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%.     

Viability of the R, S and M variants of Mycobacterium lacticolum under various preservation conditions

The survival rate of Mycobacterium lacticolum and the proportion between its R, S and M variants were studied in the course of 12 months under different conditions of storage (freeze-drying, under vaseline oil, in 0.85% NaCl solution and in distilled water). A high survival rate of the variants was found in cells freeze-dried in a protective medium containing 10% of sucrose +1% of gelatin as well as in a 0.85% solution of NaCl.The survival rate of te variants differed by 2--3 orders of magnitude in cells freeze-dried with sodium glutamate or suspended in distilled water. The proportion between the R, S and M variants in the population noticeable changed after storage under these conditions.     

Effect of protective agents, freezing temperature, rehydration media on viability of malolactic bacteria subjected to freeze-drying

AIMS: The effects of protective agents, rehydration media and freezing temperature on the viabilities of Lactobacillus brevis and Oenococcus oeni H-2 when subjected to freeze-drying were investigated. METHODS AND RESULTS: Several protectants and rehydration media were tested to improve the survival after freeze-drying. The cells were also frozen at -65 and -20 degrees C to check the effect of freezing temperature on the viability. CONCLUSIONS: The best protectant and rehydration medium to obtain the highest viability after freeze-drying varied with the species of bacteria. Yeast extract (4.0%) and sodium glutamate (2.5% ) gave maximum viability of L. brevis and O. oeni (67.8% and 53.6% respectively). The highest survival of L. brevis and O. oeni were obtained when rehydrated with 10% sucrose and MGY medium respectively. When the bacterial cells were frozen quickly (-65 degrees C) than slowly (-20 degrees C), L. brevis and O. oeni both showed increased viability after freeze-drying. SIGNIFICANCE AND IMPACT OF THE STUDY: The viabilities of L. brevis and O. oeni after freeze-drying were shown to be strain specific and dependent on protective agents, rehydration media and freezing temperature.     

Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups

Viable counts of heterotropic soil bacteria were 3–5 times higher on low-nutrient agar media compared with a series of conventional agar media. Substantial amounts of monosaccharides and amino acids were present in solid media made from distilled water and agar powder, and a salt-solution agar medium (without organic substrates added) gave practically the same colony counts as the low nutrient soil extract agar medium. MPN values were comparable to or lower than plate counts. A search for slow-growing cells in the negative MPN tubes by fluorescence microscopical examination after 3 months incubation was negative.The viable counts were 2–4% of the total microscopical counts in different soils. Assuming that the colony-forming cells did not derive from the numerous dwarf cells present in soil, a calculated percent viability of the larger cells was about 10%. The ecological significance of the plate-counting technique is discussed.     

Occurrence and Growth of Yeasts in Yogurts

Yogurts purchased from retail outlets were examined for the presence of yeasts by being plated onto oxytetracycline malt extract agar. Of the 128 samples examined, 45% exhibited yeast counts above 10³ cells per g. A total of 73 yeast strains were isolated and identified as belonging to the genera Torulopsis, Kluyveromyces, Saccharomyces, Candida, Rhodotorula, Pichia, Debaryomyces, and Sporobolomyces. Torulopsis candida and Kluyveromyces fragilis were the most frequently isolated species, followed by Saccharomyces cerevisiae, Rhodotorula rubra, Kluyveromyces lactis, and Torulopsis versatilis. The growth of yeasts in yogurts was related to the ability of the yeasts to grow at refrigeration temperatures, to ferment lactose and sucrose, and to hydrolyze milk casein. Most yeast isolates grew in the presence of 100 μg of sorbate and benzoate preservatives per ml. Higher yeast counts from yogurts were obtained when the yogurts were plated onto oxytetracycline malt extract agar than when they were plated onto acidified malt extract agar.     

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.     

