Freeze-drying Yeasts

Figures 4.2.1 and 4.2.2 [4.9] show sorption isotherms 1, for such products which become less hygroscopic with increased temperature, or 2, for glucose, fats and oils which become more hygroscopic with increased temperature. Since freeze drying lowers aw values, the growth of bacteria, fungus and yeast below aw = 0.8 is reduced, or impossible. On the other hand, the Maillard reaction increases with decreasing aw up to a maximum at aw 0,6-0,7,...

When in later years Krebs reviewed the major points which had to be established if the cycle was to be shown to be operative in cells, the obvious needs were to find the presence of the required enzymes and to detect their substrates. As the substrates are present in the cycle in catalytic amounts their accumulation required the use of inhibitors. Krebs also stressed that rates of oxidation of the individual substrates must be at least as fast as the established rates of oxygen uptake in vivo, an argument first used by Slator (1907) with reference to fermentation A postulated intermediate must be fermented at least as rapidly as glucose is. (See Holmes, 1991). This requirement did not always appear to be met. In the early 1950s there were reports that acetate was oxidized by fresh yeast appreciably more slowly than the overall rate of yeast respiration. It was soon observed that if acetone-dried or freeze-dried yeasts were used in place of fresh yeast, rates of acetate oxidation were increased more than enough to meet the criterion. Acetate could not penetrate fresh yeast cell walls sufficiently rapidly to maintain maximum rates of respiration. If the cell walls were disrupted by drying this limitation was overcome, i.e. if rates of reaction are to be... 

In the Accusphere test a freeze-dried sphere containing the test organism Streptococcus thermophilus and bromocresol purple as indicator disperses into the test milk sample. The acidification test is very similar the milk sample is heated to be further inoculated with a Streptococcus thermophilus culture containing yeast extract, bromocresol purple indicator, and trimethoprim. It is then incubated for 2.5 h at 45 C (33). In the presence of inhibitory substances, the organism growth is suppressed, acid production is reduced or eliminated, and the color of the indicator remains unchanged. Addition of penicillinase to a positive milk sample results in change of the color of the indicator from purple to yellow when only -lactams are present. 

Fig. 3.16.2. Effect of additives on survival of Saccharomyces cerevisae CBS 1171 (SC 1171) after freeze-drying. CFU, colony-forming units A, CFU/mL before freeze-drying O, yeast suspension without additives 1, maltose 2, tre-...

Saccharomyces cerevisiae WRP45073A1 survives freeze drying and yeast survival rates were dependent on the nature of the cryoprotectants 13.1 1.8%, 9.5 6.0%,... 

Influence of Cryoprotectants on Viability and Heterologous Activity of Lyophilized Yeasts in Simulated Gastrointestinal Conditions To evaluate the influence of cryoprotectants on both the survival rate and CA4H activity of WRP45073A1 in simulated gastrointestinal conditions, 1010 viable freeze-dried yeasts and 200 pmol of franx-cinnamic acid were simultaneously introduced into the TIM. Yeast cells were lyophilized in the presence of the milk protein-trehalose mix, trehalose, lactose, or maltose, as previously explained (see above). The freeze-dried samples were introduced into the artificial stomach suspended in 300 mL of yeast culture medium without any storage period. The number of viable cells introduced into the TIM was determined from previously obtained survival rates (cf. Section 5.5.3.1). 

In addition, no significant difference was observed between the ileal recovery profiles of yeasts with the various cryoprotectants, showing their lack of influence on the survival of freeze-dried WRP45073A1 in the TIM. 

A few studies have evaluated the survival rate of freeze-dried S. cerevisiae spp. in human volunteers following their oral administration. Nevertheless, comparison between in vitro results in the TIM and these in vivo data is hampered by the fact that yeast survival had been evaluated only in feces (and not at the end of the ileum) after a single or multiple oral administration of the microorganisms. Klein et al. [49] found a fecal recovery of 0.12 0.04% (n = 8) after a single dose of 1 g of S. boulardii [10104 colony-forming units (CFU)] to healthy volunteers, and Blehaut et al. [50] measured a steady-state fecal recovery of 0.36 + 0.31% (n = 8) after oral administra-... 

Conclusion Although the impact of freeze drying on both the survival rate and heterologous activity of yeasts in the artificial digestive system was found to be adverse, lyophilization appears to be a convenient technique for the dehydration of recombinant S. cerevisiae. Among the tested cryoprotectants, the association of milk proteins and trehalose was the most efficient to maintain the CA4H activity of... 

Berny, J. F., and Hennebert, G. L. (1991), Viability and stability of yeast cells and filamentous fungus spores during freeze-drying Effects of protectants and cooling rates, Mycologia, 83, 805-815. 

Abadias,M.,Benabarre,A.,Teixido,N.,Usall, J.,and Vias I. (2001), Effect of freeze-drying and protectants on viability of the biocontrol yeast Candida sake, Int. J. Food Microbiol., 65,173-182. 

MARA is an innovative bioassay devised for the evaluation of toxicity of chemicals and environmental samples. The assay utilizes a taxonomically diverse array of ten bacterial species (prokaryotes) and a yeast (eukaryote). The assay is performed in a 96 well micro titre plate and involves exposure of the microorganisms provided in a freeze-dried state. The toxicity of the test sample using a concentration gradient is determined with the employment of the redox dye tetrazolium red (TZR). The dye is transformed from a soluble colourless state to a red insoluble form upon reduction. The dye is a growth indicator and detects enzyme systems by acting as an electron acceptor. 

Yeast biomass was freeze-dried and grounded. Fatty acid extraction and preparation of methyl esters were carried out according to Lepage and Roy [25]. Samples (100 mg) were transmethylated with 5 ml of methanol/acetyl chloride (95 5 v/v). The mixture was sealed in a light-protected Teflon-lined vial under nitrogen atmosphere and heated at 80 °C for 1 h. The vial contents were then cooled, diluted with 1 ml water, and extracted with 2 ml of n-heptane. The heptane layer was dried over Na2S04, evaporated to dryness under nitrogen atmosphere and redissolved in heptane, which contained the methyl esters. 

In the case of yeast, control of freeze-drying was defined from the literature for Saccharomyces cerevisiae (Mazur 1970 Souzu 1999). It was performed by freezing at -40°C with a vacuum level of 30 Pa and a heating plate temperature of 25°C. Survival ratio was determined as the most relevant quality parameter. 

Transport into the cell nucleus is through a nuclear pore. Our collaborators, C. Akey and Q. Yang, are doing a 3D image reconstruction of yeast nuclear pore complexes. They needed to determine the mass of the complexes to aid in their analyses. Two micrographs of freeze-dried complexes, which have masses of approximately 60 MDa, are shown in Fig. 6A. 

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