Effect of cryopreservation and lyophilization on viability and growth of strict anaerobic human gut microbes

Strict anaerobic gut microbes have been suggested as ‘next‐generation probiotics’ for treating several intestinal disorders. The development of preservation techniques is of major importance for therapeutic application. This study investigated cryopreservation (?80°C) and lyophilization survival and storage stability (4°C for 3 months) of the strict anaerobic gut microbes Bacteroides thetaiotaomicron, Faecalibacterium prausnitzii, Roseburia intestinalis, Anaerostipes caccae, Eubacterium hallii and Blautia obeum. To improve preservation survival, protectants sucrose and inulin (both 5% w/v) were added for lyophilization and were also combined with glycerol (15% v/v) for cryopreservation. Bacterial fitness, evaluated by maximum growth rate and lag phase, viability and membrane integrity were determined using a standardized growth assay and by flow cytometry as markers for preservation resistance. Lyophilization was more detrimental to viability and fitness than cryopreservation, but led to better storage stability. Adding sucrose and inulin enhanced viability and the proportion of intact cells during lyophilization of all strains. Viability of protectant‐free B. thetaiotaomicron, A. caccae and F. prausnitzii was above 50% after cryopreservation and storage and increased to above 80% if protectants were present. The addition of glycerol, sucrose and inulin strongly enhanced the viability of B. obeum, E. hallii and R. intestinalis from 0.03–2% in protectant‐free cultures to 11–37%. This is the first study that quantitatively compared the effect of cryopreservation and lyophilization and the addition of selected protectants on viability and fitness of six strict anaerobic gut microbes. Our results suggest that efficiency of protectants is process‐ and species‐specific.     

Effect of cultivation conditions on trehalose content and viability of brewing yeast following preservation via filter paper or lyophilization methods

The effects of variations in cultivation conditions on trehalose concentration and the viability of brewing yeasts following preservation by filter paper or lyophilization methods were evaluated. In case of filter paper preservation, the cultivation period had no affect on yeast viability, while agitation and aeration during cultivation had a positive effect regarding viability of the bottom-fermenting strains, Rh and Frank. For effective preservation, it was necessary to harvest yeast cells from the stationary phase during cultivation. For lyophilization preservation, the yeast strains tested showed a negative effect on viability, independent of strain or cultivation method. No significant correlation was found between trehalose concentration and yeast viability following either filter paper or lyophilization preservation. However, the filter paper preservation method was suitable for both bottom and top brewing yeast strains with regard to feasibility, viability, and maintenance of the yeast’s specific character.     

The relationship between fungal preservation method and secondary metabolite production in Metarhizium anisopliae and Fusarium oxysporum

The effects of preservation regime on secondary metabolite production in two genera of economically important fungi, Metarhizium anisopliae and Fusarium oxysporum, was assessed using thin layer chromatography and high performance liquid chromatography over a 2-year testing period. Five preservation regimes, commonly used in microbial culture collections throughout the world were investigated: continual sub-culture, lyophilization, storage of mycelial plugs in water, storage at –20 °C and cryopreservation with liquid nitrogen. Preservation regime influenced secondary metabolite production in the test fungi. Changes in secondary metabolite profiles occurred after relatively short storage periods in most strains, irrespective of the preservation treatment used. Although no preservation treatment can be guaranteed to provide total stability of secondary metabolite production, cryopreservation was the best of the methods tested. Response to preservation and storage also differed between strains of the same species. Therefore, there is a need to develop new and existing preservation criteria with an emphasis on strain-specific criteria in order to reduce the prospects of instability in secondary metabolite production.     

Brachypodium distachyon as alternative model host system for the ergot fungus Claviceps purpurea

To investigate its susceptibility to ergot infection, we inoculated Brachypodium distachyon with Claviceps purpurea and compared the infection symptoms with those on rye (Secale cereale). We showed that, after inoculation of Brachypodium with Claviceps, the same disease symptoms occurred in comparable temporal and spatial patterns to those on rye. The infection rate of Claviceps on this host was reduced compared with rye, but the disease could be surveyed by fungal genomic DNA quantification. Mutants of Claviceps which were virulence attenuated on rye were also affected on Brachypodium. We were able to show that pathogenesis‐related gene expression changed in a typical manner for biotrophic pathogen attack. Our results indicated that the Claviceps–Brachypodium interaction was dependent on salicylic acid, cytokinin and auxin. We consider Brachypodium to be a suitable and useful alternative host; the increased sensitivity compared with rye will be valuable for the identification of infection mechanisms. Future progess in understanding the Claviceps–plant interaction will be facilitated by the use of a well‐characterized model host system.     

