Freeze-drying of live attenuated Vibrio anguillarum mutant for vaccine preparation

Vibrio anguillarum MVAV6203 is a mutant strain as a candidate of live attenuated vaccine. In vaccine preparation, the freeze-drying conditions of the strain were investigated to improve the survival after freeze-drying, including the protectant, rehydration medium, freezing temperature, and initial cell concentration. Vibrio anguillarum MVAV6203 is sensitive to freeze-drying and the viability was only 0.03% in the absence of protectant. Of the tested protectants, 5% trehalose with 15% skimmed milk gave the highest viability of 34.2%. Higher cell survival was obtained by quick freezing at -80 degrees C than slow freezing at -20 degrees C. Initial cell concentration was another important factor, preferable for 1-3 x 10(10)CFU/ml. The supplementation of 10% skimmed milk in rehydration medium improved obviously freeze-drying viability. The combination of the optimal conditions achieved 51.4% cell viability after freeze-drying.     

Preservation of frozen yeast cells by trehalose.

Two different methods commonly used to preserve intact yeast cells-freezing and freeze-drying-were compared. Different yeast cells submitted to these treatments were stored for 28 days and cell viability assessed during this period. Intact yeast cells showed to be less tolerant to freeze-drying than to freezing. The rate of survival for both treatments could be enhanced by exogenous trehalose (10%) added during freezing and freeze-drying treatments or by a combination of two procedures: a pre-exposure of cells to 40 degrees C for 60 min and addition of trehalose. A maximum survival level of 71.5 +/- 6.3% after freezing could be achieved at the end of a storage period of 28 days, whereas only 25.0 +/- 1.4% showed the ability to tolerate freeze-drying treatment, if both low-temperature treatments were preceded by a heat exposure and addition of trehalose to yeast cells. Increased survival ability was also obtained when the pre-exposure treatment of yeast cells was performed at 10 degrees C for 3 h and trehalose was added: these treatments enhanced cell survival following freezing from 20.5 +/- 7. 7% to 60.0 +/- 3.5%. Although both mild cold and heat shock treatments could enhance cell tolerance to low temperature, only the heat treatment was able to increase the accumulation of intracellular trehalose whereas, during cold shock exposure, the intracellular amount of trehalose remained unaltered. Intracellular trehalose levels seemed not to be the only factor contributing to cell tolerance against freezing and freeze-drying treatments; however, the protection that this sugar confers to cells can be exerted only if it is to be found on both sides of the plasma membrane.     

Freeze-drying of Lactobacillus coryniformis Si3--effects of sucrose concentration, cell density, and freezing rate on cell survival and thermophysical properties

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/ml. At 10(11) CFU/ml, these thermophysical values decreased possibly due to increased amounts of cell-associated unfrozen water.     

Response Surface Optimization of Lyoprotectant for Lactobacillus bulgaricus During Vacuum Freeze-Drying

The individual and interactive effects of skimmed milk powder, lactose, and sodium ascorbate on the number of viable cells and freeze-drying survival for vacuum freeze-dried powder formulation of Lactobacillus bulgaricus were studied by response surface methodology, and the optimal compound lyoprotectant formulations were gained. It is shown that skim milk powder, lactose, and sodium ascorbate had a significant impact on variables and survival of cultures after freeze-drying. Also, their protective abilities could be enhanced significantly when using them as a mixture of 28% w/v skim milk, 24% w/v lactose, and 4.8% w/v sodium ascorbate. The optimal freeze-drying survival rate and the number of viable cells of Lactobacillus bulgaricus were observed to be (64.41 ± 0.02)% and (3.22 ± 0.02) × 10¹¹ colony-forming units (CFU)/g using the optimal compound protectants, which were very close to the expected values 64.47% and 3.28 × 10¹¹ CFU/g.     

