Influence of modified atmosphere and preservatives on the growth of Zygosaccharomyces rouxii isolated from dried fruits

International

Journal

of

Food Microbiology 33 (1996) 219-229

Influence of modified atmosphere and preservatives on the growth of Zygosaccharomyces rouxii isolated from dried fruits A. El HalouaP,

J.M. Debevereb,*

“Department of’ Food Science, Ecole Nationale dilgriculture, Meknes, Morocco bDepartment of Food Science and Nutrition, Faculty qf’ Agricultural and Applied Biological Sciences, University of‘ Ghent, Coupure Links 653, 9000 Ghent, Belgium Received

22 August

1995; revised

26 March

1996; accepted

28 July 1996

Abstract The influence of water activity (a,), preservatives, modified atmosphere and their combinations on the growth of Z. rouxii was determined by cultivating two strains isolated from raisins and prunes in culture media under different conditions and by counting the colony forming units. Yeast extract glucose broths or agars were adjusted to the desired a, by means of glucose. Preservatives added to the media (O-600 ppm) were either K-sorbate, Na-benzoate or their mixture. Modified atmospheres were carried out by packing culture plates or flasks in plastic bags under different CO,-N, gas mixture. Response surface design was carried out to optimize the growth inhibition of Z. rouxii by the mentioned factors. Although Z. rouxii is osmotolerant, the strains studied could not grow at a, 0.79. They also showed a high tolerance to CO,; even 80% CO, seems to not inhibit growth. However, CO, atmosphere at high pHs and low preservative concentrations stimulated yeast growth. At pH 4.0 and under modified atmosphere (80% CO,-20% NJ, no growth was observed at any a, in the range of 0.80-0.90 when using a preservative concentration of 220 ppm Ksorbate or 280 ppm Na-benzoate Keywords:

K-sorbate;

* Corresponding

016%1605/96/$15.00

Modified

author.

Tel.:

atmosphere;

+ 32 9 2646164;

Na-benzoate;

fax:

+ 32 9 2252510.

0 1996 El sevier Science B.V. All rights

PII SO168-1605(96)01158-O

Water

reserved

activity

220

A. El Halouat, J.M. Debevere / ht. J. Food Microbiology 33 (1996) 219-229

1. Introduction

yeasts which comprise Zygosaccharomyces species (2. rouxii, are the main spoilage organisms in foods with low water activity (a,) such as dried fruits (Nury et al., 1960). Contamination of these foods could originate from inadequate sanitation during processing, packaging and/or in storage facilities. The spoiled product is characterized by a strong alcoholic odour and swelled package due to CO, production. Walter (1977) reported that Z. rouxii; the most common spoilage organism of the osmotolerant yeasts, causes spoilage of products of low a, (0.80-0.85). This yeast can tolerate, in the culture medium, concentrations of up to 22% NaCl and 80% glucose or sucrose (Onishi, 1963). The role of carbon dioxide in affecting yeast metabolism is not clear, although inhibition is generally found at high concentrations. If the effects of CO, upon the membrane lipids appear to be the most likely causes of the growth inhibition of yeasts, a combination of both membrane and metabolic effects may be the cause for other microorganisms (Jones and Greenfield, 1982). This interaction of CO, with lipids of the cell membrane alters the membrane properties by decreasing the ability of the cell to take up various anions (Sears and Eisenberg, 1961), as well as by disturbing the activities of the membrane and cytoplasm enzymes (Jones and Greenfield, 1982). Thus, the membrane disruptive function of carbon dioxide appears to be very important in yeast inhibition. Another inhibiting effect of CO, could be the dissipation of energy resulting from the active transport of the protons from the cell to maintain its internal pH (Dixon et al., 1988). Zygosaccharomyces species are high preservative tolerant yeasts. This tolerance to weak acid preservatives has been reported (Moon, 1983; Suihko and Mackinen, 1984). According to their effectiveness, the choice of weak acid preservatives is restricted mainly to sorbic and benzoic acids and their salts (Bills et al., 1982; Restaino et al., 1982). Preservatives are more effective in an acidic than in a neutral media. Lowering the pH of the medium increases the proportion of the undissociated acid molecules and thus increases its inhibition effectiveness (Debevere, 1987; Jermini and Schmidt-Lorenz, 1987). In low pH environment, weak acids enter the yeast cells in undissociated form. The dissociation occuring inside the cell produces an acidification and accumulation of the preservative anion (Pampulha and Loureiro-Dias, 1989). The undissociated form of the acids, according to their mechanisms, has an important role in the yeast inhibition process. However, Eklund (1983) and Moon (1983) demonstrated some inhibitory capacity of the anions for sorbic and propionic acids. At low pH, the preservative anion concentration increases in the cytoplasm and consequently a transport system is induced to remove both H-ions and preservative anions from the cell. Warth (1988) attributed Osmotolerant

