alternatives to pilot plant experiments in cheese-ripening studies

Vol 54, No 4 November 2001 International Journal of Dairy Technology

121

REVIEW

*Author for correspondence. E-mail: [email protected]

© 2001 Society of Dairy Technology

Blackwell Science Ltd

Alternatives to pilot plant experiments in cheese-ripening studies

SHAKEEL-UR-REHMAN,

1

PATRICK F FOX,

2,

* PAUL L H McSWEENEY,

2

SABRY A MADKOR

3

and NANA Y FARKYE

1

1

Dairy Products Technology Centre, California Polytechnic State University, San Luis Obispo, CA 93407, USA,

2

Food Chemistry, Department of Food Science, Food Technology and Nutrition, University College, Cork, Ireland, and

3

Novo

Enzymes North America, Inc., 77 Perry-Chapel Church Road, PO Box 576, Franklinton, NC 27525-0576, USA

Experimental studies on cheese have several objectives, from assessing the influence of the microfloraand enzymes indigenous to milk to evaluating starters and adjuncts. Several studies have beenundertaken to evaluate the influence of an individual ripening agent in the complex environment ofcheese. Cheesemaking experiments, even on a pilot scale, are expensive and time-consuming, and whencontrolled bacteriological conditions are needed, pilot plant experiments are difficult to perform. Cheesecurd slurries are simple models that can be prepared under sterile conditions in the laboratory and canbe used as an intermediate between test tubes and cheese trials, but probably cannot replace the latter.Miniature model cheeses are similar to pilot plant cheeses and can be manufactured under sterileconditions. Several approaches to assess the role of cheese-ripening agents are reviewed in this paper.

Keywords

Cheese ripening, Cheese slurries, Model systems, Nonstarter bacteria, Rennet, Starter.

I N T R O D U C T I O N

The vast majority of rennet-coagulated cheeses areripened for a period ranging from a few weeks totwo or more years, during which time the flavourand textural characteristics of the particular cheesevariety develop. The basic composition and struc-ture of cheeses are determined by the curd manu-facturing operations, but it is during ripening thatthe individuality and unique characteristics of eachcheese variety develop, as influenced by the com-position of the curd and other factors such as themicroflora established during manufacture and/orduring ripening.

1

During ripening, cheese under-goes numerous biochemical changes, which leadto the development of the appropriate flavour andaroma.

2

The complex cheese-ripening processinvolves three primary biochemical events (pro-teolysis, lipolysis and glycolysis) that are catalysedby enzymes indigenous to milk, starter bacteria,coagulant, secondary starter and the adventitiousnonstarter microflora. The contributions of theseenzymes to cheese ripening and flavour developmenthave been studied extensively.

1,3–5

Assessment of the contribution of the principalripening agents involved in flavour developmentduring the ripening of hard and semihard cheeses isan expensive and time-consuming process becauseof the long period (~6–12 months) required forfull flavour development. This severely limits thenumber of investigations that can be completedin a given time. Thus, a model system in which

ripening can be assessed rapidly appears desirable,provided that the cheese-ripening conditions can bereplicated closely. Various simple systems (e.g. solu-tions of sodium caseinate or individual caseins inwater or buffer or 5% NaCl) at various pH valueshave been used to study the proteolytic activity ofvarious enzyme systems or cultures or cell-freeextracts.

6

Typically, the contribution of one individualripening agent is studied by the development of amodel cheese system in which the action(s) of oneor more of the other ripening agents is eliminatedand the resulting cheese is studied. The role ofstarter bacteria in cheese ripening has been studiedby substituting chemical acidification (using glu-conic acid-

δ

-lactone [GDL] ) for biological acidifi-cation by the starter in cheese manufacture;

7–11

some studies on the role of starter bacteria werealso performed in the 1960s.

1

Several model cheesesystems have been used to exclude adventitiousnonstarter lactic acid bacteria (NSLAB) fromcheese curd and thus study their contribution tocheese ripening; these methods were reviewed byShakeel-Ur-Rehman

et al

.

