The effect of pH, sodium chloride, sucrose, sorbate and benzoate on the growth of food spoilage yeasts

Food Microbiology, 1997, 14, 459–468

ORIGINAL ARTICLE

The effect of pH, sodium chloride, sucrose, sorbate and benzoate on the growth of food spoilage yeasts W. Praphailong and G. H. Fleet* The effects of pH, concentration of NaCl, concentration of sucrose and concentrations of sorbic and benzoic acids on growth were examined for 30 strains of food spoilage yeasts, representing Debaryomyces hansenii, Yarrowia lipolytica, Kloeckera apiculata, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Kluyveromyces marxianus, Pichia membranaefaciens, Pichia anomala and Saccharomyces cerevisiae. Zygosaccharomyces bailii did not grow at pH 7·0 and Z. rouxii, Kl. apiculata and P. membranaefaciens did not grow at pH 8·0. Only Kl. apiculata grew at pH 1·5–2·0. The remaining species grew at pH 2·5–8·0. None of the species grew at 20% NaCl but strains of D. hansenii, Z. rouxii and P. anomala grew at 15% NaCl. S. cerevisiae and K. marxianus were the least salt tolerant, showing no growth at 7·5% NaCl and 10% NaCl, respectively. Medium pH influenced growth in the presence of NaCl. For Y. lipolytica, D. hansenii and S. cerevisiae, greatest tolerance of NaCl occurred at pH 5·0–7·0, but for Kl. apiculata, D. membranaefaciens and Z. bailii best tolerance occurred at pH 3·0. K. apiculata grew in the presence of 12·5% NaCl at pH 2·0. All yeasts grew at 50% sucrose, with Z. rouxii, Z. bailii and P. anomala and D. hansenii growing at 60–70% sucrose. Medium pH in the range 2·0–7·0 had little effect on ability to grow in the presence of high sucrose concentrations. Z. bailii and Y. lipolytica were the species most tolerant of sorbate and benzoate preservatives (750–1200 mg l−1) at pH 5·0.  1997 Academic Press Limited

Introduction Yeasts are significant as spoilage microorganisms, especially in foods of low pH, high sugar content, high salt content and in those containing sorbate or benzoate preservatives (Deak 1991, Fleet 1992, Tudor and Board 1993). Many environmental factors affect the growth of yeasts but the response to any particular condition varies with the species (Rose 1987, Fleet 1992). Thus, Zygosaccharomyces bailii is well known for its resistance to sorbate and benzoate preservatives (Thomas and Davenport 1985), Debaryomyces hansenii *Corresponding author. 0740-0020/97/050459+10 $25·00/0/fd970106

grows in the presence of high concentrations of sodium chloride (Seiler and Busse 1990, Besanc¸on et al. 1992) and Zygosaccharomyces rouxii is recognized for its tolerance of high concentrations of sugars (Tokuoka 1993). For many yeasts, however, the environmental conditions that limit their growth have not been systematically examined. The growth response to any one condition is also determined by interaction with other environmental factors (Deak and Beuchat 1993, 1994). Environmental pH is particularly significant in determining the growth of yeasts in the presence of weak organic acids (Pitt 1974, Cole and Keenan 1986) and it may also affect their responses to high concentrations of salt

Received: 9 July 1996 Department of Food Science and Technology, The University of New South Wales, Sydney, New South Wales 2052, Australia

 1997 Academic Press Limited

460 W. Praphailong and G. H. Fleet

or sugar (Tokuoka 1993) although such interactions have not been examined in depth. By recording the growth or non-growth responses in the wells of a microtitre tray, this paper reports the effects of pH, concentration of salt (NaCl), concentration of sugar (sucrose) and concentration of preservatives (benzoic and sorbic acids) upon the growth of nine species (30 strains) of food spoilage yeasts. The interactive effects of pH with NaCl, sucrose, benzoate and sorbate on the growth response are also reported.

