Yeast Cell Microcapsules as a Novel Carrier for Cholecalciferol Encapsulation: Development,Characterization and Release Properties

In this study Saccharomyces cerevisiae yeast cells was used as a novel vehicle for encapsulation of vitamin D₃. The effects of initial cholecalciferol concentration (100,000 and 500,000 IU/g yeast), yeast cell pretreatment (plasmolysis with NaCl) and drying method (spray or freeze drying) on microcapsules properties were investigated. It was found that the vitamin concentration and drying method had significant influence on encapsulation efficiency (EE) and size of yeast microcapsules. Furthermore, EE values were more increased by the plasmolysis treatment. The highest EE was obtained for plasmolysed and spray dried yeast cells prepared using initial cholecalciferol concentration of 2.5 mg per gram of yeast cells (76.10?±?6.92%). The values of mean particle size were 3.43–7.91 μm. The presence of cholecalciferol in yeast microcapsules was confirmed by X-ray diffraction (XRD) and Fourier transform-infrared (FT-IR) analyses. The in vitro cholecalciferol release from yeast microcapsules in phosphate buffer saline solution (PBS) followed a controlled release manner consistent with a Fickian diffusion mechanism. In addition, the release studies in simulated gastrointestinal tract showed sustained release of cholecalciferol in the stomach condition and significant release in intestinal medium.     

The Influence of Heat Processing and Mechanical Disintegration on Yeast for Single-Cell Protein

Yeast was processed by means of different technical drying procedures, heating in water suspension, and mechanical disintegration. The influence on the ultrastructure, the nutritive value and on the availability of the cell nitrogen-containing compounds to chemical extractants was studied. On micrographs no cell wall disrupture could be observed after any of the heat treatments. The internal cell structure was affected at the higher temperatures. After drum drying this structure was destroyed to a large extent. The heat treatments increased the nutritive value compared to unheated yeast cells but did not increase the availability of the cell content to chemical extractants. Mechanical disintegration increased both the nutritive value and the availability to chemical extractants. Heat processes and mechanical disintegration give high nutritive value to the yeast. Mechanical disintegration is advantageous when processing steps such as extraction with chemicals are necessary for obtaining specific protein products.     

Engineering factors in single-cell protein production. II. Spray drying and cell viability

One important economical method for producing singlecell protein is to spray dry the cultured cells. This study presents some preliminary data on the effects of spray drying on cell viability. Under conditions similar to those for the production of spray-dried milk, 4–5 log cycles destruction occurred. The results indicate that, the activation energy for thermal destruction of yeast was reduced from the normal heat treatment value of 84 kcal/°K mole to about 38 kcal/°K mole.     

Formulation and stabilisation of the biocontrol yeast Pichia anomala

The yeast Pichia anomala has antifungal activities and its potential in biocontrol and biopreservation has previously been demonstrated. To practically use an organism in such applications on a larger scale the microbe has to be formulated and stabilised. In this review we give an overview of our experience of formulating and stabilising P. anomala strain J121 in a wider perspective. The stabilisation techniques we have evaluated were liquid formulations, fluidised bed drying, lyophilisation (freeze-drying) and vacuum drying. With all methods tested it was possible to obtain yeast cells with shelf lives of at least a few months and in all cases the biocontrol activity was retained. Fluidised bed drying was dependent on the addition of cottonseed flour as a carrier during the drying process. In liquid formulations a sugar, preferentially trehalose, was a required additive. These two kinds of microbial stabilisation are easily performed and relatively inexpensive but in order to keep the cells viable the biomaterial has to be stored at cool temperatures. However, there is room for optimization, such as improving the growth conditions, or include preconditioning steps to enable the cells to produce more compatible solutes necessary to survive formulation, desiccation and storage. In contrast, lyophilisation and vacuum drying require a lot of energy and are thus expensive. On the other hand, the dried cells were mostly intact after one year of storage at 30°C. Inevitably, the choice of formulation and stabilisation techniques will be dependent also on the intended use.     

Influence of system and molecular parameters upon fractionation of intracellular proteins from Saccharomyces by aqueous two-phase partition.

