Freezing and freeze-drying of the bacterium Rahnella aquatilis BNM 0523: study of protecting agents, rehydration media and freezing temperatures

Aims: The aim of the present study was to evaluate and compare freezing and freeze‐drying treatments for conserving Rahnella aquatilis (BNM 0523) with the goal to achieve an adequate commercial formulation of this biocontrol agent. Methods and Results: The effect of several protective agents, rehydration media and freezing temperatures on the viability and functional activity of the R. aquatilis was investigated. The storage stability at 3 months and 4 years was determined by checking the viability of the cells and their biocontrol capability against Botrytis cinerea by measuring the percentage of reduction of disease severity on apple. The best results were obtained by the freeze‐drying of the cells using a mixture of skimmed nonfat milk 10%, yeast extract 0·5% and glucose 1% as the protecting and rehydrating medium, and a quickly freezing (?70°C) before the freeze‐drying. In this case, the viability of the cells after 4 years was 98%, and their antagonistic ability showed a little decrease with respect fresh cells. Conclusions: The studies showed that R. aquatilis was resistant to freezing and freeze‐drying when it was used a mixture of cryoprotectants and that it was possible to obtain inoculums with high viability and good effectiveness for reduction of decay caused by B. cinerea. Significance and Impact of the study: This study is probably the first report about the resistance of R. aquatilis to freezing and freeze‐drying treatments and shows that these operations could be useful for obtaining a commercial formulation of this biocontrol agent.     

Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying

Aims:  To investigate the effects of the medium and cryoprotective agents used on the growth and survival of Lactobacillus plantarum and Lactobacillus rhamnosus GG during freeze drying.
Methods and Results:  A complex medium was developed consisting primarily of glucose, yeast extract and vegetable-derived peptone. Trehalose, sucrose and sorbitol were examined for their ability to protect the cells during freeze drying. Using standardized amount of cells and the optimized freeze drying media, the effect of the growth medium on cell survival during freeze drying was investigated. The results showed that glucose and yeast extract were the most important growth factors, while sucrose offered better protection than trehalose and sorbitol during freeze drying. When the cells were grown under carbon limiting conditions, their survival during freeze drying was significantly decreased.
Conclusions:  A clear relationship was observed between cell growth and the ability of the cells to survive during the freeze drying process.
Significance and Impact of the Study:  The survival of probiotic strains during freeze drying was shown to be dependent on the cryoprotectant used and the growth medium.     

Heat and mass transfer scale-up issues during freeze-drying, III: Control and characterization of dryer differences via operational qualification tests

The objective of this research was to estimate differences in heat and mass transfer between freeze dryers due to inherent design characteristics using data obtained from sublimation tests. This study also aimed to provide guidelines for convenient scale-up of the freeze-drying process. Data obtained from sublimation tests performed on laboratory-scale, pilot, and production freeze dryers were used to evaluate various heat and mass transfer parameters: nonuniformity in shelf surface temperatures, resistance of pipe, refrigeration system, and condenser. Emissivity measurements of relevant surfaces such as the chamber wall and the freeze dryer door were taken to evaluate the impact of atypical radiation heat transfer during scale-up. “Hot” and “cold” spots were identified on the shelf surface of different freeze dryers, and the impact of variation in shelf surface temperatures on the primary drying time and the product temperature during primary drying was studied. Calculations performed using emissivity measurements on different freeze dryers suggest that a front vial in the laboratory lyophilizer received 1.8 times more heat than a front vial in a manufacturing freeze dryer operating at a shelf temperature of −25°C and a chamber pressure of 150 mTorr during primary drying. Therefore, front vials in the laboratory are much more atypical than front vials in manufacturing. Steady-state heat and mass transfer equations were used to study a combination of different scaleup issues pertinent during lyophilization cycles commonly used for the freeze-drying of pharmaceuticals.     

