International Journal of Food Microbiology 131 (2009) 280–282

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International Journal of Food Microbiology

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Short communication

Inhibition of spoilage yeasts in cheese by killer yeast Williopsis saturnusvar. saturnus☆

Shao-Quan Liu a,⁎, Marlene Tsao b

a Food Science and Technology Programme, Department of Chemistry, National University of Singapore, Singaporeb Food Science Programme, School of Chemical and Life Sciences, Nanyang Polytechnic, Singapore

☆ Part of this research was conducted at the Fonterra⁎ Corresponding author. Tel.: +65 6516 2687; fax: +

E-mail address: chmLsq@nus.edu.sg (S.-Q. Liu).

0168-1605/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.ijfoodmicro.2009.03.009

a b s t r a c t

a r t i c l e i n f o

Article history:Received 14 December 2008Received in revised form 25 February 2009Accepted 15 March 2009

Keywords:BiopreservationYeastsCheese

Williopsis saturnus var. saturnus is a known killer toxin-producing yeast. The effects of this yeast as abiopreservative against spoilage yeasts (galactose fermenting) were investigated in cheeses made underlaboratory conditions. At an inoculation level of ∼106 CFU/g of cheese, this killer yeast inhibited growth oflactose non-fermenting but galactose-fermenting yeast Saccharomyces cerevisiae VL1 inoculated at∼103 CFU/g; it also inhibited growth of lactose-fermenting and galactose-fermenting yeast Kluvyveromycesmarxianus ATCC8640 inoculated at ∼103–104 CFU/g in the cheeses manufactured with galactose-producingstarter culture Streptococcus thermophilus. In contrast, the two spoilage yeasts grew to ∼106 CFU/g from theinitial cell count of ∼103 CFU/g without the killer yeast. This study indicated that W. saturnus var. saturnuscould be an effective biopreservative for cheese spoilage control.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Yeasts can play a positive or negative role in fermented dairyproducts by contributing to cheese ripening or by causing productspoilage (Fleet, 1990; Jakobsen and Narvhus, 1996). Yeasts serve assecondary cultures of smear-ripened cheeses for appearance andaroma development (Bockelmann et al., 2005). Yeasts also promotemould growth inmould-ripened cheeses by inducing open texture dueto gas production (Hansen and Jakobsen, 2001; Hansen et al., 2001).However, yeasts can cause spoilage of other cheese types as a result ofgas (open texture or slits) and off-flavour formation (Seiller, 2002).

Cheeses made with thermophilic starters such as Streptococcusthermophilus are particularly vulnerable to spoilage by galactose-fermenting yeasts such as the majority of yeasts from the Kluyvero-myces and Saccharomyces genera. This is because some thermophiliclactic acid bacteria metabolise the glucose moiety of lactose but notgalactose which is excreted into the medium (Lourens-Hattingh andViljoen, 2001). Even cheeses made with mesophilic starters such asLactococcus lactis are susceptible to spoilage by galactose-fermentingyeasts, because lactococci can excrete galactose produced from lactoseinto cheese (Liu et al., 1998), just like some of the above-mentionedthermophilic lactic acid bacteria. To control cheese spoilage due toyeast growth, chemical- and antibiotic-based preservatives arecommonly used but are facing consumer resistance due to safetyconcerns. Natural preservation offers a better alternative and as yet hasbeen relatively under-exploited.

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Some yeasts possess antagonistic property (killer activity) towardmoulds and other yeasts by producing killer toxins (Magliani et al.,1997). This property has been exploited in the biological control ofpostharvest diseases of fruits caused by moulds (Fleet, 2003; Spadaroand Gullino, 2004). A number of killer toxin-producing yeasts areantagonistic toward not only other yeasts andmoulds but also bacteria(Michalcakova et al., 1993; Suzzi et al., 1995; Izgu and Altinbay, 1997).The species and strains of the genus Williopsis are well known killertoxin-producers, especially W. saturnus var. saturnus, W. saturnus var.mraki (Nomoto et al., 1984; Michalcakova et al., 1993).