The interaction of molecular characteristics of proteins with the physicochemical properties of PEG-phosphate aqueous two-phase systems has been studied. This has involved characterization of protein molecular weight, charge, and hydrophobicity and study of PEG molecular weight and concentration, phosphate concentration, and pH. System characterization has been conducted in the context of limited stage fractionation procedures for protein recovery from baker's yeast. Results are presented which show that the degree of purification achieved is dependent on macromolecular surface properties rather than system operating conditions. A simple conceptual model of partitioning in PEG-phosphate aqueous two-phase systems is presented which is applicable in the rational design of fractionation procedures and serves to limit the amount of empirical experimentation necessary for the establishment of practical operations.     

Spraydrying of yeast-lytic enzymes from Arthrobacter sp.

Summary Spray drying processes are widely used for large scale preservation of biological materials e.g milk, whey, yeast, egg white, etc. Nevertheless, there is an increasing tendency for drying of catalytic active proteins. The paper presents results from experiments which were carried out to test the applicability of spray drying for the preservation of several bacterial enzymes used for the lysis of yeast cell-walls (-1,3-glucanase, -mannanase, chitinase and amylase) and formed by an Arthrobacter species. Enzyme solutions were obtained by concentrating the cell free cultivation liquid by cross-flow ultrafiltration. Due to the low protein content of the liquid preparation, several substances were added as stabilizers or carriers (eg. sucrose, KCl, MgSO₄) and the effect was studied in further experiments. Spray drying was carried out at inlet air temperatures of 110 – 120°C and outlet air temperatures of 65 – 72°C. Best results were obtained by the addition of 10% KCl, the preparation having a crystalline consistency and a 60% yield in activity.     

Yeast methylotrophy and autophagy in a methanol-oscillating environment on growing Arabidopsis thaliana leaves

The yeast Candida boidinii capable of growth on methanol proliferates and survives on the leaves of Arabidopsis thaliana. The local methanol concentration at the phyllosphere of growing A. thaliana exhibited daily periodicity, and yeast cells responded by altering both the expression of methanol-inducible genes and peroxisome proliferation. Even under these dynamically changing environmental conditions, yeast cells proliferated 3 to 4 times in 11 days. Among the C1-metabolic enzymes, enzymes in the methanol assimilation pathway, but not formaldehyde dissimilation or anti-oxidizing enzymes, were necessary for yeast proliferation at the phyllosphere. Furthermore, both peroxisome assembly and pexophagy, a selective autophagy pathway that degrades peroxisomes, were necessary for phyllospheric proliferation. Thus, the present study sheds light on the life cycle and physiology of yeast in the natural environment at both the molecular and cellular levels.     

Air drying optimization of Saccharomyces cerevisiae through its water-glycerol dehydration properties

AIMS: This study describes the different stages of optimization in an original drying process for yeasts, which allows the retrieval of dried samples of Saccharomyces cerevisiae CBS 1171 with maximum viability. METHODS AND RESULTS: The process involves the addition of wheat flour to yeast pellets, followed by mixing and then air-drying in a fluidized bed dryer. The sensitivity to the osmotic stress was first studied in a water-glycerol solution and the observed results were then applied to the drying process. This study have shown that the yeast was quite resistant to osmotic stress and pointed out the existence of zones of sensitivity where viability dramatically decrease as function of final osmotic pressure and temperature of the treatment. Thus, for dehydration until low osmotic pressure (133 MPa, i.e. a(w) = 0.38) results have shown that viability was better when temperature of the treatment was less than 8 degrees C or higher than 25 degrees C. Moreover, kinetic of dehydration was found to greatly influence cells recovery. CONCLUSIONS: These observations allowed the choice of parameters of dehydration of yeasts with an original drying process which involve the mix of the yeasts with wheat flour and then drying in a fluidized bed. SIGNIFICANCE AND IMPACT OF THE STUDY: This process dried rapidly the yeasts to less than 220 MPa (aw < or = 0.2) with whole cell recovery and good fermentative capabilities.     

The flocculation of yeast with chitosan in complex fermentation media: The effect of biomass concentration and mode of flocculant addition

Summary The minimum effective dose of chitosan necessary to flocculate a suspension of yeast in a complex medium is not proportional to the yeast concentration in the medium and is dependent upon the mode of addition of the flocculant to the suspension.     

