Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2005
Volume 8
Issue 1
Topic:
Biotechnology
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Juszczyk P. , Wojtatowicz M. , Żarowska B. , Chrzanowska J. , Malicki A. 2005. GROWTH AND SPORULATION OF PENICILLIUM ROQUEFORTI STRAINS IN THE PRESENCE OF YEASTS SELECTED AS CO-STARTERS FOR CHEESE PRODUCTION, EJPAU 8(1), #10.
Available Online: http://www.ejpau.media.pl/volume8/issue1/art-10.html

GROWTH AND SPORULATION OF PENICILLIUM ROQUEFORTI STRAINS IN THE PRESENCE OF YEASTS SELECTED AS CO-STARTERS FOR CHEESE PRODUCTION

Piotr Juszczyk1, Maria Wojtatowicz2, Barbara Żarowska2, Józefa Chrzanowska1, Adam Malicki3
1 Faculty of Food Science, Agricultural University of Wrocław, Poland
2 Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Poland
3 Department of Food Hygiene and Consumer Safety, Agricultural University of Wrocław, Poland

 

ABSTRACT

The influence of yeasts (5 strains of Candida famata, 3 strains of C. sphaerica, 2 strains of C. kefyr, 2 strains of C. lipolytica) added to culture medium on growth and sporulation of 4 industrial strains of Penicillium roqueforti was examined. Mixed cultures of each strain of yeasts and mould were carried out under conditions mimicking cheese environment (cheese agar, temperature 14 °C, aw from 0.997 to 0.978 at 0% and 5% NaCl, respectively). Most of the yeasts examined slowed down the rate of vegetative growth of P. roqueforti strains, delayed the process of their sporulation (by 1-3 days) and lowered the conidia number / Petri dish by 1-10%. C. lipolytica strains, compared to representatives of other yeast species, exhibited the strongest negative influence on the growth and sporulation of P. roqueforti. However, this effect was significantly reduced in the presence of salt (5%). Interactions between the yeasts and P. roqueforti were strain dependant and affected by aw.

Key words: yeast starter cultures, Penicillium roqueforti, interaction, growth, sporulation.

INTRODUCTION

Positive aspect of yeast occurrence in cheeses, especially in soft and mould cheeses, is of interest of both researchers and cheese producers. In many research centers all over the world the role of yeasts in cheese ripening processes is examined and yeast strains most suitable for cheese making as potential co-starters are selected.

Two yeast species: Debaryomyces hansenii (and its anamorphic form, Candida famata) and Yarrowia lipolytica (anamorphic form - Candida lipolytica) are most commonly indicated as the best starter cultures for cheese making [Tempel and Jakobsen, 1998a; Tempel and Jakobsen,1998b; Cosentino et al., 2001; Czajgucka et al., 2003; Suzzi et al., 2003; Juszczyk et al., 2004]. Representatives of the first species are characterised by high tolerance of low temperature, high NaCl concentrations and ability to utilise lactose and lactic acid. Moreover, some of Debaryomyces hansenii strains reveal proteolytic and lipolytic activities, which are extremely high within Yarrowia lipolytica species.

Representatives of other yeast species: Candida sphaerica / Kluyveromyces marxianus ssp.lactis, C. kefyr / K. marxianus ssp. marxianus and Saccharomyces cerevisiae are also worth attention due to their physiological properties and enzymatic activities [Hansen and Jakobsen, 2001; Czajgucka et al., 2003; Juszczyk et al., 2004].

The use of some selected strains of Candida famata, C. lipolytica, C. sphaerica, C. kefyr and S. cerevisiae in the model cheeses production appeared to stimulate the growth of lactic bacteria, accelerate ripening process and improve the sensory quality of the final product [Guerzoni et al., 1998; Wyder et al., 1999; Bockelmann and Hoppe-Seyler, 2001; Hansen et al., 2001; Szołtysik et al., 2002a; Szołtysik et al., 2002b; Ferreira and Viljoen, 2003].

Besides biochemical abilities of yeasts as potential co-starters, their interactions with other primary and secondary cheese starter cultures should also be taken into consideration.

In blue-veined cheeses, Penicillium roqueforti fungi play an important role in sensory and rheological features development during ripening process [Contarini and Toppino,1995; Zarmpoutis et al., 1997]. Reduction of growth, metabolic activity and/or sporulation of P. roqueforti strains may result in poor taste and aroma of cheese [Cichosz, 1997]. Therefore, positive interactions between yeasts and industrial strains of Penicillium roqueforti are of importance.

