Effects of trehalose on stress tolerance and biocontrol efficacy of Cryptococcus laurentii

AIMS: To investigate the effects of internal trehalose on viability and biocontrol efficacy of antagonistic yeast Cryptococcus laurentii under stresses of low temperature (LT), controlled atmosphere (CA) and freeze drying. METHODS AND RESULTS: The content of trehalose in C. laurentii was increased by culturing the yeast in trehalose-containing medium. Compared with yeast cells with low trehalose level, the yeast cells with high level of internal trehalose not only obtained higher viability, but also showed higher population and better biocontrol efficacy against Penicillium expansum on apple fruit both at 1 degrees C and in CA condition (5% O(2), 5% CO(2), 1 degrees C). After freeze drying, survival of the yeast with high trehalose level was markedly increased when stored at 25 degrees C for 0, 15 and 30 days. Meanwhile, high integrity of plasma membrane was detected in the freeze-dried yeast with high trehalose level by propidium iodide staining. CONCLUSIONS: Induced accumulation of internal trehalose could improve viability and biocontrol efficacy of C. laurentii under stresses of LT and CA. Moreover, survival of the yeast was also increased as internal trehalose accumulation after freeze drying, and one of the reasons might be that trehalose gave an effective protection to plasma membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this experiment show a promising way to improve the biocontrol performance of antagonistic yeasts under the commercial conditions.     

Liquid formulation of the biocontrol agent Candida sake by modifying water activity or adding protectants

AIMS: To evaluate the effect of modification of water activity (aw) and the addition of protective substances in the preservation medium of liquid formulations of the biocontrol agent Candida sake stored at 4 and 20 degrees C. METHODS AND RESULTS: The aw of the preservation medium of C. sake was modified from 0.72 to 0.95 by adding glycerol or polyethylene glycol (PEG). Moreover, several protectant substances at different concentrations were evaluated. Modification of lower aw-levels (0.721-0.901) with glycerol did not maintain the viability of the yeast cells. Higher aw-levels (0.93-0.95) with either glycerol or PEG improved the viability but not at acceptable viability levels. C. sake cells maintained viabilities >60% when sugars, such as trehalose, and polyols, such as glycerol and PEG were used as protectants in liquid formulations. Moreover, liquid formulations of C. sake stored at 4 degrees C showed higher number of viable counts than at 20 degrees C. When different sugars were tested, all of them, except 10% fructose, resulted in a viability higher than 50% of the C. sake formulations. Biocontrol of liquid formulation treatments was similar to fresh cells in controlling Penicillium expansum on wounded apples. CONCLUSIONS: Sugars such as lactose and trehalose could be considered as good protectants in order to obtain liquid formulations of C. sake cells as they maintain the viability >70% for 4 months at 4 degrees C. SIGNIFICANCE AND IMPACT OF STUDY: This study shows that a suitable liquid formulation for commercial application can be produced with high viability and conservation of biocontrol efficacy. Moreover, if 10% lactose is the protectant used in the formulation, the economic costs would not be limiting for industrial production.     

Survival of the biocontrol yeast Pichia anomala after long-term storage in liquid formulations at different temperatures, assessed by flow cytometry

AIMS: Investigate the survival of liquid formulations of the biocontrol yeast Pichia anomala J121 at different temperatures, and develop a system for comparative studies of different storage conditions and formulations. METHODS AND RESULTS: The survival of P. anomala in liquid formulations with lactose, starch and trehalose amendments was measured during prolonged storage at temperatures ranging from -20 to +30 degrees C. The relative survival of the stored cells was rapidly estimated by flow cytometry. After 4 weeks incubation at 4 and 10 degrees C, 75-90% of the cells were viable, with no significant differences between the various formulations. Supplementing the storage buffer with lactose or trehalose increased the survival after longer incubations (8 and 12 weeks) at all temperatures (-20 to 30 degrees C). Trehalose was the most effective protectant at 20 and 30 degrees C (>20% viable cells after 12 weeks at 20 degrees C). The biocontrol activity was maintained after formulation and prolonged storage of P. anomala. CONCLUSIONS: The storage potential of liquid formulated P. anomala cells can be increased by supplementation with lactose or trehalose. The combination of a custom made incubation chamber and flow cytometry was suitable to evaluate stability of P. anomala formulations. SIGNIFICANCE AND IMPACT OF THE STUDY: Liquid formulated P. anomala have a long shelf life. The developed test system can be used to study different formulations of other biocontrol agents.     

