Lait (1989) 69, 305-314© Elsevier/INRA

305

Original article

Acceleration of blue cheese ripening by cheese slurryand extracellular enzymes of Penicillium roqueforti

A.M. Rabie *

Food Science Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt

(received 25 November 1988, accepted 8 March 1989)

Summary - An attempt has been made to accelerate flavour development of blue cheese. Ripenedcheese slurry has been incorporated into blue cheese curd at a rate of 1 and 2% respectively. Amixture of proteinases and lipases (1:1) of P. roqueforti has been incorporated into blue cheese curdat the proportions of 0.01 and 0.02 respectively. Blue cheese has also been made without additives(control).

Addition of either cheese slurry or enzyme mixtures to blue cheese curd has accelerated thedevelopment of the characteristic flavour of blue cheese. These additives have stimulated theformation of soluble nitrogenous compounds, free amino acids, volatile fatty acids and total carbonylcompounds.

Blue cheeses containing 2% ripened slurry or 0.02% P. roqueforti enzymes have higherorganoleptic properties than the control after 45 d of ripening. The concentrations of the previouslymentioned compounds in the same period of ripening are higher than those found in control cheeseafter 60 d.

ripening - blue cheese - protease - lipase - Penicillium roqueforti - proteolysis -Iipolysis - flavour

Résumé - Accélération de l'affinage du fromage bleu par du "caillé hydraté» homogénéiséet par des enzymes extracellulaires de Penicillium roqueforti. Un essai a été fait pour accélérerle développement de la flaveur du fromage bleu. Du «caillé hydraté» homogénéisé a été incorporédans du caillé de fromage bleu à raison de 1à 2%.

Un mélange de protéases et de lipases (1:1) de P. roqueforti a été également incorporé dans ducaillé de fromage bleu à raison de 0.01 et 0.02%. Le fromage bleu a été aussi fabriqué sans additifs(témoin).

L'addition soit du «caillé fluide» homogénéisé, soit du mélange d'enzymes, a accéléré ledéveloppement des flaveurs caractéristiques.

Ces addifits ont stimulé la formation de composés azotés solubles, d'acides aminés libres,d'acides gras volatils et des composés carbonylés totaux au cours de l'affinage.

Les fromages bieus. contenant soit 2% de «caillé fluide» homogénéisé soit 0,02% d'enzymes deP. roqueforti possèdent de meilleures propriétés organoleptiques que le témoin après 45 jours dematuration. Les concentrations des composés antérieurement mentionnés sont plus importantesdans la même période d'affinage que celles trouvées dans le témoin après 60 jours.

affinage - fromage bleu - protéase - lipase - Penicillium roqueforti - protéolyse -lipolyse - arôme

• Present address : Institut Supérieur d'Agriculture, BP 35, Gitega, Burundi.

306

INTRODUCTION

Cheese ripening is a complex processwhich involves several biochemicalreactions leading to the fermentation oflactose, breakdown of casein andhydrolysis of milk fat (Law, 1984).

Blue cheese undergoes extensiveproteolysis during ripening (Kinsella andHwang, 1976a; Hewedi and Fox, 1984)and the free fatty acids and methylketones produced are major contributorsto the characteristic flavour of bluecheese (Lawrence, 1966; JoUy andKosikowski, 1975; Kinsella and Hwang,1976a,b; King and Clegg, 1979).

Several methods have been proposedto improve the quality of different types ofcheese and shorten their ripeningperiods, such as increasing the ripeningtemperature, addition of modified starter(heat shocked), cheese slurry, traceelements or addition of enzymaticpreparations (Hofi et al., 1973; AbdelSalam et al., 1981; Abdel Baky et al.,1982, 1986). Jolly and Kosikowski (1975)accelerated the development of bluecheese flavour by adding microbiallipases.

