudomonas fluorescens or Bacillus coagu-lans could be detected by an electronic nose. The odor changescorrelated with microbial and sensory data. Maillard browning, asa function of heat treatment given to milk, can be detected byfront-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 ofheat treatment, since it is not affected by milk storage before orafter UHT processing. Heat treatment involves 2 reactions: type 1reactions involve the denaturation, degradation, and inactivationof whey proteins, enzymes, and vitamins. Type 2 reactions involve

the formation of lactulose, HMF, furosine, and others, which arenot detected in the raw milk (Morales and others 2000). Singh(2004) stated that the heat stability of the milk is its ability toundergo high heat treatment without coagulating or gelling. So-lutions to improve heat stability include preheating the productin the UHT processor, adjusting pH to the ideal heat stabilitymaximum, and adding phosphate, buttermilk, or phospholipids.

Chemical changesDirect heat processing imparts less adverse chemical changes

compared to indirect heat processing (Elliott and others 2003). Inan 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 dairyfoods undergo the following chemical changes to varying degrees:flavor, acidity (decreases following direct UHT process), enzymeinactivation, and vitamin decomposition. The heated flavor afterUHT processing is due to sulfhydryl (S–H) groups, which oxidize5 to 10 d after processing. The oxidation then gradually reducesthe cooked flavor (Hsu 1970). Heating has little effect on milksalts with 2 exceptions, carbonates and calcium phosphates. Mostof 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 withincreasing temperature. On heating, soluble calcium phosphateprecipitates onto the casein micelles, with a concomitant decreasein the concentration of calcium ions and pH. Milk oxidative ran-cidity is the reaction of oxygen on milkfat components resultingin short-chain aldehyde and ketone volatiles (Solano-Lopez andothers 2005). Enzyme inactivation is a positive chemical changeof UHT processing. Fat-soluble vitamins are affected minimallyby 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 hasbeen reported under the influence of different milk processing

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treatments (Asadullah and others 2010). UHT processing reducesB vitamins by 10%, folic acid by 15%, and vitamin C by 25%. Thenutritional value of proteins, minerals, and fats is affected mini-mally by UHT processing (Holdsworth 1992) and is correlated tothe storage temperatures, initial oxygen content, and packagingchoice (Dunkley and Stevenson 1987).

Physical changesThe unwanted physical attributes associated with UHT milk are

brown color, sedimentation, protein destabilization, and fat sepa-ration (Hsu 1970). Direct heating after homogenization appears tocause reagglomeration of the small fat globules with the formationof a solid fat layer during storage. To prevent this fat separation, ho-mogenization in direct UHT plants takes place in the downstreamposition (after the final heating step and vacuum cooling). Theproduction of free fatty acids during storage is more noticeable inmilk with higher fat content and is greater in milk produced in di-rect rather than indirect systems (Schmidt and Renner 1978). Milkproteins change more than any other milk constituent due to UHTprocessing that contributes to loss of color, flavor, and nutrition,as well as gelation and sedimentation. Denatured whey proteinsform complexes with other whey proteins, caseins, and fat globules(Dunkley and Stevenson 1987). The amount of β-lactoglobulinassociated with the casein micelle increases with the heating timeand the trend is similar for α-lactalbumin but to a lesser extent(Elfagm and Wheelock 1978). α-Lactalbumin can interact withκ-casein only in the presence of β-lactoglobulin, possibly throughthe 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 wheyprotein begins unfolding at the same time and this gives a greateropportunity 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 micellesis pH-dependent and decreases as the pH increases in the rangeof pH 6.3 to 7.3. The micellar size shows a similar dependence inrange of pH 6.5 to 6.7 (Skelte and Yuming 2003). Thermal in-activation of a transglutaminase (TG) inhibitor provides improvedcross-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. Dairyfoods with greater quantities of reducing sugars have more issueswith browning and it increases with process severity and storagetemperature (Dunkley and Stevenson 1987). These reactions areknown as Maillards reactions, and consist of a series of changeswhose 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 treatmentapplied to milk, as it is minimally affected by storage conditions.Actually, the proposed limit (600 mg/L) seems to be effective forindirect UHT milk, while in the case of direct UHT milk, itdoes not give clear evidence of unjustified overprocessing. Whencombined with furosine, lactulose also allows assessing authenticityof this type of fluid milk (Cattaneo and others 2008). Sedimentis more prevalent in products that are more severely processed,that have a targeted pH of <6.6, and that have undergone directinstead for indirect UHT processing (Holdsworth 1992). Otherfactors affecting sedimentation include homogenization pressurethat is used to control fat separation, time, and temperature pro-

file that is used to ensure product sterility, and formulations thatcan increase product variability (Hsu 1970). For example, sodiumcitrate inhibits sedimentation, whereas calcium salts increase sedi-mentation.

Factors Influencing Shelf Life of UHT MilkThe factors that influence the quality of milk that have an effect

on the gelation behavior of UHT milk are discussed here:

Age of cowMilk from older cows gels faster than that of young cows (Datta

and Deeth 2003).

Stage of lactationAuldist and others (1996) reported that early-lactation UHT

milk gelled in 5 to 6 mo, while late-lactation milk did not gelduring the 9 mo after their experiment. The reason behind thephenomenon is that greater amount of denatured whey proteinis complexed with casein in late-lactation milks as compared toearly-lactation counterparts.

MastitisMastitic milk (that is, milk with high somatic cell count, SCC)

subjected to UHT treatment is more susceptible to gelation thannormal milk (Swartling 1968). This has been attributed to in-creased proteolytic activity resulting from an elevated level of plas-min.

SeasonSeasonal variations in the composition of milk may indirectly

affect the gelation behavior of UHT-sterilized milk. Spring andlate autumn milk shows more age gelation problems than milkproduced in other seasons; this can be attributed to the mineralcomposition of milk (Hardham and Auldist 1996). Zadow andChituta (1975) observed that milk produced between August andOctober was more prone to gel during storage than that producedduring the remainder of the year. Gel time was ranging from 77to 140 d as compared with 120 to 180 d. Spring milk has highervalues of pH, lactose, soluble phosphate, and micellar hydrationthan milk collected in autumn, while spring milk has low fat andheat stability (Gaucher and others 2008a).

Microbiological quality of raw milkMilk with a high preprocessing microbial count is more suscep-

tible to gel formation than milk with a low count. Microorganismsthat produce heat-stable enzymes cause the most serious gelationproblems. Longer refrigeration times prior to sterilization allow in-creased growth of psychrotropic microorganisms and concomitantproduction of heat-stable enzymes, especially proteinases and li-pases. In work by Law and others (1977), when the psychrotrophicbacterial count was less than 8 × 106 CFU/mL, shelf life was ofthe order of 6 mo, whereas at higher counts, a marked reductionin the time to onset of gelation was observed (see Table 1).

Table 1–Effect of psychrotrophic bacteria count in raw milk on gelationtime of UHT milk.

Bacterial count/CFU/mL Gelation time, days<8.0 × 106 >1408.0 × 106 ∼635.0 × 107 ∼12

Source: Law and others 1977.



Storage temperatureThe temperature of storage markedly influences the time of

gelation of UHT-sterilized milk. In general, gelation occurs morereadily 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 temperaturesto be 30 > 25 > 20 > 15 > 10 > 2 > 40, 50 ◦C. Samel andothers (1971) suggested that, at 37 ◦C, gelation may be inhibitedif regions of proteins that could take part in protein–protein inter-actions are blocked by casein–lactose interactions involving lysineresidues. Such interactions precede browning in UHT milk storedat temperatures above 30 ◦C. This hypothesis is supported by Hilland Cracker (1968) who observed that when lysine and arginineresidues of κ-casein molecules were blocked, there was a resultantloss of sensitivity to rennet coagulation, indicating that Maillardbrowning may lead to an inhibition of κ-casein hydrolysis.

Fat contentUHT-processed skim milk is more susceptible to gelation than

UHT whole milk. This can be attributed to an enhanced actionof plasmin and bacterial proteinases in skim milk over whole milk.An explanation for the effect is that the fat in whole milk hindersaccess of the enzymes to their casein substrates. It has also beensuggested that the higher proportion of denatured whey proteinsnot attached to the micelle surface of skim milk may be a reasonfor its lower resistance to gelation. Gaucher and others (2008b)examined the effects of storage up to 6 mo at different temper-atures (4, 20, and 40 ◦C) of partially defatted UHT milk on itsparticular physicochemical characteristics, and an increase of stor-age temperature essentially affects the rate and degree of individualchanges.

Hydrolysis of lactoseTossavainen and Kallioinen (2007) studied proteolytic changes

in lactose-unhydrolyzed and lactose-hydrolyzed direct UHT-treated milks for a storage period of 12 wk. Enzymatichydrolysis was performed either before (prehydrolyzed) or after(posthydrolyzed) UHT treatment. The enzymatic hydrolysis oflactose resulted in an increase in proteolysis, compared to unhy-drolyzed milk, during the storage regardless whether hydrolysis wasperformed before or after the UHT treatment. The highest degreeof proteolysis was found at the highest storage temperature tested(45 ◦C), while proteolysis was almost nonexistent at the loweststorage temperature of 5 ◦C as measured by α-amino nitrogen/total nitrogen or as changes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. Proteolysis was alsonoticed in unhydrolyzed milk where it was caused by the plasminenzyme system and possibly by the microbial contaminants in milk.The lactase dosage in prehydrolyzed milk was 30 times higher thanin posthydrolyzed milk but proteolysis was only slightly strongerthan in posthydrolyzed milk. This means that most of the prote-olytic side activity of the lactase was destroyed during the UHTtreatment of prehydrolyzed milk. Thus, increasing the heat treat-ment during the UHT process could destroy more of the harmfulproteolytic activities in milk. However, this may lead also to in-creased protein damage due to enhanced Maillard reaction andbrowning of the product.

