Biologicals 35 (2007) 265e269www.elsevier.com/locate/biologicals

Freeze-drying of live attenuated Vibrio anguillarum mutantfor vaccine preparation

Lu Yang, Yue Ma, Yuanxing Zhang*

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China

Received 5 September 2006; revised 2 March 2007; accepted 2 March 2007

Abstract

Vibrio anguillarum MVAV6203 is a mutant strain as a candidate of live attenuated vaccine. In vaccine preparation, the freeze-dryingconditions of the strain were investigated to improve the survival after freeze-drying, including the protectant, rehydration medium, freezingtemperature, and initial cell concentration. Vibrio anguillarum MVAV6203 is sensitive to freeze-drying and the viability was only 0.03% inthe absence of protectant. Of the tested protectants, 5% trehalose with 15% skimmed milk gave the highest viability of 34.2%. Higher cell sur-vival was obtained by quick freezing at �80 �C than slow freezing at �20 �C. Initial cell concentration was another important factor, preferablefor 1e3 � 1010 CFU/ml. The supplementation of 10% skimmed milk in rehydration medium improved obviously freeze-drying viability. Thecombination of the optimal conditions achieved 51.4% cell viability after freeze-drying.� 2007 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

Keywords: Freeze-drying; Vibrio anguillarum; Protectant; Viability; Vaccine

1. Introduction

Vibrio anguillarum is an important fish pathogen [1], whichcauses a high fatal hemorrhagic septicemia in many fish spe-cies, like Atlantic salmon (Salmo salar L.) and rainbow trout(Oncorhynchus mykiss), halibut (Paralichthys olivaceus) andJapanese seaperch (Lateolabrax japonicus). It often leadsfish to death in large scales and results in substantial econom-ical losses in aquaculture. Therefore, using vaccine to preventfish from vibriosis is important. V. anguillarum MVAV6203 isa live attenuated mutant strain which was constructed by ourlaboratory [2].

Freeze-drying is widely used to preserve bacteria both inresearch and industry. However, damages will be producedduring the processes of freezing, drying, and storage, which re-sult in the death of cells [3e6]. In general, the freeze-dryingviability is species-dependent [7]. Furthermore, many factors

* Corresponding author. Tel.: þ86 21 6425 3065; fax: þ86 21 6425 3025.

E-mail address: yxzhang@ecust.edu.cn (Y. Zhang).

1045-1056/07/$32.00 � 2007 The International Association for Biologicals. Pub

doi:10.1016/j.biologicals.2007.03.001

also have important effects on freeze-drying viability, includ-ing protectants [8e10], freezing temperature [11,12], initialcell concentration [5,13], freeze-drying parameters, and rehy-dration conditions [14e16].

Protectants play a significant role in the conservation ofviability and an appropriate protectant should prevent cellsagainst freeze-drying damage and improve the stability of stor-age. Generally, freeze-drying protectants include non-reducingdisaccharides, sugar alcohols, amino acids, and complex mix-tures such as skimmed milk [9,14].

The initial cell concentration has an important influence onfreeze-drying viability. Most of the bacteria have an optimalinitial cell concentration. In recent research, it has been re-ported that increasing cell concentration (up to 1011 CFU/ml)leads to higher survival rate [5,15].

Freezing rate affects the viability directly when bacteriawere frozen. Water will flow out of the cell by osmosis, form-ing ice crystals extracellular with slow cooling rate, and thenremoved in the process of drying. Thus, the extracellular andintracellular osmotic pressures become imbalanced. In con-trast, rapid cooling leads to the formation of intracellular ice

lished by Elsevier Ltd. All rights reserved.

266 L. Yang et al. / Biologicals 35 (2007) 265e269

crystals, which cause lethal damage on cell [4]. The regulationof freezing rate is crucial in freeze-drying.

Cells are subjected damage in a certain extent in the pro-cess of freeze-drying, though many of them are not dead.These sublethal injuries can be repaired under appropriate re-hydration condition [17]. Rehydration media can effectivelycontrol the rate of recovery to keep the normal physiologicalstate of cells [18,19].

In order to obtain a stable freeze-drying product of Vibrioanguillarum, the effects of protectants, freezing rate, initialcell concentration, and rehydration medium on the stabilityof preserved V. anguillarum MVAV6203 were studied in thework.