The incorporation into an aseptic curdof aspartyl protease of Penicilliumroqueforti or metaUoprotease ofP. camemberti resulted in formation ofhigh and low molecular weight peptideswithout Iiberation of amine acids. Theliberation of amine acids resulted from theaction of exopeptidases (Gripon et al.,1977).

Recently, the Polish National Commit-tee of the IDF (1987) concluded that themost suitable method of acceleratingcheese ripening is the use of enzymaticpreparations obtained from the naturalmicroflora of cheeses.

Therefore, the present investigationwas carried out to accelerate blue cheese

A.M. Rabie

ripening, using either ripened blue cheeseslurry or a mixture of crude proteolytic andlipolytic enzymes (1:1) secreted byP. roqueforti.

MATERIALS AND METHODS

Materials

Fresh cow's milk of 3.5% fat and 9% S.N.F.was used in this investigation. A rennet powdsr(1:100.000) was obtained from L.C. GladCompany A/S, Copenhagen, Denmark.

Pure cultures of S. lactis andS. diacetylactis were obtained from Hansen'sLaboratory. The cultures were activated beforebeing used. P. roqueforti strain R64A waskindly supplied by Dr Lenoir (INRA, Laboratoirede Recherche de la Chaire de Technologie,France).

Preparation of cheese slurry

The methods described by King and Clegg(1979) for preparing aseptic curd slurry wereadopted.

Cow's milk of 3.5% fat and 9% S.N.F. washeated 10 71-?2°C for 15 s, rapidly cooled 10 asetting temperature of 35°C and acidified to pH5.6 with lactic acid. Rennet was Ihen added tocoagulate the milk (40 min). The coagulum wascut and then it was ladled in cheese clothes forcomplete drainage over 24 h. The resultantcurd was firstly mixed with 4% NaCI, andtransfered in to 500 ml conical flasks. The curdwas sterilized at 120°C for 15 min. Sierilizedcurd was aseptically inoculated with 0.5% ofPenicillium roqueforti mycelium and incubatedal 25°C for 15 days.

Production ofpreparations

crude enzyme

P. roqueforti slrain R64A was cultured at 25°Cfor 5 days in Czapek-tryplicase modified

Acceleration of blue cheese ripening 307

medium (Lenoir et el; 1979). The pH of themedium was adjusted to 4.0 for the productionof aspartyl protease and acid carboxy-peptidase, 6.5 for the production of metallo-protease, aminopeptidase and neutral carboxy-peptidase, and 7.5 for the production of lipase(Lamberet and Lenoir, 1972).

The media were inoculated withP. roqueforti strain R64A. At the end of theincubation period, the vegetative growth wasremoved by centrifugation followed by filtering.The cell free extracts containing the enzymeswere centrifuged at 2000 9 for 20 min. Equalvolumes of extracts containing proteolyticenzymes were mixed and the enzymes wereprecipitated by acetone at a concentration of80%. The mixture was immediately centrifuged(20 min x 2000 g) and the supernatant wasdiscarded. The same procedure was followedwith precipitation of Iipolytic enzymes. Theprecipitate resulting from the extractscontaining either proteolytic enzymes orlipolytic enzymes was weighed and dissolved in0.025 M Na-citrate buffer pH 4.0. Insolublematerial was removed by centrifugation (30 minx 6000 g). The mixture of crude proteolytic andIipolytic enzymes (1:1) was incorporated intothe blue cheese curd at a level of 0.01 and0.02%.

Cheesemaking

Blue cheese was made from cow's milkcontaining 3.5% fat. Cow's milk was pasteu-rized at 71-72°C for 15 s, and cooled to 30°C.A starter culture consisting of Streptococcuslactis and Streptococcus diacetylactis (1:1) at arate of 2% was added to cheese milk. Cheesemaking was completed as described byKosikowski (1977). The resultant curd wasinoculated with P. roqueforti and divided into 5equal parts. The first part was left withoutadditives and served as a control. Ripenedcheese slurry was incorporated into cheesecurd of the second and the third parts at a rateof 1 and 2% respectively, A mixture of crudeproteolytic and Iipolytic enzymes of P. roque-forti (1:1) was added at a rate of 0.01 and0.02% of curd weight to the fourth part of thecurd respectively. Cheese was ripened at8-10°C and 95% RH for 60 days.

