Survival of spray-dried <Emphasis Type="Italic">Lactobacillus kefir</Emphasis> is affected by different protectants and storage conditions

Survival of two Lactobacillus kefir strains after spray drying in reconstituted skim milk with or without the addition of 12.5 g monosodium glutamate/l, 20 g sucrose/l, or 20 g fructo-oligosaccharides (FOS)/l and during subsequent storage under different conditions of temperature (20 and 30°C) and relative humidity (RH) (0, 11 and 23%) was evaluated. After being dried, L. kefir 8321 and L. kefir 8348 had a decrease in viability of 0.29 and 0.70 log cfu/ml respectively, while the addition of different protectants improved the survival of both strains significantly. During storage, bacterial survival was significantly higher under lower conditions of RH (0–11%), and monosodium glutamate and FOS proved to be the best protectants.     

Freezing and desiccation tolerance in the moss Physcomitrella patens: an in situ Fourier transform infrared spectroscopic study

In situ Fourier transform infrared spectroscopy (FTIR) was used in order to obtain more insights in the underlying protective mechanisms upon freezing and drying of ABA-treated tissues of the moss Physcomitrella patens. The effects of different treatments on the membrane phase behaviour, glassy state, and overall protein secondary structure were studied. We found that growth on ABA resulted in the accumulation of sucrose: up to 22% of the tissue on a dry weight basis, compared to only 3.7% in non-ABA-treated tissues. Sucrose functions as a protectant during freezing and drying, but accumulation of sucrose alone is not sufficient for survival. ABA-treated tissue survives a freeze-thaw cycle down to -80 degrees C only after addition of an additional cryoprotectant (DMSO). Survival correlates with preservation of membrane phase behaviour. We found that ABA-treated P. patens can survive slow but not rapid drying down to water contents as low as 0.02 g H(2)O per g DW. Rapidly and slowly dried ABA-treated tissues were found to have similar sugar compositions and glass transition temperatures. The average strength of hydrogen bonding in the cytoplasmic glassy matrix, however, was found to be increased upon slow drying. In addition, slowly dried tissues were found to have a higher relative proportion of alpha-helical structures compared to rapidly dried tissues.     

Freezing and desiccation tolerance in the moss Physcomitrella patens: An in situ Fourier transform infrared spectroscopic study

In situ Fourier transform infrared spectroscopy (FTIR) was used in order to obtain more insights in the underlying protective mechanisms upon freezing and drying of ABA-treated tissues of the moss Physcomitrella patens. The effects of different treatments on the membrane phase behaviour, glassy state, and overall protein secondary structure were studied. We found that growth on ABA resulted in the accumulation of sucrose: up to 22% of the tissue on a dry weight basis, compared to only 3.7% in non-ABA-treated tissues. Sucrose functions as a protectant during freezing and drying, but accumulation of sucrose alone is not sufficient for survival. ABA-treated tissue survives a freeze–thaw cycle down to −80 °C only after addition of an additional cryoprotectant (DMSO). Survival correlates with preservation of membrane phase behaviour. We found that ABA-treated P. patens can survive slow but not rapid drying down to water contents as low as 0.02 g H₂O per g DW. Rapidly and slowly dried ABA-treated tissues were found to have similar sugar compositions and glass transition temperatures. The average strength of hydrogen bonding in the cytoplasmic glassy matrix, however, was found to be increased upon slow drying. In addition, slowly dried tissues were found to have a higher relative proportion of α-helical structures compared to rapidly dried tissues.     

海藻糖对乳酸菌的抗逆保护研究

研究了在冷冻干燥、高温及冻融等胁迫条件下,海藻糖对嗜热链球菌（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｔｈｅｒ－ ｍｏｐｈｉｌｌｕｓ）和植物乳杆菌（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍ）菌体细胞的保护作用。结果表明在冷冻干燥过程 中,海藻糖保护的细胞存活率分别达７５％和３３％,而对照分别为１９％和ｌ％;用９０℃高温处理干燥 状态和溶液状态的嗜热链球菌,证明海藻糖能明显提高细胞的耐热性;用冻融法反复处理嗜热链球 菌４次和８次,加海藻糖保护的细胞存活率显著高于对照。在扫描电镜下观察这     