2. Materials and methods

2.1. Organism and growth

Vibrio anguillarum MVAV6203, a deletion mutant straindeprived of siderophore synthesis related genes, was con-structed in our laboratory and cultivated in Luria broth (LB)with 2.5% (w/v) NaCl at 28 �C. When the cells had grownup to early stationary phase, the bacterial cells were harvestedby centrifugation at 5000 � g for 20 min at 4 �C, washed inpH 7.4 phosphate buffer containing 2.5% NaCl, and subse-quently suspended in freeze-drying protectants or in distilledwater as control.

2.2. Freeze-drying

Serum bottles (10 ml) were filled with 3 ml of bacterial sus-pension produced as described above and frozen at �80 �C.Then, the frozen samples were freeze-dried at a condensertemperature �45 �C, and chamber pressure 0.15 mbar for20 h in a freeze-dryer (ULVAC, Japan). The final sample tem-perature was 28 �C. After freeze-drying, the serum bottleswere sealed under vacuum.

2.3. Preparation of protectants

Trehalose (Stream Co., Ltd, Japan), sucrose, lactose, man-nitol (Lingfeng Chemical Reagent Co., China), skimmed milk(Guangming Milk Co., China) and serum (Hangzhou SijiqingBiotechnology Co., China) were mixed with distilled waterwith certain concentrations and autoclaved at 121 �C for15 min before mixing with a volume of washed cells of theV. anguillarum.

2.4. Rehydration medium

Ten percent (w/v) sucrose, 10% (w/v) trehalose, 5% (w/v)tryptone (Oxoid), 1% (w/v) tryptone, 1% (w/v) yeast extract(Oxoid), 10% (w/v) skimmed milk, LB medium, phosphatebuffer containing 0.80% (w/v) Na2HPO4, 0.14% (w/v)KH2PO4 and 2.5% NaCl, and distilled water were used as re-hydration media, respectively. The freeze-dried V. anguillarumsample were rehydrated to original volume (3 ml) with each

rehydration medium. Rehydrated samples were incubated at25 �C for 15 min before calculating the viable cells.

2.5. Freezing temperature

Five percent trehalose þ15% skimmed milk was used asthe protectant. The sample, with an initial bacterial con-centration of 7.4 � 109 CFU/ml, was equally dispensed intotwo sterile serum bottles. One bottle was directly frozen at�20 �C for 24 h. Another was frozen at �80 �C for 4 h andthen transferred to �20 �C where samples were kept for24 h. The viabilities after freezing and freeze-drying were cal-culated respectively.

2.6. Preparation of cell suspension

When the cells were harvested by centrifugation at 5000 � gfor 20 min at 4 �C, washed the bacterial cells in phosphatebuffer (pH 7.4) containing 2.5% NaCl, and subsequently sus-pended in 5% trehaloseþ15% skimmed milk protectant to orig-inal volume. Suspension was 2-fold diluted and concentratedrespectively, using the same protectant as solutions. The numberof viable cells was determined before each sample was freeze-dried. Then viabilities after freeze-drying were calculated.

2.7. Determination of cell viability

The number of viable cells before freezing, after freezingand freeze-drying was determined as colony forming units(CFU). Samples were diluted in phosphate buffer (pH 7.4)containing 2.5% NaCl (before freezing) or rehydration me-dium (after rehydration) through 10-fold serial dilutions. Sub-sequently, 100 ml diluted cell suspension was plated onto thesurface of LB plate with 2.5% NaCl and 2.0% agar (10-cmdiameter) and distributed on the agar by using sterile glassspreader per plate. After 48-h incubation at 28 �C, the numberof colonies was determined. Cell viability was calculated asCFU/ml after freezing or freeze-drying divided by CFU/ml be-fore freezing (initial cell concentration), multiplied by 100 andexpressed as percentage viability.

2.8. Reproducibility

All results presented in this work are the average of threeindependent replicate assays. Standard deviations were calcu-lated and there were significant differences ( p < 0.05) be-tween different samples according to T test.