The effects of preservation procedures on amniotic membrane's ability to serve as a substrate for cultivation of endothelial cells

Amniotic membrane (AM) has been used as a scaffold for the ex vivo expansion of different types of cells and a cell delivery matrix in regenerative medicine. Since the preservation procedures can influence the AM properties for experimental and clinical purposes, this study was established to investigate the feasibility of using the AM after different preservation methods to serve as substrates for endothelial cell expansion ex vivo. The effects of cryopreservation and lyophilization were evaluated on mechanical and histological characteristics of the AM, and the results were compared with the fresh AM. The ECM components of the basement membrane were well conserved in all groups. Although lyophilization resulted in more histological changes and lower level of physical variables including thickness, F_max, elongation at break and suture retention than the fresh and cryopreserved AM, endothelial cells grown on the lyophilized AM were better attached to the basement membrane. Cytotoxicity assay by MTT showed that the lyophilized AM is a compatible substrate for endothelial cells cultivation. The findings of this study suggest that the lyophilized AM is a suitable matrix for cultivation of endothelial cells due to this fact that lyophilization led to exposure of basement membrane of the AM.     

A new freeze-drying method for the preservation of nitrogen-fixing and other fragile bacteria

A simple effective and compact freeze-drying method involving skim milk 20% (w/v) and glutamate 5% or meso-inositol 5% or honey 10% or raffinose 5% for the long-term preservation of bacteria is described. As a case example more than 160 strains representing 36 species of nitrogen-fixing bacteria, 11 species of chemolithorutotrophic bacteria and five species of Aquaspirillum were successfully preserved. All tested strains proved viable and showed about 10–100% survival after freeze-drying and during 2–3 years of storage at +9°C. In such lyophilized cultures no loss in plasmids or other desirable characters was observed. The method is also suitable for the preservation of other fragile and difficult microorganisms as several other strains including bacteria with introduced plasmids could equally survive well and retained plasmids after lyophilization with this method.     

Studies onClaviceps parasitic onPanicum species in India

Panicum repens andP. antidotale were found to be infected withClaviceps sp. This is the first report of ergot onP. repens. The pyrenomycete produced abundant sclerotia on the host plants. The sclerotia contained 0.71 and 0.68 % alkaloids, respectively, which predominantly consisted of chanoclavine, festuclavine and agroclavine. The infected grasses were possibly mycotoxic. Submerged cultures ofClaviceps strain isolated fromPanicum spp produced significant amount of chanoclavine, festuclavine and agroclavine. No pharmaceutically important alkaloid was found in sclerotia or in submerged culture.     

Investigation of Lactobacilli Strains during Lyophilization

The Freezing Points of three lactobacilli strains cultivated in skim milk and MRS-broth were determined. Their survival was analyzed after lyophilization. A maximal preservation of the cell number of the R1 strain was found. The fermentation activity of the laclobacilli was investigated before and after lyophilization in order to characterize their species identification. Copyright 1993, 1999 Academic Press.     

Selection of Ergot Alkaloid Producers by Induced Mutagenesis

Using the induced mutagenesis technique, a series of genetically modified Clavicepssp. VKM F-2609 strains that display high levels of agroclavine and elymoclavine synthesis were selected by induced mutagenesis. Compared to the parent strain, c106 displayed a 40-fold higher level of agroclavine synthesis, and c66 displayed an eightfold higher level of elymoclavine synthesis. The levels of synthesis of other alkaloids were decreased in these strains. The effects of various carbohydrates on the strain growth and ergot alkaloid biosynthesis was then investigated in both the parent strain and c106. The largest amount of agroclavine was synthesized by c106 strain growing on a medium with maltose.     

