Maintenance of biodegradation capacities of aerobic bacteria during long-term preservation

Six strains of aerobic Gram negative bacteria degrading toluene, 2,4-dichlorophenoxyacetate, 2,2-dichloropropionate or 3-chlorobenzoate were freeze-dried and liquid-dried in the presence or absence of a protective agent. Survival and maintenance of the biodegradation capability was checked before and after drying, and after storage of the ampoules for one year at 4° or 25°C. In many cases, stability of the degradation potential was low although viability was high. Survival and stability of all strains was always highest after preservation by liquid drying in the presence of myo-inositol and activated charcoal as protective agents. Losses of biodegradation abilities were highest after freeze-drying using no protective agents. Cells grown on complex medium were less sensitive to drying than cells grown under selective pressure (on mineral medium with a special compound as the sole carbon source). A choice of the most appropriate preservation method and the use of an effective protectant is recommended to avoid genetic alterations, and to maintain biodegradation capacities during long-term preservation.     

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.     

Effects of freeze-drying on cytology, ultrastructure, DNA fragmentation, and fertilizing ability of bovine sperm

Freeze-drying sperm is an alternative to cryopreservation. Although sperm from various species has been freeze-dried, there are few reports for bovine sperm. The primary objective of this study was to evaluate the protective effect of various freeze-drying media on the structural and functional components of bovine sperm. The media tested were composed of TCM 199 with Hanks salts supplemented with 10% fetal calf serum (FCS) and TCM 199 with Hanks salts supplemented with 10% FCS and 0.2 M trehalose and EGTA solution. The efficiency of each medium on the preservation of freeze-dried sperm structures was evaluated with conventional and electron microscopy, DNA integrity was analyzed by a TUNEL assay, and fertilizing ability of lyophilized sperm was determined with ICSI. Although the plasma membrane was damaged in all media tested, mitochondria were similarly preserved in all freeze-drying treatments. The acrosome was best preserved in the media that contained trehalose (other treatments also conserved this structure). In contrast, media containing EGTA or trehalose most effectively preserved the nuclei in freeze-dried sperm, with only 2 and 5%, respectively, of cells with fragmented DNA. Furthermore, sperm conserved with these media also had higher (P<0.05) rates of sperm head decondensation (32.5 and 27.5%), pronucleus formation (37.5 and 45.0%) and blastocyst formation (19.4 and 18.3%) than medium supplemented with FCS (15.0, 20.0 and 10.2%, respectively). In conclusion, media with EGTA and trehalose adequately protected bovine sperm during freeze-drying by preserving the viability of their nuclei.     

Effect of culture age, protectants, and initial cell concentration on viability of freeze-dried cells of Metschnikowia pulcherrima

The effect of freeze-drying using different lyoprotectants at different concentrations on the viability and biocontrol efficacy of Metschnikowia pulcherrima was evaluated. The effects of initial yeast cell concentration and culture age on viability were also considered. Yeast cells grown for 36 h were more resistant to freeze-drying than were 48 h cells. An initial concentration of 10? cells·mL?1 favoured the highest survival after freeze-drying. When maltose (25%, m/v) was used as protectant, a high cell viability was obtained (64.2%). Cells maintained a high viability after 6 months of storage at 4 °C. The biocontrol efficacy of freeze-dried cells was similar to the activity of fresh cells on 'Gala' apples and was slightly lower on 'Golden Delicious' apples. After optimizing freeze-drying conditions, the viability of M. pulcherrima cells was similar to that obtained in other studies. The results constitute a first step towards the commercial development of M. pulcherrima as a biocontrol agent.     

