Dry formulations of the biocontrol agent Candida sake CPA-1 using fluidised bed drying to control the main postharvest diseases on fruits

BACKGROUND
The biocontrol agent Candida sake CPA-1 is effective against several diseases. Consequently, the optimisation of a dry formulation of C. sake to improve its shelf life and manipulability is essential for increasing its potential with respect to future commercial applications. The present study aimed to optimise the conditions for making a dry formulation of C. sake using a fluidised bed drying system and then to determine the shelf life of the optimised formulation and its efficacy against Penicillium expansum on apples.


RESULTS
The optimal conditions for the drying process were found to be 40 °C for 45 min and the use of potato starch as the carrier significantly enhanced the viability. However, none of the protective compounds tested increased the viability of the dried cells. A temperature of 25 °C for 10 min in phosphate buffer was considered as the optimum condition to recover the dried formulations. The dried formulations should be stored at 4 °C and air-packaged; moreover, shelf life assays indicated good results after 12 months of storage. The formulated products maintained their biocontrol efficacy.


CONCLUSION
A fluidised bed drying system is a suitable process for dehydrating C. sake cells; moreover, the C. sake formulation is easy to pack, store and transport, and is a cost-effective process. © 2017 Society of Chemical Industry.

Accepted Article assay with detached grape berries 10 and in field applications, different biologically based strategies significantly reduced the incidence and severity of botrytis rot under Mediterranean conditions 11 and under Atlantic climatology. 12 Sour rot of grapes was also reduced with some treatments including C. sake. 13 A formulation process must principally achieve four basic objectives: It must i) stabilise the organism during production, distribution and storage; ii) aid in the handling and application of the product; iii) protect the agent from harmful environmental factors; and finally iv) enhance the activity of the organism at the site of use. 14 The maintenance of cell viability is fundamental for commercial BCA formulations but suggested minimum shelf life changes depending on the authors from six 15 to 12 months 16 or 18 months 2 .
The efficacy of BCAs is dependent on the degree of success of the formulation, so it is necessary to carefully evaluate every detail of the formulation process to obtain high survival rates of viable microorganisms and to achieve an equal or even better efficacy as with fresh cells.
Different formulations have been developed for C. sake in previous years. Abadias et at. studied a freeze-dried C. sake formulation but obtained an efficacy significantly lower than that of fresh cells in all treatments. 17 The stability of the formulation decreased 10-fold after two months storage at 4 °C. Liquid formulation of BCA by modifying the water activity or adding protectants was also investigated, and the results were more satisfactory because the efficacy of the formulation was similar to that of fresh cells for P. expansum on apples, and the addition of sugars such as lactose and trehalose as protectants improved cell viability, which was greater than 70% after four months at 4 °C. 18 Furthermore, a liquid formulation in an isotonic solution based on trehalose (0.96 M) was developed and stored for seven months at 4 °C, and it retained its viability and efficacy against blue mould rot in apples. 19 A spray-drying system was evaluated as a dehydration method for CPA-1, but it was not satisfactory because of low cell survival, poor product recovery and low efficacy. 20 The induction of

Accepted Article
The aim of this research was to optimise the conditions of fluidising-bed dehydration to produce a dry formulation of C. sake by studying several factors that could affect cell viability, shelf life, and efficacy against P. expansum on apples.
The specific objectives were: (i) the optimisation of drying conditions such as temperature and drying time, (ii) the selection of a carrier to mix with C. sake cell paste to facilitate extrusion, (iii) the determination of the effect of protectants on cell survival,

