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Microstructure of cheese: Processing,
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microbiological
technological andconsiderationsmicrobiological indicators), and quality and safety of t
specific molecular compositions and spatial arrangemen
Corresponding author.
924-2244/$ - see front matter � 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.tifs.2009.02.006
Gomes and F. Xavier Malcata
F/E
tolic
el.:
Claudia I. Pereira, Ana M.P.
CBQ
Ca
(T
Cheese is a classical dairy product, which is strongly judged by
its appearance and texture; hence, a renewed interest in its mi-
crostructure has been on the rise, as sophisticated techniques
of analysis become more and more informative and widely
available. Processing parameters that affect microstructure
play a dominant role upon the features exhibited by the final
product as perceived by the consumer; rational relationships
between microstructure (which includes biochemical and
he
products are accordingly required. Subsequent to that extra
fundamental knowledge, technological innovations may even-
tually improve current cheesemaking processes, and permit
mechanistic design of novel ones. This review thus focuses
on recent advances pertaining to the microstructure of cheese,
and discusses them in a logical and critical manner.
IntroductionCheese is a highly regarded food in most human cultures,
and has accordingly been present throughout ages in man-kind daily life. The variety of cheeses currently available islarge, because a myriad of topical advances e encompassingmanufacture and ripening, have cumulatively taken place
over time. In addition to the somewhat intrinsic (andunpredictable) variability within each cheese type, tailor-made cheese matrices have been proposed based on specificmicrostructures e which have emerged side by side withintroduction of alternative (or improved) methodologies incheesemaking.
Microstructure is not a static concept; it evolves insteadalong the food processing chain, and eventually leads to ma-jor transformations relative to the original microstructure ofthe milk feedstock itself. This realization thus encompasses
ts.
scola Superior de Biotecnologia, Universidade
a Portuguesa, Rua Dr. Antonio Bernardino de
Almeida, P-4200-072 Porto, Portugal
D351 22 558 0004; fax: D351 22 509 0351;
e-mail: [email protected])
On the other hand, it should be emphasized that, besidesthe supporting proteinaceous/fatty matrix, microorganismsare also an integral part of cheese. Microbial activity pro-duces indeed major transformations of the cheese matrix,which will affect the final microstructure as well. Hence, itis also crucial to elucidate microorganismematrix interac-tions, in attempts to understand the whole picture.
Consumer acceptance of a cheese product dependsdirectly on its appearance, flavour and texture e whichare in turn originated by a thorough combination of micro-biological, biochemical and technological parameters, thataffect microstructure directly or indirectly. Note that theultimate success of any food product relies on consumers’reactions: in fact, human perception of organoleptic charac-teristics is closer to the consumer status at the moment ofdecision than data generated by any type of analyticalinstrumentation (Adhikari, Heymann, & Huff, 2003) edespite its constraints in repeatability and objectiveness.
Texture is intrinsically related to the arrangement ofvarious chemical components within distinct micro- andmacrostructure levels e e.g. proteic network or fat fraction;it is the external manifestation of such structures that even-tually determines the uniqueness and distinctive characterof a cheese product. However, cheeses are particularlycomplex systems, so full and meaningful assessment ofthe effects of microstructure (and texture) upon flavourand appearance is still incipient.
This paper discusses a number of fundamental aspectspertaining to microstructure of cheese e and specificallyfocuses on issues associated with microstructural effectsarising from technological and processing approaches.
Manipulation of physical properties of milkA huge variety of dairy products exists at present,
depending on the deliberate alterations of the original
characteristics of milk e which always start with someform of bulk concentration (Malcata, 1999). A set of down-stream operations are normally applied to milk e which areintended to alter, strengthen or create structure; they areaimed at minimization (or even elimination) of potentialrisk factors, e.g. product decay via metabolism of contam-inating microflora. Attempts to improve yield, as well as todesign novel products with better organoleptic features,also lead to changes at the microstructure level.
The most important unit operations in dairy food physi-cal manipulation, that impinge upon microstructure, arelisted in Table 1, and are discussed below in further detail.