Factors affecting viable cell counts of freeze-driedCryptococcus terricolus cells

Freeze-drying ofCryptococcus terricolus cells in distilled water resulted in a survival of only 0.1% of the cells. The viability could be increased to 16% by the use of a dextran-sucrose-sodium glutamate solution as suspending medium.For freeze-dried material with both low and high survival rates, and for five as well as for ten days old cultures, malt extract solution was the superior reconstitution medium. Less, but still distinct, protective action was found with a synthetic glucose-urea-salt solution.The viable cell counts of cells freeze-dried in dextran-sucrose-sodium glutamate solution were independent of the medium used for plating. When distilled water was used as a medium for freeze-drying, two to four times higher counts were obtained with malt extract agar than with synthetic glucose-urea-salt agar.Of the twenty different media tried for freeze-drying, sucrose solution gave the best protection. The viability was greatly influenced by the concentration used, maximum values being obtained when more than 10% of sucrose was added. The survival rate increased with the age of the cells until the fifth day, but was independent of the concentration of cells in the suspension. Under optimum conditions a survival rate of more than 80% was reached.     

Drying of Epicoccum nigrum conidia for obtaining a shelf-stable biological product against brown rot disease

AIMS: The effects of freeze-drying, spray-drying and fluidized bed-drying on survival of Epicoccum nigrum conidia were compared. METHODS AND RESULTS: Viability of E. nigrum conidia (estimated by measuring its germination) was 100% after fluidized bed-drying and freeze-drying, but it was determined that skimmed milk must be added in the case of freeze-drying conidia. Addition of other protectants (Tween-20, peptone, sucrose, glucose, starch and peptone + starch) to skimmed milk before freeze-drying did not improve the conidial viability which was obtained with skimmed milk alone. Glycerol had a negative effect on the lyophilization of E. nigrum conidia. Epicoccum nigrum conidia freeze-dried with skimmed milk, or fluidized bed-dried alone maintained an initial viability for 30 and 90 days, respectively, for storage at room temperature. Epicoccum nigrum conidial viability after spray-drying was lower than 10%. CONCLUSIONS: The best method to dry E. nigrum conidia was fluidized bed-drying. Conidia without protectants dried by this method had 100% viability and survived for 90 days at room temperature. SIGNIFICANCE AND IMPACT OF STUDY: This paper deals with methods for the potential formulation of a biocontrol agent which is being tested for eventual commercialization.     

海藻糖载入红细胞及其冷冻干燥的实验研究

冷冻干燥保存是长期保存人体红细胞的理想方案之一。冻干保护剂海藻糖渗入细胞内后,对细胞膜和细胞内物质有保护作用,其中的一个作用是增加细胞质的浓度,使冻干过程容易形成稳定的玻璃态。应用高渗法处理红细胞,通过考察胞内海藻糖含量、红细胞冻干后的存活率、腺苷三磷酸酶(ATPase)、超氧化物歧化酶(SOD)活力以及细胞形态变化,研究胞内海藻糖含量对红细胞冻干后活性的影响。结果显示:海藻糖对红细胞冻干具有明显的保护作用,随胞内海藻糖浓度升高,其保护性能逐渐增强;43.8mmol/L的胞内海藻糖浓度对红细胞保护最好,细胞存活率达到53.6%,形态保持良好,ATP和SOD活力均在正常的范围内。     

Effect of protective agents, rehydration media and initial cell concentration on viability of Pantoea agglomerans strain CPA-2 subjected to freeze-drying

The effect of initial cell density, protective agents and rehydration media on the viability of biocontrol agent Pantoea agglomerans CPA-2 when subjected to freeze-drying was studied. Several additives were tested as protective agents against freeze-drying injury. Maximum viability of the bacterial cells was obtained with disaccharides (survival levels > 60%). Freeze-dried samples were rehydrated with several media; the highest percentage viability was obtained with 10% non-fat skim milk (100%+). The effect of initial bacterial load on the final recovery was dependent on protectant but not on rehydration media. Sucrose was an effective protectant when a high initial concentration (10(10) cfu ml(-1) was used; the opposite occurred with non-fat skim milk. The use of 10(10) cfu ml(-1) as an initial concentration, sucrose as a protectant and non-fat skim milk as a rehydration medium enabled 100% of P. agglomerans viability to be conserved after freeze-drying. Results suggest the possibility of achieving a good formulation system for the studied biocontrol agent with a high number of viable cells to be used toward pathogens, which is desirable for the industrial development of the product.     