State transitions and physicochemical aspects of cryoprotection and stabilization in freeze-drying of Lactobacillus rhamnosus GG (LGG)

Aims: The frozen and dehydrated state transitions of lactose and trehalose were determined and studied as factors affecting the stability of probiotic bacteria to understand physicochemical aspects of protection against freezing and dehydration of probiotic cultures. Methods and Results: Lactobacillus rhamnosus GG was frozen (–22 or –43°C), freeze‐dried and stored under controlled water vapour pressure (0%, 11%, 23% and 33% relative vapour pressure) conditions. Lactose, trehalose and their mixture (1 : 1) were used as protective media. These systems were confirmed to exhibit relatively similar state transition and water plasticization behaviour in freeze‐concentrated and dehydrated states as determined by differential scanning calorimetry. Ice formation and dehydrated materials were studied using cold‐stage microscopy and scanning electron microscopy. Trehalose and lactose–trehalose gave the most effective protection of cell viability as observed from colony forming units after freezing, dehydration and storage. Enhanced cell viability was observed when the freezing temperature was ?43°C. Conclusions: State transitions of protective media affect ice formation and cell viability in freeze‐drying and storage. Formation of a maximally freeze‐concentrated matrix with entrapped microbial cells is essential in freezing prior to freeze‐drying. Freeze‐drying must retain a solid amorphous state of protectant matrices. Freeze‐dried matrices contain cells entrapped in the protective matrices in the freezing process. The retention of viability during storage seems to be controlled by water plasticization of the protectant matrix and possibly interactions of water with the dehydrated cells. Highest cell viability was obtained in glassy protective media. Significance and Impact of the Study: This study shows that physicochemical properties of protective media affect the stability of dehydrated cultures. Trehalose and lactose may be used in combination, which is particularly important for the stabilization of probiotic bacteria in dairy systems.     

Effect of culture age, protectants, and initial cell concentration on viability of freeze-dried cells of Metschnikowia pulcherrima

The effect of freeze-drying using different lyoprotectants at different concentrations on the viability and biocontrol efficacy of Metschnikowia pulcherrima was evaluated. The effects of initial yeast cell concentration and culture age on viability were also considered. Yeast cells grown for 36 h were more resistant to freeze-drying than were 48 h cells. An initial concentration of 10? cells·mL?1 favoured the highest survival after freeze-drying. When maltose (25%, m/v) was used as protectant, a high cell viability was obtained (64.2%). Cells maintained a high viability after 6 months of storage at 4 °C. The biocontrol efficacy of freeze-dried cells was similar to the activity of fresh cells on 'Gala' apples and was slightly lower on 'Golden Delicious' apples. After optimizing freeze-drying conditions, the viability of M. pulcherrima cells was similar to that obtained in other studies. The results constitute a first step towards the commercial development of M. pulcherrima as a biocontrol agent.     

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.     

Enhancing viability of two biocontrol yeasts in liquid formulation by applying sugar protectant combined with antioxidant

Survivals of Cryptococcus laurentii and Pichia membranaefaciens in liquid formulations with sugar protectants (trehalose and galactose) and L-ascorbic acid (Vc) were investigated during storage at 4°C and 25°C. When galactose or trehalose was used alone as protectant, C. laurentii maintained relatively high viability in potassium phosphate buffer. Addition of Vc to trehalose improved its protective effect. P. membranaefaciens maintained viability >60% after 90 days at 4°C when 5% galactose served as a protectant, and its combination with Vc was the most effective at maintaining viability. Moreover, liquid formulation kept higher viability of the two yeasts at 4°C than at 25°C. Biocontrol efficiency of the two yeasts was maintained after formulation and storage. The results indicate that trehalose is considered as a suitable protectant for liquid formulation of C. laurentii, while galactose is better for P. membranaefaciens. Combining Vc with the sugars improves the protective efficiency.     

Optimisation and comparison of liquid and dry formulations of the biocontrol yeast <Emphasis Type="Italic">Pichia anomala</Emphasis> J121

The biocontrol yeast Pichia anomala J121 can effectively reduce mould growth on moist cereal grains during airtight storage. Practical use of microorganisms requires formulated products that meet a number of criteria. In this study we compared different formulations of P. anomala. The best way to formulate P. anomala was freeze-drying. The initial viability was as high as 80%, with trehalose previously added to the yeast. Freeze-dried products could be stored at temperatures as high as 30 °C for a year, with only a minor decrease in viability. Vacuum-drying also resulted in products with high storage potential, but the products were not as easily rehydrated as freeze-dried samples. Upon desiccating the cells using fluidised-bed drying or as liquid formulations, a storage temperature of 10 °C was required to maintain viability. Dependent on the type of formulation, harvesting of cells at different nutritional stresses affected the initial viabilities, e.g. the initial viability for fluidised-bed-dried cells was higher when the culture was fed with excess glucose, but for freeze-drying it was superior when cells were harvested after depletion of carbon. Using micro-silos we found that the biocontrol activity remained intact after drying, storage and rehydration for all formulations.     