Z.

bailii,

Z.

bisporus)

A. El Halouat, J.M.

Debevere /ht.

J. Food Microbiology 33 (1996) 219-229

221

the reduction in the growth rate of yeasts exposed to preservative to the expenditure of the energy which is used to reduce the cell preservative concentration and to maintain cellular pH rather than to be available for growth. Warth (1991) reported that the differences in resistance among the species could be due to differences in the rate of penetration of the cell by the preservative, the capacity of the system to remove this preservative, or an intrinsic sensitivity to preservative or to its anion. The optimum use of yeast inhibiting factors such as low pH, low a,, CO, atmosphere and weak acid preservatives could improve their efficacy. Despite this, there are no studies available on the combined action of these factors on the yeast inhibitory effect. The purpose of these investigations was to determine, in culture media with low a, and low pH, the effects of CO,, K-sorbate and Na-benzoate individually and in combination on the growth of Z. rouxii.

2. Material and methods 2.1. Organisms

Two strains of Z. rouxii, isolated from Moroccan raisins (Z. rouxii (R)) and prunes (Z. rouxii, (P)) and identified by Dr. Samson (Centralbureau voor Schimmelcultures, Baarn, The Netherlands), were used in these investigations. Stock cultures were maintained on slants of yeast extract agar supplemented with 40% (w/w) D( + )-glucose (YEG40 agar) and stored at 4°C. They were transferred every 5 months. 2.2. Preparation of media and diluent 2.2.1. Growth media

Either yeast extract glucose (YEG) broth or (YEG) agar media were used for Z. Different yeast extract glucose broths or agars; YEG30, YEG40, YEG45, YEGSO, YEG55, YEG57, YEG60, YEG62, YEG65, YEG67, and YEG70 were prepared by dissolving 30, 40, 45, 50, 55, 57, 60, 62, 65, 67 and 70% (w/w) D( + ) glucose (Sigma, St Louis, USA), respectively, in 0.5% (w/w) aqueous solution of yeast extract (Amersham, Bury, England), alone for broth media and containing 1.5% (w/w) agar (Amersham, Bury, England) for agar media. After autoclaving and pH adjustment, the a, values of the media were measured at 30°C (Table 1). rouxii growth.

2.2.2. Plating media and diluent Medium and diluent prepared for yeast enumeration were supplemented with sucrose to reduce osmotic shock. Yeast extract sucrose (YES 40) agar (u, 0.96, pH 6.75) was prepared by dissolving 40% (w/w) sucrose and 1.5% (w/w) agar in 0.5% (w/w) aqueous solution of yeast extract. Diluent solution (DS 20) (a, 0.98, pH 6.97) was prepared by dissolving 20% (w/w) sucrose, 0.1% (w/w) peptone (Oxoid, Unipath LTD, Hampshire, England) and 0.85% (w/w) NaCl (UCB, Leuven, Belgium) in distilled water.