12

Various techniques havealso been used to inactivate residual coagulant incheese curd in order to study its role in cheeseripening.

1,3

Visser

13

used aseptically manufacturedrennet-free, starter-free and rennet- and starter-freeGouda cheese to study the role of cheese-ripeningagents.

Many other model systems have been developedrecently to assess the performance of enzymes,

IDT025.fm 21 Thursday, October 11, 2001 6:43 PM

Vol 54, No 4 November 2001


122

starters or adjunct starters during cheese ripening. Theseinclude cheese slurries,

14

cheese pastes

15

or cheese-like products.

16

Although such model cheese sys-tems closely replicate the biochemical events duringcheese maturation in only a few days, they do nottypically represent the body, texture and wateractivity of normal cheese. Miniature cheeses havebeen developed with a composition typical of thecheese variety under investigation to evaluate theactual contribution of different agents during cheeseripening.

17

Miniature cheeses are cheap and a largenumber can be readily produced, but the normalripening period is required. The modified minia-ture cheese system of Hynes

et al

.

18

required only28 days for ripening.

C H E E S E S L U R R I E S

Kristoffersen

et al

.

19

developed a rapid method forproducing cheese flavour by incubating cheesecurd slurries, containing about 40% solids, for 4–5 days at 30–35

°

C. This procedure resulted in asemisolid paste with a flavour intensity similar tothat of mature cheese. Because of the high moisturecontent and high incubation temperature used inthis system, it underwent accelerated biochemicalchanges.

20,21

This system permits the controlleddevelopment of characteristic cheese flavours involv-ing selected bacteria, additives and enzyme prepara-tions. Several workers have since used cheesecurd slurries to study the biochemistry of cheesematuration.

22

Singh and Kristoffersen

23

studied theinfluence of lactic cultures and curd milling acidityon the ripening of Cheddar curd. They reportedbitterness in slurries produced from curds madewith certain mixed-strain cultures. Using Cheddarcheese slurries, various enzymes (rennets, acid pro-teases, neutral proteases, acid protease-peptidases,lipases and decarboxylases) were screened bySood and Kosikowski

24

for their ability to accel-erate cheese ripening at 20 or 32

°

C, as assessed byproteolysis, lipolysis and flavour development.Ponce-Trevino

25

used a cheese curd slurry to assessthe role of lactic acid bacteria in the production ofsulphur compounds in Cheddar cheese and reportedthat the slurry prepared from directly acidifiedcurd contained only carbonyl sulphide, whereasmethanethiol, dimethyl sulphide and hydrogen sul-phide developed in the slurry produced from curdacidified by lactic acid cultures.

Farkye

et al

.

14

prepared a modified version ofthe cheese slurry of Kristoffersen

et al

.

19

by blend-ing unsalted curd, heat-shocked lactic acid bac-teria, NaCl and sterile distilled water to give a slurrycontaining 57–70% moisture, 18–23% protein, 3–4% NaCl-in-moisture and with a pH of 4.85–5.32.They reported that a cheese slurry was a reliablesystem in which to evaluate the contribution ofvarious lactic cultures and enzymes to cheese

ripening and flavour development. Although the com-position of the dry matter of this system was similarto that of cheese and it ripened quickly, it had a farhigher moisture content than hard cheeses: thismight reasonably be expected to alter the ripeningpattern.

Madkor

et al

.

21

used the cheese slurry system ofFarkye

et al

.

14

to study the autolytic properties andenzymatic activities of adjunct cultures of lactoba-cilli that influence the ripening aspects and flavourprofile of Cheddar cheese. They reported that cheeseslurries are suitable systems in which to selectadjunct cultures and enzymes according to theirproteolytic and lipolytic properties for cheeseripening.

Roberts

et al

.

26

developed an aseptic cheese curdslurry system containing 73% (w/w) moisture and3% salt-in-moisture, which remained free of con-taminating bacteria for 15 days at 30

°

C. This methodmade it possible to investigate the effects of indi-vidual micro-organisms on cheese ripening with-out interference from adventitious micro-organismsand was particularly suitable for studying the effectof starter bacteria on the development of cheeseflavour. Presumably, it suffers from the same limita-tions as the method of Farkye

et al.

