drozdzeCandida liofilizacja dodatki.pdf

International Journal of Food Microbiology 65 2001 173–182

www.elsevier.nlrlocaterijfoodmicro

Effect of freeze drying and protectants on viability of the

biocontrol yeast Candida sake

´ ˜

Posthar Õ est Unit, CeRTA Centre UdL-IRTA, 177 Ro Õ ira Roure A Õ enue, 25198, Lleida, Catalonia, Spain.

M. Abadias ) , A. Benabarre, N. Teixido, J. Usall, I. Vinas

Received 10 June 2000; received in revised form 29 September 2000; accepted 29 November 2000

Abstract

freeze drying using 10% skim milk as a protectant 28.9% survival . Liquid nitrogen freezing caused the highest level of

damage to the cells with viability -10%. Different concentrations of exogenous substances including sugars, polyols,

polymers and nitrogen compounds were tested either alone or in combination with skim milk. There was little or no effect

when additives were used at 1% concentration. Galactose, raffinose and sodium glutamate at 10% were the best protective

agents tested alone but the viability of freeze-dried C. sake cells was always -20%. Survival of yeast cells was increased

from 0.2% to 30–40% by using appropriate protective media containing combinations of skim milk and other protectants

Keywords: Viability; Survival; Preservation; Protectants; Formulation; Freeze drying

1. Introduction

pathogens of apples and pears Teixido et al., 1998;

˜ To be of practical use, microbial agents must be

formulated as products capable of storage, distribu-

tion and application in the agricultural marketplace,

requiring different approaches from traditional agro-

Biological control using microbial antagonists has

attracted much interest as an alternative to chemical

methods of controlling pre- and post-harvest plant

pathogens and pests of agricultural and horticultural

chemical product design Rodham et al., 1999 . For-

mulation is necessary in order to present the product

in a usable form and in order to optimize the effi-

cacy, stability, safety and ease of application of the

1989; Wilson and Wisniewski, 1989 . Recent studies

have shown that a strain of Candida sake K. Saito

and M. Ota N. van Uden and H.R. Buckley CPA-1

is an effective antagonist to the major fungal

product Rhodes, 1993 .

Freeze drying is the most convenient and success-

ful method of preserving bacteria, yeasts and spo-

rulating fungi Berny and Hennebert, 1991 . The

advantages of freeze drying are protection from con-

tamination or infestation during storage, long viabil-

) Corresponding author. Tel.: q34-973-702-647; fax: q34-973-

238-301.

E-mail address: isabel.abadias@irta.es M. Abadias .

ity and ease of strain distribution Smith and Onions,

Ž .

The effects of freezing method, freeze drying process, and the use of protective agents on the viability of the biocontrol

yeast Candida sake were studied. Freezing at y208C was the best method to preserve the viability of C. sake cells after

Vinas et al., 1998; Usall et al., 2000 .

crops Janisiewicz, 1988, 1990; Wilson and Chalutz,

PII: S 0168-1605 00 00513-4

174

M. Abadias et al. r International Journal of Food Microbiology 65 2001 173–182

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)

1983 . However, not all strains survive the process

and, among those surviving, quantitative viability

2. Materials and methods

rates as low as 0.1% have been reported Atkin et

al., 1949; Kirsop, 1955; Smith and Onions, 1983;

2.1. Yeast

Berny and Hennebert, 1991 . Substances such as

polymers, sugars, albumin, milk, honey, polyols and

amino acids have been tested for their protective

The yeast used in this study was the strain CPA-1

of C. sake obtained from UdL-IRTA Catalonia,

Spain . It is deposited in Coleccion Espanola de

effect during freeze drying e.g., Pedersen, 1965;

Meryman et al., 1977; Womersley, 1981; Font de

Cultivos Tipo, CECT-10817 Universidad de Valen-

Valdez et al., 1983a . Skim milk has been used alone

cia, Campus de Burjasot, Burjasot, Valencia, Spain .