222

A. El Halouai, J.M. Debevere /ht.

J. Food Microbiology 33 (1996) 219-229

2.3. Physical methods 2.3.1. pH-adjustment

After autoclaving, the pH of the culture media containing glucose (Table 1) was adjusted with 1 N HCl (UCB, Leuven, Belgium) using a Knick pH-meter (type 763, Knick, Berlin, Germany) with Ingold electrode (MG-DXK57, Urdorf, SZ). 2.3.2. Sterilization of media and solutions All media and diluent were sterilized by autoclaving at 121°C for 10 min. HCl (1 N), and preservative solutions: 1000 ppm K-sorbate or Na-benzoate (Merck, Darmstadt, Germany) were sterilized by membrane filtration (Disposable sterile bottle top filter, 0.22 pm, Corning). 2.3.3. Water activity (a,,.) measurement After pH adjustment, the (a,) values were taken at 30°C with a hydrometer (Thermoconstanter Humidat-TH2, Novasina Defensor AG-Plaffikon, S.Z.). The calibration of the apparatus was done frequently at 25°C by using three salts of known a,, delivered by the instrument company. 2.3.4. Gas packaging Immediately after inoculation, the plates (agar) or flasks (broth) were packed in plastic bags (Sidamil UCB Belgium; permeability: 6 ml O,, 15 ml CO, and 2 ml N, per m* in 24 h at 25°C and 100% RH). The bags were filled with the desired mixture of CO, and N, gases (Air Products, Belgium) in the ratio product/gas mixture of l/3 (v/v). The gas packaging was done in Multivac (Haggemiiller K.G. Germany) by creating a vacuum in the bag followed by flushing the gas mixture at one bar pressure before heat sealing. The gas packed inoculated media were stored at 30°C. 2.3.5. Gas analysis The gas atmosphere in the head space of the bags was analysed using a Servomex Food Package O&O, analyser (Serie 1402 Servomex, Sussex, UK).

Table 1 Water activity (a,) at 30°C of different yeast extract media containing increasing glucose concentrations Yeast Extract Glucose (YEG) Glucose (‘XI)

30

40

45

50

55

57

60

62

65

67

70

Broth (a,) Agar (a,)

0.96

0.94 0.93

0.92 0.92

0.90 0.90

0.88 0.87

0.85 -

0.84 0.83

0.82 0.82

0.80 0.79

0.79 -

0.76 0.74

A. El Halouat, J.M. Debevere /ht.

J. Food Microbiology 33 (1996) 219-229

223

2.4. Microbiological methods 2.4.1. Preparation of the inocula YEG40 broth was inoculated with stock yeast culture on an agar slant (YEG40 agar) and incubated at 30°C for 3 days. The yeast inocula were transferred to a new culture broth (YEG40) and incubated for 4 days at 30°C to reach lo6 cfu of yeast per ml which was used for experiment inoculation. 2.4.2. Determination of growth at dgferent a, values To find minimum a, values for growth of 2. rouxii, each strain (2. rouxii(R) or Z. rouxii(P)) was inoculated in 60 ml yeast extract glucose broths with pH 4.0 and 5.0 and with different a, in the range of 0.76 to 0.96 and incubated at 30°C. Counts (cfu/ml) were enumerated at 4 and 8 days. The samples showing no growth were checked for yeast growth after 21 day incubation. The inoculum contained approximately lo4 cfu/ml. 2.4.3. Determination of growth under CO,-atmosphere To study the behavior of the yeast under CO,-atmospheres, Z. rouxii(R) strain was investigated. The 250 ml Fernbach flasks containing 60 ml YEG broths with different a, values 0.84, 0.90 and 0.94 were inoculated with approximately lo2 cfu/ml. The flasks were put in plastic bags which were filled with the desired mixture of CO2 and N,. As controls, broth samples with the same a, values as the packed flasks were inoculated in the same conditions and exposed only to air. All samples were incubated at 30°C for a maximum of 24 days. Every 4 days, samples from each combination were removed to determine yeast counts. 2.4.4. Determination of growth using CO,/preservative combinations The yeast growth was studied in YEG45 agar (a, 0.92) having 3 different pHs; 5.8, 5.0 and 4.0 in combination with 10 different concentrations (0, 50, 100, 150, 200, 250, 300, 400, 500, and 600 ppm) of K-sorbate, Na-benzoate or their mixture (l-l). The plates containing 20 g of medium were inoculated with approximately lo3 cfu/g and incubated at 30°C for 21 days in air and under CO,-atmosphere (80% CO*-20% N2). 2.4.5. Response surface methodology investigations A response surface design experiment (Optimization Software version 5.0 int’L Qual-Tech. Minnesota, USA) was used to assess the effect of culture a, values (0.80-0.90) and preservative concentrations (O-300 ppm K-sorbate or O-400 ppm Na-benzoate), as well as CO,-atmospheres (O-80% CO,) at pH 4. The 20 indicated combinations given by the program for three independent variables were prepared using YEG agar media with the desired a, and preservative concentration. The plates were inoculated with approximately lo3 cfu/g agar and incubated at 30°C for 21 days. The experiment was carried out with both Z. rouxii strains.