Types of UHT Processing Systems for MilkUHT plants became commercially available in 1960 when asep-

tic filling technology, which is a necessity to maintain the com-mercial sterility of the UHT-treated product, was developed. The

Standardized milk

Pasteurization

20 s 80 °C

Homogenization

20 MPa

Steam Injection

2 s 150 °C

De-aerationDirect expansion cooling

80 °C

Sterilization

15 s 142 °C cool to 20 °C

Aseptic homogenization

40 MPa

Packaging material Cool to 20 °C

Aseptic

packagingSterilization

Aseptic

packaging

Figure 3–Examples of the manufacture of UHT-sterilized milk (indirect ordirect heating) with aseptic packaging.

purpose of a UHT processing plant is to heat the product to thesterilization temperature (in the range 135 to 150 ◦C), hold it therefor a few seconds, and then cool it to a suitable filling temperature.There are 2 main technologies distinguished by the medium usedfor heating to the UHT, direct and indirect systems (Figure 3).Steam, hot water, and electricity are heating methods for UHTequipment. The sterilizers utilizing steam or hot water can be sub-categorized as direct or indirect heating systems. In the indirectsystem, the product and heating medium do not have contact, asa barrier (stainless steel) is present (Burton 1988). Direct heatingmodes include steam injection, steam infusion, and scraped sur-face. Indirect heating modes include indirect spiral tubes, indirecttubes, indirect plate, scraped surface, and electricity. Indirect heat-ing with electricity includes electric elements, conductive heating,and friction (Burton 1988). Table 2 lists commercial UHT systemsand their respective heating modes.

Direct heating systems include steam injection (steam into milk)and steam infusion (milk into steam). The culinary steam must beof high quality and must not impart any off-flavors to the milkproduct. The product temperature increases almost instantly due tothe latent heat of vaporization. The condensed steam that dilutesthe milk is removed later as the heated milk is cooled in a vacuumchamber. Plate or tubular heat exchangers are 2 heating modes forindirect heating. Heating in the indirect system occurs at a slowerrate; therefore, the milk is subjected to the overall heat treatmentfor a longer time. The heat transfer coefficient is greater with plateheat exchangers due to turbulence (Datta and others 2002). Thepotential for contamination due to pinholes in the stainless steelbarrier is minimized by maintaining a greater product pressure onthe sterile side compared to the raw side. The comparisons of time-temperature curves characteristic for treatment of milk in directand indirect systems are shown in Figure 4. The thermal process



Table 2–Commercial UHT systems and heating modes.

Commercial UHT sterilizer Heating mode

Actijoule Indirect electrically heatedGerbig, sterideal system Indirect heat with tubesHigh heat infusion, tetra therm

aseptic plus 2Combined heating modes

Languilharre system, thermovac,palarisator, steritwin UHTsterilizer, ultra therm, Da-SiSterilizer

Direct heat with steam infusion

Rotatherm Direct heat with scraped surfaceSpiratherm Indirect heat with spiral tubesUltramatic, ahlborn process, sordi

sterilizer, UHT steriplak-R, dualpurpose sterilizer

Indirect heat with plates

Votator scraped surface heater,thermutator heater

Indirect heat with scraped surface

VTIS, ARO-VAC process, uperiser,grindrod

Direct heat with steam injection

Adapted from Datta and Deeth 2007.

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

is dependent upon factors such as, product (pH, water activity,viscosity, specific gravity); microbial profile (number, type, heatresistance); equipment design, and package.

AP Systems for MilkIn AP, raw or unprocessed product is heated, sterilized by hold-

ing at high temperature for a predetermined period, then cooledand delivered to a packaging unit for packaging. Packaging mate-rial and equipment surface may be sterilized by various methodssuch as heat, H2O2, irradiation, infrared light, and combinationsof methods (Ansari and Datta 2003). AP systems fill the sterileproduct into sterile packages within the confines of the sterilezone of the filler. The aseptic zone/sterile zone extends from the

Table 3–Aseptic packaging systems.

System Package Sterilant

Asepak Bags HeatASTEC Bins and tanks Pressurized steamCKD Cups H2O2Combibloc Cartons H2O2 + heatDole Aseptic Canning

SystemSteel/aluminum

cans and lidsSuperheated steam

DuPont Canada Bags, pouches H2O2ERCA Cups H2O2 +heatEvergreen Cartons H2O2 +heatGasti Cups High-pressure steamGaulin Bags Ethylene oxideHamba manufacturing Cups Ultraviolet raysHassia Cups H2O2 + heat or

pressurized steamIngko Bags Chlorine solution + heatInpaco Pouches H2O2 + heatInternational Paper Co. Rectangular

packagesH2O2 + heat

Lieffeld & Lemke Cups H2O2 + heatLiqui-Box Corp. Bags Gamma radiationManccini Bags Gamma radiationMead Packaging Co. Cups Citric acid + heatMetal-Box Freshfill

(Autoprod)Cups H2O2 + heat

Pure-Pak, Inc. Cartons H2O2 + heat, oxoniaPurity Packaging Co. Cups H2O2Remy Cups H2O2 +heatRemy Bottles H2O2 or oxoniaScholle Corp. Bags Gamma radiation or

ethylene oxideSerac Bottles H2O2Tetra Pak, Inc. Cartons H2O2Wright Sel Bags Gamma radiation or

ethylene oxide

Source: David and others 1996.

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

Types of milk AP linesThere are 5 basic types of AP lines:

(1) Fill and seal: preformed containers made of thermoformedplastic, glass, or metal are sterilized, filled in aseptic environ-ment, and sealed.

(2) Form, fill, and seal: roll of material is sterilized, formed insterile 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 soupcans.

(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 manufacturersof aseptic equipment. AP systems available for dairy foods in-clude drum and bin systems, heat during blow-molding, cartonpackaging machines, bag-in-box packaging systems, bulk tanksand containers, plastic cups/pots/cartons, and pouches/sachets(Holdsworth 1992).

Filler and container sterilizationAseptic fillers have sections containing sterile contact pipes and

valves along with noncontact sections (sterile chambers). Bothsections must be sterilized prior to production and must maintainsterility throughout production (Burton 1988). Rippen (1969)stated aseptic fillers and associated pipes are sterilized typically



with heat in the form of steam. Wet heat sterilization using satu-rated steam is the most dependable sterilant, as microorganisms aremore resistant to dry heat, which necessitates higher temperatures(Burton 1988). Sterilants are applied uniformly to the aseptic zoneby misting equipment, whereas packaging typically is sterilized bymisting or passing through sterilant bath.

Sterilization of packaging material is a critical step in the AP sys-tem. Therefore, the sterilization process should meet the followingrequirements for sterilization of packaging materials:

(1) Rapid microbiocidal activity;(2) compatibility with surfaces treated, especially packaging ma-

terial 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 unavoid-

able 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, H2O2, and UV light(David and others 1996). Some of these methods are listed inTable 4. The H2O2 is now the only chemical sterilant for ster-ilization of packaging materials that has been proved to be ac-ceptable in the United States. The FDA regulations specify that amaximum concentration of 35% H2O2 may be used for sterilizing

Table 4–Methods for sterilizing aseptic packages.

Advantages/Methods Application disadvantages Reference

Superheatedsteam

Metal containers High temperatureat atmosphericpressure.Microorganismsare moreresistant than insaturated steam

Collier andTownsend(1956)

Dry hot air Metal orcompositejuice andbeveragecontainers

High temperatureat atmosphericpressure.Microorganismsare moreresistant than insaturated steam

Denny andMathys(1975)

Hot hydrogenperoxide

Plasticcontainers,laminated foil

Fast and efficientmethod

Denny andothers(1974)

Hydrogenperoxide/UVlightcombination

Plasticcontainers(preformedcartons)

UV increaseseffectiveness ofhydrogenperoxide

Bayliss andWaites(1982)

Ethylene oxide Glass and plasticcontainers

Cannot be usedwhere chloridesare present orwhere residualswould remain

Blake andStumbo(1970)

Heat fromcoextrusionprocess

Plasticcontainers

No chemicals used –

Radiation Heat-sensitiveplasticcontainers

Can be used tosterilizeheat-sensitivepackagingmaterials.Expensive.Problems withlocation ofradiation source

–

Source: Ansari and Datta 2003.

food contact surfaces. In a properly designed APs system, a goodmicrobiocidal effect using H2O2 can be achieved and the level ofresidue can be effectively controlled to within permissible limits.The residual level of H2O2 is regulated with a maximum level of0.5 ppm. Infrared radiation and vaporized H2O2 have been stud-ied as sterilants for packaging materials (Kulozik and Guilmineau2003). There are many other chemicals such as peracetic acid,beta propiolactone, alcohol, chlorine, and its oxide, and ozonethat have been suggested as having potential for use in sterilizingAP materials (Ansari and Datta 2003).

Postprocess Contamination Concerns in UHT MilkThe problem of posttreatment contamination of in container

sterilized product is well known. The contamination can eitherthrough poor seal or through pinhole in the container. Post treat-ment contaminants in UHT milk may be either spores, whichwould not be expected to be heat resistant enough to survive theheat treatment or nonheat-resistant vegetative organisms. Organ-isms of the 1st type will probably have entered from the ineffec-tively sterilized plant downstream from the heat treatment stageof the process, which includes spores of Bacillus cereus (Wilson andothers 1960; Davies 1975) and Bacillus licheniformis (Wilson andothers 1960). Organisms of the 2nd type will probably have en-tered through a poorly sealed container after aseptic filling (Hassanand others 2009). Postprocess contamination of the aseptic zonecan be attributed to several variables: environmental bioburden,positive air pressure, processing equipment or line turbulence, sys-tem gasping, indexing operations, condensate accumulation, un-sterile product entry, or bacteriological seeding (David and others1996). Postprocess contamination occurs in individual cartons ifpackage integrity is compromised. Contamination from isolatedpackage integrity issues occurs more frequently than processingcontamination.

Rippen (1969) cited typical spoilage in UHT-AP production ata defect rate of 1 of 1000. Manufacturers of aseptic fillers targeta defect rate of ≤1/1000 or ≤1/3000, whereas ≤1/10000 is anindustry standard for aseptically packaged low-acid foods in rigid,semi-rigid, and flexible containers (David and others 1996). Thefollowing 7 potential failure modes exist for aseptic processing andpackaging of foods:

(1) Type 1 failure results from raw ingredient, handling, storage,or batching issues.

(2) Type 2 failure results from processor and filler cleaning inplace, sanitation, preventive maintenance, and presteriliza-tion issues.