Physiological manipulation and formulation of the biocontrol yeast Pichia anomala for control of Penicillium verrucosum and ochratoxin A contamination of moist grain

The major hurdle in the production of commercial biocontrol agents (BCAs) has been the lack of production of appropriate formulations. Of particular importance is the conservation of viability and ecological competence after application. With this in mind studies were conducted to develop formulations of P. anomala which would have these attributes. Cells were grown in molasses-based medium modified with proline to different water availability levels (0.98 and 0.96) which significantly increased (up to 50%) the content of trehalose and arabitol in the yeast cells during liquid broth fermentation. The use of isotonic solutions for harvesting the yeast cells further increased the endogenous content of these compatible solutes as well as glycerol. Fluidised bed drying of cells at 30–80°C was carried out for 10 and 20 min and showed that viability was significantly decreased at 70–80°C. A temperature of 50°C for 20 min was found to be best for viability (70%) and moisture content of <10%. Several additives for conservation of viability showed that cotton seed flour+skimmed milk was the best treatment when dried at 50°C. The biocontrol efficacy of formulated P. anomala cells was tested in laboratory scale studies and this showed that they inhibited growth of Penicillium verrucosum and reduce ochratoxin A production in moist wheat grain under some combinations of water availability. Physiologically modified formulated yeast cells with increased levels of trehalose and arabitol gave similar efficacy as fresh cells. This suggests that ecophysiological manipulation of such BCAs can result in improved ecological competence of such formulations and effective biocontrol.     

Enhancement of the biocontrol agent Candida oleophila (strain O) survival and control efficiency under extreme conditions of water activity and relative humidity

The objective of this work is to evaluate the ability of some additive substances in protecting the biocontrol agent Candida oleophila (strain O) against the adverse effects of environmental factors, such as water activity (a_w, 0.93 and 0.98) and relative humidity (75% and 98%). The protection obtained with various protectant substances, skimmed milk (SM), peptone, maltose, sucrose, sorbitol, lactose and polyethylene glycol was assayed under in vitro and in vivo conditions. The yeast cells with the highest level of protecting agents (1%) had higher viability than those with low protectant levels (0.1% and 0.5%). SM, sucrose and sorbitol improved significantly the C. oleophila survival on apple fruit surface by 80.8%, 42.26% and 37.27% and gave a significant protection (from 96% to 100%) against Penicillium expansum under dried conditions. The highest strain O density and efficacy was obtained with SM. Under experimental conditions reflecting practical conditions, SM applied in combination with the strain O resulted in improved biocontrol efficacy by 74.65%. Therefore, SM could be used as material substrate with the best sugar protectants during the formulation process of this antagonistic yeast for eventual pre-harvest application.     

Combined effects of endo- and exogenous trehalose on stress tolerance and biocontrol efficacy of two antagonistic yeasts

Effects of endo- and exogenous trehalose on viability of two antagonistic yeasts, Cryptococcus laurentii (Kuffer.) Skinner and Rhodotorula glutinis (Fresen.) Harrison, were investigated after being treated with rapid-freezing, slow-freezing and freeze-drying, respectively. The accumulation of intracellular trehalose in the two yeasts was induced by culturing the yeast cells in trehalose-containing medium, which significantly enhanced viabilities of both yeasts in the slow-freezing test. Trehalose, as an exogenous protectant, at the concentration of 5% or 10% could markedly increase survivals of the two yeasts when subjected to freeze-drying. When combined with exogenous trehalose as a protective substance, the yeasts containing high intracellular trehalose level showed higher viabilities as compared to those containing low levels under both freezing and freeze-drying stresses. The highest survival of C. laurentii and R. glutinis were 90% and 97% after freeze-drying, respectively, compared to 63% and 28% for the yeasts with lower intracellular trehalose levels. These results may be due to the fact that a combined effect occurred between endo- and exogenous trehalose of yeast cells. The combined effect on C. laurentii and R. glutinis also resulted in the highest level of biocontrol efficacy against blue mold in apple fruit caused by Penicillium expansum Link, and reduced the disease indexes to 45 and 56, respectively, compared to 94 and 81 in the untreated control. Meanwhile, the combination of endo- and exogenous trehalose significantly increased population of both yeasts in apple wounds, especially at the first 48 h after inoculation, which might explain the reason of the improvement in biocontrol effects of the two yeasts.     