224

A. El Halouat, J.M.

Debevere /ht.

J. Food Microbiology 33 (1996) 219-229

Table 2 Effects of a,,, levels on the growth (log, cfu/ml) of 2. rouxii(R) in yeast extract glucose broths at pH 4.0 and 5.0 during 4 and 8 day incubation periods” a, (30°C)

0.96 0.94 0.90 0.88 0.84 0.80 0.79 0.76

pH 5.0

pH 4.0 4 days

8 days

4 days

8 days

7.30 8.15 7.60 7.00 5.70 5.48 ngb ng

7.00 7.60 7.70 7.30 6.02 5.30 ng ng

7.60 7.60 7.60 7.30 5.65 5.30 ng ng

7.48 7.70 8.00 7.00 6.65 5.00 ng ng

“Initial count lo4 cfu/ml. bNo growth at 4, 8 and 21 days of incubation

2.4.6. Enumeration procedures The 20 g of 2. rouxii culture

agar contained in each plate were removed aseptically and homogenized for 1 min in 180 ml of DS20 using a Colworth stomacher. From this homogenate or from broth growth media after yeast clumps dispersion by shaking, 1 ml volume was pipetted for decimal dilution series in the diluent DS20 and plated in duplicate on yeast extract sucrose YES40 agar. Plates were incubated at 30°C for a minimum of 5 days. Counts were transformed to log,, cfu/ml of broth or g of agar.

3. Results and discussion 3.1. Growth of Z. rouxii at different a, values The results of the a, investigation (Table 2) confirmed the finding that Z. rouxii is a high osmotolerant yeast (Bills et al., 1982). In contrast to previous studies which extended the a, tolerance limits of Z. rouxii to the value of 0.760 (Anand and Brown, 1968 and Jermini and Schmidt-Lorenz, 1987) and which found the minimum a, for Z. rouxii growth in sucrose-glycerol broths between 0.62 and 0.64 (Tapia De Daza et al., 1995), the strains tested did not grow at a, 0.79 and lower within the incubation time (21 days) used in this study, either at pH 4.0 or at pH 5.0. The a, range (0.95-0.96), reported to be the optimum for Z. rouxii growth (Bills et al., 1982), could be much wider according to the present results since the growth was very high between a, 0.96-0.88. As reported by Restaino et al. (1983), these disagreements could be due to different experimental conditions including the type of solute used to reduce a, of growth media, as well as slight genetic variations within the species.

A. El Halouat, J.M.

Debevere /ht.

225

J. Food Microbiology 33 (1996) 219-229

3.2. Growth of Z. rouxii under CO,-atmospheres

Several investigations with both Z. rouxii strains (unpublished data) showed their high tolerance to CO, conditions. In the present experiment, using Z. rouxii(R) strain in YEG broths with pH 4.0 (Table 3), the final count in all CO, combinations was higher than the inoculum (lo2 cfu/ml). After 24 days incubation, no significant difference in the yeast count was detected between different CO, combinations and the control (air) at any a, value tested. Although after 4 days of incubation at a, 0.94, the growth count under CO, conditions were higher than in the control. Oura et al. (1980) reported that CO, stimulates yeast growth. Lowering the a, of the test media to 0.83 was more effective in delaying the appearance of the yeast growth in the presence of CO, and, consequently, increasing the lag phase than in influencing the final growth count. 3.3. Effects of CO,, preservatives and pH Analysis of variance results indicated a significant CO, x preservative x pH level interaction (p < 0.05). This investigation was conducted using growth media with a, reduced to 0.92. Restaino et al. (1983) reported that Z. rouxii displayed maximal resistance to sorbate at a, 0.92. The results, in Table 4, showed that the preservatives either under air or modified atmospheres have been, in general, more effective Table 3 Growth (log,,,, cfu/ml) of 2. rouxii(R) strain within the CO, range of 20-80% (v/v) in yeast extract glucose broths with pH 4.0 and different a, during incubation for up to 24 days at 30°C” a, (3OOC)

co,

(%)b

Incubation

period

(days)

