The use of carbon dioxide in the processing and packaging of milk and dairy products: A review

REVIEWThe use of carbon dioxide in the processing andpackaging of milk and dairy products: A review

PREETI SINGH,1* ALI ABAS WANI,1 ,2* A A KARIM3 andHORST-CHRISTIAN LANGOWSKI11Chair of Food Packaging Technology, Technical University of Munich, Weihenstephaner Steig 22, D-85350 Freising-Weihenstephan, Germany, 2Department of Food Technology, Islamic University of Science and Technology, Awantipora,J&K, India, and 3Food Technology Division, School of Industrial Technology, 11800, Universiti Sains Malaysia, Penang,Malaysia

*Author forcorrespondence. E-mails:[email protected]; [email protected]

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The upswing in consumer demand for fresh and high quality preservative-free foods has led to the devel-opment of modified atmosphere packaging (MAP). Increasingly, MAP is being used with high carbondioxide (CO2) concentration as well as CO2 ⁄N2 gas mixes. Modified atmosphere packaging or ‘gas flush-ing’ as it is also known is an increasingly popular technique used to extend the shelf life (both qualityand safety) of a number of dairy products. Carbon dioxide is an active constituent of MAP, naturallypresent in freshly drawn raw milk. Addition of CO2 to raw milk or flushing the package headspace hasproved to be a simple and cost-effective method, depending upon the initial microbiological quality of thefood product. Carbon dioxide addition through MAP or direct injection as an economically affordableshelf life extension strategy is used commercially worldwide for some dairy products. The development offood packaging machines with integrated gas flushing capabilities and the supply of ‘food grade’ gasesallow dairy foods manufacturers to enhance the quality of their products. This review presents a broadspectrum of current research and the current trends with respect to CO2 as a natural microbial hurdlewith special focus on its precise mechanism and its role in quality improvement of dairy products.

Keywords Milk, Modified atmosphere packaging, Dairy products, Cheese, Shelf life, Thermal process-ing.

INTRODUCT ION

Scientific and technological advances in processingand packaging of dairy foods have influenced con-sumers and processors of dairy products. The con-sumer demands include convenient dairy productswith enhanced nutrition and specific product func-tionalities. These prerequisite together with theseverity of the traditional food processing technol-ogies was driving forces for the improvements inexisting technologies as well as development ofnew techniques (Pereira and Vicente 2010; Singhet al. 2011). Thermal food processing is a well-known conventional technique for destroyingspoilage and pathogenic micro-organisms of liquidfoods with high water activity (aw) including milkand milk products. The changing lifestyle, eco-nomic growth and food preferences have cata-pulted the market to grow for nutritious, functionalfoods and fresh food products. This has brought a

significant increase in the food quality has of non-thermal processed foods including MAP (Singhet al. 2010a). In the recent years, the modifiedatmosphere packaging (MAP) market has shownten fold growth, and the scientific publications areincreasing in this area of study (Singh et al. 2011).However, microbial food safety aspects are of con-cern for the use of nonthermal processing technolo-gies for fresh products.Food safety regulatory authorities have recom-

mended the food industries to reduce the chemicaland microbial overload in foods. These stringentregulations along with consumer preferences fornatural and healthy products free of preservativeswith extended shelf life have led the food industryto develop new packaging concepts. Among them,MAP in combination with refrigeration has provensuccessful to increase the shelf life of milk andmilk products (Rodriguez-Aguilera et al. 2011a,b).Carbon dioxide (CO2) gas, a natural antimicrobial

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agent, is effective against the growth of some psychrotropicmicro-organisms (Hotchkiss and Chen 1996). It is naturallypresent in raw milk and is considered generally recognised assafe (Mermelstein 1997). Freshly drawn milk contains a signifi-cant amount of dissolved CO2 ranging from 40 to 1100 ppm,which quickly dissipates during thermal processing. Therefore,preservation of milk and milk products with CO2 would bepopularly acceptable both to the regulatory agencies and theconsumers. Depending on the initial quality, processing condi-tions and post process handling, the shelf life of nonsterile dairyproducts is generally restricted to 1–3 weeks (Salvador andFiszman 2004), whereas ultra-high temperature (UHT)-pro-cessed dairy products achieve a shelf life of 3 months–1 yearwithout refrigerated storage (Ma et al. 2000; Boor and Murphy2002). Following UHT, the most deleterious quality defectsresult from the release of proteolytic and lipolytic enzymes thatcause the breakdown of the protein and ⁄or hydrolysis of lipids(Espie and Madden 1997; Boor and Murphy 2002). This pro-cess results in release of bitter peptides and short chain fattyacids, which adversely affects the consumer acceptability. Forthese reasons, CO2 has been used as an alternate by the foodindustries, particularly for the preservation of highly perishableand certain high-value commodities (Table 1). Carbon dioxidewhen properly added in sufficient quantities extends the shelflife of dairy products by inhibiting the growth of microbial con-taminations, specifically aerobes and possibly limiting oxida-tive rancidity (Arvanitoyannis et al. 2011a,b; Mexis et al.2011). Among the available nonthermal preservation technolo-gies, MAP with CO2 has led the evolution of fresh and mini-mally processed foods for the past two decades and maintainsthe same quality for a longer time (Figure 1; Jayas and

Jeyamkondan 2002; Merriman et al. 2003; Jakobsen and Risbo2009). The continuous growth of the MAP processed dairyproducts is consumer demand driven because of its high protec-tion of nutrients and the sensory preferences. The other growthfactors leading to the growth of this market include concern onthe use of chemical preservatives, increasing cost of labour andenergy and choice for fresh like appearance.

CARBON DIOX IDE AS MICROBIAL HURDLEAND ANT IMICROB IAL AGENT

Shelf life of milk and milk products is limited because of theirhigh aw and favourable pH for the microbial growth (Muir1996a,b,c). The rapid spoilage adversely affects the flavour andtexture along with visual colour changes of refrigerated rawand pasteurised milk, cottage cheese and similar products. Theresponsible micro-organisms include psychrotrophic Gram-negative bacterial species (Pseudomonas, Acinetobacter, Flavo-bacterium, Enterobacter, Klebsiella, Aerobacter, Escherichia,Serratia, Proteus, Aeromonas and Alcaligenes), yeasts andmoulds (Geotrichum, Scopulariopsis, Mucor, Alternaria andPenicillium; Boor and Murphy 2002; Chambers 2002). Frommand Boor (2004) reported the presence of heat-resistantpsychrotrophic Gram-positive rods Paenibacillus, Bacillus andMicrobacterium as predominant spoilage organisms in the pas-teurised milk samples from three commercial dairy plants. Ithas been estimated that 25% of milk shelf life is limited by ther-moduric psychrotrophs, primarily Bacillus spp. (Sorhaug andStepaniak 1997). These organisms produce extracellular prote-ase and lipase activity, which reduce the functionality of milkproteins and fat and often produce undesirable aromas, many of

Table 1 Thermal processing versus carbon dioxide (CO2) processing

Thermal processing CO2 processing

Methods MethodsPasteurisation Direct addition of CO2

Sterilisation ⁄ ultra-high temperature (UHT) Modified atmosphere packagingExpensive technology; high infrastructure costs Cost-effective technologyNo combination; sole application of each method Improve the efficiency and end-quality of processing methods when used

in combinationNegative effects on nutritional, sensory and technological features Maintain physical, nutritional and organoleptic featuresMilk sterilised by the use of UHT affects probiotics and caseinstructure as well as whey proteins during storage