花菇的冷冻干燥技术研究

实验研究用板层导热法研究了花菇的冻干特性,获得了新鲜花菇的冻干曲线,分析了花菇冻干过程,测定和比较了新鲜花菇和冻干花菇的营养成份。证实试验机的适应性并确定了花菇的冻干工艺,为工业生产提供了理论依据和参考价值。     

Screening of freeze-dried protective agents for the formulation of biocontrol strains, Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans 2Re40

Aims: The effects of different freeze‐drying protective agents on the viabilities of biocontrol strains Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans 2Re40 were investigated. Method and Results: Several concentrations of protective and rehydration media were tested to improve the survival of biocontrol agents after freeze‐drying. The subsequent survival rates during storage and rehydration media of freeze‐dried biocontrol strains were also examined. Conclusions: The results indicated that cellobiose (5%) and d ‐galactose (5%) gave maximum viability of strains Bu. vietnamiensis B418 and P. agglomerans 2Re40 (98 and 54·3% respectively) while the perfect one (100%) of strain B. cereus AR156 was obtained with sucrose (5%) during freeze‐drying, and the highest survival of the three strains was reached when they were rehydrated with 10% nonfat skim milk. In the following storage, the survival rates showed that B. cereus AR156 could still reach 50% after 12 months. Significance and Impact of the study: This study showed that freeze‐drying could be used to stabilize cells of these three biocontrol strains. Further studies should focus on the scale‐up possibilities and formulation development.     

The Preservation of Plant Pathogenic Bacteria

Some 160 cultures were preserved by freeze drying, under mineral oil and in soil. After storage for 5 years all freeze dried cultures were viable; most cultures of xanthomonads were viable under oil and in soil; pseudomonads survived well in soil but only moderately well under oil; soft-rotting Erwinia spp. survived poorly but storage under oil was better than in soil; other Erwinia spp. and most Corynebacterium spp. survived well in soil and under oil. The mean half lives in years ( h ) calculated for freeze dried cultures of groups of closely related bacteria were: Erwinia 'chrysanthemi group', 0·40; Erwinia 'carotovora group', 0·51; Pseudomonas 'syringae group', 0·50; Xanthomonas spp., 0·84 years. Estimated half lives for Corynebacterium spp. ranged from 1·8 to 6·5 years. There was no evidence that bacteria which had been in culture for more than 3 years before being freeze dried had a longer storage life than those freeze dried within 3 years of isolation. Cultures of the Pseudomonas 'syringae group'had a longer storage life when freeze dried by Greaves'method ( h = 0·73) than when freeze dried by Annear's method ( h =0·50). There appeared to be no general correlation between half life in storage and either the proportion of cells surviving the freeze drying process or the viable cell count immediately after freeze drying. Most of the variation in the results could be attributed to variation in viable cell count between different ampoules of the same batch of a culture.     

EVALUATION OF PIGMENT EXTRACTION METHODS AND A RECOMMENDED PROTOCOL FOR PERIPHYTON CHLOROPHYLL a DETERMINATION AND CHEMOTAXONOMIC ASSESSMENT1

Many methods have been proposed to extract and quantify algal pigments. Comparative studies have found that pigment extraction efficiency varies among solvent and mechanical disruption protocols due to differential cellular resistance, thereby, leading to potential misinterpretation of pigment data. When the type or resistance of algae are unknown, a method is required that efficiently extract pigments from all taxonomic groups. The objective of this study was to develop a simple and efficient one stage periphyton pigment extraction protocol by comparing the extractability of four solvents (acetone, methanol, methanol/acetone, and methanol/acetone/N,N‐dimethylformamide), the effects of grinding, and the effects of freeze‐drying. The best overall extraction was obtained using freeze‐dried samples extracted with methanol/acetone/DMF/water (MAD). Eighty‐six percent more chlorophyll was extracted when the sample was freeze‐dried relative to fresh/frozen samples extracted with 90% acetone. Freeze‐drying greatly improved the extraction of both polar and non‐polar (lipophilic/hydrophobic) pigments while MAD increased the extractability of polar pigments and improved peak resolution of all pigments. Chemotaxonomic assessment differed between samples that were fresh/frozen or freeze‐dried before extraction. The relative abundance of cyanobacteria was greater for freeze‐dried material compared with fresh/frozen due to the improved extractability of cyanobacterial pigments. Based on the results of this study, the traditional approach of 90% acetone as a solvent is not recommended for periphyton samples containing cyanobacteria or when the composition of the mat is unknown. The combination of freeze‐drying and MAD was sufficient for the extraction of pigments from a periphyton mat containing filamentous cyanobacteria, green algae, and diatoms.     