Materials and Methods Yeast species Thirty yeast strains, representing nine different species, were obtained from the culture collections of the Department of Food Science and Technology, The University of New South Wales, Sydney and the Division of Food Science and Technology, CSIRO, North Ryde, Sydney. The identity of each strain was confirmed by the method of Heard and Fleet (1990) and with the ATB 32C system (BioMerieux, France). The yeasts were maintained on slants of malt extract agar (Oxoid, Australia) and were freshly subcultured and checked for purity before use in experiments.

Effect of environmental variables on growth The presence or absence of yeast growth in response to environmental conditions was determined in 96-welled microtitre trays (Disposable Products, Australia). Medium (0·3 ml) was aseptically dispensed into the wells of the tray and each well was inoculated with 0·04 ml of yeast suspension. The trays were incubated at 25°C for 7–14 days and growth was recorded visually as the presence or absence of turbidity in the wells. Inoculum cultures (10 ml) were grown in tubes of yeast nitrogen base (NB; Difco, U.S.A.) with 5% glucose for 24 h at 25°C on a roller drum operating at 100 rev min−1, then diluted in 0·1% peptone water to give a suspension of 102–103 cfu ml−1 for inoculation. Experiments were repeated at least two to three times.

Unless indicated otherwise, YNB+5% glucose was used as the basal medium for growth. In some experiments, glucose was used at 0·5% instead of 5%, and yeast extract (0·5%) (Oxoid, Australia) was used in place of YNB. To examine the effect of pH on yeast growth, the medium was prepared in either of the following buffer systems: 0·1 M citric acid–0·2 M sodium dihydrogen phosphate (pH 2·0–8·0); 0·2 M KCl–0·2 M HCl (pH 1·0–2·0); 0·1 M potassium hydrogen phthalate–0·1 M HCl (pH 3·0–4·0); 0·1 M potassium hydrogen phthalate–0·1 M NaOH (pH 5·0); 0·1 M potassium dihydrogen phosphate–0·1M NaOH (pH 6·0, 6·5, 7·0, 8·0, 9·0, 10·0). Sucrose was incorporated into the basal medium of different pH values (citrate-phosphate buffer) to give final concentrations of 20, 40, 50, 60 and 70% w/v, and water activity values in the range 0·97–0·98, 0·95–0·97, 0·92–0·94, 0·90–0·92, and 0·88–0·90, respectively as measured with the VAISALA humidity indicator (model HMI 31, Finland). NaCl was added to the basal medium of different pH values (citrate-phosphate buffer) to give final concentrations of 2·5, 5·0, 7·5, 10·0, 12·5, 15·0, 20·0% w/v, and water activity values in the range of 0·98–0·99, 0·96–0·98, 0·94–0·95, 0·93–0·94, 0·92–0·93, 0·89–0·91, and 0·84–0·89, respectively. Benzoic acid (Sigma Chemicals, UK) or sorbic acid (Sigma Chemicals) were added to the basal medium of different pH values (citrate-phosphate buffer) to give final concentrations of 0, 250, 500, 750, 1000, 1200 mg l−1. To maintain a constant concentration of the basal medium, allowances were made during preparation for addition of the sucrose, salt, and preservative solutes. For the concentration of all components, allowances were made for the dilution on inoculation. The pH of media was checked before dispensing into the microtitre trays. All media were sterilized by filtration through a 0·2 µm membrane.