Maintain the bioactivity of probiotics and of the molecules of milk,colostrum and whey during storage

Failure of pasteurisation process is sometimes evident and is acompromise between fresh milk and sterilised milk

Use of CO2 in raw milk increases the lethality of pasteurisation processwhich in turn increases the consumer acceptance of dairy products

Alteration of enzymatic activity Modulation of enzymatic activityNo variation possible during processing as it leads to severe effects Variation in CO2 levels can be achieved without severe effectsLimited application Wide range of applicationDestruction of microstructure of heat-labile bioactives andcomponents

Improved microstructure through component interactions

Digestibility of the pasteurised milk is markedly impaired Digestibility is least affected

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which can be described as ‘fruity.’ Gram-positive organisms,particularly those producing lactic and acetic acids, can spoildairy foods, but the numbers of organisms required are gener-ally higher than for Gram-negative bacteria, and the changescan be less noticeable. Drop in the pH (curdling point) of pas-teurised milk is caused by the growth of heat-resistant lacticacid-producing cocci (Jay 2000).It has been reported that the product shelf life increases by

low oxygen atmospheres because of reduction in the above-specified aerobic micro-organisms. The antimicrobial effect ofCO2 occurs at or near a 10% level, and further increase in CO2

affects adversely the growth of Pseudomonas, Acenatobacterand Moraxella. The largest inhibition by CO2 occurs withGram-negative psychrotrophs, particularly Pseudomonas spp.,and the least inhibition effect is generally observed withGram-positive psychrotrophs, particularly Lactobacillus spp.(Hendricks and Hotchkiss 1997). Factors such as species, sub-strate and CO2 concentration influence the effect on pathogenicpsychrotrophs (Boor and Murphy 2002). The protective role ofCO2 is especially important to prevent the mould growth thathas significance in the cheeses. Its function in creating an anaer-obic environment with the displacement of existing molecularO2, its extra and intracellular pH decreasing effect and itsdestroying effect on the cell membrane make CO2 an inhibitorysubstance towards micro-organisms. There are three generalmechanisms by which CO2 inhibits micro-organisms (Clarkand Takacs 1980; Eklund 1984), namely, displacement of oxy-gen, influence of pH, and by cellular penetration. Although themechanism of antimicrobial effect of CO2 is not clear, itappears that CO2 extends the lag phase in many ways: it pene-trates the microbial cell wall and alters the permeability, solubi-lises inside the cells and produces carbonic acid, which reducesthe pH of the cell and interferes with several enzymatic and bio-chemical pathways inside the microbial cells. At the sameinstance, the use of very high CO2 levels may favour thegrowth of anaerobic species like Clostridium botulinum,Listeria monocytogens and Yersenia enterocolitica spp. in

refrigerated products. The maintenance of refrigerated condi-tions is a prerequisite to avoid the growth of these pathogenicmicro-organisms. The other advantage of the use of CO2 is toretard the oxidative rancidity in the milk and milk products.

Displacement of oxygenThis 1st mechanism for the CO2 action is displacement of someor all of the O2 available for aerobic microbial metabolism,thereby reducing the growth to a proportional amount.Although reducing available O2 may have some inhibitoryeffect on bacterial growth, it does not appear to be the most lim-iting factor (Daniels et al. 1985). As CO2 is more soluble thanO2, it readily displaces O2 and may also minimise variousdegradation reactions (Hotchkiss and Chen 1996).

Influence of pHThe 2nd mechanism is a lowering of the pH in the medium orfood because of the dissolution of CO2 and formation of car-bonic acid in the aqueous phase of the food. Most studies onCO2 atmosphere and bacterial growth show that the pH of thegrowth medium is decreased. One of the theories postulated forthe inhibitory effect of CO2 on aerobic spoilage bacteriaincludes the alteration of intracellular pH and consequenteffects on intracellular enzyme activities and substrate transport(Wolfe 1980). Several researchers have suggested that whengaseous CO2 is applied to a biological tissue, it is first dissolvedinto the liquid phase of the tissue and then absorbed as carbonicacid in the undissociated form (Mitsuda et al. 1980). The undis-sociated form of carbonic acid is not recognised by the protonmotive force of the microbial cell transport system, dissociatesinside the cell that in turn results in enzyme inhibition anddecreased microbial growth.

Cellular penetrationThe 3rd mechanism is a direct effect on the metabolism ofmicro-organisms as opposed to the indirect effects of pH reduc-tion and displacement of O2 (Daniels et al. 1985). Carbon diox-ide is an antimicrobial agent itself acting as weak, organic acid(upon dissolution) penetrating plasma membrane and acidifyingthe cell’s interior. Specific mode of action is not well under-stood; however, several contributory factors considered include,changes in the physical properties of the plasma membraneadversely affecting solute transport; inhibition of key enzymes,particularly those involving carboxylation ⁄decarboxylationreactions in which CO2 is reactant and reaction with proteinamino groups causing changes in their properties and activity(Adams and Moss 1995; Hong and Pyun 2001). The most com-mon experimental setup in defined media involves replacingsome portion of the air surrounding the growth media withCO2. Unfortunately, the growth media have not always beenbuffered to negate large shifts in pH because of CO2 dissolutionand formation of carbonic acid in the media, so it is unclearwhether the effect is simply because of a reduction in pH or ifCO2 has an inhibitory effect not associated with reduced pH.

High ----------------------------------------------------------------------------

Low -----------------------------------------------------------------------------

Product quality

Storage period

Air MAP

Figure 1 Quality of food products over length of storage.

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Furthermore, these experiments are often conducted in filmpouches that allow the permeation of both O2 and CO2 and thecomposition of the atmospheric changes over the course of theexperiment. Microbial and ⁄or fruit and vegetable respirationalso contributes to the atmospheric changes. The effects ofCO2-modified atmospheres on the growth of Pseudomonasfluorescens and Listeria monocytogenes in highly bufferednutrient solution under either constant O2 (20%) and varyingconcentrations of CO2 (0–80%) or constant CO2 (20%) andvarying concentrations of O2 (0–40%; balance N2) have beeninvestigated. Bacterial suspensions were incubated at 7 �Cunder a continuous flowing atmosphere of each gas mixture tobetter understand the relative significance of pH, O2 depletion,and direct effects of CO2 on growth (Hendricks and Hotchkiss1997). The results showed that CO2 suppresses growth, evenwhen the amount of O2 in the atmosphere is held constant at20% and the medium does not change pH. Fahestil et al.(1963) reported that CO2 stimulated mitochondrial ATPaseactivity and that such an action would have an uncouplingeffect on oxidative phosphorylation, resulting in a decreasedlevel of energy and inhibited certain decarboxylation enzymesthrough a mass action effect.

FACTORS INFLUENCING THEANT IMICROBIAL EFFECT OF CO2

The antimicrobial effect of CO2 is dependent on many factors,including the partial pressure, application time, concentration ofCO2 and temperature of the medium (Blickstad et al. 1981);volume of headspace gas, acidity, aw of the medium and thetype of organism present (Davidson and Juneja 1990); the typeof food product and microbial growth phase (Ogden 1997) andstorage temperature (Lambert et al. 1991).

