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Preservation of aerial conidia and biomasses from entomopathogenic fungi


Journal of Invertebrate Pathology 105 (2010) 16–23

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Journal of Invertebrate Pathology
journal homepage: www.elsevier.com/locate/jip

Preservation of aerial conidia and biomasses from entomopathogenic fungi Beauveria brongniartii and Metarhizium anisopliae during lyophilization
Stefan Toegel, Sharareh Salar-Behzadi, Andrea Horaczek-Clausen, Helmut Viernstein *
Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria

a r t i c l e

i n f o

a b s t r a c t
In this study, we assessed the stability provided by different formulations to aerial conidia or biomasses (conidia, blastospores, and mycelia) of Beauveria brongniartii and Metarhizium anisopliae subjected to lyophilization. First, the impact of the freezing and drying processes on spore survival was evaluated. Whereas unprotected B. brongniartii spores showed high cryosensitivity, those of M. anisopliae were markedly harmed by the drying process. Then, the protective ef?ciency of 14 excipients was systematically evaluated and optimized regarding required concentrations. Fructose, glucose, and saccharose signi?cantly enhanced viabilities for B. brongniartii and M. anisopliae spores following lyophilization, especially as a result of their cryoprotective effects. In addition, the effect of various bulking agents on spore survival was studied and dextran 4 was selected to enhance the physical properties of the lyophilized products. The combination of fructose and dextran 4 was further applied to prepare lyophilized biomasses of both fungi. In comparison to freshly harvested biomasses, the lyophilized products showed similar growth rates and a comparable production of virulent secondary metabolites such as destruxin A, destruxin B, or oosporein, suggesting their applicability as biological control agents. ? 2010 Elsevier Inc. All rights reserved.

Article history: Received 10 December 2009 Accepted 4 May 2010 Available online 8 May 2010 Keywords: Metarhizium anisopliae Beauveria brongniartii Cryoprotection Lyoprotection Imbibitional damage Formulation

1. Introduction Agricultural pests such as scarab beetles, weevils, locusts or grasshoppers are considered a major threat to agricultures, forests and horticultures worldwide. Up to the present, control strategies are dependent on the use of synthetic chemical insecticides such as organochlorine compounds or organophosphates. Given the increasing public sensitivity to environmental pollution, however, biological control using entomopathogenic fungi is becoming an interesting alternative to chemical control (Maniania et al., 2008; Shah and Pell, 2003; Vega et al., 2008). In this context, Beauveria brongniartii and Metarhizium anisopliae are known to effectively cause epizootics of soil dwelling pests with a limited host range and minimal impact on non-target species (Amiri et al., 1999; Erlacher et al., 2006; Hadapad and Zebitz, 2006). Although the use of entomopathogenic fungi appears promising, it is crucial, from an industrial point of view, to provide economically competitive products for the agricultural market. Production processes for fungal biopesticides must be low-cost and provide viable, virulent, and persistent propagules at the same time (Jackson, 1997; Kassa et al., 2008). Therefore, the optimization of both mass production and subsequent formulation strategies

* Corresponding author. Fax: +43 1 4277 9554. E-mail address: helmut.viernstein@univie.ac.at (H. Viernstein). 0022-2011/$ - see front matter ? 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.05.004

allowing storage, distribution and application are of particular importance (Rhodes, 1993). Despite its practical relevance, little is known about the impact of technological operations on the viability and metabolism of B. brongniartii and M. anisopliae. In a previous study, we have evaluated the applicability of spray drying as a preservation method for fungal conidia of B. brongniartii (Horaczek and Viernstein, 2004a,b). We found that microencapsulation using spray drying represents a useful and economic way to formulate aerial conidia of B. brongniartii with high viability rates. Freeze-drying is an additional option to process microorganisms. Given the bene?cial characteristics of lyophilized products, freeze-drying is often used for the preservation and pre-formulation of biological samples. It is discussed as a feasible alternative for stabilizing yeast, bacteria and as well as sporulating fungi (Berny and Hennebert, 1991). However, not all fungi tolerate this process to the same extent and among those surviving, viability rates as low as 0.1% have been reported (Smith and Onions, 1983). Therefore, it appears essential to adapt the process to the particular requirements and limitations of each particular species to maintain optimal cell viability. In general, microbial cell survival during freeze-drying can be optimized by factors such as the cooling rate (Tan et al., 1994), the composition of the protective medium (Berny and Hennebert, 1991), the initial microorganism concentration (Bozoglu et al., 1987), and the rehydration conditions (Font de Valdez et al., 1985). In particular, the addition of certain excipients such as sugars, amino acids,