4

8

12

16

20

24

0.94

Air 20 40 60 80

5.98 7.12 7.04 6.97 7.04

7.95 6.95 6.97 7.00 6.78

6.40 6.95 6.17 6.57 6.95

6.34 7.23 6.57 4.97 6.57

5.56 6.74 6.04 6.11 5.99

5.51 5.69 5.62 5.60 5.98

0.90

Air 20 40 60 80

5.81 5.89 5.83 5.96 5.36

6.90 6.83 6.61 6.20 6.81

6.49 7.08 6.51 6.57 6.76

6.20 7.04 6.77 6.18 6.85

6.26 6.75 6.46 6.84 6.45

6.28 6.75 6.54 6.08 6.64

0.83

Air 20 40 60 80

3.54 2.10 2.11 2.00 1.90

6.63 4.90 5.42 4.86 4.43

7.30 6.40 6.70 6.89 6.80

7.18 6.83 6.54 6.70 6.65

5.74 6.48 6.54 6.51 5.95

6.96 6.86 6.68 6.36 6.53

used

as modified

“Initial count (loo-X)0/,)

IO* cfu/ml.bMixtures

of CO*-N,

gases

atmospheres

(CO,:X%,

N,:

A. El Halouat, J.M. Debevere 1 ht. J. Food Microbiology 33 (1996) 219-229

226

Table 4 Effects of CO,, preservatives, and pH on growth (log,,, extract glucose (YEG45), incubated at 30°C for 21 days” PH

Preserv.b

Concen.

(ppm)

K-sorbate

cfu/g

agar)

of Z. rouxii(R) strain

in yeast

Na-benzoate

Sorbate + Benzoate

(ppm)

Air

80% COC,

Air

80% CO,

Air

80% co,

5.8

0 50 100 150 200 250 300 400 500 600

6.65 6.33 4.78 5.48 5.00 4.88 5.74 6.11 5.93 6.40

8.42 7.85 6.81 6.90 6.46 6.22 6.18 6.04 5.95 5.23

6.65 5.93 5.93 5.88 5.81 5.88 6.49 7.15 7.13 7.00

8.42 6.57 6.30 6.51 5.81 6.02 5.88 6.42 6.29 5.58

6.65 6.04 6.42 5.70 6.04 6.36 6.61 6.81 6.78 5.78

8.42 6.59 7.24 6.56 6.28 6.02 6.13 6.34 6.22 5.45

5.0

0 50 100 150 200 250 300 400 500 600

7.40 7.56 7.57 7.60 7.60 7.74 7.98 5.63 5.42 4.94

7.04 7.42 6.54 6.88 6.38 6.90 2.95 2.78

7.40 7.60 7.45 7.34 7.46 6.93 6.60 5.18 5.02 4.10

7.04 7.30 6.85 7.33 7.32 6.57 6.32 5.93 5.35 4.74

7.40 6.78 7.48 7.40 7.39 7.08 6.56 6.22 6.00 5.95

7.04 7.31 6.53 5.78 6.26 6.29 6.65 5.60 5.30 2.78

0 50 100 150 200 250 300 400 500 600

6.65 6.99 7.93 7.22 6.90 6.42 4.47 2.57

6.22 6.57 6.41 5.64 3.04 ng ng ng ng

6.22 6.34 6.08 6.15 6.15 4.95 4.85 2.50 2.04 2.30

6.65 6.28 6.58 5.95 6.13 6.00 4.88 3.00 3.90 1.74

6.22 6.62 6.10 6.24 6.08 5.16 2.90 1.70

ng ng

6.65 7.08 6.93 7.04 6.42 6.35 6.32 6.26 4.81 4.35

4.0

“Initial count 10s cfu/g agar. bMedia concentration of salt preservative ‘CO, atmosphere: 80% CO,+20% N,.

1.98 2.30

1.40

added

rather

than equivalent

ng ng

acid.

at low pHs. The treatments showed significant differences in the responses to K-sorbate, Na-benzoate and their mixture in both atmospheres (air and CO,) and at each preservative concentration tested. In air conditions, the 2. rouxii (R) strain was found to be resistant to Nabenzoate. This strain tolerates 600 ppm of preservative at pH 5.8, pH 5.0 and even at pH 4.0. While under modified atmosphere (80% CO,-20% NJ and at pH 4.0, only 400 ppm Na-benzoate was needed to inhibit growth. The high resistance of 2. rouxii to Na-benzoate was reported by Marco et al. (1987).

