高密度发酵和真空冷冻干燥工艺对乳酸菌抗冷冻性的影响

经真空冷冻干燥得到的乳酸菌发酵剂存活率和后期的低温贮藏稳定性与诸多因素相关。本文综述了制备乳酸菌发酵剂过程中高密度发酵和真空冷冻干燥工艺的不同对乳酸菌抗冷冻性的影响,其中高密度发酵过程中的培养基组分、培养温度、发酵恒定pH、中和剂的选择、菌体收获时期和发酵结束后处理以及真空冷冻干燥过程中保护剂的添加、预冷冻处理等是影响乳酸菌抗冷冻性的重要因素。通过对这些相关因素的综述分析,为提高乳酸菌发酵剂的冻干存活率和后期的低温贮藏稳定性提供新的思路,且应用抗冷冻性强、活力高的乳酸菌发酵剂对有效提高乳制品的质量和企业的经济效益意义重大。     

Survival curves for microbial species stored by freeze-drying

The survival of a variety of species of microorganism following storage for up to 20 years has been analyzed. The organisms were freeze-dried, sealed in ampoules under vacuum (<1 Pa) and stored in the dark at 5 degrees C. The yeast that was tested, Saccharomyces cerevisiae, showed only 8% survival when recovered shortly after freeze-drying, but subsequent loss during storage was the least among all the tested microorganisms. The decrease in the logarithm of survival per year (log survival) was -0.010, which corresponds to a survival rate of 97.7% per year. The Gram-negative bacteria tested, Escherichia coli, Pseudomonas putida, and Enterobacter cloacae, showed 42.6, 33.5, and 50.8% survival shortly after freeze-drying, which was higher than the corresponding survival of S. cerevisiae, but the subsequent loss during storage was greater than S. cerevisiae, the log survival figures being -0.041, -0.058, and -0.073 per year. These values correspond to survival rates of 91.0, 87.5, and 84.5% each year. The Gram-positive bacteria tested, Lactobacillus acidophilus and Enteroccoccus faecium, showed 62.5 and 85.2% survival shortly after freeze-drying, which was even higher than that of the Gram-negative species, and these organisms also showed better survival during storage than Gram-negative bacteria; their log survival rates were -0.018 and -0.016 per year, which corresponded to survival rates of almost 96% per year. Comparison of these results with other published data for different drying conditions suggests that survival during storage is strongly influenced by the degree of vacuum under which the ampoules were sealed. The excellent survival after freeze-drying of each species might be attributable to the high level of desiccation and to sealing under vacuum.     

Viability of micrococci and lactobacilli upon freezing and freeze-drying in the presence of different cryoprotectants

Studies were conducted on the viability of Micrococcus varians strain M₉₅ and Lactobacillus plantarum strain L₄ upon freezing and freeze-drying using five cryoprotectants (sucrose, lactose, sodium glutamate, peptone, dry nonfat milk) singly or in combinations with gelatin, glutamic acid, and sodium acetate. The number of survivals was determined immediately after treatment and after storage at room temperature or refrigeration temperatures, under vacuum or in air. Dry nonfat milk and peptone introduced at the levels of 8 and 5%, respectively, to broth culture, were found to be the best cryoprotectants providing a 100% viability determined immediately after the treatment of the strains under investigation.Immediately after freezing and freeze-drying, the numbers of viable micrococci remain high, the percentage viability in the presence of almost all the protectants used being 100%. During storage, those numbers decrease rapidly, reaching zero in 3 months upon storage at room temperature in air. The storage ability of lactobacilli is considerably better and, regardless of the fact that the percentage viability decreases, sufficient numbers of viable cells remain after 6 months of storage at both test temperatures.The best results are obtained on storing the microoganisms under vacuum in ampoules under reduced temperatures (+5 °C).     