冷冻干燥保藏曲霉菌种的效果

本文报告了用冷冻干燥法保藏曲霉属（Aspergillus）5种8株曲霉的效果,并分别对这些曲霉菌株的糖化酶活力进行检测。这些曲霉菌株经过冷冻干燥保藏8年后全部保持生活能力,其培养及形态特征除一株生长稍差外,其余菌株均保留原有形状,测定其糖化酶活力未有明显变化。     

冻干乙型脑炎减毒活疫苗的冻干工艺优化研究

为了研究优化冻干乙型脑炎减毒活疫苗的冻干工艺,运用正交试验法L934考察不同冻干参数对该疫苗成品质量的影响,以外观、残余水分、病毒滴度以及热稳定性为直观分析指标,并对病毒滴度进行方差分析。结果显示,主干燥时间和主干燥真空压力对病毒滴度的影响有显著统计学意义(P<0.05)。优化的冻干参数为预冻温度-35℃、时间2h;主干燥温度从-35℃升温至-10℃,再由-10℃升温至33℃,总耗时16h,真空压力0.220mbar;二次干燥的温度维持于30℃,真空压力为0.001mbar,终点测试压力无变化时结束。对冻干乙型脑炎减毒活疫苗冻干参数筛选优化后,可得到较佳的冻干曲线,经验证该曲线适用于生产。     

Near‐infrared spectroscopic evaluation of lyophilized viral vaccine formulations

This article examines the applicability of near‐infrared spectroscopy (NIRS) to evaluate the virus state in a freeze‐dried live, attenuated vaccine formulation. Therefore, this formulation was freeze‐dried using different virus volumes and after applying different pre‐freeze‐drying virus treatments (resulting in different virus states): (i) as used in the commercial formulation; (ii) without antigen (placebo); (iii) concentrated via a centrifugal filter device; and (iv) stressed by 96 h exposure to room temperature. Each freeze‐dried product was measured directly after freeze‐drying with NIR spectroscopy and the spectra were analyzed using principal component analysis (PCA). Herewith, two NIR spectral regions were evaluated: (i) the 7300–4000 cm^?1 region containing the amide A/II band which might reflect information on the coated proteins of freeze‐dried live, attenuated viruses; and (ii) the C–H vibration overtone regions (10,000–7500 and 6340–5500 cm^?1) which might supply information on the lipid layer surrounding the freeze‐dried live, attenuated viruses. The different pre‐freeze‐drying treated live, attenuated virus formulations (different virus states and virus volumes) resulted in different clusters in the scores plots resulting from the PCA of the collected NIR spectra. Secondly, partial least squares discriminant analysis models (PLS‐DA) were developed and evaluated, allowing classification of the freeze‐dried formulations according to virus pretreatment. The results of this study suggest the applicability of NIR spectroscopy for evaluating live, attenuated vaccine formulations with respect to their virus pretreatment and virus volume. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1573–1586, 2013     

Evidence of membrane damage in Lactobacillus bulgaricus following freeze drying

The mechanism of inactivation of Lactobacillus bulgaricus due to freeze drying was investigated. Cells were freeze-dried in skim milk powder, maltodextrin, glycerol, trehalose and water. Results are presented confirming previous authors'observations regarding membrane damage during freeze drying. In an attempt to define more clearly the nature of this damage, further experiments were carried out. Results show that following freeze drying changes occur in the unsaturated: saturated fatty acid ratio, a decrease in the activity of the membrane-bound enzyme ATPase and a loss of ΔpH.     

