Improved co-oxidation of beta-carotene to beta-ionone using xanthine oxidase-generated reactive oxygen species in a multiphasic system.

beta-Ionone, an aroma compound exhibiting flower notes, can be obtained from beta-carotene in a cooxidation system utilizing xanthine oxidase-generated reactive oxygen species (ROS). ROS have to be controlled as, although they can give rise to beta-ionone, they may also degrade it. In this work, the biotransformation of beta-carotene into beta-ionone was investigated in systems containing variable proportions of decane to extract beta-ionone before degradation. The use of 50% or 90% decane resulted in increased production yields. Tween 80, which was added to further improve the production, slightly decreased the reactivity of the medium and the extraction of beta-carotene, but increased the extraction of beta-ionone. In total, the addition of Tween 80 significantly improved the yields of conversion, which reached 34% with 50% decane and 2.5 g/L Tween 80 compared to 10% without decane and Tween 80. These results show that it is possible to control a ROS-mediated reaction by the addition of a solvent phase and by modifing the medium composition.

Effect of acyl-CoA oxidase activity on the accumulation of gamma-decalactone by the yeast Yarrowia lipolytica: a factorial approach.

beta-Oxidation is a cyclic pathway involved in the degradation of lipids. In yeast, it occurs in peroxisomes and the first step is catalyzed by an acyl-CoA oxidase (Aoxp). The yeast Yarrowia lipolytica possesses several genes (POX) coding for Aoxps. This study is based on the factorial analysis of results obtained with the many POX derivative strains that have been constructed previously. The effect of interactions between Aoxps on the acyl-CoA oxidase (Aox) activity was important even at the second order. We then investigated the effect of Aox activity on growth and lactone production. Aox activity was correlated with acidification of the medium by cells and with cellular growth but not with lactone production, although Aox activity on short chains was inversely correlated with lactone accumulation. Due to the poor correlation between Aox activity and lactone production, the modeling of this parameter gave no satisfactory results but growth depending on Aox activity was modeled.

Toxicity of fatty acid hydroperoxides towards Yarrowia lipolytica: implication of their membrane fluidizing action.

Linoleic acid hydroperoxide (HPOD), substrate of hydroperoxide lyase, an enzyme of the lipoxygenase pathway, can be transformed into many aromatic compounds, the so-called "green notes". The presence of linoleic acid hydroperoxide in the culture medium of Yarrowia lipolytica, the yeast expressing the cloned hydroperoxide lyase of green bell pepper, undoubtedly exerted an inhibition on the growth and a toxic effect with 90% of yeast cells died after 120 min of exposition in 100 mM HPOD solution. The increase in cell membrane fluidity evaluated by measuring fluorescence generalized polarization with the increasing concentration of HPOD in the medium confirmed the fluidizing action of HPOD on yeast membrane. In addition, we determined by infrared spectroscopy measurement that this compound rapidly diffused into model phospholipids [1, 2-Dimyristoyl-D54-sn-Glycero-3-Phosphocholine (DMPC-D54)] bilayer, modifying their general physical state and their phase transition. In the presence of various concentrations of HPOD, the phase transition of DMPC-D54 occurred with an increase of both the corresponding wave number shift and the temperature range but the phase transition temperature was not modified. These results show that the toxic effects of HPOD on the yeast Yarrowia lipolytica may be initially linked to a strong interaction of this compound with the cell membrane phospholipids and components.

Metabolism of phenylalanine and biosynthesis of styrene in Penicillium camemberti.

The occurrence of styrene in food may be an important aroma defect (celluloid odour), even at very low concentrations (Miltz et al. 1980) causing consumer rejection and is therefore a problem for the food industry. We examined the biosynthetic pathway leading to styrene formation by Penicillium camemberti using labelled compounds. As styrene is strongly hydrophobic and volatile, we first had to develop a continuous extraction process. Using resins XAD2 it was reasonable to suspect phenylalanine (Phe) as the precursor. The addition of Phe marked with 13C on the ring provokes the accumulation of labelled styrene. The enzyme activities involved were also tentatively measured. Styrene appears to be synthesized from phenylalanine by phenylalanine ammonia-lyase activity followed by a decarboxylation catalysed by a cinnamic acid decarboxylase.

Relationship between the presence of the citrate permease plasmid and high electron-donor surface properties of Lactococcus lactis ssp. lactis biovar. diacetylactis.

Some strains of Lactococcus lactis subspecies possess a citrate permease that enables them to utilize citrate and to produce diacetyl. Such strains are classified as diacetylactis biovariants (L. lactis ssp. lactis biovar. diacetylactis). We investigated the electron-donor surface properties of L. lactis strains and observed that the diacetylactis biovariants presented increased adhesion to electron-acceptor solvents (microbial adhesion to solvents electron-donor characteristics of cells of <27% for L. lactis and about 50% for L. lactis ssp. lactis biovar diacetylactis). We investigated the properties of a pCitP- derivative and observed for a diacetylactis biovariant strain a loss of the electron-donor characteristics falling from 47% for a pCitP+ strain to 8% for its pCitP- derivative. This suggests that the presence of high electron-donor characteristics on the surface of L. lactis results to a large extent from the presence of the citrate permease plasmid.