Despite the potential effectiveness of Williopsis yeasts as biopre-servatives in food spoilage control, there have been no attempts to usethese yeasts in dairy product preservation. Given the need to developnatural means of food preservation due to consumer demand, weattempted to investigateW. saturnus var. saturnus as a biopreservativeto control cheese spoilage yeasts using cheese as a model system. Theresults of this study are communicated in this brief report.

2. Materials and methods

Microorganisms were from the culture collection (kept frozen at−80 °C) held at the Fonterra Research Centre, Palmerston North,New Zealand. W. saturnus var. saturnus CBS254 was originally fromCentraalbureau voor Schimmelcultures (Utrecht, the Netherlands).K. marxianusATCC8640was originally from the American Type CultureCollection (VA, USA). S. cerevisiae VL1 was originally from LallemandInc (Ontario, Canada). S. thermophilus B2522 was a Fonterra-ownedcheese starter culture (Palmerston North, New Zealand).

Yeasts were grown in YEPD broth comprised of the followingcomponents (g/L): Bacto-yeast extract (Difco, BD-Diagnostic Systems,

Table 1Inhibition of galactose-fermenting yeast S. cerevisiae VL1 in cheeses by the killer yeastW. saturnus var. saturnus CBS 254.

Total yeast cell count (CFU/g of cheese)

Day 0 Day 21

− killer yeasta + killer yeastb − killer yeasta + killer yeastc

Trial 1 8.0×103 2.0×106 2.0×106 6.0×105

Trial 2 2.0×103 2.0×106 4.0×106 3.0×105

a S. cerevisiae VL1 only.b S. cerevisiae VL1 (∼103 CFU/g) + W. saturnus var. saturnus CBS 254 (∼106 CFU/g).c W. saturnus var. saturnus CBS 254 only, as S. cerevisiae VL1 was not detectable.

Table 2Inhibition of galactose-fermenting yeast K. marxianus ATCC8640 in cheeses by the killeryeast W. saturnus var. saturnus CBS 254.

Total yeast cell count (CFU/g of cheese)

Day 0 Day 21

− killer yeasta + killer yeastb − killer yeasta + killer yeastc

Trial 1 1.0×104 2.0×106 1.0×106 2.0×105

Trial 2 1.0×103 1.0×106 4.0×105 3.0×104

a K. marxianus ATCC8640 only.b K. marxianus ATCC8640 (∼103 CFU/g) + W. saturnus var. saturnus CBS 254

(∼106 CFU/g).c W. saturnus var. saturnus CBS 254 only, as K. marxianus ATCC8640 was not

detectable.

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Sparks, Maryland, USA), 10; Bacto-peptone (Difco), 10; dextrose(Merck, Whitehouse Station, New Jersey, USA), 20. The ingredientswere dissolved in deionised water and pH was adjusted to 5.0,followed by autoclaving at 121 °C for 15 min. Yeast cultures wereincubated at 30 °C for up to 48 h without aeration and used toinoculate milk together with cheese starter (see below). Cheesestarter culture S. thermophilus B2522 was grown in reconstituted skimmilk (RSM, pre-autoclaved at 115 °C for 15min) and incubated at 37 °Cfor 24 h. This culture was used to inoculate milk for cheesemaking(see below). Yeasts were plated following the conventional platingmethod (spreading) on oxytetracycline–glucose yeast extract (Oxoid,Hampshire, UK) agar containing 0.1 g/L of chloramphenicol and plateswere incubated at 25 °C for 2–4 days. W. saturnus var. saturnusCBS254 was differentiated from S. cerevisiae VL1 and K. marxianusATCC8640 by their colony appearance: the colony of strain CBS254appeared dull, wrinkled and rough, whereas the colony of strains VL1and ATCC8640 was creamy, shiny and smooth.