Type of micro-organismsCO2 may exert a bactericidal or bacteriostatic effect on somemicro-organisms and stimulatory effect on other micro-organ-isms. Gram-negative bacteria are reported to be more sensitiveto CO2 than Gram-positive bacteria (Lambert et al. 1991).Gram-negative Pseudomonas species and Acinobacter morax-ella are inhibited by 20% CO2. Gill and Tan (1980) reportedBacillus thermosphacta is largely unaffected by CO2, while lac-tobacilli are very resistant to CO2 and can tolerate and grow in100% CO2 (Shaw and Nicol 1969). Anaerobic food-poisoningorganisms are not significantly affected by CO2. The growth ofpathogens, such as Salmonella spp., Staphylococcus aureus,Campylobacter, Y. enterocolitica and L. monocytogenes, is lessaffected by high CO2 concentration (Hintlian and Hotchkiss1986).

Time of applicationThe antimicrobial effects of CO2 also depend on the applicationtime. Exposure of micro-organism to CO2 causes an increasedlag phase duration of the micro-organisms (Gill and Tan 1980).

As bacteria enter the logarithmic phase of growth, the inhibi-tory effect of CO2 is reduced. For example, if CO2 is appliedimmediately to fresh meat while the spoilage bacteria are stillin their lag phase, shelf life can be extended by 50%, however,if CO2 is applied later, i.e. once bacterial growth has com-menced, the shelf life extension is reduced to 30% (Gill andTan 1980).

Gas concentrationMicro-organisms vary in their sensitivity to CO2. The concen-tration between 5% and 50% (v ⁄v) CO2 causes the inhibitionof yeasts and moulds (Y&M) and bacteria. The applied gascompositions for packaging of dairy products can vary from10% to 100% CO2, typically balanced with N2 as an inert fillergas, preventing package collapse as a result of CO2 solubilisa-tion in the cheese product. Subsequently, the CO2 is able tointerchange dynamically between the product and the head-space gas. The degree of this exchange is expected to dependon material properties such as the CO2 solubility in the specificcheese as well as on the product to headspace volume ratio andthe initial concentration of CO2 in both the packaging gas andthe product. This equilibration process is known to be relativelyfast and is expected to occur within the first day after packaging(Jakobsen and Bertelsen 2002). During transportation and stor-age, the CO2 will continue to exchange as a response onchanges in temperature conditions. In the case of MAP cheese,the applied gas volume ⁄headspace is often too small, comparedwith the product volume, to act as an adequate buffer for thisCO2 equilibration, and large changes in both headspace volumeand gas composition can occur. The fact that packagings areoften flexible ⁄ semiflexible results in either contraction (col-lapse ⁄ snug down) or expansion (swelling) of the package. Insome cases, this contraction is fully intentional creating anapparent vacuum package (e.g. packages of block cheese); inother cases, these volume changes are unwanted and cause con-sumer rejection, e.g. retail packages of sliced cheese (Fava andPiergiovanni 1992).

Storage temperatureThe storage temperature of product should be kept as low aspossible because the solubility of CO2 decreases dramaticallywith increasing temperature. The increased efficiency of CO2 atlower temperatures is related to the dissolution of CO2 in theaqueous phase of the produce, which is greater at lower temper-atures (Genigeorgis 1985). Gill and Tan (1980) observed thatthe inhibitory effect of a constant concentration of CO2

increased with decreasing temperature. The effects of holdingraw milk under CO2 pressures of 68–689 kPa at temperaturesof 5, 6.1, 10 and 20 �C on the indigenous microbiota wereinvestigated by Rajagopal et al. (2005). They concluded thatcombining low CO2 pressure and refrigeration would improvethe microbiological quality and safety of raw milk and may bean effective strategy for shipping raw single strength or concen-trated milk over long distances.


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EFFECTS OF CARBON DIOX IDEPROCESS ING AND PACKAGING

Modified atmosphere packaging (MAP) versus directinjection of CO2

Among the packaging technologies developed for the foodindustry, MAP has led the evolution of fresh and minimallyprocessed food preservation. Modified atmosphere packagingis defined as the replacement of the headspace gas surround-ing a food product with a gas mixture different from air.Modified atmosphere packaging is widely practiced for foodstorage and distribution (Brody 1995). In addition to alteringthe gas composition surrounding the food, a barrier packag-ing material is often employed to retard the dissipation ofthe modified atmosphere through the package material. Mod-ified atmosphere packaging (indirect method) surrounds thefood products in high-barrier packaging materials, in whichgaseous environment is modified, which dissolves CO2

rapidly in food products and can create a vacuum inside ofrigid packages (Parry 1993).Modified atmosphere packaging can be applied to dairy

products to control some of the fungal problems and extendtheir shelf life. Low level of CO2 by the application of MAPcould significantly improve the shelf life of some cheeses.Owing to soft texture, soft cheeses are more successfully pack-aged under MAP because of the cushioning effect of the gas.Half-fat soft cheese packaged in the metallic films also benefitsfrom gas flushing of the headspace (Addington 1991). For dairyproducts, MAP may not provide sufficient control and the shelflife of the product may be inconsistent (Moir et al. 1993). Aninjection of 5.68–22.7 mM CO2 directly into products coupledwith high-barrier packaging has been developed as a method toinhibit undesirable micro-organisms in these dairy products toextend shelf life (Chen and Hotchkiss 1991a,b). A process thatis practiced commercially includes injecting liquefied CO2 gasdirectly into a flowing stream of dairy product via a gas-sparg-ing unit and the equipment consists of a sintered stainless steelfrit with porosity of 7–30 lm. The process has been termed‘direct addition of CO2’ to distinguish it from conventionalMAP. The net effect is similar to that of MAP; the gas is addedto the product for the purpose of increasing shelf life by inhibit-ing microbial activity. The cost of the addition of CO2 to dairyfoods via this method is generally economically feasible. Maand Barbano (2003a) studied freezing point and pH of milkwith different fat levels (0%, 15%, 30%) when injected withCO2 at 0 and 40 �C. At 0 and 40 �C, milk fat was mostly solidand liquid, respectively. Milk with a higher fat content showeda lower pH and freezing point with CO2 injection at 0 �C. Atall fat levels, milk exhibited same pH levels at 40 �C.

Effect of packaging materials used in MAPThe principal function of packaging is to delay microbiologi-cal spoilage by restricting the growth of spoilage organisms,but to be commercially useful, nonmicrobial deterioration

must also be controlled (Gill and Molin 1991; Hutton 2003).When packaging materials are properly applied, they providea means for substantially extending the range of productsthat can be prepared at the packer or processor level, andthe geographical area over which these products can be dis-tributed and merchandised. However, nonpackage factorssuch as initial microbiological load, product temperature his-tory and product composition often determine the effective-ness of preservative packagings (Gill 1991). Therefore, bothpackaging and nonpackaging factors are important and mustbe considered during the selection of a packaging system fora particular application. One of the most important factorsaffecting the use of direct addition of CO2 to dairy productshas been the lack of sufficient barrier in the packaging mate-rials. There is little benefit of CO2 addition to a product ifthe gas is allowed to dissipate. Therefore, packaging is theprincipal means of preserving the original concentration ofCO2 within the product. As per the observations of Moiret al. (1993), CO2 level has been found to decline rapidlyduring storage when dissolved in cottage cheese samples(packaged in conventional polystyrene plastic tubs) becauseof the probable reason that conventional tubs used in cottagecheese packaging are highly permeable to CO2. This loss ofdissolved gases can be overcome by placing a high CO2

barrier foil ⁄polyolefin laminant seal over the opening of aconventional cheese tubs (Gorski 1996). In addition, activepackaging, when used in combination with MAP, can furtherprovide continued inhibition for product stabilisation byaltering the package film permeability and selectively absorb-ing food components or releasing compounds to the foodproduct.