Results Effect of pH Initially, the growth of yeasts was screened

Growth of food spoilage yeasts

over the range pH 1·0–10·0, using various inorganic buffer systems (Table 1). All species exhibited strong growth in the range pH 4·0–7·0, except the strains of Z. bailii which did not grow at pH 7·0. Strains of Y. lipolytica, D. hansenii, K. marxianus, S. cerevisiae and P. anomala grew at pH 8·0 but Z. rouxii, P. membranaefaciens and Kl. apiculata did not grow at this pH. None of the yeasts grew at pH 9·0 or higher pH values. Most strains grew at pH 3·0, with notably stronger responses by Kl. apiculata and Z. bailii. None of the strains of K. marxianus grew at pH 3·0 and only one out of the four strains of D. hansenii grew at this pH. The strains of Kl. apiculata were the most acid tolerant, with strong growth (turbidity) being evident at pH 1·5–2·0. The remaining species gave either weak or no growth at pH 2·0. Table 2 shows the effect of pH on yeast growth over the range pH 2·0–8·0, using citrate–phosphate buffer to control pH. The growth responses at pH 8·0 were similar to those found using inorganic buffers, namely, Y. lipolytica, D. hansenii, K. marxianus, S. cerevisiae, P. anomala grew at this pH whereas Kl. apiculata, P. membranaefaciens, Z. bailii, Z. rouxii did not grow. None of the strains of Z. bailii grew at pH 7·0, but all other yeasts grew at this pH. In contrast to the analyses in inorganic buffers, all strains grew at pH 3·0 and generally exhibited stronger growth at this pH than in the inorganic buffers. Moreover, with the exception of three strains of D. hansenii, all yeasts grew at pH 2·5. Whereas none of the strains of K. marxianus grew in inorganic buffer at pH 3·0, they grew at pH 3·0 and pH 2·5 in citrate-phosphate buffer. Again, strains of Kl. apiculata were the most acid tolerant and gave strong growth at pH 2·0 in citrate-phosphate buffer. None of the other species grew at pH 2·0, with the exception of weak growth of one strain of Y. lipolytica. Growth at different pH values was determined in two basal media, namely, 0·5% yeast extract and YNB, and at two glucose concentrations, namely, 0·5% and 5·0% (Tables 3 and 4). For strains of Y. lipolytica, D. hansenii, K. marxianus, Z. rouxii and P. anomala, growth responses at the different pH values were the same on all media and

corresponded to the data of Tables 1 and 2 (data not shown). For Kl. apiculata, S. cerevisiae and Z. bailii, growth or better growth occurred at the lower and higher pH values when YNB was used as the basal medium (Tables 3 and 4). For example, Z. bailii grew at pH 2·5 when YNB, but not yeast extract, was the basal medium. Also, this species did not grow at pH 6·0 with yeast extract in the medium, but grew in YNB at this pH (Table 4). For both basal media, growth response to pH was the same at 0·5 and 5·0% glucose but greater turbidity, indicating more growth, occurred in media containing 5% glucose.

Effect of NaCl concentration The growth of yeasts at different concentrations of NaCl varied with the species (Table 5). None of the yeasts grew in the presence of 20% NaCl, but there were strains of D. hansenii, Z. rouxii and P. anomala which grew at 15% NaCl. Saccharomyces cerevisiae and K. marxianus exhibited the weakest tolerance of NaCl and did not grow at concentrations greater than 7·5 and 10% NaCl, respectively. Some variation was observed for strains within a species. One strain of D. hansenii did not grow at 15% NaCl and two strains of K. marxianus did not grow at concentrations exceeding 7·5% NaCl. The pH of the medium affected growth in the presence of NaCl (Table 5). For Y. lipolytica, D. hansenii and S. cerevisiae, greatest tolerance to salt occurred at the higher pH values of 5·0–7·0. However, several other species exhibited greatest tolerance to salt at the lower pH values. For Kl. apiculata, P. membranaefaciens and Z. bailii, best tolerance was at pH 3·0 and for Z. rouxii and P. anomala it occurred at pH 3·0–5·0. For all yeasts except Kl. apiculata, ability to grow in the presence of NaCl was decreased at pH 2·0. All strains of Kl. apiculata grew in the presence of 12·5% NaCl at pH 2·0. Interestingly, this species grew at pH 7·0, but not in the presence of the lowest concentration of salt (2·5% NaCl) tested. Also, species of P. anomala, P. membranaefaciens, and Z. bailii that did not grow at pH 2·0 in the absence of salt (Table 2), grew at this pH when salt (2·5% NaCl) was included in the medium.