221

A. El Halouat, J.M. Debevere 1Int. J. Food Microbiology 33 (1996) 219-229

In comparing data (Table 4), high levels of K-sorbate were much more effective than the same levels of Na-benzoate or the mixture, especially at pH 4.0 either in air or under CO, atmosphere (80% CO,-20% N2). As reported by Liick (1980), an additive effect in inhibiting 2. rouxii was observed at low pH with the combination of K-sorbate and Na-benzoate. At high pH and low preservative concentrations; up to 300, 250 or 200 ppm of K-sorbate, Na-benzoate or their mixture, respectively, the CO, seems to stimulate Z. rouxii growth. However, at low pH, the effectiveness of any preservative was influenced by 80% CO,-20% N,. At pH 4.0 and after 21 days of incubation under CO, condition, the final Z. rouxii(R) counts were much lower than inoculum count (lo3 CFU/g) in test media containing 250, 400 or 300 ppm K-sorbate, Na-benzoate or mixture, respectively. Thus, at low pH when used in combination with 80% CO,-20% N,, antimicrobial agents such as K-sorbate and Na-benzoate can affect significally the growth of Z. rouxii. 3.4. Optimization

of

Z. rouxii growth inhibition factors

In the optimization experiment, both strains of Z. rouxii were tested in YEG agar media at pH 4 using a,, CO, and preservative as variable factors. The inhibition of Z. rouxii growth depends upon complex interations between all factors, as well as upon individual effects of each factor. Model equations estimating these effects on Z. rouxii growth are shown in Table 5. Since both experimented strains exhibited a similar trend, only the results connected with the experiment carried out with Z. rouxii(R) strain were presented in Fig. 1. Table 5 Model equations

of Z. rouxii response

surface

study

Strain

Preservative

Model

equations

R A I s I N

K-sorbate

Log(cfu/ml) = -232.63+532.07A-294.36A”-0.24ABf0.22B -4.0610-4B2-2.5310-2C

Na-benzoate

Log(cfu/ml) = -220.69+507.75A-280.82A’-4.9410 +2.4810-3B-7.1010~4B2+1.3910~4BD

P R U N E s

K-sorbate

Log(cfu/ml)= -245.90+562.12A-312.05A’-0.18AB+O.l5B -2.1510-SB2-2.1810~*C

Na-benzoate

Log(cfu/ml) = +86.43-220.70A+ 149.66A’+0.24AB-9.41 -0.22B-5.3410-4B-2-l.6010-4BD+5.7810-2D

A: B: C: D:

a,. CO1 (%I) D: Na-benzoate K-sorbate (ppm). Na-benzoate (ppm).

(ppm).

AD

IO-’

AD

228

A. El Halouat.

J.M. Debevere / Inr. J. Food Microbiology

33 (1996) 219-229

Fig. 1. Response surface contour plots Z. rouxii (R) strain growth in yeast extract glucose agar at pH 4, different a,, different preservative and CO* concentrations, and incubating at 30°C for 21 days. Initial count: lo3 cells/g agar.

The growth was reduced with reducing media a,, increasing preservative concentration and/or incubating the cultures under high COz concentrations ( > 50%). The curves (Fig. 1) confirmed, as reported by Oura et al. (1980) and observed before in this study, that low concentrations of CO2 tend to stimulate yeast growth. The results showed also a synergistic effect when inhibiting factors were used in combination; with reducing a, the inhibiting effect of preservatives and CO, mainly increases. Bills et al. (1982) reported that the toxicity of sorbic acid is determined by the a, value; the further the a, value is from the osmotic optimum of a yeast the more effective is the preservative. Under 80% CO, atmosphere, the inhibition amounts of preservative were reduced by 40 to 50%. Under this atmosphere and at any a, value in the studied range (O.SO-0.90) approximately 150 ppm Ksorbate or 200 ppm Na-benzoate were only needed to get the final Z. rouxii count, after 21 days, lower than the inoculum count (lo3 cfu/g). While, no growth was observed when the preservative concentration was increased to 220 ppm K-sorbate or 280 ppm Na-benzoate.

