| 1. |
- Al-Essawe, Essraa M, et al.
(author)
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Improved cryosurvival of stallion spermatozoa after colloid centrifugation is independent of the addition of seminal plasma
- 2018
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In: Cryobiology. - : Elsevier BV. - 0011-2240 .- 1090-2392. ; 81, s. 145-152
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Journal article (peer-reviewed)abstract
- Addition of seminal plasma (SP) prior to cryopreservation may influence stallion sperm cryosurvival. The objective of this study was to investigate the addition of pooled SP from "good" or "bad" freezer stallions to spermatozoa selected by single layer centrifugation (SLC) prior to cryopreservation on post-thaw sperm quality. Semen from 12 stallions was collected; 5 mL was frozen as control (C) and the remainder was processed by SLC to remove SP and was divided into three aliquots: i) SLC sample without SP (SLC); ii) SLC plus pooled SP from "good freezer" stallions (SLC-GF); iii) SLC plus pooled SP from "bad freezer" stallions (SLC-BF). After thawing, the following parameters were evaluated: chromatin integrity (DNA fragmentation index; ßI), mitochondria) membrane potential (MMP), membrane integrity (MI), reactive oxygen species (ROS) and sperm kinematics. The ßI was reduced (P < 0.0001) in SLC samples compared to controls. The SLC group showed a lower proportion of spermatozoa with low MMP and a higher proportion of spermatozoa with high MMP than other groups (P < 0.0001), and had lower hydrogen peroxide content than control. Sperm kinematics were not different. In conclusion, selection by SLC prior to cryopreservation improved post-thaw sperm quality; inclusion of SP from "good" and "bad" freezer stallions did not have an additional beneficial effect.
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| 2. |
- Alkmin, Diego V., et al.
(author)
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Boar sperm cryosurvival is better after exposure to seminal plasma from selected fractions than to those from entire ejaculate
- 2014
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In: Cryobiology. - : Elsevier. - 0011-2240 .- 1090-2392. ; 69:2, s. 203-210
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Journal article (peer-reviewed)abstract
- Boar bulk ejaculates are now being collected instead of usual sperm-rich fractions (SRF) for artificial insemination purpose. The present study evaluated the influence of holding boar sperm samples before freezing surrounded in their own seminal plasma (SP), from either fractions/portions or the entire ejaculate, on post-thawing sperm quality and functionality. Ejaculates collected as bulk (BE) or as separate (first 10 mL of SRF [P1] and rest of SRF [P2]) from 10 boars were held 24 h at 15-17 degrees C and then frozen. Some bulk ejaculate samples were frozen immediately after collections as Control. In addition, epididymal sperm samples from the same 10 boars were collected post-mortem and extended in SP from P1 (EP1), P2 (EP2) and post SRF (EP3), and also held 24 h before freezing for a better understanding of the influence of SP on boar sperm cryopreservation. The sperm quality (motility, evaluated by CASA, and viability, evaluated by flow cytometry) and functionality (flow cytometry assessment of plasma membrane fluidity, mitochondrial membrane potential and intracellular generation of reactive oxygen species [ROS] in viable sperm) were evaluated at 30, 150 and 300 min post-thaw. Post-thawing sperm quality and functionality of P1 and P2 were similar but higher (p less than0.01) than BE samples. Control samples showed higher (p less than 0.01) post-thaw sperm quality and functionality than BE samples. Post-thawing sperm quality and functionality of EP1 and EP2 were similar but higher (p less than 0.05) than EP3. These results showed that boar sperm from BE are more cryosensitive than those from the SRF, particularly when held 24 h before freezing, which would be attributable to the cryonegative effects exerted by the SP from post SRF.
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| 3. |
- Alvarez-Rodríguez, Manuel, et al.
(author)
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Brown bear sperm double freezing : Effect of elapsed time and use of PureSperm(®) gradient between freeze-thaw cycles.