461

462 W. Praphailong and G. H. Fleet

Table 1. Effect of pH (inorganic buffer)a on the growth response of yeast species pH Yeast species D. hansenii (three strains) (one strain) Y. lipolytica (five strains) P. anomala (one strain) P. membranaefaciens (two strains) S. cerevisiae (two strains) K. marxianus (four strains) Kl. apiculata (four strains) Z. bailii (six strains) Z. rouxii (two strains)

1·5

2·0

3·0

4·0

5·0

6·0

6·5

7·0

8·0

− −

− +

− +

+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

−

+

+

+++

+++

+++

+++

+++

+++

−

−

++

+++

+++

+++

+++

+++

+++

−

+

++

+++

+++

+++

+++

+++

−

−

−

+

+++

+++

+++

+++

+++

++

−

−

−

++

++

++

++

++

++

+++

+++

+++

+++

+++

+++

+++

−

−

+++

+++

+++

+++

+++

−

−

−

−

++

+++

+++

+++

+++

+++

−

++ −

a

Buffer systems were: 0·2 M KCl–0·2 M HCl (pH 1·0–2·0); 0·1 M potassium hydrogen phthalate–0·1 M HCl (pH 3·0–4·0); 0·1 M potassium hydrogen phthalate–0·1 M NaOH (pH 5·0); 0·1 M KH2PO4–0·1 M NaOH (pH 6·0, 6·5, 7·0, 8·0). Growth reaction: −, no growth; +, weak growth; ++, good growth; +++, strong growth as determined by relative amount of turbidity (visual observation) in wells of microtitre trays.

Table 2. Effect of pH (citrate-phosphate buffer) on the growth response of yeast species pH Yeast species D. hansenii (three strains) (one strain) Y. lipolytica (five strains) P. anomala (one strain) P. membranaefaciens (two strains) (S. cerevisiae (two strains) K. marxianus (two strains) (two strains) Kl. apiculata (three strains) (one strain) Z. bailii (six strains) Z. rouxii (two strains)

2·0

2·5

3·0

4·0

5·0

6·0

6·5

7·0

7·5

8·0

− −

− +

+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

+

+++

+++

+++

+++

+++

+++

+++

+++

+++

−

++

+++

+++

+++

+++

+++

+++

+++

+++

−

++

+++

+++

+++

+++

+++

+++

+

−

−

+

+++

+++

+++

+++

+++

+++

+++

+

− −

+ ++

+ +++

++ +++

++ +++

++ +++

++ +++

++ +++

++ +++

++ +++

+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

− +

− −

−

++

+++

+++

+++

+++

−

−

−

−

++

+++

+++

+++

+++

+++

+++

++

− −

Growth reaction: −, no growth; +, weak growth; ++, good growth; +++, strong growth as determined by relative amount of turbidity (visual observation) in wells of microtitre trays.

Growth of food spoilage yeasts

Effect of sucrose concentration All strains grew in the presence of 50% sucrose, but the growth response at higher concentrations varied with the species (Table 5). Z. rouxii, Z. bailii, and P. anomala were the most tolerant of sucrose, growing at con-

centrations up to 70%. D. hansenii exhibited growth in the presence of 60% sucrose. For most strains, there was no variation in tolerance to sucrose concentrations at either pH 3·0, 5·0, and 7·0, and for some (Y. lipolytica, Kl. apiculata, P. membranaefaciens and P. anomala), this also included pH 2·0. P.