Influence of the growth phase and culture medium on the survival of Mannheimia haemolytica during storage at different temperatures

AIMS: To quantify the influence of the growth phase, storage temperature and nutritional quality of the plate count medium on the apparent viability of Mannheimia haemolytica during storage at different temperatures. METHODS AND RESULTS: Mannheimia haemolytica was grown in shake flasks and in aerobic continuous culture to investigate factors affecting cell viability during storage, which was determined using plate counts on different media and epifluorescence microscopy. The high specific death rates of cells harvested after cessation of exponential growth and stored at 22, 4, -18 and -75 degrees C could be related to the rapid onset of exponential death in batch cultures. Yeast extract supplementation of the culture medium increased the viability of cells at most of the above-mentioned storage temperatures. Of the total cell count in continuous culture, only 48% could be recovered on brain-heart infusion agar, whereas supplementation of the agar medium with foetal calf serum increased the plate count to 71% of the total count. CONCLUSIONS: Mannheimia haemolytica cells harvested from the exponential growth phase had the highest survival rate during storage at low temperatures. Plate count values also depended on the nutritional quality of the agar medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Results presented here impact on the procedures for culture preservation and plate count enumeration of this fastidious animal pathogen.     

Malt Extract as Protective Medium for Lactic Acid Bacteria in Cryopreservation

S ummary : Malt extracts prepared from barley and sorghum malts were used as media for preserving bacteria at -196°. The results are compared with those obtained with other protective substances such as dimethylsulphoxide, glycerol, malic acid, sucrose and sodium glutamate. The malt extract medium is recommended for use in the food industry.     

Freeze-drying of live attenuated Vibrio anguillarum mutant for vaccine preparation

Vibrio anguillarum MVAV6203 is a mutant strain as a candidate of live attenuated vaccine. In vaccine preparation, the freeze-drying conditions of the strain were investigated to improve the survival after freeze-drying, including the protectant, rehydration medium, freezing temperature, and initial cell concentration. Vibrio anguillarum MVAV6203 is sensitive to freeze-drying and the viability was only 0.03% in the absence of protectant. Of the tested protectants, 5% trehalose with 15% skimmed milk gave the highest viability of 34.2%. Higher cell survival was obtained by quick freezing at -80 degrees C than slow freezing at -20 degrees C. Initial cell concentration was another important factor, preferable for 1-3 x 10(10)CFU/ml. The supplementation of 10% skimmed milk in rehydration medium improved obviously freeze-drying viability. The combination of the optimal conditions achieved 51.4% cell viability after freeze-drying.     

Enhancement in the viability and biosensing activity of freeze-dried recombinant bioluminescent bacteria

The genetically-engineeredEscherichia coli strain, DPD2540, which contains afabA::luxCDABE fusion gene, gives a bioluminescent output when membrane fatty acid synthesis is needed. For more practical application of this strain in the field as biosensor, freeze-drying was adopted. A 12% sucrose solution with Luria-Bertani (LB) broth, as determined by the viability after freeze-drying, was found to be the most effective composition for lyophilization solution among various compositions tested. Rapid freezing with liquid nitrogen also gave the best viability after freeze-drying as compared to samples frozen at −70°C and −20°C. The biosensing activities of the cells showed a greater sensitivity when the cells from the exponential phase were freeze-dried. Finally, the optimum temperature for use of the freeze-dried cells in the biosensor field was determined.     

Freeze-drying of live oral streptomycin dependent mutant Shigella vaccines: survival of streptomycin-dependent Shigella sonnei after freeze-drying

The resistance to freeze-drying damage of three strains of S. sonnei has been studied. Over 90% survival was obtained with all strains when suspended in a solution containing sucrose and peptone. However in distilled water only the virulent streptomycin sensitive strain retained its resistance unchanged. This extreme stability may be associated with its colonial morphology.     

