Vol 58, No 2 May 2005 International Journal of Dairy Technology

89

ORIGINAL RESEARCH

*Author for correspondence. E-mail: guerzoni@foodsci.unibo.it

© 2005 Society of Dairy Technology

Blackwell Publishing, Ltd.

Evaluation of the ability of

Yarrowia lipolytica

to impart strain-dependent characteristics to cheese when used as a ripening adjunct

ROSALBA LANCIOTTI,

1

LUCIA VANNINI,

1

CLEMENCIA CHAVES LOPEZ,

2

MARCO GOBBETTI

3

and M ELISABETTA GUERZONI

1

*

1

Dipartimento di Protezione e Valorizzazione Agroalimentare, University of Bologna, via Fanin 46, Bologna, I-40127, Italy,

2

Dipartimento di Scienze degli Alimenti, University of Teramo, via Spagna 1, Mosciano Stazione, Teramo, I-64023, Italy, and

3

Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi di Bari, via Amendola, 165,

Bari, I-70125, Italy

Four

Yarrowia lipolytica

strains were tested as cheese-ripening adjuncts with milk culture in cheeseproduction to evaluate their effects on the microbiological and biochemical features of the cheeses. The

Y. lipolytica

strains were able to overcome the other naturally occurring yeasts and were compatible withlactic acid bacteria (LAB). Fourier transform infrared (FTIR) profiles and analysis of free fatty acids(FFAs) released during ripening showed that the strains induced a marked lipolysis and gave rise todifferent FFAs accumulation over time with respect to the control. Strain-dependent protein breakdownpatterns were identified and these biochemical differences resulted in different cheese organolepticcharacteristics.

Keywords

Cheese ripening, Lipolytic activity, Proteolytic activity, Ripening adjunct,

Yarrowia

lipolytica.

*

Author for correspondence. E-mail: guerzoni@foodsci.unibo.it

I N T RO D U C T I O N

The occurrence and growth patterns of a restrictedgroup of yeast species during cheesemaking andripening have been described for different cheesetypes (Walker and Ayres 1970; Fleet 1990; Deak1991; Rhom

et al

. 1992; Tudor and Board 1993;Cosentino

et al

. 2001). Specific surveys haveshown that the most frequently occurring speciesin cheeses are

Debaryomyces hansenii

,

Yarrowialipolytica

,

Pichia membranaefaciens

,

Pichia fer-mentans

,

Candida famata

,

Kluyveromyces lactis

,

Saccharomyces cerevisiae

and

Geotrichum candi-dum

(Nakase and Komagata 1977; Fleet andMain 1987; Tzanetakis

et al

. 1987; Jakobsen andNarvhus 1996; van den Tempel and Jakobsen2000). The mechanisms by which yeast growthmight influence the maturation process are: fer-mentation of residual lactose, utilization of lacticacid (with consequent pH increase), proteolyticand lipolytic activities, and release of autolysisproducts (Ferreira and Viljoen 2003). However, thecontribution of yeasts to cheese flavour develop-ment during ripening is generally underestimated.Evaluation of the importance of their activityremains incomplete, because the assessment oftheir contribution to spoilage or flavour improve-ment is complicated by subjective judgement of

the detrimental or beneficial changes inducedduring ripening.

Although the deliberate inoculum of speciessuch as

D. hansenii

,

S. cerevisiae

and

Y. lipolytica

(Fatichenti

et al

. 1983; van den Tempel and Jakobsen2000; Guerzoni

et al

. 2001; Hansen and Jakobsen2001; Hansen

et al

. 2001; Ferreira and Viljoen2003) as a starter has been proposed, the dominanceof yeast species in the various steps of cheese-making is generally a simple effect of natural envi-ronmental selection. Among the yeast species thatmight have the attributes necessary to assist thelactic acid bacteria (LAB) and participate in cheeseripening,

Y. lipolytica

has been regarded as asuitable candidate (Guerzoni

et al

. 1998; van denTempel and Jakobsen 2000).

Yarrowia lipolytica

has key properties offering competitive advantagesfor growth and predominance in dairy productssuch as tolerance to high salt concentration andlow temperature, lactate and citrate assimilationand production of extracellular proteolytic andlipolytic enzymes (Fleet 1990; Guerzoni

et al

. 1993,2001; Sinigaglia

et al

. 1994; Vannini

et al

. 1996,2001; van den Tempel and Jakobsen 2000; Suzzi

et al

. 2001). However, the enzymatic activities of

Y. lipolytica

strains, and consequently their realcontribution to cheese ripening, are strongly affectedby the physico-chemical and environmental

Vol 58, No 2 May 2005

© 2005 Society of Dairy Technology

90

conditions (Guerzoni

et al

. 2001; van den Tempeland Jakobsen 2000). As already achieved withLAB, a preliminary selection of the strainssuitable to be inoculated as ripening agents isnecessary. As the deliberate use of these speciesis not a common practice, selection criteria inrelation to the desired cheese attributes have to beidentified.

A survey of a relevant number of strains havingdifferent environmental origins showed a highbiodiversity within the species

Y. lipolytica

for pro-teolytic and lipolytic activities, as well as for themembrane fatty acid composition (Sinigaglia

et al

.1994; Vannini

et al

. 1996, 2001; Guerzoni

et al

.1998, 2001; Suzzi

et al

. 2001). In particular, arelevant number of strains had been isolated andcharacterized for lipolytic and proteolytic activities(Vannini

et al

. 1996, 2001; Guerzoni

et al

. 2001;Suzzi

et al

. 2001). Moreover, a preliminary inves-tigation of the potential use of

Y. lipolytica

as acheese ripening agent identified different patternsof protein breakdown on the basis of the

in vitro

activity on

α

s1

- and

β

-caseins (Guerzoni

et al

.1998).

The aim of the present work was the evaluationof the ability of four

Y. lipolytica

strains, represent-ative of different

in vitro

protein breakdown pat-terns, to be used as costarters with milk culture forcheese production. In particular, the technologicalcharacteristics taken into consideration for eachstrain were the ability to predominate over wildyeasts, the compatibility with LAB, the productionof extracellular proteolytic and lipolytic enzymesand the impact on the product’s sensory properties.In addition, an instrumental approach based onFourier transform infrared (FTIR) spectroscopy wasused to provide information on the individualfeatures of the strains and the biochemical changesduring cheese ripening.

M AT E R I A L S A N D M E T H O D S

Microorganisms

The following strains of

Y. lipolytica

, from thecollection of the Dipartimento di Protezione e Valo-rizzazione Agroalimentare, University of Bologna,were used: A1, Y3, PO17 and PZ67.

Milk and starter culture preparation

Milk culture was prepared from raw cow’s milk,heated to 60

°

C for 30 min and incubated at roomtemperature (20–25

°

C) for 10 h. The pH after 10 hwas 5.4.

