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.     

Response Surface Optimization of Lyoprotectant for Lactobacillus bulgaricus During Vacuum Freeze-Drying

The individual and interactive effects of skimmed milk powder, lactose, and sodium ascorbate on the number of viable cells and freeze-drying survival for vacuum freeze-dried powder formulation of Lactobacillus bulgaricus were studied by response surface methodology, and the optimal compound lyoprotectant formulations were gained. It is shown that skim milk powder, lactose, and sodium ascorbate had a significant impact on variables and survival of cultures after freeze-drying. Also, their protective abilities could be enhanced significantly when using them as a mixture of 28% w/v skim milk, 24% w/v lactose, and 4.8% w/v sodium ascorbate. The optimal freeze-drying survival rate and the number of viable cells of Lactobacillus bulgaricus were observed to be (64.41 ± 0.02)% and (3.22 ± 0.02) × 10¹¹ colony-forming units (CFU)/g using the optimal compound protectants, which were very close to the expected values 64.47% and 3.28 × 10¹¹ CFU/g.     

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.     

Survival of spray-dried <Emphasis Type="Italic">Lactobacillus kefir</Emphasis> is affected by different protectants and storage conditions

Survival of two Lactobacillus kefir strains after spray drying in reconstituted skim milk with or without the addition of 12.5 g monosodium glutamate/l, 20 g sucrose/l, or 20 g fructo-oligosaccharides (FOS)/l and during subsequent storage under different conditions of temperature (20 and 30°C) and relative humidity (RH) (0, 11 and 23%) was evaluated. After being dried, L. kefir 8321 and L. kefir 8348 had a decrease in viability of 0.29 and 0.70 log cfu/ml respectively, while the addition of different protectants improved the survival of both strains significantly. During storage, bacterial survival was significantly higher under lower conditions of RH (0–11%), and monosodium glutamate and FOS proved to be the best protectants.     

Influence of formulation additives on the desiccation tolerance and storage stability of blastospores of the entomopathogenic fungus Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes)

Blastospores of the entomopathogenic fungus Paecilomyces fumosoroseus were formulated with 10% lactose/1% bovine serum albumin (BSA) or various compositions of Fantesk™, a starch-oil composite prepared by jet-cooking an aqueous dispersion of starch and oil. Storage stability studies with wet blastospore formulations showed that maximum blastospore survival was achieved during low-temperature storage at -20°C with lactose/BSA formulations or starch-oil formulations supplemented with sucrose, zein protein, and whole milk. Under conditions of wet storage at -20°C, the addition of whole milk to starch-oil formulations significantly improved blastospore stability while the addition of sucrose or zein protein had no effect. In freeze-drying studies, no significant differences were seen in blastospore desiccation tolerance or in stability during storage at either 4 or -20°C when blastospores of P. fumosoroseus were formulated with lactose/BSA or starch-oil formulations with sucrose, zein protein, and whole milk. Freeze-dried blastospore formulations stored at 4°C showed no loss in blastospore viability after 3 months storage and blastospore formulations stored at -20°C showed no loss in viability during the entire 12-month study. For freeze-dried, starch-oil formulations, sucrose was shown to improve blastospore survival during the freeze-drying process. The addition of whole milk to starch-oil formulations significantly improved the stability of freeze-dried blastospores stored at 4°C. Compared to unformulated blastospore suspensions that showed blastospore settling after 30 min, suspensions of blastospores formulated with lactose/BSA or starch-oil composites remained stable for up to 2 h after mixing.     