,

14

i.e. a highmoisture content.

Smit

et al

.

15

prepared a cheese paste by homog-enizing grated Gouda cheese (6 weeks old or amixture of older and younger cheeses) in water togive a final moisture level of 50–55% in the paste.The slurry was heated at 80

°

C for 3 min and cooledto 30

°

C to give a paste with a solid consistencywhen cold. It was claimed that the product pre-pared by this method could be liquified by heatingto 55

°

C without the separation of fat, thus allowingthe incorporation of NSLAB or enzymes; the prod-uct regained its original structure on cooling. Thissystem had a composition and texture similar tothose of natural cheese, but had the disadvantagethat the heat treatment given to the curd wouldchange its chemical and physical characteristics.The consistency of this model cheese was differentfrom that of real cheese.

Muehlenkamp-Ulate and Warthesen,

27

who studieddifferences in the proteolytic capability of dif-ferent species and strains of NSLAB in Cheddarcheese slurries (59% moisture, 4.3% salt-in-moisture and 15.5% protein), suggested that thisapproach would be suitable for screening the abilityof NSLAB to produce peptides and amino acids.The slurry had a far higher moisture content thanCheddar cheese, which may make this model un-suitable for studying the significance of NSLAB inCheddar cheese. Wyder and Puhan,

28

who studiedthe role of selected yeasts in cheese ripening inaseptic curd slurries, reported that yeasts were pro-teolytic and produced a cheesy aroma. Dias andWeimer

29

studied the production of volatile sulphur

IDT025.fm Page 122 Thursday, October 11, 2001 6:43 PM


123


compounds in Cheddar cheese slurries and reportedthat starter bacteria increased the production ofmethanethiol.

Cheese slurries have been used to assess thesuitability of proteinases, lipases or peptidases toaccelerate flavour development,

30

offering the advan-tage of time and cost savings. The mechanism offlavour development in slurries is unclear and thewhole process is difficult to control. Cheese slurrymodels can be prepared easily but their body, tex-ture and water activity differ from those of normalcheese.

C H E E S E - L I K E P RO D U C T S

Salles

et al

.

16

developed a model cheese as a basein which to study the sensory attributes of flavourcompounds extracted from various varieties ofcheese. The base was made from native calciumphosphocaseinate (170 g), deodorized anhydrousmilk fat (190 g), low-heat skimmed milk powder(30 g), NaCl (2 g), water (600 mL) and GDL (8 g);the mixture (1000 g) was heated at 33

°

C for 2.5 h,0.15 mL rennet extract was added and the mixturewas homogenized, held at 33

°

C for 1 h and storedat 15

°

C until use. The resulting cheese-like productcontained 60–61% moisture and 23.4–23.8% pro-tein. The rheological behaviour of the cheese model,measured by a compression test, was similar to thatof some hard cheeses and its flavour was assessedas neutral. Compared with some mildly flavouredcheeses, this model cheese was found to be slightlysalty, bitter or sour, and had only a light milkyaroma. This model system could be prepared easilybut its body, texture and water activity were differ-ent from those of normal cheese.

The effect of fat globule size and shape on thedevelopment of Cheddar cheese flavour was stud-ied by Wijesundera

et al

.

31

in a fat-substituted modelcheese. Cheddar flavour in the cheese containing ahigh-melting fat fraction, after maturation at 8

°

Cafter 9 months, was significantly lower than that incheese containing unfractionated anhydrous milkfat or a low-melting fat fraction.

AQ U E O U S S Y S T E M S S I M I L A R TO C H E E S E

Many authors

32–36

have used aqueous systems thatsimulate cheese to study the role of starters /enzymesin cheese. The main advantage of these systems isthat various parameters are defined more easilythan in cheese, but they represent neither the com-bined stress of cheese manufacturing conditionsnor the complexity of the changing cheese environ-ment during ripening.