Yarrowia lipolytica

strains A1, Y3, PO17and PZ67, used in the different trials, were culturedin 1.5 L Sabouraud Broth (Difco Laboratories,Detroit, MI, USA), under agitation at 28

°

C for 48 h.Cells were collected by centrifugation and rinsedtwice with sterile deionized water containing NaCl(9 g/L).

Cheesemaking

Five different types of cheese were made fromcow’s milk pasteurized at 60

°

C for 30 min:

1

model cheeses made with the milk culture (TC).These model cheeses had a flow sheet similar tothat of typical Italian cheeses such as Caciotta andCasatella;

2

cheeses made with the milk culture and with

Y. lipolytica

A1;

3

cheeses made with the milk culture and with

Y. lipolytica

Y3;

4

cheeses made with the milk culture and with

Y. lipolytica

PO17;

5

cheeses made with the milk culture and with

Y. lipolytica

PZ67.

A total of 15 batches (three repetitions for eachcheese type) were produced in a cheese dairy onthree consecutive days with milk from the samefarm. The technological and microbiological factorswere kept as similar as possible for the 15 batches.

For each cheese type, raw cow’s milk was sub-jected to physical depuration and then transferredto a vat containing 300 L of milk. The milk was thenpasteurized at 60

°

C for 30 min and cooled to 38

°

C.Milk culture or milk culture plus

Y. lipolytic

wassubsequently inoculated. The initial levels of LAB,expressed as log cfu/g, ranged between 6.50 and6.70 for the curds of the control samples (TC) andfor the samples co-inoculated with

Y. lipolytica

strains. The levels of

Y. lipolytica

in the curds ofthe latter were

ca

6 log cfu/g. Commercial rennet(1 : 10 000) was added to the milk at the same time.Coagulation occurred within 35 min. After curd breakup and resting (15 min), the curds were extractedfrom whey without cooking and placed in perforatedhoops (plastic cheese moulds) for 2 h. During thisperiod the cheeses were pressed with a Perspex discweighted to 100 g, turned every 20 min and further100 g weights added until a total of 500 g rested oneach cheese. They were then salted for 12 h at 20

°

Cin brine (NaCl 100 g/L) and ripened for 60 days at15

°

C. For each cheese type 15 cheeses were pro-duced. Before ripening, each cheese weighed 2.0 kg.At each sampling time during ripening, microbio-logical and physico- chemical analyses were per-formed in duplicate on three samples (produced onthree different days) for each cheese type.

Microbiological analysis

For all curds and cheeses, 10 g samples, taken atthe surface and in the inner part of each cheese,were placed in 90 mL of sterile saline water (9 g/L)and homogenized in a stomacher (Laboratory-blender 80, Pbi International) for 2 min. Decimaldilutions of the homogenate were prepared insterile saline water and samples (0.1 mL) of appro-priate dilutions were spread onto the surface of Plate

© 2005 Society of Dairy Technology

91

Vol 58, No 2 May 2005

Count Agar (Difco). The plates were incubated at30

°

C for 48 h, and then the total number of mes-ophilic bacteria colonies were counted. Sabourauddextrose agar (Difco) was used for counts of viable

Y. lipolytica

cells by surface spreading and incubationat 28

°

C for 1 week. Counts of the LAB were made byplating appropriate dilutions of the samples on MRSagar (Difco) and incubating at 37

°

C for 72 h inanaerobic jars containing H

2

and CO

2

gases (gen-erated by an Oxoid BR38 kit). Violet Red Bile Agar(Difco) was used for total and faecal coliforms,incubated for 24 h at 37 and 44

°

C, respectively.The microbiological analyses of the cheeses were

carried out at time 0 (immediately after salting)and after 2, 6, 11, 15, 30 and 60 days of storage.The results are the mean of three replicates (threecheeses produced on different days) and two re-petitions (two cheeses produced on the same day).

For yeast identification, two or three colonieswith different morphologies were selected fromeach Sabouraud plate and characterized accordingto Kurtzman (1998).

FTIR spectroscopic analysis

Curds and cheese samples of each cheese typewere subjected to FTIR analyses at time 0 and after30 and 60 days of storage, using an ATI MattsonFTIR spectrometer, equipped with a horizontalzinc selenide (ZnSe) ATR sampling accessory inthe sample compartment. Each spectrum, recordedbetween 700 and 4000 cm

−

1

at a resolution of4 cm

−

1

, consists of 200 interferograms. For eachsample the spectra were recorded in triplicate assample single-beam spectra and compared with thesingle-beam spectrum of water, before conversioninto absorbance units. Triangular apodization wasused. Bands were identified using known standardsand by acquiring spectral data on standard sets(Fairbrother

et al

. 1991).

Extracellular proteolytic activity

The pH 4.6 insoluble total nitrogen from the curdsand from the 1- and 2 month-old cheeses was

analysed by urea–polyacrylamide gel electrophoresis(urea–PAGE) according to the method of Andrews(1983). The gels were stained as described byBlakesley and Boezi (1977).

Analysis of free fatty acids

Free fatty acids (FFAs) were extracted from thecheese and quantified according to the methodof Caboni

et al

. (1990), at time 0 and after 30 and60 days of storage. The analysis of FFAs, as methylesters, was carried out using a Carlo Erba HRGC5160 Mega Series instrument, equipped with a30 m

×

0.32 mm i.d. fused silica capillary columncoated with a film of thickness 0.20

µ

m (Supelco).The oven temperature was programmed from 30 to320

°

C, with a rate of increase of 10

°

C/min, and thedetector (FID) temperature was 350

°

C. The resultsare the mean of three repetitions and two replicates.The variability coefficients, expressed as the per-centage ratio between the standard deviations andthe mean values, were lower than 10%.

Sensory evaluation

According to a nonstructured sensory analysis,the sensory attributes of the different cheese typeswere assessed after 60 days by 15 untrained evalu-ators. The samples were served at 25

°

C. A sensoryvocabulary comprising five attributes was used.The assessors were asked to rate each attributeon a 0–5-point scale (0 = low intensity to 5 = highintensity). In addition, the assessors were askedto rate each cheese type in terms of overall accept-ability (0 = poor to 5 = excellent). The sensorycriteria included flavour and aroma, texture (creamyand compact), taste (piquant and sweetness) andbitter aftertaste.

R E S U LT S

Evolution of yeasts during cheesemaking and ripening

Five cheese types, obtained with and without

Y. lipolytica

strains, were compared. In particular,the four

Y. lipolytica

strains A1, Y3, PO17 andPZ67 were inoculated in milk with a milk culture.The cheeses obtained were compared with controlmodel cheeses made only with milk culture (TC).