Cryopreservation studies of Campylobacter

Seven strains of Campylobacter fetus ss. fetus, one of Campylobacter fetus ss. venerealis, and one of Campylobacter jejuni were preserved using a variety of cryopreservation methods. Organisms were frozen to -150 degrees C in a liquid nitrogen refrigerator, in the freezer compartment of a refrigerator (-20 degrees C), and in a mechanical freezer (-65 degrees C). In the latter two cases, viabilities of the organisms were compared after being frozen in Brucella Albimi broth and 10% glycerol. Viabilities were also examined after Campylobacter species were freeze-dried using rapid or slow cooling, using sucrose or skim milk as cryoprotective agents and in bulb-type vials on a manifold or batch vials. Preservation in liquid nitrogen resulted in no loss in viability after 4 years storage. When Campylobacter species were frozen at -20 degrees C, no cells were recovered after 1 month storage in Brucella Albimi broth or seven months in glycerol. A 6.5 log decrease in viability resulted after organisms were frozen at -65 degrees and subsequently stored at the same temperature for 2 years. In this case, glycerol had no protective advantage over Brucella Albimi broth. Postpreservation viability of organisms cooled slowly was two logs higher than those cooled rapidly prior to freeze-drying. When skim milk or sucrose were employed as cryoprotective agents during freeze-drying, equal viabilities resulted. Equivalent viabilities were also demonstrated when the bulb type or "batch" vials were utilized for freeze-drying. No significant differences were observed between the viabilities of the three species when a given cryopreservation method was employed.     

Cell viability of microencapsulated Bifidobacterium animalis subsp. lactis under freeze-drying, storage and gastrointestinal tract simulation conditions

The purpose of this study was to improve the survival of Bifidobacterium animalis subsp. lactis 10140 during freeze-drying process by microencapsulation, using a special pediatric prebiotics mixture (galactooligosaccharides and fructooligosaccharides). Probiotic microorganisms were encapsulated with a coat combination of prebiotics–calcium-alginate prior to freeze-drying. Both encapsulated and free cells were then freeze-dried in their optimized combinations of skim milk and prebiotics. Response surface methodology (RSM) was used to produce a coating combination as well as drying medium with the highest cell viability during freeze-drying. The optimum encapsulation composition was found to be 2.1 % Na-alginate, 2.9 % prebiotic, and 21.7 % glycerol. Maximum survival predicted by the model was 81.2 %. No significant (p?>?0.05) difference between the predicted and experimental values verified the adequacy of final reduced models. The protection ability of encapsulation was then examined over 120 days of storage at 4 and 25 °C and exposure to a sequential model of infantile GIT conditions including both gastric conditions (pH 3.0 and 4.0, 90 min, 37 °C) and intestinal conditions (pH 7.5, 5 h, 37 °C). Significantly improved cell viability showed that microencapsulation of B. lactis 10140 with the prebiotics was successful in producing a stable symbiotic powdery nutraceutical.     

Improving survival and storage stability of bacteria recalcitrant to freeze-drying: a coordinated study by European culture collections

The objective of this study is to improve the viability after freeze-drying and during storage of delicate or recalcitrant strains safeguarded at biological resource centers. To achieve this objective, a joint experimental strategy was established among the different involved partner collections of the EMbaRC project (www.embarc.eu). Five bacterial strains considered as recalcitrant to freeze-drying were subjected to a standardized freeze-drying protocol and to seven agreed protocol variants. Viability of these strains was determined before and after freeze-drying (within 1 week, after 6 and 12 months, and after accelerated storage) for each of the protocols. Furthermore, strains were exchanged between partners to perform experiments with different freeze-dryer-dependent parameters. Of all tested variables, choice of the lyoprotectant had the biggest impact on viability after freeze-drying and during storage. For nearly all tested strains, skim milk as lyoprotectant resulted in lowest viability after freeze-drying and storage. On the other hand, best freeze-drying and storage conditions were strain and device dependent. For Aeromonas salmonicida CECT 894^T, best survival was obtained when horse serum supplemented with trehalose was used as lyoprotectant, while Aliivibrio fischeri LMG 4414^T should be freeze-dried in skim milk supplemented with marine broth in a 1:1 ratio. Freeze-drying Campylobacter fetus CIP 53.96^T using skim milk supplemented with trehalose as lyoprotectant resulted in best recovery. Xanthomonas fragariae DSM 3587^T expressed high viability after freeze-drying and storage for all tested lyoprotectants and could not be considered as recalcitrant. In contrary, Flavobacterium columnare LMG 10406^T did not survive the freeze-drying process under all tested conditions.     

