ORIGINAL PAPER

Postharvest Control of Gray Mold in Apples with LyophilizedFormulations of Cryptococcus laurentii: the Effect of Cold Stressin the Survival and Effectiveness of the Yeast

Leonardo G. Navarta & Juan Calvo & Paola Posetto &

Soledad Cerutti & Julio Raba & Delia Benuzzi &María I. Sanz

Received: 20 October 2013 /Accepted: 16 March 2014 /Published online: 4 April 2014# Springer Science+Business Media New York 2014

Abstract Cryptococcus laurentii BNM 0525 adapted to coldwas used for developing a lyophilized formulation for con-trolling Botrytis cinerea (gray mold) in apples. For cold stress,the yeast was grown at 1 °C for 48 h. The trehalose content ofstressed cells reached 109 mg g−1 after 24 h, but it did notshow significant modification in unstressed cells which weregrown at 28 °C. The skimmed nonfat milk 10 %, yeast extract0.5 %, and glucose 1 % (SMYG) medium was chosen asfreeze-drying protectant for stressed and unstressed cells.After the freeze-dried process, stability of stressed yeast cellswas significantly higher along 90 days of storage at 4 °C thanthat of unstressed cells. The effectiveness in protection againstB. cinerea was also improved. When apple protection wasperformed with freeze-dried cells, the maximum protectionwas obtained with stressed cells. In this case, decay reductionpercentage was 79.30 %, and there were no significant differ-ences when a lyophilized formulation stored for 90 days wasused. Unstressed cells were less effective immediately afterthe freeze-dried process (69.12 %) and less resistant to stor-age. The percentage of decay reduction was less to 60%whenapplied with unstressed freeze-dried cells stored during90 days. Cold stress increased the trehalose content inC. laurentii cells and improved the behavior of the yeast infront of preservation operations and also its effectiveness forcontrolling B. cinerea.

Keywords Cryptococcus laurentii . Cold stress . Botrytiscinerea .Biocontrol . Freeze-drying .Graymold . Postharvestdiseases

Introduction

Postharvest losses of fruit and vegetables can reach very highvalues, representing more than 25 % of the total production inindustrialized countries and more than 50 % in developingcountries (Nunes 2012). Postharvest diseases limit the storageperiod and marketing life of fruits and vegetables. The wound-invading fungus Botrytis cinerea is one of the most importantpostharvest pathogens affecting pome fruits. This fungus isthe causal of gray mold in apples. The control of postharvestmold rots relies on the use of synthetic fungicides, but thedemanding requirements in sustainable agriculture, integratedcropmanagement, and organic production have resulted in theneed of developing other methods to control postharvest de-cays (He et al. 2003; Sansone et al. 2005; Nunes 2012).Several antagonistic microorganisms have been shown asbiological controllers because they reduce postharvest fungaldecay on pome fruits (Janisiewicz and Korsten 2002; Calvoet al. 2010); however, nowadays, there are only few biologicalproducts available in the market. The major obstacle in thecommercialization of biocontrol products is the developmentof a shelf-stable formulated product (Coulibaly et al. 2010;Droby et al. 2008). Freeze-drying under vacuum is the mostconvenient and successful method of preserving bacteria,yeast, and fungi (Ming et al. 2009). However, not all strainsare able to survive in quantitative rate and or retain biocontrolactivity after a freeze-drying process (Bonaterra et al. 2005;Janisiewicz and Korsten 2002). This is critical since a high cellconcentration is necessary in order to obtain a good formulat-ed product for commercial application that, moreover, can be

L. G. Navarta : J. Calvo : P. Posetto :D. Benuzzi :M. I. SanzLaboratorio de Microbiología Industrial, Área de TecnologíaQuímica y Biotecnología, Departamento de Química, Facultad deQuímica, Bioquímica y Farmacia, Universidad Nacional de San Luis,Ejército de los Andes 950, 5700 San Luis, Argentina

S. Cerutti : J. Raba :M. I. Sanz (*)Instituto de Química San Luis, INQUISAL, CONICET, UniversidadNacional de San Luis, Ejército de los Andes 950, 5700 San Luis,Argentinae-mail: msanz@unsl.edu.ar

