Influence of lyophilization, fluidized bed drying, addition of protectants, and storage on the viability of lactic acid bacteria

Aims:  The present study focuses on the impact of two different drying technologies and the influence of protectants on process survival and storage stability of the two lactic acid bacterial strains Enterococcus faecium and Lactobacillus plantarum .
Methods and Results:  After incubation with the protectants glucose, sucrose, trehalose, and maltodextrin the concentrated bacterial suspensions were subjected to fluidized bed drying and lyophilization and subsequently stored at 4, 22, and 35°C for half a year. Lactobacillus plantarum turned out to be more sensitive to both drying methods than Ent. faecium . Without the addition of a protectant cells of both strains suffered higher losses during fluidized bed drying. Elevated storage temperatures correlate with a higher decline of viable bacterial cells.
Conclusions:  Although survival rates varied between the strains, the nonreducing disaccharides revealed overall best protection for both investigated lactic acid bacteria during processing and storage. The addition of protective carbohydrates can prevent the decline in viability during fluidized bed drying.
Significance and Impact of the Study:  The influence of protectants proved to be species specific and therefore needs to be determined on a case-to-case basis. Survival rates, duration, and energy consumption appear to be the crucial parameters to evaluate the economy of production processes for industrial starter cultures.     

Influence of storage temperature on infectious hematopoietic necrosis virus detection by cell culture isolation and RT-PCR methods

The detection of infectious hematopoietic necrosis virus (IHNV) in infected rainbow trout Oncorhynchus mykiss and in cell culture supernatants stored under different conditions was studied. IHNV-positive fish visceral organ homogenates and cell culture supernatants were incubated at 4 and 25 degrees C. Virus titre was measured by virus isolation on epithelioma papulosum cyprini (EPC) cells and the IHNV RNA was detected by RT-PCR and semi-nested RT-PCR. The influence of repeated freezing and thawing on the virus isolation from organ homogenates and from cell culture supernatants was studied as well. It was possible to isolate the virus from IHNV-positive organ material during the 3 d of incubation at 4 degrees C but, only on the first day of incubation at 25 degrees C. Viral RNA could be amplified during the incubation period of 35 d at 4 degrees C but only during 8 d of incubation at 25 degrees C. In IHNV-infected cell culture supernatant stored at 4 degrees C, it was possible to detect virus for 36 and 16 d in supernatant stored at 25 degrees C. Viral RNA could be followed by using molecular methods during the entire experimental period of 123 d. Each cycle of freezing and thawing of samples resulted in a reduction of IHNV titre in the suspension of visceral organs, while the virus titre in cell culture supernatant remained almost the same following 33 freezing-thawing cycles. The present results show that rapid laboratory processing and storage of potentially virus-containing tissue samples as well as the use of different detection methods are very important for efficient IHNV diagnosis.     

Effect of storage duration, storage temperature, and diluent on the viability and fertility of fresh ram sperm

Cervical artificial insemination (AI) in sheep with fresh semen yields a much higher pregnancy rate than when frozen-thawed semen is used, and consequently frozen semen is only acceptable for laparoscopic insemination. The short life span of fresh semen is a major constraint on the use of AI in genetic improvement programs for sheep. The main objective of this study was to examine the effects of storage conditions on viability and fertilization ability of fresh ram (Ovis aries) semen up to 72 h postcollection. Experiment 1 was designed to evaluate the effect of diluent type (standard skim milk, AndroMed, OviPro, and INRA 96) and storage temperature (5 °C and 15 °C) on the motility and viability of fresh ram semen. Storage temperature, irrespective of diluent, had a significant effect on both motility and viability. Storage at 5 °C maintained acceptable motility and viability up to 72 h compared with that of storage at 15 °C. In Experiment 2, the penetrating ability of fresh ram semen, diluted in either skim milk, AndroMed, or INRA 96, was assessed using artificial mucus. Flat capillary tubes containing artificial mucus were suspended in 250 μL semen at a sperm concentration of 20 × 10⁶/mL. Semen was stored at 5 °C and tested after 6, 24, 48, and 72 h. There was a significant diluent by time interaction. In Experiment 3, the fertilizing ability of fresh ram semen stored at 5 °C was evaluated in vitro. Fresh semen (diluted in either skim milk, AndroMed, or INRA 96) was added to matured ewe oocytes at 6, 24, or 72 h after semen collection. Cleavage rate was recorded at 48 h postinsemination, and blastocyst development was recorded on Days 6 to 9. There was a significant treatment effect on cleavage and blastocyst rates; insemination of semen stored for 24 h resulted in higher rates than those for storage at 72 h. In Experiment 4, the fertilizing ability of fresh ram semen was evaluated in vivo. Semen was diluted in INRA 96, stored at 5 °C, and used to inseminate ewes on the day of collection or at 24, 48, and 72 h postcollection. Multiparous ewes were cervically inseminated at a synchronized estrus. Fertility rate decreased linearly (P < 0.001) up to 72 h after semen collection.     