Thermal treatmentsThermal treatment is the most common operation used with
milk; this is due to the tight safety that is enforced in foodplants, including industrial cheese manufacture. Several dis-tinct heat treatments have been successfully applied inindustrial practice e ranging from mild to severe ones. Asexpected, the more severe the heat treatment, the more exten-sive the damages brought about thereby e as the thermalhistory of milk affects the properties of the coagulum, andthus the development afterwards of microstructure; heat-treated (and homogenized) milk may in fact lead to off-flavours and weak clotting features (Singh & Wauguna, 2001).
Another consequence of milk heating is whey proteindenaturation, followed by aggregation; or interaction withcaseins, in the first place. The fraction of whey proteinstaken up may reach 50e70%, depending on their degreeof denaturation. A high pre-heating temperature improvesyield and stability of the final product, as well as texturee which will become smoother if short-time heating is em-ployed, because of increased water binding to the denaturedwhey proteins (Hinrichs, 2001).
Table 1. Processing applications in cheesemaking milk, and effects thereo
Treatment parameters Microstructural effects
High temperatures No effect on structure of casein micelles
Weakened clotting properties
Denaturation of serum proteins
Interactions between k-casein and whey protein
Interactions between milk proteins and fat mem
High pressures Effect on milk protein intramolecular bonds
Change of coagulation characteristics
No effect on activity of native milk enzymes
Reduction of syneresis
Increase of cheese yield
Closer-packed structure
Acceleration of cheese ripening
Enzymes (TGase) Modification of rheological and renneting prope
Prevention of coalescence between milk fat glo
Incorporation of whey proteins
Influence on primary and secondary stages of c
Membranes Reduction of rennet coagulation time
Increase in firmness of coagulum
More compact structure of casein
Softer texture and altered flavour characteristics
Pressure treatmentsHigh pressure (HP) has been proposed as a suitable tech-
nology for milk treatment to substitute, or in addition tothermal treatment. Its relatively recent development hasbeen associated mainly with the possibility of inactivatingundesired microorganisms and/or enzymes in milk; how-ever, one cannot disregard the effect of such a practiceupon milk constituents themselves, and consequently onthe final characteristics of ripened cheese e beyond im-provement of microbial safety and extension of shelf-life.
So far, the most important concern in terms of HP-inducedchanges pertains to the physicochemical properties of caseinmicelles and whey proteins e as HP affects intramolecularbonds, either reinforcing or weakening them. Lopez-Fandino, Carrascosa, and Olano (1996) reported that pressur-ization of cheesemaking milk at 300e400 MPa for 30 mincaused an increase of 14e20% in curd weight, and a decreaseof 7.5e15% in protein loss in whey. Huppertz, Fox, de Kruif,and Kelly (2006), Lopez-Fandino (2006) and Considine,Patel, Anema, Singh, and Creamer (2007) have comprehen-sively reviewed the molecular changes induced by HP inmilk proteins, whereas Trujillo, Capellas, Saldo, Gervilla,and Guamis (2002) highlighted the principles of HP and theirimplications on the final properties of dairy products.However, a thorough description of the aforementionedchemical and molecular mechanisms, and of the underlyingtheoretical issues is not within the scope of this review e butrather the implications of such a type of process on the struc-tural features of cheese.