Influences of protectants,rehydration media and storage on the viability of freeze-dried <Emphasis Type="Italic">Oenococcus oeni</Emphasis> for malolactic fermentation

Malolactic fermentation (MLF) is an important process in wine production. To achieve successful MLF, expanding interest in ready-to-use Oenococcus oeni starter cultures has placed greater emphasis on developing starter production and preservation methods. In this study, influences of protectants, rehydration media and storage on the viability of O. oeni H-5 when subjected to freeze-drying were investigated. It was found that sodium glutamate (2.5%) was the best protectant, giving the cell viability 72.4%. Adding polysaccharides and disaccharides in suspension media also improved significantly the cell viability. Rehydration is an important step in recovery after freeze-drying. When freeze-dried O. oeni was rehydrated in GYM medium, the highest viability (87.1%) was obtained. Rehydration in the disaccharide solutions tested made the cell viability obviously decrease. After 6 months storage at 4°C, loss of viability occurred, the extent of which depended on protectants used, sodium glutamate again being the most effective.     

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.     

Proceedings: Freezing and freeze-drying of Spirulina platensis

A species of blue-green alga, Spirulina platensis, is extremely susceptible to freezing and drying. However, young cells grown autotrophically with a high intensity of light were resistant to freeze-thawing if the rate of temperature change in the operation was in the range of 20–50 °C/min. Several amino acids, gum arabic, and gelatin were effective in protecting cells from injury caused by freezing.In the case of drying only gum arabic and gelatin could protect cells from injury. The gum arabic-plug method of freeze-drying was shown to be the most suitable method for the maintenance of viability in this alga.The freeze-thawed or freeze-dried cells grew to form abnormally long cells after a period of long lag in the first stage of transfer.     

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.     

The Structure of Destruxin B,a Toxic Metabolite of Oospora destructor

The viability of freeze-dried Lactobacillus bulgaricus B-1 was affected by rehydration temperature, and maximum recovery of the viable cells was obtained when they were rehydrated at 20 to 25°C. Cellular ribonucleotides leaked out from the freeze-dried cells during rehydration, but there was no correlation between the viability of cells and the amount of leaked substances. Rehydration of the freeze-dried cells in the presence of RNase caused marked loss of viability. These results suggest that the cell surface was damaged by freeze-drying and its selective permeability was lost to some extent.     

Effect of various growth media upon survival during storage of freeze-dried Enterococcus faecalis and Enterococcus durans

AIMS: The effects of three different growth media (MRS, M17 and Lee's) on survival during freeze-drying and subsequent storage of six strains of Enterococcus faecalis and two strains of E. durans were investigated. METHODS AND RESULTS: Distinct Enterococcus spp. strains were grown on M17, MRS and Lee's broth, freeze-dried and stored at 20 degrees C in air under darkness. At regular intervals throughout storage, freeze-dried samples were rehydrated and then plated on M17 agar. CONCLUSIONS: A higher survival rate during storage of dried E. durans was obtained when growth occurred in MRS. The same effect was not observed, however, for the majority of E. faecalis strains, which clearly survived better in the dried state when this organism had been grown in M17 or Lee's medium. SIGNIFICANCE AND IMPACT OF STUDY: The survival of the dried Enterococcus spp. tested during storage was shown to be strain-specific and dependent on the growth medium.     

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.     

Factors affecting viable cell counts of freeze-driedCryptococcus terricolus cells

Freeze-drying ofCryptococcus terricolus cells in distilled water resulted in a survival of only 0.1% of the cells. The viability could be increased to 16% by the use of a dextran-sucrose-sodium glutamate solution as suspending medium.For freeze-dried material with both low and high survival rates, and for five as well as for ten days old cultures, malt extract solution was the superior reconstitution medium. Less, but still distinct, protective action was found with a synthetic glucose-urea-salt solution.The viable cell counts of cells freeze-dried in dextran-sucrose-sodium glutamate solution were independent of the medium used for plating. When distilled water was used as a medium for freeze-drying, two to four times higher counts were obtained with malt extract agar than with synthetic glucose-urea-salt agar.Of the twenty different media tried for freeze-drying, sucrose solution gave the best protection. The viability was greatly influenced by the concentration used, maximum values being obtained when more than 10% of sucrose was added. The survival rate increased with the age of the cells until the fifth day, but was independent of the concentration of cells in the suspension. Under optimum conditions a survival rate of more than 80% was reached.     