This experiment was conducted using cheese prepared on alaboratory scale.1.2 L of pasteurisedmilk (pH∼6.6–6.7)wasmeasuredinto a sterile 2 L beaker coveredwith aluminium foil. Themilkwas thenwarmed up to 32 °C inwater bath, followed by adding 2.0% v/v cheesestarter S. thermophilus B2522 (∼107 CFU/mL of cheesemilk), whichwas pre-cultured in RSM as described above. This was followed byinoculating the milk with 1% v/v each of W. saturnus var. saturnusCBS254 (∼105 CFU/mL of cheesemilk) and an appropriately dilutedtarget yeast (∼102 CFU/mL of cheesemilk) (Saccharomyces cerevisiaeVL1 or Kluyveromyces marxianusATCC8640). The control had no addedyeast CBS254. All yeasts were pre-cultured in YEPD broth describedabove. The inoculated milk was incubated at 30 °C for 30 min and pHwas measured (expected to be 6.5). 120 µL of rennet was then addedandmixed into themilk. Incubationwas continued at 32 °C until a firmcoagulumwas formed (about 30–45 min later). The curd was cut andthe water bath temperature was raised to and kept at 38 °C for 2 min.The curd-whey mixture was stirred slowly for 10 min to break uplarge curds. The whey pH was measured every 30 min until it reached6.2–6.3 (about 90 min). Approximately 80% of whey was then drainedoff, curds broken up and returned to water bath. The curd was cutfurther, about 1/3 of the remaining whey drained off, and pH me-asured every 30 min until reaching 5.3. The beaker was then removedfrom the water bath and all the remaining whey drained off. Curd wastransferred to a sterile cheesecloth and excess moisture removed bysqueezing manually. The squeezed curd was placed into sterilecentrifuge tubes and weighed. Food grade salt (NaCl) was added at1.8%w/wandmixed thoroughlymanually. The curdwas centrifuged at22 °C in a swing bucket centrifuge for 60min (1550 g). The cheese curdwas then vacuum-packed in oxygen non-permeable plastic bags andstored at 20 °C for threeweeks to accelerate the process. Samplesweretaken at day zero and day 21 for microbiological analysis.

Compositional analysis of cheese was performed using standardmethods. Protein was determined according to the Kjeldahl method(Barbano et al., 1990). Fat was analysed using the Babcock method(APHA,1985). Saltwas determined as described by Jeffery et al. (1989).Moisturewas determined following themethod ofMIF (1959). Lactose

and galactose were analysed using GLC (Marcy and Carroll, 1982).Analysis of volatiles was conducted using SPME-GC/MS as describedelsewhere (Liu et al., 2003). Data given were average values of dupli-cate determinations.

3. Results and discussion

The cheeses had an average gross composition of 37.0+1.0% w/wprotein, 26.0±1.0%w/w fat, 1.85±0.2%w/w salt, and 44.5±1.5%w/wmoisture. After the cheeses were made at day 0, the cheeses contained0.95±0.05% w/w galactose but no detectable level of lactose.

W. saturnus var. saturnus CBS254 does not ferment lactose andgalactose and hence, was not expected to grow in the cheesesmanufactured with galactose-producing starter S. thermophilus. Onthe contrary, the target yeasts S. cerevisiae VL1 (galactose fermenting)and K. marxianus ATCC8640 (both lactose and galactose fermenting)were expected to grow in the cheeses.

As shown in Tables 1 and 2, both spoilage yeasts S. cerevisiae VL1 andK. marxianus ATCC8640 grew to ∼106 CFU/g from the initial cell countof ∼103 CFU/g in the cheeses without the added killer yeast CBS254(control). When the added killer yeast CBS254 was present, the totalyeast cell count declined to ∼105 CFU/g from the initial inoculumconcentration of ∼106 CFU/g. This indicated that the target yeast didnot grow or was inhibited in the cheeses, because microbiologicalanalysis (plating) showed that only colonies of the killer yeast CBS254were present on the agar plates (see Materials and methods). Theseresults suggest that the killer yeast CBS254 inhibited growth of lactoseand/or galactose-fermenting spoilage yeasts in cheese. The inhibition ofcheese spoilage yeasts by the killer yeast CBS254 was also supported bya lack of gas formation (no bag bulging) in the cheeses with the killeryeast, whereas there was gas formation (bag bulging) in the controlcheeses (no killer yeast), as the cheeses were vacuum-packed inoxygen-impermeable plastic bags.

