Effects of di- and polysaccharide formulations and storage conditions on survival of freeze-dried Sphingobium sp.

In this study we have compared the ability of the organic polymers Ficoll and hydroxyethylcellulose (HEC) and the disaccharides sucrose and trehalose to support cell survival during freeze-drying and subsequent storage of a gram-negative Sphingobium sp. In addition to determination of viability rates, cell integrity was evaluated using lipid peroxidation and RNA quality assays for the different storage conditions and formulation compositions. All formulations resulted in high initial cell survival rates after freeze-drying. However, the disaccharide formulations were superior to the polymer-based formulations in supporting cell survival during storage with the exception of Ficoll that upon storage under vacuum yielded bacterial survival rates equal to that of sucrose. Storage in the presence of both oxygen and moisture was detrimental for bacterial survival in all formulations tested, however, lipid peroxidation or RNA damages were not the controlling mechanisms for cell death in this system. The ability of Ficoll and HEC to support cell survival during freeze-drying show that organic polymers, expected to lack the water replacing capability of e.g. disaccharides, can successfully be used as lyoprotectants. For storage under vacuum conditions we suggest that the intracellular amount of sugars (i.e. trehalose), or other protective native cell components, is sufficient for a basic protection inside the bacteria cell and that the amorphous state is the most important aspect of the formulation excipient. However, when exposed to oxygen and moisture during storage this protection is not sufficient to prevent cell degeneration.     

Microencapsulating aerial conidia of Trichoderma harzianum through spray drying at elevated temperatures

Conidia of Trichoderma harzianum produced from either solid or liquid fermentation must be dried to prevent spoilage by microbial contamination, and to induce dormancy for formulation development and prolonged self-life. Drying conidia of Trichoderma spp. in large scale production remains the major constraint because conidia lose viability during the drying process at elevated temperatures. Moreover, caking must be avoided during drying because heat generated by milling conidial chunks will kill conidia. It is ideal to dry conidia into a flow-able powder for further formulation development. A method was developed for microencapsulation of Trichoderma conidia with sugar through spray drying. Microencapsulation with sugars, such as sucrose, molasses or glycerol, significantly (P < 0.05) increased the survival percentages of conidia after drying. Microencapsulation of conidia with 2% sucrose solution resulted in the highest survival percentage when compared with other sucrose concentrations and had about 7.5 × 10¹⁰ cfu in each gram of dried conidia, and 3.4 mg of sucrose added to each gram of dried conidia. The optimal inlet/outlet temperature setting was 60/31 °C for spray drying and microencapsulation. The particle size of microencapsulated conidia balls ranged from 10 to 25 μm. The spray dried biomass of T. harzianum was a flow-able powder with over 99% conidia, which could be used in a variety of formulation developments from seed coatings to sprayable formulations.     

Engineering Desiccation Tolerance in Escherichia coli

Recombinant sucrose-6-phosphate synthase (SpsA) was synthesized in Escherichia coli BL21DE3 by using the spsA gene of the cyanobacterium Synechocystis sp. strain PCC 6803. Transformants exhibited a 10,000-fold increase in survival compared to wild-type cells following either freeze-drying, air drying, or desiccation over phosphorus pentoxide. The phase transition temperatures and vibration frequencies (PO stretch) in phospholipids suggested that sucrose maintained membrane fluidity during cell dehydration.     

A case study on stress preconditioning of a Lactobacillus strain prior to freeze-drying

Freeze-drying of bacterial cells with retained viability and activity after storage requires appropriate formulation, i.e. mixing of physiologically adapted cell populations with suitable protective agents, and control of the freeze-drying process. Product manufacturing may alter the clinical effects of probiotics and it is essential to identify and understand possible factor co-dependencies during manufacturing. The physical solid-state behavior of the formulation and the freeze-drying parameters are critical for bacterial survival and thus process optimization is important, independent of strain. However, the maximum yield achievable is also strain-specific and strain survival is governed by e.g. medium, cell type, physiological state, excipients used, and process. The use of preferred compatible solutes for cross-protection of Lactobacilli during industrial manufacturing may be a natural step to introduce robustness, but knowledge is lacking on how compatible solutes, such as betaine, influence formulation properties and cell survival. This study characterized betaine formulations, with and without sucrose, and tested these with the model lactic acid bacteria Lactobacillus coryniformis Si3. Betaine alone did not act as a lyo-protectant and thus betaine import prior to freeze-drying should be avoided. Differences in protective agents were analyzed by calorimetry, which proved to be a suitable tool for evaluating the characteristics of the freeze-dried end products.     