Comprehensive optimization of composite cryoprotectant for Saccharomyces boulardii during freeze-drying and evaluation of its storage stability

Abstract

Saccharomyces boulardii (S. boulardii) is widely adopted in the diarrhea treatment for humans or livestock, so guaranteeing the survival rate of S. boulardii is the critical issue during freeze-drying process. In this study, the survival rate of S. boulardii with composite cryoprotectants during freeze-drying procedure and the subsequent storage were investigated. With the aid of response surface method, the composite cryoprotectants were comprehensively optimized to be lactose of 21.24%, trehalose of 22.00%, and sodium glutamate of 4.00%, contributing to the supreme survival rate of S. boulardii of 64.22?±?1.35% with the viable cell number of 9.5?±?0.07?×?10⁹ CFU/g, which was very close to the expected rate of 65.55% with a number of 9.6?×?10⁹ CFU/g. The accelerated storage test demonstrated that the inactivation rate constant of the freeze-dried S. boulardii powder was k_?18?=?8.04?×?10^?6. In addition, the freeze-dried goat milk powder results exhibited that the inactivation rate constants were k₄?=?4.48?×?10^?4 and k₂₅?=?9.72?×?10^?3 under 4 and 25?°C, respectively. This work provides a composite cryoprotectant formulation that has a good protective effect for the probiotic S. boulardii during freeze-drying process, possessing the potential application prospect in food, medicine, and even feed industry.     

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.     

Optimization of a protective medium for enhancing the viability of freeze-dried Lactobacillus delbrueckii subsp. bulgaricus based on response surface methodology

Response surface methodology (RSM) was used to optimize a protective medium for enhancing the cell viability of Lactobacillus delbrueckii subsp. bulgaricus LB14 during freeze-drying. Using a previous Plackett–Burman design, it was found that sucrose, glycerol, sorbitol and skim milk were the most effective freeze-drying protective agents for L. bulgaricus LB14. A full factorial central composite design was applied to determine the optimum levels of these four protective agents. The experimental data allowed the development of an empirical model (P<0.0001) describing the inter-relationships between the independent and dependent variables. By solving the regression equation, and analyzing the response surface contour and surface plots, the optimal concentrations of the agents were determined as: sucrose 66.40 g/L, glycerol 101.20 g/L, sorbitol 113.00 g/L, and skim milk 130.00 g/L. L. bulgaricus LB14 freeze-dried in this medium obtained a cell viability of up to 86.53%.     

Evaluation of Tris–citric acid,skim milk and sodium citrate extenders for liquid storage of Punjab Urial (Ovis vignei punjabiensis) spermatozoa

The Punjab Urial (Ovis vignei punjabiensis) is an endangered subspecie of ovidae, distributed as small scattered populations in the forest belt of the Himalayan foothills of Pakistan and in the areas enclosed by the Indus and the Jhelum rivers. The present study was conducted to evaluate the liquid storage of Punjab Urial spermatozoa in different extenders for use in future in situ conservation activities. Semen was collected by electro-ejaculation from three captive Punjab Urial rams. Suitable ejaculates of individual animals were pooled and divided into three aliquots for dilution with the experimental extenders (Tris–citric acid, skim milk and sodium citrate) at 37 °C. Extended semen was cooled from 37 °C to 5 °C in 2 h, and stored for three days at 5 °C. Sperm motility (%), viability (%; live/dead), acrosome integrity (%) and plasma membrane integrity (%) were assessed on days 1, 2 and 3 of storage. On day 1, sperm motility, viability as well as acrosome and plasma membrane integrity were similar (p > 0.05) in all three experimental extenders. On day 2, sperm motility, viability, acrosome and plasma membrane integrity were higher (p < 0.05) in Tris–citric acid extender compared to sodium citrate based extender. On day 3 of storage, the values of motility, viability and acrosome integrity were higher (p < 0.05) in Tris–citric acid extender than in skim milk and sodium citrate based extenders. In conclusion, Tris–citric acid extender appears to be a better option compared with skim milk and sodium citrate extenders for liquid storage of Punjab Urial semen.     