Influence of lyophilization, fluidized bed drying, addition of protectants, and storage on the viability of lactic acid bacteria

Aims:  The present study focuses on the impact of two different drying technologies and the influence of protectants on process survival and storage stability of the two lactic acid bacterial strains Enterococcus faecium and Lactobacillus plantarum .
Methods and Results:  After incubation with the protectants glucose, sucrose, trehalose, and maltodextrin the concentrated bacterial suspensions were subjected to fluidized bed drying and lyophilization and subsequently stored at 4, 22, and 35°C for half a year. Lactobacillus plantarum turned out to be more sensitive to both drying methods than Ent. faecium . Without the addition of a protectant cells of both strains suffered higher losses during fluidized bed drying. Elevated storage temperatures correlate with a higher decline of viable bacterial cells.
Conclusions:  Although survival rates varied between the strains, the nonreducing disaccharides revealed overall best protection for both investigated lactic acid bacteria during processing and storage. The addition of protective carbohydrates can prevent the decline in viability during fluidized bed drying.
Significance and Impact of the Study:  The influence of protectants proved to be species specific and therefore needs to be determined on a case-to-case basis. Survival rates, duration, and energy consumption appear to be the crucial parameters to evaluate the economy of production processes for industrial starter cultures.     

Influence of oligosaccharides on the viability and membrane properties of Lactobacillus reuteri TMW1.106 during freeze-drying

Freeze-drying is a process commonly used in starter culture preparation. To improve the survival rate of bacteria during the process, cryoprotectives are usually added before freezing. This study investigated the influence of the addition of sucrose, fructo-oligosaccharides (FOS), inulin and skim milk on the viability and membrane integrity of Lactobacillus reuteri TMW1.106 during freezing, freeze-drying and storage. The effect of drying adjuncts on survival was correlated to their interaction with bacterial membrane by determination of the parameters membrane fluidity and membrane lateral pressure. Sucrose, FOS and skim milk significantly enhanced survival of exponential-phase cells of L. reuteri during freeze-drying. Cellular viability during storage of exponential-phase cells remained highest for cells dried in the presence of skim milk and inulin. Membranes of these cells were completely permeabilized after freeze-drying. The application of FOS significantly improved survival of stationary phase cells of L. reuteri TMW1.106 after freeze-drying and storage. This increased viability of L. reuteri TMW1.106 in the presence of FOS correlated to improved membrane integrity. Fructo-oligosaccharides and fructans, but not gluco-oligosaccharides interacted with membrane vesicles prepared from L. reuteri TMW1.106 as indicated by increased membrane lateral pressure in the presence of FOS and fructans. Increased membrane integrity of stationary phase L. reuteri TMW1.106 was attributed to direct interactions between FOS and the membrane which leads to increased membrane fluidity and thus improved stability of the membrane during and rehydration.     

Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying.

The microorganisms Escherichia coli DH5 alpha and Bacillus thuringiensis HD-1 show an increased tolerance to freeze-drying when dried in the presence of the disaccharides trehalose and sucrose. When the bacteria were dried with 100 mM trehalose, 70% of the E. coli and 57% of the B. thuringiensis organisms survived, compared with 56 and 44%, respectively, when they were dried with sucrose. Only 8% of the E. coli and 14% of the B. thuringiensis organisms survived drying without the sugars. Fourier transform infrared spectroscopy was used to investigate the role of membrane phase transitions in the survival of the organisms during drying and rehydration. Both E. coli and B. thuringiensis showed an increase of 30 to 40 degrees C in the temperature of their phospholipid phase transition when dried without the sugars, while phase transition temperatures of those dried with the sugars remained near those of the hydrated cells. A Fourier transform infrared spectroscopy microscope made it possible to investigate the effects of drying on the protein structure in the intact cells. The amide II peak shifts from 1,543 cm-1 in the hydrated cells to about 1,533 cm-1 in the cells dried without sugar. There is no shift in the amide II peak when the cells are dried with trehalose or sucrose. We attribute the increased survival to the sugars' ability to lower the membrane phase transition temperature and to protect protein structure in the dry state.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Dehydration-induced conformational transitions in proteins and their inhibition by stabilizers.