Okumura and Kinsella

37

studied the formationof methyl ketones by

Pencillium camemberti

inmodel systems prepared in the following medium:

500 mL homogenized milk diluted with 1 L 0.15

m

phosphate buffer (i.e. buffer containing 15 mLCzapaks’ solution—a nutrient for moulds—and 45 gsucrose).

Pencillium camemberti

, incubated in amodel system containing milk lipids, hydrolysed thelipids with subsequent oxidation of the free fattyacids to carbonyl compounds, of which approximately60% were methyl ketones (mainly 2-nonanoneand 2-undecanone). Incubation in the presenceof the sodium laurate (45 m

m

) reduced myceliumgrowth, but extensive synthesis of undecanoneoccurred. It was reported that

P. camemberti

my-celium behaved similarly to

Penicillium roqueforti

in the model system. The authors proposed usingthis model to develop a flavour concentrate inorder to accelerate the development of Camembertflavour.

Youssef

38

made a cheese-like product (syntheticmodel cheese) by blending calcium

para

-caseinate(calcium phosphate complex), lactic acid, NaCl andnumerous buffers to simulate the aqueous phaseof cheese; the enzyme system(s) to be studiedwas then added. This cheese-like product had anaqueous phase similar to that of cheese, but its com-position differed in several respects from that ofnatural cheese. The models were used to study pro-teolysis by

Lactococcus lactis

ssp.

cremoris

HP asa function of various ripening variables. Proteolysiswas monitored by several methods. More aminoacids were produced at pH 6.2 than at pH 4.7 or 5.2and at an NaCl content of 0 or 4% than at 8%;much larger quantities of amino acids were pro-duced from

para

-casein predigested by rennet thanfrom untreated

para

-casein at 30

°

C than at 20

°

C. Aratio of

para

-casein to water of 1 : 2.5 or 1 : 3.5 didnot significantly affect the formation of aminoacids by the starter. Compared with intact cells,lysed cells caused increased production of aminoacids. De Jong and de Groot-Mostert

39

foundthat proteolysis in simple model substrates (e.g.solutions of casein,

para

-casein or synthetic pep-tides or milk) did not reflect the actual progressof proteolysis in cheese. The texture of all of theabove model cheeses was different from real cheeseand their ripening characteristics have not beencompared with those of real cheese.

Pripp

et al

.

40

studied the contribution of differ-ent proteolytic agents to primary proteolysis in asodium caseinate solution under cheese-like condi-tions. The sodium caseinate solutions were treatedwith rennet and either a sonicated cell suspension of

Lc. lactis

ssp.

cremoris

SK11 or a heat-inactivatedsonicated cell suspension of this strain. Proteolysiswas assessed after 3, 10 and 17 days’ incubationat 8

°

C by urea-polyacrylamide gel electrophoresis(PAGE) of the pH 4.6-insoluble fraction. Principalcomponent analysis of the urea-PAGE electro-phoretograms indicated that rennet and the sonicatedcells were responsible for > 90% and ~8% of




124

primary proteolysis, respectively. Bacterialproteolytic enzymes showed an interactive effectwith rennet by degrading

β

-CN (f1–192) in rennet-treated samples.

Pripp

et al

.

41

studied the contribution of

Lc. lactis

ssp.

cremoris

223, 227, SK11, AM1, Wg2 and

Lc. lactis

ssp.

lactis

UC 317 by individually addingsonicated cells of these strains to sodium caseinatesolutions under the conditions found in Cheddar orGouda cheese during ripening (pH 5.25 and 5.0%NaCl). Proteolysis was assessed by urea-PAGE ofthe pH 4.6- and ethanol (70%)-insoluble fractionsand reverse-phase high-performance liquidchromatography (RP-HPLC) of the ethanol (70%)-soluble fraction, and the resulting peptide profileswere analysed by principal component analysisand hierarchical cluster analysis. Statistical analysisof peptide profiles of the ethanol (70%)-solublefraction from 2-, 9-, 17- or 23-day-old samplesgrouped the strains into three clusters that weresimilar to those found for the same strains in experi-ments with miniature Cheddar-type cheeses.