In Table 1 the growth data of the yeasts obtained,analysing the cheeses over 60 days of ripening, areshown. The yeast counts were lower in the controls,where they did not exceed 5.5 log cfu/g. Such yeastlevels are common in cheeses. In fact, high yeastcounts, exceeding 6 log cfu/g, have been reportedfor several cheese types, even though they were notadded as starter culture (Lenoir 1963; Chavarri

et al

. 1985; Garcia

et al

. 1987; Kivanc 1989; Ash-enafi 1990; Mor-Mur

et al

. 1992; van den Tempeland Jakobsen 2000; Addis

et al

. 2001). In the curdsfrom milk inoculated with A1, Y3, PO17 and PZ67

Table 1

Yeasts cell counts (log cfu/g) during storage of cheeses obtained from milk inoculated with milk culture (TC) or with milk culture and four different strains of

Yarrowia lipolytica

(A1, Y3, PO17 and PZ67)

Time (days) TC A1 Y3 PO17 PZ67

0a 2.90 ± 0.36 6.00 ± 0.21 5.99 ± 0.18 6.00 ± 0.29 6.20 ± 0.412 4.00 ± 0.11 7.17 ± 0.18 7.27 ± 0.12 7.11 ± 0.18 7.35 ± 0.126 4.50 ± 0.09 6.87 ± 0.37 7.40 ± 0.09 7.00 ± 0.08 7.40 ± 0.1911 5.30 ± 0.27 6.84 ± 0.15 7.40 ± 0.11 6.80 ± 0.23 7.25 ± 0.3115 5.50 ± 0.21 7.04 ± 0.26 7.48 ± 0.17 7.60 ± 0.17 7.60 ± 0.1530 4.55 ± 0.57 6.70 ± 0.31 6.80 ± 0.45 6.72 ± 0.22 6.90 ± 0.4560 4.05 ± 0.16 5.63 ± 0.44 5.43 ± 0.18 5.25 ± 0.19 5.38 ± 0.31

aImmediately after salting

Vol 58, No 2 May 2005

© 2005 Society of Dairy Technology92

with milk culture, the yeast number increased up toabout 7.0–7.5 log cfu/g within 2 days and remainedalmost at the same level over 30 days. At the endof storage decreases ranged between 1.0 and1.5 log cfu/g, depending on the Y. lipolytica strain.The Y. lipolytica strains tested were able to over-come the other naturally occurring yeasts. After15 days in the cheeses obtained from milk inocu-lated with Y. lipolytica A1, Y3, PO17 and PZ67,this species accounted for about 95%, 91%, 93%and 89% of the total yeast population, respectively.Yarrowia lipolytica presents a readily recognizablecolony morphology (Kurtzman 1998) following aweek of incubation, allowing an easy identifica-tion of the colonies. The other isolates belongedto D. hansenii, S. cerevisiae and Kluyveromycesmarxianus species.

In the control curds only D. hansenii andK. marxianus were isolated. During the ripeningthe wild yeast population was represented in thecontrol cheeses by D. hansenii and S. cerevisiae.

Modification of the microbial ecosystem induced by the Y. lipolytica co-inoculumRaw milk used in the process contained a maximumof 5 log cfu/mL of mesophilic bacteria and less than2 log cfu/mL of Enterobacteriaceae. After heattreatment, a reduction of about 2 log cfu/mL of mes-ophilic bacteria was observed. Enterobacteriaceaewere under the detection limit (data not shown).

The evolution over time of faecal and totalcoliforms was analysed during ripening at 20°C.The growth of these microbial groups was notremarkably affected by a co-inoculum with theY. lipolytica strains (data not shown). In particular,the faecal coliforms were at levels lower than2 log cfu/g over the whole ripening period in allthe cheese types. The total coliforms were belowthe detection limit in all the studied curds. Theyreached levels ranging between 2 and 3 log cfu/g in30-day-ripened cheeses regardless of the costarterstrain used. At the end of ripening they were underthe detection limit.

To assess the compatibility of Y. lipolytica strainswith LAB, the four strains were co-inoculated inmilk with milk culture. The co-inoculum of yeaststrains with milk culture seems to stimulate theLAB growth, particularly in the early phase, withrespect to the control inoculated with milk culture(Table 2). This result indicates that the four differ-ent strains of Y. lipolytica can enhance the growthof the naturally occurring LAB to various degrees.The stimulating effect of Y. lipolytica on LAB canbe attributed to the yeast proteolytic and lipolyticactivities, or to citric acid production in the wholemass of the cheese. Moreover, the co-inoculumwith Y. lipolytica, which consumes oxygen for itsgrowth, also renders the outer cheese layers morefavourable for microaerobic LAB.

Proteolytic activityTo compare the ability of the four yeasts strains toinduce strain-specific dependent differences in theprotein breakdown during ripening, the curds andalso 1- and 2-month-old cheeses, obtained fromcontrol milk (inoculated with the milk culture) andfrom the milk co-inoculated with milk culture andthe four strains of Y. lipolytica, were examined withtwo techniques, FTIR analysis and urea–PAGE.

FTIR spectroscopy provides spectral data onwater, protein, acids, lipids and carbohydrateswith a single analysis (Belton et al. 1987). Rapidtechniques for determination of fat, protein andtotal solids in milk by IR absorption spectroscopyhave been widely adopted in the dairy industry(McGann 1978) and IR analysis is an approvedstandard method of the Association of OfficialAnalytical Chemists (AOAC) (Biggs 1972). McQueenet al. (1995) and Chen and Irudayaraj (1998) havereported the use of FTIR spectroscopy for cheeseanalysis.

Superimpositions of the spectra relative to 1- and2-month-old cheeses, obtained from the strains PO17and Y3, as well as the control curd and cheeses arepresented in Figure 1a–c. Well-separated bandswere observed in all the spectra. In particular, thesecondary structure of proteins was reflected in theFTIR spectra due to the absorbance of the bandsin the Amide I region between 1620 and 1690 cm−1,attributed primarily to the stretching vibration ofthe carbonyl group (Garland 1994). The absorbanceband around 1670 cm−1 can be associated withthe β structure, while bands close to 1650 and1640 cm−1 are associated with the helical and ran-dom portions of proteins, respectively (Susi andByler 1988).