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peptides, or proteins to suspending ?uids is known to improve survival (Tan et al., 1995). In the present study, we examined the protective activity of 14 excipients during lyophilization of aerial conidia of the entomopathogenic fungi M. anisopliae and B. brongniartii. Dividing the lyophilization process into its two components – freezing and drying – the susceptibility of the conidia to the different stress sources was evaluated allowing to identify appropriate protectants for the two fungi. Given the need to prepare economic products of good physical structure, we further investigated the applicability of various bulking agents regarding their impact on spore viability. In addition, the most suitable formulation containing fructose as lyoprotectant and dextran 4 as bulking agent was studied regarding its applicability to preserve and formulate fungal biomasses of M. anisopliae and B. brongniartii. 2. Material and methods 2.1. Fungal isolates An isolate belonging to the M. anisopliae complex (isolated from Cydia pomonella in Denmark) and B. brongniartii (isolated from pasture soil infested with Melolontha melolontha in Austria) were kindly provided by Dr. Hermann Strasser. 2.2. Preparation of conidia suspensions M. anisopliae and B. brongniartii were grown at 25 °C in the dark on Sabouraud-4%-Glucose (S4G) agar and Sabouraud-2%-Glucose (S2G) agar, respectively. Conidia suspensions were prepared by ?ooding 14–20 days cultures with sterile 0.1% aqueous Tween? 80 solutions and scraping the surface with a bent glass rod. The resulting conidial suspensions were sonicated for 3 min and vortexed for 1 min to ensure homogeneity. Afterwards, suspensions were sieved through a 45 lm mesh and conidia concentrations were determined using a Thoma haemocytometer. Spore concentrations were adjusted to equal concentrations for all assays. The effect of the initial spore concentration on conidia viability in the lyophilisates was tested at 106, 107 and 108 conidia/ml. 2.3. Cryoprotectants and bulking agents Table 1 summarizes the lyoprotectants and bulking agents used as excipients for lyophilization. Lactose was obtained from Kwizda (Vienna, Austria) whereas maltose, raf?nose and sorbitol were purchased from Sigma–Aldrich (Vienna, Austria). Dextran 4 was from Serva (Heidelberg, Germany) and cysteine was obtained from Roth