Food Bioprocess Technol (2014) 7:2962–2968DOI 10.1007/s11947-014-1303-0

handled using the normal channels of distribution and storage(Sharma et al. 2009). The microbial cell viability and efficacyresulting after the freeze-drying process are dependent onmany factors, including the initial microorganism condition(Morgan et al. 2006), the protective medium, the freezingtemperature, and the rehydration conditions (Hubálek 2003).Physiological manipulation of biocontrol agents can be usedto improve the behavior in front of preservation operations,i.e., freeze-drying process, or to allow a best adaptation toenvironmental conditions when they are applied. Moreover,physiological manipulation may also enhance mechanisms ofbiocontrol. Osmotic shocks using modified growth mediawith NaCl (Texeidó et al. 2005) exposition to high tempera-tures (Hernández-Oropeza and Aranda-Barrada 2011) or lim-itations of nutrients (Aranda et al. 2004) are the different waysused for improving the survival and effectiveness of the bio-control agents.

Another study (Kandror et al. 2004) showed that below10 °C, yeasts have an adaptive response that protects viabilityto subsequent exposure to low or freezing temperatures. Morerecently, it was shown that cells of industrial strains growing at15 °C displayed enhanced freeze and frozen storage resistancethan those grown at 30 °C. The adaptation of yeast cells to lowtemperatures implies a change in gene expression with con-sequences at the level of metabolism, membrane physico-chemical properties, and expectedly the production and accu-mulation of trehalose (Kandror et al. 2004; Tulha et al. 2010).

One potential microorganism that could be developed forcommercial applications is the naturally occurring yeastCryptococcus laurentii (Kuffer) strain BNM 0525, whichwas isolated from the surface of apple fruits in a commercialorchard in Cuyo region (Argentina) (Calvo et al. 2003). Theprincipal goal of this study was to develop a formulation withC. laurentii BNM 0525 adapted to cold, with the aim of usingit against gray mold decay in apples. For reaching this objec-tive, the proposal was to (1) investigate the effect of cold stresson intracellular trehalose levels in the yeast, (2) select theadequate cryoprotectant for freeze-dried process, and (3) as-sess the viability and efficacy of lyophilized formulations incontrolling gray mold in apples.

Material and Methods

Microorganisms

C. laurentii BNM 0525 was isolated and identified in ourlaboratory from microbial consortiums obtained by pickingthe surface of apple fruit throughout the growing season(Calvo et al. 2007). The strain was deposited in the NationalBank of Microorganisms (WDCM938) of the Facultad deAgronomía, Universidad de Buenos Aires (FAUBA),Argentina. Stock cultures of C. laurentii were stored at 4 °C

on yeast extract 5 g L−1, glucose 10 g L−1, and agar-agar20 g L−1 (YGA).

B. cinerea was a kind gift to us from National Institute ofAgricultural Technology (INTA, Lujan de Cuyo, Argentina).It was maintained on potato dextrose agar (PDA) medium(200 mL of extract of boiled potatoes, 20 g of dextrose, 20 gof agar, and 800 mL of water). To maintain virulence, it wasperiodically grown on apples and reisolated. For conidialproduction, B. cinerea was grown on PDA at 20–25 °C.When the mycelium appeared, cultures were kept at 15 °Cfor inducing sporulation. After a week, spores were harvestedand suspended in 10 mL of sterile distilled water containing0.05% (v/v) Tween 80. The concentration of spore suspensionwas determined with a Neubauer chamber and adjusted withsterile distilled water to 1×105 CFU mL−1.

Cold Stress

C. laurentii cells were grown using yeast extract 5 g L−1 andglucose 10 g L−1 (YG) broth. Cultures were made in 1-LErlenmeyer flasks with baffles containing 250 mL of mediumat 28 °C on rotary shaker (2.5 eccentricity—140 rpm). Cellswere harvested at the beginning of the stationary phase bycentrifugation 10,000 rpm for 10 min in a Sorvall SS-3(DuPont Instruments). Then, yeast cells at 1×108 CFU mL−1

in YG broth were exposed to cold stress. The cultures inErlenmeyer flask on rotary shaker (120 rpm) were placed incold rooms at 1 °C and were sampled at 12, 24, and 48 h.Trehalose and colony-forming unit per milliliter were deter-mined at these times. A culture of the yeast at 28 °C was usedas control.