3. Results

3.1. Selection of protectant

V. anguillarum MVAV6203 show different viabilities withvarious protectants (Table 1). The viability was only 0.03%in the absence of protectant and significantly enhanced by us-ing protectants of all kinds. The viability of 6.7% in 20%skimmed milk was the lowest of all protectants. Trehalose

267L. Yang et al. / Biologicals 35 (2007) 265e269

had a better protection than the two other disaccharides (su-crose and lactose). Three combined disaccharides resulted insimilar freeze-drying viability, but significantly better eachof them used individually, although the best protectant was10% trehalose þ20% skimmed milk. Mannitol and serum incombination had no significant effect on protection of V. an-guillarum MVAV6203.

The concentrations of trehalose and skimmed milk werefurther optimized to improve freeze-drying viability (Fig. 1).The highest viability, 34.2%, was obtained in 5% trehaloseþ15% skimmed milk, whereas concentrated trehalose washarmful to cell survival.

3.2. Effect of freezing temperature on the survival afterfreezing and freeze-drying

Different freezing temperature caused different cooling rateand finally resulted in diverse viabilities. The effects of

Table 1

With different protectants, the viability of V. anguillarum MVAV6203 after

freeze-drying and rehydrating with phosphate buffer

Protectant Concentration (%) Viability (%) � S.D.

Control d 0.03 � 0.02

Sucrose 10 11.7 � 0.6

Trehalose 10 13.2 � 0.8

Lactose 10 9.3 � 0.7

Skimmed milk 20 6.7 � 1.3

Trehaloseþ 10 27.6 � 0.2

skimmed milk 20

Trehaloseþ 10 27.2 � 0.2

skimmed milkþ 20

mannitol 3

Trehaloseþ 10 26.8 � 0.3

skimmed milkþ 20

serum 5

Initial cell concentrations were in the range of 6e8 � 109 CFU/ml.

40

30

20

10

010 15 20

Fig. 1. Freeze-drying viabilities versus trehalose and skimmed milk concentra-

tions. The initial cell concentration was in the range of 6e8 � 109 CFU/ml

and freeze-drying products were rehydrated with phosphate buffer. Trehalose

concentration: white 5%, gray 10%, and black 15%.

freezing temperature on the viability of V. anguillarumMVAV6203 are showed in Table 2. Survival after freezingand freeze-drying was higher when the cells were frozen at�80 �C compared to �20 �C.

3.3. Effect of initial cell concentration

Initial cell concentrations ranging from 1.6 � 108 to8 � 1010 CFU/ml were investigated to determine their influ-ences on survival of V. anguillarum MVAV6203 after freeze-drying (Fig. 2). High viability was obtained when the initialcell concentrations ranged from 1 to 3 � 1010 CFU/ml, andthe highest survival was 45.3% with a cell concentration of1.6 � 1010 CFU/ml. However, the viability decreased sharplywhen cell concentrations were below 1.0 � 1010 CFU/ml andabove 3.0 � 1010 CFU/ml.

3.4. Effect of rehydration medium

Samples were rehydrated to the original volume (3 ml)after freeze-drying in different rehydration media. Therewas significant difference in the viability of V. anguillarumMVAV6203, depending on the rehydration medium used(Table 3). Rehydration in 10% skimmed milk gave the bestcell restoration of 51.4% among the rehydration media tested.In contrast, the survival rate was only 29.4% when a samplewas rehydrated in distilled water control. Other rehydrationmedia showed different enhancements of viability.

4. Discussion

In this study, the effects of protectant, freezing temperature,initial cell concentration, and rehydration medium on V.anguillarum MVAV6203 viability after freeze-drying wereinvestigated. Disaccharides, as common components of pro-tectants, are capable of hydrating membranes, and then replacethe structural water, which is removed in the process of drying[20,21]. In addition, disaccharides prevent proteins from un-folding and aggregate by formation of hydrogen bonds [22].Mixtures, such as skimmed milk, contain many kinds of aminoacids. The protection of amino acids was thought to be the re-sult of a reaction between the carboxyl groups of the bacterialproteins and the amino group of the protectant, stabilizing theproteins structure [23]. Different bacteria need different pro-tectants. Pseudomonas chlororaphis was shown to have high-est freeze-drying viability of 25% when 10% sucrose was usedas the protectant, however, the survival rate was only 4.3% in

Table 2

At different freezing temperatures the viabilities of V. anguillarum

MVAV6203 after freezing and freeze-drying

Freezing

temperature

Viability after

freezing (%) � S.D.

Viability after freeze-

drying (%) � S.D.