Storage stability and sourdough acidification kinetic of freeze-dried Lactobacillus curvatus N19 under optimized cryoprotectant formulation

In this study, the response surface methodology was used to optimize the cryoprotective agent (skimmed milk powder, lactose and sucrose) formulation for enhancing the viability of Lactobacillus curvatus N19 during freeze-drying and storage stability of cells freeze-dried by using optimum formulation was evaluated. Our results showed that the most significant cryoprotective agent influencing the viability of L. curvatus N19 to freezing and freeze-drying was sucrose and skim milk, respectively. The optimal formulation of cryoprotective agents was 20 g/100 mL skim milk, 3.57 g/100 mL lactose and 10 g/100 mL sucrose. Using the optimum formulation during freeze-drying, the cell survival was found more than 98%. Under the optimal conditions, although only storage of the cells at 4 °C for 6 month retained the maximum stability (8.85 log cfu/g), the employed protectant matrix showed promising results at 25 °C (7.89 log cfu/g). The storage stability of cells under optimized conditions was predicted by accelerated storage test, which was demonstrated that the inactivation rate constant of the freeze-dried L. curvatus N19 powder was 9.74 × 10⁻⁶ 1/d for 4 °C and 2.08 × 10⁻³ 1/d for 25 °C. The loss of specific acidification activity after the storage at 4 and 25 °C was determined.     

Optimisation and comparison of liquid and dry formulations of the biocontrol yeast <Emphasis Type="Italic">Pichia anomala</Emphasis> J121

The biocontrol yeast Pichia anomala J121 can effectively reduce mould growth on moist cereal grains during airtight storage. Practical use of microorganisms requires formulated products that meet a number of criteria. In this study we compared different formulations of P. anomala. The best way to formulate P. anomala was freeze-drying. The initial viability was as high as 80%, with trehalose previously added to the yeast. Freeze-dried products could be stored at temperatures as high as 30 °C for a year, with only a minor decrease in viability. Vacuum-drying also resulted in products with high storage potential, but the products were not as easily rehydrated as freeze-dried samples. Upon desiccating the cells using fluidised-bed drying or as liquid formulations, a storage temperature of 10 °C was required to maintain viability. Dependent on the type of formulation, harvesting of cells at different nutritional stresses affected the initial viabilities, e.g. the initial viability for fluidised-bed-dried cells was higher when the culture was fed with excess glucose, but for freeze-drying it was superior when cells were harvested after depletion of carbon. Using micro-silos we found that the biocontrol activity remained intact after drying, storage and rehydration for all formulations.     

Effect of water activity and protective solutes on growth and subsequent survival to air-drying of Lactobacillus and Bifidobacterium cultures

Probiotic cultures of Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei and Lactobacillus acidophilus were grown in media having water activities (a _w) adjusted between 0.99 and 0.94 with NaCl or with a mixture of glycerol and sucrose in order to find conditions of osmotic stress which would still allow for good growth. Cultures grown at a _w?=?0.96 or 0.99 were then recovered by centrifugation, added to a sucrose–phosphate medium and air-dried. In some assays, a 2-h osmotic stress was applied to the cell concentrate prior to air-drying. Assays were also carried out where betaine, glutamate and proline (BGP) supplements were added as protective compounds to the growth or drying media. For most strains, evidence of osmotic stress and benefits of BGP supplementation on growth occurred at a _w?=?0.96. Growing the cells in complex media adjusted at a _w?=?0.96 did not enhance their subsequent survival to air-drying, but applying the 2-h osmotic stress did. Addition of the BGP supplements to the growth medium or in the 2-h stress medium did not enhance survival to air-drying. Furthermore, addition of BGP to a sucrose–phosphate drying medium reduced survival of the cultures to air-drying. This study provides preliminary data for producers of probiotics who wish to use air-drying in replacement of freeze-drying for the stabilization of cultures.     