The major differences for the various absorbancescan be observed in the spectral region between1300 and 1700 cm−1, corresponding both to theAmide I vibration of the α helix and to the Amide IIvibration of the β sheet of proteins. The absorbanceof these bands was very low in all the curds

Table 2 Lactic acid bacteria counts (log cfu/g) during storage of cheeses obtained from milk inoculated with milk culture (TC) or with milk culture and four different strains of Yarrowia lipolytica (A1, Y3, PO17 and PZ67)

Time (days) TC A1 Y3 PO17 PZ67

0a 6.70 ± 0.25 6.67 ± 0.31 6.55 ± 0.27 6.53 ± 0.27 6.60 ± 0.222 7.43 ± 0.19 7.84 ± 0.18 8.54 ± 0.11 8.25 ± 0.19 8.50 ± 0.076 7.95 ± 0.14 8.10 ± 0.22 8.70 ± 0.15 8.45 ± 0.11 8.60 ± 0.1111 8.57 ± 0.09 8.60 ± 0.11 8.90 ± 0.12 8.70 ± 0.15 8.75 ± 0.2315 8.20 ± 0.10 8.32 ± 0.09 8.50 ± 0.09 8.80 ± 0.10 8.40 ± 0.1830 8.00 ± 0.16 8.15 ± 015 8.26 ± 0.19 8.43 ± 0.15 8.15 ± 0.2460 6.80 ± 0.18 7.50 ± 033 7.10 ± 0.20 7.70 ± 0.32 7.00 ± 0.45

aImmediately after salting

© 2005 Society of Dairy Technology 93

Vol 58, No 2 May 2005

and increased during cheese ripening, presumablybecause of the increase in peptide content. However,while the absorbance of the band correspondingto the Amide II attained similar values in all the

five cheese types after 2 months, the absorbanceof the Amide I band, which appeared as a largeshoulder on the Amide II peak in the control cheeses(Figure 1a), was considerably reduced in the cheeses

Figure 1 Superimposition of the FTIR spectra of curds (thin line), 30-day- (broken line) and 60-day- (thick line) old cheeses obtained from milk inoculated with milk culture (a) or milk culture and Y. lipolytica strain PO17 (b) or Y3 (c).

Vol 58, No 2 May 2005

© 2005 Society of Dairy Technology94

obtained using A1 and PO 17 strains as costarters(Figure 1b). On the other hand, in the cheesesobtained using the strains Y3 and PZ67 as costarters,the absorbance of the two bands of Amide I andAmide II decreased, following a slight increaseduring the first month (Figure 1c). In particular, theabsorbance of the Amide I band fell below thebaseline. It is probable that some peptides formedduring the breakdown were highly hydrophobic,because of the predominance of aromatic aminoacids, and were no longer water soluble.

Relevant strain-dependent differences can also beobserved in the zone corresponding to the Amide IIIof proteins (1230–1330 cm−1). The most intenseband in this region is near 1300 cm−1, with a weakercomponent at about 1245 cm−1. The absorbance ofboth the bands increased over time in all the samples.However, different patterns were observed in the 2-month-old cheeses. In particular, the absorbance ofthe band at 1245 cm−1 increased up to values of about0.20 in cheeses obtained with strains Y3 and PZ67.However, the absorbance of this band presented alower increase in the control as well as in cheesesobtained with PO17 and A1 strains. Moreover, itcan be observed that the relative ratio between thetwo bands at 1330 and 1450 cm−1, whose natureshould be more fully investigated, presented differ-ent values for the cheeses obtained with the Y3 andPZ67 or A1 and PO17 strains.

Figure 2 shows that, with regard to the urea–PAGE of the pH 4.6 insoluble nitrogen, all thecheeses were characterized by strong proteolysis.In fact, αs1- and β-caseins were completely hydro-lysed after 2 months of ripening. However, somedifferences in the Y. lipolytica strain features werefound. At the beginning of ripening the electro-phoretograms of the cheeses obtained using A1strain plus milk culture showed additional bandswith either high or low electrophoretic mobility. After

1 month of ripening it seemed that Y. lipolyticastrain Y3 had caused a slightly higher hydrolysisof αs1- and especially β-caseins compared to theother strains.

Lipolytic activityIn the FTIR spectra three regions accounting forthe changes in fatty acids can be identified: thezone from 2800 to 3100 cm−1, corresponding toC–H vibration; the band at 1740 cm−1, correspond-ing to carbonyl absorption of the triglyceride esterlinkage; and the spectral zone around 900 cm−1,corresponding to C=C–H bending vibrations ofdouble bonds. Major differences can be observedin the 60-day-old cheeses. The absorbance ofthe bands between 2800 and 3100 cm−1 attained avalue of 0.7 in the cheeses inoculated with PZ67and Y3 with milk culture. In the control and incheeses inoculated with A1 and PO17 the absorb-ance values of these bands did not exceed 0.45. Theband at 1740 cm−1 accounts for both lactic acid andfree fatty acids. In the control cheeses its absorb-ance increased only during the first month up to 0.2and then decreased down to 0.1, probably becauseof lactic acid consumption by wild moulds andyeasts. This decrease was confirmed by theabsorbance decrease of the band at 1160 cm−1 cor-responding to the carbohydrate zone. However, theabsorbances of the bands at 1160 and 1740 cm−1

continued to increase over time in all the cheesesinoculated with Y. lipolytica, particularly with strainsY3 and PZ67. Another similarity between thecheeses inoculated with A1 and PO17 and thecontrol is the absorbance increase of a band at960 cm−1 after 2 months of ripening.

The contents of individual FFAs in the lipid frac-tions of the 30- and 60-day-old cheeses obtainedusing the four Y. lipolytica strains as costarters arereported in Table 3.

In the control curds, low concentrations (lowerthan 50 ppm) of capric (C10:0), lauric (C12:0),myristic (C14:0), palmitic (C16:0), stearic (C18:0)and oleic (C18:1∆9) acids were detected. All thecurds obtained using Y. lipolytica as costarter con-tained traces of isopentadecanoic (iso-C15:0) andisomargaric (iso-C17:0) acids (data not shown).

The total content of FFAs after 2 months ofripening markedly increased with respect to thecontrol when the strains PZ67 and Y3 were used.This result is in agreement with the increase of thebands at 2800 and 3000 cm−1observed withFTIR spectroscopy. Moreover, the four strainsinduced quantitative and qualitative differencesin the content of the individual fatty acids withrespect to the control. The proportion of short-chainFFAs, namely butyric (C4:0), capric and undeca-noic (C11:0) acids, in cheeses co-inoculated withY. lipolytica did not differ appreciably from that ofthe control. Only cheeses obtained with Y. lipolytica

Figure 2 Urea–PAGE electrophoresis of pH 4.6 insoluble nitrogen of cheeses. Lanes 1–5: curds obtained from milk inoculated with milk culture or milk culture and Y. lipolytica strain A1, PO17, Y3 or PZ67. Lanes 6–10: 30-day-old cheeses obtained from milk inoculated with milk culture or milk culture and Y. lipolytica strain A1, PO17, Y3 or PZ67. Lanes 11–15: 60-day-old cheeses obtained from milk inoculated with milk culture or milk culture and Y. lipolytica strain A1, PO17, Y3 or PZ67.

© 2005 Society of Dairy Technology 95

Vol 58, No 2 May 2005

strains A1 and PZ67 had a higher content ofcaprylic acid (C8:0).