Heat resistance of Paecilomyces variotii in sauce and juice

It has been demonstrated that some anamorphic fungi ( Paecilomyces variotii, Fusarium sp) could cause spoilage of food products after pasteurisation. Four food-borne and one clinical isolate of P. variotii were cultivated on one solid medium and three liquid media. Their survival after heating at 80–100˚C for 0.25–15 min in sterile distilled water and curry sauce or fruit juice was investigated. Heat resistance was determined by the thermal death method in a thermostatically-controlled oil bath. The most resistant spores of P. variotii from curry sauce cultivated on malt extract agar survived 100˚C for 0.5 min in sauce; cultivated in curry sauce survived 100˚C for 15 min in water and cultivated in malt broth survived 100˚C for 5 min in water and sauce. The most resistant spores of P. variotii from juice cultivated on malt extract agar were able to survive 100˚C for 15 min in water; cultivated in juice survived 100˚C for 0.5 min in juice and suspensions from cultivation in malt broth survived 100˚C for 1.5 min in juice. Spores of the clinical strain of P. variotiifrom malt extract agar survived 95˚C for 0.33 min in water, and orange juice cultures survived 96˚C for 10 min in orange juice. It was thus found that P. variotii strains cultivated in food were better adapted to heat stress, suggesting that fungal biomass suspensions were able to survive the higher temperatures for longer time intervals than spore suspensions. Journal of Industrial Microbiology & Biotechnology (2000) 24, 227–230. Received 02 June 1999/ Accepted in revised form 05 December 1999     

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.     

Viability of lyophilized anaerobes in two media

Viability of 15 species of anaerobes was followed after freeze-drying and storage for 1 year. Organisms maintained in a 12% sucrose concentration in chopped meat carbohydrate broth survived longer and maintained higher viability counts than those organisms in double-strength skim milk.     

Metabolism of vanillic acid by Micromycetes

The ability of 953 strains of Micromycetes to grow with vanillic acid (0.5 g/l) was investigated. Toxicity assays were performed on malt extract/agar medium, while consumption was estimated by growing fungi on solid synthetic medium with vanillic acid as sole carbon source. More than half of the tested strains grew in both conditions. After cultivation on solid media, 296 strains were selected and cultivated in liquid synthetic medium. These experiments allowed division of the Micromycetes into different groups according to their consumption of the phenolic compound and the appearance of new metabolites. Results were related to the taxonomic position of the strains.     

Preservation of Mycoplasma Strains by Freezing in Liquid Nitrogen and by Lyophilization with Sucrose

Procedures for maintaining Mycoplasma strains are described. Liquid nitrogen storage provided an adequate means for keeping stock cultures stable. Over 74 strains of approximately 26 species have been preserved in this way, some for as long as 9 years. Mycoplasma strains can also be recovered satisfactorily from the freeze-dried state when the procedure described includes the use of 12% (v/v) sucrose as an additive. Fifteen strains representing 12 or more species were subjected to a freeze-drying program with and without sucrose added to concentrated cell suspensions in growth medium. Cell counts indicate improved survival with sucrose.     

Effect of protective agents and previous acclimation on ethanol resistance of frozen and freeze-dried Lactobacillus plantarum strains

The aim of this work was to study the protective effect of sucrose, trehalose and glutamate during freezing and freeze-drying of three oenological Lactobacillus plantarum strains previously acclimated in the presence of ethanol. The efficiency of protective agents was assessed by analyses of membrane integrity and bacterial cultivability in a synthetic wine after the preservation processes. No significant differences in the cultivability, with respect to the controls cells, were observed after freezing at −80 °C and −20 °C, and pre-acclimated cells were more resistant to freeze-drying than non-acclimated ones. The results of multiparametric flow cytometry showed a significant level of membrane damage after freeze-drying in two of the three strains. The cultivability was determined after incubation in wine-like medium containing 13 or 14% v/v ethanol at 21 °C for 24 h and the results were interpreted using principal component analysis (PCA). Acclimation was the most important factor for preservation, increasing the bacterial resistance to ethanol after freezing and freeze-drying. Freeze-drying was the most drastic method of preservation, followed by freezing at −20 °C. The increase of ethanol concentration from 6 to 10% v/v in the acclimation medium improved the recovery of two of the three strains. In turn, the increase of ethanol content in the synthetic wine led to a dramatic decrease of viable cells in the three strains investigated. The results of this study indicate that a successful inoculation of dehydrated L. plantarum in wine depends not only on the use of protective agents, but also on the cell acclimation process prior to preservation, and on the ethanol content of wine.     