Reduced leaching of the herbicide MCPA after bioaugmentation with a formulated and stored Sphingobium sp.

The use of pesticides on sandy soils and on many non-agricultural areas entails a potentially high risk of water contamination. This study examined leaching of the herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) after bioaugmentation in sand with differently formulated and stored Sphingobium sp. T51 and at different soil moisture contents. Dry formulations of Sphingobium sp. T51 were achieved by either freeze drying or fluidised bed drying, with high initial cell viability of 67–85 %. Storage stability of T51 cells was related to formulation excipient/carrier and storage conditions. Bacterial viability in the fluidised bed-dried formulations stored at 25 °C under non-vacuum conditions was poor, with losses of at least 97 % within a month. The freeze-dried formulations could be stored substantially longer, with cell survival rates of 50 %, after 6 months of storage at the same temperature under partial vacuum. Formulated and long-term stored Sphingobium cells maintained their MCPA degradation efficacy and reduced MCPA leaching as efficiently as freshly cultivated cells, by at least 73 % when equal amounts of viable cells were used. The importance of soil moisture for practical field bioaugmentation techniques is discussed.     

Stabilization of a recombinant human epidermal growth factor parenteral formulation through freeze-drying

Development studies were performed to design a pharmaceutical composition that allows the stabilization of a parenteral rhEGF formulation in a lyophilized dosage form. Unannealed and annealed drying protocols were tested for excipients screening. Freeze-dry microscopy was used as criterion for excipients and formulation selection; as well as to define freeze-drying parameters. Excipients screening were evaluated through their effect on freeze-drying recovery and dried product stability at 50 °C by using a comprehensive set of analytical techniques assessing the chemical stability, protein conformation and bioactivity. The highest stability of rhEGF during freeze-drying was achieved by the addition of sucrose or trehalose. After storing the dried product at 50 °C, the highest stability was achieved by the addition of dextran, sucrose, trehalose or raffinose. The selected formulation mixture of sucrose and dextran could prevent protein degradation during the freeze-drying and delivery processes. The degradation rate assessed by RP-HPLC could decrease 100 times at 37 °C and 70 times at 50 °C in dried with respect to aqueous formulation. These results indicate that the freeze-dried formulation represents an appropriate technical solution for stabilizing rhEGF.     

Optimisation of initial cell concentration enhances freeze-drying tolerance of Pseudomonas chlororaphis

The freeze-drying tolerance of Pseudomonas chlororaphis, an antifungal bacterium used as biocontrol agent was investigated. P. chlororaphis is freeze-drying sensitive and the viability drops more than 3 log units in the absence of protective freeze-drying medium. Of the freeze-drying media tested, lactose, sucrose, trehalose, glutamate, sucrose with glutamate, skimmed milk, and skimmed milk with trehalose, skimmed milk gave the lowest survival (0.6+/-0.2%) and sucrose the highest (6.4+/-1.2%). Cellular accumulation of sucrose from the freeze-drying medium and the protective effect of sucrose were dependent on sucrose concentration. The effect of initial cell concentration, from 1 x 10(7) to 5 x 10(10) CFU/ml, on survival after freeze-drying was studied for carbon starved cells with sucrose as freeze-drying medium. The highest freeze-drying survival values, 15-25%, were obtained for initial cell concentrations between 1 x 10(9) and 1 x 10(10) CFU/ml. For cell concentrations outside this window more than 10 times lower survival values were observed. P. chlororaphis was cultivated to induce stress response that could confer protection against freeze-drying inactivation. Carbon starvation and, to a lesser extent, heat treatment enhanced freeze-drying tolerance. By combining optimal cell concentration, optimal sucrose concentration and carbon starvation the survival after freeze-drying was 26+/-6%.     