Organoleptic properties

Cheese sampies were organolepticallyevaluated by the method of Spreer (1978) withmaximum score points of 4.5, 3.5., 2.0 and10.0 for external appearance, internaiappearance, odour and taste respectively.

Chemical analysis

Cheese samples were analysed after salting,then after 30, 45 and 60 days, for moisture, fat,salt, total nitrogen, soluble nitrogen and pH asdescribed by Ling (1963). Amino acid nitrogenwas determined according to the method ofStadhouders (1959).

Total volatile fatty acids

Free volatile fatty acids were determined by themethod of Kosikowski (1977).

Tyrosine and tryptophane

Tyrosine and tryptophane were spectrophoto-metrically determined by the method describedby Vakaleris and Priee (1959).

Total carbonyl compounds

The total carbonyl concentration of blue cheesewas determined by preparing 2,4-dinitrophenylhydrazones of carbonyl compounds by themethod of Lawrence (1965) and measuring theoptical density at 340 nm, using Safas MonacoType 210 Spectrophotometer. Total carbonylcontent was quantified using the molarextinction coefficient of 22,500, since methylketones accounted for more than 90% of totalcarbonyls in solution.

Statistical analysis

The effects on scores of blue cheese of theaddition of both ripened cheese slurry and

308

Table 1.Chemical composition of ripened bluecheese slurry.

Components Values

Moisture % 55.75

Fat % 20.20

Salt % 3.90

Total N % 3.55

Soluble N * 35.80

Tyrosine ** 150.40

Tryptophan ** 120.70

T.V.F.A.*** 40.20

pH 5.80

* Values are expressed as % of total nitrogen.** Values are expressed as mgl100 9 of cheese.*** T.V.F.A. : Total volatile fatty acids are expressed asml of 0.01 N NaOH/100 9 of cheese.

proteolytic and lipolytic enzymes to bluecheese curd were statistically analysed by theF test. In cases of significant F, differencesbetween treatments were examined byDuncan's multiple range test (Snedecor andCochran, 1957).

RESULTS AND DISCUSSION

Cheese slurry

Table 1 shows the chemical compositionof ripened blue cheese slurry. Ripenedblue cheese slurry contained higher levelsof soluble nitrogenous compounds, freeamino acids and carbonyi compoundsafter 15 days of incubation. The flavour of

A.M. Rabie

typical blue cheese has developed duringthis period. Farag (1987) found thatinoculation of aseptic blue cheese curdwith different P. roqueforti strains showeda typical blue cheese flavour after 2weeks of incubation at 25°C.

Gross chemical compositionTable Il shows that either addition ofripened cheese slurry or a mixture ofproteolytic and Iipolytic enzymes (1:1) tocheese curd had a slight effect on themoisture, fat and salt contents of bluecheese after salting and during ripening.Blue cheese containing either ripenedslurry or enzymes mixture had differentpH values compared with the control. Thechanges in the pH values between thecontrol cheese and experimental cheeseswere small after salting. As ripeningproceeded, cheese containing either ripe-ned slurry or enzymes mixture showedhigher pH values than the control. On theother hand, cheese made with ripenedslurry had slightly higher pH values thanthose made with enzyme mixtures after45 days of cheese ripening. This may bedue to the different rate of caseindegradation at this stage of cheeseripening (Table III).

Generally, the pH values of both con-trol cheese and ail experimental cheesesgradually increased but at different ratesduring ripening. The observed differencebetween the control cheese and cheesecontaining slurry or enzyme mixtures maybe due to the different rate of proteolysisoccuring in blue cheeses containing theabove mentioned additives (Table III):Similar results were obtained by Farag(1987).