Effect of protective agents, rehydration media and initial cell concentration on viability of Pantoea agglomerans strain CPA-2 subjected to freeze-drying

The effect of initial cell density, protective agents and rehydration media on the viability of biocontrol agent Pantoea agglomerans CPA-2 when subjected to freeze-drying was studied. Several additives were tested as protective agents against freeze-drying injury. Maximum viability of the bacterial cells was obtained with disaccharides (survival levels > 60%). Freeze-dried samples were rehydrated with several media; the highest percentage viability was obtained with 10% non-fat skim milk (100%+). The effect of initial bacterial load on the final recovery was dependent on protectant but not on rehydration media. Sucrose was an effective protectant when a high initial concentration (10(10) cfu ml(-1) was used; the opposite occurred with non-fat skim milk. The use of 10(10) cfu ml(-1) as an initial concentration, sucrose as a protectant and non-fat skim milk as a rehydration medium enabled 100% of P. agglomerans viability to be conserved after freeze-drying. Results suggest the possibility of achieving a good formulation system for the studied biocontrol agent with a high number of viable cells to be used toward pathogens, which is desirable for the industrial development of the product.     

A novel cold-active β-d-galactosidase with transglycosylation activity from the Antarctic Arthrobacter sp. 32cB – Gene cloning,purification and characterization

A gene encoding a novel β-d-galactosidase from the psychrotolerant Antarctic bacterium Arthrobacter sp. 32cB was isolated, cloned and expressed in Escherichia coli. The active form of recombinant β-d-galactosidase consists of two subunits with a combined molecular weight of approximately 257 kDa. The enzyme's maximum activity towards o-nitrophenyl-β-d-galactopyranoside was determined as occurring at 28 °C and pH 8.0. However, it exhibited 42% of maximum activity at 10 °C and was capable of hydrolyzing both lactose and o-nitrophenyl-β-d-galactopyranoside at that temperature, with K_m values of 1.52 and 16.56 mM, and k_cat values 30.55 and 31.84 s⁻¹, respectively. Two units of the enzyme hydrolyzed 90% of the lactose in 1 mL of milk at 10 °C in 24 h. The transglycosylation activity of Arthrobacter sp. 32cB β-d-galactosidase was also examined. It synthesized galactooligosaccharides in a temperature range from 10 to 30 °C. Moreover, it catalyzed the synthesis of heterooligosaccharides such as lactulose, galactosyl-xylose and galactosyl-arabinose, alkyl glycosides, and glycosylated salicin from lactose and the appropriate acceptor at 30 °C. The properties of Arthrobacter sp. 32cB β-d-galactosidase make it a candidate for use in the industrial removal of lactose from milk and a promising tool for the glycosylation of various acceptors, especially those which are thermosensitive.     

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.     

Viability of micrococci and lactobacilli upon freezing and freeze-drying in the presence of different cryoprotectants

Studies were conducted on the viability of Micrococcus varians strain M₉₅ and Lactobacillus plantarum strain L₄ upon freezing and freeze-drying using five cryoprotectants (sucrose, lactose, sodium glutamate, peptone, dry nonfat milk) singly or in combinations with gelatin, glutamic acid, and sodium acetate. The number of survivals was determined immediately after treatment and after storage at room temperature or refrigeration temperatures, under vacuum or in air. Dry nonfat milk and peptone introduced at the levels of 8 and 5%, respectively, to broth culture, were found to be the best cryoprotectants providing a 100% viability determined immediately after the treatment of the strains under investigation.Immediately after freezing and freeze-drying, the numbers of viable micrococci remain high, the percentage viability in the presence of almost all the protectants used being 100%. During storage, those numbers decrease rapidly, reaching zero in 3 months upon storage at room temperature in air. The storage ability of lactobacilli is considerably better and, regardless of the fact that the percentage viability decreases, sufficient numbers of viable cells remain after 6 months of storage at both test temperatures.The best results are obtained on storing the microoganisms under vacuum in ampoules under reduced temperatures (+5 °C).     