Effects of nanofibrillated cellulose hydrogels on adipose tissue extract and hepatocellular carcinoma cell spheroids in freeze-drying

The aim of this study was to evaluate the effects of two nanofibrillated cellulose (NFC) hydrogels on two human derivatives during freeze-drying. Native NFC hydrogel is a suitable platform to culture 3D cell spheroids and a hydrogel processed further, called anionic NFC (ANFC) hydrogel, is an excellent platform for controlled release of proteins. Moreover, it has been shown to be compatible with freeze-drying when correct lyoprotectants are implemented. Freeze-drying is a method, where substance is first frozen, and then vacuum dried trough sublimation of water in order to achieve dry matter without the loss of the original three-dimensional structures.The first chosen human derivative was adipose tissue extract (ATE) which is a cell-free growth factor-rich preparation capable of promoting growth of regenerative cells. The release of growth factors from the freeze-dried mixture of ATE and ANFC was compared to that of non-freeze-dried control mixtures. The release profiles remained at the same level after freeze-drying. The second derivative was hepatocellular carcinoma (HepG2) cell spheroids which were evaluated before and after freeze-drying. The 3D structure of the HepG2 cell spheroids was preserved and the spheroids retained 18% of their metabolic activity after rehydration. However, the freeze-dried and rehydrated HepG2 cell spheroids did not proliferate and the cell membrane was damaged by fusion and formation of crystals.     

阪崎肠杆菌标准品制备中冻干工艺的优化

本研究对阪崎肠杆菌(Enterobacter sakazakii)冻干工艺进行优化,旨在为E.sakazaki定性标准品和定量标准品的制备提供技术基础,同时为其他肠杆菌科标准品的制备工艺提供理论指导.实验结果表明:最佳冻干保护剂组合为海藻糖3%,脱脂奶粉8%,谷氨酸钠1.5%;最佳预冻温度和预冻时间分别为-20℃,4 ...     

Survival rate of microbes after freeze-drying and long-term storage

The survival rates of 10 species of microorganisms were investigated after freeze-drying and preserving in a vacuum at 5 degrees C. The survival rates varied with species. The survival rates immediately after freeze-drying were different among yeast, gram-positive bacteria, and gram-negative bacteria, and the change in the 10-year survival rate was species-specific. The survival rate of yeast, Saccharomyces cerevisiae, was about 10% immediately after drying, and the rate did not decrease significantly during the 10-year storage period. Survival rates after the drying of gram-positive bacteria, i.e., Brevibacterium flavum, B. lactofermentum, Corynebacterium acetoacidophilum, C. gultamicum, and Streptococcus mutans, were around 80%. The survival rate of Brevibacterium and Corynebacterium did not decrease greatly during the storage period, whereas the rate of S. mutans decreased to about 20% after 10 years. Survival rates after the drying of gram-negative bacteria, i.e., Escherichia coli, Pseudomonas putida, Serratia marcescens, and Alcaligenes faecalis, were around 50%. The survival rate decreased for the first 5 years and then stabilized to around 10% thereafter.     

Preservation of influenza virosome structure and function during freeze-drying and storage

Virosomes derived from influenza virus are reconstituted viral envelopes, which retain the receptor-binding and cell entry properties of the native virus, but lack the viral genetic material. These virosomes are of interest because of their potential use as vaccines or cellular delivery systems. However, in aqueous dispersion influenza virosomes have a relatively poor stability. Although freeze-drying of the virosomes could improve their stability, a lyoprotectant is required to preserve the structure and function of the virosomes during the lyophilization process as well as during subsequent storage of the dry powder formulation. In this study, inulin, a medium-chain oligosaccharide, was identified as an effective stabilizer of influenza virosomes. When inulin was added to an aqueous virosomal dispersion, the vesicular structure of the virosomes, with spike proteins protruding from the virosomal surface, as well as their membrane fusion activity were completely preserved during freeze-drying. When the freeze-drying process was performed from dispersions lacking a lyoprotectant, both structure and fusogenic properties of the virosomes were lost. Moreover, it was shown that the immunogenicity of inulin-stabilized virosomes was preserved. For example, dry powder formulations of virosomes retained HA potency for at least 12 weeks at 20 degrees C. Virosomes with encapsulated pDNA encoding for the eGFP reporter gene were also found to be stabilized by inulin. The fusion capacity and the transfection efficacy (determined in BHK-21 cells) could be preserved for 12 weeks during storage at 4 degrees C. It is concluded that freeze-drying in the presence of inulin as a lyoprotectant completely preserves the structure and function of influenza virosomes.     