42

M I N I AT U R E M O D E L C H E E S E S Y S T E M

Shakeel-Ur-Rehman

et al

.

17

used miniature Cheddar-type cheeses (20 g) to study the contribution ofstarters, enzymes or adjuncts to cheese proteolysis.The miniature cheeses manufactured within abatch of six cheeses in different batches or ondifferent days were similar statistically in termsof gross composition and pH and were similarto pilot-scale Cheddar cheese in terms of theseparameters, as well as flavour and proteolysis. Thismodel system is very convenient and will reducethe cost of studying the effects of cheese-ripeningagents. The model is better than the syntheticmodels or milk, which do not reflect accurately theactual progress of proteolytic pathways in cheese.Although the miniature cheeses were brine-saltedand centrifugation (1700

×

g

) was used to expelmoisture, the salt and moisture levels in the minia-ture cheeses were similar to those of conventionalCheddar cheese, suggesting very close similaritybetween the two. Shakeel-Ur-Rehman

et al

.

42

usedthis miniature cheese system to study the cheese-ripening properties of 11 single strains of

Lc. lactis

ssp. Primary proteolysis, as determined by urea-PAGE of water-insoluble fractions of cheeses(WISF), and the level of water-soluble nitrogen(WSN) were similar in all cheeses made with dif-ferent strains of

Lc. lactis

. Secondary proteolysis,as determined by urea-PAGE of the water-solublefraction (WSF), RP-HPLC of ethanol (70%)-solubleand -insoluble fractions of the WSF, individual aminoacids and total amino acids, showed differencesbetween cheeses made with different lactococcalstrains. The cheese made with

Lc. lactis

ssp.

cremoris

Wg2 differed considerably from other strains andthe 4-month-old cheese had the poorest quality.

Using this miniature model cheese system,Shakeel-Ur-Rehman

et al

.

43

showed that it is pos-sible to inactivate residual coagulant in the curd bypepstatin, a potent competitive inhibitor of aspartylproteinases, in order to study the role of coagulantin cheese ripening. Since the cheesemaking pro-tocol for the miniature cheeses was identical to thatfor Cheddar up to the point of whey drainage, itmay be assumed that pepstatin will also inhibitresidual coagulant in conventionally made Cheddarcheese. The coagulant was shown to be responsiblefor the degradation of

α

s1

-casein and the productionof most of the WSN. The technique of using pep-statin to inhibit the residual coagulant in cheese isthe easiest and most effective method developed todate for producing cheese curd free from residualcoagulant activity.

The miniature cheese model of Shakeel-Ur-Rehman

et al

.

17

was modified by Hynes

et al

.

18

tomanufacture miniature washed-curd cheeses undercontrolled microbiological conditions. Forty mini-ature cheeses (40 g) were produced over 10 work-ing days and ripened for 28 days. Gross composition(dry matter, salt-in-moisture and pH) of the 1-day-oldcheeses did not differ significantly between cheese-making days: average values were 45.16, 2.46 and5.15%, respectively. The adventitious

Lactobacillus

population remained at less than 200 cfu g

–1

through-out ripening, and phage were absent. This modeloffers the possibility of testing single bacterialstrains in a real washed-curd cheese environmentwithout the uncertainties caused by contaminationwith adventitious NSLAB or phage attack.

C O N C L U S I O N S

Although all the models discussed in this revieware useful for studying cheese ripening, we believethe miniature cheese systems are best as they arecloser to cheese than any other model proposedto date.

In vitro

experiments involving the use ofaqueous solutions similar to cheese cannot be usedto assess the impact of a given factor in the complexenvironment of cheese, while cheese slurries canbe used only as an intermediate between test tubesand cheese experiments.

R E F E R E N C E S

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© 2001 Society of Dairy Technology126

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alternatives to pilot plant experiments in cheese-ripening studies

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