Table 3. Effect of basal medium and glucose concentration on the growth responses of yeast species at different pH values (inorganic buffersa) pH Yeast species Kl. apiculata (four strains) YE+0·5% glucose YE+5·0% glucose YNB+0·5% glucose YNB+5·0% glucose S. cerevisiae (two strains) YE+0·5% glucose YE+5·0% glucose YNB+0·5% glucose YNB+5·0% glucose Z. bailii (six strains) YE+0·5% glucose YE+5·0% glucose YNB+0·5% glucose YNB+5·0% glucose

1·5

2·0

− − ++ ++

− + ++ +++

− − − − − − − −

3·0

4·0

5·0

6·0

6·5

7·0

8·0

− + ++ +++

++ +++ ++ +++

++ +++ ++ +++

++ +++ ++ +++

++ +++ ++ +++

++ ++ ++ +++

− − − −

− − − −

− − − +

++ +++ ++ +++

++ +++ ++ +++

++ +++ ++ +++

++ +++ ++ +++

++ +++ ++ +++

++ +++ + ++

− − − −

− − ++ +++

++ +++ ++ +++

++ +++ ++ +++

++ +++ ++ +++

++ +++ ++ +++

− − − −

− − − −

a

Buffer systems were: 0·2 M KCl–0·2 M HCl (pH 1·5–2·0); 0·1 M potassium hydrogen phthalate–0·1 M HCl (pH 3·0–4·0); 0·1 M potassium hydrogen phthalate–0·1 M NaOH (pH 5·0); 0·1 M KH2PO4–0·1 M NaOH (pH 6·0, 6·5, 7·0, 8·0). Growth reaction: −, no growth; +, weak growth; ++, good growth; +++, strong growth as determined by relative amount of turbidity (visual observation) in wells of microtitre trays.

Table 4. Effect of basal medium and glucose concentration on the growth responses of yeast species at different pH values (citrate–phosphate buffer) pH Yeast species Kl. apiculata (four strains) YE+0·5% glucose YE+5·0% glucose YNB+0·5% glucose YNB+5·0% glucose S. cerevisiae (two strains) YE+0·5% glucose YE+5·0% glucose YNB+0·5% glucose YNB+5·0% glucose Z. bailii (six strains) YE+0·5% glucose YE+5·0% glucose YNB+0·5% glucose YNB+5·0% glucose

2·5

3·0

4·0

5·0

6·0

6·5

7·0

7·5

8·0

+ + ++ +++

++ +++ ++ +++

++ +++ ++ +++

++ +++ ++ +++

++ ++ ++ +++

+ + ++ +++

− − + +

− − − −

− − − −

− − + +

++ +++ ++ +++

++ +++ ++ +++

++ +++ ++ +++

+ + ++ +++

+ + ++ +++

+ + ++ +++

+ + ++ +++

− − + +

− − + ++

++ +++ ++ +++

++ +++ ++ +++

++ +++ ++ +++

− − ++ +++

− − − −

− − − −

− − − −

− − − −

Growth reaction: −, no growth; +, weak growth; ++, good growth; +++, strong growth as determined by relative amount of turbidity (visual observation) in wells of microtitre trays.

463

464 W. Praphailong and G. H. Fleet

anomala, P. membranaefaciens, Z. bailii and one strain of D. hansenii did not grow at pH 2·0 (Table 2), but grew at this pH in the presence of 50% sucrose (Table 5).

at pH 3·0. At pH 5·0, Y. lipolytica and Z. bailii were the species most resistant to sorbate, growing at 1000 and 750 mg l−1, respectively. At pH 7·0, all strains except Z. bailii, grew at 1200 mg l−1 of sorbic acid.

Effect of preservatives With the exception of Y. lipolytica and Z. bailii, all yeasts were inhibited by 250 mg l−1 of benzoic acid at pH 3·0 (Table 6). At pH 5·0, all species grew in the presence of benzoic acid, although the maximum concentration tolerated varied with the species. Both Y. lipolytica and Z. bailii were the most tolerant at this pH, and grew at 1200 mg l−1 which was the greatest concentration tested. D. hansenii, K. marxianus and P. anomala were most sensitive to benzoic acid and did not grow at concentrations greater than 500 mg l−1 at pH 5·0. At pH 7·0, all strains, except Z. bailii, grew at 1200 mg l−1 benzoic acid. As noted already, Z. bailii does not grow at pH 7·0. All the yeasts were inhibited by lower concentrations of sorbic acid than benzoic acid at pH 3·0 and pH 5·0 (Table 6). Only Z. bailii grew in the presence of 250 mg l−1 sorbic acid