- 2013
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In: Cryobiology. - : Elsevier. - 0011-2240 .- 1090-2392. ; 67:3, s. 339-346
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Journal article (peer-reviewed)abstract
- The use of sexed spermatozoa has great potential to captive population management in endangered wildlife. The problem is that the sex-sorting facility is a long distance from the semen collection place and to overcome this difficulty two freeze-thaw cycles may be necessary. In this study, effects of refreezing on brown bear electroejaculated spermatozoa were analyzed. We carried out two experiments: (1) to assess the effects of the two freezing-thawing cycles on sperm quality and to analyze three different elapsed times between freezing-thawing cycles (30, 90 and 180 min), and (2) to analyze the use of PureSperm between freezing-thawing cycles to select a more motile and viable sperm subpopulation which better survived first freezing. The motility, viability and undamaged acrosomes were significantly reduced after the second thawing respect to first thawing into each elapsed time group, but the elapsed times did not significantly affect the viability and acrosome status although motility was damaged. Our results with the PureSperm gradient showed higher values of viability in freezability of select sample (pellet) respect to the rest of the groups and it also showed a significant decrease in the number of acrosome damaged. In summary, the double freezing of bear semen selected by gradient centrifugation is qualitatively efficient, and thus could be useful to carry out a sex-sorting of frozen-thawed bear spermatozoa before to send the cryopreserved sample to a biobank. Given the low recovery of spermatozoa after applying a selection gradient, further studies will be needed to increase the recovery rate without damaging of the cell quality.
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| 4. |
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| 5. |
- Axelsson, Stina, et al.
(author)
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Cryopreserved peripheral blood mononuclear cells are suitable for the assessment of immunological markers in type 1 diabetic children
- 2008
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In: Cryobiology. - : Elsevier BV. - 0011-2240 .- 1090-2392. ; 57:3, s. 201-208
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Journal article (peer-reviewed)abstract
- Cryopreserved peripheral blood mononuclear cells (PBMC) are commonly used when assessing immune responses in clinical trials, both for practical reasons and to minimize interassay variation, as samples are often collected and studied over time. This study investigated the effect of cryopreservation on cytokine and chemokine secretion, and on expression of regulatory T-cell associated markers, in samples from children with type 1 diabetes. PBMC were cultured before and after cryopreservation either with GAD(65) or PHA. Secretion of cytokines (IL-5, -6, -10, -12, -13 -17, IFN-gamma and TNF-alpha) and chemokines (IP-10, MCP-1, MIP-1 alpha, MIP-1 beta and RANTES) was analysed in cell supernatants using multiplex fluorochrome technique (Luminex). Expression of FOXP3 and TGF-beta mRNA was detected by multiplex real-time RT-PCR. Increased spontaneous secretion of IL-6, -10, -12, -13, IFN-gamma and MCP-1, and mRNA expression of FOXP3 and TGF-beta, was detected after cryopreservation. Stimulation with GAD65 induced higher levels of IL-6, IFN-gamma, TNF-alpha and MIP-1 alpha, whereas lower secretion was found for IL-10 and IL-13 in cryopreserved PBMC. Stimulation with PHA induced lower secretion of IP-10, MCPA and RANTES and FOXP3 mRNA expression after cryopreservation. Thus, cryopreserved PBMC were suitable to assess the immunological markers included in this study, even though their expression could differ from freshly handled cells.
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| 6. |
- Bergenholtz, Sa Schoug, et al.
(author)
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A case study on stress preconditioning of a Lactobacillus strain prior to freeze-drying
- 2012
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In: Cryobiology. - : Elsevier BV. - 0011-2240 .- 1090-2392. ; 64:3, s. 152-159
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Journal article (peer-reviewed)abstract
- 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.
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| 7. |
- Bergqvist, Ann-Sofi
(author)
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Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate
- 2011
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In: Cryobiology. - : Elsevier BV. - 0011-2240 .- 1090-2392. ; 63, s. 137-144
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Journal article (peer-reviewed)abstract
- Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10 mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PIP and intracellular calcium ([Ca(2+)]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between PI and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (P<0.05) in SRF-P1 compared to P1. There were no significant differences in percentages of spermatozoa with high [Ca(2+)]i between PI and SRF-P1 in fresh as well as in frozen-thawed semen. A higher (P<0.001) proportion of spermatozoa displayed PIP during the course of cryopreservation indicating a definite effect of the cryopreservation process on sperm PTP. The proportion of spermatozoa with PTP did not differ significantly between portions of the boar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32 kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF. (C) 2011 Elsevier Inc. All rights reserved.