Alternative methods for sampling and preservation of photosynthetic pigments and tocopherols in plant material from remote locations

Current methods for the study of pigments involve freezing in liquid nitrogen and storage at −80°C or lyophilization until HPLC analysis. These requirements greatly restrict ecophysiological research in remote areas where such resources are hardly available. We aimed to overcome such limitations by developing several techniques not requiring freezing or lyophilization. Two species with contrasting foliar characteristics (Olea europaea and Taraxacum officinale) were chosen. Seven preservation methods were designed, optimized and tested in a field trial. These protocols were compared with a control immediately frozen after collection. Pigments and tocopherols were analysed by HPLC. Main artefacts were chlorophyll epimerization or phaeophytinization, carotenoid isomerization, altered de-epoxidation index and tocopherol degradation. Among all methods, sample desiccation in silica gel provides robust samples (pigment composition was unaffected by storage time or temperature) and almost unaltered pigment profiles, except for a shift in epoxidation state. Although liquid nitrogen freezing and subsequent lyophilization or freezer storage were preferred, when these facilities are either not available or not suitable for long-distance transport, desiccation with silica gel, passive extraction in acetone and/or storage of fresh samples in water vapour saturated atmospheres enable a complete pigment characterization. Silica gel is advisable for long-term sample conservation.     

去甲基万古霉素菌种低温冷冻、冷干保藏条件优化及10 年菌种保藏效果

【目的】针对去甲基万古霉素产生菌不耐保藏的问题,改进菌种保藏方法,对超低温液氮保藏、-80°C低温冷冻保藏、冷干保藏方法跟踪考察10年保藏稳定性,评价不同保藏方法对去甲基万古霉素产生菌的保藏适用性。【方法】采用甘油作基础保护剂进行超低温液氮保藏和-80°C低温冷冻保藏,采用脱脂牛奶作基础保护剂进行冷干保藏,针对超低温液氮保藏进行降温速率考察,研究非渗透性冷冻保护剂海藻糖、聚乙烯吡咯烷酮(PVP)等对3种保藏方法的冻存影响,对优选出的保藏方法进行10年跟踪考察。【结果】3种保藏方法冻后菌种存活率依次为:-80°C低温冷冻保藏超低温液氮保藏冷干保藏。液氮保藏最适降温速率为快速冷冻。优选出最佳保护剂配方:超低温液氮保藏为甘油8.0%,海藻糖3.5%;-80°C低温冷冻保藏为甘油6.0%,PVP 5.0%;冷干保藏为脱脂牛奶,6.0%海藻糖。采用优化保藏条件,液氮保藏10年存活率稳定在70.6%,菌种发酵水平为入藏水平的92.9%。【结论】在优化条件下,尤以超低温液氮保藏适合于去甲基万古霉素产生菌长期保藏。     

Survival or revival: long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria

Knowledge on long-term preservation of micro-organisms is limited and research in the field is scarce despite its importance for microbial biodiversity and biotechnological innovation. Preservation of fastidious organisms such as methane-oxidizing bacteria (MOB) has proven difficult. Most MOB do not survive lyophilization and only some can be cryopreserved successfully for short periods. A large-scale study was designed for a diverse set of MOB applying fifteen cryopreservation or lyophilization conditions. After three, six and twelve months of preservation, the viability (via live-dead flow cytometry) and culturability (via most-probable number analysis and plating) of the cells were assessed. All strains could be cryopreserved without a significant loss in culturability using 1% trehalose in 10-fold diluted TSB (TT) as preservation medium and 5% DMSO as cryoprotectant. Several other cryopreservation and lyophilization conditions, all of which involved the use of TT medium, also allowed successful preservation but showed a considerable loss in culturability. We demonstrate here that most of these non-culturables survived preservation according to viability assessment indicating that preservation induces a viable but non-culturable (VBNC) state in a significant fraction of cells. Since this state is reversible, these findings have major implications shifting the emphasis from survival to revival of cells in a preservation protocol. We showed that MOB cells could be significantly resuscitated from the VBNC state using the TT preservation medium.     