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.     

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.     

Determination of Growth and Glycerol Production Kinetics of a Wine Yeast Strain Saccharomyces cerevisiae Kalecik 1 in Different Substrate Media

Summary Glycerol has been known as an important by-product of wine fermentations improving the sensory quality of wine. This study was carried out with an endogenic wine yeast strain Saccharomyces cerevisiae Kalecik 1. The kinetics of growth and glycerol biosynthesis were analysed at various initial concentrations of glucose, fructose, and sucrose in a batch system. Depending on the determined values of Monod constants, glucose (K_s = 28.09 g/l) was found as the most suitable substrate for the yeast growth. Initial glucose, fructose and sucrose concentrations necessary for maximum specific yeast growth rate were determined as 175 g, 100 l, and 200 g/l, respectively. The yeast produced glycerol at very high concentrations in fructose medium. Fructose was determined as the most suitable substrate for glycerol production while the strain showed low tendency to use it for growth. S. cerevisiae Kalecik 1 could not produce glycerol below 200 g/l initial sucrose concentration. When natural white grape juice was used as fermentation medium, maximum glycerol concentration and dry weight of the yeast were determined as 9.3 g/l and 11.8 g/l, respectively.     

Effect of cultivation conditions on trehalose content and viability of brewing yeast following preservation via filter paper or lyophilization methods

The effects of variations in cultivation conditions on trehalose concentration and the viability of brewing yeasts following preservation by filter paper or lyophilization methods were evaluated. In case of filter paper preservation, the cultivation period had no affect on yeast viability, while agitation and aeration during cultivation had a positive effect regarding viability of the bottom-fermenting strains, Rh and Frank. For effective preservation, it was necessary to harvest yeast cells from the stationary phase during cultivation. For lyophilization preservation, the yeast strains tested showed a negative effect on viability, independent of strain or cultivation method. No significant correlation was found between trehalose concentration and yeast viability following either filter paper or lyophilization preservation. However, the filter paper preservation method was suitable for both bottom and top brewing yeast strains with regard to feasibility, viability, and maintenance of the yeast’s specific character.     

Iodometric method for detection of beta-lactamase activity in yeast cells carrying ampicillin resistance gene in chimeric plasmids

In order to determine whether an ampicillin resistance gene in a chimeric plasmid is active in transformed yeast cells, it is necessary to have a simple and quick assay procedure. We describe here a procedure for achieving this goal using an iodometric color reaction. This method is based on the fact that the ampicillin resistance gene product, beta-lactamase, can hydrolyze penicillin G and release a reducing product, which can be visualized by the discoloration of a dark blue iodine-starch complex. We have improved this method so that the assay can be carried out on agar plate and in liquid culture. It permits the detection of the beta-lactamase enzyme activity in yeast liquid culture at a concentration as low as 1 X 10(5) cells/ml within 12 h. This method is especially useful for certain yeast transformation systems, such as industrial yeast cultures, where the transformants can be selected only by drug resistance.     

An efficient method for the extraction of astaxanthin from the red yeast Xanthophyllomyces dendrorhous

This study investigated an efficient method for the extraction of astaxanthin from the red yeast Xanthophyllomyces dendrorhous. The extraction process comprised three steps: (1) cultivating the yeast; (2) treating the yeast culture suspension with microwaves to destroy the cell walls and microbodies; and (3) drying the yeast and extracting the astaxanthin pigment using ethanol, methanol, acetone, or a mixture of the three as the extraction solvent. Ultimately, various treatment tests were performed to determine the conditions for optimal pigment extraction, and the total carotenoid and astaxanthin contents were quantified. A frequency of 2,450 MHz, an output of 500 watts, and irradiation time of 60 s were the most optimum conditions for yeast cell wall destruction. Furthermore, optimal pigment extraction occurred when using a cell density of 10 g/l at 30 C over 24 h, with a 10% volume of ethanol.     