Microorganisms
Yeast isolate The C. sake strain CPA-1 used in this investigation belongs to the collection of the University of Lleida-IRTA, Catalonia, Spain and it was deposited in the Colección Española de Cultivos Tipo (CECT-10817) at the University of Valencia, Burjassot, Spain. It was isolated from the surface of apples.
C. sake stock cultures were stored at 4 °C on nutrient yeast dextrose agar plates (NYDA: nutrient broth, 8 g l -1 ; yeast extract, 5 g l -1 ; dextrose, 10 g l -1 ; and agar, 15 g l -1 ) and were sub-cultured on NYDA plates at 25 °C for 48 h before use. Sub-cultured cells were transferred to potassium phosphate buffer (pH 6.5; KH 2 PO 4 0.2 mol l -1 , 70 ml; K 2 HPO 4 0.2 mol l -1 , 30 ml and deionised water, 300 ml) to obtain the suspension used as the starter inoculum for biomass production in liquid bioreactors and for the in vivo efficacy assays.
Cells were produced in a liquid production system with a 5 l working volume (BIOSTAT-A modular bioreactor, Braun Biotech International, Melsungen, Germany) at Accepted Article fluidised bed dryer 350S (Burkard Manufacturing Co. Ltd, Hertfordshire, UK). After the fluidised bed drying was complete, 0.05 g of dry extruded particles were weighed and rehydrated with 5 ml of phosphate buffer, then shaken for 1 min and allowed to rehydrate for 9 min. The final concentration of dehydrated cells of C. sake was determined by dilution plating on NYDA. Viability after the fluidised bed drying process was expressed as log (N/N 0 ), where N 0 represents CFU per gram of extruded dough before drying and N is the CFU per gram of the same quantity of wet extruded dough after fluidised bed drying.
To determine the dry matter of the initial wet dough, 1 g of the product was placed in duplicate aluminium weighing boats and dried in a convection oven at 105 ± 1 °C for 24 hours. The same process was carried out with the dry extruded product but only 0.5 g were weighed.
During the fluidised bed drying process, the room temperature was controlled at 20 ºC and relative humidity at 40% to achieve the same conditions for all assays. All fluidised bed drying experiments were completely random designs.

Optimisation of time and temperature of drying
A range of drying temperatures from 30 °C to 50 °C were used to study the effect of inlet air temperatures of 30, 40, 45 and 50 °C. Drying times of: 5, 10, 15, 20, 30, 45 and 60 min were also evaluated. Potato starch was used as a carrier in studies to optimise the drying time and temperature. Three replicates of each formulate were weighted for each time and temperature, and the viability was the mean of them. The experiment was repeated twice.

Optimisation of the carrier
Four substances were tested to identify an optimal carrier for a dry formulation of C. sake using fluidised bed drying, natural silicate (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), potato starch (Panreac Química S.A., Barcelona, Spain), corn Accepted Article starch (Fluka, Sigma-Aldrich Chemie GmbH) and rice starch (Sigma-Aldrich Chemie GmbH). The survival of the BCA was the mean of three replicates. The fluidised bed drying conditions for this assay were 40 °C for 45 min.
This optimisation was carried out with different assays on different days. Three formulations with different protective compounds and a formulation without a protective substance (the control) were tested in each assay. The effect of protective compounds on the survival of the C. sake yeast cells after fluidised bed drying was expressed as: [log (N f with protectant /N 0 with protectant ) -log (N f control /N 0 control )], where N 0 is the CFU per gram of the extruded dough before drying and N f is the CFU per gram of the same quantity of wet extruded dough after fluidised bed drying. Each protective compound was tested at least in two drying processes. Two replicates of each formulate were weighted to evaluate the survival.

Optimisation of the rehydration media and the rehydration conditions
The rehydration process is an important factor because it can improve the viability of the final product. Two aspects of the rehydration process were considered: i) the composition of the rehydration media because it could facilitate the repair of cell Accepted Article damage and the restoration of physiological function, and ii) the rehydration temperature and time, which could affect the cell viability after rehydration.
Phosphate buffer and skimmed milk (100 g l -1 ) were tested as rehydration media for dried formulations. Rehydration times of 10 min, 2 h, 5 h and 24 h and rehydration temperatures of 6, 15, 25, 30 and 35 °C were studied with the previously optimised formulation. Three replicates of each formulate were weighted for each condition of rehydration and the viability was the mean of them. The experiment was repeated at least twice.

Shelf life of formulated products
To study the stability of the optimised formulation, 72 glass vials containing approximately 0.05 g of the formulation were sealed with laboratory film (Parafilm "M").
Then 36 vials were kept in an airtight container filled with silica gel to avoid sample humidification. The other 36 vials were stored under vacuum. The vials were stored at 4 °C and 25 °C, and 3 vials were removed at different times to determine survival by dilution plating on NYDA with phosphate buffer at room temperature for 10 min as the rehydration media. The resulted survival for each condition was the mean of the three vials. Shelf life assays were conducted for twelve months.