(3) Type 3 failure results from the thermal process heating cycleincluding regeneration.

(4) Type 4 failure results from the cooling cycle including surgetanks.

(5) Type 5 failure results from sterilization issues with the pack-age.

(6) Type 6 failure results from sterility loss in the aseptic zone orfrom environmental load.

(7) Type 7 failure results from loss of package integrity (Davidand others 1996).

Commercial Sterility Testing of UHT Milk ProcessScheduled processes in retort operations and UHT processes in-

activate vegetative cells and spores of pathogenic bacteria. The gen-era Bacillus and Clostridium are the primary sporeforming spoilagemicrobes (Ravishankar and Maks 2007). Spoiled packages are



identified as “flat sours” or swells. Spoilage organism identificationis useful in troubleshooting the cause of spoilage and the originof contamination (Burton 1988). Underprocessing is indicated byspoilage 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 UHTmilk microbial counts should be <100 CFU/g following 15 d at30 ◦C. Hsu (1970) stated that souring and/or coagulation wouldbe identified after incubating UHT-AP products 7 to 10 d at 37 ◦C.An incubation and inspection program is recommended by FDAto verify sterility of aseptically packaged products. Sampling plansare more extensive when commissioning aseptic filler than duringroutine production (Burton 1988). Sampling between 0.1% and1.0% for routine production is recommended with samples takenat 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 viewedas an additional verification quality program and is completedthrough traditional and rapid methods (Dunkley and Stevenson1987). Visual inspections, sensory analysis, and pH measurementsare done in conjunction with rapid methods to verify productquality (Grow 2000). Quantitative methods include direct enu-meration and viable enumeration. Viable cells are counted usingstandard plate counts, most probable number, membrane filtration,plate loop methods, or spiral plating. Qualitative methods includemeasuring metabolic activity or cellular constituents (Goff 2008).The Cellscan Innovate System by Celsis uses bioluminescence tomeasure adenosine triphosphate found in living microorganisms(Grow 2000).

UHT Milk Container IntegrityPackaging plays an important role in the food manufacturing

process as it makes food more convenient. It gives the food greatersafety assurance from microorganisms and biological and chemicalchanges such that the packed foods can have longer shelf life. Foodpackaging for shelf-stable products must provide barrier propertiesand physical strength so that they can withstand handling, distri-bution, and storage. It is intriguing, however, that the number offailures (nonsterile product) associated with UHT plants aroundthe world is an almost constant number, namely 1 to 4 in 104.The reasoning widely accepted for this is that nonsterility arisesthrough failure of packaging material or package seals or a plantbeing “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 inflationtest, compression test, decompression test, biotesting, ultrasoundimaging, mechanical tests, and headspace indicators. The mechan-ical tests on filled packages include stress testing, stack testing, loadvibration, and impact resistance.

Plasmin ActivitySince the adoption of refrigerated bulk tanks for the collec-

tion and storage of milk, the predominant organisms in milk arenow psychrotrophic bacteria. These are organisms that are able togrow at temperatures below 7 ◦C, although their optimum growthtemperature may lie between 20 and 30 ◦C. The majority of thepsychrotrophic bacteria (excluding Bacillus spp.) are destroyed bypasteurization, but they produce extracellular enzymes that are ex-tremely thermostable. The most important of these enzymes fromthe commercial viewpoint are the proteases and lipases, both ofwhich are able to withstand high temperature short time and UHT

treatments. Proteolysis in UHT milk can cause the developmentof bitter flavor and lead to an increase in viscosity, with eventualformation of a gel during storage, which is a major factor lim-iting its shelf life and market potential. The enzymes responsiblefor the proteolysis are the native milk alkaline proteinase, plas-min, and heat-stable extracellular bacterial proteinases producedby psychrotrophic bacterial contaminants in raw milk. Proteol-ysis of casein caused by plasmin is responsible for gelation andbitterness of UHT milk during storage.

Psychrotrophic bacteria in milkPsychrotrophic 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.) andGram-positive bacteria (such as Bacillus, Clostridium, Corynebac-terium, Streptococcus, Lactobacillus, and Microbacterium spp.). Further-more, refrigeration conditions under which raw milk is storedselects 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 absenceof viable bacterial cells. A variety of psychrotrophic organisms,including P. fluorescens, P. putida, P. fragi, P. putrefaciens, Acinetobacterspp., Achromobacter spp., Flavobacterium spp., Aeromonas spp., andSerratia marcescens, produce heat-stable extracellular proteases andsome of them also produce heat-stable extracellular lipases. Pseu-domonas spp. usually represents no more than 10% of the microfloraof freshly drawn milk; however, they are the most important ofthe psychrotrophs that dominate the microflora of raw or pasteur-ized milk at the time of spoilage. This genus is also known to bestrongly lipolytic (Sørhaug and Stepaniak 1997).

Thermoresistant psychrotrophs. Among the microorganismsthat survive pasteurization (the thermoduric microorganisms),sporeforming Bacillus spp. dominate those that are also psy-chrotrophs. Other thermoduric psychrotrophs are representedin the genera Arthrobacter, Microbacterium, Streptococcus, Corynebac-terium, and Clostridium. Clostridium is also sporeformer and anaero-bic. Psychrotrophic Bacillus spp. secrete heat-resistant extracellularproteinases, lipases, and phospholipase (lecithinase) that are ofcomparable heat resistance to those of Pseudomonads. Bacillussporothermodurans is a mesophilic spore-former that produces highlyheat-resistant spores and was first detected in UHT milk in 1985in southern Europe and in UHT milk in Germany in 1990 (Pet-tersson and others 1996). The spores survive the heat process andthen multiply to a maximum of about 105/mL of milk duringincubation at 30 ◦C for 5 d, but cause no noticeable spoilage. Thespores of B. sporothermodurans are more resistant than the spores ofmany thermophiles (Brown 2000).

Secretion and biochemical properties of proteinases andlipases 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 ofgrowth. The maximum concentrations of proteinases get accumu-lated in shaken milk than those in static culture, but variable effectsof aeration on the production of lipases have been reported. Mostof 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 readilyable to degrade κ-, αs1- and β-casein, and have low activity onnondenaturated whey proteins (Sørhaug and Stepaniak 1997).



Plasmin, plasminogen, plasminogen activators (PAs), PAinhibitors (PAIs), and plasmin inhibitors (PIs)

Plasmin (EC 3.4.21.7), the main native protease in milk, ispart 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 ofplasmin (Nielsen 2003). Plasmin is an alkaline serine proteinasewith pH optimum of 7.5, which readily hydrolyzes β-casein, αs2-casein, and (more slowly) αs1-casein (Fox and McSweeney 1996).Its enzymatic reactions result in desired and undesired effects ondairy products. Plasmin plays a positive role in cheese ripening formany varieties of cheeses (such as Emmental, Romano, Swiss, andGouda); however, its enzymatic action during milk clotting andstorage of UHT milk can affect the products adversely.

Plasmin activity is higher in mastitis and late-lactation milk dueto 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 finalplasmin activity of milk and, subsequently, milk casein hydroly-sis, would depend not only on the amount of plasminogen andPA but also on the quantity of PAI. The occurrence in milk ofblood serum trypsin inhibitors with both high and low molecularweight has been documented. They presumably would interferewith the function of serine proteinases and therefore, with plasminand PA activity. However, other milk constituents, for example,β-lactoglobulin also could inhibit these enzymes (Korycka-dahland 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 conversioninto 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 PAare known to be present in fresh milk: a tissue type associated withcasein and a urokinase type associated with the somatic cells. PIsand PAI are located mainly in milk serum; however, the inhibitorsmight appear in several different forms, possibly due to formationof complexes with other milk proteins (Precetti and others 1997).α1-Antitrypsin, α2-antiplasmin, and PAI have been isolated frommilk serum and partially characterized (Weber and Nielsen 1991).

Little is known about the heat stability of PAI and PI, and it hasbeen generally proposed that the inhibitors are thermally unsta-ble. Richardson (1983) suggested that PAI is inactivated by mildthermal treatments. An increase in activity of PL and a subsequentdecrease in concentration of PG were observed in pasteurizedmilk compared to raw milk after incubation at 37 ◦C for up to80 h. Effect of heat treatment on inhibitors of both PA and plas-min was studied by (Prado and others 2006) and fractions of milkwith inhibitory activities against PA and plasmin were isolated.Thermal inactivation of PA inhibitor (81.1%) was considerablydifferent 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 Richardson1983).

75 ◦C for 15 s. The PI in the isolated fractions was suggested tobe α2-antiplasmin, since it reacted immunochemically with poly-clonal goat antihuman α2-antiplasmin and competitively inhibitedplasmin. Results showed that PA inhibitor is less heat stable thanPI, indicating that plasminogen activation could overcome anyinhibition of plasmin resulting after milk pasteurization.

Hard gel formation was caused by Pseudomonas proteinase andpartial digestion of the casein by plasmin in the samples incubatedat 40 ◦C for 3 h. The clarified samples indicated extensive break-down of casein as evident by turbidity, while the gelled samplesindicated limited proteolysis. The characteristics of hard gel aresimilar 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 thecasein 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 astrong 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 theactivators are known to be heat stable (Richardson 1983; Lu andNielsen 1993). Consequently, heat treatment of milk alters thenatural balance between the activators and inhibitors in favor ofthe activators. This can lead to enhanced proteolysis in heatedmilk (Deharveng and Neilsen 1991). Driessen and van der Waals(1978) reported the D142◦C for milk proteinase (plasmin) to be18 s. Rollema and Poll (1986) reported 28%, 6%, 4%, and 1.3%plasmin/plasminogen remaining after indirect heating for 5 s at110, 120, 140, and 147 ◦C, respectively, but none after heating at147 ◦C for 10 s.

If proteases survive UHT sterilization, milk containing themand stored without refrigeration would be a good environmentfor their activity. The optimum pH for one protease ranges frompH 7 to 8 with 85% to 90% of maximum activity at pH 6.5,the pH of milk (Speck and Adams 1976). The production ofdifferent phospholipases has been reported for Gram-negative andGram-positive psychrotrophs. The “bitty cream” defect (floatingclumps of fat) occurs in products that contain high numbers ofBacillus cells, suggesting a unique ability of phospholipases fromthis microorganism to damage the fat globule membrane (Sørhaugand Stepaniak 1997).