EFFECTS OF CO2 ON MILK CONST ITUENTSAND OTHER PROPERT IES

In milk at pH 6.3–6.5, approximately 88% of CO2 exists as dis-solved CO2 gas, 2% as carbonic acid and the remaining 10% asbicarbonates. Carbonate has a buffering capacity (pKa � 6.4),and it can modify salt balance by the formation of calcium car-bonate salt. The integrity of casein micelles is dependent on thebalance between hydrophobic interactions and electrostaticrepulsions, and it is well accepted that casein micelles are steri-cally stabilised by the j-casein ‘hairy’ layer (Tuinier and DeKruif 2002). It has been observed that carbonation increases theviscosity of milk and reduced the size of casein micelles(Chang and Zhang 1992). Gevaudan et al. (1996) and Guil-laume et al. (2004a,b) conducted reversible acidifications ofmilk by means of carbonation, injecting pressurised CO2 as theacidifying agent instead of HCl ⁄NaOH, to reduce the pH to avalue in the range, 5.8–4.8 (at 5 ± 1 �C). When compared withHCl ⁄NaOH, carbonation has the benefit to leave the ionicstrength of the milk constant after restoring the original pH.These authors found that mineral and protein partition wasrestored and no change of micelle size or charge was observed


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after milk carbonation in the studied pH range. However, car-bonation to a pH below 5.8 reported improvement in rennetingproperties with a reduction in the rennet clotting time andchange in buffering curves. Gevaudan et al. (1996) attributedthese phenomena that results in a change in micellar calciumphosphate salt form. Mineral equilibrium was achieved after30–60 min of acidification (Gastaldi et al. 1994; Law andLeaver 1998); whereas in general, protein equilibrium reachedafter 24 h, although Law and Leaver (1998) showed that a largequantity of casein dissociates from the micelles at pH 5.5 and5.2 within 1 h after acidification. Addition of CO2 to milk thusinduces qualitative and quantitative changes in buffering capac-ity. Carbonated milk has two maximal buffering peaks at pH4.95 and 5.4 compared with only one at pH 5.1 for control milk(De La Fuente et al. 1998). At 15 �C, concentrations of CO2

added to raw milk in the range of 0.4–33.6 mM lowered thepH from 6.8 to approximately 6.1 (Martin 2002). Similarly, forautoclaved raw milk amended at lower temperatures (4 �C)with 0–35 mM CO2, the pH value was found to be 5.9 (low-ered from 6.70; Loss 2001).The effect of CO2 itself or combined with a heat treatment

was reported on caseins and whey proteins (Eie et al. 1987;Chang and Zhang 1992; Olano et al. 1992; Ruas-Madiedoet al. 1996), carbohydrate fraction (Olano et al. 1992), andorganic acids and volatile compounds (Ruas-Madiedo et al.1996). A study by Sierra et al. (1996) demonstrated thatafter 7 days, all-trans-retinol, tocopherols, and b-caroteneremained stable in refrigerated raw milk acidified with CO2

(pH 6.0 and 6.4). Eie et al. (1987) reported that when milkwas acidified with CO2 to pH 6.0 and used for the manufac-turing of dried milk, whey proteins were denatured to greaterextent. On contrary, it has been reported that the acidificationof milk with CO2 from pH 6.9 to pH 6.2 did not increasewhey protein denaturation or lactose isomerisation duringheating and remained stable during sterilisation (Olano et al.1992). Sensory score of acidified milk (with CO2 to pH 6.4and heated at 70 �C for 30 min) was found to be comparablewith the control sample (Olano et al. 1992). After degassing,pH returned to its initial state and no changes occurred in theconcentration of inorganic phosphate, calcium or magnesiumin the aqueous phase. Casein micelle stability and reactivitywere assessed on milk subjected to reversible acidification bycarbonation (Raouche et al. 2007). In this study, pressurisedCO2 was injected at 4 �C, leading to controlled acidificationfrom 6.63 ± 0.02 to a target pH (5.5 or 5.2). Upon CO2

treatment, calcium and protein partition, zeta potential andsize of casein micelles were restored directly after neutralisa-tion. The rheological properties of the gel obtained by theacid coagulation of CO2-treated milk did not change as aresult of carbonation. Micelle hydration increased after neu-tralisation and during storage. Milk buffering capacity in thepH range of 4.5–5.5 decreased after the neutralisation of milkacidified by carbonation but increased during chilled storageof this milk.

PRESERVAT ION OF DAIRY PRODUCTS BYCO2 ADDIT ION

Raw milkCarbonated milk has the potential to meet the needs of health-conscious consumers for nutritious and pleasant beverages(Chang and Zhang 1992). Carbon dioxide can reduce the levelof enzyme activity in raw milk. This minimises the develop-ment of off-flavours during transportation and storage. One pro-cedure to prevent the proliferation of micro-organisms involvesthe addition of CO2 to refrigerated raw milk (Roberts andTorrey 1988; Amigo et al. 1995; Ruas-Madiedo et al. 1996;Espie and Madden 1997). This is an affordable method toextend cold milk storage at the farm or processing plant. Acidi-fication of raw milk to pH 6.2 with periodic pH adjustments bygas bubbling has proved to be very efficient for extending thecold storage. Vacuum degasification prior to pasteurisation ren-dered milk acceptable for liquid consumption (Skudra 1983;Amigo et al. 1995; Ruas-Madiedo et al. 1996) as well as tominimise the build up of deposits on the walls of the pasteuriser(Calvo and Derafael 1995). Carbon dioxide can be incorporatedinto milk by sparging in milk line or by bubbling into milk silothrough suitable gas-sparging system fitted at the bottom of thesilos. Before processing of milk, degassing is to be performedeither by using vacuum degasifier or by sparging flowingstream of nitrogen gas into milk prior to HTST pasteurisation(Gevaudan et al. 1996; Ruas-Madiedo et al. 1996; Ganguli2001; Thongoupakarn 2001; Santos et al. 2003; Rajagopalet al. 2005). Refrigeration of raw milk reduces the growth rateof mesophilic bacteria but favours the proliferation of psychro-tropic micro-organisms (Kraft 1992). More investigations havedocumented the chemical and microbiological effects of lowlevels of added CO2 in raw milk (Table 2).

Pasteurised milkShelf life of pasteurised milk is 10–20 days when stored at6.1 �C (Labuza 1982). Its shelf life could be extended by atleast 25–200% at CO2 concentration near the sensory threshold(Hotchkiss et al. 1999). The sensory threshold as determinedby a trained panel for CO2 in pasteurised milk with 2% fat is>2.8 mM, and milk remains sensorially fit up to the level<9.1 mM (Hotchkiss et al. 1999). They added 1.8–3.2 mMCO2 to whole pasteurised milk that was stored in paperboardcartons at 6 �C for up to 14 days. The sensory threshold forCO2 in full fat pasteurised milk was 16.8 mM, which was wellabove the highest CO2 concentration tested. They also observedthat the threshold appeared to increase as the fat contentincreased, possibly because CO2 is more soluble in fat thanwater. A low concentration of CO2 inhibits the growth of psy-chrotrophic bacterial species as well as provides a reasonableshelf life, and the effect was greater at 4 �C than at 7 �C (Chenet al. 1992; Hotchkiss et al. 1999).Bacillus cereus is an aerobic spore former commonly found

in milk and milk-based products and can cause either emetic or


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Table 2 Shelf life and quality of raw milk under carbon dioxide (CO2): summary of various studies

Treatment Effects References

50 atm of CO2 to raw milk Reduction in growth rate of microbial species Hoffman (1906)10 atm of CO2 to raw milk Delayed lactic fermentation

No curdling even after 72 hVan Slyke and Bosworth (1907)

10–40 mM CO2 to raw andbacterial-inoculatedsterilised skim milk

Increasing CO2 concentration and decreasing temperatures shown toinhibit bacterial growth

Initial microbial quality affects the efficiency of CO2

Difference between SPC of treated and control was as much as3 log units after 6 days

Effect was directly because of presence of CO2, not because of loweringof pH or to the displacement of oxygen

King and Mabbitt (1982)Mabbitt (1982)Law and Mabbitt (1983)Mabbitt (1982)

20–30 mM CO2 to sterilemilk (inoculated withproteolytic psychotropicbacteria)

Lag time increased and exponential growth rate decreasedRefrigerated storage life could be extended 1–3 days by the additionof low levels of CO2

Roberts and Torrey (1988)

Acidification of inoculated,sterilised and raw milk byCO2

pH lowered down till 6.0Increased generation times and decreased growth rates for severalpseudomonas spp.