Dehydration of proteins results in significant, measurable conformational changes as observed using Fourier-transform infrared spectroscopy and resolution-enhancement techniques. For several proteins these conformational changes are at least partially irreversible, since, upon rehydration, denaturation and aggregation are observed. The presence of certain stabilizers inhibited these dehydration-induced transitions; the native structure was preserved in the dried state and upon reconstitution. Conformational transitions were also observed in a model polypeptide, poly-L-lysine, after lyophilization and were inhibited with the addition of stabilizing cosolutes. The ability of a particular additive to preserve the aqueous structure of dehydrated proteins and poly-L-lysine upon dehydration correlates directly with its ability to preserve the activity of lactate dehydrogenase, a labile enzyme, during drying.     

Dehydration-induced conformational changes of poly-l-lysine as influenced by drying rate and carbohydrates

The conformation of hydrated and air-dried poly-l-lysine in thin films was studied using Fourier transform IR spectroscopy in the amide-I region. Hydrated poly-l-lysine has a random coil conformation. Upon slow drying of small droplets of the polypeptide solution over a period of several hours, an extended β-sheet conformation is adopted. This conformational transition can be prevented by fast air-drying within 2–3 min. Slow air-drying in the presence of sucrose also preserves the aqueous conformation and results in the formation of a glassy state. Comparison of shifts of the OH band with temperature indicates that sucrose/poly-l-lysine mixtures form a molecularly more densely packed glassy matrix, having a higher glass transition temperature (T_g), than sucrose alone. Whether direct interaction of sugar and polypeptide or glass formation is involved in the stabilization during slow air-drying was studied by drying in the presence of glucose or dextran. Compared with dextran (and sucrose to a lesser extent), glucose gives superior protection. Dried glucose has the lowest T_g and the best interacting properties. We conclude that either immobilization by fast air-drying or sufficient interaction with a protectant through hydrogen bonding (slow drying) plays the leading role in the preservation of the aqueous protein structure.     

A Fourier-transform infrared spectroscopy study of sugar glasses

Fourier-transform infrared spectroscopy (FTIR) was used to study the hydrogen-bonding interactions that take place in vitrified carbohydrates of different chain lengths. The band position of the OH stretching band (vOH) and the shift in band position as a function of temperature were determined from the FTIR spectra as indicators for the length and strength of intermolecular hydrogen bonds, respectively. Differential scanning calorimetry (DSC) was used to corroborate the FTIR studies and to measure the change in heat capacity (delta C(p)) that is associated with the glass transition. We found that with increasing T(g), the band position of vOH increases, the wavenumber-temperature coefficient of vOH in the glassy state, WTC(g), increases, whereas (delta C(p) decreases. The positive correlation that was found between vOH and the glass transition temperature, T(g), indicates that the length of the hydrogen bonds increases with increasing T(g). The increase in WTC(g) with increasing T(g) indicates that the average strength of hydrogen bonding decreases with increasing T(g). This implies that oligo- and polysaccharides (high T(g)) have a greater degree of freedom to rearrange hydrogen bonds during temperature changes than monosaccharides (low T(g)). Interestingly, WTC(g) and delta C(p) showed a negative linear correlation, indicating that the change in heat capacity during the glass transition is associated with the strength of the hydrogen-bonding network in the glassy state. Furthermore, we report that introduction of poly-L-lysine in glassy sugar matrices decreases the average length of hydrogen bonds, irrespective of the size of the carbohydrate. Palmitoyl-oleoyl-phosphatidylcholine (POPC) vesicles were found to only interact with small sugars and not with dextran.     

Production and survival during storage of spray-dried Bradyrhizobium japonicum cell concentrates

Production of Bradyrhizobium japonicum cell concentrates by spray-drying in skim milk plus sucrose medium and the feasability of storing dried inocula over long periods were investigated. Storage of spray-dried cells under mild vacuum was equivalent to storage under nitrogen. Oxygen and ambient temperature were found detrimental for survival of dried cells. High initial cell concentration and storage under low relative humidities (< 23% RH) at 4°C increased the longevity of the inocula (> 10⁹ cfu g^-1 during at least a 25 week storage period) without altering the symbiotic properties of B. japonicum.     