(Karlsruhe, Germany). All other chemicals were purchased from Merck (Darmstadt, Germany). All excipients were studied regarding their cryoprotective and lyoprotective potential (protection against freezing or freezing and drying, respectively). Therefore, excipients were added to conidia suspensions at indicated concentrations prior to incubation at room temperature for 30 min allowing cell adaption. The in?uence of the additive concentration on conidia viability was investigated using 5%, 10% or 20% solutions in case of dextran 4 and skim milk as well as 0.14 M, 0.28 M and 0.56 M solutions in case of all other excipients. 2.4. Freeze-drying of conidial suspensions and viability assessment Five hundred ll of conidial suspensions were frozen at ?80 °C for 24 h. Afterwards, samples were freeze-dried using a CHRIST BETA 1–8 K (Christ; Osterode am Harz, Germany) lyophilizer operating at 0.090 mbar. Within 20 h, the temperature was gradually raised from –35 °C to 25 °C. Freshly harvested, frozen, and lyophilized conidia were dispersed in 0.1% aqueous Tween? 80 solution. The samples were vortexed for 1 min and incubated for 10 min. Then, 50 ll of the resulting suspension were spread on agar plates (1 g/l glucose, 0.5 g/l peptone, 1.5 g/l agar and 0.5 g/l yeast extract, supplemented with 30 mg/l streptomycin sulfate and 50 mg/l chloramphenicol) and incubated for 24 h at 25 °C. The viability of 300 spores per plate was assessed after staining with lactophenol cotton blue using a Nikon 104 light microscope. Only spores with germ tubes longer than their width were considered to have germinated. By comparison of germinated spores before and after freezing or freeze-drying the viability or the loss of viability was calculated. Following this protocol, the in?uence of three different rehydration media was also investigated: 0.9% sodium chloride, 0.1% Tween? 80, and Merck maximum recovery diluent (0.1% peptone and 0.9% sodium chloride). 2.5. Development of conidia-containing formulations In order to develop lyophilized conidia formulations suitable for industrial use, 10 composite suspending media comprising a cyroprotectant (fructose) and bulking agents were investigated regarding spore viability and physical structure of the resulting lyophilisates. The 10 formulations are described in Table 2. Fructose at 0.28 M and 0.56 M was used as stabilizing agents for B. brongniart-

Table 2 In?uence of composite suspending media on spore viability following lyophilization. Formulation M. anisopliae Bulking agent – Mannitol Raf?nose Dextran 4 Glutamine – Mannitol Raf?nose Dextran 4 Glutamine Fructose 0.56 M 0.56 M 0.56 M 0.56 M 0.56 M 0.28 M 0.28 M 0.28 M 0.28 M 0.28 M Viability (% ± SD) 35 ± 3 27 ± 8 32 ± 12 33 ± 7 12 ± 6* 79 ± 9 35 ± 9** 56 ± 11 61 ± 11 20 ± 3***

Table 1 Stabilizing and bulking agents used as excipients for lyophilization. Substance classes Sugars Excipient Fructose Glucose Ribose Lactose Maltose Saccharose Raf?nose Dextran 4 Sorbitol Mannitol Cysteine Glutamine NaCl Skim milk MW 180.16 180.16 150.13 342.3 342.3 342.3 504.44 4000–6000 182.17 182.17 121.16 146.15 58 Function Stabilizing agent Stabilizing agent Stabilizing agent Stabilizing agent Stabilizing agent Stabilizing agent Stabilizing agent Bulking agent Stabilizing agent Bulking agent Bulking agent Stabilizing agent Bulking agent Stabilizing agent

B. brongniartii

Amino acids Salts Others

Dextran 4 was used at a concentration of 10%, all other bulking agents at 0.28 M. Statistics were performed using one-way ANOVA (M. anisopliae: degrees of freedom: 4; F: 4.306; B. brongniartii: degrees of freedom: 4; F: 19.72). Signi?cant differences from the control without bulking agent are marked with asterisks. * p < 0.05. ** p < 0.01. *** p < 0.001.

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ii and M. anisopliae, respectively. Additionally, either 10% dextran 4, 0.28 M glutamine, 0.28 M mannitol or 0.28 M raf?nose were studied as bulking agent.

3. Results 3.1. Cryoprotective impact of the excipients on spore viability Fig. 1 shows that freezing and subsequent thawing of B. brongniartii induced a loss of viability of 58 ± 8% when no cryoprotectant was used. Whereas NaCl did not show any cryoprotecting effect (p > 0.05) and the addition of cysteine even impaired spore viability (p < 0.001), all other investigated protectants signi?cantly reduced the loss of viability encountered during freezing (p < 0.001). Of note, only 4 ± 3% of the B. brongniartii spores were lost using fructose as cryoprotectant. In case of M. anisopliae (Fig. 2), freezing and thawing in the absence of cryoprotectants reduced the spore viability by 17 ± 7%. In comparison, the addition of glucose (p < 0.01), maltose (p < 0.001), saccharose (p < 0.001), raf?nose (p < 0.001), sorbitol (p < 0.001) or cysteine (p < 0.05) signi?cantly protected the conidia during the freezing process. Interestingly, mannitol and NaCl (both p < 0.001) markedly harmed M. anisopliae spores during this ?rst part of lyophilization, resulting in germination rates of only about 1%. 3.2. Lyoprotective impact of the excipients on spore viability