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.     

Development of Freeze-Dried Bacteriocin-Containing Preparations from Lactic Acid Bacteria to Inhibit Listeria monocytogenes and Staphylococcus aureus

There has been a recent movement to produce and consume ??minimally processed?? and more ??natural?? foods through the use of fewer chemical preservatives. The shift to more ??natural?? foods has resulted in a great interest in the use of bacteriocins from lactic acid bacteria as natural biopreservatives. The objective of this comparative study was to identify bacteriocins that can be produced in low-cost or no-cost dairy-based media (DBM), concentrated using freeze-drying, and applied to Cheddar cheese samples to concurrently inhibit Listeria monocytogenes and Staphylococcus aureus. Select bacteriocin producers were grown in DBM, their cell-free supernatants (CFS) were frozen, and the frozen CFS samples were freeze-dried to produce bacteriocin-containing powders. Cheddar cheese samples were challenged with L. monocytogenes or Staph. aureus cells. The challenged samples were exposed to buffered solutions of freeze-dried powders containing bacteriocins, incubated at 4?°C for 24?C72?h, and plated onto appropriate selective media. All freeze-dried bacteriocin-containing powders tested were active against L. monocytogenes and Staph. aureus. Our research findings indicated that low-cost or no-cost DBM could successfully be used for production of bacteriocin-containing preparations. In addition, freeze-drying was determined to be a feasible approach to prepare concentrated and stable bacteriocin-containing powders for prospective food applications. The prevention of even a very small percentage of foodborne illnesses via the use of bacteriocins as natural biopreservatives would help reduce the number of foodborne illness-related hospitalizations, deaths, and financial loss due to medical expenses, lost income/productivity, cost of litigation/penalties, and loss of trade.     

Survival of cultured cells in the cold : A kinetic study with special reference to the effect of serum in culture media

The survival at 4 °C of mouse fibroblasts (strain L-929) and rat liver cells (strain JTC-25·P5) was kinetically analysed after they had been pre-incubated at 37 °C in medium with or without supplement of serum. Both the composition of medium used for preincubation at 37 °C and that employed for storage at 4 °C had influence on the survival period.When the cells had been grown at 37 °C in Eagle minimal essential medium (MEM) alone, they rapidly lost their viability at 4 °C from the beginning. However, when grown at 37 °C in MEM supplemented with calf serum, they maintained viability at 4 °C for about 16 days and 8 days for L cells and JTC-25·P5 cells respectively, before the initiation of rapid loss of viability. The presence of macromolecular fraction of calf serum in the medium during preincubation was found to be responsible for the prolongation of survival at 4 °C.     

Effect of carbohydrates and related compounds on the long-term preservation of freeze-dried bacteria

A selection of bacteria were freeze-dried in horse serum containing various carbohydrates and related compounds. An accelerated storage test at elevated temperatures was used to determine the long-term viability of the dried organisms with the assumption that the results of accelerated storage reflect long-term viability. The results suggest that meso-inositol, non-reducing disaccharides and certain polyalcohols are the most suitable of the compounds tested for incorporation into suspending media for use in freeze-drying.     

Effects of Freeze-Drying on Permeability and Respiration of Germinating Lily Pollen

The germination of lily pollen (Lilium longiflorum cv. Ace) was impaired by freeze-drying. This loss of viability was associated with a modified pattern of respiration and an increased leakage of soluble carbohydrates, phosphate, and ninhydrin-positive material into the culture medium. 2,4-Dinitro-phenol, (DNP), an uncoupler of oxidative phosphorylation showed a decreased ability to stimulate O₂ uptake in freeze-dried pollen. The altered viability, respiration, and permeability resulted from drying under vacuum and not the initial freezing of the pollen. Mature lily pollen contained approximately 0.3 “Ai phosphate, of which 15 % was inorganic phosphate and about 50 %> was acid soluble organic phosphate of unknown identity.     