The controls were characterized by a highpresence of oleic, linoleic (C18:2) and linolenic(C18:3) fatty acids in comparison with the cheeses

inoculated with the strains A1 and PO17. Theformer was present at the highest level only in thecheeses co-inoculated with the Y3 strain, whilethe C18:2 and C18:3 acids were absent in the chee-ses prepared with A1. Arachidic acid (C20:0) wasabsent in PO17 cheeses and behenic acid (C22:0)was present only in the control and Y3.

The four strains imparted specific characteris-tics to the free medium-chain fatty acid composi-tion of the 2-month-old cheeses. In particular, PZ67and Y3 gave rise to a high presence of the saturatedfree fatty acids C12:0, C14:0, C16:0 and C18:0. Inthe cheeses inoculated with the strain PZ67, a con-siderable amount of iso-C17:0 was also detected.

The use of A1 resulted in an increased productionof tridecanoic (C13:0), pentadecenoic (C15:1),isoheptadecenoic (C17:1) and undecenoic (C11:1)acids. The strain PO17 gave rise to a relatively highpercentage of C11:1, C12:1, iso-C15:0, iso-C17:0and iso-C17:1.

Sensory assessmentThe results of the sensory assessment are reportedin Table 4. The cheeses obtained from milk inocu-lated with Y. lipolytica strains A1 and PO17 received

Table 3 Free fatty acid content (mg/kg) of 30- and 60-day-old cheeses obtained from milk inoculated with milk culture (TC) or with milk culture and four different strains of Yarrowia lipolytica (A1, Y3, PO17 and PZ67)

30-day-old cheese 60-day-old cheese

FFA TC A1 Y3 PO17 PZ67 TC A1 Y3 PO17 PZ67

C4:0 690.00 606.00 614.61 496.37 663.13 662.02 635.34 645.47 641.29 697.15C8:0 727.73 1 128.54 520.54 580.59 496.37 573.71 2 143.30 496.37 611.64 2 188.10C10:0 1 707.45 496.70 614.80 1 459.17 610.24 1 792.12 538.11 756.18 1 665.43 967.92C11:0 596.67 0.00 921.91 581.33 1 614.50 527.89 0.00 3 477.45 496.37 4 336.61C11:1 0.00 505.39 546.01 1 700.20 585.79 0.00 2 962.95 673.17 2 738.01 748.83C12:0 1 847.25 0.00 989.95 537.83 1 930.07 2 876.88 0.00 5 211.65 561.26 6 313.87C12:1 0.00 496.37 496.37 3 309.48 542.20 0.00 566.83 601.60 7 616.41 634.41C13:0 534.11 2 672.57 513.76 544.06 549.45 566.18 7 478.28 635.90 641.47 678.00C14:0 3 805.19 535.79 1 754.85 792.89 4 269.30 7 678.87 624.28 14 960.74 1 187.66 17 641.30C14:1 563.11 752.46 515.43 604.20 1 078.44 618.89 1 171.49 773.09 855.91 827.93ante-C15:0 894.02 586.82 622.42 738.98 922.47 1 154.01 826.63 1 827.73 1 140.63 2 150.27ante-C:15:1 645.10 686.37 553.91 496.37 654.21 785.18 1 140.16 1 258.21 530.86 570.64iso-C:15 798.28 496.37 608.66 5 311.66 823.19 1 085.51 496.37 1 833.96 17 364.67 2 063.83C15:1 507.90 5 066.18 496.37 556.70 496.37 531.70 16 672.18 570.46 608.94 593.42C16:0 7 075.71 872.46 2 852.34 908.90 7 970.56 15 477.84 11 970.75 31 459.74 1 805.23 38 132.30C16:1 496.37 496.37 510.41 508.64 591.19 593.05 756.64 775.60 615.82 1 122.22ante-C17 1 357.95 0.00 663.04 551.40 577.99 1 617.28 0.00 2 864.89 743.26 1 031.97C17:1 651.70 496.37 518.22 496.37 0.00 605.04 647.70 812.23 496.37 0.00iso-C17 609.59 0.00 521.01 1 369.01 2 191.17 682.65 0.00 959.56 4 981.78 10 441.14iso-C17:1 496.37 1 622.22 496.37 4 530.68 0.00 1 137.65 4 679.96 496.37 20 802.59 0.00C18:0 2 124.62 6 460.27 905.36 1 027.97 9 452.59 4 099.76 16 692.16 6 912.02 3 215.04 43 801.30C18:1 8 764.18 1 445.79 2 368.24 597.13 1 677.70 15 552.20 2 584.17 25 637.68 992.74 6 077.59C18:2 1 614.77 0.00 775.79 562.00 768.45 2512.97 0.00 3 834.84 914.66 1 502.30C18:3 721.41 590.81 556.61 636.55 675.03 831.19 0.00 1 061.99 686.65 1 385.00C20:0 657.37 503.53 523.05 0.00 527.42 781.55 0.00 911.41 0.00 496.37C22:0 550.01 0.00 546.48 0.00 0.00 0.00 0.00 572.69 0.00 0.00Total 38 436.99 26 517.16 21 006.65 28 898.62 39 667.94 62 744.22 72 587.39 11 0021.10 71 415.45 14 1681.70

Table 4 Sensory evaluation scoresa of 60-day-old cheeses obtained from milk inoculated with milk culture (TC) or with milk culture and four different strains of Yarrowia lipolytica (A1, Y3, PO17 and PZ67)

Sensory criteria TC A1 Y3 PO17 PZ67

Flavour and aromab 3.0 ± 0.83 4.8 ± 0.45 4.0 ± 0.71 4.6 ± 0.89 3.6 ± 0.55Textureb

Creamy 2.2 ± 0.45 4.8 ± 0.45 4.6 ± 0.55 4.8 ± 0.45 4.4 ± 0.55Compactness 4.8 ± 0.45 2.0 ± 1.0 2.0 ± 1.0 1.8 ± 0.84 2.4 ± 0.55

Tasteb

Piquant 2.2 ± 0.45 4.8 ± 0.45 4.4 ± 0.55 3.0 ± 0.71 4.8 ± 0.45Sweetness 2.4 ± 0.55 2.8 ± 0.83 3.0 ± 0.71 4.6 ± 0.89 2.8 ± 0.83

Bitter aftertasteb 2.4 ± 0.55 1.2 ± 0.45 2.0 ± 1.0 1.2 ± 0.45 3.2 ± 0.45Overall assessmentc 3.2 ± 1.0 4.8 ± 0.45 4.0 ± 0.71 4.6 ± 0.89 4.0 ± 0.71

aValues are means ± SE for analyses performed by 15 assessorsbFive-point sensory scale (from 0 = low intensity to 5 = high intensity)cFive-point scale (from 0 = poor to 5 = excellent)

Vol 58, No 2 May 2005

© 2005 Society of Dairy Technology96

the highest mean overall grade. However, while thetaste of the cheeses obtained with the strain PO17was defined as ‘sweet’, the taste of those obtainedusing the strain A1 was defined as ‘piquant’. Thecreamy texture was the sensory attribute that mostlycontributed to the rating of both cheese types,which presented similar FTIR profiles, particularlyin the spectral zone corresponding to the Amide I,Amide II and Amide III signals of proteins. Thus asimilar protein breakdown might be affecting thewater-binding capacity of the protein or peptidesand, consequently, in part the microstructure of thecheeses. However, the FFAs released after 30 daysin cheeses obtained with the strains A1 and PO17were different. In particular, in the A1 cheeses arelatively high content of odd fatty acids, suchas C13:0, C15:1 and C17:1, was observed. Thesefatty acids do not occur frequently in cheesesand their presence can be specifically regarded asdepending on the metabolic activity of the strains.It is known that some strains of Y. lipolyticainclude in their phospholipids relevant percentagesof odd fatty acids.