Effect of protective agents, freezing temperature, rehydration media on viability of malolactic bacteria subjected to freeze-drying

AIMS: The effects of protective agents, rehydration media and freezing temperature on the viabilities of Lactobacillus brevis and Oenococcus oeni H-2 when subjected to freeze-drying were investigated. METHODS AND RESULTS: Several protectants and rehydration media were tested to improve the survival after freeze-drying. The cells were also frozen at -65 and -20 degrees C to check the effect of freezing temperature on the viability. CONCLUSIONS: The best protectant and rehydration medium to obtain the highest viability after freeze-drying varied with the species of bacteria. Yeast extract (4.0%) and sodium glutamate (2.5% ) gave maximum viability of L. brevis and O. oeni (67.8% and 53.6% respectively). The highest survival of L. brevis and O. oeni were obtained when rehydrated with 10% sucrose and MGY medium respectively. When the bacterial cells were frozen quickly (-65 degrees C) than slowly (-20 degrees C), L. brevis and O. oeni both showed increased viability after freeze-drying. SIGNIFICANCE AND IMPACT OF THE STUDY: The viabilities of L. brevis and O. oeni after freeze-drying were shown to be strain specific and dependent on protective agents, rehydration media and freezing temperature.     

正交法优化嗜酸氧化亚铁硫杆菌冷冻干燥保护剂

利用正交实验方法,以甘油、海藻糖、蔗糖和牛血清蛋白为因素,对嗜酸氧化亚铁硫杆菌（Acididfiobacillus ferrooxidans,A．ferrooxidans）冷冻干燥保护剂的最优化配比进行了研究。直观分析、因素指标分析和方差分析的结果表明：由甘油、海藻糖、蔗糖和牛血清蛋白组成的冷冻干燥保护剂中,对存活率影响的主次顺序依次为：甘油〉海藻糖〉牛血清蛋白〉蔗糖。保护剂的最优化组合为甘油5％、海藻糖15％、蔗糖18％、牛血清蛋白10％。经过验证,该组合的保护剂可使冷冻干燥嗜酸氧化亚铁硫杆菌的存活率达到94％。     

Bioluminescent assay of total bacterial contamination of raw milk]

A rapid (30-35 min) bioluminescence assay of total bacterial contamination (TBC) of raw milk was optimized. This method includes incubation of milk samples in the presence of Neonol-10 and medical purity grade pancreatin with further removal of nonbacterial ATP by filtration through a membrane filter, cell disruption by treatment with dimethyl sulfoxide, and measurement of ATP concentration in a reaction with the bioluminescent reagent Immolum. The TBC detection threshold is 0.5 x 10(5) colony-forming units (CFU) per ml milk. Coefficients of correlation between the standard plate count method and bioluminescence assay (R) and residual standard deviations (Sxy) in raw milk samples (n = 140) were 0.83 and 0.54, respectively. In sterilized milk samples artificially contaminated with pure cultures of the main representatives of milk microflora (coli-forms, Staphylococcus aureus, Streptococcus thermophilus, and Streptococcus group D), these values were 0.89-0.99 and 0.09-0.29, respectively. The specific content of ATP was found to be (0.8 +/- 0.1) x 10(-18) mol/CFU in coli-forms; (12.0 +/- 8.1) x 10(-18) mol/CFU in S. aureus; (35.2 +/- 16.9) x 10(-18) mol/CFU in S. thermophilus; and (42.5 +/- 1.3) x 10(-18) in Streptococcus group D.     