Assessing the viability of three Lactobacillus bacterial species protected in the cryoprotectants containing whey and maltodextrin during freeze-drying process

Freeze-drying of bacteria associates with different stresses such as osmotic pressure, temperature and oxidation, and decreases bacterial viability, which seem to reduce by applying cryoprotectants. The present study evaluated the effect of four cryoprotectants on decreasing the stress caused by freeze-drying process among three Lactobacillus species. Additionally, it highlighted the use of whey and maltodextrin as a substitute for peptone and sucrose in cryoprotectants respectively. The viability of lactobacilli was measured after freeze-drying, 1 month of storage at 25 and 4°C. Based on the results, the viability rate of bacteria in protectants during freeze-drying stage was dependent on their strains. The best viability of Lacticaseibacillus rhamnosus GG and Ligilactobacillus salivarius 20687 was, respectively, observed in the protectants containing sucrose and whey, while Lactiplantibacillus plantarum NRRL B-14768 viability was equal in all protectants. The number of live bacteria reduced significantly by storing bacteria for 1 month at 25°C compared to the 4°C storage. During the storage period, the viability of L. salivarius improved by adding sucrose in protectant. Due to the positive effect of whey and sucrose in the drying and storage stage, on bacterial viability, the protectant consisting of whey and sucrose is suggested for all of the species under study.     

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.     

Effect of drying on conidial viability of Penicillium frequentans, a biological control agent against peach brown rot disease caused by Moniliniaspp

The effects of drying methods (freeze-, spray-, and fluid bed-drying) on viability of Penicillium frequentans conidia were compared. Viability, estimated by germination of fluid bed- and freeze-dried conidia, was similar to that of fresh conidia. Skimmed milk alone, or in combination with other protectants, was added to conidia before freeze-drying. After the freeze-drying process, all protectants used, except glycerol improved conidial viability. Freeze-dried P. frequentans conidia did not maintain viability after 30 days of storage at room temperature, while conidia dried by fluid bed-drying showed 28% viability following 180 days after drying. This work also demonstrated a relationship between conidial viability after 1 year of storage at room temperature, moisture content after fluid bed-drying and initial weight of sample. Conidial moisture contents must be reduced to 5-15% for optimal storage at room temperature. P. frequentans conidia dried by fluid bed-drying were as effective as fresh conidia in controlling brown rot of peaches.     

Reversible self-polymerizing high T(g) lyoprotectants

One mode of action of protectants in the storage of biological materials is by promoting the formation of a vitrified state on cooling or drying. In the case of preservation by drying, the glassy material comprises a low water content mixture of protectant and organic material. The protectant must on drying form a glassy state of glass transition temperature (T(g)) above the desired storage temperature. However, in some applications it must also be easily transported through cell membranes and this restricts the choice to a relatively limited number of small molecules, which typically exhibit very low glass transition temperatures. In this work we describe a self-polymerizing protectant comprising an inorganic salt and a small hydroxy functional molecule such as glycerol. This forms co-ordinate polymer chains of high T(g) on drying but rapidly depolymerizes into the original components on rehydration. The polymerization process is general for polyhydroxy compounds including glucose and related compounds.     

Kinetics of the alkaline phosphatase catalyzed hydrolysis of disodium p-nitrophenyl phosphate in frozen model systems

The alkaline phosphatase catalyzed hydrolysis of disodium-p-nitrophenyl phosphate was studied in four model systems comprising sucrose, maltodextrin, carboxymethylcellulose (CMC), and CMC-lactose in a temperature range of -28 to 20 degrees C. In the maltodextrin and CMC-lactose model systems, the reaction rate decreased to a very low value as the glass transition temperature was approached. In the CMC and CMC-lactose systems with low initial solute concentration, as a consequence of freeze-concentration, a rate maximum around the initial freezing temperature was observed. The Arrhenius equation described the temperature dependence of the reaction rate both in the liquid and the glassy states in all systems studied, while a slightly curved Arrhenius plot was observed in the "rubbery" state of the CMC and CMC-lactose systems. The WLF equation with system-dependent coefficients described the kinetics in the rubbery state of all the model systems except sucrose, excluding the short temperature range where reaction rate enhancement with decreasing temperature was observed.     