Heat Resistance of Enzymes Present in MilkGenerally, 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, andphospholipase (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 areadditional salt bridges, additional hydrogen bonds, tighter Ca2+-binding sites, maximized packing, shorter loops, and an ex-panded hydrophobic core. Many of these features are apparentlyalso stabilizing factors in heat-resistant proteinases, lipases, andphospholipase C from all psychrotrophic microorganisms. Lipases



Table 5–Heat resistance of enzymes from Pseudomonads.

Enzyme Source Heat resistance

Protease (s) Pseudomomas D150◦C = 90 sProtease (s) Total psychrotrophic

flora of milkSurvived 149 ◦C/10 s

Protease Pseudomomasfluorescens P26

D149◦C = 90 s

Protease Pseudomomas Survived unspecified UHTsterilization temperature

Protease Pseudomomas Survived 135 ◦C/3.5 minLipase P. fluorescens 22F D150◦C = 4.8 min

and proteinases are more sensitive to low-temperature treatmentat 50 to 60 ◦C than to heat treatment at >100 ◦C. Proteinasesare 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 inPA3679 spores and B. stearothermophilus spores, respectively. Pu-trefactive Anaerobe 3679 are the spores commonly are used forthe development of sterilization processes, and a heat treatmentof 149 ◦C for 4 s should sterilize fluid milk products effectively.These proteases showed less than 10% destruction during UHTsterilization of milk at 149 ◦C for 4 s (Speck and Adams 1976).Kishonti (1975) showed that 24 of 60 strains of psychrotrophicbacteria isolated from milk and including Pseudomonas spp., Al-caligenes spp., and Aerobacter spp. produced extracellular enzymescapable of retaining at least 75% of their activity after exposure to63 ◦C for 30 min. Stadhouders and Mulder (1960) showed thatstrains of Achromobacter spp. and Serratia spp. produced lipases thatcould 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 Requivalent) for UHT milk to have a shelf life of at least 3 mo at25 ◦C. Mitchell and Ewings (1985) determined the threshold valuefor proteinase to be about 0.3 ng/mL for a shelf life of at least4 mo at 23 ◦C.

Inactivation Kinetics of Enzymes Present in MilkUHT treatments can be sufficient to inactivate microorganisms

and obtain a sterile product but provide insufficient heat load toreduce plasmin activity and obtain a stable product. Insufficientinactivation causes bitter-tasting milk, resulting in product lossfor producers and consumers of milk. To avoid bitterness, an in-activation of 99% of the plasmin present is generally applied inindustries. Metwalli and others (1998) observed that irreversibleinactivation can be achieved at around 65 ◦C, but above 92 ◦C theinactivation rate increases only slightly with temperature due to alower 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 bovinemilk using improved enzymatic assays. Activation energies (Ea)for the heat denaturation of plasmin, plasminogen, and PAs were29, 35, and 24 kJ/mol, respectively, in the temperature range 95to 140 ◦C, and 244, 230, and 241 kJ/mol, respectively, in thetemperature range 70 to 90 ◦C (Saint Denis and others 2001).

The inactivation kinetics of plasmin in milk has been describedby Rollema and Poll (1986) and (partly) denatured β-lactoglobulinis found to affects the rate of plasmin inactivation (Crudden andothers 2005). Due to thermal processing free S–H groups willbecome available when β-lactoglobulin unfolds, which is the 1ststage of denaturation. As a result of unfolding of β-lactoglobulin,

the highly reactive S–H groups cause irreversible denaturationof plasmin (Crudden and others 2005). The above implies thata certain degree of denaturation of β-lactoglobulin is necessaryto increase the inactivation of plasmin and plasminogen. On theother hand, denaturation of β-lactoglobulin induces the forma-tion of deposit on the wall of heat treatment equipment and isalso an indicator for product degradation. This implies that anoptimized heat treatment needs to be designed where the degreeof denaturation of β-lactoglobulin is high enough to inactivateplasmin to avoid bitterness but low enough to minimize the for-mation of deposit and product degradation. Previous experimentsshowed 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 degreeof denaturation of >50% (Huijs and others 2004). However, theeffect of denatured β-lactoglobulin is usually not taken into ac-count when predicting/designing UHT processes in relation tothe inactivation of plasmin (Crudden and others 2005).

Gelation of UHT MilkThe 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. Themajor 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 itsglobular structure above 55 ◦C (Gough and Jenness 1962; Sawer1969); this causes rupture of cystine disulfhide bonds and a markedincrease in thiol group reactivity. Prolonged heat treatment above80 ◦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-lactoglobulinaggregates, or intermolecularly to form disulfhide bonds betweenβ-lactoglobulin and other S–H-containing molecules such as κ-casein and proteins of the milk fat globule membrane. In thecasein micelle, κ-casein exists at the surface and is available forsuch 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 thickenand 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 tothe self-association of denatured β-lactoglobulin); the release ofthe βκ-complex from the casein particle; and the cross-linkingof the βκ-complexes and associated proteins. When these asso-ciations (βκ-complexes) are disrupted, κ-casein is released alongwith its attached β-lactoglobulin. The released βκ-complexes areobserved as protuberances and tendrils on the micelle surface.The disruption of the κ-casein anchors to the other caseins canoccur through enzymic (proteinase) action or nonenzymic means(Harwalkar 1982). The order of susceptibility of the caseins tohydrolysis by bacterial proteinases and milk plasmin are κ > β >

αs1 and β = αs2 > αs1 > κ , respectively. Overall, the relationshipbetween added purified proteinase activity and gelation time canbe obtained from the following equation:

Gelation time (months) = 2.3916 × X−0.6449,

where X is proteinase activity measured by the Merck proteinasetest kit using dehydrogenase as the substrate and relating the



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-trials, Denmark) in nanogram per milliliter (Mitchell and Ewings1985).

Enzymatic mechanism of gelationAccording 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 theβκ-complex. This dissociation of βκ-complexes from the caseinmicelles by proteinases is considered to be the 1st stage in a 2-stagemechanism of age gelation. The 2nd stage involves the subsequentaggregation of the βκ-complexes and formation of a 3D networkof cross-linked proteins (Figure 6) and effect of proteolytic en-zymes on gelation in Table 6. Enright and others (1999) observedthat 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, andformation of sediments at a similar time, and of similar appearance,to those seen in raw milk. The addition of plasmin to UHT milkafter heating reduced the stability of the milk, increased proteol-ysis, and led to the early formation of sediments. The results ofthis 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

plasmin activity, which increase on storage due to activation ofplasminogen.

Nonenzymatic mechanism of gelationAndrews 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 milkat 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 preventsmicelles 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 resultsfrom changes in the free energy of casein micelles. Differences inpotential energy promote aggregation of the casein micelles, theextent of this depends upon the probability of contact and thenumber of low potential micelles, both of which increase withstorage time. Micelle aggregation leads to increased viscosity ofthe UHT milk.

Measuring Enzyme Activity in UHT MilkAnalysis of milk in the manner proposed can enable the UHT

milk manufacturer to determine if proteolysis is occurring, orhas occurred in the milk and, if so, whether it is caused by milk



plasmin, bacterial proteinase, or both. If it is caused by plasmin, it islikely that the UHT processing conditions are too mild that causesless denaturation of plasmin and whey proteins. This results inless whey protein–casein interaction and less inhibition of plasminaction on the casein that is most commonly encountered in thedirect UHT processes, steam infusion, or injection (Manji andothers 1986; Manji and Kakuda 1988). If the proteolysis is causedby bacterial proteinases, the quality of the raw milk is implicated.The most common cause is high levels of psychrotrophic bacteriain the raw milk. Inadequately cleaned equipment that supportsbacterial growth and production of proteinases can also be a cause(Driessen 1983).

Protease activity in the sterile skim milk was determinedby measuring proteolysis at weekly intervals. Single samplesfrom 3 bottles were assayed by the Hull method (1947) withFolin–Ciocalteau reagent. Measurements were continued until theincrease in absorbance exceeded 0.6 or until whey separation andgelation. The rates of proteolysis were determined by calculatingthe regression of proteolysis on time. From the slopes of the re-gression lines, the percentage inactivation of proteolysis caused bylow-temperature-inactivation (LTI) was calculated as:

Percent of inactivation = 100 (slope of control

− slope of LTI sample)/slope of control.

Table 6–Effect of proteolytic enzymes on gelation.

Results andEnzyme/enzyme Experiment conclusions

Plasmin/plasminogen(Manji and others1986)

Direct, indirectheating

Indirect inactivatedplasmin; no gelationto 182 d in indirectinactivated plasmin

Plasmin (Gruffertyand Fox 1986).

– Caused degradation ofcasein and gelation in60 d

Bacterialproteinases(Speck andAdams 1976)

pH opt, 6.5; tempopt, 45 ◦C

Caused bitter flavor andgelation

Psychrotrophproteinases(Cogan 1977)

– Responsible for gelation

Bacterial, P.fluorescens (Lawand others 1977)

– Responsible forgelation; time to geldependent on extentof growth in raw milk

Bacterial, plasmin – Both caused gelationPlasmin (Snoeren

and others 1979)Good quality milk

<2700 CFU/mLPlasmin caused gelation

in 90 dBacterial (Fox 1981;

McMahon 1996)Poor quality milk, 2

× 106 CFU/mLBacterial proteinases

cause gelation in 21 dProteinases

(Harwalkar 1982)Direct compared

with indirectMore proteolysis and

gelation in directlyheated milk

Proteinases (Sameland others 1971).

Storage at 4, 20, 30,37 ◦C

More proteolysis butless gelation at 37 ◦C;gelation not due toproteolysis

Plasmin isolatedfrom raw milk(Manji andKakuda 1988).

Unconcentratedmilk

No correlation betweenamount of proteolysisand gelation time butsome proteolysisnecessary for gelation

Proteinase inhibitors(de Koning andothers 1985)

– Direct heating Inhibitedplasmin action;retarded gelation, nogelation norproteolysis afterstorage of 9 mo at20 ◦C

A sensitive assay for protease activity based on the reaction of pri-mary amino groups of trichloroacetic acid (TCA) soluble peptidesand amino acids with fluorescamine (4-phenylspiro[furan-2(3H),1′-phthalan]-3,3′ dione) was applied to sterile milk (Chism andothers 1979). The assay was linear in the range of 2 to 50 nmolof amino groups per aliquot. The assay is suitable for determiningproteolytic activity in sterile milk products and for determiningprotease activity in general.