Formulated product efficacy
The efficacy of two optimised dry formulations was tested against P. expansum on apples. Both formulations were stored at 4 °C and air-packaged, one for one month and a half and another for six months. Both formulations were rehydrated with phosphate buffer at room temperature for 10 min, and their efficacy was compared to the efficacy of fresh cells. Fresh cells were obtained following growth in 100 ml conical flasks containing 50 ml of the same medium of formulated cells. Flasks were incubated for 48 hours at 25 °C and 150 rpm, and then the cells were centrifuged (as section 2.2.) Accepted Article (data not shown). A low temperature (30 °C) was insufficient to dry the product, so the final formulation had a high moisture content of approximately of 12.5% (data not shown). Drying the cells at a lower temperature or for less time to improve cell viability was considered, but both options increased the risk of an important decline in the shelf life of the final formulation due to excess available water in the dried product.
Consequently, these temperatures were rejected, and 40 °C for 45 min was chosen to continue the study.

Optimisation of the carrier
A carrier substance was necessary to reduce the adhesiveness of the cell paste and achieve a malleable dough that could be extruded to obtain small particles for fluidised bed drying. In addition, it was important to choose a carrier that did not damage the cells or reduce their survival.
Two of the four substances tested as carriers proved to be not useful for this BCA.
Specifically, natural silicate caused problems with the formulation process because the dough could not be extruded, and the corn starch produced non-soluble formulations during the rehydration process. Only potato starch and rice starch were useable as carriers in further experiments.
The viability of C. sake after drying the cells at 40 °C for 45 min was significantly higher with potato starch (31.3% viability) than with rice starch (18.5% viability) as the carrier.
Likewise, the reduction of C. sake cells after drying was 0.5 log units when potato starch was used as carrier whereas the reduction was 0.73 log units with rice starch.
The moisture content of the dried product was also better with potato starch (8.6%) because the dried product with rice starch had a moisture content above 10% that could compromise the shelf life of the formulated product.

Optimisation of protective compounds
Different protectant substances at different concentrations were tested to improve cell survival after the fluidised bed drying process (Fig. 2). Most of the compounds tested resulted in lower survival than the control formulation (without a protectant substance).
Improved survival was achieved with all tested percentages of carboxymethyl cellulose (10, 50 and 100 g l -1 ), 200 g l -1 sorbitan monostearate, 10 g l -1 glucose and 10 g l -1 trehalose, but no significant differences were observed. Moreover, despite of the improvement in C. sake survival, these formulations had too high moisture content for practical use ( Table 1). The worst survival was observed when 100 g l -1 sucrose and 100 g l -1 polyethylene glycol were used.

Optimisation of the rehydration media and the rehydration conditions
Laboratory experiments showed that differences among phosphate buffer and skimmed milk were rarely significant at low rehydration temperatures (6 °C and 15 °C), and no significant differences were evident among the different rehydration times at these temperatures (Fig. 3). Phosphate buffer was a significantly better medium only when rehydration was carried out at 6 °C for two hours. At room temperature (25 °C), both rehydration media showed the same tendency with time in the experiment. Two hours of rehydration yielded the highest C. sake survival, although no significant differences were observed between 10 minutes and two hours. At higher temperatures (30 °C and 35 °C), significant differences were observed among rehydration times but not between the rehydration media.
In general, the cell recovery did not decrease with time at low temperatures (6 and 15 °C), whereas at high temperatures (25, 30 and 35 °C), a notable decrease of the cell survival with time was observed.
The highest survival was obtained with skimmed milk at 35 °C for 10 minutes (70.0%) although this was not significantly different from cells rehydrated with phosphate buffer.

Accepted Article
Moreover, for both media, survival was not significantly different after 10 min of rehydration at 25 °C and 35 °C (data not shown).

Shelf life of formulated products
In general, storage temperature had a strong influence on the shelf-life viability of fluidised bed dried C. sake cells, and better results were obtained at 4 °C than at 25 °C ( Fig. 4). At 25 °C, the survival of the formulated cells stored decreased sharply under both air conditions, and after two months, the air-packaged formulation showed a survival of less than 10%, a decrease of 0.69 log units. At 4 °C, the viability remained stable for both storage conditions after 12 months, and the differences between an airvacuum and air-packaging were only significant at the first and the twelfth month of storage.