D I S C U S S I O N

The assessment of the importance of each microbialgroup in the development of both desirable andundesirable flavour and texture during cheeseripening is generally obscured by the complexsuccession and interactions of the inoculated ornaturally occurring microbial species. In particular,the specific contribution of the yeasts or secondarymicroflora to cheese ripening is generally underes-timated or not easily distinguished. The experimentalcheeses prepared did not correspond to specificor typical cheeses and have been used as modelcheeses with milk culture as a starter. The method-ological approach adopted focused on the evaluationof the contribution of Y. lipolytica to the proteoly-sis and lipolysis as well as on its ability to impartstrain-dependent characteristics to the cheese. Theresults indicate that the four Y. lipolytica strainspossess the prerequisites to be used as starters,that is the ability, when inoculated at a level of about6 log cfu/mL, to overcome the other naturallyoccurring yeasts and to be compatible with LAB.Different lipolytic and proteolytic profiles wereobserved in relation to the strain used as costarter.The lipolytic activity of Y. lipolytica has been widelystudied (Ota et al. 1984; Roostita and Fleet 1996;Freitas et al. 1999). Yarrowia lipolytica lipasesare reported to act prevalently on oleyl residues atpositions 1 and 3 of the glyceride (Hadeball 1991).Although oleic acid was the principal fatty acidreleased in butter inoculated with different strainsof Y. lipolytica, Guerzoni et al. (1998, 2001) showedthe ability of some strains to release relevant pro-portions of branched and/or polyunsaturated fatty

acids. The existence of different lipases havingdifferent positional or fatty acid specificity hasbeen hypothesized (Guerzoni et al. 2001). However,several authors have found that the in vitro charac-terization of extracellular and cell-bound lipaseactivity is not sufficient to predict the lipolysispattern under real conditions (van den Tempel andJakobsen 2000; Guerzoni et al. 2001; Suzzi et al.2001). In fact, in addition to their effect on yeastgrowth, the physico-chemical and compositionalfactors also have an indirect effect on lipase activ-ity principally because of their influence on themicrostructure of the system (Kojima et al. 1994;Guerzoni et al. 2001). The importance of environ-mental conditions, such as NaCl and lactic acidconcentrations as well as of temperature, on thelipolytic activity of some Y. lipolytica strains hasbeen shown by van den Tempel and Jakobsen (2000)and Guerzoni et al. (2001).

The strains used in this work induced a markedlipolysis and gave rise to different lipolytic patternswith respect to the control. The total FFA contentsreleased after 60 days of ripening in the cheesesobtained by co-inoculating Y. lipolytica strains weresignificantly higher than those detected in thecontrol cheeses. However, the latter also evinced amarked lipolysis attributable to superficial greenmould growth, which did not occur in any ofthe cheeses inoculated with Y. lipolytica (datanot shown). The growth of such moulds, whichare very frequent in ripening rooms, seemed tobe inhibited by Y. lipolytica inoculum. However, onthe basis of our data we cannot exclude the possi-bility of the contribution to lipolysis of othernaturally occurring microbial groups, such as ente-rococci, micrococci, nonstarter LAB and yeasts.These secondary microflora play a major role inthe ripening process of several cheese varieties(Beresford et al. 2001).

The strains PZ67 and Y3, which gave rise to sim-ilar proteolysis patterns, were able to induce themost intense lipolysis in 60-day-ripened cheeses,as shown by the total FFA values. Moreover,the data obtained showed that all the Y. lipolyticastrains preferentially released large amounts ofbranched saturated or unsaturated fatty acids, withthe exception of the strain Y3, which was able torelease an increased quantity of C18:1, C18:2 andC18:3 with respect to the control. The occurrenceof unusual levels of C15 and C17 fatty acids withrespect to the control can be in part attributableto their natural occurrence in cell phospholipidsof Y. lipolytica (Sinigaglia et al. 1994). A relevantrole in the aroma of several surface-ripened cheeseshas been attributed to the release of unsaturated fattyacids by this yeast (Jelen and Wasowicz 1998).

Two of the strains used, PZ67 and Y3, gave riseto a higher content of lauric and myristic acids.These fatty acids can contribute both to product

© 2005 Society of Dairy Technology 97

Vol 58, No 2 May 2005

safety and to organoleptic properties. Indeed,Papageorgiou and Marth (1989) and Kinderlereret al. (1996) showed that these medium-chainFFAs had antibacterial activity against Listeriamonocytogenes. Moreover, they are the precursorsof methyl ketones and secondary alcohols, whichcontribute to the sensory characterization of cheeses(Curioni and Bosset 2002).

The electrophoretic profiles of the differentcheese types were also considered, as proteolysis iscommonly used as an index of ripening (Farkyeand Fox 1990). It is also useful for the evaluationof starter culture performance (Fox 1989) becausethis lytic activity strongly contributes to flavourand texture development (Freitas et al. 1997). Theelectrophoretic profiles obtained showed differencesrelated to Y. lipolytica strains even at the beginningof ripening. In particular, the cheeses obtainedusing the A1 strain as costarter showed additionalbands with either high or low electrophoreticmobility. Moreover, a slightly higher hydrolysisof αs1- and especially β-caseins was shown for theY. lipolytica strain Y3 after 1 month of ripening. Astrong proteolytic activity of Y. lipolytica strainsalso at temperatures lower than 10°C is well docu-mented (Kobatake and Kurata 1983; Kobatakeet al. 1992; Sinigaglia et al. 1994; Wyder 1998).The ability of some Y. lipolytica strains to degradeall casein components under cheese ripening con-ditions has been documented (van den Tempel andJakobsen 2000). However, a diversity in hydrolyticpatterns of αs1- and β-caseins among Y. lipolyticastrains of different origins has also been reported(Guerzoni et al. 1998; Vannini et al. 2001). Moreover,the key role of environmental conditions on theproteolytic activity of Y. lipolytica strains has beendemonstrated (Sinigaglia et al. 1994; van denTempel and Jakobsen 2000; Guerzoni et al. 2001;Suzzi et al. 2001).