It is widely accepted that HP treatments alter milk coag-ulation characteristics e either by reducing or increasingthe gelation time (Lopez-Fandino, 2006), or by inducingproteolytic and lipolytic activities that promote accelerationof cheese ripening (Guerzoni et al., 1999). Exposure to HP
f upon microstructure of cheese
References
s
brane components
Kim and Jimenez-Flores (1995)
Grappin and Beuvier (1997)
Heinrichs (2001)
Schreiber (2001)
Singh and Wauguna (2001)
Guerzoni et al. (1999)
O’Reilly et al. (1999)
Needs et al. (2000)
Wendin et al. (2000)
Capellas et al. (2001)
Kheadr et al. (2002)
Huppertz et al. (2006)
Lopez-Fandino (2006)
rties
bules
oagulation
Cozzolino et al. (2003)
Hinz, Huppertz, Kulozik
and Kelly (2007)
Bonisch et al. (2008)
, in hard- and semi-hard cheeses
Erdem (2000)
Mistry (2004)
Benfeldt (2006)
increases cell membrane permeability (Malone, Shellhammer,& Courtney, 2002), thus favouring release of intracellularenzymes and their access to substrates. This techniquecan also be used to attenuate the viability of starter bacteriathroughout ripening, and thus enhance the release of meta-bolic compounds upon lysis (Upadhyay, Huppertz, Kelly, &McSweeney, 2007). Furthermore, HP aids in release of Ca,P and as1- and as2-caseins e depending on the operatingconditions, hence leading to destabilization of micelles(Huppertz, Fox, & Kelly, 2004; Regnault, Dumay, &Cheftel, 2006); this renders caseins more susceptible toeventual action by proteolytic enzymes.
From a microscopic point of view, HP treatments of milkproduce a closer-packed structure in cheese, due to the re-duction in average size of casein micelles (Needs, Stenning,Gill, Ferragut, & Rich, 2000); the finer structure strengthensthe cheese matrix, via establishment of caseinecasein and/orcaseinefat complexes (Kheadr, Vachon, Paquin, & Fliss,2002), thus producing firmer curds (Lopez-Fandino et al.,1996). These findings are well documented via microscopy,both electronic (Capellas, Mor-Mur, Sendra, & Guamis,2001; Kheadr et al., 2002) and optical one (Capellas et al.,2001; Wendin, Langton, Caous, & Hall, 2000).
Such a technology has been as well applied to cheese aftermanufacture (Garde, Arques, Gaya, Medina, & Nunez, 2007;Juan, Ferragut, Guamis, & Trujillo, 2008). Several authors(O’Reilly et al., 2003; O’Reilly, Murphy, et al., 2002;O’Reilly, O’Connor, Murphy, Kelly, & Beresford, 2000;O’Reilly, O’Connor, Murphy, Kelly, & Beresford, 2002)have studied in depth the effects of a number of cheese HPtreatments on key ripening characteristics of both Cheddarand Mozzarella cheeses. In general, high pressures (ca.200e400 MPa) for relatively short times (ca. 20 min)brought about changes in the protein structure itself e whichin turn improved the functional features of the final cheesematrix, viz. an increase in the flowability and fluidity, and a re-duction in the melt time upon heating. On the other hand,lower pressures (ca. 50e200 MPa) combined with longerprocessing times (up to 82 h) did not seem to affect structuralfeatures, as indicated by confocal laser scanning microscopy.
Membrane treatmentsMembrane technology is more and more widely used in
the dairy industry, because of its versatility and high ener-getic efficiency. It basically consists on a pressure-drivenoperation through a porous solid medium, aimed at separa-tion and/or concentration; it ranges from plain filtration toreverse osmosis e also known as hyperfiltration (Mistry,2004), depending on the percolating pore diameter and con-sequent particle size partition capacity, from ca. 100 mmdown to ca. 0.1 nm (Kelly, 2004).
The main goal of ultrafiltration (UF) is to concentratebulk milk via retention of whey proteins; this enhances yieldof cheese, which also contains lower concentrations of lac-tose and minerals (Hinrichs, 2001). A few physicochemicalproperties of milk are critical in cheesemaking via UF e viz.
viscosity, buffering capacity and rennet-driven coagulation.Upon performance of UF on milk, proteins and salts aresimultaneously concentrated, thus leading to an increasedbuffering capacity; this influences metabolism of lacticacid bacteria favourably, whereas coagulation time is re-duced and firmness of the resulting coagulum is increased(Mistry, 2004).
Furthermore, membrane treatments have been used forrecovery of milk components and their incorporation asnew ingredients in cheese manufacture formulations eviz. UF retentates (Govindasamy-Lucey, Jaeggi, Bostley,Johnson, & Lucey, 2004), milk protein concentrates andphosphocasein (Guinee, O’Kennedy, & Kelly, 2006).Therefore, membrane technology can be regarded as anefficient tool for development and continuous productionof cheese, via intermediate production of highly concen-trated matrices e that can be coagulated with little to noacid whey production at all (Nelson & Barbano, 2005).