Claviceps arundinis identification and its role in the die-back syndrome of Phragmites australis populations in central Italy

Common reed Phragmites australis (Cav.) Trin. ex Steud. is one of the most widely distributed angiosperms with important ecological functions. In recent decades, it has been affected by a severe decline known as reed die-back syndrome (RDBS), the causal factors of which are still under investigation. Among the biotic factors that influence the dynamic of the reed population, the role of microorganisms is still poorly understood. During surveys carried out on P. australis populations in Central Italy, Claviceps-like sclerotia were detected: is Claviceps infection related to P. australis sexual reproduction and seed production? Could Claviceps infection be involved in the RDBS? These are the questions that we address. We characterized the sclerotia at the morphological, molecular, and chemical level and we demonstrated that they belong to Claviceps arundinis Pa?outová &; M. Kola?ík. To our knowledge, this is the first report of C. arundinis on P. australis in Italy. Furthermore, the association of C. arundinis with RDBS was evaluated considering a set of macromorphological traits generally related to RDBS, such as P. australis clumping habit, culm height and diameter. No correlation was seen between the occurrence of C. arundinis and the declining status of reed populations.     

Long-Term Preservation of Fungus Cultures with Liquid Nitrogen Refrigeration

A preservation technique was tested on 162 strains of culturally fastidious fungi sensitive to lyophilization, representing five classes. The results indicated that liquid nitrogen storage of frozen specimens may be used as an alternative to lyophilization for long-term preservation of stock cultures of fungi. The fungus was frozen in 10% (v/v) glycerol-water menstruum in heat-sealed ampoules. The cooling from ambient temperatures to -35 C was controlled at a rate of approximately 1 C per minute. Further cooling to the storage temperature of -165 to -196 C was uncontrolled and took place at an accelerated rate. Frozen ampoules were thawed in a water bath at 38 to 40 C. Viable and unmutated cultures were developed from reactivated specimens after storage for as long as 5 years.     

Screening for Ergot Alkaloid Producers among Microscopic Fungi by Means of the Polymerase Chain Reaction

The potential of the polymerase chain reaction for the detection of ergot alkaloid producers among microscopic fungi of the generaPenicilliumand Clavicepswas evaluated. Twenty-three strains of various species of fungi with a previously studied capacity for alkaloid production were used. The internal fragment of the gene encoding 4-dimethylallyltryptophan synthase, the enzyme catalyzing the first step in the biosynthesis of ergot alkaloids, was amplified using degenerate primers. This approach revealed an about 1.2-kb specific DNA fragment in micromycetes synthesizing ergot alkaloids with complete tetracyclic ergoline system. Microorganisms that produce alkaloids with modified C or D ergoline rings, as well as -cyclopiazonic acid, did not yield the PCR fragment of the expected size. This fragment was also not found in fungi incapable of ergot alkaloid production.     

Application of accelerated storage test to lyophilized plant viruses

Summary The preservation of nine plant virus strains of tobamovirus and cucumovirus groups after freeze-drying in different lyophilic forms was examined. Quantitative studies on survival were performed. In tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV), accelerated storage test at 70 – 100°C was applied for screening 20 protecting media. A perspective medium, 5% sorbitol, 3.6% dextran, for plant viruses lyophilization with high cryo- and xeroprotective effects was found.     

Preservation affects the vegetative growth and fruiting body production of <Emphasis Type="Italic">Cordyceps militaris</Emphasis>

Cordyceps militaris is a model species of Cordyceps fungi, and has been traditionally used as an edible and medicinal fungus due to its richness of bioactive and pharmacological metabolites. The fruiting bodies of this fungus are widely used as healthy food and nutrition supply. In industrial production, fruiting bodies are cultivated on artificial media, but their yield and quality are usually affected by the quality of fungal strains. In this study, the effect of colony growth rate of the fungal strains, fungal age and repeated subculturing on the fungal biomass accumulation was investigated. The results indicated that the fungal biomass was positively correlated with the colony growth rate and not affected by fungal age and the repeated subculturing. The preservation conditions for stock cultures, including choice of cultures, lyophilization, temperature and protective agents were optimized based on the mycelial formation and conidia production in artificial inoculum. The development of fruiting bodies from the fungal strains stored under the optimized preservation conditions were further analyzed to determine the ideal time period of preservation. Results indicated that storing the fungus at 4 °C could maintain the fungal vitality and fruiting body producing capacity for at least 12 months. This study established practical criteria of fungal inoculum for artificial cultivation of fruiting body and provided a simple and efficient preservation method for C. militaris. The results may shed light on preservation for other Cordyceps species and other edible fungi.     