Effects of processing methods on nutritional composition and antioxidant activity of mealworm (Tenebrio molitor) larvae

We examined the effects of different processing methods on the nutritional composition and antioxidant activity of mealworms. After processing with nine methods, we calculated the contents of protein, fat, ash, carbohydrate, minerals (P, Ca, K, Fe, Na), vitamin B group (B₁, B₂, B₃), moisture, and calories. When processed by freeze drying among freeze drying, hot air drying, oven broiling, roasting, pan frying, deep frying, boiling, steaming, and microwaving, the contents of protein, some minerals, and vitamins were the highest. The content of total minerals was lowest after deep frying, and those of vitamin B₁ and B₃ were the lowest after microwaving. Antioxidant activity was then evaluated using DPPH and ABTS radical scavenging assays. DPPH assays showed that microwaving, freeze drying, deep frying, steaming, boiling, and oven broiling of mealworms yielded scavenging activities of 20.9–29.0% at 2,000 μg/mL, which was similar to the activity level (22.7–33.2%) of 40–60 μM tocopherol. ABTS assays confirmed that only freeze‐dried mealworms at 2,000 μg/mL exhibited higher activity than 10 μM tocopherol. Interestingly, similar trends were found for antioxidant activity levels and total phenolic contents in mealworms.     

Effect of intracellular trehalose in Cryptococcus laurentii and exogenous lyoprotectants on its viability and biocontrol efficacy on Penicillium expansum in apple fruit

AIMS: To improve viability and biocontrol efficacy of Cryptococcus laurentii after freeze drying and in subsequent storage. METHODS AND RESULTS: Viability of C. laurentii was improved after freeze drying and in subsequent storage at 4 or 25 degrees C by using skimmed milk (SM) and sugars (glucose, galactose, sucrose and trehalose) as protectants. Sugars and SM mixed together showed better protection than when they were used separately. Citric acid used as carbon source could induce accumulation of intracellular trehalose in the yeast. The yeast cells with high trehalose level (HT cells) had higher viability than those with low trehalose level (LT cells) after freeze drying and storage for 90 days. After storage for 90 days at 4 degrees C, the HT cells plus SM and sugars as protectant showed a similar biocontrol effect against blue mould rot in apple fruit caused by Penicillium expansum as fresh cells. CONCLUSIONS: Increasing intracellular trehalose content of C. laurentii and adding exogenous protectant (sugars + SM) could improve its viability and maintain its biocontrol efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: The results have a potential value for commercial application of C. laurentii.     

Metabolism of Klebsiella pneumoniae freeze‐dried cultures for the design of BOD bioassays

Aims: The survival rate of freeze‐dried cultures is not enough information for technological applications of micro‐organisms. There could be serious metabolic/structural damage in the survivors, leading to a delay time that can jeopardize the design of a rapid biochemical oxygen demand (BOD) metabolic‐based bioassay. Therefore, we will study the metabolic activity (as ferricyanide reduction activity) and the survival rate (as colony‐forming units, CFU) of different Klebsiella pneumoniae freeze‐dried cultures looking for stable metabolic conditions after 35 days of storage. Method and Results: Here, we tried several simple freeze‐drying processes of Kl. pneumoniae. Electrochemical measurements of ferrocyanide and survival rates obtained with the different freeze‐dried cultures were used to choose the best freeze‐drying process that leads to a rapid metabolic‐based bioassay. Conclusions: The use of milk plus monosodium glutamate was the best choice to obtain a Kl. pneumoniae freeze‐dried culture with metabolic stable conditions after storage at ?20°C without the need of vacuum storage and ready to use after 20 min of rehydration. We also demonstrate that the viability and the metabolic activity are not always directly correlated. Significance and Impact of the Study: This study shows that the use of this Kl. pneumoniae freeze‐dried culture is appropriate for the design of a rapid BOD bioassay.     

FTIR spectroscopy for the detection and evaluation of live attenuated viruses in freeze dried vaccine formulations