Despite its intrinsic advantages, UF has not yet been fullyadopted for manufacture of hard- and semi-hard cheeses ebecause of the harder texture in the final product, whencompared with conventional soft and semi-soft cheeses.Furthermore, the high water binding capacity of whey pro-teins e coupled with retarded proteolysis, influences the tex-ture of cheese, which becomes progressively firmer (i.e.more resistant to fracturing), more cohesive, grainier anddrier, whereas the structure of the protein matrix becomescoarser and more compact, thus resulting in slower softeningduring ripening (Fox, McSweeney, Cogan, & Guinee, 2000).Finally, Erdem (2000) suggested that casein micelles de-crease in size, and possibly undergo rearrangement via estab-lishment of hydrophobic bonds e thus producing a morecompact structure and an increase in elasticity; Karami, Eh-sani, Mousavi, Rezaei, and Safari (2009) provided evidencefor this fact via scanning electron microscopy. However, fun-damental knowledge on how the changes in milk proteinsand fat during UF affect cheese manufacturing is still limited.
Enzymatic treatments
Milk coagulation can be influenced by various factors, in-cluding enzyme-mediated cross-linking of casein micellesvia microbial transglutaminase (TGase) e a transferasethat forms bonds both within and between several proteins,via glutamine and lysine residues (Kashiwagi et al., 2002).Caseins are particularly suitable substrates for this activity,owing to their low level of tertiary structure (Huppertz &de Kruif, 2007).
Cross-linking of food proteins brought about by TGaseaffects their gelling, rheological, emulsifying and rennetingproperties; Bonisch, Heidebach, and Kulozik (2008) showedthat such cross-linking affects both the primary and thesecondary stages of rennet coagulation. Hinz et al. (2007)reported, in turn, that TGase-induced cross-linking of milkproteins affects considerably their emulsifying features,via increasing the stability of fat globules against coales-cence. Finally, Cozzolino et al. (2003) suggested use of
TGase as a means to incorporate whey proteins into cheese-making milk, in a way somewhat similar to UF. These fea-tures seem promising in cheese manufacture, as theyincrease yield and gel strength e thus favouringmicrostructure.
Characterization of cheese
The whole cheesemaking process contributes to developa distinct and more complex matrix than the departing feed-stock milk. During coagulation e which, in most cases, isenzyme-driven, rennet enzymes bring about breakdown ofk-casein, that is present especially on the surface of caseinmicelles. After such a chemical step, physical agglomera-tion takes place e which gives rise to a more uniform pro-tein mass, that spontaneously expresses whey; this processof compactation (termed syneresis) may be taken one stepfurther, by externally applying pressure onto the curd. Onthe other hand, fat globules normally retain their mem-branes e and are thus observed as single entities, whichmay form clusters that are entrapped within the so formedproteinaceous, three-dimensional matrix.
Physical matrixCream cheese is a spreadable soft cheese e which
structurally differs from other cheeses because of itslack of a compact protein matrix, coupled with a relativelyhighmoisture content. Its major structural component is fat(ca. 33%), in the form of clusters of globules interspersedwithin milk proteins (Kalab, 1993); its microstructure hasbeen defined as corpuscular, or composed of compact fat-casein aggregates with large spaces filled with whey(Kalab, 1985). The moisture content thereof is a factor thatpositively contributes to spreadability: if protein particlesand fat globules are packed less densely, a high-moistureproduct will result.
An increasing demand for reduced fat cheese has arisenamong more educated, health-aware consumers. However,decreasing the level of (or even totally removing) fat oftenleads to textural drawbacks, viz. a hard body with poormeltability and stretchability (Merrill, Oberg, McManus,Kalab, & McMahon, 1996), as well as a rubbery texture(Mistry, 2001). The flavour, colour and mouthfeel of cheesewill also be adversely affected, since a few compoundsdissolve preferentially in fat (Li, Marshall, Heymann, &Fernando, 1997) e although a higher smoothness mayalso be imparted to the cheese matrix (Mistry, 2001).