The effects of preservation procedures on amniotic membrane’s ability to serve as a substrate for cultivation of endothelial cells

Amniotic membrane (AM) has been used as a scaffold for the ex vivo expansion of different types of cells and a cell delivery matrix in regenerative medicine. Since the preservation procedures can influence the AM properties for experimental and clinical purposes, this study was established to investigate the feasibility of using the AM after different preservation methods to serve as substrates for endothelial cell expansion ex vivo. The effects of cryopreservation and lyophilization were evaluated on mechanical and histological characteristics of the AM, and the results were compared with the fresh AM. The ECM components of the basement membrane were well conserved in all groups. Although lyophilization resulted in more histological changes and lower level of physical variables including thickness, F_max, elongation at break and suture retention than the fresh and cryopreserved AM, endothelial cells grown on the lyophilized AM were better attached to the basement membrane. Cytotoxicity assay by MTT showed that the lyophilized AM is a compatible substrate for endothelial cells cultivation. The findings of this study suggest that the lyophilized AM is a suitable matrix for cultivation of endothelial cells due to this fact that lyophilization led to exposure of basement membrane of the AM.     

Relative stability of biological properties in encapsulated strains of Staphylococcus aureus after freezing and freeze-drying

The relative stability of the biological properties of three encapsulated strains of Staphylococcus aureus was compared after preservation for 1 year in two different vehicles, 10% glycerol and 15% honey and at two different temperatures, ?30 and ?80 °C. A third method of preservation was by lyophilization in 10% skim-milk plus 0.1% glutamic acid and 2% honey. Comparison with control stock cultures maintained by bimonthly subcultivation on brain heart infusion (BHI) agar slants indicated that viability of the organisms was best preserved in 15% honey. When freezing and freeze-drying were compared, superiority was achieved by the latter. Quantitative activities of acid phosphatase, DNase, and coagulase remained constant in all subcultures. Also, while no loss of virulence for mice was observed with these methods, some did occur with the stock subcultures on BHI agar slants. Concerning relative salt tolerance of the strains in these preparations, the lyophilized organisms surpassed the frozen ones. However, when lyophilizing time was prolonged, yellow pigmentation corresponding to β-carotene decreased. Finally, both frozen and lyophilized organisms maintained stable characteristics of growth type in serum-soft agar.     

Glycosylation of ergot alkaloids by free and immobilized cells of Claviceps purpurea

Summary A new strain, Claviceps purpurea 88-EP-47, with high invertase activity was selected. Free and Calginate immobilized cultures of this strain were used for fructosylation of ergot alkaloids. By bioconversion from their aglycones, elymoclavine-O--d-fructofuranosyl(21)-O--d-fructofuranoside, and elymoclavine-O--d-fructoside, the respective fructosides of chanoclavine, lysergol and dihydro-lysergol monofructosides were obtained. These substances are formed by -d-fructofuranosidase present in Claviceps cells. The bioconversion activity of the enzyme system (fructose transfer) is strongly dependent on pH, substrate (sucrose) concentration and the developmental profile of invertase activity. The pH optimum for elymoclavine fructosylation is 6.5, for chanoclavine 5.7, and the optimal sucrose concentration is 75 g/l. Fifteen-day-old production cultures had the best glycosylation activities. Fructosylation of alkaloids can be stimulated in production cultures of C. purpurea or C. fusiformis forming elymoclavine or chanoclavine by a pH shift to 6.5 at the end of the production phase. Glycosylating Claviceps strains producing elymoclavine eliminate the free alkaloid into glycosides. The feedback inhibition of alkaloid synthesis by elymoclavine is then strongly reduced, helping to further improve elymoclavine yields. Elymoclavine can be liberated simply by invertase activity of baker's yeast.Offprint requests to: V. Ken