Survival of Escherichia coli during drying and storage in the presence of compatible solutes

Five different compatible solutes, sucrose, trehalose, hydroxyectoine, ectoine, and glycine betaine, were investigated for their protective effect on Escherichia coli K12 and E. coli NISSLE 1917 during drying and subsequent storage. Two different drying techniques, freeze-drying and air-drying, were compared. The highest survival rate was observed when the non-reducing disaccharides sucrose (for E. coli K12) and trehalose (for E. coli NISSLE 1917) were added. The two tetrahydropyrimidines, hydroxyectoine and ectoine, gave protection to freeze-dried E. coli NISSLE 1917 whereas E. coli K12 was protected only by hydroxyectoine. Glycine betaine seemed to be harmful for both strains of E. coli with both drying techniques. Air0drying gave much better survival rates than freeze-drying. The two strains of E. coli differed in their ability to take up compatible solutes.     

Fluidised-bed spray-drying formulations of Candida sake CPA-1 by adding biodegradable coatings to enhance their survival under stress conditions

The biocontrol agent Candida sake CPA-1 has demonstrated to be effective against several diseases on fruit. However, for application of CPA-1 under field conditions, it was necessary to mix it with a food coating to improve survival under stress conditions, as well as adherence and distribution on fruit surfaces. The objective of this study was to obtain a more competitive formulation under field conditions to be applied independently of any product. To achieve this purpose, the drying process of CPA-1 by a fluidised-bed spray-drying system together with biodegradable coatings was optimised. This approach is novel for the drying system used and the formulation obtained which was able to form a film or coating on fruit surfaces. Several substances were tested as carriers and binders, and drying temperature was optimised. The addition of protective compounds was also tested to improve survival of CPA-1 during the dehydration process. Product shelf life, biocontrol efficacy on grapes against Botrytis cinerea, and the improvement of C. sake behaviour under stress conditions were tested. The optimal temperature of drying was 55 °C and two formulations that were able to develop a coating on fruit surfaces were obtained. One of the formulations was created by using a combination of native and pregelatinised potato starch; the other formulation was obtained using maltodextrin and by adding skimmed milk and sucrose as protectant compounds. The formulated products reduced the incidence and severity of B. cinerea, and CPA-1 survival rate was increased under stress conditions of temperature and humidity.

     

Engineering desiccation tolerance in Escherichia coli

Recombinant sucrose-6-phosphate synthase (SpsA) was synthesized in Escherichia coli BL21DE3 by using the spsA gene of the cyanobacterium Synechocystis sp. strain PCC 6803. Transformants exhibited a 10,000-fold increase in survival compared to wild-type cells following either freeze-drying, air drying, or desiccation over phosphorus pentoxide. The phase transition temperatures and vibration frequencies (P==O stretch) in phospholipids suggested that sucrose maintained membrane fluidity during cell dehydration.     

Formulation and stabilisation of the biocontrol yeast Pichia anomala

The yeast Pichia anomala has antifungal activities and its potential in biocontrol and biopreservation has previously been demonstrated. To practically use an organism in such applications on a larger scale the microbe has to be formulated and stabilised. In this review we give an overview of our experience of formulating and stabilising P. anomala strain J121 in a wider perspective. The stabilisation techniques we have evaluated were liquid formulations, fluidised bed drying, lyophilisation (freeze-drying) and vacuum drying. With all methods tested it was possible to obtain yeast cells with shelf lives of at least a few months and in all cases the biocontrol activity was retained. Fluidised bed drying was dependent on the addition of cottonseed flour as a carrier during the drying process. In liquid formulations a sugar, preferentially trehalose, was a required additive. These two kinds of microbial stabilisation are easily performed and relatively inexpensive but in order to keep the cells viable the biomaterial has to be stored at cool temperatures. However, there is room for optimization, such as improving the growth conditions, or include preconditioning steps to enable the cells to produce more compatible solutes necessary to survive formulation, desiccation and storage. In contrast, lyophilisation and vacuum drying require a lot of energy and are thus expensive. On the other hand, the dried cells were mostly intact after one year of storage at 30°C. Inevitably, the choice of formulation and stabilisation techniques will be dependent also on the intended use.     