Sensory properties were maintained

Amigo et al. (1995)

30–40 mM CO2 to raw milk SPC, coliforms, psychotrophs showed inhibition at 6 �C for up to7 days except lactobacilli

Espie and Madden (1997)

Addition of CO2 incombination withhigh-barrier packages

Three fold increase in shelf life of raw milk Ganguli (2001)

0.6–6.14 mM CO2 to rawand inoculated sterile milkat 15 �C

Significantly inhibits bacterial growthWith increase in CO2 concentration, the lengthening of time for maximumgrowth was observed as well as bacterial growth rate reduced

Greater effects towards Gram-negative than Gram-positiveEven at above-refrigeration temperatures, CO2 can reduce the growth ofmilk pathogenic and spoilage organisms

Martin et al. (2003)

68–689 kPa CO2 at 5–20 �C No protein precipitation observed up to 9 days of storageMicrobial quality was of high standardsAt 6.1 �C, the time to reach 4.30 log cfu ⁄mL increased by 4 days ascompared with control

Rajagopal et al. (2005)

0–54 mM CO2 pH of treated milk decreased during pasteurisation in response to increasein pressure and CO2 concentration

Beaulieu et al. (1999)

600–2400 ppm CO2 followedby heating and pressurisation

At fixed temperature pH of milk (with added CO2) decreased withincreasing CO2 concentration and pressure

At fixed CO2 concentration, the effect of pressure on pH decrease wasgreater at a higher temperature

pH depression caused by the modification of milk with up to 23 mM ofCO2 could be reversed by vacuum removal of CO2

Pasteurisation temperatures and pressures and initial CO2 content of milkare important factors to prevent milk degradation during pasteurisation

Ma and Barbano (2003c)Ma et al. (2001)

1–58 mM CO2 Dissolved CO2 between 1 and 36 mM linearly decreased the decimalreduction time at 50 �C for Pseudomonas fluorescens

CO2 concentration of 44–58 mM significantly reduced the z value for SPC(63–93 �C)

Loss (2001)

Acidification with CO2 to pH6.0–6.2 at 4 �C

Neither caseins nor whey proteins were affected by combined treatment ofCO2 addition, vacuuming and pasteurisation

Lower microbial counts as compared with controlAdditional shelf life achieved by the addition of CO2 did not affect fat-orwater-soluble vitamin or free monosaccharide content of raw milk

Little difference in terms of sensory and biochemical properties

Ruas-Madiedo et al. (1996)Ruas-Madiedo et al. (2000)


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diarrhoeal food borne illness because of toxins (Kramer andGilbert 1989; Anderson et al. 1995). Bacillus cereus may alsocause spoilage of aseptically packaged milk, canned milks andnonaseptic packaged refrigerated milk. In the absence of com-petition and over the course of an extended storage period, lowconcentration of B. cereus can cause defects, such as coagula-tion and off-odours because of proteolysis and lipolysis (Meeret al. 1991; Anderson et al. 1995). Spores of the micro-organ-isms are thermally resistant and can survive pasteurisation andstorage at refrigeration temperatures. Werner and Hotchkiss(2002) advocated that the addition of moderate levels of CO2

(11.9 mM) did not have either stimulatory or inhibitory effecton the initiation of germination and subsequent outgrowth ofB. cereus spores over long-term storage and did not increasethe risk of food borne illness. Similar conclusions were maderegarding C. botulinum (Glass et al. 1999). Smoot and Pierson(1982) demonstrated that CO2 could inhibit or stimulate or havelittle effect on germination and toxigenesis of spore formerssuch as C. botulinum.

Dry milk powdersDry whole milk has a shelf life of <6 months, which is mainlybecause of fat oxidation (De 2000). Dried milk must be consid-ered a sensitive product because it is often consumed afterreconstitution without additional heating (Richter et al. 1992).Reducing the O2 content in the package also reduces oxidativerancidity. Dry milk powders packaged in cans or drums forlong-term storage are commonly commercially packaged inmodified atmospheres, including mixed CO2 and N2, 100% N2,or reduced O2 atmospheres. Driscoll et al. (1985) investigatedthe sensory quality of instant and regular non-fat dry milk pow-ders after 4 years of storage in cans or polybags at 10, 21 and32 �C and modified atmospheres (air, 100% CO2, 100% N2).At 21 �C, milk powder stored in cans or in polybags under airwas less desirable in sensory qualities (off-flavours) than milkpowder stored under either N2 or CO2 at the same temperature.Packaging techniques have also been developed for dry milkpowders to eliminate or reduce O2 and, hence, reduce autooxi-dation. These include gas flushing and use of O2 absorbers. De(2000) observed that fat decomposition resulting in a tallowyflavour is a major storage defect in whole milk powder and sug-gested inhibitory measures by removing O2 from the headspaceof the package followed by flushing either by N2 or mixture ofN2 and CO2, the latter being restricted to 5–20%.

Hard cheesesModified atmosphere packaging, although used for a wide vari-ety of products in the dairy sector, has mainly been applied tothe packaging of cheese. The cheese category itself comprisesmany different types, varying in composition and thus in shelflife. Therefore, the packaging of each type of cheese needs tobe considered separately. Another factor to consider is thatsome cheeses are CO2 producers, while others are not. Further-more, the age of the cheese may vary from 3 months to 2 years

at the stage of packaging (McDonald 1985). When cheese ispackaged in a modified atmosphere, the optimal gas composi-tion used depends on the characteristics of the cheese (Alveset al. 1996; Eliot et al. 1998; Gonzalez-Fandos et al. 2000). Itis important that the levels of CO2 are controlled because forcertain cheeses, high levels of CO2 have been found to impartoff-flavours to the cheese (Mannheim and Soffer 1996; Gonz-alez-Fandos et al. 2000; Trobetas et al. 2008). In addition, ifthe levels of CO2 are too high without N2 in the package, theCO2 can be absorbed by the product leading to pack collapse.However, the bacteriostatic and fungistatic effect of CO2 willnot be experienced if its levels are too low (Floros et al. 2000).Juric et al. (2003), however, found that packaging atmospheresof 100% CO2 resulted in undesirable changes in texture and fla-vour of sliced Samso cheese stored under light. Cheeses storedunder CO2 contained higher concentrations of aldehydes andfatty acids and lower concentrations of alcohols and esters thancheeses stored under N2. Colour bleaching occurred only incheeses packaged under CO2 and exposed to light. The shift incolour is proposed to be due to an interaction between CO2 andhigh-intensity light, leading to the oxidation of the pigmentmolecule, bixin (Colchin et al. 2001). Hard and semisoftcheeses, such as cheddar, are commonly packed in 100% CO2

