Integrated Expression of the Oenococcus oeni mleA Gene in Saccharomyces cerevisiae

Malolactic enzyme is the function enzyme which catalyses the reaction for L-malate converting to L-lactic during malolactic fermentation （MLF）. In this paper, researches concerning the malolactic enzyme gene mleA cloned from a patent strain Oenococcus oeni SD-2a screened in Chinese wine and integrated expressing in Saccharomyces cerevisiae were performed in order to carry out both alcoholic fermentation （AF） and malolactic fermentation （MLF） during winemaking, with a view to achieving a better control of MLF in enology. To construct the expression plasmid named pYILmleA, cloned mleA gene, PGK1 promoter, and ADH1 terminator were ligated and inserted into integrating vector YIp5. Yeast transformants were screened on SD/-Ura and identified by auxotrophic test, mating type test, and colony PCR. Target protein was detected by SDS-PAGE and the targeted gene integrated to the chromosome was detected by dot bloting hybridization. After the transformant was cultured in SD/-Ura adding glucose （10%） and L-malate （5 648 mg L-1） for 4 d, the culture supernatant was collected and L-malate and L-lactic acid contents were detected by HPLC. 1 278-1 312 mg L-1 L-lactic acids were detected, while the comparative drop rates of L-malate were 20.18-20.85%. L-malate and L-lactic contents of the transformants showed extra significant difference and significant difference with the control ones by t-test respectively. The result indicated that the functional expression was achieved in recombinants S. cerevisiae.

The Effect of Acid Stress Treatment on Viability and Membrane Fatty Acid Composition of Oenococcus oeni SD-2a

To obtain ready-to-use wine malolactic starter cultures with high viability, the effects of acid stress treatments on the growth, inoculation viability, freeze-drying viability, and membrane fatty acid composition of the native Oenococcus oeni SD-2a strain were studied. The results showed that pH 3.5 and 3.2 adaptive treatments did not strongly decrease cell biomass but increased distinctly inoculation viability and freeze-drying viability. Concerning the membrane fatty acid composition, it was observed that acid stress conditions increased significantly the relative concentration of lactobacillic acid （C19cycl 1） and the unsaturated：saturated fatty acid ratio in cell membrane lipids. We assumed that acid-induced cross protective responses could be used in preparing ready-to-use O. oeni SD-2a malolactic starter cultures, and the accumulation of lactobacillic acid in the membrane of O. oeni SD-2a cells appears as an acid stress response mechanism, which might be related with the enhanced viability.

Advanced Progress on Adaptive Stress Response of Oenococcus oeni

Oenoccoccus oeni is an alcohol-tolerant, acidophilic lactic acid bacterium with its ability to perform malolactic fermentation in wine, which is of fundamental importance in oenology. As a representative of the wine bacterium with remarkable adaptability to the very harsh physicochemical conditions of wine, many studies were carded out for its applied interest and focused mainly on its stress response mechanisms of O. oeni. on both physiological and molecular levels. In this review, three main stress response mechanisms in O. oeni during culturing process were addressed. Of them, various solute transporters and secondary metabolic energy-generating systems were utilized to control the intracellular environment and the energetic status of O. oeni. The changes in cell membrane fatty acid composition profiles and synthesis of stress proteins, especially small heat shock proteins were required for active cell response to maintain membrane integrity and function under stress conditions. The study on stress response of O. oeni played an important role on culture bacteria selection, making inoculation culture and construction of other engineering bacteria.

Isolation and Screening of Oenococcus oeni and Its Medium Optimization

[Objectives] This study was conducted to further improve the application of Oenococcus oeni in the wine brewing industry. [Methods] O. oeni was isolated, screened, and identified, and medium optimization was performed to determine the best medium, temperature and oxygen conditions. [Results] O. oeni grew better in mFT80 medium, and the culture conditions were 25 ℃ and an anaerobic condition. [Conclusions] This study provides a reference for the development of China’s wine industry.

Near and Mid Infrared Spectroscopy to Detect Malolactic Biotransformation of Oenococcus oeni in a Wine-Model

The aim of this work was to verify the potential of infrared （IR） spectroscopy in near and mid regions to detect the beginning of the malolactic fermentation （MLF） occurring in a model-wine and the further cells autolysis. MLF in wine is a secondary biotransformation due to lactic acid bacteria that usually occurs spontaneou,;ly or after starter inoculation at the end of alcoholic fermentation. Nowadays, it is desirable to supply winemakers with a new rapid and non-destructive approach to monitor MLF progress and 1R spectroscopy technology appears to be suitable for this purpose. The transformation of L-malic acid into L-lactic acid was carried out by inoculating a synthetic wine with an Oenococcus oeni culture and it was monitored through microbiological and chemical methods. At the same time, Fourier transform near infrared （FT-NIR） spectral data, in diffusive transflection mode using an optic probe, and FT-IR spectra, using an germanium crystal attenuated total reflectance （ATR） cell, were collected. Principal component analysis of the spectra was able to identify absorption bands related to the key molecular modifications that took place during the L-malic acid transformation. Thus, the samples were discriminated according to the fermentation phase. Although this study is a preliminary approach, results confirm that near infrared （NIR） and mid infrared （MIR） spectroscopy could be successfully applied to detect the start of MLF and the autolysis of the lactic acid bacteria （LAB） cells.

Microbiota for production of wine with enhanced functional components

Microbial communities during winemaking are diverse and change throughout the fermentation process. Microorganisms not only drive alcohol fermentation, flavor and aroma, but also enhance wine functional components such as extraction of polyphenols from the berries, production of γ-aminobutyric acid, hydroxytyrosol and melatonin. Polyphenols such as resveratrol, catechin and quercetin determine the functional quality of the wine. Moderate wine consumption, particularly red wine has been associated with functional benefits to human health, which includes anti-inflammation, promoting healthy aging, prevention of cardiovascular diseases, cancers, type 2 diabetes and metabolic syndrome. Indeed, the management of microbiota allows the production of wine with distinct features and functional components that benefits human health. This review scrutinizes the possible contributions of wine microbiota to the production of wine with enhanced functional components and highlights the contributions of Saccharomyces and non-Saccharomyces yeasts and bacteria to enhance wine functional components during winemaking. Thus, contributing to the dissemination of the benefits of light to moderate wine intake to human health.