Inhibition of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> in Hot Dogs by Surface Application of Freeze-Dried Bacteriocin-Containing Powders from Lactic Acid Bacteria

Six lactic acid bacteria (LAB) strains, Lactococcus lactis BFE 920, L. lactis subsp. lactis ATCC 11454, L. lactis subsp. cremoris ATCC 14365, Lactobacillus curvatus L442, Lact. curvatus LTH 1174, and Lact. bavaricus MN, were grown in cheddar cheese whey supplemented with complex nutrient sources. Cell-free culture supernatants were freeze-dried, and the resulting bacteriocin-containing powders were applied on the surface of hot dogs that were inoculated (~4 log cfu/hot dog) with a five-strain Listeria monocytogenes cocktail. Hot dogs were vacuum-sealed and stored at 4 °C for 4 weeks. L. monocytogenes was enumerated, using both tryptic soy agar (TSA) and oxford listeria agar (OXA), on day 0 and at 1, 2, 3, and 4 weeks of the refrigerated storage. In hot dogs containing only the L. monocytogenes inoculum, L. monocytogenes counts increased from 4 up to 7 log cfu/hot dog. All samples containing freeze-dried bacteriocin-containing powders exhibited significantly lowered (P < 0.05) L. monocytogenes populations on the surface of hot dogs throughout the 4-week study except for bavaricin MN powder. Bacterial counts on hot dogs packed without any powder were statistically equal on day 0 when enumerated on OXA. Freeze-dried bacteriocin-containing powders from Lact. curvatus L442 and L. lactis subsp. cremoris ATCC 14365 decreased L. monocytogenes populations on the surface of hot dogs by greater than 2 log cfu/hot dog throughout the 4-week study. For the powdered bacteriocin preparations from L. lactis BFE 920, L. lactis subsp. lactis ATCC 11454, and Lact. curvatus LTH 1174, L. monocytogenes populations were determined to be approximately 3-log cfu/hot dog after 4 weeks of storage.     

Stabilization of freeze-dried Lactobacillus paracasei subsp. paracasei JCM 8130T with the addition of disaccharides,polymers, and their mixtures

Although freeze-drying is a widely used dehydration technique for the stabilizing of unstable lactic acid bacteria, Lactobacillus paracasei subsp. paracasei JCM 8130^T (L. paracasei) is destabilized after freeze-drying and subsequent storage. In order to improve the stability of freeze-dried L. paracasei, effects of disaccharides (sucrose and trehalose), polymers (maltodextrin; MD and bovine serum albumin; BSA), and their mixtures on the survival rate of freeze-dried L. paracasei were investigated. The survival rate of non-additive sample decreased slightly after freeze-drying but decreased drastically after subsequent storage at 37 °C for 4 weeks. The reduction was diminished by the addition of disaccharides and polymers. The stabilizing effect of disaccharides was not affected by the co-addition of MD. In contrast, the disaccharide–BSA mixtures had a synergistic stabilizing effect, and the survival rates were largely maintained even after storage. It is suggested that the synergistic effect originates from the conformational stabilization of the dehydrated bacteria.     

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.     

Effects of various sugars added to growth and drying media upon thermotolerance and survival throughout storage of freeze-dried Lactobacillus delbrueckii ssp. bulgaricus

The aim of this research effort was to investigate the role of various sugar substrates in the growth medium upon thermotolerance and upon survival during storage after freeze-drying of Lactobacillus bulgaricus. Addition of the sugars tested to the growth medium, and of these and sorbitol to the drying medium (skim milk) was investigated so as to determine whether a relationship exists between growth and drying media, in terms of protection of freeze-dried cells throughout storage. The lowest decrease in viability of L. bulgaricus cells after freeze-drying was obtained when that organism was grown in the presence of mannose. However, L. bulgaricus clearly survived better during storage when cells had been grown in the presence of fructose, lactose or mannose rather than glucose (the standard sugar in the growth medium). A similar effect could not be observed in terms of thermotolerance; in this case, the growth medium supplemented with lactose was found to yield cells bearing the highest heat resistance. Supplementation of the drying medium with glucose, fructose, lactose, mannose or sorbitol led in most cases to enhancement of protection during storage, to a degree that was growth medium-dependent.