Enhancement of the biocontrol agent Candida oleophila (strain O) survival and control efficiency under extreme conditions of water activity and relative humidity

The objective of this work is to evaluate the ability of some additive substances in protecting the biocontrol agent Candida oleophila (strain O) against the adverse effects of environmental factors, such as water activity (a_w, 0.93 and 0.98) and relative humidity (75% and 98%). The protection obtained with various protectant substances, skimmed milk (SM), peptone, maltose, sucrose, sorbitol, lactose and polyethylene glycol was assayed under in vitro and in vivo conditions. The yeast cells with the highest level of protecting agents (1%) had higher viability than those with low protectant levels (0.1% and 0.5%). SM, sucrose and sorbitol improved significantly the C. oleophila survival on apple fruit surface by 80.8%, 42.26% and 37.27% and gave a significant protection (from 96% to 100%) against Penicillium expansum under dried conditions. The highest strain O density and efficacy was obtained with SM. Under experimental conditions reflecting practical conditions, SM applied in combination with the strain O resulted in improved biocontrol efficacy by 74.65%. Therefore, SM could be used as material substrate with the best sugar protectants during the formulation process of this antagonistic yeast for eventual pre-harvest application.     

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.     

Combination of antagonistic yeasts with two food additives for control of brown rot caused by Monilinia fructicola on sweet cherry fruit

AIMS: To evaluate beneficial effect of two food additives, ammonium molybdate (NH4-Mo) and sodium bicarbonate (NaBi), on antagonistic yeasts for control of brown rot caused by Monilinia fructicola in sweet cherry fruit under various storage conditions. The mechanisms of action by which food additives enhance the efficacy of antagonistic yeasts were also evaluated. METHODS AND RESULTS: Biocontrol activity of Pichia membranefaciens and Cryptococcus laurentii against brown rot in sweet cherry fruit was improved by addition of 5 mmol l(-1) NH4-Mo or 2% NaBi when stored in air at 20 and 0 degrees C, and in controlled atmosphere (CA) storage with 10% O2 + 10% CO2 at 0 degrees C. Population dynamics of P. membranefaciens in the wounds of fruit were inhibited by NH4-Mo at 20 degrees C after 1 day of incubation and growth of C. laurentii was inhibited by NH4-Mo at 0 degrees C in CA storage after 60 days. In contrast, NaBi did not significantly influence growth of the two yeasts in fruit wounds under various storage conditions except that the growth of P. membranefaciens was stimulated after storage for 45 days at 0 degrees C in CA storage. When used alone, the two additives showed effective control of brown rot in sweet cherry fruit and the efficacy was closely correlated with the concentrations used. The result of in vitro indicated that growth of M. fructicola was significantly inhibited by NH4-Mo and NaBi. CONCLUSION: Application of additives improved biocontrol of brown rot on sweet cherry fruit under various storage conditions. It is postulated that the enhancement of disease control is directly because of the inhibitory effects of additives on pathogen growth, and indirectly because of the relatively little influence of additives on the growth of antagonistic yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained in this study suggest that an integration of NH4-Mo or NaBi with biocontrol agents has great potential in commercial management of postharvest diseases of fruit.     

Action of trehalose on the preservation of Lactobacillus delbrueckii ssp. bulgaricus by heat and osmotic dehydration

AIM: This work determines the efficiency of trehalose on the preservation by heat or osmotic drying of a strain of Lactobacillus delbrueckii ssp. bulgaricus. Cell recovery at different trehalose concentrations during drying correlated with the surface properties and osmotic response of cells after rehydration. METHODS AND RESULTS: Bacteria were dried in the presence of glycerol, trehalose, sucrose at 70 degrees C and at 20 degrees C. Trehalose attenuates the loss of viability at 0.25 m. At this concentration, the osmotic response and zeta potential of the bacteria were comparable with the nondried ones. CONCLUSIONS: Trehalose diminishes significantly the damage produced by dehydration both when the bacteria are dried by heating or subjected to osmotic dehydration. This effect appears related to the preservation of the permeability to water and the surface potential of the bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Dehydration occurring during heating or during osmosis appears to have similar effects. As dehydration-induced damage is in correlation with osmotic response recovery and is hindered or buffered by the presence of trehalose, it may be related to water eliminated from biological structures involved in water permeation.     