Fat substitutes e based on proteins, polysaccharides andsynthetic chemical molecules (or even alternative fats),have been tested in attempts to convey at least as satisfac-tory sensory attributes; among others, Wendin et al. (2000)proved that it is possible to create a cream cheese witha lower fat content, but possessing an organoleptic profilesimilar to that of normal cream cheeses e simply by crite-rious modification of processing parameters. A more recentstudy (Sahan, Yasar, Hayaloglu, Karaca, & Kaya, 2008)showed that low-fat cheeses containing fat replacers exhibit
a higher level of total free fatty acids than low-fat controlcheeses, with a significant increase in their texture attri-butes and meltability; however, the full-fat cheese wasawarded the best sensory scores, at all stages of ripening.Scanning electron micrographs helped elucidate how total(or partial) substitution of regular milk fat produces cheeseswith different microstructures and inherently distinct tex-tural characteristics (Lobato-Calleros et al., 2007).
Another good example of a matrix for which the interre-lationship between microstructure and final quality isapparent is processed cheese. Cheese processing was orig-inally proposed as a means to upgrade cheese of lowerquality. Processed cheeses are typically prepared by heatinga mixture of comminuted cheeses in the presence of salts(Piska & Stetina, 2004) e which usually include sodiumcitrate or phosphate, the main role of which is sequesteringcalcium from the calciumecaseinate complex and restoringthe emulsifying properties of cheese proteins. The influenceof emulsifying salts on the final processed cheese structurehas been discussed by Kalab, Modler, Caric, and Milanovic(1991) and Awad, Abdel-Hamid, El-Shabrawy, and Singh(2002), among others; those salts also aid in controllingpH and stability of disperse proteins, thus contributing tothe development of an appropriate microstructure.
Several reports are available pertaining to manufactureof processed cheeses, using lower grade White, Cheddar,Gruyere, Gouda, Kashkaval, Emmental and Feta cheeses(and other ingredients) as feedstock. For instance, Tamime,Kalab, Davies, and Younis (1990) studied the processing ofCheddar cheese with skim milk powder, and its effect uponthe microstructure of the final product. Kalab et al. (1991)found, in turn, that White cheese is suitable to manufactureprocessed cheese, as the original matrix is harder even athigh levels of cheese incorporation. Finally, Piska andStetina (2004) focused on cheese ripening and rate ofcooling of the blend.
Microbiological factorsIn microstructural studies pertaining to Serra da Estrela
cheese, Parker, Gunning, Macedo, Malcata, and Brockle-hurst (1998) found bacteria embedded in the protein matrix,and especially lining the curd junctions; they also cameacross with protein that appears as strands and free fat inthe smear e likely as a result of proteolytic and lipolyticactivities. Furthermore, cells were either directly in contactwith the fat globule membrane (as happened with most bac-teria) or located at the caseinefat interface. On the otherhand, Lopez, Maillard, Briard-Bion, Camier, and Hannon(2006) found bacteria organized as colonies in the matrixof Emmental cheese e such colonies were preferentiallylocalized at the fat/protein interface, and both fat and bac-teria were entrapped in the casein network; similar resultswere previously obtained during characterization of Mozza-rella cheese (Oberg, McManus, & McMahon, 1993).
Lactic acid bacteria have been claimed to contribute tocheese microstructure, viz. Lactococcus lactis e via their
Existing product
Incorporation of new ingredients
Reduction/avoidance of harmful-likeingredients
Incorporation of pre/probiotic ingredients
Inclusion of component(s) with nutraceuticalvalue
New product
Herbs/ham/etc.
“Light” product
“Bio” product
Vitamins/Ca2+/Peptides/etc.