or mixtures of CO2–N2 using horizontal form-fill-seal pillow-pack machines. The packaging materials used include polyv-inylidene chloride-coated cellophane or polyester ⁄polyethylene(Damske 1990), 15 lm oriented polyester ⁄50 lm low-densitypolyethylene with 4% ethylene vinyl acetate (Addington 1991)and clear polypropylene. Oyugi and Buys (2007) studied themicrobiological quality of shredded cheddar cheese packagedin different modified atmospheres with and without O2 scav-engers included in the packaging film and concluded that thefilm with O2 scavengers was more effective than the controlfilm against mould growth and 73% CO2 ⁄27% N2 atmosphereresulted in the cheese with the best microbiological qualities.Carbon dioxide treated milk has also been shown useful for

the production of several rennet-coagulated cheese without anyadverse effects on the quality (Uceda et al. 1994; McCarneyet al. 1995). Presence of gas reduced the coagulation time orthe amount of rennet for milk coagulation. Calvo et al. (1993)found that the acidification of raw milk with CO2 to pHbetween 6.0 and 6.5 reduced psychrotrophic bacteria counts,resulting in improved cheese yields. However, the differenceswere small, and the initial microbial counts were in the range of105–106 cfu ⁄mL in the controls, making it unclear whethersimilar results would be seen with lower initial counts. Otherstudies (Ruas-Madiedo et al. 1998a,b) looked at milk of lowermicrobial load and found that cheese yields from CO2-treatedand untreated stored milk did not differ significantly. McCarneyet al. (1995) have also investigated the effects of CO2 additionto milk used to make cheese. They concluded that the additionof 30 mM of CO2 reduced the time to reach psychrotrophiccounts of 106 cfu ⁄mL, which in turn improved grading scores.The cheese made from CO2-treated milk showed reduction in


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casein and lipid breakdown, presumably because of reducedproteolytic and lipolytic activity. Montilla et al. (1995) showeda 75% reduction in the amount of rennet necessary for coagula-tion along with a small reduction in proteolysis in cheeses madewith CO2-treated milk. The authors suggested that use of CO2-treated milk would not have detrimental effects on cheese prop-erties or yield and would extend the keeping quality of the rawmilk. In one study, Ruas-Madiedo et al. (2002) investigated theeffect of CO2 incorporation (to raw milk) on the production ofrennet-coagulated Spanish hard cheeses. Vacuum degassingwas carried out prior to pasteurisation process and ⁄or thecheese-making stages. They observed that when compared withcheese made from pasteurised milk, CO2-treated milk exhibitedslower initial growth of lactobacilli with lower levels of acidproduction. In follow up of this study, Ruas-Madiedo et al.(2003) evaluated the effects of these treatments on proteolysisoccurring in cheese and concluded that cheeses manufacturedfrom CO2-treated milk showed lower levels of hydrophilic pep-tides while no variation in hydrophobic peptides amounts at theend of the ripening process. During ageing, b-casein break-down was not affected while a-casein disruption was elevated.Similarly, Nelson et al. (2004a,b) observed no changes inb-casein disintegration and an increase in a-casein breakdownduring the ageing of cheese made with CO2-treated milk. How-ever, cheese produced from CO2-acidified milk showed lowertotal fat and calcium content than the control cheese, and highertotal salts with no change in total crude protein. Ma and Barb-ano (2003b,c) studied the effect of protein concentration andtype in CO2-treated ultrafiltered (UF) and microfiltered (MF)milk on freezing point and pH and observed that (i) increasingcasein or soluble protein amount increased the buffering capac-ity of milk, (ii) at low CO2 injection temperatures, pH reductionwas influenced by the type and amount of protein in milk. Stud-ies on modified skim milk (with moderate pressures of CO2) byGevaudan et al. (1996) showed that the shift in bufferingcapacity of the milk to a slightly lower range might be due toan irreversible build up of milk salts.Unattractive crystals on cheddar cheese have been investi-

gated since 1930s (McDowall and McDowell 1939), and yetthe problem remains a challenge to cheese manufacturers(Chou et al. 2003). A large number of cheddar cheeses manu-factured in the US have the problem of calcium lactate crystals(CLC; Johnson 2004). Johnson et al. (1990) observed fasterand greater crystal formation on gas-flushed (CO2) cheeses thanvacuum-packaged. Dybing et al. (1988) hypothesised that freeionic calcium combines with lactate through a mechanisminvolving carbonic acid. As CO2 is absorbed, the pH of theserum phase is reduced. It is hypothesised that low pH incheese shifts colloidal calcium to soluble calcium (Hassan et al.2004) and increased serum calcium concentration facilitatesCLC formation. Calcium lactate crystals have also beenobserved on vacuum-packaged cheeses in which the packagehas lost integrity (Johnson et al. 1990). Because cheese serumtends to move to the surface of the cheese or to cracks and

crevices inside the cheese during ageing, lactic acid concentra-tion in those spaces increases. In loosely packaged cheese, thesurface of the cheese dries because of evaporation, formingnucleation sites that accelerate crystal formation. Residual cal-cium and lactose, after pressing, also contribute to CLC. Lac-tose is metabolised to lactic acid and can react with calcium incheese to form CLC (Sutherland and Jameson 1981). Elevatedlevels of lactose (more than 4.8%) in cheese milk increase lac-tose content in cheese, which can be used by starter bacteria orNSLAB to produce lactate in cheese and potentially increasecalcium lactate concentrations (Dybing et al. 1988). However,Blake et al. (2005) showed that high lactose content in cheesemilk does not guarantee CLC formation. Recent study by Agar-wal et al. (2005) also confirmed that gas flushing (regardless ofgas composition), milk composition and presence of nonstarterlactic acid bacteria can contribute to the development of CLCon cheese surfaces.

Soft cheesesSoft cheeses made from pasteurised milk and having highmoisture content are common craft products in various coun-tries. These cheeses have limited shelf life, in most cases requir-ing controlled refrigerated conditions during distribution andsale. An alternative, for conventional packaging, to increaseshelf life of cheese is to use MAP that could be an interestingpossibility leading to an additive-free product (Floros and Mat-sos 2005).CO2 acts both directly on moulds and indirectly by displac-

ing O2; moulds have an absolute requirement for O2. Vacuumpackaging (VP) does not remove all of the O2, and thus, Y&Mgrowth can still occur (Hocking and Faedo 1992), particularlyin regions of the food product-packaging interface where pack-age wrinkling occurs. Carbon dioxide is also absorbed into thecheese and creates a vacuum within the pouch that in turnretards the chemical changes (Rodriguez-Alonso et al. 2011).Sliced and grated cheeses can be pillow-packed under MAP(Damske 1990; Pluta et al. 2005). The gas mixture typicallyused is 70% N2:30% CO2 to inhibit mould growth, to keep thepackage from collapsing around the shreds, and to preventshred matting. Alves et al. (1996) have compared 100% N2

and 100% CO2 with 50% N2:50% CO2 for packaging slicedmozzarella cheese in high-barrier laminated films. Theyreported that atmospheres of ‡50% CO2 were more effectivethan air or 100% N2 in improving shelf life of sliced mozzarellacheese. Eliot et al. (1998) found similar benefits of CO2 inshredded mozzarella cheese. The major effect of CO2 on thesecheeses is the inhibition of surface mould growth (Maniar et al.1994), although high CO2 MAP atmospheres have been shownto inhibit growth of lactic and mesophilic bacteria as well (Eliotet al. 1998). Different workers observed an increase in the shelflife of about 240% in buffalo milk mozzarella cheese by MAP(Sarantopoulos et al. 1995). Carbon dioxide enriched atmo-spheres (100% CO2) showed a significant role in maintainingthe overall sensorial characteristics of shredded mozzarella