The general trend of chemical compo-sition of blue cheese containing ripenedslurry agreed with that observed by AbdelBaky et al. (1982). They found that theutilization of cheese slurry to accelerate

Acceleration of blue cheese ripening 309

Table Il. Gross chemical composition of blue cheese as affected by cheese slurry and enzymesmixture of P. roqueforti *.Composition chimique globale du fromage bleu influencée par le caillé hydraté et des mélangesd'enzymes de P. roqueforti *.

Components Ripeningperiod(days)

Withoutadditives(control)

Additives

Cheese slurry Enzyme mixtures1% 2% 0.01% 0.02%

F 48.7 48.8 48.4 48.8 48.8Moisture 30 45.6 45.7 45.8 45.6 44.6

45 44.5 44.6 44.7 44.4 44.660 44.3 44.4 44.4 44.2 44.4

F 45.6 45.9 46.1 45.7 45.7Fat (DM) 30 45.4 45.8 46.0 45.6 45.6

45 45.4 45.8 45.9' 45.5 45.560 45.4 45.8 45.8 45.6 45.4

F 3.6 3.7 3.8 3.7 3.8Salt 30 3.7 3.8 3.9 3.8 3.8

45 3.8 3.9 3.9 3.9 3.960 3.8 3.9 3.9 3.9

F 4.8 4.9 4.9 4.9 4.9pH 30 5.2 5.4 5.6 5.3 5.5

45 5.8 6.2 6.4 5.9 6.1460 6.2 6.7 6.8 6.5 6.7

F = alter salting. DM : dry matter. * = average of 3 determinations.

ripeninq of Egyptian hard cheese (Ras)has a slight effect on the gross chemicalcomposition of cheese.

Soluble nitrogen compounds andaminoacids

Ripening indicesTable III indicates that the addition ofripened blue cheese slurry or enzymesmixture of P. roqueforti to blue cheesecurd stimulated the formation of solublenitrogenous compounds, tyrosine andtryptophan. However, the levels of thesecompounds were higher in cheesecontaining ripened blue cheese slurry

Changes in soluble nitrogen, amino acidnitrogen, soluble tyrosine and tryptophancontents, free volatile fatty acids and totalcarbonyl compounds were taken asindices of testing blue cheese ripening.

cheese containing ripened cheese slurryor mixture of proteolytic and Iipolyticenzymes could be explained on the basisthat cheese slurry contained P. roqueforti,which secretes a range of very activeendo and exopeptidases which catalysethe degradation of casein (Zevaco et et.,1973; Lenoir et al., 1979).

On the other hand, cheese slurrycontains a range of soluble, nitrogenousand amine acids compounds whichprobably stimulate the growth ofP. roqueforti and enhance the formation offlavour compounds (Table 1).

The results obtained agreed with thosereported by Desmazeaud et al. (1976).

310 A.M. Rabie

Table III. Soluble nitrogen compounds and amine acids in blue cheese as affected by cheese slurryand enzymes mixture of P. roqueforti during ripening".Composés azotés solubles et acides aminés dans le fromage bleu influencés par le caillé hydratéet des mélanges d'enzymes de P. roqueforti au cours de l'affinage '.

Components Ripening Without Additivesperiod additives(days) (control) Cheese slurry Enzyme mixtures

1% 2% 0.01% 0.02%

F 9.14 9.84 10.65 9.24 10.20Soluble N"" 30 17.44 25.55 28.74 23.24 25.75

45 28.64 38.90 40.90 35.74 37.9060 32.56 45.44 50.70 44.79 45.74

F 1.44 1.66 1.84 1.72 1.84Amino N"" 30 5.60 7.40 7.94 6.94 7.50

45 7.40 9.64 10.49 8.55 9.4060 9.77 11.84 12.50 11.26 12.25

F 25.45 28,94 32.40 40.40 45.90Tyrosine """ 30 150.40 187.20 240.60 230.55 260.50