The results of the present work indicate thatFTIR spectroscopy is a promising tool for monitor-ing the compositional changes in cheeses duringripening as well as those in the overall secondarystructure of proteins induced by microbial species.This spectroscopic method is nondestructive, moreflexible and less time-consuming than many othertechniques and does not require any sample pre-paration. The FTIR spectroscopic analyses provideddifferent but coherent information with respect tothe methods most frequently used to analyse prote-olytic and lipolytic activities. They showed thatindividual strains within the species Y. lipolyticaare characterized by proteolytic and lipolytic enzymesable to give rise to different protein breakdown anddifferent FFA accumulation over time. In partic-ular, the FTIR analysis showed that two differentprotein breakdown patterns can be identified, onesimilar to the control and the other completelydifferent, at least after 2 months of ripening. The

possibility of recognizing the yeast strain used onthe basis of FTIR ‘fingerprints’ emphasizes howthe Y. lipolytica strain used as costarter can conferstrain-dependent features on cheese.

The different and specific lipolytic and proteolyticpatterns of each cheese type resulted in differentsensory properties as indicated by sensory analysis,which assigned the highest score to the productsobtained using the strain A1 as costarter. On theother hand, it is well known that the typical sensorycharacteristics of ripened cheese are principallydue to proteolysis and lipolysis and to the subse-quent transformation of their final products (DiCagno et al. 2003).

The consequence of biochemical diversity for theorganoleptic and textural characteristics of cheesesshould be better analysed in relation to the specificquality criteria established for well-defined dairyproducts. In addition, the bioactivity of the peptidesproduced by the various strains should be investi-gated and possibly exploited (Xu 1998). Alterna-tively, the strain diversity can be exploited for theproduction of new types of cheeses for whichthe proteolytic and lipolytic pathways can be com-pletely redesigned to obtain particular organolepticcharacteristics.

R E F E R E N C E S

Addis A, Fleet G H, Cox J M, Kolak D and Leung T (2001)The growth, properties and interactions of yeasts andbacteria associated with the maturation of Camembertand blue-veined cheese. International Journal of FoodMicrobiology 69 25–36.

Andrews P (1983) Proteinases in normal bovine milk and theiraction on casein. Journal of Dairy Research 50 45–52.

Ashenafi M (1990) Microbiological quality of Ayib, a tradi-tional Ethiopian cottage cheese. International Journal ofFood Microbiology 10 263–268.

Belton P S, Saffa A M and Wilson R H (1987) Use of Fouriertransform infrared spectroscopy for quantitative analysis:a comparative study of different detection methods.Analyst 12 1117–1120.

Beresford T P, Fitzsimons N A, Brennan N L and Cogan T M(2001) Recent advances in cheese microbiology. Interna-tional Dairy Journal 11 259–274.

Biggs D A (1972) Precision and accuracy of infrared milkanalysis. Journal of the Association of Official AnalyticalChemists 55 488–497.

Blakesley R and Boezi J (1977) A new staining technique forprotein in polyacrylamide gels using Coomassie BrilliantBlue G250. Analytical Biochemistry 82 580–583.

Caboni M F, Zannoni M and Lercker G (1990) Fat lipolysisin Parmiggiano-Reggiano cheese. Scienze e TecnologiaLattiero-Casearia 41 289–297.

Chavarri F J, Nùnez J A, Bautista L and Nùnez M (1985)Factors affecting the microbiological quality of Burgosand Villalon cheese at the retail level. Journal of FoodProtection 48 865–869.

Chen M and Irudayaraj J (1998) Sampling techniques forcheese analysis by FTIR spectroscopy. Journal of FoodScience 63 96–99.

Vol 58, No 2 May 2005

© 2005 Society of Dairy Technology98

Cosentino S, Fadda M E, Deplano M, Mulargia A F andPalmas F (2001) Yeasts associated with Sardinian ewe’sdairy products. International Journal of Food Microbiol-ogy 69 53–58.

Curioni P M G and Bosset J O (2002) Key odourants in variouscheese types as determined by gas chromatography–olfactometry. International Dairy Journal 12 959–984.

Deak T (1991) Food-borne yeast. Advances in AppliedMicrobiology 36 180–277.

Di Cagno R, Banks J, Sheehan L, Fox B, Brechany E Y,Corsetti A and Gobbetti M (2003) Comparison of themicrobiological, compositional, biochemical, volatile profileand sensory characteristics of three Italian PDO ewes’milk cheeses. International Dairy Journal 13 961–972.

Fairbrother P, George W O and Williams J M (1991) Wheyfermentation: on-line analysis of lactose and lactic acid byFTIR spectroscopy. Applied Microbiology and Biotech-nology 35 301–305.

Farkye N Y and Fox P F (1990) Objective indices of cheeseripening. Trends in Food Science and Technology 1 37–40.

Fatichenti F, Bergere J L, Deiana P and Farris G A (1983)Antagonistic activity of Debaryomyces hansenii towardsClostridium tyrobutyricum and Cl. butyricum. Journal ofDairy Research 50 449–457.

Ferreira A D and Viljoen B C (2003) Yeasts as adjunct startersin matured Cheddar cheese. International Journal of FoodMicrobiology 86 131–140.

Fleet G H (1990) A review: yeasts in dairy products. Journalof Applied Bacteriology 68 199–211.

Fleet G H and Main M A (1987) The occurrence and growthof yeasts in dairy products. International Journal of FoodMicrobiology 4 145–155.

Fox P F (1989) Proteolysis during cheese manufacture andripening. Journal of Dairy Science 72 1379–1400.

Freitas A C, Fresno J B, Prieto B, Malcata F X and CarballoF J (1997) Effects of ripening time and combination ofovine and caprine milks on proteolysis of Picante cheese.Food Chemistry 2 219–229.

Freitas A C, Pintado A E, Pintado M E and Malcata F X(1999) Role of dominant microflora of Picante cheese onproteolysis and lipolysis. International Dairy Journal 9593–603.

Garcia M C, Otero A, Garcia M L and Moreno B (1987)Microbiological quality and composition of two types ofSpanish sheep’s milk cheese (Manchego and Burgosvarieties). Journal of Dairy Research 54 551–557.

Garland B (1994) FT-IR of protein secondary structure inaqueous and dried states. Nicolet Application Note,AN9479, Nicolet Analytical, Madison, WI, USA.

Guerzoni M E, Lanciotti R and Marchetti R (1993) Survey ofthe physiological properties of the most frequent yeastsassociated with commercial chilled foods. InternationalJournal of Food Microbiology 17 329–341.

Guerzoni M E, Gobbetti M, Lanciotti R, Vannini L andChaves Lopez C (1998) Yarrowia lipolytica as a potentialripening agent in milk products. In Yeasts in the DairyIndustry: Positive and Negative Aspects, pp 23–33.Jakobsen M, Narvhus J and Vilijoen B C, eds. Brussels:International Dairy Federation.