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| 8. |
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| 9. |
- Hernandez, Marta, et al.
(author)
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Cryo-scanning electron microscopy (Cryo-SEM) of semen frozen in medium-straws from good and sub-standard freezer AI-boars
- 2007
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In: Cryobiology. - : Elsevier. - 0011-2240 .- 1090-2392. ; 54:1, s. 63-70
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Journal article (peer-reviewed)abstract
- A major limiting factor for commercial cryopreservation of boar semen for artificial insemination (AI) is the large individual variation to cooling, where the degree of cell dehydration during ice (re)shaping seems to play a major role. This study investigated, in the frozen state, the degree of dehydration and ice crystal distribution in boar semen doses whose spermatozoa displayed different viability after thawing. Cross-sectioned medium-straws (0.5 mL, n = 10) from a total of 10 stud boars classified as "good" (n = 5) or sub-standard (e.g., "bad" freezers, n = 5) by conventional analyses (computer assisted motility and sperm viability) were examined by Cryo-scanning electron microscopy (Cryo-SEM) to determine whether differences between groups could be already distinguishable prior to thawing. The degree of hydration was monitored in relation to the areas of ice crystal formed extracellularly (lakes), the areas of frozen, concentrated extender (veins) where spermatozoa were located and the degree of compartmentalization (number of lakes) present. Irrespectively of the region studied, the gradient of main dehydration (as lakes) observed along the cross-section area of the straws was very irregular. Most spermatozoa were enclosed in the freezing extender matrix and no obvious signs of external membrane damage were observed. None of the Cryo-SEM variables significantly correlated with post-thaw sperm parameters (p greater than 0.05). However, we identified significant differences (p less than 0.0001) among boars for all ultrastructure variables studied, including the size of the veins, where differences in solute concentration is expected. We concluded that despite the large variability in ice crystal formation during the conventional freezing process among boars, this is unrelated to inter-boar post-thaw sperm differences. (c) 2007 Elsevier Inc. All rights reserved.
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| 10. |
- Kvarnström, Maria, 1971-, et al.
(author)
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Effect of cryopreservation on expression of Th1 and Th2 cytokines in blood mononuclear cells from patients with different cytokine profiles, analysed with three common assays: an overall decrease of interleukin-4 : An overall decrease of interleukin-4
- 2004
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In: Cryobiology. - : Elsevier BV. - 0011-2240 .- 1090-2392. ; 49:2, s. 157-168
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Journal article (peer-reviewed)abstract
- Studies on cytokine expression in blood cells are commonly performed on cryopreserved cells. Previous studies show that cryopreservation affects cytokine expression, but the findings are not consistent. This may be due to divergent effects of freezing on different cytokines, different stimuli, and different patient groups or to the use of different assays in the studies. This study was designed to investigate the effect of freezing on spontaneous, auto-antigen, allergen, and mitogen induced cytokine secretion from peripheral blood mononuclear cells from several groups of patients expressing different cytokine profiles; multiple sclerosis, atopic children, non-atopic children, and pregnant women. The expression of IFN-γ, IL-4, IL-5, IL-9, IL-10, and IL-13 was analysed with ELISA, ELISPOT and/or real time RT-PCR. Our data provide evidence that the process of cryopreservation and thawing does affect the expression of cytokines, both at the protein and the mRNA level. Moreover, the effect varied among different cytokines, different stimuli, and different patient groups, which partly may be explained by differences in optimal freezing conditions for non-activated and activated cells. An increase of allergen and PHA stimulated IFN-γ secretion in atopic children was found following cryopreservation, but no such increase in auto-antigen induced IFN-γ was seen in MS-patients. The most consistent finding was that expression of IL-4 was generally decreased in spontaneous and auto-antigen/allergen induced expression in cryopreserved cells. In conclusion, this study points out the importance of investigation of the effects of freezing for each cytokine, stimuli and patient group before using frozen cells in studies of in vitro cytokine secretion.
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| 11. |
- Li, Junwei, et al.