Discussion It is generally reported that most yeasts initiate growth within the range, pH 3·0–7·0 (Walker 1977, Miller 1979, Deak 1991). The data of Tables 1–4 support this conclusion, but also reveal new information. Most species did not grow or gave weak growth at pH values less than 2·5. The exception was Kl. apiculata, where all strains exhibited good growth at pH 2·0 and 1·5. The prevalence of this species in the acidic environments of fruits is well known (Recca and Mrak 1952, Spencer et al. 1992) and its particularly strong tolerance of low pH could account for these observations. Unlike other species, however, the strains of Kl. apiculata were not tolerant of alkaline pH values and gave poor growth or no growth at values greater than 7·5. The ability of P. membranaefaciens to

Table 5. Effect of sodium chloride and sucrose concentrations on the growth response of yeasts at different pH values Maximum NaCl (%w/v) giving growth Yeast species D. hansenii (three strains) (one strain) Y. lipolytica (five strains) P. anomala (one strain) P. membranaefaciens (two strains) S. cerevisiae (two strains) K. marxianus (two strains) (two strains) Kl. apiculata (four strains) Z. bailii (six strains) Z. rouxii (two strains)

Maximum sucrose (%w/v) giving growth

pH 2

pH 3

pH 5

pH 7

pH 2

pH 3

pH 5

pH 7

NG 2·5

10·0 10·0

15·0 10·0

15·0 10·0

NG 50·0

60·0 60·0

60·0 60·0

60·0 60·0

2·5

10·0

12·5

12·5

50·0

50·0

50·0

50·0

2·5

15·0

15·0

12·5

70·0

70·0

70·0

70·0

2·5

12·5

5·0

5·0

50·0

50·0

50·0

50·0

NG

5·0

7·5

7·5

NG

50·0

50·0

50·0

NG NG

7·5 10·0

5·0 10·0

2·5 10·0

NG NG

50·0 50·0

50·0 50·0

50·0 50·0

12·5

12·5

10·0

G

50·0

50·0

50·0

50·0

2·5

12·5

5·0

NG

50·0

70·0

70·0

NG

NG

15·0

15·0

7·5

NG

70·0

70·0

70·0

G, growth in the absence of NaCl; NG, no growth in the absence of NaCl or sucrose.

Growth of food spoilage yeasts

grow at pH values around 2·0 has been reported by Pitt (1974), and Spencer et al. (1992). The pH limits for the growth of Y. lipolytica appear not to have been reported, but the data of Tables 1 and 2, suggest it exhibits good tolerance of both acid and alkaline environments. Growth of Z. bailii at pH 2·5–3·0 has been reported (Thomas and Davenport 1985, Cole and Keenan 1986, Warth 1986a, b), but less recognized, is its inability to grow at pH 7·0, and this was a unique response among the species examined. Growth of some species at the extremes of pH was affected by the buffer system used and the composition of the basal medium. Stronger growth occurred in citrate phosphate buffer than in the inorganic buffers (Tables 1 and 2). This would not be due to the potential of the cultures to metabolize citric acid, as some of the species showing better growth at low pH in the presence of citrate (Kl. apiculata, P. membranaefaciens, S. cerevisiae, Z. bailii and Z. rouxii) are not able to utilize this substrate (Barnett et al. 1990). Pitt (1974), however, has noted that several yeast species were more tolerant of low pH in inorganic buffer than organic buffer because