Differential effects of polymers PVP90 and Ficoll400 on storage stability and viability of Lactobacillus coryniformis Si3 freeze‐dried in sucrose

Aims: To investigate the effect of freeze‐dried Lactobacillus coryniformis Si3 on storage stability by adding polymers to sucrose‐based formulations and to examine the relationship between amorphous matrix stability and cell viability. Methods and Results: The resistance to moisture‐induced sucrose crystallization and effects on the glass transition temperature (T_g) by the addition of polymers to the formulation were determined by different calorimetric techniques. Both polymers increased the amorphous matrix stability compared to the control, and poly(vinyl)pyrrolidone K90 was more effective in increasing amorphous stability than Ficoll 400. The viability of Lact. coryniformis Si3 after storage was investigated by plate counts following exposure to different moisture levels and temperatures for up to 3 months. The polymers enhanced the cellular viability to different degrees, dependent upon polymer and storage condition. Conclusions: Polymers can be used to enhance the stability of freeze‐dried Lact. coryniformis Si3 products, but cell viability and matrix stability do not always correlate. The general rule of thumb to keep a highly amorphous product 50° below its T_g for overall stability seemed to apply for this type of bacterial products. We showed that by combining thermal analysis with plate counts, it was possible to determine storage conditions where cell viability and matrix stability were kept high. Significance and Impact of the Study: The results will aid in the rational formulation design and proper determination of storage conditions for freeze‐dried and highly amorphous lactic acid bacteria formulations. We propose a hypothesis of reason for different stabilizing effects on the cells by the different polymers based on our findings and previous findings.     

Effect of Sugars, Surfactant, and Tangential Flow Filtration on the Freeze-Drying of Poly(lactic acid) Nanoparticles

Poly(d,l-lactic acid) nanoparticles were freeze-dried in this study. With respect to drying, effect of protective excipients and purification from excess surfactant were evaluated. The nanoparticles were prepared by the nanoprecipitation method with or without a surfactant, poloxamer 188. The particles with the surfactant were used as such or purified by tangential flow filtration. The protective excipients tested were trehalose, sucrose, lactose, glucose, poloxamer 188, and some of their combinations. The best freeze-drying results in terms of nanoparticle survival were achieved with trehalose or sucrose at concentrations 5% and 2% and, on the other hand, with a combination of lactose and glucose. Purification of the nanoparticle dispersion from the excess surfactant prior to the freeze-drying by tangential flow filtration ensured better drying outcome and enabled reduction of the amount of the protective excipients used in the process. The excess surfactant, if not removed, was assumed to interact with the protective excipients decreasing their protective mechanism towards the nanoparticles.     

Influence of freeze-drying and spray-drying preservation methods on survivability rate of different types of protectants encapsulated Lactobacillus acidophilus FTDC 3081

ABSTRACT

The aims of this study were to compare the effectiveness of different drying methods and to investigate the effects of adding a series of individual protectant such as skim milk, sucrose, maltodextrin, and corn starch for preserving Lactobacillus acidophilus FTDC 3081 cells during spray and freeze-drying and storage at different temperatures. Results showed a remarkable high survival rate of 70–80% immediately after spray- and freeze-drying in which the cell viability retained at the range of 10⁹ to 10¹⁰ CFU/mL. After a month of storage, maltodextrin showed higher protective ability on both spray- and freeze-dried cells as compared to other protective agents at 4°C, 25°C, and 40°C. A complete loss in viability of spray-dried L. acidophilus FTDC 3081 was observed after a month at 40°C in the absence of protective agent.     

Formulation of the biocontrol agent <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> CPA-8 using different approaches: liquid,freeze-drying and fluid-bed spray-drying

The present work focuses on the assessment and comparison of three different formulation technologies and the effect of protectants on cell viability, storage stability and antagonistic activity of the biocontrol agent Bacillus amyloliquefaciens CPA-8. Cultures were concentrated with different protective substances such as MgSO₄, sucrose and skimmed milk (SM) and subjected to liquid formulation, freeze-drying and fluid-bed spray-drying. Results showed that CPA-8 freeze-dried cells without protectants or amended with SM suffered the highest losses in cell viability (0.41?0.48 log). Moreover, the cell viability of the tested freeze-dried products decreased after four months of storage at both tested temperatures (4 and 22 °C). Otherwise, liquid and fluid-bed spray-dried products were stable for four months at 4 °C and for 12 months at 22, 4 and ?20 °C, respectively, and no effect of the protectants was observed. The most suitable CPA-8 products were then tested against Monilinia laxa and M. fructicola in artificially wounded nectarines and in all cases the antagonistic activity was maintained similar to fresh cells. The efficacy results revealed that the formulation process did not affect the biocontrol potential of CPA-8. This work led us to conclude that effective formulations with final concentrations ranging from 1.93 × 10⁹–2.98 × 10⁹ CFU ml^?1 and from 4.76 × 10⁹–1.03 × 10¹⁰ CFU g^?1 were obtained for liquid and dried products, respectively. Additionally, the suitability of the fluid-bed spray drying technology should be taken into account to develop a stable and effective CPA-8 product for practical applications to control brown rot in stone fruit.     