This article examines the applicability of Fourier Transform Infrared (FTIR) spectroscopy to detect the applied virus medium volume (i.e., during sample filling), to evaluate the virus state and to distinguish between different vaccine doses in a freeze dried live, attenuated vaccine formulation. Therefore, different formulations were freeze dried after preparing them with different virus medium volumes (i.e., 30, 100, and 400 µl) or after applying different pre‐freeze‐drying sample treatments (resulting in different virus states); i.e., (i) as done for the commercial formulation; (ii) samples without virus medium (placebo); (iii) samples with virus medium but free from antigen; (iv) concentrated samples obtained via a centrifugal filter device; and (v) samples stressed by 96h exposure to room temperature; or by using different doses (placebo, 25‐dose vials, 50‐dose‐vials and 125‐dose vials). Each freeze‐dried product was measured directly after freeze‐drying with FTIR spectroscopy. The collected spectra were analyzed using principal component analysis (PCA) and evaluated at three spectral regions, which might provide information on the coated proteins of freeze dried live, attenuated viruses: (i) 1700–1600 cm^?1 (amide I band), 1600–1500 cm^?1 (amide II band) and 1200–1350 cm^?1 (amide III band). The latter spectral band does not overlap with water signals and is hence not influenced by residual moisture in the samples. It was proven that FTIR could distinguish between the freeze‐dried samples prepared using different virus medium volumes, containing different doses and using different pre‐freeze‐drying sample treatments in the amide III region. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1107–1118, 2015     

Ultrastructural observations on tissues processed by a quick-freezing,rapid-drying method: Comparison with conventional specimen preparation

A quick-freeze, rapid-dry method for processing unfixed tissue for electron microscopy has been developed. The technique employs freezing on a cryogenchilled metal surface and drying in a cryosorption vacuum apparatus that allows osmium-vapor fixation and epoxy-resin embedment under high vacuum. Liver, kidney, bone marrow, and monolayer cultures of ventricular myocytes were selected as tissue specimens representing a wide range of physical properties, to demonstrate the practical aspects of achieving good ultrastructural morphology by freeze drying. A comparison was made between freeze drying and conventional processing using aldehyde fixation and alcohol dehydration. The preservation of cellular ultrastructure achieved by freeze drying allowed the identification of specific cell types within each specimen. Membranous organelles were well preserved, surrounded by cytoplasmic ground substance devoid of ice crystal damage. Electron-dense material was observed within the rough endoplasmic reticulum and Golgi cisternae and vesicles of frozen-dried, but not conventionally processed cells. This suggests the preservation by freeze drying of cytoplasmic components otherwise extracted from the cell by solvent exposure.     

影响生物制品冻干粉针剂水分的探讨

探讨生物制品冻干粉针剂样品放置一时间后残余水分增高的原因。进行了水分测定,真空度检测,二甲硅油和丁基橡胶药用瓶塞干燥失重的检测。冻干后每只丁基橡胶药用瓶塞平均含水分0.00224g。结果表明丁基橡胶药用瓶塞灭苗,干燥和冻干过程中去除水分不彻底是引起样品中水分升高的直接原因。     

Preservation of Bacteria by Circulating-Gas Freeze Drying

Water-washed Serratia marcescens and Escherichia coli were freeze dried in a circulating-gas system at atmospheric pressure. This convective procedure resulted in a substantially higher survival of organisms than could be obtained by the vacuum method of freeze drying. There was little or no decrease in cell viability during convective drying when the residual moisture content was 15% or higher. Below this level, survival declined with decreasing moisture content. A detailed comparison of the convective and vacuum methods indicated that the advantage gained by freeze drying bacteria in air accrues in the early period of sublimation, at which time cells were found to be sensitive to vacuum drying but insensitive to air drying. An explanation for this difference is proposed, based upon the kinetics of water removal in the two processes. In brief, it is suggested that the convective method permits samples to be dried more uniformly; and regional over-drying, which may be deleterious even if transient, is thus avoided in achieving the optimal level of moisture.     

Influence de l'âge de la culture sur la survie de Rhizobium meliloti à la lyophilisation et à la conservation après lyophilisation

Summary Survival after freeze drying of Rhizobium meliloti grown on different media was higher in young cultures when cells were in their logarithmic phase than in the old which were in their stationary phase. On the contrary the ability of the freeze dried organisms to survive during storage at 30°C was better for cells from old cultures than from young ones.     

Preservation of Vibrio fetus by Freeze Drying

S ummary . Vibrio fetus can be successfully freeze dried using the growth from thioglycollate blood agar. This medium is unsatisfactory for estimating the numbers of surviving organisms after freeze drying and storage. For this purpose a liquid medium containing 0.05% agar has been satisfactory. The long term storage of lyophilized preparations of V. fetus has not been fully investigated.