45 277.60 380.44 420.70 395.44 425.7060 302.42 442.74 490.40 455.60 485.50

F 15.45 20.44 24.45 26.70 30.40Tryptophan """ 30 95.65 115.55 140.60 125.88 155.50

45 185.40 210.62 270.40 220.70 235.3560 210.74 250.44 295.36 240.44 279.40

F = alter salting .• = average of 3 determinations .•• = % of total nitrogen .••• = mg/100 9 of cheese.

than those containing enzyme mixtures orcontrol blue cheese during ripening. Theincreased rate of both soluble nitrogenouscompounds and amine acids wasproportional to the amount of addedcheese slurry and P. roqueforti enzymemixtures. Addition of ripened blue cheeseat a rate of 2% to the curd was moreeffective in this respect. Concentrations ofsoluble nitrogen, amine acid nitrogen andamine acids of blue cheese containing2% of ripened slurry or 0.02% of P.roqueforti enzyme mixtures, at 45 days ofcheese ripening, were higher than thoseof control at the end of the ripeningperiod. The high rate of proteolysis in blue

Acceleration of blue cheese ripening 311

Table IV. Free volatile fatty acids of blue cheese as affected by cheese slurry and enzymes mixtureof P. roqueîotti : during ripening.Acides gras volatils du fromage bleu influencés par le caillé hydraté et des mélanges d'enzymes deP. roqueforti • au cours de l'affinage.

Components AdditivesRipeningperiod(days)

Withoutadditives(control) Cheese slurry Enzyme mixtures

1% 2% 0.01% 0.02%

• = average 01 3 determinations.•• T.V.F.A. = total volatile latty acids are expressed as ml 01 0.01 N NaOH/l00 g 01cheese.••• T.C.C. = total carbonyl compounds are expressed as .umoV1 00 g 01 cheese.

T.V.FA ••F 2.430 18.445 36.260 56.8

F 25.4030 185.6245 245.7060 300.42

T.C.C .r:

They found that the proteolysis is veryintensive in cheese curd inoculated with astrain of P. roqueforti. At the end ofripening, the soluble nitrogen at pH 4.6,non-protein nitrogen and amino acidnitrogen, calculated as percent of totalnitrogen, were 50, 30, and 10% respec-tively.

Free tatty acids and carbonylcompqunds

Table IV shows the changes of freevolatile fatty acids and carbonyl corn-pounds in blue cheese containing ripenedslurry or a mixture of proteolytic andlipolytic enzymes of P. roqueforti. Resultsshowed that control blue cheesecontained lower concentrations of freevolatile fatty acids and carbonylcompounds than experimental cheesesmade with either added cheese slurry orenzymes mixtures. Incorporation into

3.235.655.771.4

4.542.465.292.8

4.150.874.486.7

3.830.460.479.2

32.45220.42450.74495.42

36.70250.40750.37790.45

35.40245.34759.40759.40

34.40225.40412.87438.35

cheese curd ripened slurry or enzymemixtures accelerated the formation of freevolatile fatty acids (C2-G1O) and totalcarbonyl compounds. These results couldbe explained on the basis that cheeseslurry and enzyme mixtures contain acidlipases which are responsible for theIiberation of short chain fatty acids fromtriglycerides of milk fat. Penicilliumroqueforti can produce at least 2 lipasesthat are distinguished by their optimumpH. Short-chain fatty acid specificities ofthese lipases influence the organolepticqualities of blue veined cheese, becausethe amounts of each lipase vary widelyfrom one cheese to another, beingparticularly related to the strain used andthe ripening (Imamura and Kataoka,1963). These volatile fatty acids aretransformed in methyl ketones by eitherthe spores or the mycelium ofP. roqueforti (Dwivedi and Kinsella, 1974;Kinsella and Hwang, 1976b). Addition ofripened blue cheese slurry was more

312 A.M. Rabie

Table V. Organoleptic properties • of blue cheese as affected by cheese slurry and enzymes mixtureof P. roqueforti.Propriétés organoleptiques • du fromage bleu influencées par le caillé hydraté et les mélangesd'enzymes de P. roqueforti.