Plasmin and plasminogen can also be determined spectrophoto-metrically using the chromogenic substrate H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide (VLLN) as described by Korycka-dahl andothers (1983) with some modifications. Milk samples must bedefatted at 10 ◦C by centrifugation (5000 × g for 20 min) andskimmed milk obtained must be incubated with 50-mM ε-amino-n-caproic acid (EACA) for 2 h at room temperature to dissociateplasmin and plasminogen from casein micelles. A plasmin activ-ity was measured without adding urokinase- and plasminogen-derived activity and calculated as total activity minus plasminactivity and was expressed in the same units. One unit of activitywas defined as the amount of enzyme that produces a change inabsorbance (path length 1 cm) at 405 nm of 0.0001 in 1 min at pH7.4 and 37 ◦C in the defined reaction mixture (Manji and others1986). Plasmin activity in dairy products can also be measuredwith synthetic chromogenic or fluorogenic substrates. Syntheticsubstrates can be made by linking Lys peptides to chromogenic orfluorogenic tags. VLLN is a synthetic substrate that can be used todetermine plasmin activity as it hydrolyzes this peptide to release4-nitroaniline, a compound that absorbs light at 405 nm (Bastianand others 1993).

The most sensitive methods reported, including enzyme-linkedimmunosorbent assays (ELISAs), were able to detect enzyme ac-tivity or the presence of enzyme when the number of Pseudomonascells present was about 106 CFU/mL. ELISAs that can detect 0.25ng/mL of proteinases or 20 ng/mL of lipase from Pseudomonasspp. have been reported. An ELISA that detects proteolysis bymeasuring the accumulation of glycomacropeptide released fromκ-casein by proteinases from Pseudomonas spp. has also beendeveloped (Sørhaug and Stepaniak 1997). Datta and Deeth (2003)differentiated the peptides produced by enzymes responsible forthe proteolysis from the native milk alkaline proteinase, plasmin,and heat-stable, extracellular bacterial proteinases produced bypsychrotrophic bacterial contaminants in the milk prior to heatprocessing. In order to differentiate, these peptide products,reversed-phase high-performance liquid chromatography(HPLC), and the fluorescamine method were used to analyzethe peptides soluble in 12% TCA and those soluble at pH 4.6.The TCA filtrate showed substantial peptide peaks only if themilk was contaminated by bacterial proteinase, while the pH 4.6filtrate showed peptide peaks when either or both bacterial andnative milk proteinases caused the proteolysis. Results from thefluorescamine test were in accordance with the HPLC resultswhereby the TCA filtrate exhibited significant proteolysis valuesonly when bacterial proteinases were present, but the pH 4.6filtrates showed significant values when the milk contained eitheror both types of proteinase. A procedure based on these analysescan be proposed as a diagnostic test for determining which typeof proteinase-milk plasmin, bacterial proteinase, or both areresponsible for proteolysis in UHT milk.

Plasmin Deactivation in UHT MilkHeat-stable proteinases produced by psychrotrophic bacterial

contaminants of raw milk are also capable of causing gelation



of UHT milk. Several authors have attempted to correlate thelevel of bacterial proteinase with the time to gelation duringstorage of UHT milk and then to make recommendations onthe maximum advisory level of proteinase to ensure a long shelflife. The severity of heat treatments during preheating and steril-ization is a very important consideration in retarding gelation inUHT milk, especially where this is initiated by plasmin-catalyzedproteolysis.

Samuelson and Holm (1966) observed that increasing the ster-ilization temperature from 142 to 152 ◦C and time from 6 to12 s, allowed milk to be stored longer without gelation. Zadowand Chituta (1975) confirmed that an increase in gelation timeis observed when the sterilization temperature is increased from135 to 140 ◦C along with holding time from 3 to 5 s. Adamsand 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 produceabout 10 or more units of heat-stable protease that would shortenthe shelf life of sterile milk significantly (Speck and Adams 1976).In contrast, heat-resistant protease activity at 40 ◦C did not appearcorrelated with the bacterial populations in raw milk. Correlationbetween Standard Plate Count, psychrotrophic, and proteolytic-psychrotroph 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 psy-chrotrophic bacterial and SCCs, proteinase, and plasmin activity)and UHT temperature (145 or 150 ◦C for 4 s) on proteolysis inUHT milk processed by a direct (steam injection) system duringstorage at 25 ◦C for 180 d. High proteinase activity was measuredin low-quality raw milk, which had high SCC, bacterial count,and plasmin activity. Sterilization at 150 ◦C extended the shelf lifeof the UHT milk by reducing proteolysis, gelation, and bitterness.

Driessen (1983) suggested that proteolysis by bacterial enzymesis accompanied by an increase of nonprotein N and the formationof para-κ-casein, while the plasmin produces an increase of non-casein N and the formation of γ -caseins. This also has diagnosticvalue for the detection of the cause of gelation and bitterness.Mottar and others (1985) reported that changes in the quantityof 2 specific protein breakdown components during refrigeratedstorage of raw milk show a significant correlation with the bacte-rial count. Raw milk containing these compounds at higher thana 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 innonprotein 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 wasattributable to the action of proteinases from psychrotropic bacteriaon κ-casein, releasing the sialic acid-containing glycomacropep-tide. They concluded that the determination of free sialic acid is auseful method for monitoring proteolysis by bacterial proteinasesin UHT milk during storage and to provide an early warning ofthe onset of gelation. Manji and others (1986), while attributingthe greater propensity to gelation of milk sterilized by direct UHTprocesses (compared with indirectly sterilized milk) to higher plas-min and plasminogen activities, found no correlation between theshelf life of the directly sterilized milk and the extent of proteoly-sis. It should also be noted that the difference in gelation times ofUHT milks stored at different temperatures cannot be explainedby the level of proteolysis; more proteolysis occurs at 40 ◦C than

at 30 or 20 ◦C (Renner 1988), but gelation is retarded in milksstored at 40 ◦C (Kocak and Zadow 1985b).

Manji and others (1986) observed that there is a correlationbetween the extent of proteolysis and plasmin activity. Rate oftransformation of plasminogen to plasmin was similar for sam-ples stored at 37 ◦C and 22 to 25 ◦C but significantly slower at4 ◦C. They also found that the extensively degraded proteins wereunable to form a gel matrix and if any gel structure that mayhave formed may have been degraded by continued proteolyticactivity, thus reducing the apparent viscosity. Manji and Kakuda(1988) observed that milks with 28% whey protein denaturationgelled after 115 d, while more severely heat-treated milk with 66%denatured whey protein gelled after 150 d. These results indicatethat the formation of complexes between whey proteins and ca-seins, which accompanies denaturation, plays an important role indetermining the onset of gelation. McMahon (1996) concludedthat milk treated by indirect UHT processes is more stable thanmilk treated by direct UHT processes.

Farrell and Thompson (1990) reported that β-lactoglobulininhibited phosphoprotein phosphatases, including mammaryalkaline phosphatase. The ability of β-lactoglobulin to inhibitphosphatase activity is influenced by acetate, calcium ions, andgenetic variants of β-lactoglobulin. They also hypothesized thatphosphatase activity in milk regulated phosphate content and thatphosphatase, in turn, is regulated by β-lactoglobulin, calcium,potassium, and sodium. Thus, β-lactoglobulin could have a phys-iological role in enzyme regulation.

Garcıa-Risco and others (2003) observed the effects of highpressure (up to 400 MPa), applied at room temperature, on nativeproteinase 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 onlydecreased around 20% to 30% total plasmin activity after plas-minogen activation. In milk, plasmin activity was shown to resistat least 400 MPa applied for 30 min at 25 ◦C (Lopez- Fandino andothers 1996), while these conditions reduced the total plasminactivity after plasminogen activation by 25% (Garcıa-Risco andothers 1998). Pressurization at higher temperatures considerablyincreased plasmin inactivation in milk, which reached 86.5% aftertreatments at 60 ◦C. Plasmin was also baro stable in buffer at roomtemperature, 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 MilkTo date, fluid product shelf life extension has focused primarily

on reducing and controlling the presence of bacterial contaminantsto achieve better product quality for longer periods. The extendedshelf life and shelf stability are definite advantages of UHT milk.Shelf life is the storage time before quality drops to an unacceptablelevel with subjective attributes that include taste, color, odor, gela-tion, sedimentation, separation, and viscosity. These attributes canbe affected by raw product quality, pretreatment process, processtype, homogenization pressure, deaeration, postprocess contami-nation, AP, and package barriers. Spoilage of raw milk prior toprocessing can occur from poor sanitation and inadequate storagetemperatures. Some of the ways to improve shelf life of UHT milkare described as follows:

Quality of raw milkThe use of high-quality raw milk is of utmost importance for

achieving a long shelf life of UHT milk (Law and others 1977).



Storage of raw milk at low temperature (<4 ◦C) for a mini-mum period of time (≤48 h) minimizes growth of psychrotrophicbacteria and, consequently, the amount of extracellular bacterialproteinases produced in the milk before heat treatment. Speck andAdams (1976) suggested that preventing contamination of the rawmilk with psychrotrophic bacteria would be difficult and expen-sive. The heat resistance of the enzymes precludes their destructionat UHT. If the raw milk bacterial count is <25000 CFU/mL, thenthe raw milk SCC will be the most important determinant of shelflife in pasteurized extended shelf life milk with respect to devel-opment of off-flavors when postpasteurization bacterial growth iscontrolled (such as below 500000 CFU/mL) (Barbano and others2006).

