The production of preconditioned freeze-dried Oenococcus oeni primes its metabolism to withstand environmental stresses encountered upon inoculation into wine.

Oenococcus oeni is the most resistant lactic acid bacteria species to the environmental stresses encountered in wine, particularly the acidity, presence of ethanol and phenolic compounds. Indigenous strains develop spontaneously following the yeast-driven alcoholic fermentation and may perform the malolactic fermentation whereby improving taste, aroma, and the microbial stability of wine. However, spontaneous fermentation is sometimes delayed, prolonged or incomplete. In order to better control its timing and quality, O. oeni strains are selected and developed to be used as malolactic starters. They are prepared under proprietary manufacturing processes to survive direct inoculation and are predominantly provided as freeze-dried preparations. In this study, we have investigated the physiological and molecular alterations occurring in O. oeni cells prepared by an industrial process that consists of preconditioning protocols and freeze-drying, and compared them to the same strain grown in a grape juice medium. We found that compared to cultured cells, the industrial production process improved survival under extreme conditions, i. e. at low pH or high tannin concentrations. In contrast, cultured cells resumed active growth more quickly and strongly than freeze-dried preparations in standard pH wines. A proteomic analysis showed that during the industrial production most non-essential metabolic processes are shut down and components of the general and the stringent stress response are upregulated. The presence of major components of the stress response facilitates protein homeostasis and physiological changes that further ensure the integrity of cells.

Display of active beta-glucosidase on the surface of Schizosaccharomyces pombe cells using novel anchor proteins.

Here, we demonstrate display of beta-glucosidase (BGL) on the surface of Schizosaccharomyces pombe cells using novel anchor proteins. A total of four candidate anchor proteins (SPBC21D10.06c, SPBC947.04, SPBC19C7.05, and SPBC359.04c) were selected from among almost all of S. pombe membrane proteins. The C-terminus of each anchor protein was genetically fused to the N-terminus of BGL, and the fusion protein was expressed using S. pombe as a host. The highest cell surface-associated BGL activity (107 U/10(5) cells was achieved with SPBC359.04c serving as the anchor, followed by SPBC947.04 (44 U/10(5) cells) and SPBC21D10.06c (38 U/10(5) cells). S. pombe displaying BGL with SPBC359.04c as an anchor showed the highest growth on 2 % cellobiose (10.7 × 10(7) cells/mL after 41 h of cultivation from an initial density of 0.1 × 10(7) cells/mL). Additionally, culturing BGL-displaying S. pombe in medium containing cellobiose as the sole carbon source did not affect protein expression, and ethanol fermentation from cellobiose was successfully demonstrated using BGL-displaying S. pombe. This is the first report describing a cell surface display system for the functionalization of S. pombe.

[Food-dependent exercise-induced anaphylaxis that was difficult to evoke by a provocation test].

As a result of ingesting wheat- and soybean-based food products in school meals, an 8-year-old boy repeatedly experienced dyspnea and urticaria while exercising. Based on the symptoms, he was assumed to have been experiencing a food-dependent exercise-induced anaphylactic reaction. Based on the Japanese pediatric guideline for oral food challenge in food allergy 2009, examination using various combinations of food products (wheat and soybeans), medicine (aspirin), and exercise was performed. However, the examination failed to elicit any symptoms. Although we eliminated the food products from the examination, dyspnea caused by exercising after ingesting only wheat products was observed again. Thereafter, we performed a provocation test using wheat products, but symptoms were observed only on increasing the amount of ingested food and the momentum of exercise, without administering aspirin. The possibility that wheat is a more potent inducing factor than aspirin in increasing the momentum of exercise and amount of ingestion in food-dependent exercise-induced anaphylaxis was suggested.