Guerzoni M E, Lanciotti R, Vannini L, Galgano F, Favati F,Gardini F and Suzzi G (2001) Variability of the lipolyticactivity in Yarrowia lipolytica and its dependence onenvironmental conditions. International Journal of FoodMicrobiology 69 79–89.

Hadeball W (1991) Production of lipases by Yarrowia lipolytica.Acta Biotechnologica 11 159–167.

Hansen T K and Jakobsen M (2001) Taxonomical andtechnological characteristics of Saccharomyces spp.associated with blue veined cheese. International Journalof Food Microbiology 69 59–68.

Hansen T K, van den Tempel T, Cantor M D and Jakobsen M(2001) Saccharomyces cerevisiae as a starter culture inMycella. International Journal of Food Microbiology 69101–111.

Jakobsen M and Narvhus J (1996) Yeasts and their possiblebeneficial and negative effects on the quality of dairyproducts. International Dairy Journal 6 755–768.

Jelen H and Wasowicz E (1998) Volatile fungal metabolitesand their relation to the spoilage of agricultural commod-ities. Food Review International 14 391–426.

Kinderlerer J L, Matthias H E and Finner P (1996) Effect ofmedium-chain fatty acids in mould ripened cheeses onthe growth of Listeria monocytogenes. Journal of DairyResearch 63 593–606.

Kivanc M (1989) A survey on the microbiological quality ofvarious cheeses in Turkey. International Journal of FoodMicrobiology 9 73–77.

Kobatake M and Kurata H (1983) Proteolytic and lipolyticactivities of psychophilic bacteria and psychotrophicyeasts isolated from chilled household foods and rawseafood. Journal of the Food Hygiene Society of Japan 24532–539.

Kobatake M, Kreger-van Rji N J W, Placido T L C and vanUden N (1992) Isolation of proteolytic psychotrophicyeasts from fresh raw seafoods. Letters in Applied Micro-biology 14 37–42.

Kojima Y, Yokoe M and Mase T (1994) Purification andcharacterisation of an alkaline lipase from Pseudomonasfluorescens AK 102. Bioscience, Biotechnology and Bio-chemistry 58 1564–1568.

Kurtzman C P (1998) Yarrowia van der Walt and von Arx. InThe Yeasts—a Taxonomic Study, pp 420–422. KurtzmanC P and Fell J W, eds. Amsterdam, the Netherlands: Else-vier Science B.V.

Lenoir J (1963) La flore microbienne du Camembert et sonevolution au cours de la maturation (1). (Microflora asso-ciated to camembert and its evolution during cheese rip-ening) (1). Le Lait 347 262–270.

McGann T C A (1978) Automated physico-chemical methodsfor the analysis of milk. A review of major advances(1966–1978). Irish Journal of Food Science and Technol-ogy 2 141–152.

McQueen D H, Wilson R, Kinnunen A and Jensen E P (1995)Comparison of two infrared spectroscopic methods forcheese analysis. Talanta 42 2007–2012.

Mor-Mur M, Carretero C, Pla R and Guamis B (1992) A sur-vey on the microbiological quality of a semi-soft on-farm manufactured goat cheese. Food Microbiology 9345–352.

Nakase K and Komagata K (1977) Microbiological studies oncheese. I. Yeast flora in cheese imported from Europe andNorth America. Journal of the Food Hygiene Society ofJapan 18 346–352.

Ota Y, Oikawa S, Morimoto Y and Minoda Y (1984)Nutritional factors causing mycelial development ofSaccharomycopsys lipolytica. Agricultural and BiologicalChemistry 48 1933–1939.

Papageorgiou D K and Marth E H (1989) Fate of Listeria

© 2005 Society of Dairy Technology 99

Vol 58, No 2 May 2005

monocytogenes during the manufacture and ripening ofblue cheese. Journal of Food Protection 52 459–465.

Rhom H, Eliskases-Lechner F and Brauer M (1992) Diversityof yeasts in selected dairy products. Journal of AppliedBacteriology 72 370–376.

Roostita R and Fleet G F (1996) Growth of yeasts in milk andassociated changes to milk composition. InternationalJournal of Food Microbiology 31 205–219.

Sinigaglia M, Lanciotti R and Guerzoni M E (1994) Bio-chemical and physiological characteristics of Yarrowialipolytica strains in relation to isolation source. CanadianJournal of Microbiology 40 54–59.

Susi H and Byler M D (1988) Protein structure by Fouriertransform spectroscopy in protein conformation studies.In Methods for Protein Analysis, pp 235–255. Cherry J Pand Barford R A, eds. Champaign, IL, USA: AmericanOil Chemistry Society.

Suzzi G, Lanorte M T, Galgano F, Andrighetto F, Lombardi A,Lanciotti R and Guerzoni M E (2001) Molecular charac-terisation and metabolic activities of Yarrowia lipolyticaisolated from cheese. International Journal of FoodMicrobiology 69 147–152.

van den Tempel T and Jakobsen M (2000) The technologicalcharacteristics of Debaryomyces hansenii and Yarrowialipolytica and their potential as starter cultures for produc-tion of Danablu. International Dairy Journal 10 263–270.

Tudor D A and Board R G (1993) Food spoilage yeasts. In The

Yeasts, Vol. 5, pp 436–516, Rose A H and Harrison J S,eds. London: Academic Press.

Tzanetakis N, Litopoulou-Tzanetaki E and Manolkidis K(1987) Microbiology of Kopanisti, a traditional Greekcheese. Food Microbiology 4 251–256.

Vannini L, Lanciotti R, Criscuoli G C and Guerzoni M E(1996) Yarrowia lipolytica come potenziale agente dimaturazione nei prodotti lattiero-caseari. (Yarrowia lip-olytica as potential ripening agent of dairy products). InProceedings of the AAA Biotec – Advances in Biotechnol-ogy, Agriculture, Nutrition and Environment Congress,pp 493–501, Ferrara Italy.

Vannini L, Baldi D and Lanciotti R (2001) Use of Fouriertransform infrared (FTIR) spectroscopy to evaluate theproteolytic activity of Yarrowia lipolytica and its contri-bution to cheese ripening. International Journal of FoodMicrobiology 69 113–123.

Walker H W and Ayres J C (1970) Yeasts as spoilage organisms.In The Yeasts, Vol. 5, pp 464–527, Rose A H and HarrisonJ S, eds. London: Academic Press.

Wyder M T (1998) Identification and characterisation of theyeast flora in kefir and smear-ripened cheese. PhD Thesis,p 152, Swiss Federal Institute of Technology (ETH),Zurich, Switzerland.

Xu R J (1998) Bioactive peptides in milk and their biologicaland health implications. Food Reviews International 11–16.