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cheese in pizza samples (baked and unbaked; Singh and Goyal2010; Singh et al. 2010b,c).Modified atmosphere packaging with CO2 under refrigera-

tion temperature is also effective in extending the shelf life ofRequeijao, a Portugueses whey cheese. Pintado and Malcata(2000) studied the effect of MAP on the microbial flora in Req-ueijao and reported that the viable numbers of Enterobacteria,Staphylococci, yeasts and spore forming bacteria in the experi-mental whey cheeses did not increase within 15 days at 4 �Cunder 100% CO2. Authors also reported inhibitory effectagainst Bacillus, Pseudomonoas, Lactobacillus and Streptococ-cus species. Rosenthal et al. (1991) studied the effect of CO2

on the shelf life of Quarg cheese and found that no growth ofY&M. Also, no changes in the pH value for 67 days of storageat 4 �C under CO2 enriched atmosphere in cheeses allowed ashelf life of up to 98 days; whereas in air, the shelf life of thisproduct was only a few days (Sarantopoulos et al. 1995). Pier-giovanni et al. (1993) compared Tallegiio cheese packagedunder four modified atmospheres and stored at 6 �C to conven-tional paper wrapping and found that samples packaged inMAP had satisfactory quality. Westall and Filternberg (1998)studied the influence of yeasts on the spoilage of decorated softcheese packaged in MAP and found that the increase in theconcentration of CO2 affected the growth of spoilage yeasts.Gammariello et al. (2008) evaluated the shelf life of Stracciatel-la cheese packaged in four different CO2:N2:O2 gas mixtures at8 �C and showed that the MAP, in particular 50:50:0 and95:5:0, prolonged the sensorial acceptability limit by delayedgrowth of spoilage bacteria, without affecting the dairy microfl-ora. Favati et al. (2007) evaluated the shelf life of portionedProvolone cheese packaged in protective atmosphere usingdifferent CO2 ⁄N2 gas mixtures. The gas mixture (30%CO2 + 70% N2) guaranteed portioned Provolone cheese thebest preservability, as it was able to slow down the proteolyticand lipolytic phenomena typical of cheese ripening. ‘Anthotry-ros’ cheese (Greek cheese) was packaged under 30%CO2 ⁄70% N2 modified atmosphere and exhibited delayedmicrobial growth compared with VP as well as extended shelflife (by 10 days) with good sensory characteristics (Papaioan-nou et al. 2007).Whitley et al. (2000) studied the effect of MAP on the growth

of L. monocytogenes in mould ripened cheeses at 2–8.3 �C for6 weeks. MAP with a CO2 (£20%) concentration allowedgrowth to occur only when O2 was incorporated. This resultedin reduced lag time from 3 to 2 weeks and subsequent growthwas also faster, producing an increase in cell numbers of 1.4 logcycles over the incubation period. A recent study by Del Nobileet al. (2009) suggest that MAP of Ricotta cheese with 95% CO2

inhibits microbial growth without a significant effect on lacticacid bacteria, probably due to their facultative anaerobic natureand also maintains the natural colour of Ricotta cheese. Theeffect of MAP on the growth of L. monocytogenes in inoculatedand noninoculated Cameros cheese (goat cheese) was evaluatedby Olarte et al. (2002) at 4 �C. A concentration of 100% CO2

showed the lowest microbial counts. L. monocytogenes growthwas lower when the CO2 concentration increased but even after28 days, the L. monocytogenes population was only 1.3 logunits lower in inoculated cheeses packaged at 100% CO2 thanfor those packaged in air. L. monocytogenes were not found inany of the noninoculated sample. Dermiki et al. (2008) studiedthe effect of MAP on shelf life extension of whey cheese ‘Myzi-thra Kalathaki’ and concluded that 40% CO2 ⁄60% N2 and 60%CO2 ⁄40% N2 gas mixtures were most effective for inhibitinggrowth of aerobic microflora, Y&M, Enterobacteriaeceae andpsychrotrophs as well as maintaining the sensory characteristicsuntil 40 days at 4 ± 0.5 �C.The use of CO2 has been found to be commercially benefi-

cial in the preservation of cottage cheese. Creamed cottagecheese sealed in flexible containers following CO2 flushing andstorage at 4 �C showed depressed growth of psychrotrophs,yeasts and moulds (Kosikowski and Brown 1973). Fresh fla-vour was maintained for 73 days, but because of the high levelof CO2, the cottage cheese had a ‘fizzy’ character. Mann(1991) reported a significant inhibitory effect on pseudomonasin cottage cheese by bubbling CO2 through the cheese beforepackaging. Cups are flushed with CO2 before filling, and at theend, the headspace is again flushed with CO2. The tubs aresealed with aluminium foil and capped. Moir et al. (1993) sug-gested that the addition of CO2 throughout the cheese beforepackaging was necessary to inhibit psychrotrophs both on thesurface and within the depth of the cheese. They reported a sig-nificant difference in the microbial counts between the surfaceand the interior of cottage cheese packaged in conventional,thermoformed, high-impact polystyrene cups.Many of the problems associated with CO2 in cottage cheese

have been overcome by the direct addition of CO2 into thecream dressing prior to mixing with the curd to form cream-style cottage cheese (Chen and Hotchkiss 1991a,b, 1993). Thequality of CO2-containing cottage cheese packaged in polysty-rene tubs overwrapped with a high-barrier heat-shrinkable filmcan be maintained for 63 and 42 days at 4 and 7 �C, respec-tively (Chen and Hotchkiss 1991b). The commercial procedurefor manufacturing cottage cheese with a low level of CO2

involves injecting CO2 into the cream dressing via an in-linesparging unit designed for food applications. Several parame-ters should be controlled, including the size of the CO2 bubbles,backpressure within the line, residence time in the line, temper-ature and the filling process (Hotchkiss and Lee 1996). Usingtrained sensory panellists in triangle tests, they have found thatthe lowest threshold for CO2 in milk is between 4.54 and9.10 mM (Chen et al. 1992; Lee 1996). The flavour thresholdfor untrained ‘consumer’ panels is likely to be higher. Moiret al. (1993) found that 10 mM CO2 injected into cottagecheese cream dressing and package headspace could signifi-cantly increase shelf life while not affecting pH or flavour.Mermelstein (1997) also advocated the potential of the com-mercialisation of the use of CO2 in the curd dressing for cottagecheese to improve its shelf life. Commercial product has the


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shelf life of 8 weeks as compared with normal shelf life of3 weeks.

Ice cream mix and sour creamDirect addition of CO2 combined with high-barrier packaginghas been shown to extend the shelf life of ice cream mix andsour cream. Dairy mixes, such as ice cream mix, which is pro-duced in one plant for further processing in another, may alsoget benefit from the addition of CO2. A level of up to1000 ppm CO2 increases the shelf life of ice cream mix by 75–125%. The ice cream mix is further processed in the secondplant, and hence, there are no concerns about the CO2 affectingmouth feel (Henzler and Paradis 1997). Carbonation of icecream mix was found to have no appreciable effect on the bac-teria (Prucha et al. 1922; Rettger et al. 1922). It was furtherdemonstrated that CO2 at atmospheric pressure had no bacterio-static or bactericidal effect on organisms originally present inthe ice cream mix.