Extraction and Determination of Trehalose

Yeast cells exposed to cold stress (stressed cells (SCs)) wereharvested by centrifugation at 5,000 rpm for 5 min andwashed three times with cold distilled water in order to re-move residual medium. Cells cultured at 28 °C (unstressedcells (UCs)) were used as control. The yeast paste wasdried in stove at 40 °C overnight, and then, 10 mg wastaken for trehalose determination. Trehalose was extractedfrom the dry yeast with water and determined usinghigh-pressure liquid chromatography. An Acquity™ UltraHigh-Performance LC system (Waters, Milford) equippedwith autosampler injection was used. The mobile phase wasformic acid 0.1 % at 0.5 mL min−1. The determinations wererepeated twice.

Sample Preparation for Lyophilization

SCs and UCs were harvested by centrifugation at 10,000 rpmfor 10 min in a Sorvall SS-3 (DuPont Instruments). Thegrowth medium was decanted and the cell paste suspended

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in 15 mL of 0.05 M potassium phosphate buffer (PB), pH 6.5,and centrifuged again. The resulting paste was suspended inthe protecting medium, and the initial cell concentration ofeach suspension was adjusted to 109 CFU mL−1 by the stan-dard count method on YGA plates.

Selection of Protecting Medium

A basic protecting medium (SM10) was formulated with 10 %(w/v) of powdered skimmed nonfat milk (SM) in deionizedwater. SM10was used as protective agent, alone (SM10) or withadditives. The additives were sugars such as trehalose, fructose,and sucrose. These were tested in concentration of 10%. Also, itwas assayed by other protecting mixture that consisted of SM10 %, yeast extract 0.5 %, and glucose 1 % (SMYG).

For each protectingmedium to assay, three autoclaved vialswere filled with 5 mL of sample (SC or UC) and placed at−70 °C overnight. Then, the vials were desiccated in chamberof a freeze-drier (Labconco7740030; Labconco Corp., KansasCity, MO, USA), operating at −45 °C and 0.05 mbar for 12 h.

The freeze-dried samples were rehydrated until reachingthe original volume (5 mL) with the medium to test and wereleft at room temperature for 20 min. Then, serial dilutionswere performed employing the rehydration solution. Thenumber of CFU per milliliter was determined by the platingas described previously. Plates were incubated at 28 °C for24 h. Then, the number of viable cells (survival level) wascalculated by comparing them before (N0) and after (Nf)freeze-drying and rehydration processes. The determinationof Nf was carried out 24 h after freeze-drying treatments andwas repeated twice.

Stability of Freeze-dried Cells

Stability of freeze-dried cells (UC and SC) was assessed bydetermining their viability after 90 days of storage (at 4 °C).The viability was determined as described previously.

Determination of Antagonistic Activity of Freeze-driedC. laurentii Cells Against B. cinerea on Apples in ColdStorage

Antagonistic activity of freeze-dried C. laurentii cells (SC andUC) against B. cinerea was tested on apples cv. Red Delicious.The treatments applied consisted in SCs andUCs before and 24 hafter the freeze-dried processes and 90 days after this operation.The fruit came from a commercial orchard in San Luis,Argentina, and was selected free of wounds and rots and, asmuch as possible, homogeneous in maturity and size. Before theassay, apple surfaces were disinfected by immersion for 1 min ina dilute solution of sodium hypochlorite (1 % active chlorine),washed two times by immersion in distilled water, and left in adry place to remove excesswater off the surface. Then, the apples

werewounded (2×2×2mm) in two places (midway between thecalyx and the stem end) with a punch. A 20-μL suspension(106 CFU mL−1) of freeze-dried or fresh C. laurentii cells wasput in each wound. After 3 h at room temperature, the treatedwounds were inoculated with 20 μL of B. cinerea spores (1×105 CFUmL−1). Immediately, the apples were stored at 4 °C and90±5 % RH. The lesion diameters (severity) were determinedafter 40 days of cold storage. Then, decay reduction percentagewas calculated. Fifty fruits constituted a single replicate, and eachtreatment was replicated three times.

Population Dynamics of C. laurentii

Growth curves were done in fruit (Red Delicious apples). Theapples were wounded in the equatorial zone (2×2×2mm)with apunch. The wounds were inoculated with 20 μL of stressed orunstressed yeast cells (24 h after freeze-dried process) in suspen-sion of known concentration (106 CFU mL−1) and incubated for35 days at 4 °C and 90±5%RH. For monitoring the population,the wounded tissue was extracted by using a cork borer (6-mminternal diameter), was suspended in 50 mL of sterile distilledwater, and was shaken on a rotatory shaker for 20 min at240 rpm. Serial dilutions of the washings were made and platedon YGA plates. The colonies were counted after 48 h of incu-bation at 28 °C. There were three replicates of three fruit pertreatment, and the experiment was repeated twice.