2.6. Preparation of lyophilized biomasses from B. brongniartii and M. anisopliae Conidia from 14–20 days cultures were suspended in sterile 0.1% aqueous Tween? 80 followed by inoculation into 100 ml liquid S2G and S4G medium for B. brongniartii and M. anisopliae, respectively. The ?nal conidia concentration in the liquid medium was 106/ml. Cultures were incubated at 25 °C on a gyratory shaker (GFL, Burgwedl, Germany) at 200 rpm for 8 days. Then, the biomass containing a mixture of different morphological forms such as mycelia, conidia and blastospores was separated by centrifugation and resuspended in medium containing 0.56 M fructose as lyoprotectant as well as 5% dextran 4 as bulking agent. Resulting biomass suspensions were frozen by dipping in liquid nitrogen and subsequently subjected to the freeze-drying process as described above.

2.7. Growth rates and metabolism of lyophilized fungal biomasses The freeze-dried biomass was resuspended and washed three times with 100 ml distilled water to remove protectants. Depending on the fungal species, 20 ml of S2G or S4G medium were inoculated with 1–2 mg biomass (dry weight) and incubated at 25 °C on a gyratory shaker at 200 rpm for 8 days. 1–2 mg of untreated biomass (harvested after 8 days in submerge culture) were prepared as control. Growth rates of the fungal biomass was determined each day as follows. Fungal biomasses were separated by centrifugation and the dry weight was determined using a Sartorius MA 30 moisture content analyzer (Sartorius, G?ttingen, Germany). The supernatant was sterile ?ltered and stored at –20 °C until further use. The pH of the supernatant was analyzed using a pH-meter and the production of destruxin A, destruxin B, and oosporein was detected using high pressure liquid chromatography (HPLC; Agilent HP1090) and ultraviolet diode array detection (UV-DAD) as reported previously with minor modi?cations (Seger et al., 2005, 2006). Brie?y, destruxin A and destruxin B were assessed using a Merck RP-18 guard column, an Agilent Zorbax SB-C18 column and a water/acetonitrile gradient elution. Oosporein was assessed using a Phenomenex RP-18 guard column, a Phenomenex SYNERGI Hydro column and a water/acetonitrile gradient (both solvents containing 0.1% (v/v) acetic acid and 0.9% (v/v) formic acid). Samples were ?ltered through a 10 kD membrane by a VIVASPIN 2 ml concentrator 10000 MWCO CTA (Sartorius; G?ttingen, Germany). Samples of B. brongniartii were diluted with methanol in a ratio of 1:10 whereas those of M. anisopliae were used directly. Secondary metabolites were quanti?ed using standard curves of destruxin A, destruxin B, and oosporein (Sigma, Vienna, Austria).

2.8. Statistics Statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, USA) and the Microsoft Excel p integrated analysis tools. Data were arcsine p transformed and analyzed using one-way ANOVA with post hoc Tukey test crosscomparing all study groups. Comparison among two means (metabolite production using HPLC) was performed using the Student’s t-test. Values of p < 0.05 were considered signi?cant. All experiments were repeated at least three times on different dates using different batches of fungal preparations.