Optimization of a Bifidobacterium longum production process

Bifidobacteria are used as probiotics mainly in the dairy industry as cell suspensions or as freeze-dried additives. So far there have been no reports on a thorough investigation on factors influencing the production process or a statistical approach to the optimization thereof. A 2(8-4) fractional factorial design was used in determining the critical parameters influencing bioreactor cultivations of Bifidobacterium longum ATCC 15707. Glucose, yeast extract and l-cysteine concentrations were found critical for the cultivation of this strain. Glucose and yeast extract concentrations were further studied together with temperature in a three factor central composite design. The optimized cultivation conditions were temperature 40 degrees C, yeast extract concentration 35 gl(-1) and glucose concentration 20 gl(-1). Freeze-drying of frozen cell suspensions of B. longum was studied first in controlled temperatures and thereafter with temperature programming experiments. The results were statistically evaluated. A temperature program with a 2 h temperature gradient from -10 to 0 degrees C, a 10 h temperature gradient from 0 to +10 degrees C and a 12 h temperature hold at +10 degrees C was found best for the freeze-drying process. Temperature programming reduced drying times by over 50% and improved the product activity by over 160%.     

Phenotypic plasticity for life history traits in Drosophila melanogaster. I. Effect on phenotypic and environmental correlations

All 36 possible crosses among 6 homozygous lines of Drosophila melanogaster were tested for their phenotypic response in developmental time and dry weight at eclosion to variation in temperature and yeast concentration. This method was chosen because it allows one to produce the same heterozygous offspring repeatedly for testing under more conditions than could be handled at once. We estimated the effects of yeast concentration and temperature and their interaction on both the phenotypic and the environmental components of variation and covariation of the two traits. Development was slower at low temperatures and yeast concentrations, and dry weight and viability were lower at higher temperatures and lower yeast levels. Interactions of the two factors with the crosses and with each other indicated that there were genetic differences in plasticity and that the sensitivity of a trait to one factor depended on the level of the other. The covariation of the two traits was generally weak within an environment. Across environments, its sign depended on the factor that changed between the environments: positive for temperature, negative for yeast concentration. These findings can be explained in terms of an established growth model for Drosophila larvae. We conclude that for plastic traits with moderate or low heritability, the relationship between the phenotypic and genetic covariance matrices may be a complex function of the environmental factors that affect the traits. Some implications for the prediction of the evolution in fluctuating environments are outlined.     

Autoregulated changes in stability of polyribosome-bound beta-tubulin mRNAs are specified by the first 13 translated nucleotides.

The expression of tubulin polypeptides in animal cells is controlled by an autoregulatory mechanism whereby increases in the tubulin subunit concentration result in rapid and specific degradation of tubulin mRNAs. We have now determined that the sequences that are necessary and sufficient to specify mouse beta-tubulin mRNAs as substrates for this autoregulated instability reside within the first 13 translated nucleotides (which encode the first four beta-tubulin amino acids Met-Arg-Glu-Ile). This domain has been functionally conserved throughout evolution, inasmuch as sequences isolated from the analogous region of human, chicken, and yeast beta-tubulin mRNAs also confer autoregulation. Further, for an RNA to be a substrate for regulation, not only must it carry the 13-nucleotide coding sequence, but it must also be ribosome bound and its translation must proceed 3' to codon 41.     

Synthesis of acid phosphatase during dehydration of the yeast Saccharomyces cerevisiae

Summary During the dehydration of exponentially growing yeast cells for 24 h at 37° C, a 2–3 fold increase in the activity of acid phosphatase was observed. This increase is inhibited by cycloheximide and 2-deoxy-D-glucose and therefore is indicative of de novo synthesis. The presence of exogenous orthophosphate during drying does not affect the specific activity of this enzyme, thus indicating the constitutive character of the newly formed acid phosphatase.Freeze-etching showed some rearrangement of the plasmalemma structure of yeast cells during dehydration.     

An infrared drying technique as a rapid reliable means of cell concentration estimation in fermentation systems

Summary A rapid infrared drying cell concentration estimation technique for use in fermentation systems was developed based on commercially available equipment. This system gave accurate results when compared to the traditional oven drying technique, and enabled results to be obtained within 15–30 minutes. This technique overcomes some of the problems associated with cell concentration estimation based on microwave or convention oven drying.