The aim of the present study was to examine the effect of yeasts selected as potential cheese starter cultures on the growth and sporulation of industrial Penicillium roqueforti strains.

MATERIALS AND METHODS

Microorganisms: Twelve strains, representatives of four yeast species: Candida famata (AII4b, BI6a, EII2b, KI2a, MI1a), C. sphaerica (BI6c, BII1b, FII7a), C. kefyr (KI1a and PII1b) and C. lipolytica (PII6a, PII6c) were used in this study. Yeasts were isolated from blue veined Rokpol cheese and identified as described by Wojtatowicz et. al. 2001. The strains were maintained on YMPG-agar slopes [0.3 % Yeast Extract, Difco; 0.3 % Malt Extract, Difco; 0.5 % Bacto Peptone, Difco; 1.0 % Glucose, POCH, Poland; 1.5 % Agar-agar, Merck], at 4 °C.

Four strains of Penicillium roqueforti (P. roq PR 1, P. roq PR 2, P. roq PR 3, P. roq PR 4), applied in Rokpol cheese production, were included in interaction studies. The strains were maintained on slants of CYE-agar (Czapek Yeast Extract Agar, Difco), at 4 °C.

All yeast and fungal strains are kept in the microrganism collection of the Department of Biotechnology and Food Microbiology, Agricultural University of Wrocław.

Radial growth of P. roqueforti in the presence of yeast: Growth experiments were carried out on MSG-agar [3% Malt Extract, Difco; 0.3% Soy Peptone, Merck; 2% Glucose, POCH, Poland; 1.5% Agar-agar, Merck; pH 5.6]. The media were adjusted by adding of NaCl (0% and 5% w/v) and the corresponding water activities (aw), measured with Novasina Thermoconstanter (Novasina AG, Switzerland), were 0.997 and 0.962, respectively.

Inocula of yeasts (1*105 cells / mL) and P. roqeforti (1*105 conidia / mL) were prepared according to Tempel and Nielsen [2000]. Suspensions of each yeast strain tested (0.1 mL) were spread onto the surface of above media and allowed to dry for 3h. One point inoculation of Penicillium roqueforti spore suspension (0.01mL ) was made into each Petri dish. For control the fungal strains were inoculated into yeast-free plates. After 11 days incubation at 14 °C in the dark P. roqueforti colony diameters were measured manually. The data are shown as percentage of inhibition or stimulation of radial growth of P. roqueforti in yeasts presence. The examinations were carried out in triplicate and the mean values and standard deviations are reported.

Sporulation studies for P. roqueforti: Sporulation tests were performed on cheese agar, pH 5.6, prepared as described by Hansen and Jakobsen [1998]. The media were adjusted by adding of NaCl (0% and 5% w/v) and the corresponding water activities (aw) were 0.995 and 0.978, respectively. Yeast inocula and inocula of P. roqueforti conidia were prepared according to Hansen & Jakobsen [2001].

One mL of P. roqueforti spore suspension (1*106 conidia / mL) and 1 mL of yeast suspension (1*106 cells / mL) were mixed with 20 mL melted (45 °C) cheese agar and poured into Petri dishes. The plates were incubated at 14 °C for 11 days. During incubation, the plates were inspected daily and the time when blue-coloured P. roqueforti spores appeared was noted. Comparisons were made to agar plates containing P. roqueforti alone. For counting of conidia, the contents of one Petri dish were transferred to a stomacher sterile bag, 50 mL of SPO [0.1% Peptone, Difco; 0.85% NaCl; 0.03% Na2HPO4+2H2O; 1mL Tween 80, Merck; pH 7.0] was added and homogenised in a Stomacher (Lab Blender 400 Seward Medical, London UK) for 2 min. at high speed. For appropriate dilutions, the conidia were counted using Thom´s chamber. The data are shown as log of conidia number per plate and standard deviation of the mean value. The examinations were carried out in duplicate.

RESULTS

The effect of 12 strains, representatives of four yeast species: Candida famata, C. sphaerica, C. kefyr and C. lipolytica on growth of 4 industrial Penicillium roqueforti strains is shown in Fig. 1.

Fig. 1. Relative growth of four Penicillium roqueforti strains: PR 1 (■), PR 2 (),
PR 3 (), PR 4 (□) on MSG-agar at water activity 0.997 (A) and 0.962 (B) in presence of various Candida famata, C. sphaerica, C. kefyr and C. lipolytica strains.
The results are given as average values and SD of three replications

Generally, yeasts inhibited the vegetative growth of all P. roqueforti strains. Interactions between the microorganisms were strain dependant and were influenced by aw. The reduction of radial growth of P. roqueforti was the lowest, within 1-15%, in the presence of 3 out of 5 Candida famata strains (BI6a, EII2b, KI2a), 1 out of 3 C. sphaerica strains (BII1b) and 1 out of 2 C. kefyr strains (KI1a).