In addition to the stress caused by freezing, the process of drying contributes to the reduction of spore activity during lyophilization. Figs. 1 and 2 indicate the extent of the cell damage caused by drying for B. brongniartii and M. anisopliae, respectively, resulting in an overall (cumulative) loss of viability induced by the freeze-drying process. In case of B. brongniartii, the total loss of viability in medium without protectant was 65 ± 11% (about 7% due to drying). Ribose, mannitol, glutamine and NaCl failed to act as lyoprotectant since comparable values were yielded (p > 0.05; Fig. 1). In contrast, all other excipients showed signi?cantly reduced losses of viability, indicating their lyoprotective potential although none of them was capable of markedly avoiding loss of viability during the drying step. Fructose, glucose, maltose, saccharose, raf?nose and sorbitol yielded germination rates above 70% (all p < 0.001), whereas lactose, dextran 4 and skim milk maintained a spore viability of 68% (p < 0.05), 67% (p < 0.05) and 61% (p < 0.01), respectively. As illustrated in Fig. 2, 96 ± 4% of M. anisopliae spores perished during lyophilization without protectant (about 79% due to drying). Although variable degrees of cryopreservative action were noticed among lyoprotectants, none of them provided considerable protection during the entire process of lyophilization. Only fructose (p < 0.001), glucose (p < 0.001) and saccharose (p < 0.05) yielded viability losses <85%. In general, lyophilization of conidia in the presence of increasing additive concentrations resulted in enhanced germination rates after rehydration (Figs. 3 and 4). A signi?cant concentration-dependent lyoprotective effect was observed in case of maltose (p < 0.001; F: 34.76), saccharose (p < 0.05; F: 9.864) and raf?nose (p < 0.01; F: 19.78) for B. brongniartii (Fig. 3) and in case of fructose (p < 0.05; F: 6.190), ribose (p < 0.001; F: 78.54), maltose (p < 0.001; F: 39.79), saccharose (p < 0.05; F: 7.509) and raf?nose (p < 0.05; F: 5.061) for M. anisopliae (Fig. 4). In the case of the lyophilization-sensitive spores of M. anisopliae, markedly improved protection was achieved using 0.56 M fructose, which resulted in a two times higher spore survival (44 ± 10%) as compared to using 0.28 M (23 ± 3%). Other strategies to increase the spore viability after lyophilization failed: different initial conidia loads and different rehydration media did not reveal signi?cant improvements (data not shown). 3.3. Testing composite media comprising stabilizing and bulking agents Preliminary studies have shown that the lyophilization of most mono- and disaccharides, including fructose, resulted in

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ta nt fru ct os e gl uc os e

cc ha ro se ra ffi no se de xt ra n 4 so rb ito l m an ni to l cy st ei ne gl ut am in e

e la ct os e

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ec

ly op

Fig. 1. The cryoprotective and lyoprotective effects of excipients on the viability of B. brongniartii spores. Dextran 4 and skim milk were used at 10% whereas all other excipients were used at 0.28 M. B. brongniartii spores were subjected to –80 °C for 24 h prior to lyophilization. Grey bars show the loss of viability after freezing, whereas the p height of the columns (i.e., grey plus white bars) show the cumulative loss of viability after freeze-drying. Statistics were performed using one-way ANOVA of arcsine p transformed data (degrees of freedom: 14; F: 25.22). Asterisks mark signi?cant differences of the total loss of viability as compared to samples without protectant (p < 0.05).

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al to se

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sa

ec

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N

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Fig. 2. The cryoprotective and lyoprotective effects of excipients on the viability of M. anisopliae spores. Dextran 4 and skim milk were used at 10% whereas all other excipients were used at 0.28 M. M. anisopliae spores were subjected to ?80 °C for 24 h prior to lyophilization. Grey bars show the loss of viability after freezing, whereas the p height of the columns (i.e., grey plus white bars) show the cumulative loss of viability after freeze-drying. Statistics were performed using one-way ANOVA of arcsine p transformed data (degrees of freedom: 14; F: 15.63). Asterisks mark signi?cant differences of the total loss of viability as compared to samples without protectant (p < 0.05).