Examples Innovations
Improvement/development of novelflavours
Flavouring agents
Fig. 1. Schematic formulation path of a novel dairy product.
role in proteolysis (Centeno, Tomillo, Fernandez-Garcıa,Gaya, & Nunez, 2002), and Lactobacillus casei e via its ca-pacity to improve cheese body and texture (Merrill et al.,1996). Strains of Lactobacillus bulgaricus and Streptococ-cus thermophilus have also been currently used in cheesemanufacture to increase moisture content and improvemelting properties (Low et al., 1998; Peterson, Dave,McMahon, Oberg, & Broadbent, 2000).
The hyphae of Penicillium camemberti, Penicilliumroqueforti, Penicillium glaucum or Mucor rasmussen pene-trate the protein matrix, but their sporangia develop onlyon the surface (Kalab, 1993); e.g. in Camembert cheese,P. camemberti spores germinate on the cheese surface andpenetrate the curd, with a thick white mat of sporangia grow-ing on the cheese surface (Rousseau, 1984). Brooker (1987)found that deamination of amino acids by moulds on thesurface increases pH, and consequently leads to precipita-tion of calcium phosphate from the aqueous phase e which,in turn, produces a gradient between the surface and the bulkof the cheese; this promotes a sandy mouthfeel, which isdirectly related to microstructure. Furthermore, such anincrease in pH facilitates the action of plasmin, thus contrib-uting further to cheese softening (Everett & Auty, 2008).
On the other hand, yeasts in cheese have been claimed(Jakobsen & Narvhus, 1996) to assist in growth of P. roque-forti by producing gas e which leads to curd openness; it thusaffects microstructure, besides supporting said growth e viasecretion of suitable nutrients thereinto.
As a vector to protect bacteria from the putativeunfriendly conditions found in cheese environments, immo-bilization of cells e via e.g. microencapsulation, appears tobe a promising technique. According to Ozer, Kirmaci,Sxenel, Atamer, and Hayaloglucth (2009), microencapsula-tion does not adversely affect appearance or texture ofexperimental cheeses with immobilized bacteria, yet itimpacts significantly upon aroma and flavour attributes.
ConclusionsAt present, several food trends encompass creating desir-
able, distinctive or novel textures, framed by attractiveappearance and appealing odour and taste. In particular,innovation in the dairy field relies nowadays mainly on: i) in-corporation of nonconventional ingredients; ii) reduction incontent, or avoidance of harmful-like ingredients; iii)incorporation of pre- or/and probiotic vectors; iv) inclusionof components with nutraceutical value; and v) enhancementof existing, or development of alternative flavours (Fig. 1).Consumers are indeed calling for improvements in the sec-ondary (or organoleptic, including texture perception) roleof foods, but are simultaneously becoming more and moreaware of the health-promotion and safety constraints associ-ated with (the tertiary role of) foods. In all cases, microstruc-tural development and mastering is crucial e as the waytexture is perceived depends on the actual components ofthe cheese matrix, and on their chemical and spatial interac-tions. The physically heterogeneous continuum of cheese
affects, in turn, development of microbial populations ewhich are implicated in flavour development or may evenbe vectors of intoxications. If microstructure issues are notproperly addressed, then technological improvement offoods will be commercially hampered by inadequate sensoryand safety features. Furthermore, incorporation in cheese ofbacteria that have proven in vitro to be beneficial to healthrequires adequate microenvironments e which are stronglydependent on the mutual effects of the proteinaceous net-work and the milk fat globule distribution.
A new era in dairy food processing appears to be about tobegin; the chief concern of industry e which is obviouslydriven by consumer preference, will hereafter be coupledwith fundamental knowledge made available via sophisti-cated analytical apparata and imaging techniques; this willeventually lead to rational design of novel cheeses and ratio-nal improvement of existing ones. Further complementarystudies on microstructure subjects are thus fully warranted.
AcknowledgmentsFinancial support for author C. I. P. e provided via
a PhD fellowship (ref. SFRH/BD/18258/2004), issued byprogram POCI 2010, supervised by author F. X. M. andadministered by Fundac~ao para a Ciencia e a Tecnologia(Portugal), is hereby gratefully acknowledged.
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