(author)
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Post-thaw boar sperm motility is affected by prolonged storage of sperm in liquid nitrogen. A retrospective study
- 2018
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In: Cryobiology. - : Elsevier. - 0011-2240 .- 1090-2392. ; 80, s. 119-125
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Journal article (peer-reviewed)abstract
- Owing to the quick genetic turnover of the pig industry, most AT-boar sires live 2-3 yr, a period during which for 1-2 yr their semen is extended and used in liquid form for AI. Despite showing low cryosurvival, affecting fertility after AI, boar semen is frozen for easiness of transport overseas and reposition of valuable genetics. For the latter, semen is stored in liquid nitrogen (LN2, cryostorage) for many years, a controversial practice. Here we studied how length of cryostorage could affect sperm quality. Straws (0.5 mL) frozen using the same cryopreservation protocol at one specific location from AI-sires of proven fertility were stored in LN2 for up to 8 yr. Post thaw sperm quality was evaluated after 2, 4 or 8 yr of cryostorage, always compared to early thawing (15 d after freezing). Sperm motility and kinematics were evaluated post-thaw using CASA and sperm viability was cytometrically evaluated using specific fluorophores. Sperm viability was not affected by length of cryostorage, but total and progressive sperm motility were lower (p amp;lt; 0.01) in sperm samples cryostored for 4 or 8 yr compared to those thawed 15 d after freezing. Cryostorage time affected sperm kinetics, but with greater intensity in the samples cryostored for 4 yr (p amp;lt; 0.001) than in those for 2 yr (p amp;lt; 0.01). The fact that the major phenotypic characteristic of boar spermatozoa, motility, is constrained by time of cryostorage should be considered when building cryobanks of pig semen. Attention should be placed on the finding that amp;gt; 2 yr of cryostorage time can be particularly detrimental for the post-thaw motility of some sires, which might require increasing sperm numbers for AI.
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| 12. |
- Morrell, Jane
(author)
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Single layer colloid centrifugation technique improves motility, viability and chromatin integrity of ram spermatozoa after thawing
- 2019
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In: Cryobiology. - : Elsevier BV. - 0011-2240 .- 1090-2392. ; 86, s. 77-83
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Journal article (peer-reviewed)abstract
- The cell membrane of ram spermatozoa is more sensitive to the freezing process than in other species due to its composition. As a result, the quality and viability of frozen thawed ram spermatozoa are often poor, which together with the specific structure of the ewe's cervix are the main reasons for lower fertility in ewes after intracervical insemination. In the present study we investigated the effects of semen centrifugation through a single layer of a species-specific colloid (Androcoll-O) on post-thaw quality of ram spermatozoa. Motility, viability and morphology were analysed 0, 6, 12 and 24 h after thawing. DNA fragmentation index (ßI) of the samples was assessed 0 h after thawing, by SCSA (TM). Membrane and acrosome integrity of spermatozoa were analysed by Sybr-14/PI/PNA test 0 h after thawing.The proportion of motile spermatozoa was significantly higher in SLC - selected samples in comparison to control (not SLC - selected) samples at 0, 6, 12 (P < 0.001) and 24 h (P < 0.05). The proportion of viable spermatozoa was also significantly higher in SLC - selected samples in comparison to control samples at all times (P < 0.001). The proportion of abnormal acrosomes and morphologically abnormal spermatozoa (MAS) were significantly lower in SLC selected samples compared to control samples at all times (P < 0.001). Analysis of chromatin stability revealed significantly lower ßI values in SLC - selected samples compared to control samples (P < 0.001). The SYBR-14/PI/PNA test also revealed significantly better values in SLC - selected compared to control samples (P < 0.05). In conclusion, single layer colloid centrifugation significantly improved post-thaw quality and longevity of ram spermatozoa, making it suitable for artificial insemination initiatives.
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| 13. |
- Najafi, Davood, et al.