益生菌冷冻干燥高活性保护机制研究进展

摄取足量益生菌有助于维持肠道微生物群落的稳态,对维持人体肠道健康具有重要意义。然而,在工业化应用中,益生菌抗逆能力较弱且对储存条件要求高,导致益生菌产品对运输和活性维持条件要求较高,这些产业需求对高活力益生菌的制备工艺提出了挑战。干燥处理常应用于保持益生菌活性和稳定性,其中冷冻干燥技术应用最广泛,但冻干过程中益生菌会受到各类环境压力的刺激,引起细胞损伤甚至死亡。因此,可以显著提高益生菌存活率的冻干保护剂成为目前益生菌工业应用的研究热点。本文从益生菌常用及新发现的冻干保护剂种类及其作用机制进行了系统归纳,对菌株冻干后细胞存活率的影响因素进行全面综述,并对冻干保护剂研究方向进行了展望,旨在为高活力益生菌冻干菌粉的研制提供理论支持。     

Determination of parameters for tobacco mosaic virus cryogenic treatment and freeze-drying

Summary Studies on determination of appropriate conditions for cryogenic treatment and lyophilization of tobacco mosaic virus (TMV) in infected leaf tissue, plant sap and purified preparation were carried out. The found parameters were: freezing to minus 22°C (for 1.5 hours); twelve-hours-sublimation at 50 Pa vacuum until 20°C temperature of the sample and secondary drying at 1–2 Pa for 5–6 hours. Fifteen variants of cryoprotection were applied. TMV showed a survival higher than 89% after freeze-drying in glucose, fructose, maltose, lactose, sorbitol and the combinations with dextran. A technological scheme for automatic processing and standard work was developed.     

Surface coats on human lymphocytes: freeze-drying and staining with cations.

Surface coats can be demonstrated on human peripheral blood lymphocytes by staining with ruthenium red, alcian blue, Thorotrast, and cationized ferritin, which are similar in distribution to a 40- to 65-nm layer of amorphous extracellular material recently reported on fixed, freeze-dried lymphocytes. Several additional lines of evidence, including X-ray microanalysis, suggest that the latter is not a contaminant added by freeze-drying. Freeze-drying may provide the means for a morphological assessment of the lymphocyte surface, including the extracellular coat, which may give additional insight into the immune response.     

Key composition optimization of meat processed protein source by vacuum freeze-drying technology

Vacuum freeze-drying technology is a high technology content, a wide range of knowledge of technology in the field of drying technology is involved, it is also a method of the most complex drying equipment, the largest energy consumption, the highest cost of drying method, but due to the particularity of its dry goods: the freeze-drying food has the advantages of complex water performance is good, cooler and luster of freezing and drying food to maintain good products, less nutrient loss, light weight, easy to carry transportation, easy to long-term preservation, and on the quality is far superior to the obvious advantages of other dried food, making it become the forefront of drying technology research and development. The freeze-drying process of Chinese style ham and western Germany fruit tree tenderloin is studied in this paper, their eutectic point, melting point and collapse temperature, freeze-drying curve and its heat and mass transfer characteristics are got, then the precool temperature and the highest limiting temperature of sublimation interface are determined. The effect of system pressure on freeze-dried rate in freeze-drying process is discussed, and the method of regulating pressure circularly is determined.     

香蕉果实冻干过程参数优化的研究

对香蕉果实冻干生产工艺中的物料厚度、冻结方法、加热板温度、干燥室真空度等参数进行比较试验,结果表明,各过程参数对香蕉冻干品质量和产量均有显著的影响。香蕉冻干过程中参数较佳的工艺条件建议为物料厚度选取5~7 mm,采用速冻方法冻结,加热板温度设定45℃,干燥室真空度控制于20~30 Pa。     

Effects of freeze-preservation on some pollen enzymes: II. Freezing and freeze-drying stresses