Formulated product efficacy
The optimised dry formulation process using fluidised bed drying was used to determine the efficacy of formulated C. sake cells stored at 4 °C against P. expansum in Golden Delicious apples. The efficacy experiments showed that both C. sake formulations significantly inhibited development of blue mould (Fig. 5). Two different storage times (one and a half and six months) were tested to demonstrate that the efficacy did not decrease during storage. Both the incidence and severity of P. expansum on apples treated with C. sake cells were reduced up to 52 and 72%, respectively. No significant differences were found between both storage formulations and fresh cells.

DISCUSSION AND CONCLUSIONS
The present study demonstrated that a dry formulation of the biocontrol agent Candida sake CPA-1 using fluidised bed drying is appropriate for cell survival and efficacy. The optimised formulation was dried at 40 ºC for 45 min using potato starch as carrier and without protectant compounds. The chosen rehydration media was phosphate buffer at Accepted Article content was satisfactory. According to our goal, a high survival rate immediately after drying is less important than low inactivation during storage, an aspect which is crucial for commercial exploitation 31 .
The optimum rehydration medium and rehydration conditions are phosphate buffer at 25 °C for 10 min. Nevertheless, a combination that produced a higher survival rate, Muller et al. reported that the reconstitution technique had a significant effect on the bacterial recovery and described the rehydration process as a vital step for the achievement of maximal viability. 33 A critical parameter of the rehydration process is the temperature of the rehydration medium 29 which is usually from 20 °C to 30 °C for fluidised bed drying 34,35 . Nevertheless, cell survival during rehydration is affected by many factors such as the strain, the temperature, the rehydration kinetics or the pH of the medium 29 .
The results obtained in this study showed that an easy to recover dry product with a high viability had been achieved, but knowledge about the time that it could be stored without undergoing a significant decrease in survival was necessary. This study demonstrated that the survival of the fluidised bed dried C. sake cells is closely related to the storage temperature, and is better at 4 °C than at 25 °C. Fu and Chen Accepted Article associated the loss of cell viability at elevated temperature with the degradation of lifeessential macromolecules during storage. 29 Previous studies with this BCA showed that a cold storage temperature for C. sake was better to keep metabolic activity low and maintains stability with time [17][18][19] . In this study, a significant difference between vacuum conditions and air-packaging was only seen after 12 months of storage at 4 °C; but during this time, the dried cell survival stored with air-packaging decreased by Nevertheless, this liquid formulation can be stored for one year (unpublished data).
Additionally, we have proven that this dry formulation retains its efficacy against P. expansum on Golden Delicious apples after six months of storage and is similar to fresh C. sake cells. Both the incidence and severity were reduced by 42 and 64%, respectively. Previous studies of freeze-dried C. sake cells with P. expansum on apples showed that this formulation was not as effective as fresh cells. 17 Furthermore, spraydried cells were not as effective as fresh cells against blue mould on pome fruit. 20 Therefore, this is the first dry formulation of C. sake CPA-1 that retained its efficacy against blue mould on apples. Other fluidised bed dried BCAs, such as Penicillium frequentans 36 or P. anomala 28 , retained an efficacy similar to fresh cells. Also efficacy of freeze-dried Pseudomonas spp. was nearly identical to that of the fresh cells. 37 In summary, we have demonstrated that a fluidised bed drying system is suitable for dehydrating C. sake cells because the dry formulation had satisfactory solubility, Accepted Article retained its viability for 12 months and was as effective as fresh cells against P. expansum on apples. In addition, it is easy to package, store and transport and is cost effective. The cell concentration after 12 months is very satisfactory because just over one kilogram of formulation can treat a 1000 litre drencher, which is a reasonable amount of product to use.
Based on these findings, we assume that this dry formulation overcomes most of the shortcomings hindering the commercialisation of this biocontrol product. However, the extrusion of the yeast dough is an involved process that must be performed manually, so further research should focus on the development a similar formulation using an automatic process. Optimisation of a fluid-bed spray-drying process for C. sake that takes advantage of the benefits of this method could be the next step.
Accepted Article Table 1. Moisture of the formulations with the best protective compounds after fluidised bed drying.