Low-temperature inactivation of proteinasesBarach and others (1976) reported that heat-resistant enzymes in

milk could be inactivated by treatment at low temperature (about55 ◦C) for a prolonged period of holding (30 to 60 min). Theeffectiveness of such “low-temperature-inactivation” treatment isindependent of proteinase concentration and does not significantlyalter the flavor of the milk. The method can be applied before orafter sterilization or to aged sterile milk and is most effective whenused in milk at least 1 d after UHT treatment. LTI at 55 ◦C is onlyeffective up to 60 min; thereafter, the inactivation rate of LTI whencombined 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 proteinaseundergoes a unique conformational change followed by aggrega-tion of altered proteinase with casein to form an enzyme-caseincomplex that causes inactivation of the enzyme. The combinationof LTI and UHT sterilization can prolong the shelf life (up to 3times) of sterile skimmilk containing psychrotrophic bacterial pro-teinase. Furthermore, some proteinases are quite resistant to heattreatment at 55 ◦C for an hour, so its usefulness for age gelationmay be limited. LTI did not alter the flavor or protein content ofthe milk and κ-casein was affected by the protease. Such a process,if feasible on a commercial scale, could offer the best solution tothe 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. Theextent of protease inactivation appeared to be independent of pro-tease concentration and, therefore, could occur at the low proteasethat might be in raw milk.

Heat treatment during preheating and sterilizationAdequate heating is required for denaturation of most of the

β-lactoglobulin and complexation with casein. Such high heattreatment also inactivates plasmin. For the same bactericidal ef-fect, indirect heating produces milk that is more stable to gelationthan that produced by direct heating. Lu and Nielsen (1993) addedserine 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 notobserved after 9 mo of storage at 20 ◦C. Recently, 2 proteinaseinhibitors, PA inhibitor-1 and alpha 2-antiplasmin, were isolatedfrom bovine milk. Since these proteinase inhibitors are heat labile,their use in control of the plasmin system would only be possibleif they were added (aseptically) after heat processing. The shelf lifeof UHT milk could be enhanced by addition of PA inhibitor-1 at a concentration of 125 mg/mL; at this level, no PA couldbe 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-LactoglobulinA, α-lactalbumin, and bovine serum albumin at concentrationsof 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 retardingage gelation. However, it is unfortunate that heating conditionsthat minimize age gelation cause the most cooked flavor in UHTmilk, 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 storedat 2 ◦C for 120 to 168 h and processed the milk at 140 ◦C for4 s. The addition of 0.05% calcium chloride or 0.1% SHMP tomilk before UHT processing resulted in a considerable increase instability, with no gelation evident after 500 d at 25 ◦C. Additionof a low level of SHMP would facilitate bridging between ion-ized groups of casein micelles that would not otherwise form anionic bond. This would hold the κ-casein more tightly to the mi-celle and delay release of the βκ-complex, thus retarding gelationof UHT milk during storage. The results suggest that polyphos-phates, such as SHMP, inhibit the 2nd stage of gelation involvingprotein coagulation. Addition of sodium phosphate and sodiumcitrate accelerate gelation in UHT milk, while polyphosphates,such as SHMP, delay gelation (Samuelson and Holm 1966).

ISI heatingThe 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 shownin Figure 7. In the ISI, the product is pumped through a pipe with anarrow end (nozzle, 1 to 2 mm). The wall of this pipe contains sev-eral small openings through which high-pressure steam is injected,



enabling very fast heating of the product. The milk can be heatedat 80 ◦C (during different residence times) before (preheated) orafter (postheated) the heat treatment with the ISI (0.2 s, 180 ◦C).After heating, the product can be instantaneously cooled usingflash cooling (van Asselt and others 2008). The heat treatment inindustrial applications is much shorter, but a higher temperatureis applied. For example, Tetra Pak’s Tetra Therm Aseptic Plus 2(Deeth and Datta 2003) includes a preheating of approximately45 s at 90 ◦C. With respect to plasmin inactivation, this heat loadis 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% denaturedβ-lactoglobulin was chosen as previous experiments with the ISIshowed that application of ISI-heating resulted in that level ofdenaturation (Huijs and others 2004). The results showed that apostheat treatment is sufficient to reduce the amount of plasminbelow 1% of its initial level. By applying these new kinetics, theheat load for currently applied UHT treatments of milk can bereduced while obtaining a sufficient inactivation of plasmin (thatis, <1%) and to achieve a 6 decimal reduction of B. sporothermod-urans. This opens the way for the production of extended shelflife milk with even less product degradation (that is, <50% de-naturation of β-lactoglobulin) compared with currently availableUHT products (that is, >50% denaturation of β-lactoglobulin)and improved taste characteristics (van Asselt and others 2008).

Addition of the sulfhydryl (SH) group-blocking agentN-ethylmaleimide (NEM) added to milk before heating inhibits

denaturation of whey proteins and interaction of these proteinswith caseins. Hong and others (1984) showed that UHT milk,containing 0.5 g/L NEM, and processed by direct heating, gelledlater (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 2milks is unexplained. In concentrated milks, NEM had little effecton 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-bindingsite present in the kringles of plasmin/plasminogen. It also causesdissociation 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 itsuse as a practical measure for controlling plasmin activity in milk(Bramley 1998).

Treatment of milk with carbon dioxide or nitrogenThis treatment can effectively inhibit the growth of different

psychrotrophs (Sørhaug and Stepaniak 1997).

Membrane processing of UHT milkMilk 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 UHTheating. Hirichs (2000) used ultrafiltration (UF) and reverse os-mosis concentrates made from milk with differing fat and proteincontents that were sheared in defined flow conditions to establishthe critical concentration of the constituents beyond which flowproperties and heat stability change. The heat coagulation timeat 140 ◦C of milk concentrates was dramatically influenced bysteric interactions if the whole volume fraction of fat and protein

exceeded 0.5. The higher the fat content with the same wholevolume fraction, the lower the heat stability was because visibleflocs were formed earlier. Increased heat stability was detected forUF > Nanofiltration (NF) > Reverse Osmosisi (RO) concentratebecause of the reduced ash content (UF < NF < RO). The stor-age stability can be improved if the ash content is reduced, whichcan be achieved by using electrodialysis, or nano or UF.

High-pressure treatmentsThe plasmin system is very pressure stable at room temperature.

A synergistic effect of high pressure and temperature is observedin the 300 to 600 MPa and 35 to 65 ◦C ranges, and a stabiliza-tion effect could be observed for pressures above 600 MPa. Bordaand others (2004) concluded that particular attention could begiven to the stability of the plasmin system at pressures above 600MPa and the possibilities of high-pressure thermal inactivation ofplasmin in the 300 to 600 MPa and 36 to 65 ◦C range. Anothertype of high-pressure processing that has been developed is high-pressure homogenization (HPH). The principle of the operationis similar to that of conventional homogenizers used in the dairyindustry except that it works at higher pressures (up to 400 MPa).This technology is also called ultra-HPH (UHPH) depending onthe pressure achieved. Milk treated at 200 MPa at 30 ◦C had thelongest microbial shelf life (about 21 d) and achieved an outlettemperature of about 80 ◦C for 0.7 s, which means that the ther-mal effect on milk was less than that of the high-pasteurizationtreatment. However, at 200 MPa and 30 ◦C, a marked decreaseof milk pH was observed. With the other UHPH treatments, amicrobial shelf life between 14 and 18 d, similar to that observedfor high-pasteurized milk, was obtained. Therefore, the micro-bial data indicate the possibility of obtaining UHPH-treated milkwith equal or better microbial shelf life than high-pasteurized milk.The UHPH treatment, besides achieving a reduction in microbialcounts, generated changes in the physicochemical properties suchas color, viscosity, pH, and acidity. Color, texture, and mouthfeelare important signals that determine consumer perception of thefreshness of milk (Pereda and others 2007).

ConclusionUHT and AP of milk is a well-established technology in many

countries. Direct and indirect heating systems are used along withsterile packages and form-fill-seal systems. Advantages of UHTmilk include reduced energy consumption, extended shelf life, andambient storage and distribution conditions. Commercial successof UHT is affected by factors such as, postprocess contamination,customer acceptance, chemical/physical changes resulting fromheat treatment, and extended storage. Age gelation is a majorfactor limiting the shelf life of UHT milk. It can be explainedby a 2-stage process involving formation, during heating, of aβ-lactoglobulin–κ-casein complex that cross-links after partial orcomplete release from the casein micelle to form a protein networkgel. Proteolysis, by native milk plasmin or bacterial proteinases, ac-celerates gelation by facilitating release of the complex from themicelle. Factors that influence the shelf life of UHT milk are ageof cow, stage of lactation, seasonal changes, microbial quality ofraw milk, storage temperature, fat content of milk, and hydrol-ysis of lactose. The shelf life of UHT milk can be increased byconsidering the above-mentioned factors and along with new im-proved manufacturing methods. Thus, by increasing the shelf lifeof UHT milk by using techniques such as ISI-heating, LTI, mem-brane processing, UHPH, consumers will have more choices inthe marketplace.



References

Adams DM, Barach JT, Speck ML. 1975. Heat-resistant proteases producedin milk by psychrotrophic bacteria of dairy origin. J Dairy Sci 58:828–34.

Andrews AT, Cheeseman GC. 1972. Properties of aseptically packedultra-high-temperature milk. II. Molecular weight changes of the caseincomponents during storage. J Dairy Res 39:395–408.

Ansari IA, Datta AK. 2003. An overview of sterilization methods forpackaging materials used in aseptic packaging systems. Trans IChemE81:57–65.

Asadullah, Khair-un-Nisa, Omer MT, Syed AA, Khalid J, Askari Begum.2010. Study to evaluate the impact of heat treatment on water-solublevitamins in milk. J Pak Med Assoc 60:909–12.

Auldist MJ, Coats SJ, Sutherland BJ, Hardham JF, McDowell GH, RogersGL. 1996. Effect of somatic cell count and stage of lactation on the qualityand storage life of ultra-high-temperature milk. J Dairy Res 63:377–86.

Barach JT, Adams DM, Speck ML. 1976. Low temperature inactivation inmilk of heat-resistant proteinases from psychrotrophic bacteria, J Dairy Sci59:391–5.

Barbano DM, Ma Y, Santos, MV. 2006. Influence of raw milk quality onfluid milk shelf life. J Dairy Sci 89(Suppl E):E15–9.

Bastian ED, Hansen KG, Brown RJ. 1993. Inhibition of plasmin bybeta-lactoglobulin using casein and a synthetic substrate. J Dairy Sci76:3354–61.