Heckly, 1961 or with other compounds Butterfield

et al., 1974; Smith and Onions, 1983; Berny and

2.2. Cultures

Hennebert, 1991 . Components of the suspending

media have two main functions in preserving viabil-

ity of freeze-dried cells. The first is to provide a dry

residue with a definite physical structure acting as a

support material and as a receptor in rehydration, and

the second is to protect the living cells biochemically

Stock cultures were stored at 48C and had been

subcultured on nutrient yeast dextrose agar NYDA ,

which contained nutrient broth, 8 g l

Biokar

Diagnostics, BK003, Beauvois, France ; yeast ex-

tract, 5 g l , Biokar Diagnostics, 112002 ; dex-

trose, 10 g l , Rectapur, 24 379.294, Prolabo,

against damage during freezing andror drying Berny

Fontenay SrBois, France and agar, 15 g l

and Hennebert, 1991 . These workers found that by

using skim milk as a support material in combination

with two compounds from honey, sodium glutamate,

trehalose or raffinose, the viability of Saccha -

romyces cere Õ isiae cells was increased from 30% to

96–98%.

The cooling rate of the cells during the cooling

phase is a critical factor in the freeze drying process.

The optimum cooling rate appears to be that at

which the cells do not lose water and reaches the

NOKO, RG-99112318, Asturias, Spain .

2.3. Cell production

broth NYDB , which was NYDA without agar.

Cultures were grown in a 5-l fermentor Gallenkamp,

Loughborough, Leicestershire, UK containing 4 l of

medium at 25"18C with constant stirring and aera-

tion for 38 h. Cells were harvested at the beginning

of the stationary phase by centrifugation at 8315= g

for 10 min at 108C in an Avantie J-25 centrifuge

eutectic frozen point in an amorphous state Berny

and Hennebert, 1991 . If cooling is slow enough,

water will have time to flow out of the cell by

osmosis dehydrating the cell and thus avoiding freez-

ing. If the cells do not lose water quickly enough to

maintain equilibrium, ice crystals eventually form

Beckman; Palo Alto, CA . The growth medium was

decanted and the cell paste resuspended in 10–15 ml

of potassium phosphate buffer PB, 70 ml 0.2 M

intracellularly Mazur, 1977 .

Few studies have been carried out to evaluate the

efficacy of such treatments for conserving viability

of cells of postharvest biological control agents such

as C. sake . This is critical since a high cell concen-

tration is necessary in order to obtain a good formu-

lated product for commercial application. Thus, the

objectives of this study were to compare the effi-

ciency of different freezing methods, and compare

the additions of a range of individual additives and

combinations with powdered skimmed milk as pro-

tectants for preserving the viability of C. sake cells

during freeze drying.

KH PO Rectapur, 26 923.298, Prolabo q30 ml

0.2 M K HPO Rectapur, 26 930.293, Prolabo , pH

2 4

6.5, and centrifuged again. The resulting cell paste

was stored at 48C and used the same day. Usually,

about 15 g of cell paste with a moisture content of

about 75% wrw were obtained per liter of NYDB

medium.

2.4. Freezing treatments

C. sake cells were produced as described above

and the cell paste was then resuspended in powdered

Ž w

The growth medium was nutrient yeast dextrose

skimmed non-fat milk SM, Sveltesse , Nestle Es-

M. Abadias et al. r International Journal of Food Microbiology 65 2001 173–182

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175

used as a control. Ten-fold dilutions of this suspen-

sion were made and spread plated in duplicate onto

the surface of 9-cm Petri plates in order to calculate

the initial concentration. Plates were incubated at

25"18C for 48 h and the initial number of colony

in three autoclaved vials and were frozen at y208C

overnight. Then samples were connected to the

freeze-drier for 24 h. Each sample of freeze-dried C.

sake cells was rehydrated and plated as described

above. The percentage of survival was determined

from the viable yeast counts made before and after

freeze drying.

forming units per mililiter CFU ml

y1 .

was calcu-

lated.