References Anand, J.C. and Brown, A.D. (1968) Growth rate patterns of so-called osrnophilic and non osmophilic yeasts in solutions of polyethylene glycol. J. Gen. Microbial. 52, 2055212. Bills, S., Restaino, L. and Lenovich, L.M. (1982) Growth response of an osmotole-rant sorbate-resistant yeast, Saccharomyces roux& at different sucrose and sorbate levels. J. Food Protect. 45, 1120- 1124.

A. El Halouat, J.M.

Debevere 1 Int. J. Food Microbiology 33 (1996) 219-229

229

Debevere, J.M. (1987) The use of buffered acidulant systems to improve the microbiological stability of acid foods. Food Microbial. 4, 105-I 13. Dixon, N.M., Lovitt, R.W., Morris, J.G. and Kell, D.B. (1988) Growth energetics of Clostridium sporogenes NCIB 8053 in defined media. J. Appl. Bacterial. 63, 171-l 82. Eklund, T. (1983) The antimicrobial effect of dissociated and undissociated sorbic acid at different pH levels. J. Appl. Bacterial. 54, 383-389. Jermini, M.F. and Schmidt-Lorenz, W. (1987) Growth of osmotolerant yeasts at different water activity values. J. Food Protect. 50, 4044410. Jones, R.P. and Greenfield, P.F. (1982) Review. Effect of carbon dioxide on yeast growth and fermentation. Enzyme. Microb. Technol. 4, 210-223. Luck, E. (1980) Antimicrobial Food Additives. Springer-Verlag, New York, pp. 9833990. Marco, F., Jermini, M. and Schmidt-Lorenz, W. (1987) Activity of Na-benzoate and Ethyl-Paraben against osmotolerant yeasts at different water activity values. J. Food Protect. 50, 920-927. Moon, N. (1983) Inhibition of growth of acid tolerant yeasts by acetate, lactate, and propionate and their synergistic mixtures. J. Appl. Bacterial. 55, 453-460. Nury, F.S., Miller, M.W. and Brekke, J.E. (1960) Preservative effect of some antimicrobial agents on high moisture dried fruits. Food Technol. 14, 113- 115. Onishi, H. (1963) Osmophilic yeasts. Adv. Food Res. 12, 53394. Oura, E., Haarasilta, S. and Londesborough, J.J. (1980) Carbon dioxide fixation by Baker’s yeast in variety of growth conditions. J. Gen. Microbial. 118, 51-58. Pampulha, M.E. and Loureiro-Dias, M.C. (1989) Combined effect of acetic acid, pH and ethanol on intracellular pH of fermenting yeasts. Appl. Microbial. Biotechnol. 31, 547-550. Restaino, L., Bills, S., Tsherneff, K. and Lenovich, L. (1983) Growth characteristics of Succharomyces rouuii isolated from chocolate syrup. Appl. Environ. Microbial. 45, 1614&1621. Restaino, L., Lenovich, L.M. and Bills, S. (1982) Effect of acids and sorbate combinations on the growth of four osmophilic yeasts, J. Food Protect, 45, 1138-1142. Sears. D.F. and Eisenberg, R.M. (1961) A model representing the physiological role of COZ, at the cell membrane. J. Gen. Physiol. 44, 8699887. Suihko, M.L. and Mackinen, V. (1984) Tolerance of acetate, propionate and sorbate by Saccharomyces crrevisiue and Torulopsis holmii. Food Microbial. 1. 10% 1 IO. Tapia De Daza, M.S., Aguilar, C.E., Roa, V. and Diaz De Tablante, R.V. (1995) Combined effects on growth of Zygosaccharomyce:ces roux-ii from an intermediate moisture papaya product. J. Food Sci. 60(2), 3566359. Walter, H.W. (1977) Spoilage of food by yeasts. Food Technol. 31, 57761. Wdrth, (1991) Effect of benzoic acid and glycolytic metabolite levels and intracellular pH in Saccharomyces cerevisiae. Appl. Environ. Microbial. 57, 3415-3417. Warth, (1988) Effect of benzoic acid on growth yield of yeasts differing in their resistance to preservatives. Appl. Environ, Microbial. 54, 2091-2095.