The influence of water activity was the strongest in common cultures of P. roqueforti and C. lipolytica. At aw = 0.997 (0% NaCl), both strains of C. lipolytica completely inhibited the development of substrate mycelium and conidia formation of P. roqueforti strains (Fig. 1A). This negative effect of C. lipolytica on P. roqueforti was reduced significantly when aw dropped down to aw = 0.962 at 5% NaCl. Fungi colonies relative diameter (as compared to control culture without yeast) reached the level of 57-78% (Fig. 1B).

Apart from the negative interactions between the microorganisms, growth stimulation of one of P. roqueforti strains (PR 1) by one of C. kefyr strains (PII1b) was observed. This, however, took place only in the medium without salt.

Penicillium roqueforti strains, growing on cheese agar at temp. 14 °C, formed reproductive mycelia in the 5-6 day of incubation when water activity was high (0.995), whereas at low aw (0.978), conidia appeared on the 7th day of cultivation (Table 1).

Table 1. Visible sporulation time (days) of four Penicillium roqueforti strains in the presence of Candida famata, C. sphaerica, C. kefyr, C. lipolytica strains in cheese agar at various water activity

Yeast strain

P. roq PR 1

P. roq PR 2

P. roq PR 3

P. roq PR 4

w a t e r                        a c t i v i t y

0.995

0.978

0.995

0.978

0.995

0.978

0.995

0.978

Control-without yeast

5

7

6

7

6

7

6

7

                 

C. famata AII4b

7

8

6

7

8

ns

9

9

C. famata BI6a

6

7

6

9

6

7

8

9

C. famata EII2b

6

7

6

9

8

7

8

9

C. famata KI2a

6

9

6

9

8

8

8

9

C. famata. MI1a

7

8

6

7

6

8

9

11

                 

C. sphaerica BI6c

7

8

6

7

8

8

ns

11

C. sphaerica BII1b

6

9

6

9

6

7

6

9

C. sphaerica FII7a

5

8

6

7

6

8

9

11

                 

C. kefyr KI1a

6

7

6

7

6

7

6

7

C. kefyr PII1b

5

8

6

7

6

8

9

9

                 

C. lipolytica PII6a

7

8

ns

9

ns

ns

ns

9

C. lipolytica PII6c

ns

7

ns

9

ns

8

ns

ns

ns - not visible sporulation during 11 days of incubation at 14 °C

In the presence of yeasts in the culture medium, mould sporulation process was delayed by, most often, 1-3 days, except for P. roqueforti PR 2 strain, which formed conidia in both control and inoculated with C. famata, C. sphaerica and C. kefyr strains media on the same day of incubation.

In a few cases green spores of P. roqueforti were either observed on the 11th day or they did not appear at all during cultivation period lasting 11 days. This was most often the case in mixed cultures of P. roqueforti and C. lipolytica, especially in the medium without NaCl (aw= 0.995). At lowered aw 0.978 (5% NaCl), the negative influence of the yeast on the process of fungi sporulation was significantly weaker.

The results presented in Table 2 show the influence of yeast presence on the number of conidia formed by P. roqueforti strains in cheese agar.

Table 2. Number of Penicillium roqueforti conidia in the presence of Candida famata, C. sphaerica, C. kefyr and C. lipolytica yeast strains in cheese agar at different water activitiy

Yeast strain

P. roq PR 1

P. roq PR 2

P. roq PR 3

P. roq PR 4

w a t e r                       a c t i v i t y

0.995

0.978

0.995

0.978

0.995

0.978

0.995

0.978

Control-without yeast

9.5+/-0.01

10.3+/-0.0

10.3+/-0.0

10.4+/-0.1

10.5+/-0.2

10.2+/-0.1

10.0+/-0.0

9.7+/-0.1

                 

C. famata AII4b

9.4+/-0.1

10.2+/-0.0

9.6+/-0.0

9.8+/-0.1

10.0+/-0.1

ns

10.5+/-0.0

9.9+/-0.0

C. famata BI6a

9.8+/-0.1

9.9+/-0.0

10.1+/-0.0

10.1+/-0.0

9.7+/-0.1

9.7+/-0.0

10.1+/-0.0

10.0+/-0.1

C. famata EII2b

9.6+/-0.0

9.9+/-0.0

9.7+/-0.1

9.5+/-0.0

9.9+/-0.1

9.6+/-0.0

10.0+/-0.0

9.7+/-0.1

C. famata KI2a

9.5+/-0.0

10.0+/-0.1

9.6+/-0.0

9.7+/-0.0

9.8+/-0.0

9.4+/-0.0

10.0+/-0.1

9.9+/-0.1

C. famata MI1a

9.3+/-0.0

9.8+/-0.1

9.4+/-0.0

9.7+/-0.0

9.8+/-0.1

10.3+/-0.0

9.8+/-0.0

9.8+/-0.0

                 