Thereafter, 5-ml samples were distributed in 24

Suspensions of sugars glucose, fructose, galac-

tose, trehalose, lactose, sucrose and raffinose ; poly-

autoclaved vials 10 ml, Serum Type Reaction Vial,

ols glycerol, mannitol, sorbitol, inositol and ado-

Supelco, Bellefonte, PA in order to evaluate the

effect of four different freezing methods on C. sake

viability. The first and second methods consisted of

freezing directly at y128C and y208C, respectively,

and maintaining the samples at these temperatures

overnight. Progressive freezing consisted of refriger-

ating samples at 48C for 2 h, then freezing at y128C

for 8 h, and maintaining the samples at y208C

overnight. Liquid nitrogen freezing consisted in sub-

Ž.

nitol ; polymers dextran, starch and polyethylene

glycol 200 PEG ; peptone and sodium glutamate

were made with de-ionized water. Concentrations of

1%, 5% and 10% wrv were prepared. Moreover, a

10% suspension of SM was studied in order to

compare with the other treatments.

Solutions were autoclaved at 1158C for 15 min,

except adonitol, which was sterilised by filtration

using a 0.20-mm diameter filter GS Type, Millipore,

merging the samples in liquid nitrogen N and then

maintaining the cells at y208C overnight. There-

after, three of the six samples of each treatment were

thawed at room temperature and viability was calcu-

lated by the standard plate count method as de-

scribed above in order to evaluate the effect of

freezing on C. sake cell viability. The other three

Ireland .

Ž.

D q -Sucrose, anhydrous D q -glucose, starch

Ž.

and glycerol 98% were provided by Rectapur Pro-

.Ž. Ž.

labo ; D q -galactose, D q -raffinose pentahydrate,

Ž.

D -mannitol, D q -trehalose dihydrate, dextran,

meso-inositol and adonitol by Sigma-Aldrich, Stein-

. Ž .

heim, Germany ; D y -fructose, lactose-1-hydrate,

Ž.

vials were connected to a freeze-drier Cryodos,

PEG200, D y -sorbitol and sodium L -glutamate-1-

Telstar, Terrassa, Spain operating at 1 Pa and y458C

for 24 h. Each sample of freeze-dried C. sake was

hydrate by Panreac Quımica, Montcada i Reixac,

Barcelona, Spain .

Glucose, fructose, galactose, trehalose, sucrose,

lactose, raffinose, glutamate, dextran, starch, sorbitol

rehydrated to its original volume 5 ml with PB for

10 min at room temperature, and then CFU ml y1

was determined as described above. The survival

level was determined for frozen, and freeze-dried

cell treatments, and quantitative comparisons made

CFU ml before and after freezing and freeze

drying, respectively. The experiments were all re-

peated twice.

Table 1

Viability of C. sake cells after freezing and freeze drying with

different methods using 10% of skim milk as a protective agent

Ž.

Viability %

After freezing

2.5. Effect of protecti Õ e agents

After freeze drying

Freeze

Mean

Standard

Mean

Standard

Cell paste obtained by centrifugation as described

above was resuspended in the protective medium to

make a total volume of 10% of the initial one,

containing approximately 1.0=10 9 to 5.0=10 9

CFU ml y1 . The initial cell concentration of each

protective suspension was calculated by the standard

method

deviation

y128C

89.7

3.4

19.3

4.1

y208C

85.1

5.3

28.9

1.1

Progressive

71.3

1.1

20.7

1.9

Liquid nitrogen

52.1

4.5

9.2

2.1

plate count method. Aliquots 5 ml were distributed

Means with different letters within columns are significantly

pana, Barcelona, Spain at 10% wrv which was

different according to Duncan’s Multiple Range Test P -0.05 .

176

M. Abadias et al. r International Journal of Food Microbiology 65 2001 173–182

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and adonitol were combined at 5 and 10% wrv

3. Results

with SM at 5 and 10% wrv concentration.

3.1. Comparison of freezing treatments

2.6. Data analysis

The resulting colonies from samples taken before

and after freeze drying were counted and the per-

centages of surviving C. sake were also estimated.

Percentages of viability were analysed by a General

y128C and y208C, with survival )85% Table 1 .

After freeze drying, the viability of the yeast cells

was drastically decreased to -30% in all cases,

with the best results obtained at the y208C freezing

treatment. The viability of the cells after freezing,

and freeze drying when the cells were frozen with N 2

Lineal Model GLM procedure with SAS software

SAS Institute, version 6.12, Cary, NC, USA . Statis-

tical significance was judged at the level P s0.05.