不同培养基对酒酒球菌SD-2a存活率及膜脂肪酸组分的影响

[目的] 为获得高效的葡萄酒乳酸菌发酵剂,本文研究了3种具有不同pH缓冲能力的培养基对酒酒球菌接种存活率、冻干存活率及细胞膜脂肪酸组分的影响.[方法]采用平板计数法测定菌体的接种存活率、冻干存活率;并采用GC/MS色谱方法测定收获菌体细胞膜脂肪酸组分.[结果]实验结果表明,没有添加苹果酸的ATB培养基,其pH缓冲能力弱.分别与FMATB和MATB培养基相比,ATB培养基培养获得的菌体,其接种模拟酒培养基后的存活率提高了20.3%和40.2%,其冷冻干燥存活率提高了48.5%和68.3%,其细胞膜中C19cyc11的相对含量提高了10.0%和36.8%,其细胞膜U/S值提高了20.4%和45.2%.[结论]本文推测ATB培养基培养所得菌体,由于自我酸胁迫反应,增强了其对葡萄酒胁迫因素及冷冻干燥的抗性,而该反应与菌体细胞膜脂肪酸组分的变化密切相关.故ATB培养基更适合于酒酒球菌SD-2a发酵剂的制备.     

Optimising the viability during storage of freeze-dried cell preparations of Campylobacter jejuni

Freeze-dried cultures of Campylobacter jejuni are used in the food and microbiological industry for reference materials and culture collections. However, C. jejuni is very susceptible to damage during freeze-drying and subsequent storage and it would be useful to have longer-lasting cultures. The survival of C. jejuni during freeze-drying and subsequent storage was investigated with the aim of optimising survival. C. jejuni was freeze-dried using cultures of different age (24-120 h), various lyoprotectants (10% phytone peptone, proteose peptone, peptonized milk, trehalose, soytone and sorbitol), various storage (air, nitrogen and vacuum) and re-hydration (media, temperature and time) conditions. One-day-old cultures had significantly greater survival after freeze-drying than older cultures. The addition of trehalose to inositol broth as a lyoprotectant resulted in almost 2 log(10) increase in survival after 2 months storage at 4 degrees C. Storage in a vacuum atmosphere and re-hydration in inositol broth at 37 degrees C increased recovery by 1-2 log(10) survival compared to re-hydration in maximal recovery diluent (MRD) after storage at 4 degrees C. Survival during storage was optimal when a one-day-old culture was freeze-dried in inositol broth plus 10% (w/v) trehalose, stored under vacuum at 4 degrees C and re-hydrated at the same incubation temperature (37 degrees C) in inositol broth for 30 min. The results demonstrate that the survival of freeze-dried cells of C. jejuni during storage can be significantly increased by optimising the culture age, the lyoprotectant, and the storage and re-hydration conditions. The logarithmic rate of loss of viability (K) followed very well an inverse dependence on the absolute temperature, i.e., the Arrhenius rate law. Extrapolation of the results to a more typical storage temperature (4 degrees C) predicted a very low K value of 1.5 x 10(-3). These results will be useful to the development of improved reference materials and samples held in culture collections.     

Some observations in freeze-drying of recombinant bioluminescent Escherichia coli for toxicity monitoring

A recombinant bioluminescent bacteria, containing a fabA::luxCDABE fusion gene, has been used to characterize freeze-drying methods, which may be conveniently used as a tool for the development of a portable biosensor. Through residual water, viability, biosensing activity and scanning electron microscopy analyses, the characteristics that four cryoprotectants, trehalose, sucrose, sorbitol, and mannitol, conferred on freeze-dried samples were elucidated, including the morphology, water content and activity of the cells. It was found that trehalose showed the best freeze-drying efficiency among the tested cryoprotectants and it might have a specific capacity limitation in protection of the cells during the freeze step. Humidity might result in damage to the cells, according to the viability, when exposed to air during storage, while the water remaining post freeze-drying showed good correlation with damage to the freeze-dried cells when under air-tight storage conditions. The results with other recombinant bioluminescent bacteria indicated that these findings might be general features of the freeze-drying processes.