Effects of cryoprotectants on viability of <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> CICC6226

Freeze-drying is commonly used to preserve probiotics, but it could cause cell damage and loss of viability. The cryoprotectants play an important role in the conservation of viability during freeze-drying. In this study, we investigated the survival rates of Lactobacillus reuteri CICC6226 in the presence of cryoprotectants such as sucrose, trehalose, and reconstituted skim milk (RSM). In addition, we determined the activities of hexokinase (HK), pyruvate kinase (PK), lactate dehydrogenase (LDH), and ATPases immediately following the freeze-drying. The results showed that the differences in HK and PK activities with and without the cryoprotectants during freeze-drying were not significant, but cell viability and activities of LDH and ATPase were significantly different (P<0.01) prior to and after freeze-drying. Meanwhile, the results showed that the maintenance of the membrane integrity and fluidity was improved in the presence of the 10% trehalose or 10% RSM than other treatments during freeze-drying. These results have provided direct biochemical and metabolic evidence of injured cell during freeze-drying. Freeze-drying damaged membrane structure and function of cell and inactivated enzymes (LDH and ATPases). The results imply that LDH and ATPases are key markers and could be used to evaluate the effect of cryoprotectants on viability and metabolic activities of L. reuteri CICC6226 during freeze-drying.     

Effect of protective agents and previous acclimation on ethanol resistance of frozen and freeze-dried Lactobacillus plantarum strains

The aim of this work was to study the protective effect of sucrose, trehalose and glutamate during freezing and freeze-drying of three oenological Lactobacillus plantarum strains previously acclimated in the presence of ethanol. The efficiency of protective agents was assessed by analyses of membrane integrity and bacterial cultivability in a synthetic wine after the preservation processes. No significant differences in the cultivability, with respect to the controls cells, were observed after freezing at −80 °C and −20 °C, and pre-acclimated cells were more resistant to freeze-drying than non-acclimated ones. The results of multiparametric flow cytometry showed a significant level of membrane damage after freeze-drying in two of the three strains. The cultivability was determined after incubation in wine-like medium containing 13 or 14% v/v ethanol at 21 °C for 24 h and the results were interpreted using principal component analysis (PCA). Acclimation was the most important factor for preservation, increasing the bacterial resistance to ethanol after freezing and freeze-drying. Freeze-drying was the most drastic method of preservation, followed by freezing at −20 °C. The increase of ethanol concentration from 6 to 10% v/v in the acclimation medium improved the recovery of two of the three strains. In turn, the increase of ethanol content in the synthetic wine led to a dramatic decrease of viable cells in the three strains investigated. The results of this study indicate that a successful inoculation of dehydrated L. plantarum in wine depends not only on the use of protective agents, but also on the cell acclimation process prior to preservation, and on the ethanol content of wine.     

Influence of freeze-drying and spray-drying preservation methods on survivability rate of different types of protectants encapsulated Lactobacillus acidophilus FTDC 3081

ABSTRACT

The aims of this study were to compare the effectiveness of different drying methods and to investigate the effects of adding a series of individual protectant such as skim milk, sucrose, maltodextrin, and corn starch for preserving Lactobacillus acidophilus FTDC 3081 cells during spray and freeze-drying and storage at different temperatures. Results showed a remarkable high survival rate of 70–80% immediately after spray- and freeze-drying in which the cell viability retained at the range of 10⁹ to 10¹⁰ CFU/mL. After a month of storage, maltodextrin showed higher protective ability on both spray- and freeze-dried cells as compared to other protective agents at 4°C, 25°C, and 40°C. A complete loss in viability of spray-dried L. acidophilus FTDC 3081 was observed after a month at 40°C in the absence of protective agent.     