When the analysis was statistically significant, Dun-

can’s Multiple Range Test was used for separation of

means.

was significantly lower P -0.05 than that obtained

with the other freezing methods. Consequently, this

method was rejected and freezing at y208C was

Table 2

Viability of C. sake % after freeze drying using different concentrations of protective agents dissolved in deionized water

Concentration, % wrv

Ž.

10%

Protectant

Mean

Standard

Mean

Standard

Mean

Standard

deviation

Monossaccharides

Glucose

0.2

def

0.0

2.8

0.3

8.6

1.2

Fructose

0.2

def

0.1

4.3

0.4

5.9

0.7

Galactose

1.0

0.3

6.4

1.0

16.6

6.5

Dissaccharides

Sucrose

0.7

cdef

0.2

6.2

0.7

11.4

1.4

Lactose

0.8

cde

0.2

5.9

0.6

12.2

2.7

Trehalose

1.5

0.0

6.1

1.8

12.5

1.4

Tri-saccharides

Raffinose

1.7

0.5

13.2

1.6

19.1

3.6

Polymers

Dextran

1.7

0.9

3.4

0.8

3.7

1.5

Starch

2.1

0.5

7.0

3.0

11.9

2.0

PEG

-0.1

0.2

0.1

0.2

0.1

Polyols

Glycerol

0.3

cdef

0.1

0.3

0.4

Mannitol

0.1

1.0

0.3

6.4

1.0

Sorbitol

0.6

cdef

0.1

2.6

0.4

13.0

1.7

Adonitol

0.8

0.6

1.5

0.2

8.6

0.7

Inositol

0.6

cdef

0.5

-0.1

Nitrogen compounds

Glutamate

0.6

cdef

0.2

5.4

0.4

19.8

1.3

Peptone

-0.1

Different letters within the same column indicate that means are significantly different P -0.05 according to a Duncan’s Multiple Range

Test.

The highest viability of C. sake cells after freez-

ing was obtained when samples were frozen at

M. Abadias et al. r International Journal of Food Microbiology 65 2001 173–182

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177

chosen as the best treatment with which to carry out

further experimentation.

additives were all ineffective in conserving viability.

At 5%, the best protective agent was raffinose, with

13.2% of cells remaining viable. Best viabilities

were observed with 10% glutamate, raffinose and

galactose, with 19.8%, 19.1% and 16.6% survival,

respectively.

Among the sugars, the trisaccharide raffinose was

found to be the best protectant giving cell viabilities

of 13.2% and 19.1% at concentrations of 5% and

10%, respectively. No significant differences were

3.2. Effect of protecti Õ e agents

The additives tested as protective agents against

freeze drying in this study may be divided in four

groups: sugars, polyols, polymers and nitrogen com-

pounds. In this experiment all additives were tested

using deionized water as a suspending medium. Af-

ter freeze drying, cell viability in the absence of the

protective agents was very low, 0.2% and 0.3% for

observed P )0.05 between the dissacharides su-

crose, lactose and trehalose at both of the higher

concentrations tested. Galactose was the best

monosaccharide treatment tested and showed statisti-

cally higher survival of C. sake cells than glucose

and fructose. At 10% concentration, raffinose and

galactose showed the best protective effect, signifi-

cantly different from the other sugars.

The protective effect of polyols was in general

deionized water and PB, respectively data not

shown . The viability of C. sake cells freeze dried in

10% SM was 22% data not shown . Results with the

additives are shown in Table 2.

An increase in viability was observed when the

concentration of the protective agent was increased

from 1% to 10%, except for dextran, glycerol, inosi-

tol, PEG and peptone. At 1% concentration, the

lower than that obtained with sugars. Sorbitol 10%

Fig. 1. Viability of C. sake cells after freeze drying using combinations of: A glucose, B fructose, C trehalose and D sucrose at 0%

Ž. Ž . Ž.

Ž. Ž. Ž.

Ž.

I , 5% or 10% B with SM. Within the same figure, different letters indicate significant differences P -0.05 according to

Duncan’s Multiple Range Test.

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