Impact of fermentation pH and temperature on freeze-drying survival and membrane lipid composition of <Emphasis Type="Italic">Lactobacillus coryniformis</Emphasis> Si3

During the industrial stabilization process, lactic acid bacteria are subjected to several stressful conditions. Tolerance to dehydration differs among lactic acid bacteria and the determining factors remain largely unknown. Lactobacillus coryniformis Si3 prevents spoilage by mold due to production of acids and specific antifungal compounds. This strain could be added as a biopreservative in feed systems, e.g. silage. We studied the survival of Lb. coryniformis Si3 after freeze-drying in a 10% skim milk and 5% sucrose formulation following different fermentation pH values and temperatures. Initially, a response surface methodology was employed to optimize final cell density and growth rate. At optimal pH and temperature (pH 5.5 and 34 °C), the freeze-drying survival of Lb. coryniformis Si3 was 67% (±6%). The influence of temperature or pH stress in late logarithmic phase was dependent upon the nature of the stress applied. Heat stress (42 °C) did not influence freeze-drying survival, whereas mild cold- (26 °C), base- (pH 6.5), and acid- (pH 4.5) stress significantly reduced survival. Freeze-drying survival rates varied fourfold, with the lowest survival following mild cold stress (26 °C) prior to freeze-drying and the highest survival after optimal growth or after mild heat (42 °C) stress. Levels of different membrane fatty acids were analyzed to determine the adaptive response in this strain. Fatty acids changed with altered fermentation conditions and the degree of membrane lipid saturation decreased when the cells were subjected to stress. This study shows the importance of selecting appropriate fermentation conditions to maximize freeze-drying viability of Lb. coryniformis as well as the effects of various unfavorable conditions during growth on freeze-drying survival.     

Maintenance of quaternary structure in the frozen state stabilizes lactate dehydrogenase during freeze-drying

Sugars inhibit protein unfolding during the drying step of lyophilization by replacing hydrogen bonds to the protein lost upon removal of water. In many cases, polymers fail to inhibit dehydration-induced damage to proteins because steric hindrance prevents effective hydrogen bonding of the polymer to the protein's surface. However, in certain cases, polymers have been shown to stabilize multimeric enzymes during lyophilization. Here we test the hypothesis that this protection is due to inhibition of dissociation into subunits during freezing. To test this hypothesis, as a model system we used mixtures of lactate dehydrogenase isozymes that form electrophoretically distinguishable hybrid tetramers during reversible dissociation. We examined hybridization and recovery of catalytic activity during freeze-thawing and freeze-drying in the presence of polymers (dextran, Ficoll, and polyethylene glycol), sugars (sucrose, trehalose, glucose), and surfactants (Tween 80, Brij 35, hydroxy-propyl beta-cyclodextrin). The surfactants did not protect LDH during freeze-thawing or freeze-drying. Rather, in the presence of Brij 35, enhanced damage was seen during both freeze-thawing and freeze-drying, and the presence of Tween 80 exacerbated loss of active protein during freeze-drying. Polymers and sugars prevented dissociation of LDH during the freezing step of lyophilization, resulting in greater recovery of enzyme activity after lyophilization and rehydration. This beneficial effect was observed even in systems that do not form glassy solids during freezing and drying. We suggest that stabilization during drying results in part from greater inherent stability of the assembled holoenzyme relative to that of the dissociated monomers. Polymers inhibit freezing-induced dissociation thermodynamically because they are preferentially excluded from the surface of proteins, which increases the free energy of dissociation and denaturation.