Comprehensive optimization of composite cryoprotectant for Saccharomyces boulardii during freeze-drying and evaluation of its storage stability

Abstract

Saccharomyces boulardii (S. boulardii) is widely adopted in the diarrhea treatment for humans or livestock, so guaranteeing the survival rate of S. boulardii is the critical issue during freeze-drying process. In this study, the survival rate of S. boulardii with composite cryoprotectants during freeze-drying procedure and the subsequent storage were investigated. With the aid of response surface method, the composite cryoprotectants were comprehensively optimized to be lactose of 21.24%, trehalose of 22.00%, and sodium glutamate of 4.00%, contributing to the supreme survival rate of S. boulardii of 64.22?±?1.35% with the viable cell number of 9.5?±?0.07?×?10⁹ CFU/g, which was very close to the expected rate of 65.55% with a number of 9.6?×?10⁹ CFU/g. The accelerated storage test demonstrated that the inactivation rate constant of the freeze-dried S. boulardii powder was k_?18?=?8.04?×?10^?6. In addition, the freeze-dried goat milk powder results exhibited that the inactivation rate constants were k₄?=?4.48?×?10^?4 and k₂₅?=?9.72?×?10^?3 under 4 and 25?°C, respectively. This work provides a composite cryoprotectant formulation that has a good protective effect for the probiotic S. boulardii during freeze-drying process, possessing the potential application prospect in food, medicine, and even feed industry.     

Engineering trehalose synthesis in Lactococcus lactis for improved stress tolerance

Trehalose accumulation is a common cell defense strategy against a variety of stressful conditions. In particular, our team detected high levels of trehalose in Propionibacterium freudenreichii in response to acid stress, a result that led to the idea that endowing Lactococcus lactis with the capacity to synthesize trehalose could improve the acid tolerance of this organism. To this end, we took advantage of the endogenous genes involved in the trehalose catabolic pathway of L. lactis, i.e., trePP and pgmB, encoding trehalose 6-phosphate phosphorylase and β-phosphoglucomutase, respectively, which enabled the synthesis of trehalose 6-phosphate. Given that L. lactis lacks trehalose 6-phosphate phosphatase, the respective gene, otsB, from the food-grade organism P. freudenreichii was used to provide the required activity. The trehalose yield was approximately 15% in resting cells and in mid-exponential-phase cells grown without pH control. The intracellular concentration of trehalose reached maximal values of approximately 170 mM, but at least 67% of the trehalose produced was found in the growth medium. The viability of mutant and control strains was examined after exposure to heat, cold or acid shock, and freeze-drying. The trehalose-producing strains showed improved tolerance (5- to 10-fold-higher survivability) to acid (pH 3) and cold shock (4°C); there was also a strong improvement in cell survival in response to heat shock (45°C), and no protection was rendered against dehydration. The insight provided by this work may help the design of food-grade strains optimized for the dairy industry as well as for oral drug delivery.     

Survival of freeze-dried bacteria.

The aim of this study was to investigate the survival of freeze-dried bacterial species stored at the International Patent Organism Depository (IPOD) and to elucidate the characteristics affecting survival. Bacterial strains were freeze-dried, sealed in ampoules under a vacuum (<1 Pa), and stored in the dark at 5 degrees C. The survival of a variety of species following storage for up to 20 years was analyzed. The survival of freeze-dried species was analyzed in terms of two stages, freeze-drying and storing. Nonmotile genera showed relatively high survival after freeze-drying. Motile genera with peritrichous flagella showed low survival rates after freeze-drying. Vibrio and Aeromonas, which produce numerous flagella, showed very low survival rates. In Lactobacillus, non-trehalose-fermenting species showed better survival rates after freeze-drying than did fermenting species, and those species with teichoic acid in the cell wall showed lower survival rates during storage than species with teichoic acid in the cell membrane. Human pathogenic species of Corynebacterium, Bacillus, Streptococcus, and Klebsiella showed lower survival rates during storage than nonpathogenic species within the same genus. Among Pseudomonas species, P. chlororaphis, the only species tested that forms levan from sucrose, showed the lowest survival rate during storage in the genus. Survival rates of Gram-negative species during storage tended to be lower than those of Gram-positive species, though Chryseobacterium meningosepticum had stable survival during storage. The conclusion is that smooth cell surfaces (i.e., no flagella) and lack of trehalose outside the cytoplasm improved survival rates after freeze-drying. Because desiccation is important for survival during storage, the presence of extracellular polysaccharides or teichoic acids is disadvantageous for long-term survival. The lower survival rates of freeze-dried Gram-negative bacteria compared with those of Gram-positive bacteria may be attributed to the thinner peptidoglycan layer and the presence of lipopolysaccharides on the cell wall in the former species.     