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freeze-dried cakes of very poor physical properties, whereas lactose, raf?nose, dextran 4, mannitol, cysteine, glutamine, sodium chloride and skim milk provided freeze-dried products with a light and porous structure (data not shown). Thus, we investigated whether the addition of bulking agents (mannitol, raf?nose, dextran 4, or glutamine) to the fructose-containing medium would result in altered viability of entomopathogenic spores (Table 2). In order to minimize

the adverse effect of fructose with respect to the physical properties of the formulation, we decided to use fructose at only 0.28 M for B. brongniartii, as this concentration yielded satisfactory protection for that fungus (Fig. 3). For M. anisopliae, in contrast, we decided to apply 0.56 M fructose to achieve higher germination rates (Fig. 4). In the case of M. anisopliae, the presence of glutamine resulted in signi?cantly reduced viability rates as compared to experiments

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viability (%)

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0 fructose glucose lactose maltose saccharose raffinose dextran 4 sorbitol skim milk

Fig. 3. Effect of excipient concentration on spore viability of B. brongniartii after lyophilization. Dextran 4 and skim milk were used at 5% (white bars), 10% (grey bars), and 20% (black bars) whereas all other excipients were used at 0.14 M (white bars), 0.28 M (grey bars), and 0.56 M (black bars). Statistics were performed using one-way ANOVA of p arcsine p transformed data (degrees of freedom: 2). Asterisks mark signi?cant differences as compared to the respective samples with 5% or 0.14 M protectants (p < 0.05).

60

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viability (%)

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0 fructose glucose ribose maltose saccharose raffinose

Fig. 4. Effect of excipient concentration on spore viability of M. anisopliae after lyophilization. Excipients were used at 0.14 M (white bars), 0.28 M (grey bars), and 0.56 M p (black bars). Statistics were performed using one-way ANOVA of arcsine p transformed data (degrees of freedom: 2). Asterisks mark signi?cant differences as compared to the respective samples with 0.14 M protectants (p < 0.05).

carried out with fructose alone (p < 0.05). In contrast, the formulations including mannitol, raf?nose or dextran 4 yielded similar results as compared to samples without bulking agent (p > 0.05). In the case of B. brongniartii, the use of composite media comprising glutamine or mannitol as bulking agents resulted in signi?cantly reduced viability rates as compared to experiments carried out with fructose alone (p < 0.001 and p < 0.01, respectively). In contrast, the formulations with raf?nose or dextran 4 yielded similar results as compared to samples without bulking agent (p > 0.05). 3.4. Freeze-drying of fungal biomasses of B. brongniartii and M. anisopliae Finally, the formulation containing fructose as lyoprotectant and dextran 4 as bulking agent was tested regarding its impact on the entomopathogenic activity of B. brongniartii and M. anisopliae biomasses. Therefore, the growth rate, the pH of the medium as

well as the production of secondary metabolites were determined and compared to those in freshly harvested biomasses. Fig. 5 shows that the biomass of freshly harvested B. brongniartii increased about 3-fold (from 4.0 ± 2.6 mg/ml to 11.7 ± 6.0 mg/ml) within the ?rst 4 days in culture (Fig. 5A). At the same time, the pH of the medium decreased from 5.5 to 3.8. Both pH and weight of the biomass, however, did not further change signi?cantly in prolonged cultures. In comparison to the freshly harvested biomass of B. brongniartii (Fig. 5A), we found that the lyophilized biomass formulated with fructose and dextran 4 (Fig. 5B) showed comparable growth characteristics indicated by the signi?cant increase in the biomass weight from 3 ± 0.2 mg/ml to 15.4 ± 1.9 mg/ml within 96 h of culture (Fig. 5B). Of note, the lyophilized product containing B. brongniartii biomass (Fig. 5D) exhibited a similar production pattern of oosporein as compared to the freshly harvested biomass (Fig. 5C). In both cases, the metabolite oosporein signi?cantly increased during the

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(A)

(B)

(C)

(D)

Fig. 5. Growth rate and pH values (A, B) and oosporein production (C,D) of B. brongniartii in submerged culture (20 ml S2G medium; 25 °C, 200 rpm). The lyophilized biomass of B. brongniartii using a composite solution containing 10% fructose and 5% dextran 4 (B, D) is compared with the freshly harvested biomass of the strain (A, C).