Influence of inoculum size, incubation temperature, and cell culture density on virus detection in environmental samples

The influence of inoculum size and cell culture density on virus titer by cytopathic effect or plaque assay was studied using poliovirus type 1 and BGM (Buffalo green monkey) cells as a model for this evaluation. With a plaque assay system, a linear relationship was observed for an inoculum size of up 1 mL/25 cm2; a marked decrease in the number of plaques was observed when over 1 mL of sample was inoculated on this surface area. Cell culture density also affected virus titer; maximal titers were observed when cells were seeded at 25 000 to 75 000 cells/mL and incubated for 6 days before infection with the virus. Viral density, evaluated as most-probable-number and measured by cytopathic effect under liquid overlay, revealed that the viral titer was similar up to 1 mL inoculum and increased only when over 1 mL was inoculated. Cell density had no significant effect on the viral titer measured by the most-probable-number method and cytopathic effect. Inactivation of inoculum due to an incubation temperature of 37 degrees C for a short period was shown to be minimal for poliovirus type 1, reovirus type 2, coxsackievirus B-5, and the simian rotavirus SA-11. Longer inactivation time led to a 2 logs reduction of the infectious titer of coxsackievirus B-5 (in 48 h) while the other viruses showed a significant reduction in titer only after 96 h.     

A tissue culture method for propagation and low temperature storage of Trifolium repens genotypes

Proliferating shoot cultures of Trifolium repens L . cv. Grasslands Huia have been established on Murashige and Skoog's medium containing 3% sucrose and 0.2 mg l⁻¹ benzylamino purine. The shoots could be readily rooted on media without cytokinin and transferred to potting mix with 100% survival. The cultured shoots have been stored for 10 months, at 5°C in darkness without adversely affecting their potentiality for rapid multiplication. The significance of this approach for genotype storage is discussed.     

Influence of microencapsulation and spray drying on the viability of Lactobacillus and Bifidobacterium strains

Improved production methods of starter cultures, which constitute the most important element of probiotic preparations, were investigated. The aim of the presented research was to analyse changes in the viability of Lactobacillus. acidophilus and Bifidobacterium bifidum after stabilization (spray drying, liophilization, fluidization drying) and storage in refrigerated conditions for 4 months. The highest numbers of live cells, up to the fourth month of storage in refrigerated conditions, of the order of 10(7) cfu/g preparation were recorded for the B. bifidum DSM 20239 bacteria in which the N-Tack starch for spray drying was applied. Fluidization drying of encapsulated bacteria allowed obtaining a preparation of the comparable number of live bacterial cells up to the fourth month of storage with those encapsulated bacteria, which were subjected to freeze-drying but the former process was much shorter. The highest survivability of the encapsulated L. acidophilus DSM 20079 and B. bifidum DSM 20239 cells subjected to freeze-drying was obtained using skimmed milk as the cryoprotective substance. Stabilization of bacteria by microencapsulation can give a product easy to store and apply to produce dried food composition.     

Growth induction by serum or polyoma virus inhibits the aggregation of trypsinised suspensions of BHK21 tissue culture fibroblasts

BHK21 hamster tissue culture fibroblasts, when brought into suspension with trypsin, aggregate spontaneously in serum-free medium. The amount of aggregation appears to depend on the proportion of quiescent (growth inhibited) cells which were present in the culture. It is greatest in cells from cultures whose growth is inhibited, either by high cell density or by low serum concentration. When serum is added to low serum cultures, growth is induced and S phase and mitosis follow at 14 and 20 h respectively. It has now been found that aggregation ceases less than 2 h after the addition of serum. Growth can also be induced in such cultures by infection with high multiplicities of polyoma virus. Infected cells enter S phase about 28 h after infection. It has been found that aggregation ceases between 12 and 20 h after infection. Thus in both these cases loss of aggregation precedes S phase by about 12 h. It is concluded that the absence of aggregation in suspensions derived from polyoma virus-transformed lines of BHK21 cells is a consequence of their resistance to density-dependent inhibition of growth. Failure to undergo spontaneous aggregation appears to be an indicator of an early surface change associated with the induction of growth.     

Influence of environmental storage relative humidity on biological indicator resistance, viability, and moisture content.

The effect of environmental storage relative humidity (RH) on the moisture content, viability, and moist heat and gaseous ethylene oxide (EO) resistance of biological indicators (BIs) was evaluated. No statistically significant difference was observed between the initial Bacillus stearothermophilus spore population and the spore population of BIs stored at 20 degrees C and 0, 20, 44, of 55% RH or under ambient, 4 degrees C, or -20 degrees C conditions after 12 months. A statistically significant decrease in moist heat resistance from initial starting levels was found for BIs stored at 20 degrees C and either 0 or 20% RH. There was a statistically significant decrease in the B. subtilis BI spore population, compared with initial levels, when the BIs were stored at 20 degrees C and 0% RH concomitant with a significant increase in their EO resistance. BI storage at 20 degrees C and 20 or 44% RH, or under ambient, 4 degrees C, or -20 degrees C conditions, had no significant effect on EO resistance. BIs stored at 20 degrees C and 66% RH demonstrated a significantly lower EO resistance compared with starting levels.     