Bayliss CE, Waites WM. 1982. Effect of simultaneous high-intensityultraviolet irradiation and hydrogen peroxide on bacterial spores. J FoodTechnol 17:467–70.

Blake DF, Stumbo CR. 1970. Ethylene oxide resistance of microorganismsimportant in spoiling of acid and high acid foods. J Food Sci 35:26–9.

Bonisch MP, Lauber S, Kulozik U. 2004. Effect of ultra-high-temperaturetreatment on the enzymatic cross-linking of micellar casein and sodiumcaseinate by transglutaminase. J Food Sci 69:398–404.

Borda D I, Smout C, Van Loey A, Hendrickx M. 2004. High-pressurethermal inactivation kinetics of a plasmin system. J Dairy Sci 87:2351–8.

Bramley AJ. 1998. The role of lysine as an inhibitor of plasmin on proteinquality. NEDFRC Annual Report. Available from: http://www.foodscience.cornell.edu/dairycenter/qs18.htm. Accessed Dec 15, 2010.

Brown KL. 2000. Control of bacterial spores. Br Med Bull 56:158–71.Burton H. 1988. Ultra-high temperature processing of milk and milkproducts. New York. Elsevier Science Publishing Co., Inc. New York.

Cattaneo S, Masotti F, Pellegrino L. 2008. Effects of overprocessing on heatdamage of UHT milk. Eur Food Res Technol 226:1099–106.

Cerf O, Davey KR. 2001. An explanation of non-sterile (leaky) milk packsin well-operated UHT plant. Trans IChemE 79:219–22.

Chism GW, Huang AE, Marshall JA. 1979. Sensitive assay for proteases insterile milk. J Dairy Sci 62:1798–800.

Cogan TM. 1977. A review of heat-resistant lipases and proteinases and thequality of dairy products. Ir J Food Sci Technol 1:95–105.

Collier CP, Townsend CT. 1956. The resistance of bacterial spores tosuperheated steam. Food Technol 10:477–81.

Crudden A, Kelly AL. 2003. Studies of plasmin activity in whey. Int Dairy J12:987–93.

Crudden A, Oliveira JC, Kelly AL. 2005. Kinetics of changes in plasminactivity and proteolysis on heating milk. J Dairy Res 72:493–504.

Datta N, Deeth HC. 2003. Diagnosing the cause of proteolysis in UHTmilk. Lebensm Wiss Technol 36:173–82.

Datta N, Deeth HC. 2007. Advances in thermal and non-thermal foodpreservation. Ames, Iowa: Blackwell Publishing. p 63–90.

Datta N, Elliott AJ, Perkins ML, Deeth HC. 2002. Ultra-high-temperature(UHT) treatment of milk: comparison of direct and indirect modes ofheating. Aust J Dairy Tech 57:211–27.

David JRD, Graves RH, Carlson VR. 1996. Aseptic processing andpackaging of food: a food industry perspective. New York: CRC Press, Inc.

Davies FL. 1975. Heat resistance of Bacillus species. J Soc Dairy Tech28:69–72.

Deeth HC, Datta N. 2003. Heating systems. In Roginski H, Fuquay JW, FoxPF, editors. Encyclopedia of dairy sciences, Vol. 4 London, U.K.: AcademicPress. p 2642–52.

Deharveng G, Nielsen SS. 1991. Partial purification and characterization ofnative plasminogen activators from bovine milk. J Dairy Sci 74:2060–72.

de Koning PJ, Kaper J, Roellama HS, Driessen FM. 1985. Age-thinning andgelation in unconcentrated and concentrated UHT-sterilized skim milk.Effect of native milk proteinase. Neth Milk Dairy J 39:71–87.

Denny CB, Brown CK, Yao MG. 1974. NCA tests of prime pak asepticcanning machine using hydrogen peroxide for equipment and containersterilization, March 20–23 and 26–28, 1973. Research Report nr 1–74,Washington, D.C.: Natl. Canners Assn.

Denny CB, Mathys AW. 1975. NCA test on dry heat as a means ofsterilization of containers, lids and a closing unit for aseptic canning. FinalReport on RF4614, December 1975. Washington, D.C.: Natl. CannersAssn. Research Foundation.

Driessen FM. 1983. Lipases and proteinases in milk: occurrence, heatinactivation, and their importance for the keeping quality of milk products[PhD thesis]. Wageningen Agricultural Univ.

Driessen FM, van der Waals CB. 1978. Inactivation of native milk proteinaseby heat treatment. Neth Milk Dairy J 32:245–54.

Dunkley WL, Stevenson KE. 1987. Ultra-high-temperature processing andaseptic packaging of dairy products. J Dairy Sci 70:2192–202.

Elfagm AA, Wheelock JV. 1978. Heat interaction between α-lactalbumin,β-lactoglobulin and casein in bovine milk. J Dairy Sci 61:159–63.

Elliott AJ, Dhakal A, Datta N, Deeth HC. 2003. Heat-induced changes inUHT milks—part 1. Aust J Dairy Tech 58:3–10.

Enright E, Bland AP, Needs EC, Kelly AL. 1999. Proteolysis andphysicochemical changes in milk on storage as affected by UHT treatment,plasmin activity and KIO3 addition. Int Dairy J 9:58–91.

Farahnik S. 1982. A quality control program recommendation for UHTprocessing and aseptic packing of milk and milk byproducts. Dairy Food San2:454–7.

Farrell HM, Thompson MP. 1990. β-Lactoglobulin and α-lactalbumin aspotential modulators of mammary cellular activity. Protoplasma 159:157–67.

Fox PF. 1981. Proteinases in dairy technology. Neth Milk Dairy J 35:233–53.Fox P, McSweeney PLH. 1996. Proteolysis in cheese. Food Rev Int12:457–509.

Garcıa-Risco MR, Cortes E, Carrascosa AV, Lopez-Fandino R. 1998.Microbiological and chemical changes in high-pressure- treated milk duringrefrigerated storage. J Food Prot 61:735–7.

Garcıa-Risco MR, Recio I, Molina, E, Lopez-Fandino R. 2003. Plasminactivity in pressurized milk. J Dairy Sci 86:728–34.

Gaucher I, Boubellouta T, Beaucher E, Piot M, Gaucheron F, Dufour E.2008a. Investigation of the effects of season, milking region, sterilisationprocess and storage conditions on milk and UHT milk physico-chemicalcharacteristics: a multidimensional statistical approach. Dairy Sci Technol88:291–312.

Gaucher I, Molle D, Gagnaire V, Gaucheron F. 2008b. Effect of storagetemperature on physicochemical characteristics of semi-skimmed UHTmilk. Food Hydrocolloids 22:130–43.

Gedam K, Prasad R, Vijay VK. 2007. The study on UHT processing of milk:a versatile option for rural sector. World J Dairy Food Sci 2: 49–53.

Goff D. 2008. University of Guelph, Canada. Dairy Chemistry and Physics.Available from: http://www.foodsci.uoguelph.ca/dairyedu. Accessed Dec15, 2010.

Gosta B. 2003. Dairy processing handbook. Chapter 9, 2nd ed. Lund,Sweden: Tetra Pak Processing Systems, S-221 86. p 227–44.

Gough P, Jenness R. 1962. Heat denaturation of beta-lactoglobulins A andB. J Dairy Sci 45:1033–9.

Grow KP. 2000. Quality control manual. Arkansas City, Kans.: Kan-Pak,LLC.

Grufferty MB, Fox PF. 1986. Potassium iodate-induced proteolysis inultra-high-heat treated milk during storage: the role of β-lactoglobulin andplasmin. J Dairy Res 53:139–77.

Hardham J, Auldist M. 1996. The effect of stage of lactation and somatic cellcount on the age-gelation of UHT milk. Heat treatments and alternativemethods. Vienna, Austria: Intl. Dairy Federation—ref S.I.9602. p 350–3.

Harrington R. 2009. Asia and UHT milk to lead strong growth in asepticpackaging. Available from: http://www.foodproductiondaily.com/content/view/print/250203. Accessed Dec 15, 2010.

Harwalkar VR. 1982. Age gelation of sterilized milks. In: Fox PF, editor.Development in dairy chemistry, Vol 1: proteins. London: Elsevier SciencePublishers Ltd. p 229–69.

Hassan A, Amjad I, Mahmood S. 2009. Microbiological and physicochemicalanalysis of different UHT milks available in the market. African J Food Sci3:100–6.



Hedrick TI, Harmon LG, Chandan RC, Seiberling D. 1981. Dairy productsindustry in 2006. J Dairy Sci 6:959–70.

Hill RD, Cracker BA. 1968. The role of lysine residues in the coagulation ofcasein. J Dairy Res 35:13–9.

Hirichs J. 2000. UHT-processed milk concentrates. Lait 80:15–23.Holdsworth SD. 1992. Aseptic processing and packaging of food products.New York: Elsevier Science Publishing Co., Inc. p 1–300.

Hong YH, Guthy K, Klostermeyer H. 1984. On the influence of SH-groupsin UHT milk during storage. Milchwissenschaft 39:285–7.

Horak P. 1980. Uber die Reaktionskinetik der Sporenabtoetung undchemischer Veraenderungen bei der thermischen Haltbarmachung vanMilch. Thesis, Munich: Technical Univ.

Hsu DS. 1970. Ultra-high-temperature (U.H.T.) processing and asepticpackaging (A.P.) of dairy products. New York: Damana Tech, Inc.

Huijs G, van Asselt AJ, Verdurmen REM, de Jong P. 2004. High-speed milk.Dairy Ind Int 69:30–2.

Hull ME. 1947. Studies on milk protein. II. Calorimetric determination ofthe partial hydrolysis of the protein in milk. J Dairy Sci 30:881–4.

Jovanka VP, Nada SL, Jovanka GL, Miroljub BB, Visnja MS. 2008. Colorchanges of UHT milk during storage. Sensors 8:5961–74.

Kelly AL, Foley J. 1997. Proteolysis and storage stability of UHT milk asinfluenced by milk plasmin activity, plasmin/β-lactoglobulin complexation,plasminogen activation and somatic cell count. Int Dairy J 7:411–20.

