Freeze-drying and cryopreservation of bacteria

Perry, Stephen F.

doi:10.1007/bf02752697

Cited by 30 publications

(24 citation statements)

References 9 publications

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“…In this work, we studied the influence of mentioned factors on bovine ruminal bacterial composition with the special attention to Firmicutes and Bacteroidetes, which are the two dominant phyla populating the rumen [19,20]. Previous investigations of preservation procedures for rumen contents have been evaluated in the most part on the basis of culture viability [11,12,21,22], fermentation patterns [23,24] or on polysaccharide-degrading activity [25]. In general, the outcome of these investigations indicate no major influence of storage conditions, namely freezing at different temperatures with or without additives, on fermentative activity and the biodiversity of microbial populations in rumen inoculum.…”

Section: Discussionmentioning

confidence: 99%

“…Gram-negative bacteria, which predominate in the rumen, are considered to be particularly sensitive to freezing and thawing [10]. Therefore, freezing samples with glycerol as cryoprotectant has been recommended to minimise cell damage and maintain viability after freezing [11,12]. However, several reports indicate that bacterial diversity of rumen fluid is not substantially altered during preservation [13].…”

Section: Introductionmentioning

confidence: 99%

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Effect of DNA extraction and sample preservation method on rumen bacterial population

et al. 2014

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The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Effect of DNA extraction and sample preservation method on rumen bacterial population

et al. 2014

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“…This freezing temperature may have resulted in bigger ice crystals that resulted in cell injury. Hongpattarakere et al (2012) and Perry (1998) noted that slow freezing occurring at temperatures above −30°C may result in poor survival due to loss of water from the cell while increasing the effects of solute concentration. Exposure of L. plantarum culture to extremely low freezing temperature of -196°C have shown to have the significantly lower freeze-drying viability loss of 0.18 log (CFU/g) as compared to -80 and -20°C with cell reduction of 0.57 and 0.38 log (CFU/g), respectively (Hongpattarakere et al, 2012).…”

Section: Resultsmentioning

confidence: 99%

Storage stability of freeze-dried Lactobacillus plantarum S20 starter culture as affected by various formulations of drying medium, and its fermentation characteristics in mung bean (Vigna radiata L.) slurry was studied by Sulabo et al.

Sulabo¹,

Villasanta²,

Hermo³

et al. 2020

Food Res.

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Development of an active starter culture is essential in the production of safe and highquality fermented foods. Freeze-drying is an effective microbial cell immobilization technology intended to produce active and stable starter cultures for long-term storage. To determine the influence of various drying medium formulations with mung bean powder and disaccharides on the viability of L. plantarum S20 during freeze-drying and subsequent storage at 6±2°C and 30±1°C, three treatments of drying medium consisting of mung bean powder without disaccharides (MBP), MBP with 5% (w/v) trehalose (MBP+T), and MBP with 5% (w/v) sucrose (MBP+T) were evaluated. Results showed that L. plantarum S20 viability after freeze-drying ranged from 9.56 to 10.03 log (CFU/g) with viability loss ranging from 0.20 to 0.51 log (CFU/g), with MBP being the least effective (p<0.05) in minimizing viability loss. Supplementing MBP with either 5% sucrose or 5% trehalose improved (p<0.05) survival of freeze-dried L. plantarum S20; and both disaccharides exhibit similar (p>0.05) cryoprotective effects on L. plantarum S20 during freeze-drying and subsequent storage. Storage at 6±2°C for 180 days resulted in significant decreases of 2.15, 2.17, and 3.27 log (CFU/g) in L. plantarum S20 population in MBP+T, MBP+S, and MBP, respectively. A more drastic decline [>6.0 Log (CFU/g)] in L. plantarum S20 population was observed in freeze-dried culture stored at 30±1°C for 60 days. Fermentation characteristics of freeze-dried L. plantarum S20 in mung bean slurry were also determined. Freeze-dried L. plantarum S20 in MBP with 5% (w/v) sucrose was able to acidify mung bean slurry from pH 6.8 to pH 3.8 after 8 hrs with maximum LAB population of 9.2 log (CFU/mL). Acidification of mung bean slurry by fresh culture (non-freeze-dried) on the other hand was relatively slower where pH dropped from 6.8 to pH 3.72 after 24 hrs. Current work suggested that lyophilized L. plantarum S20 could be used as a starter culture for mung bean-based fermentations but further research on this area is needed.

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“…Thus, preservation of ruminal fluid might overcome this limitation as it allows the use of inoculum without having to keep donor animals (7,8,9) . This is conducted by using glycerol to minimizes microbial cell damage (10,11,12) and maintain the microbial community (13,14) .…”

Section: Introductionmentioning

confidence: 99%

Evaluación de métodos nutricionales para reactivar inóculo ruminal preservado analizado a través de cinética de fermentación y digestibilidad de forrajes in vitro

Domínguez-Ordóñez

Miranda‐Romero

Martı́nez-Hernández

et al. 2019

Rev. Mex. Cienc. Pecu.

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Se evaluó el efecto del medio de cultivo y tiempo de pre-incubación para reactivar inóculo ruminal preservado, evaluando parámetros de la cinética de fermentación y digestibilidad in vitro de materia seca (DIVMS). En el primer experimento, los tratamientos fueron 1) CONTROL, líquido ruminal fresco; 2) BAJO24, inóculo reactivado pre-incubándolo durante 24 h en una solución base; 3) MODE24, inóculo reactivado pre-incubándolo durante 24 h en una solución base, extracto de levaduras y peptona de caseína; y 4) ALTO24, inóculo reactivado pre-incubándolo durante 24 h en una solución base, extracto de levaduras, peptona de caseína y carbohidratos. En el segundo experimento, los tratamientos fueron 1) CONTROL, líquido ruminal fresco, y 2) ALTO12, similar a ALTO24, pero el inóculo fue pre-incubado durante 12 h. Se realizaron tres réplicas. Se midió el volumen máximo de gas (Vm), la fase lag (L), la tasa de producción de gas (S) y DIVMS usando cuatro substratos. Se analizaron los efectos principales de inóculos y substratos e interacciones. Comparado con el CONTROL, la preservación del inóculo afectó (P<0.01) Vm, L y S. Sin embargo, ALTO24 mejoró (P<0.01) la cinética de fermentación y DIVMS comparado con el tratamiento MODE24 o BAJO24. En el segundo experimento, DIVMS fue menor (P<0.01) para el tratamiento ALTO12 a las 72 h de fermentación comparado con el CONTROL. La alfalfa y el pasto ovillo tuvieron mayor (P<0.01) Vm y DIVMS comparado con el cocuite y el pasto de Guinea. La reactivación del inóculo ruminal preservado por pre-incubación durante 24 h en medio que contiene extracto de levaduras, peptona de caseína y carbohidratos tuvo un desempeño mejor que la pre-incubación por 12 h; sin embargo, la cinética de fermentación y DIVMS no fueron comparables al líquido ruminal fresco.

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Freeze-drying and cryopreservation of bacteria

Cited by 30 publications

References 9 publications

Effect of DNA extraction and sample preservation method on rumen bacterial population

Effect of DNA extraction and sample preservation method on rumen bacterial population

Storage stability of freeze-dried Lactobacillus plantarum S20 starter culture as affected by various formulations of drying medium, and its fermentation characteristics in mung bean (Vigna radiata L.) slurry was studied by Sulabo et al.

Evaluación de métodos nutricionales para reactivar inóculo ruminal preservado analizado a través de cinética de fermentación y digestibilidad de forrajes in vitro

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