Preservation of probiotic strains isolated from kefir by spray drying

Aims:  This work aims to investigate the survival of Lactobacillus kefir CIDCA 8348, Lactobacillus plantarum CIDCA 83114 and Saccharomyces lipolytica CIDCA 812, all isolated from kefir, during spray drying and subsequent storage.
Methods and Results:  Micro-organisms were grown in De Man, Rogosa, Sharpe (MRS) or yeast medium (YM) medium and harvested in the stationary phase of growth. The thermotolerance in skim milk ( D and Z values), the survival of spray drying at different outlet air temperatures and subsequent storage in different conditions during 150 days were studied. The resistance to the heat treatments was higher in Lact. plantarum compared to Lact. kefir and S. lipolytica . The three micro-organisms studied varied considerably in their ability to survive to spray drying processes . Lactobacillus plantarum showed the highest survival rate for all the tested outlet air temperatures and also to the further storage in the dried state. The survival rates of Lact. kefir and S. lipolytica through drying and subsequent storage in the dried state decreased when the drying outlet air temperatures increased.
Conclusions:  Spray drying is a suitable method to preserve micro-organisms isolated from kefir grains. A high proportion of cells were still viable after 80 days of storage at refrigerated temperatures
Significance and Impact of Study:  It is the first report about spray-dried probiotic strains isolated from kefir grain and contributes to the knowledge about these micro-organisms for their future application in novel dehydrated products.     

Kinetics of the pectin methylesterase catalyzed de-esterification of pectin in frozen food model systems

The pectin methylesterase (PME) catalyzed de-esterification of pectin was studied in four frozen food model systems based on sucrose, fructose, maltodextrin, and carboxymethylcellulose (CMC) in a temperature range from -24 to 20 degrees C, with the aim of elucidating the applicability of the theory of "food polymer science" on the kinetics. The rate substantially decreased around the glass transition temperature in the case of CMC, while very low rates were observed far above the glass transition temperature in the case of maltodextrin, fructose, and sucrose model systems. In general, the kinetics of this reaction was found to be influenced more by factors such as the characteristics of the component solutes, freeze concentration, the possible viscosity enhancement due to a particular combination of solutes, and the molecular size of the substrate molecule rather than the glass transition process. The Arrhenius equation described the temperature dependence of kinetics both in the liquid state of all the systems studied (r(2) > or = 0.97) and the glassy state of CMC (r(2) = 0.95). A clear break in the Arrhenius plot was observed as the temperature decreased to subfreezing temperatures. The Arrhenius equation could describe the kinetics reasonably well in the rubbery state for fructose and sucrose model systems (r(2) > 0.992). In the case of maltodextrin and CMC, the Arrhenius plots showed a slight curvature followed by a break at the glass transition temperature for CMC. The WLF equation with system-dependent coefficients better described the kinetics in the rubbery state of the CMC and part of the maltodextrin system. A linear relationship between the logarithm of the rate and T - Tg' described the kinetics in the sucrose as well as fructose model systems (r(2) = 0.9928 and 0.993, respectively).     

Studies on Destabilization of Caseinate Complex during Frozen Storage of Milk

Unpasteurized skim milk was storaged in a frozen state at ?7°C or ?20°C for up to several months. There was no increase of non casein and non protein nitrogens, but a slight increase of free tyrosine and a slight decrease of alkaline phosphatase activity were detected when storage period was prolonged. Destabilization occurred solely in caseinate complex, but non micellar casein appeared to be stable.

The contents of calcium and inorganic phosphate in the caseinate complex separated by ultracentrifugation were increased appreciably after frozen storage. The viscosity characteristics of frozen storaged skim milk was also investigated.

Caseinate complex was ultracentrifugally separated from skim milk before and after frozen storage, and then lyophilized. Skim milk itself was also lyophilized before and after frozen storage. Dispersibility was examined on the reconstituted suspension of the lyophilized samples.

The lyophilized sample from frozen storaged milk was much less dispersible than the lyophilized control sample prepared before frozen storage. However, when lyophilized samples were once resolved with reagents such as urea and potassium oxalate and then dialyzed against fresh milk, stable micelle resulted in both samples prepared before and after frozen storage.

Some reduction of dispersibility occurred during lyophilization and subsequent storage in a dried state in the caseinate complex prepared before frozen storage. This reduction was small when skim milk was lyophilized and stored.