Kennedy A, Kelly AL. 1997. The influence of somatic cell count on the heatstability of bovine milk plasmin activity. Int Dairy J 7:717–21.

Kessler HG. 1981. Food engineering and dairy technology. FreisingGermany: Verlag A Kessler.

Kessler HG, Horak P. 1981. Objective evaluation of UHT-milk heating bystandardization of bacteriological and chemical effects. Milchwissenschaft36:129–33.

Kishonti E. 1975 Influence of heat-resistant lipases and proteases inpsychrotrophic bacteria on product quality. Ann Bull Intl Dairy FederationNr 86:121–4.

Kocak HR, Zadow JG. 1985a. Age gelation of UHT whole milk asinfluenced by storage temperature. Aust J Dairy Technol 40:14–21.

Kocak HR, Zadow JG. 1985b. Controlling age gelation of UHT milk withadditives. Aust J Dairy Technol 40:58–64.

Korel F, Balaban MO. 2002. Microbial and sensory assessment of milk withan electric nose. J Food Sci 67:758–64.

Korycka-dahl, M, Ribadeau Dumas B, Chene N, Martal Z. 1983. Plasminactivity in milk. J Dairy Sci 66:704–11.

Kulozik U, Guilmineau F. 2003. Food process engineering and dairytechnology at the Technical University of Munich. Int J Dairy Tech56:191–8.

Labropoulos AE, Varzakas TH. 2008. A computerized procedure forestimating chemical changes in thermal processing systems. Am J FoodTech 3:174–82.

Law BA, Andrews AT, Sharpe ME. 1977. Gelation ofultra-high-temperature-sterilized milk by proteinases from a strain ofPseudomonas fluorescence isolated from raw milk. J Dairy Res 44:145–8.

Lewis MJ. 1999. Microbiological issues associated with heat-treated milks. IntJ Dairy Tech 52:121–5.

Lopez-Fandino R, Carrascosa, AV, Olano. A. 1996. The effects of highpressure on whey protein denaturation and the cheesemaking properties ofraw milk. J Dairy Sci 79:929–36.

Lu DD, Nielsen SS. 1993. Isolation and characterization of native bovinemilk plasminogen activators. J Dairy Sci 76:3369–83.

Lyster RLJ. 1964. The free and masked sulphydryl groups of heated milkpowder and a new method for their determination. J Dairy Res 31:41–51.

Manji B, Kakuda Y. 1988. The role of protein denaturation, extent ofproteolysis, and storage temperature on the mechanism of age gelation in amodel system. J Dairy Sci 71:1455–63.

Manji B, Kakuda Y, Arnott DR. 1986. Effect of storage temperature on agegelation of ultra-high-temperature milk processed by direct and indirectheating systems. J Dairy Sci 69:2994–3001.

McMahon DJ. 1996. Heat-induced changes in β-lactoglobulin and thestability of UHT milk. 2nd Bienn UHT milk symposium. U.S.A: Utah StateUniv. p 1–6.

Metwalli AAM, de Jongh HHJ, van Boekel MAJS. 1998. Heat inactivation ofbovine plasmin. Int Dairy J 8:47–56.

Mitchell GE, Ewings KN. 1985. Quantification of bacterial proteolysiscausing gelation in UHT-treated milk. NZ J Dairy Sci Technol 20:65–76.

Morales FJ, Romero C, Jimenez-Perez S. 2000. Characterization of industrialprocessed milk by analysis of heat-induced changes. Int J Food Sci Tech35:193–200.

Mottar J, van Renterghem RV, de Vilder J. 1985. Evaluation of the rawmaterial for UHT milk by determining the degree of protein breakdownthrough HPLC. Milchwissenschaft 40:717–21.

Nielsen SS. 2003. Plasmin system in milk. In: Roginskin H, Fuquay, JW, FoxPF, editors. Encyclopedia of dairy sciences. Vol II. London, U.K.: AcademicPress. p 929–34.

Oldfield DJ, Singh H, Taylor MW. 1998. Association of β-lactoglobulin andβ-lactalbumin with the casein micelles in skim milk heated in anultra-high-temperature plant. Int Dairy J 8:765–70.

Oupadissakoon G. 2007. Comparison of the sensory properties ofultra-high-temperature (UHT) milk from different countries and preferencemapping of UHT milk between U.S. and Thai consumers [MS thesis].Kans.: Kansas State Univ.

Pereda J, Ferragut V, Quevedo JM, Guamis B, Trujillo AJ. 2007. Effects ofultra-high pressure homogenization on microbial and physicochemical shelflife of milk. J Dairy Sci 90:1081–93.

Pettersson B, Lembke F, Hammer P, Stackebrandt E, Priest FG. 1996.Bacillus sporothermodurans, a new species producing highly heat-resistantendospores Int J System Bacteriol 46:759–64.

Politis I, Zavizion B, Barbano DM, Gorewit RC. 1993. Enzymatic assay forthe combined determination of plasmin plus plasminogen in milk: revisited.J Dairy Sci 76:1260–7.

Prado BM, Sombers SE, Ismail B, Hayes KD. 2006. Effect of heat treatmenton the activity of inhibitors of plasmin and plasminogen activators in milk.Int Dairy J 16:593–9.

Precetti AS, Oria MP, Nielsen SS. 1997. Presence in bovine milk of twoprotease inhibitors of the plasmin system. J Dairy Sci 80:1490–6.

Ravishankar S, Maks N. 2007. Advances in thermal and non-thermal foodpreservation. Ames, Iowa: Blackwell Publishing. p 3–31.

Renner E. 1988. Storage stability and some nutritional aspects of milkpowders and ultra-high-temperature products at high ambient temperatures.J Dairy Res 55:125–42.

Richardson BC. 1983. The proteinases of bovine milk and the effect ofpasteurization on their activity. NZ J Dairy Sci Technol 18:223–45.

Rippen AL. 1969. Aseptic packaging of grade “A” dairy products. J DairySci 53:111–5.

Rollema HS, Poll JK. 1986. The alkaline milk proteinase system: kinetics andmechanism of heat-inactivation Milchwissenschaft 41:536–40.

Saint Denis T, Humbert G, Gaillard J. 2001. Heat inactivation of nativeplasmin, plasminogen and plasminogen activators in bovine milk: a revisitedstudy. Lait 81:715–29.

Samel R, Weaver RWV, Gammack DB, 1971. Changes on storage in milkprocessed by ultra-high-temperature sterilization. J Dairy Res 38:323–32.

Samuelson EG, Holm S. 1966. Technological principles for ultra-high-heattreatment of milk. 17th International Dairy Congress. Munich, Germany,Section B(1):57–65.

Sawer WH. 1969. Complex between beta-lactoglobulin and κ-casein. Areview: interaction in beta-lactoglobulin J Dairy Sci 52:1347–55.

Schamberger GP, Labuza TP. 2006. Evaluation of front-face fluorescence forassessing thermal processing of milk. J Food Sci 71:69–74.

Schmidt R, Renner E. 1978. Sensoric and chemical changes during thestorage of sterilized milk kinds. II. Chemical changes. Lebensem WissTechnol 11:244–8.

Scollard PG, Beresford, TP, Murphy PM, Kelly. AL. 2000. Barostability ofmilk plasmin activity. Lait 80:609–19.

Singh H. 2004. Heat stability of milk. Int J Dairy Tech 57:111–9.Singh RK. 2007. Advances in thermal and non-thermal food preservation.Ames, Iowa: Blackwell Publishing. p 43–61.

Skelte G A, Yuming Li. 2003. Association of denatured whey proteins withcasein micelles in heated reconstituted skim milk and its effect on caseinmicelle size. J Dairy Res 70:73–83.

Snoeren THM, van der Spek CA, Dekker R, Both P. 1979. Proteolysisduring the storage of UHT-sterilized whole milk. I. Experiments with milkheated by the direct system for 4 seconds at 142 ◦C. Neth Milk Dairy J33:31–9.



Solano-Lopez CE, Ji T, Alvarez VB. 2005. Volatile compounds and chemicalchanges in ultrapasteurized milk packaged in polyethylene terephthalatecontainers. J Food Sci 70:407–12.

Sørhaug T, Stepaniak L. 1997. Psychrotrophs and their enzymes in milk anddairy products: quality aspects. Trends Food Sci Technol 8:35–41.

Speck ML, Adams DM. 1976. Impact of heat-stable microbial enzymes infood processing heat-resistant proteolytic enzymes from bacterial source. JDairy Sci 59:786–9.

Stadhouders S, Mulder H. 1960 Fat hydrolysis and cheese flavour, IV. Fathydrolysis in cheese from pasteurized milk. Neth Milk Dairy J 14:141–7.

Swartling P. 1968. UHT-sterilization and aseptic packaging of milk. TechPubl Aust Soc Dairy Technol 19:18–25.

Topcu A, Numanoglu E, Saldaml I. 2006. Proteolysis and storage stability ofUHT milk produced in Turkey. Int Dairy J 16:633–8.

Tossavainen O, Kallioinen H. 2007. Proteolytic changes in lactose-hydrolysedUHT milks during storage. Milchwissenschaft 62:410–5.

van Asselt AJ, Sweere APJ, Rollema HS, de Jong P. 2008. Extremehigh-temperature treatment of milk with respect to plasmin inactivation. IntDairy J 18:531–8.

Weber BA, Nielsen SS. 1991. Isolation and partial characterization of a nativeserine-type protease inhibitor from bovine milk. J Dairy Sci 74:764–71.

West FB, Adams DM, Speck ML. 1978. Inactivation of Heat ResistantProteases in normal ultra-high-temperature sterilized skim milk by alow-temperature treatment. J Dairy Sci 61:1078–84.

Westhoff DC. 1981. Microbiology of ultrahigh temperature milk. J Dairy Sci64:167–73.

Wilson HK, Herreid E, Whitney RM. 1960. Ultracentrifugation studies ofmilk heated to sterilization temperatures. J Dairy Sci 43:165–74.

Zadow JG, Chituta F. 1975. Age gelation of ultra-high-temperature milk.Aust J Dairy Technol 30:104–6.

Zalazar C, Palma S, Candioti M. 1996. Increase of free sialic acid andgelation in UHT milk. Aust J Dairy Technol 51:22–3.


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