The isozymes of lily and corn pollen esterases and acid phosphatase were studied in relation to freeze-drying and vacuum-drying. Fresh samples of Lilium longiflorum L. and Zea mays L. pollen were frozen at rates ranging between 200 and 100 °C/min and freeze-dried at temperatures from 0 to ?70 °C for approximately 48 to 70 hr. Freeze-dried samples were rehydrated slowly (10% relative humidity) and rapidly (90% relative humidity). Vacuum-drying was performed at room temperature (22 °C).Soluble pollen enzymes were analyzed by disc electrophoresis on polyacrylamide gels stained with substrate specific reagents. The stained gels were evaluated by densitometry for migration rate, isozyme pattern, and relative activity. The numerical data generated in this manner were then statistically analyzed.The following conclusions resulted from this study: (i) freeze-drying and freeze-thawing treatments were comparable except for corn esterases; (ii) freeze-drying induced alterations in enzyme activity except for corn acid phosphatase; (iii) the freezing rate, the final freezing temperature, and exposure to various relative humidities produced few changes in freeze-dried material; (iv) freeze-drying was less detrimental than vacuum-drying to the enzyme characteristics of corn; (v) freeze-drying yielded higher viabilities than vacuum-drying; and (vi) acid phosphatase alterations appeared to be related to pollen viability in most cases.     

Proceedings: Preservation of bacteriophages by freezing and freeze-drying

Losses in titer determined before and after freezing and freeze-drying of bacteriophages were matched with the following known characters of the phages: particle morphology, size, chloroform sensitivity, nucleic acid content, and “osmotic” sensitivity. A high percentage of morphological type A phages are unusually sensitive to freezing and freeze-drying; type B phages are less sensitive. Large phages do not freeze-dry as well as small ones. Osmotically sensitive phages are not preserved as successfully as osmotically resistant ones.     

Successful long-term preservation of rat sperm by freeze-drying

Background

Freeze-drying sperm has been developed as a new preservation method where liquid nitrogen is no longer necessary. An advantage of freeze-drying sperm is that it can be stored at 4°C and transported at room temperature. Although the successful freeze-drying of sperm has been reported in a number of animals, the possibility of long-term preservation using this method has not yet been studied.

Methodology/Principal Findings

Offspring were obtained from oocytes fertilized with rat epididymal sperm freeze-dried using a solution containing 10 mM Tris and 1 mM EDTA adjusted to pH 8.0. Tolerance of testicular sperm to freeze-drying was increased by pre-treatment with diamide. Offspring with normal fertility were obtained from oocytes fertilized with freeze-dried epididymal sperm stored at 4°C for 5 years.

Conclusions and Significance

Sperm with –SS– cross-linking in the thiol-disulfide of their protamine were highly tolerant to freeze-drying, and the fertility of freeze-dried sperm was maintained for 5 years without deterioration. This is the first report to demonstrate the successful freeze-drying of sperm using a new and simple method for long-term preservation.     

Optimisation of initial cell concentration enhances freeze-drying tolerance of Pseudomonas chlororaphis

The freeze-drying tolerance of Pseudomonas chlororaphis, an antifungal bacterium used as biocontrol agent was investigated. P. chlororaphis is freeze-drying sensitive and the viability drops more than 3 log units in the absence of protective freeze-drying medium. Of the freeze-drying media tested, lactose, sucrose, trehalose, glutamate, sucrose with glutamate, skimmed milk, and skimmed milk with trehalose, skimmed milk gave the lowest survival (0.6+/-0.2%) and sucrose the highest (6.4+/-1.2%). Cellular accumulation of sucrose from the freeze-drying medium and the protective effect of sucrose were dependent on sucrose concentration. The effect of initial cell concentration, from 1 x 10(7) to 5 x 10(10) CFU/ml, on survival after freeze-drying was studied for carbon starved cells with sucrose as freeze-drying medium. The highest freeze-drying survival values, 15-25%, were obtained for initial cell concentrations between 1 x 10(9) and 1 x 10(10) CFU/ml. For cell concentrations outside this window more than 10 times lower survival values were observed. P. chlororaphis was cultivated to induce stress response that could confer protection against freeze-drying inactivation. Carbon starvation and, to a lesser extent, heat treatment enhanced freeze-drying tolerance. By combining optimal cell concentration, optimal sucrose concentration and carbon starvation the survival after freeze-drying was 26+/-6%.     