Impact of mild heat treatments on induction of thermotolerance in the biocontrol yeast Candida sake CPA-1 and viability after spray-drying

Aims: The objective of this study was to examine the induction of thermotolerance in the biocontrol agent Candida sake CPA‐1 cells by mild heat treatments to enhanced survival of formulations using spray‐drying. The possible role of heat‐shock proteins (HSPs) biosynthesis in induced thermotolerance and the role of sugars and sugar alcohols were also determined. Methods and Results: Studies were conducted on C. sake cells grown in molasses medium and exposed to mild temperatures of 30 and 33°C during mid‐ (16 h), late‐exponential (24 h), early‐ (30 h) and mid‐stationary (36 h) growth phases. The effect on viability was determined both before and after spray‐drying. Cycloheximide and chloramphenicol were used to examine the role of HSPs and HPLC was used to analyse the accumulation of sugar and sugar alcohols. The results indicate that both temperatures induced thermotolerance in cells of C. sake. Mild heat‐adapted cells at 33°C in the early‐ or mid‐stationary phases had survival values after spray‐drying significantly higher (P ≤ 0·05) than nonadapted cells. However, viabilities were not high enough to be considered for commercial use with values up to 17%. HSPs were not implicated in thermotolerance acquired by mild heat‐adapted cells as similar viabilities were obtained in the presence of protein inhibitors. Little change was observed in sugar and sugar alcohols with an increase in glucose and arabitol in some treatments. Conclusions: This study suggests that it is possible to induce thermotolerance in biocontrol yeasts such as C. sake. However, this does not improve survival of cells exposed to spray‐drying sufficiently to consider this a suitable formulation method for this biocontrol agent. HSPs, sugars and sugar polyols were not directly responsible for induced thermotolerance in yeast cells. Significance and Impact of the Study: This type of information can be effectively applied to improve the viability of cells in the process of formulation.     

三种拮抗酵母菌对苹果采后青霉病的抑制效果

从苹果果实上分离获得的50余种酵母菌中筛选出了能够有效地抑制苹果青霉病(Penicilium expansum Link)的丝孢酵母(Trichosporon pullulans (Lindner.) Diddens and Lodder)、罗伦隐球酵母(Cryptococcus laurentii (Kuffer.) Skinner)和粘红酵母(Rhodotorula glutinis (Fresen.) F. C. Harrison).其中,抑病效果最好的T. pullulans 是一种用于采后果实生物防治的新型拮抗菌.研究了这三种拮抗菌不同浓度处理和外加营养物质以及与钙配合使用对苹果青霉病的抑病效果.实验结果表明:酵母菌浓度越高,抑病作用越强;外源营养物质的加入削弱了酵母菌的拮抗效果;在C. laurentii的细胞悬浮液中加入0.18 mol/L 的CaCl2能显著提高其抑病能力,但增加CaCl2 对T. pullulans 和R. glutinis 的抑病效果却没有明显作用.     

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.     

Effect of air-blast drying and the presence of protectants on the viability of yeast entrapped in calcium alginate beads with an aim to improve the survival rate

Five yeast strains, Saccharomyces cerevisiae D8, M12, and S13; Hanseniaspora uvarum S6; and Issatchenkia orientalis KMBL5774, isolated from Korean grapes, were entrapped in Ca-alginate beads, which are non-toxic, simple to use, and economical. Ca-alginate beads containing yeast cells were soaked in protective solutions, such as skim milk, saccharides, polyols, and nitrogen compounds, before air-blast drying to improve the yeast survival rate and storage ability. The results showed that both entrapment in Ca-alginate beads and soaking in protective agents favorably affected the survival of all strains. The microenvironment formed by the beads and protective agents can protect the yeast cells from harsh environmental conditions, such as low water (below 10 %). All the yeast strains entrapped in Ca-alginate beads showed greater than 80 % survival and less than 11 % water content after air-blast drying at 37 °C for 5 h. In addition, air-blast dried cells of S. cerevisiae D8, M12, S13; H. uvarum S6; and I. orientalis KMBL5774 entrapped in 2 % Ca-alginate beads and soaked in protective agents (10 % skim milk containing 10 % sucrose, 10 % raffinose, 10 % trehalose, 10 % trehalose, and 10 % glucose, respectively) after air-blast drying at 37 °C for 5 h showed 90, 87, 92, 90, and 87 % viability, respectively. All dried entrapped yeast cells showed survival rates of at least 51 % after storage at 4 °C for 3 months.