Data Analysis

Survival percentages of C. laurentii were estimated on thebasis of CFU per milliliter counted before (fresh cells) andafter freeze-dried treatments:

% Viability ¼ N f=N0ð Þ � 100:

The percentage of decay reduction was calculated on thebasis of lesion diameters (ϕ) as follows:

% Reduction decay ¼ ϕcontrol−ϕtreatment=ϕcontrolð Þ � 100:

Differences in percentages were analyzed by one-wayanalysis of variance (ANOVA) followed by multiple compar-ison test of Tukey (P<0.05).

Data from trehalose content were compared in a Student’s ttest. Significance was judged at level P<0.05.

Results

Intracellular Trehalose Content of C. laurentii After ColdStress

The content of trehalose in SC and UC cells of C. laurentiiwasmeasured, and the results are shown in Fig. 1. Trehalose

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content of SC was significantly increased from 60 to109 mg g−1 after 24 h of cold stress, while the contents of thisdisaccharide in UC did not show significant modificationsalong the time of incubation. Taking into account that theaccumulation of trehalose was observed at 24 h, all studieswere carried out with cells harvested at this time.

Effect of PreservationMedia on Viability ofC. laurentiiBNM0525 After Freeze-drying

The viability of cells (UC and SC) of C laurentii after freeze-drying using different preservation media is shown in Fig. 2.As can be seen in Fig. 2, significant differences in the viabilityof cells after freeze-drying were observed depending on the

protectant and the internal content of trehalose. The viabilityof C laurentii cells in SM10 was, for UC and SC, 11.40 and16.10 %, respectively, while in SM10 plus trehalose, theviability reached 36 and 43 % for UC and SC, respectively.The best protection was given by the SMYG (81.91 % for UCand 96 % for SC). In all cases, the viability of cells with hightrehalose content (SC) was higher, independent of the cryo-protectants used.

Taking into account that SMYG medium gave the bestresults, all following assays were carried out using SMYGas freeze-drying protectant.

Stability of Freeze-dried Cells

Viability of cells stored during 90 days was compared with thecells recently processed (24 h after freeze-dried process)(Fig. 3). Results showed that the SCs were more resistantand more stable than the unstressed ones. After 90 days ofstorage, there was no significant difference between the SCs,while the UCs showed a statistically significant decrease intheir viability.

Antagonistic Activity of Freeze-dried C. laurentii CellsAgainst B. cinerea on Apples in Cold Storage

After 40 days of cold storage, apples inoculated withB. cinerea and protected with freeze-dried C. laurentii cells(UC and SC) were examined, and the lesion diameter wasregistered to calculate percentage of decay reduction. Freshcells (UC and SC) were used as control. Results are shown inFig. 4. Decay reduction percentage was of 91.62 % whenapples were protected with fresh cells, and there was no

Fig. 1 Effect of cold stress on intracellular trehalose content ofC. laurentii. Data from trehalose content were compared in a Student’s ttest. Significance was judged at level P<0.05

Fig. 2 Effect of preservationmedia on viability of C. laurentiiBNM 0525(SC and UC) afterfreeze-drying. Differences inpercentages were analyzed byone-way analysis of variance(ANOVA) followed by multiplecomparison test of Tukey(P<0.05)

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significant difference between SCs and UCs.When protectionwas performed with freeze-dried cells, the maximum protec-tion was obtained with SC. In this case, decay reductionpercentage was 79.30 %, and a similar result was obtainedwith freeze-dried SC (no significant differences) stored for90 days. UCs were less effective immediately after the freeze-dried process (69.12%) and less resistant to storage. As can beseen in Fig. 4, decay reduction percentage was less to 60 %when applied with freeze-dried UC with storage of 90 days.

Population Dynamics of C. laurentii

Assay of population dynamics in apple wounded at 4 °Cshowed an evident difference in the lag period and a

significant difference in number of cells between UCs andcold SCs. The lag period for UCwas 12 days while for SC, thelag period was only 7 days (Fig. 5).