?rst 48 h of culture, yielding 37.7 ± 12.9 lg/ml in the fresh cultures and 31.9 ± 0.8 lg/ml in the cultures started from lyophilized biomasses. These two metabolite levels were not signi?cantly different (unpaired t-test: p = 0.48; degrees of freedom: 4). Fig. 6 indicates that comparable results were obtained from freshly harvested and lyophilized M. anisopliae biomasses regarding growth rates and production of destruxin A and destruxin B. The biomasses in both culture systems gradually gained weight within 96 h, yielding 13.7 ± 6.7 mg/ml in the fresh cultures and 16.5 ± 5.0 mg/ml in the cultures started from lyophilized biomasses (Fig. 6A and B, respectively). In addition, culturing freshly harvested M. anisopliae biomasses for 96 h resulted in the accumulation of 40.5 ± 22.0 lg/ml destruxin A, whereas culturing the lyophilized product yielded 28.2 ± 11.9 lg/ml destruxin A (unpaired t-test: p = 0.44; degrees of freedom: 4). Regarding destruxin B production in these cultures, 11.3 ± 6.5 lg/ml (Fig. 6C) and

18.1 ± 5.6 lg/ml (Fig. 6D) were found, respectively (unpaired ttest: p = 0.24; degrees of freedom: 4). 4. Discussion In recent years, the demand for dried products containing entomopathogenic fungi has increased due to environmental advantages of biological control agents over traditional pesticides (Butt et al., 2001). Lyophilization might represent a way for the stabilization of commercial mycoinsecticides, however, controlled and ef?cient processing of the fungi using lyophilization still remains a technological challenge. Several studies have reported on the development of formulations and freeze-drying protocols for fungal propagules (Burges, 1998; Kassa et al., 2004; Lomer et al., 1995 and Lomer et al., 2001; Voyron et al., 2009). In this context, Kassa et al. have demonstrated the possibility to effectively dry

(A)

(B)

(C)

(D)

Fig. 6. Growth rate and pH values (A, B) and destruxin A and B production (C,D) of M. anisopliae in submerged culture (20 ml S2G medium; 25 °C, 200 rpm). The lyophilized biomass of M. anisopliae using a composite solution containing 10% fructose and 5% dextran 4 (B, D) is compared with the freshly harvested biomass of the strain (A, C).