C. sphaerica BI6c

9.6+/-0.0

10.1+/-0.1

10.1+/-0.1

9.7+/-0.1

10.1+/-0.0

10.2+/-0.1

ns

10.7+/-0.0

C. sphaerica BII1b

9.5+/-0.0

10.0+/-0.1

10.0+/-0.0

9.8+/-0.1

10.1+/-0.1

9.7+/-0.0

9.7+/-0.0

9.6+/-0.0

C. sphaerica FII7a

9.7+/-0.1

10.4+/-0.0

9.8+/-0.1

10.3+/-0.0

9.9+/-0.1

10.1+/-0.1

10.5+/-0.1

9.9+/-0.0

                 

C. kefyr KI1a

10.1+/-0.0

10.0+/-0.0

10.1+/-0.1

10.0+/-0.0

10.2+/-0.0

9.9+/-0.0

10.2+/-0.0

9.7+/-0.0

C. kefyr PII1b

9.8+/-0.0

10.3+/-0.0

9.7+/-0.1

9.9+/-0.0

9.7+/-0.0

10.5+/-0.0

10.4+/-0.0

10.2+/-0.0

                 

C. lipolytica PII6a

9.7+/-0.0

10.2+/-0.0

ns

10.1+/-0.0

ns

ns

ns

9.8+/-0.3

C. lipolytica PII6c

ns

9.8+/-0.0

ns

9.4+/-0.0

ns

9.6+/-0.0

ns

ns

Note: Data are given as log conidia/plate and are the average of two replications +SD; ns - no spores

In control cultures (without yeast), P. roqueforti strains produced from 9.5 Log to 10.5 Log conidia per Petri dish. Water activity (0.995 and 0.978) did not affect significantly conidia count in the case of PR 2, PR 3 and PR 4 strains (the difference in the number of spores did not exceed 0.3 Log). Only PR 1 strain of P. roqueforti produced more spores (10.3 Log ) at lower water activity than at higher (9.5 Log).

The introduction of C. famata, C. sphaerica or C. kefyr cells into cheese agar did not significantly affect spore number produced by fungi. The difference in spore harvest between control and mixed culture only in a few cases reached 1.0 Log.

However, both C. lipolytica strains examined completely inhibited the sporulation process of P. roqueforti strains at high water activity up to 11 day of incubation. Moreover, no conidia were produced by P. roqueforti PR3 strain in presence of C. famata AII4b and by P. roqueforti PR4 strain in presence of C. sphaerica BI6c at water activity of 0.978 and 0.995, respectively (Table 2).

DISCUSSION

In production of blue veined cheese varieties, mesophilic lactic acid bacteria are used as primary starter cultures and filamentous fungi Penicillium roqueforti as secondary starters. Non-starter microflora also develops in these cheeses during ripening. Yeasts are a significant part of that microflora. They exhibit big variations in species and numbers [Tempel and Jakobsen, 1998b; Wojtatowicz et al., 2001; Chrzanowska et al., 2003]. Depending on the profile of yeast populations, they may positively or negatively affect the growth and activity of starter microflora and cheese quality. Selection of yeast strains to be used as co-starters in production of mould cheeses requires detailed studies on their interactions with industrial strains of Penicillium roqueforti.

The strains of C. famata, C. sphaerica, C.kefyr and C. lipolytica species examined in the present study had been selected as potential secondary starter cultures for cheese making based on their physiological properties and enzymatic abilities [Czajgucka et al., 2003; Juszczyk et al., 2004]. When used as co-starters in cheese production, they stimulated the growth of primary starter with lactic acid bacteria and accelerated the process of fat and proteins degradation during ripening [Szołtysik et al., 2002a; Szołtysik et al., 2002b].

The results of the present study show that in the presence of these yeasts the vegetative growth of four industrial P. roqueforti strains was restricted to a various degree. Under conditions mimicking cheese environment during ripening at 14 °C (cheese agar, water activity from 0.995 to 0.978, at NaCl concentration 0% and 5%, respectively), most of yeast strains tested also delayed the process of mould sporulation which, however, did not significantly affect the yield of spores.