(author)
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Effect of Achillea millefolium-loaded nanophytosome in the post-thawing sperm quality and oxidative status of rooster semen
- 2018
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In: Cryobiology. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 0011-2240 .- 1090-2392. ; 82, s. 37-42
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Journal article (peer-reviewed)abstract
- The aim of this study was to compare the effectiveness of antioxidants including Achillea millefolium extract (AmE) (n0t1.5: 1.5, n0t3: 3 and n0t4.5: 4.5 mg/L) and AmE loaded in nano phytosome (n1t1.5: 1.5, n1t3: 3 and n1t4.5: 4.5 mg/L) in the freezing of Ross 308 rooster semen. Sperm motility (CASA), membrane integrity (HOS test), viability, total abnormality and enzymatic parameters (SOD, CAT and GPx) were assessed after thawing. AmE-loaded nano phytosome at a concentration of 3 mg/l resulted in significantly (P amp;lt; 0.05) higher total motility (MOT) (73.78 +/- 2.92) and at concentrations of 1.5 mg/L and 3 mg/L in progressive motility (PROG) (14.12 +/- 0.38, 16.78 +/- 0.38) in comparison with the control group (MOT: 58.48 +/- 2.92; PROG: 9.08 +/- 0.38). Sperm viability (Vi) was higher (P amp;lt; 0.05) in n1t3 (74.62 +/- 1.55) and membrane integrity (Mi) in n0t3 and n1t3 groups (65.91 +/- 1.91, 63.73 +/- 1.91, respectively) compared to the control groups (Vi: 66.85 +/- 1.55; Mi: 53.18 +/- 1.91). Moreover, the lowest percentage of MDA was measured in n1t3 group (1.31 +/- 0.31). There was no significant difference for SOD and CAT values with the use of various extenders. In conclusion, we suggest that AmE loaded in nano phytosome at 3 mg/l dose can be added to basic extender for improving rooster sperm motility, viability and oxidative stress values during the freezing procedure.
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| 14. |
- Schoug, Åsa, et al.
(author)
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Freeze-drying of Lactobacillus coryniformis Si3--effects of sucrose concentration, cell density, and freezing rate on cell survival and thermophysical properties
- 2006
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In: Cryobiology. - Maryland Heights, USA : Elsevier BV. - 0011-2240 .- 1090-2392. ; 53:1, s. 119-127
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Journal article (peer-reviewed)abstract
- Freeze-drying is commonly used to stabilize lactic acid bacteria. Many factors have been reported to influence freeze-drying survival, including bacterial species, cell density, lyoprotectant, freezing rate, and other process parameters. Lactobacillus coryniformis Si3 has broad antifungal activity and a potential use as a food and feed biopreservative. This strain is considered more stress sensitive, with a low freeze-drying survival, compared to other commercialized antifungal lactic acid bacterial strains. We used a response surface methodology to evaluate the effects of varying sucrose concentration, cell density and freezing rate on Lb. coryniformis Si3 freeze-drying survival. The water activity of the dry product, as well as selected thermophysical properties of importance for freeze-drying; degree of water crystallization and the glass transition temperature of the maximally freeze concentrated amorphous phase (Tg') were determined. The survival of Lb. coryniformis Si3 varied from less than 6% to over 70% between the different conditions. All the factors studied influenced freeze-drying survival and the most important factor for survival is the freezing rate, with an optimum at 2.8 degrees C/min. We found a co-dependency between freezing rate and formulation ingredients, indicating a complex system and the need to use statistical tools to detect important interactions. The degree of water crystallization decreased and the final water activity increased as a function of sucrose concentration. The degree of water crystallization and Tg' was not affected by the addition of 10(8)-10(10) CFU/mI. At 10(11) CFU/ml, these thermophysical values decreased possibly due to increased amounts of cell-associated unfrozen water.
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| 15. |
- Tao, D L, et al.
(author)
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Active oxygen scavengers during cold acclimation of Scots pine seedlings in relation to freezing tolerance
- 1998
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In: Cryobiology. - 0011-2240 .- 1090-2392. ; 37:1, s. 38-45
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Journal article (peer-reviewed)abstract
- Freezing injury of plants may be caused by the deleterious reactions of active oxygen species, and free-radical scavenging systems may be important in the alleviation of freezing stress. To test the feasibility of this hypothesis, enzymes and metabolites that cooperatively scavenge O-2(-) and H2O2 were analyzed in Scots pine (Pinus sylvestris L.) seedlings during a stepwise cold acclimation procedure. Elevated levels of enzymatic scavengers such as ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehy droascorbate reductase were found, along with increased freezing tolerance during cold acclimation, supporting the hypothesis. Induction of the scavenging systems during acclimation is discussed in relation to freezing tolerance. (C) 1998 Academic Press.
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