Effects of protective agents on membrane fluidity of freeze-dried Lactobacillus delbrueckii ssp. bulgaricus

AIMS: To evaluate the effect of protective agents upon survival of Lactobacillus delbrueckii ssp. bulgaricus during freeze-drying and storage, and selective amino acids on cell membrane fluidity. METHODS AND RESULTS: The protective effect of amino-acids and sugars at different concentrations was studied by determining the viability of lyophilized cells after storage under air at 30 degrees C. Survival following freeze-drying was improved by all compounds. During storage, neither proline nor maltose had protective effects on lyophilized Lact. delbrueckii ssp. bulgaricus. Glutamate 5% and aspartate 5% showed similar protection capability during freeze-drying (94-95%) and after storage (92-99%). Fluorescence probes (DPH and TMA-DPH) were used to study the effect of both amino acids on membrane fluidity. Polarization decreased with increasing concentrations of glutamate or aspartate. Lowest values were observed with TMA-DPH. CONCLUSIONS: Glutamate 5% and aspartate 5% allowed maintaining high viability rates during freeze-drying and storage of Lact. delbrueckii ssp. bulgaricus because of an increase of the membrane fluidity by inserting in the interfacial region of bacterial plasma membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results show the first evidence of the mechanisms underlying glutamate and aspartate as lyoprotectors.     

Influence of oligosaccharides on the viability and membrane properties of Lactobacillus reuteri TMW1.106 during freeze-drying

Freeze-drying is a process commonly used in starter culture preparation. To improve the survival rate of bacteria during the process, cryoprotectives are usually added before freezing. This study investigated the influence of the addition of sucrose, fructo-oligosaccharides (FOS), inulin and skim milk on the viability and membrane integrity of Lactobacillus reuteri TMW1.106 during freezing, freeze-drying and storage. The effect of drying adjuncts on survival was correlated to their interaction with bacterial membrane by determination of the parameters membrane fluidity and membrane lateral pressure. Sucrose, FOS and skim milk significantly enhanced survival of exponential-phase cells of L. reuteri during freeze-drying. Cellular viability during storage of exponential-phase cells remained highest for cells dried in the presence of skim milk and inulin. Membranes of these cells were completely permeabilized after freeze-drying. The application of FOS significantly improved survival of stationary phase cells of L. reuteri TMW1.106 after freeze-drying and storage. This increased viability of L. reuteri TMW1.106 in the presence of FOS correlated to improved membrane integrity. Fructo-oligosaccharides and fructans, but not gluco-oligosaccharides interacted with membrane vesicles prepared from L. reuteri TMW1.106 as indicated by increased membrane lateral pressure in the presence of FOS and fructans. Increased membrane integrity of stationary phase L. reuteri TMW1.106 was attributed to direct interactions between FOS and the membrane which leads to increased membrane fluidity and thus improved stability of the membrane during and rehydration.     

Injury and Death of Streptococcus lactis due to Freezing and Frozen Storage

Cells of Streptococcus lactis were harvested in the early stationary phase, washed, and resuspended in either skim milk (10% nonfat milk) or buffered distilled water (0.0003 m dipotassium phosphate, pH 7.2). Samples of each suspension were frozen and stored at -20 C for intervals up to 28 days. Colony counts of the frozen culture were made using lactic agar and a “restricted” lactic agar medium (Tryptone reduced to 0.5%) to determine injury and death. Death was determined by the difference in plate counts on lactic agar before and after freezing. Injured cells were determined by the difference in plate counts on the two plating media. Greatest injury of the cells occurred during early stages of frozen storage and decreased with time, and death continuously increased. Injury and death were more pronounced when cells were frozen in water than when frozen in 10% nonfat milk solids. Certain cultures survived better when frozen rapidly, whereas with others survival was greater when freezing was slow. Successive freezing, thawing, and propagation of the culture gradually eliminated cells which showed injury by freezing.