Discussion

The cold stress ofC. laurentiiBNM 0525 improved its survivaland effectiveness against B. cinerea perhaps because to theincrement in intracellular trehalose. Different authors havereported accumulation of trehalose in various microorganismsin response to heat shock and osmotic shock (Kandror et al.2004) or induced by a culture medium formulated with citricacid (Li and Tian 2006) or trehalose ( Kandror et al. 2002; Liet al. 2008). Also, trehalose is considered as a suitable protectantfor liquid formulation of C. laurentii. Moreover, combiningL-ascorbic acid with this sugar improved the protectiveefficiency (Liu et al. 2009). Adaptation to cold of differentmicroorganisms, among them yeasts, is manifested with theaccumulation to intracellular trehalose (Kandror et al. 2004)demonstrated an adaptive response in yeasts that was activatedbelow 10 °C and increased tolerance to low temperatures andfreezing. This response involved induction of trehalose-synthesizing enzymes. Aguilera et al. (2007) revised themechanisms of response to cold stress in Saccharomycescereviseae, among them, the induction of enzymes relatedwith trehalose synthesis. However, so far, there were noreported works with induction of trehalose as a response tocold stress in C. laurentii as we report here. In the presentstudy, the maximum accumulation of trehalose in cold SCswas reached at 24 h, and the level was similar to one reportedby Li and Tian (2006). An interesting fact was the decrease ofthis sugar after 24 h which coincided with the beginning of

Fig. 3 Stability of freeze-dried C. laurentii cells (SC and UC). Differ-ences in percentages were analyzed by one-way analysis of variance(ANOVA) followed by multiple comparison test of Tukey (P<0.05)

Fig. 4 Antagonistic activity of freeze-dried C. laurentii cells (SC andUC) against Botrytis cinerea on apples in cold storage (40 days at 4 °C).Differences in percentages were analyzed by one-way analysis of vari-ance (ANOVA) followed by multiple comparison test of Tukey (P<0.05)

Fig. 5 Population dynamics ofC. laurentiiBNM0525 (SC andUC; 24 hafter freeze-dried process) on the surface of wounded apples incubated at4 °C for 35 days

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growth in cold conditions while at this same time, at 28 °C, theyeast reached the stationary phase (data not shown). Studiescarried out with the yeast S. cerevisiae determined that thebiological function of trehalose is protecting certain cellstructures such as membranes and proteins in the cell cytosolwhen culture conditions are not optimal. Furthermore, thesugar is hydrolyzed during the budding which would meanthat it also has a function as reserve carbohydrate for thereproduction of the yeast (Hernández-Oropeza and Aranda-Barrada 2011). Stability of SC was significantly higher along90 days of storage at 4 °C than that of UCs. In fact, cold stresssignificantly improved the survival of the yeast. Also,effectiveness against B. cinereawas improved. The best resultswere obtained with SC in SMGYas protecting medium. Theseresults suggested two important points to consider: The firstpoint is related to the internal accumulation of trehaloseinduced by adaptation to coldwhich significantly improved thesurvival and the ability of cells for growth in the apple wounds,decreasing the lag period and increasing the number of cellsreached after 35 days of incubation at 4 °C. Consequently, theeffectiveness against B. cinerea was improved since the mainmode of action of yeasts as biological control agents iscompetition for space and nutrients (Sharma et al. 2009).The second point is the nature of the cryoprotectant used.SMYG is a mixture of cryoprotectants, which showed tobe a good vehicle for using in lyophilized formulationsof biological control agents since similar results wereobtained previously when SMYG was used for freeze-dried process of Rahnella aquatilis (Navarta et al. 2011).

Conclusions

The results reported here showed a promising way to preserveantagonist yeast as C. laurentii for commercial application.The cold stress combined with SMYG as cryoprotectantseems to be a good tool for reaching an adequate stability oflyophilized formulations of the yeast for application to bio-logical control of B. cinerea. Moreover, adaptation to coldshowed to be a good strategy for improving the effectivenessof C. laurentii as a controller of this fungus.

Acknowledgments The support by the Universidad Nacional de SanLuis, the Agencia Nacional de Promoción Científica y Tecnológica, andthe Consejo Nacional de Investigaciones Científicas y Técnicas(CONICET) is gratefully acknowledged.

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