The interactions between the microorganisms used in the study depended on the strain and water activity of environment. The results are in agreement with earlier observations of Tempel and Jakobsen [2000] who studied the interactions between D. hansenii and Y. lipolytica yeasts and industrial cultures of P. roqueforti.

The negative effect of yeasts on the development of filamentous fungi was most visible in case of the two C. lipolytica strains used in the study. The strains completely inhibited the development of both vegetative and reproductive mycelium of P. roqueforti, but only at high water activity. However, at low aw 0.978 in the presence of 5% NaCl, their effect on P. roqueforti strains was weaker. On the contrary, Tempel and Jakobsen [2000] noticed strong inhibition of P. roqueforti growth by Y. lipolytica strains in medium (cheese agar) supplemented with 4% NaCl.

It was suggested that the inhibition of P. roqueforti by yeasts may have resulted from competition for nutrients present in the medium [Tempel and Jakobsen, 2000; Tempel and Nielsen, 2000].

Competition for living space is another mechanism pointed out by Björnberg and Schnűrer [1993] and Petersson and Schnűrer [1998]. The authors observed a faster colonisation of medium by Pichia anomala yeast, which created a barrier for the development of P. roqueforti moulds. Similar observations were made in the present study. C. lipolytica strains, growing faster than filamentous fungi, covered all the surface of agar on Perti dish during the first 1-2 days and blocked the development of P. roqueforti. At 5% NaCl in the medium, the growth of C. lipolytica was slowed down, which enabled the development of P. roqueforti.

Additionally, Hansen and Jakobsen [1998], who observed strong inhibition of P. roqueforti by both, growing cells of Kluyveromyces marxianus ssp. lactis and post-culture liquids, suggested that the antagonism resulted from the ability of the yeasts to synthesise killer toxins.

No strong antagonistic activity of C. sphaerica (imperfect form of Kluyveromyces marxianus ssp. lactis) strains towards P. roqueforti was observed in the present study, although Wojtatowicz et al. [2002] showed that killer proteins are produced by strains of this species.

The positive influence of yeasts on industrial strains of P. roqueforti was rarely reported by other authors. Hansen and Jakobsen [2001] and Hansen et al. [2001] observed faster growth and more abundant sporulation of P. roqueforti, but only in the presence of Saccharomyces cerevisiae. In this work we observed that only one of four strains of P. roqueforti was stimulated by one of twelve strains of yeasts (C. kefyr PII1b). Moreover, some strains of C. famata, C. sphaerica and C. kefyr influenced the higher number of P. roqueforti spores. Tempel and Nielsen [2000] confirmed the positive influence of D. hansenii (perfect form C. famata) on the growth of industrial strain P. roqueforti only in modified atmosphere (25% carbon dioxide and 0.3% oxygen) imitating the conditions inside cheese.

The study confirmed that positive interactions between yeasts and Penicillium roqueforti should be taken into consideration in the development of yeast starter cultures to be used in production of mould cheeses.

CONCLUSIONS

  1. The interactions between examined yeasts and industrial strains of P. roqueforti were strain dependant and affected by water activity of environment.

  2. Yeast strains of C. famata, C. sphaerica, C. kefyr and C. lipolyitica inhibited at different degree the vegetative growth of all strains of P. roqueforti, delayed the process of their sporulation and lowered the conidia number.

  3. C. lipolytica strains had the most negative effect on P. roqueforti.

This research was supported by the State Committee for Scientific Research: project number: 5 PO6G 045 19.

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Piotr Juszczyk
Faculty of Food Science,
Agricultural University of Wrocław, Poland
C.K. Norwida 25, 50-375 Wrocław, Poland
email: juszczyk@ozi.ar.wroc.pl

Maria Wojtatowicz
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
C.K. Norwida 25, 50-375 Wrocław, Poland
Fax. 4871- 3284124
Phone: 48-71-3205117
email: mwojt@wnoz.ar.wroc.pl

Barbara Żarowska
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
C.K. Norwida 25, 50-375 Wrocław, Poland
email: zarowska@ozi.ar.wroc.pl

Józefa Chrzanowska
Faculty of Food Science,
Agricultural University of Wrocław, Poland
C.K. Norwida 25/27, 50-375 Wrocław, Poland
email: jch@ozi.ar.wroc.pl

Adam Malicki
Department of Food Hygiene and Consumer Safety,
Agricultural University of Wrocław, Poland
C.K. Norwida 31, 50-375 Wrocław, Poland
tel. (0-71) 320 53 99
email: malicki@ozi.ar.wroc.pl

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