Influence of medium composition, pH and temperature on the growth and viability of Lactobacillus acidophilus

The Lactobacillus acidophilus growth was investigated to find the optimal concentrations of 2 nitrogen sources (thought to be in excess) and 2 organic acids. A Plackctt and Burman experimental design was used allowing identification of the more important factors with very few experiments. In the studied range, all the factors had a linear effect and a great influence on the final viability. 20 g/L of each nitrogen sources, 3 g sodium-citrate/L and 5 g sodium acetate/L are necessary. pH, temperature and glucose had poor influence.     

Long-term storage of tissue samples for cell culture

Summary The establishment of cultured cell lines from skin biopsies stored at −196°C for periods up to 1 year has been investigated. Attempts to initiate cell cultures from the frozen tissue samples were uniformly successful. There was no alteration in chromosome constitution, morphological appearance, or specific activities of lysosomal enzymes in cells cultured from the stored samples. This process can safeguard against failure of the initial tissue culture and provide an alternate means of storing viable cells when it is impossible or impractical to initiate a cell culture immediately. This project was supported by the South Carolina Department of Mental Retardation and the Self Foundation.     

Long-term storage of tissue samples for cell culture

The establishment of cultured cell lines from skin biopsies stored at -196 degrees C for periods up to 1 year has been investigated. Attempts to initiate cell cultures from the frozen tissue samples were uniformly successful. There was no alteration in chromosome constitution, morphological appearance, or specific activities of lysosomal enzymes in cells cultured from the stored samples. This process can safeguard against failure of the initial tissue culture and provide an alternate means of storing viable cells when it is impossible or impractical to initiate a cell culture immediately.     

Effects of storage temperature on viability, germination and antioxidant metabolism in Ginkgo biloba L. seeds.

The behaviour of the Ginkgo biloba L. seeds was studied during storage at 4 and 25 degrees C. When stored at 25 degrees C, all the seeds died in 6 months. Cold temperatures preserved seed tissue viability for 1 year but did not preserve their capability to germinate, since such capability decreased after 6 months. A significant increase in lipid peroxidation occurred in the seed both in the embryo and in the endosperm. During storage a progressive deterioration of the endosperm tissues was evident. The two major water soluble antioxidants, ascorbate (ASC) and glutathione (GSH), showed different behaviour in the two conditions of storage and in the two main structures of the seed, the embryo and the endosperm. The ASC content of embryos and endosperms remained quite unchanged in the first 9 months at 4 degrees C, then increased. At 25 degrees C a significant decrease in the ASC content in the embryos was evident, whereas it remained more stable in the endosperm. The GSH pool decreased at both storage temperatures in the embryos. As far as the ASC-GSH redox enzymes are concerned, their activities decreased with storage, but changes appeared to be time-dependent more than temperature-dependent, with the exception of the endosperm ascorbate free radical (AFR) reductase (EC 1.6.5.4), the activity of which rapidly decreased at 25 degrees C. Therefore overall the antioxidant enzymes were scarcely regulated and unable to counteract oxidative stress occurring during the long-term storage.     

Effect of air drying on bacterial viability: A multiparameter viability assessment

The effect of desiccation on the viability of microorganisms is a question of great interest for a variety of public health questions and industrial applications. Although viability is traditionally assessed by plate counts, cultivation-independent methods are increasingly applied with the aim to gain more insight into why cells might not form colonies and to optimize production processes. To evaluate their usefulness, we applied in this study a multiparameter viability assay to selected bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterococcus hirae, and Staphylococcus aureus) subjected to air-drying in the absence or presence of supplements. Tests included growth on solid culture medium and the measurement of membrane integrity, membrane potential, esterase and respiratory activities using fluorescent dyes. All measured parameters were responsive to desiccation stress. Results suggested that extending plate count analysis with cultivation-independent methods can greatly enhance resolution especially for moderate stress conditions, which do not get reflected in plate counts due to cellular recovery. Whereas plate counts reflect the final effect on viability, immediate measurement of cellular functions provides a snapshot picture of the fitness status at a specific point in time. Special emphasis was given to MgCl(2) which in concentrations≥50mM dramatically increased the bacterial susceptibility to desiccation in the case of the gram-negative bacteria and to a lesser extent also for the gram-positive bacteria. The study in addition confirmed a good agreement of results obtained with the recently developed real-time viability (RTV) assay and the BacLight LIVE/DEAD method in combination with a fluorescence plate reader.