Inhibition of cheese spoilage yeasts is further supported by a lack ofproduction of volatile metabolites associated with yeast growth in thetreatment cheeses (with killer yeast), whereas there was a productionof a number of volatile metabolites associated with yeast growth inthe control cheeses (no killer yeast). In the case of S. cerevisiae VL1(without killer yeast), there was a formation of ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol, 2-phenylethanol, 3-methyl-1-butanaland ethyl acetate. In the case of K. marxianus ATCC8640 (no killeryeast), there was production of ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol, 2-phenylethanol, 3-methyl-1-butanal, benzalde-hyde, ethyl acetate, 3-methyl-1-butyl acetate and 2-phenylethyl acetate.

W. saturnus var. saturnus CBS 254 is ideal as a biopreservative sinceit is neither lactose fermenting nor galactose fermenting. This offers anovel way of controlling cheese spoilage yeasts and may replacechemical or antibiotics-based preservatives. Further work is requiredto test the effectiveness of this yeast to inhibit mould growth oncheese. There is one report that shows inhibition of several moulds oncheese by a dairy yeast Debaryomyces hansenii (Liu and Tsao, 2008).While it is beyond the scope of the present work to investigate the

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mechanism of yeast inhibition by W. saturnus var. saturnus CBS 254, itcould be due to a combination of competition for space and killer toxin,as the cell population of this yeastwas approximately 1000-fold higherthan that of the target yeasts (see Materials and methods) and thisyeast is a known killer toxin producer (see Introduction). Futurestudies should include a non-killer toxin-producing strain ofWilliopsisyeasts (preferably a strain of W. saturnus var. saturnus) to help under-stand themechanism of inhibition, at least to rule out the contributionof killer toxin(s).

Findings of this studyalso have implications for selecting secondarycultures for cheese ripening, not only for mould- and smear-ripenedcheeses but also for other cheese varieties (Hansen and Jakobsen,2001; Hansen et al., 2001; Ferreira and Viljoen, 2003; Bockelmannet al., 2005). The inclusion of killer yeasts in the secondary culturesmay retard or even inhibit growth of other yeasts and thus, affectingcheese maturation. Screening of yeasts for their killer activity orsusceptibility to killer activity may be necessary in the development ofsecondary cultures for cheeses.

Lactose-fermenting Kluyveromyces and galactose-fermenting Sac-charomyces yeasts tend to spoil young and fresh cheeses such assoft cheeses and pasta filata cheeses (e.g. Mozzarella) during thefirst few days of maturation when lactose and galactose are morereadily available. Further research is warranted to ascertain whetherW. saturnus var. saturnus CBS 254 and, indeed, other lactose non-fermenting and galactose non-fermenting but killer toxin-producingyeasts can inhibit growth of spoilage yeasts in these cheese types,especially during early stages of ripening.

Williopsis yeasts, including W. saturnus and W. californica, are partof the natural microflora of cheeses (Wyder and Puhan, 1999; Seiller,2002). They are non-pathogenic, non-proteolytic and non-lipolytic,and therefore, are not expected to impact on cheese flavour andtexture. Indeed, the killer yeastW. saturnus var. saturnus CBS254 itselfdid not produce any volatile metabolites (desirable or undesirable) inthe cheese. Williopsis yeasts may play an important role in cheesebiopreservation. Nonetheless, good hygiene and good manufacturingpractice are essential to minimize or prevent cheese spoilage.

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