Characteristics Ripening Without Additivesperiod additives(days) (control) Cheese s/urry Enzyme mixtures

1% 2% 0.01% 0.02%

External appearance (4.5) 4.0 4.0 4.0 4.0 4.0Internai appearance (3.5) 2.8 2.9 3.1 2.7 3.0Odour (2.0) 30 1.0 1.4 1.6 1.3 1.5Taste (10.0) 6.4 7.2 8.5 7.2 7.4Total (20.0) 14.2 ± 0.4 15.5 ± 0.2 17.2 ± 0.3 15.2±0.1 15.9 ± 0.3

External appearance (4.5) 3.9 3.9 3.8 3.8 3.8Internai appearance (3.5) 2.9 3.1 3.2 3.0 3.0Odour (2.0) 45 1.3 1.5 1.7 1.4 1.6Taste (10.0) 7.6 8.1 8.3 8.1 8.2Total (20.0) 15.7±0.4 16.6 ± 0.2 17.0 ± 0.1 16.3 ± 0.4 16.6 ± 0.2

External appearance (4.5) 3.8 3.8 3.8 3.7 3.7Internai appearance (3.5) 3.0 3.1 3.2 3.1 3.2Odour (2.0) 60 1.5 1.7 1.9 1.6 1.7Taste (10.0) 8.0 8.1 8.2 8.2 8.0Total (20.0) 16.3 ± 0.3 16.7±0.2 17.1±0.2 16.6 ± 0.2 16.6 ± 0.2

Total general mean •• 15.4 •• 16.3 •• 17.1 •• 16.0 •• 16.4 ••

• Mean ± SEM of 3 determinations .•• Values in the same row differ significantly (P> 0.05).

effective in this respect. The figuresobtained were higher than those found byGodinho and Fox (1981) and agreed withthose obtained by King and Clegg (1979)and Farag (1987).

Organoleptic properties

Table V shows the effect of the addition ofeither ripened cheese slurry or mixture ofproteolytic and lipolytic enzymes (1:1) ofP. roqueforti on the organoleptic pro-perties of blue cheese during ripening.Organoleptic examinations showed thatboth cheeses containing ripened slurry or

enzyme mixtures gave good organolepticqualities of blue cheese compared withthe control, but between the differenttreatments and the control cheese therewere significant differences. However,best quality cheese was obtained whenthe cheese contained 2% blue cheeseslurry. Descending order of quality wereobtained with cheese made with 0.02%enzymes mixture, cheese containing 1%slurry and cheese made with added0.01% enzymes mixture when comparedwith the control (Table V). Cheesecontaining 2% slurry was significantlysuperior to ail treatments and acquiredtypical blue cheese flavour as weil as

Acceleration of blue cheese ripening 313

good body characteristics earlier thancontrol cheese. These results could beattributed to the high concentration ofsoluble nitrogenous compounds, free fattyacids and carbonyl compounds in bluecheese containing ripened slurry, sincefree volatile fatty acids and methylketones are major flavour of blue cheese(Jolly and Kosikowski, 1975; King andClegg, 1979).

CONCLUSION

ln this investigation an attempt was madeto accelerate blue cheese ripening usingripened slurry and crude proteolytic andlipolytic enzymes secreted byP. roqueforti. These additives stimulatedthe reaction rate for the formation ofimportant flavour compounds,such asfree fatty acids, amine acids, and carbonylcompounds.

Addition of ripened blue cheese slurryor naturally produced proteolytic andlipolytic enzymes seems to be the mostsuitable method for accelerating cheeseripening. However, further studies concer-ning its application on an industrial scaleshould be investigated.

ACKNOWLEDGMENTS

The author is greatly indebted to Mr. E.Niyonzima, director of Dairy DevelopmentCentre of Kiryama, for his kind help during themanufacture of cheese.

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