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 ...     

Optimising the viability during storage of freeze-dried cell preparations of Campylobacter jejuni

Freeze-dried cultures of Campylobacter jejuni are used in the food and microbiological industry for reference materials and culture collections. However, C. jejuni is very susceptible to damage during freeze-drying and subsequent storage and it would be useful to have longer-lasting cultures. The survival of C. jejuni during freeze-drying and subsequent storage was investigated with the aim of optimising survival. C. jejuni was freeze-dried using cultures of different age (24-120 h), various lyoprotectants (10% phytone peptone, proteose peptone, peptonized milk, trehalose, soytone and sorbitol), various storage (air, nitrogen and vacuum) and re-hydration (media, temperature and time) conditions. One-day-old cultures had significantly greater survival after freeze-drying than older cultures. The addition of trehalose to inositol broth as a lyoprotectant resulted in almost 2 log(10) increase in survival after 2 months storage at 4 degrees C. Storage in a vacuum atmosphere and re-hydration in inositol broth at 37 degrees C increased recovery by 1-2 log(10) survival compared to re-hydration in maximal recovery diluent (MRD) after storage at 4 degrees C. Survival during storage was optimal when a one-day-old culture was freeze-dried in inositol broth plus 10% (w/v) trehalose, stored under vacuum at 4 degrees C and re-hydrated at the same incubation temperature (37 degrees C) in inositol broth for 30 min. The results demonstrate that the survival of freeze-dried cells of C. jejuni during storage can be significantly increased by optimising the culture age, the lyoprotectant, and the storage and re-hydration conditions. The logarithmic rate of loss of viability (K) followed very well an inverse dependence on the absolute temperature, i.e., the Arrhenius rate law. Extrapolation of the results to a more typical storage temperature (4 degrees C) predicted a very low K value of 1.5 x 10(-3). These results will be useful to the development of improved reference materials and samples held in culture collections.     

Persistence of Streptococcus mutans in stationary-phase batch cultures and biofilms

Streptococcus mutans is a member of oral plaque biofilms and is considered the major etiological agent of dental caries. We have characterized the survival of S. mutans strain UA159 in both batch cultures and biofilms. Bacteria grown in batch cultures in a chemically defined medium, FMC, containing an excess of glucose or sucrose caused the pH to decrease to 4.0 at the entry into stationary phase, and they survived for about 3 days. Survival was extended up to 11 days when the medium contained a limiting concentration of glucose or sucrose that was depleted by the time the bacteria reached stationary phase. Sugar-limited cultures maintained a pH of 7.0 throughout stationary phase. Their survival was shortened to 3 days by the addition of exogenous lactic acid at the entry into stationary phase. Sugar starvation did not lead to comparable survival in biofilms. Although the pH remained at 7.0, bacteria could no longer be cultured from biofilms 4 days after the imposition of glucose or sucrose starvation; BacLight staining results did not agree with survival results based on culturability. In both batch cultures and biofilms, survival could be extended by the addition of 0.5% mucin to the medium. Batch survival increased to an average of 26 (+/-8) days, and an average of 2.7 x 10(5) CFU per chamber were still present in biofilms that were starved of sucrose for 12 days.