of the inhibitory effects of the organic acid anions. Strains of Kl. apiculata, S. cerevisiae and Z. bailii exhibited better growth at the pH extreme when YNB rather than yeast extract was the basal medium. This difference could be due to the vitamins and amino acids present in YNB and possibly a more readily available nitrogen source in the form of ammonium sulphate. The ability of yeasts to tolerate acid pH values is related to the activity of plasma membrane ATPase which regulates intracellular pH by exporting protons (Eraso and Gancedo 1987). Presumably, yeasts which tolerate low pH values have a more efficient or stable plasma membrane ATPase system but this would need to be demonstrated experimentally by comparing the activity of this system in the different yeast species. Growth of D. hansenii, Z. rouxii, and P. anomala at the low water activities (aw 0·89–0·91) produced by NaCl at concentrations of 15% or greater is well known (Onishi 1963, Norkrans 1966, Tokuoka and Ishitani 1991, Tokuoka 1993), and is supported by the data presented in Table 5. However, none of the strains examined in our

Table 6. Effect of benzoic and sorbic acid concentrations on the growth responses of yeasts at different pH values Maximum benzoic acid (mg l−1) giving growth Yeast species D. hansenii (three strains) (one strain) Y. lipolytica (five strains) P. anomala (one strain) P. membranaefaciens (two strains) S. cerevisiae (two strains) K. marxianus (four strains) Kl. apiculata (four strains) Z. bailii (six strains) Z. rouxii (two strains)

Maximum sorbic acid (mg l−1) giving growth

pH 2

pH 3

pH 5

pH 7

pH 2

pH 3

pH 5

pH 7

NG −

− −

500 250

1200 1200

NG NG

− −

250 −

1200 1200

−

250

1200

1200

−

−

1000

1200

−

−

500

1200

NG

−

250

1200

−

−

750

1200

NG

−

250

1200

NG

−

750

1200

NG

−

250

1200

NG

−

500

1200

NG

−

250

1200

−

−

750

1200

−

−

250

1200

NG

250

1200

NG

NG

250

750

NG

NG

−

750

1200

NG

−

500

1200

NG, no growth in the absence of preservatives; −, no growth in the presence of 250 mg l−1 of preservatives.

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466 W. Praphailong and G. H. Fleet

study grew at 20–25% NaCl, as reported elsewhere (Norkrans 1966). Strain variation in response to NaCl could explain this result and, for D. hansenii, this is evident in Table 5 and was also reported by Besanc¸on et al. (1992). Y. lipolytica and Kl. apiculata are not known as salt tolerant species but, nevertheless, they gave strong growth in the presence of 12·5% NaCl. According to Guerzoni et al. (1993), Y. lipolytica can grow at the low aw of 0·89 (10% NaCl+10% sucrose). The growth of S. cerevisiae and K. marxianus at concentrations up to 7·5% NaCl and 10% NaCl, respectively (Table 5) concurs with previous reports (Hobot and Jennings 1981, Besanc¸on et al. 1992, Vivier et al. 1993, 1994). For most yeasts, tolerance of NaCl is decreased at the extremes of pH and this conclusion is reflected in the data of Table 5 and of other studies (Onishi 1957a, b, Norkrans 1966). However, some yeasts (D. hansenii, Y. lipolytica, S. cerevisiae) are more tolerant of NaCl at the higher pH values (e.g. pH 5·0–7·0) while others (Kl. apiculata, Z. bailii, K. marxianus, P. membranaefaciens) give better tolerance under more acid conditions (e.g. pH 3·0). The greater tolerance of D. hansenii and S. cerevisiae to NaCl at pH 5·0–7·0 compared with other pH values has been reported previously (Hobot and Jennings 1981). Growth of Kl. apiculata in the presence of 12·5% NaCl at pH 2·0, but not at pH 7·0 is particularly noteworthy (Table 5). The restrictive effect of sugar on yeast growth became evident at concentrations greater than 50% sucrose (aw>0·92). The ability of Z. rouxii, Z. bailii, P. anomala and D. hansenii to grow in the presence of 60–70% sucrose (Table 5) is well documented (Tokuoka and Ishitani 1991, Tokuoka 1993). Not so well recognized, however, is the growth of species such as Kl. apiculata, Y. lipolytica and K. marxianus in 50% sugar environments. Unlike the observations with salt, pH over the range 2·0–7·0 did not exert a major influence on the growth capability of yeasts at high sugar concentrations. This observation was similar to those of English (1954) who showed that Z. rouxii grew in 46% glucose for the pH range 2·0–8·7. The presence of either NaCl or sucrose had a positive effect on the ability of some species