�80 �C 94.1 � 1.2 33.2 � 1.1

�20 �C 78.6 � 1.6 26.8 � 0.9

Freeze-dried samples were rehydrated with phosphate buffer.

268 L. Yang et al. / Biologicals 35 (2007) 265e269

5% trehalose þ15% skimmed milk as protectant [5]. In thisstudy, mixtures of trehalose and skimmed milk as protectantsresulted in higher freeze-drying viability of V. anguillarumMVAV6203 than disaccharides only or skimmed milk. Similarresults have been obtained for the yeast Candida sake [14],and the highest freeze-drying viability was given in 5% treha-lose þ15% skimmed milk as protectant.

The freezing process involves complex courses that havenot been completely understood. Ice crystals will form whencells are frozen, no matter what cooling rate is. On onehand, rapid freezing leads to the formation of intracellularice crystal whose stress could damage the membrane. On theother hand, slow freezing results in the imbalance of intracel-lular and extracellular osmotic stress, which is also fatal [4]. Inthis study, quick freezing at �80 �C, showed higher viabilityof V. anguillarum MVAV6203 than slow freezing at �20 �Cboth after freezing and freeze-drying. According to this result,the influence of osmotic stress might outweigh that of ice crys-tal stress, and rapid freezing could maintain the viability effec-tively. Lactobacillus brevis and Oenococcus oeni also showedhigh freeze-drying survival when the cells were quickly frozenat �65 �C [16].

50

40

30

20

108 9 10 11

Log (CFU)

Fig. 2. Freeze-drying survival at different initial cell concentrations. Protec-

tants were 5% trehalose þ15% skimmed milk. Phosphate buffer was used

as the rehydration medium.

Table 3

Effect of rehydration medium on the viability of V. anguillarum MVAV6203

freeze-dried in 5% trehalose þ15% skimmed milk as protectant

Rehydration media Viability (%) � S.D.

10% Trehalose 36.2 � 0.7

10% Sucrose 39.1 � 1.2

5% Tryptone 48.4 � 2.3

1% Tryptone 34.7 � 1.0

1% Yeast extract 42.6 � 0.8

10% Skimmed milk 51.4 � 0.6

LB medium 31.8 � 1.6

Phosphate buffer 41.2 � 0.8

Distilled water 29.4 � 1.1

Initial cell concentrations were in the range 1.5e2 � 1010 CFU/ml.

Generally, different freeze-drying viability corresponds todifferent initial cell concentrations and most of the bacteriahave an optimal range of initial cell concentration. The experi-mental results indicated that the optimal initial cell con-centration of V. anguillarum MVAV6203 was between 1 and3 � 1010 CFU/ml when 5% trehalose þ15% skimmed milkwas used as protectant. The optimal initial concentration mightbe related to the species. The highest freeze-drying viability ofPseudomonas chlororaphis (15e26%) was obtained for initialcell concentrations between 1 � 109 and 1 � 1010 CFU/ml [5].

Rehydration is an important step, in which rehydration me-dium always plays the significant role. Similarly, the effect ofrehydration medium on freeze-drying viability is dependent onthe species. In most cases, mixtures, such as skimmed milkand tryptone, could provide various nutrients that facilitatethe recovery of cells after freeze-drying. Ray et al. presumedthat rehydration in a high osmotic pressure solution controlsthe rehydration rate and thus reduces possible damage [19].Candida sake showed high freeze-drying viability when usedthe 10% skimmed milk as hydration medium [14]. Many othercomplex rehydration media, just like peptone, tryptone andyeast extract, also conduced to good results with the Strepto-coccus lactis and Leuconostoc lactis [17]. Similar result wasobtained in this study, 10% skimmed milk was the best inall rehydration media tested, and others had different improve-ments on survival as compared with distilled water.

In conclusion, the survival of V. anguillarum MVAV6203 isdependent on protectant, freezing temperature, initial cell con-centration, and rehydration media. An appropriate selection ofthese factors is essential for obtaining the maximum viabilityof cells. A viability as high as 51.4% after freeze-drying of V.anguillarum MVAV6203 was achieved when cells, in the opti-mal initial concentration range, were frozen at �80 �C, 5%trehalose þ15% skimmed milk used as protectant and 10%skimmed milk as rehydration medium.

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