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M. acridum using spray- and freeze-drying techniques (Kassa et al., 2004). However, given the reduced viability of freeze-dried conidia in that study, the authors underlined the necessity to search for suitable protectants to further improve the stabilizing process. To our knowledge, the present study is the ?rst to investigate the cryoprotective and lyoprotective potential of various excipients for processing conidia and biomasses of B. brongniartii and M. anisopliae. During freeze-drying, the denaturation of sensitive proteins represents a crucial event accounting for the decreasing viability of many cell types (Carpenter et al., 1987). It is well known, however, that certain sugars can preserve and stabilize membranes, proteins, and living cells in the dry state for extended periods of time. Trehalose, in particular, was found to be an effective protectant for a range of applications (Crowe et al., 2001). Recently, a 31P NMR study revealed that the performance of trehalose in biopreservation is derived from the formation of a rigid hydrogen bonded network that results in dynamical slowdown and conformational restriction of biomolecules (Jain et al., 2009). However, high production costs might make this disaccharide unfeasible for industrial use. Therefore, – and in accordance with Sun and Leopold’s cytoplasmatic vitri?cation hypothesis (Sun and Leopold, 1997) – we selected a range of comparatively inexpensive excipients, potentially stabilizing membranes and macromolecules upon desiccation. Crowe et al. found that freezing and dehydration are fundamentally different stress vectors requiring different mechanisms to obviate their adverse effects (Crowe et al., 1990). Therefore, we separated the lyophilization process into its two components, freezing and drying in our current study. In agreement with previous studies we found that the two procedures differentially impaired the spore viability depending on the species of microorganisms (Font de Valdez et al., 1983). Whereas the freezing process in absence of protectants killed 58 ± 8% of B. brongniartii spores, only 17 ± 7% of M. anisopliae spores perished under the same conditions (Figs. 1 and 2). In contrast, B. brongniartii spores without lyoprotectants (control) were remarkably resistant to the drying step, as the total loss of viability only slightly increased after lyophilization. On the other hand, M. anisopliae was highly sensitive to drying (about 79% loss of viability for unprotected conidia), resulting in 96% viability loss following the whole freeze-drying process. Faria et al. (1999) reported viability losses ranging from 23% to 59% for four M. anisopliae isolates following freeze-drying with a mixture of 3% glucose and 3% gelatin as a cryoprotectant, whereas for B. bassiana isolates the loss was in the 2–16% range. We further found that the protective capacity of the selected excipients depended on both the stress source and the fungal species. Fructose markedly protected B. brongniartii spores from the whole freeze-drying process (Fig. 1), especially as a result of its cryoprotective effect. Comparable results were yielded using glucose, lactose, maltose, saccharose, raf?nose, dextran 4, sorbitol and skim milk. However, an additive that offers cryoprotection does not necessarily ensure that the organism will survive the drying step. Ribose, mannitol and glutamine, although being effective protectants against freezing could not protect B. brongniartii spores from the drying process. For M. anisopliae, none of the tested excipients was capable of reducing damage to the conidia due to drying. With respect to the entire freeze-drying process, only fructose, glucose and saccharose at a concentration of 0.28 M could signi?cantly reduce the loss of viability during lyophilization. Of note, viability rates above 40% were yielded using 0.56 M fructose (Fig. 4). Thus, M. anisopliae is either more sensitive to the drying step of lyophilization than B. brongniartii or results may be explained, at least partially, by occurrence of imbibitional damage. For instance, Faria et al. (2009) have shown that dry M. anisopliae spores plunged into water at 15 and 25 °C may have their viability reduced by ca. 70% and 30%, respec-

tively. In order to avoid imbibitional damage, dry M. anisopliae spores should be slowly rehydrated in a humidity chamber prior to suspension preparation or directly added to warm water/surfactant. Since in our study dry M. anisopliae spores were plunged into 0.1% tween solution at room temperature (25 °C), the drying step of lyophilization might not be the sole source of viability loss. As a matter of fact, imbibitional damage may help explain other poor results previously reported for M. anisopliae spores following freeze-drying (Faria et al., 1999; Horaczek and Viernstein, 2004b). The limited loss of viability observed in our study after fast rehydration of dry B. brongniartii spores suggests that this species is relatively insensitive to imbibitional damage, as previously reported to B. bassiana by Faria and colleagues. This study further indicates that dextran 4 represents a suitable bulking agent since it did not negatively in?uence the viability of both species and provided good physical characteristics and ease of handling of the lyophilized products (Table 2). Having de?ned feasible protectants and suitable bulking agents for the lyophilization of B. brongniartii and M. anisopliae conidia, we ?nally aimed to evaluate the applicability of fructose as lyoprotectant and dextran 4 as bulking agent for the formulation of a lyophilized product containing the fungal biomass of B. brongniartii or M. anisopliae. The virulence of entomopathogenic fungi has recently been shown to depend, at least in part, on the production of secondary metabolites such as oosporein (B. brongniartii) or destruxin A and destruxin B (M. anisopliae) (Seger et al., 2005 and Seger et al., 2006; Sree and Padmaja, 2008; Strasser et al., 2000; Wang et al., 2004). Given the impact of the lyophilization process on the cellular behaviour of microorganisms, a potentially altered production of these insecticidal substances appears a crucial quality criterion. Of note, however, we found that in comparison to freshly harvested biomasses, the freeze-dried fungi did not produce signi?cantly altered levels of these substances following rehydration. Therefore, we conclude that the combination of fructose as lyoprotectant and dextran 4 as bulking agent might be a suitable approach to formulate fungal biomasses of B. brongniartii or M. anisopliae maintaining production of secondary metabolites and bene?cial physical characteristics of the lyophilisates. References
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