Maintenance of quaternary structure in the frozen state stabilizes lactate dehydrogenase during freeze-drying

Sugars inhibit protein unfolding during the drying step of lyophilization by replacing hydrogen bonds to the protein lost upon removal of water. In many cases, polymers fail to inhibit dehydration-induced damage to proteins because steric hindrance prevents effective hydrogen bonding of the polymer to the protein's surface. However, in certain cases, polymers have been shown to stabilize multimeric enzymes during lyophilization. Here we test the hypothesis that this protection is due to inhibition of dissociation into subunits during freezing. To test this hypothesis, as a model system we used mixtures of lactate dehydrogenase isozymes that form electrophoretically distinguishable hybrid tetramers during reversible dissociation. We examined hybridization and recovery of catalytic activity during freeze-thawing and freeze-drying in the presence of polymers (dextran, Ficoll, and polyethylene glycol), sugars (sucrose, trehalose, glucose), and surfactants (Tween 80, Brij 35, hydroxy-propyl beta-cyclodextrin). The surfactants did not protect LDH during freeze-thawing or freeze-drying. Rather, in the presence of Brij 35, enhanced damage was seen during both freeze-thawing and freeze-drying, and the presence of Tween 80 exacerbated loss of active protein during freeze-drying. Polymers and sugars prevented dissociation of LDH during the freezing step of lyophilization, resulting in greater recovery of enzyme activity after lyophilization and rehydration. This beneficial effect was observed even in systems that do not form glassy solids during freezing and drying. We suggest that stabilization during drying results in part from greater inherent stability of the assembled holoenzyme relative to that of the dissociated monomers. Polymers inhibit freezing-induced dissociation thermodynamically because they are preferentially excluded from the surface of proteins, which increases the free energy of dissociation and denaturation.     

Synergetic effect of freeze-drying and gamma irradiation on the mechanical properties of human cancellous bone

Freeze-drying and irradiation are common process used by tissue banks to preserve and sterilize bone allografts. Freeze dried irradiated bone is known to be more brittle. Whether bone brittleness is due to irradiation alone, temperature during irradiation or to a synergetic effect of the freeze-drying-irradiation process was not yet assessed. Using a left–right femoral head symmetry model, 822 compression tests were performed to assess the influence of sequences of a 25 kGy irradiation with and without freeze-drying compared to the unprocessed counterpart. Irradiation of frozen bone did not cause any significant reduction in ultimate strength, stiffness and work to failure. The addition of the freeze-drying process before or after irradiation resulted in a mean drop of 35 and 31% in ultimate strength, 14 and 37% in stiffness and 46 and 37% in work to failure. Unlike irradiation at room temperature, irradiation under dry ice of solvent–detergent treated bone seemed to have no detrimental effect on mechanical properties of cancellous bone. Freeze-drying bone without irradiation had no influence on mechanical parameters, but the addition of irradiation to the freeze-drying step or the reverse sequence showed a detrimental effect and supports the idea of a negative synergetic effect of both procedures. These findings may have important implications for bone banking.     

A case study on stress preconditioning of a Lactobacillus strain prior to freeze-drying

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.     

Effect of pH value of freeze-drying solution on the chromosome integrity and developmental ability of mouse spermatozoa

The nuclei of freeze-dried mouse spermatozoa are able to retain their chromosome integrity and developmental potential. To optimize the conditions of freeze-drying, we examined whether pH values of the freeze-drying solution affect the chromosome integrity and developmental potential of sperm nuclei. The sperm freeze-drying solution we used contained a high concentration (50 mM) of calcium-chelating EGTA. Sperm chromosomes were examined at the metaphase of the first mitosis after injection of freeze-dried spermatozoa into matured oocytes. The developmental potential of sperm nuclei was assessed by examining the development of fetuses in midgestation. The results showed that both sperm chromosomes and sperm developmental potential are maintained better when the freeze-drying solution was slightly alkaline (pH 8.0) rather than near neutral or acidic (pH 7.4-6.0). The data indicated that the chromosome integrity and developmental ability of mouse spermatozoa are affected by the pH value of freeze-drying solution.