Yoghurt and fermented beveragesYoghurt is a flavourful and healthful food. In efforts to offervariety and competition in the market, carbonation of yoghurtmay attract new yoghurt consumers. Owing to the low pH, bac-terial growth in these products is retarded. The major factorsfor determining the shelf life and quality of yoghurt productsare Y&M growth, development of off-flavours as well assurvival of probiotic microbial species (Robinson et al. 2002;Viljoen et al. 2003). Various preservation technologies havebeen developed to extend shelf life of these products by takinginto account the effect on spoilage and desirable micro-organ-isms. Blakistone (1990) has shown experimentally that gasflushing of the headspace of yoghurts cups after filling extendsthe shelf life by retarding the growth of spoilage organisms. Onthis basis, yoghurt being a semisolid product, can also easilyaccept carbonation. Taylor and Ogden (2002) patented themethod for the carbonation of spoonable yoghurt as well asoptimised and improved process for the carbonation of differentviscous fluids. Karagul-Yuceer et al. (2001) observed that1.10–1.27 volumes of dissolved CO2 (incorporated intoyoghurt) did not affect the growth ⁄proliferation of typical ornon-typical yoghurt bacterial species as well as growth ofundesirable microorganisms. From their studies, they had alsohypothesised that the CO2 could feasibly stimulate growth ofstarter culture bacterial species thereby reducing their produc-tion time. Of all microbial species monitored during storage,L. monocytogenes was not affected by CO2 but slowly declinedin treated and untreated products. Counts of Escherichia colidecreased to nondetectable levels in CO2-treated yoghurt till60 days, while Bacillus licheniformis showed no growth underall conditions. Choi and Kosikowski (1985) reported the loweracidity in carbonated yoghurt as compared with noncarbonatedone, and typical yoghurt bacterial cultures were unaffected.This may be attributed to the suppression, by carbonation, ofgrowth of lactic psychrotropic bacteria especially responsible

for the post acidification in yoghurt. They also carbonated thesweetened plain yoghurt beverage (0.5 kg ⁄cm2 CO2) at 4 �Cand extended its shelf life to 120 days as compared with30 days for control. They observed an improved sensory qual-ity and mouth feel in the carbonated yoghurt without anydefects including bitterness and post acidification. The sensorythreshold could be used as a parameter to improve the technol-ogy of carbonated yoghurt products thereby extending shelf lifewithout affecting sensory characteristics. Wright et al. (2003)observed that the sensory threshold in carbonated yoghurt is onan average 5.97 mM. Calvo et al. (1999) indicated that yoghurtmade from CO2 treated skim milk was not different (includinglactic acid production) from control yoghurts samples. Similarfindings were also reported by Gueimonde et al. (2003) andthey also observed no changes in the evolution of organic acidsfrom experimental and control samples with little effect ongrowth of starter cultures and sensory properties.With the increasing demand for fermented dairy beverages,

CO2 has also been studied for its use in manufacture of theseproducts. It has been observed that CO2 decreased milk fermen-tation time in these products with no significant effect on theirsensory properties as well as on starter cultures (Vinderolaet al. 2000; Gueimonde and de los Reyes-Gavilan 2004). Inanother study on B. cereus-inoculated ABT (Lactobacillusacidophilus ⁄Bifidobacterium bifidum ⁄Streptococcus thermo-philus) milk at 4 �C, proteolysis and acid production werefound to be reduced in inoculated milk and CO2 can be used asan effective method for reducing the risk of B. cereus contami-nation in ABT milk with no impact on the growth of the probi-otic Bifidobacterium (Noriega et al. 2003). In another study byKaragul-Yuceer et al. (1999), sweetened low fat (1%) plainyoghurt and low fat Swiss-style strawberry and lemon yoghurtswere manufactured after the addition of cultures and thenyoghurt samples were incubated at 43 �C until desirable pHvalues of 5.0 or 4.2 were reached. Carbon dioxide (0.08–0.09 kg ⁄cm2) was incorporated and stored at 4 �C for 45 days.Their results showed that the carbonation had no significanteffect on the acceptability of yoghurt during shelf life with noalteration on the sensory characteristics.

ButterThe studies undertaken by Hunziker (1924) on the effect ofCO2 on butter indicated that carbonation could not be reliedupon as a means of destroying bacteria present in cream andrendering such cream, or the butter made from this cream, safefor human consumption. Addition of CO2 to butter during thechurning process during butter manufacture has been studied(Prucha et al. 1925). The gas was allowed to flow into thecream during the entire churning operation. No pronouncedeffect on microbial growth was observed but shown preserva-tive effect on riboflavin and carotenoids (yellowish colour) dur-ing storage under light exposure (Bosset et al. 1995). The‘sourish’ taste (undoubtedly because of residual CO2 levels thatwere above the taste threshold) of the butter immediately after


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carbonation disappeared during storage. This latter phenome-non suggests that the CO2 level was not maintained within thebutter sufficiently to have an inhibitory effect. Prucha et al.(1925) reported that bacterial growth was best retarded in car-bonated butter when packaged in airtight containers (tubs).However, it is still unclear from these studies, that why CO2

did not remain dissolved in the butter as it is generally estab-lished that it is highly soluble in nonpolar lipids (Fogg andGerrard 1991). While studying effect of CO2 injection tempera-ture on CO2 solubility, Ma and Barbano (2003a) found that athigher temperatures CO2 solubility in the fat increased and viceversa. Recent studies on butter with regard to the application ofCO2 have not been performed.

CONCLUS IONS

Modified atmosphere packaging has been traditionally used topreserve the freshness of fresh produce, meats and fish bycontrolling their biochemical metabolism. MAP dramaticallyextends the shelf life of packaged food products, and in somecases, food does not require any further treatments or anyspecial care during distribution. In recent years, MAP hasfound application in milk and milk products with the use ofCO2 injection technique. Carbon dioxide is a unique naturalantimicrobial and processing aid that has several potentialuses in the dairy industry. It is unique because it can beadded to and removed from dairy products with no deleteri-ous effects.The direct addition of CO2 to dairy products coupled with

increasing the barrier properties of the containers has been com-mercially successful and economically feasible with cottagecheese and other fluid products with a shelf life increase of200–400%.The benefits of CO2 to the cheese industry are quite clear,

and there is abundant data supporting the use of CO2 toimprove the microbial quality of raw milk. The knowledge andinformation needs to be transferred to the farm and milk collec-tion sectors of the industry. Quantifying the effects of CO2 ongrowth of spoilage organisms and pathogens through the use ofstatistical modelling will be helpful for optimising its use andensuring safe and wholesome products. Work in this area is stillin infancy needs to be demonstrated by appropriate methods.Moreover, for a MAP system to work effectively, optimal pack-aging material with proper gas permeability properties must beselected. In most cases, extending shelf life and maintainingquality requires a multiple hurdle technology system, for exam-ple, introducing temperature control as well as MAP is gener-ally essential to maintain the quality of packaged foods. Hurdletechnology is, therefore, important for MAP applications ofdairy products, because the modified atmosphere provides anunnatural gas environment that can create serious microbialproblems such as the growth of anaerobic bacteria and the pro-duction of microbial toxins. Therefore, an included temperaturecontrol system is very important for quality preservation and

microbial control. Further research on precise preservativeaction of CO2 can provide a better understanding for the pro-cessing of dairy products.

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Documents

The use of carbon dioxide in the processing and packaging of milk and dairy products: A review