to initiate growth at the pH extremes. P. membranaefaciens, Z. bailii and P. anomala gave no growth at pH 2·0 in the absence of NaCl, but grew at this pH when 2·5% NaCl was present in the medium. Z. bailii did not grow at pH 2·0 in the absence of sugar, but grew at this pH in the presence of 50% sucrose. This observation was not consistent with the findings of Onishi (1957b) who found that growth of osmophilic yeasts in NaCl-free medium occurred over the range pH 3·0–7·0, whereas in the presence of 18% NaCl, growth was limited to the range pH 4·0–5·0. Smittle (1977) reported that increasing concentrations of salt shifted the pH optimum for growth of Z. bailii and Z. acidifaciens to lower values. The mechanisms by which some yeast species tolerate high salt and high sugar (low water activity) environments have been the subject of considerable study (reviewed by Onishi 1963, Witter and Anderson 1987, Tokuoka 1993). Essentially, these species produce high concentrations of intracellular polyols (compatible solutes) such as glycerol, arabitol, mannitol and erythritol, that balance the external osmotic pressure, but other factors involved include altered composition of cell wall and cell membrane and, in the case of salt tolerance, a more active Na+/K+ ATPase system would be needed to maintain a low intracellular concentration of sodium ions (Watanabe et al. 1991, Ushio et al. 1992, Nishi and Yagi 1995). The interactive effect of salt and sugar tolerance with pH, especially at low pH as reported in this paper, has not been the subject of detailed study. It is not clear why some species (Kl. apiculata, Z. bailii, P. membranaefaciens, and K. marxianus) are more tolerant of high NaCl at low pH than at high pH and why some species (D. hansenii, Y. lipolytica, S. cerevisiae, P. anomala, P. membranaefaciens, and Z. bailii) are less tolerant of NaCl at low pH. It is not clear why the lower pH limits for growth of some yeasts are extended by the presence of either sugar or salt. Also, it is not evident why salt tolerance in yeasts is more critically affected by pH than sugar tolerance. Biochemical or molecular research aimed at explaining these observations is required.

Growth of food spoilage yeasts

The data of Table 6 confirm previous reports that some species such as Z. bailii, are tolerant of high concentrations (>750 mg l−1) of benzoate and sorbate (Cole and Keenan 1986, Warth 1986a, b) while other such as S. cerevisiae are inhibited by lower concentrations of these preservatives (Neves et al. 1994). The inhibitory response is stronger at pH values less than 5·0 where a greater proportion of the acid is present in the undissociated form (Pitt 1974, Liewen and Marth 1985, Warth 1986a, b). Table 6 also confirms the stronger inhibitory effect on yeasts of sorbate compared with benzoate as noted elsewhere (Pitt 1974). Yarrowia lipolytica is not widely recognized as a preservative resistant species but the data of Table 6 along with the report of Guerzoni et al. (1993) suggest that this species is comparable with Z. bailii in its tolerance of benzoate and sorbate preservatives. The mechanisms by which yeasts tolerate high concentrations of weak acid preservatives have been discussed by Warth (1986a) and relate to their ability to pump out (export) intracellular protons and anions that accumulate due to dissociation of the acid within the cell. In conclusion, this study has revealed confirmatory as well as new information about factors which limit the growth of some important food spoilage yeasts. It provides the basis for more definitive, quantitative studies aimed at predicting the growth responses of yeasts in food ecosystems, and it indicates directions for molecular studies to explain the diversity of yeast responses to environmental stresses.

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