Importance of bacterial surface properties to control the stability of emulsions.

In colloidal media such as emulsions or food matrixes, the stability results from physicochemical interactions. The same type of interaction is involved in the attachment processes of microorganisms, through their surface properties, to interfaces. When bacteria are present in a food matrix, it is probable that their surface interacts with the other constituents. In this paper, the involvement of bacterial surface properties of Lactococcus lactis subsp lactis biovar diacetylactis (LLD) on the stability of model emulsions has been studied. The hydrophobic and electrostatic cell-surface properties were characterized by the MATH method and by microelectrophoresis, respectively. The oil-in-water emulsions were stabilized by various surface-active compounds, CTAB, SDS or Tween 20, giving differently charged droplets. Two strains with different surface characteristics were added to the emulsion. Contrasting with emulsions made with the non-ionic surfactant, for which the stability was not modified by the addition of bacteria, the emulsions made with ionic surface-active compounds were unstable in the presence of bacteria when the bacterial surface charge was opposite to the one of the emulsion droplets. Moreover, aggregation and flocculation phenomena were observed for emulsions stabilized with the cationic surfactant, particularly for more negatively charged bacteria. The effect of bacteria on the emulsion stability depended on the strain which shows the importance of the choice of the microorganism according to of the characteristics of the colloidal media to obtain a stable system. In addition, these results suggest that the interactions between bacteria and other food components can influence the position of bacteria in food matrixes.

Surface properties of Yarrowia lipolytica and their relevance to gamma-decalactone formation from methyl ricinoleate.

The surface of the lipid-degrading yeast, Yarrowia lipolytica, was characterized by contact angle and zeta potential (zeta) measurements. The cells were mainly hydrophilic with a negative charge that was only affected from pH 2 to 4. To study the effects of the surface charges on the biotransformation of methyl ricinoleate into the aroma compound, gamma-decalactone, the zeta values of the substrate droplets were modified by adding a cationic surfactant into the medium at concentrations that did not diminish cell viability: the adhesion of the lipid substrate to the cells was increased but not the overall performance of the process, therefore the adhesion is not the rate limiting here. Our methodology offers interesting perspectives for further applications.

Increased electron donor and electron acceptor characters enhance the adhesion between oil droplets and cells of Yarrowia lipolytica as evaluated by a new cytometric assay.

The adhesion of methyl ricinoleate droplets to cells of the yeast Yarrowia lipolytica was investigated. A new cytometric method, relying on the double staining of fatty globules with Nile Red and of cells with Calcofluor, enabled us to quantify methyl ricinoleate droplet adhesion to cells precultured on a hydrophilic or on a hydrophobic carbon source. In this last case, droplet adsorption was enhanced and a MATS (microbial adhesion to solvents) test revealed that this increase was due to Lewis acid-base interactions and not to an increase in the hydrophobic properties of the cell surface. These preliminary results demonstrate that the developed cytometric method is promising for various applications concerning the study of interactions between microorganisms and an emulsified hydrophobic substrates.

Effect of cis/trans isomerism of beta-carotene on the ratios of volatile compounds produced during oxidative degradation.

beta-Carotene is, when cleaved, an important source of flavor and aroma compounds in fruits and flowers. Among these aroma compounds, the main degradation products are beta-ionone, 5,6-epoxy-beta-ionone, and dihydroactinidiolide (DHA), which are associated by flavorists and perfumers with fruity, floral, and woody notes. These three species can be produced by degradation of beta-carotene through an attack by enzyme-generated free radicals and a cleavage at the C9-C10 bond. This study investigated the influence of cis/trans isomerism at the C9-C10 bond on the production of beta-carotene degradation compounds, first with a predictive approach and then experimentally with different isomer mixtures. beta-Carotene solutions containing high ratios of 9-cis-isomers produced more DHA, suggesting a different pathway than for the transformation of all-trans-beta-carotene to ionone and DHA. These results are important in the search for financially viable processes to produce natural carotene-derived aroma compounds.

Interaction of an odorant lactone with model phospholipid bilayers and its strong fluidizing action in yeast membrane.

Some odorant lactones are naturally present in fruits or in fermented products; they can also be used as food additives and can be produced by microorganisms at the industrial scale by biotechnological processes. Gamma-decalactone was previously shown to have antimicrobial properties. We determined by infrared spectroscopy measurements that this compound rapidly diffused into model phospholipid bilayers (within 2 min), modifying the general physical state of a dimyristoyl-L-alpha-phosphatidylcholine (DMPC) film. In vivo, the lactone strongly increased membrane fluidity in the model yeast Yarrowia lipolytica, as evaluated by fluorescence anisotropy measurements. This effect was more important than that of benzyl alcohol, which is known as a fluidizing agent in living cells, and may explain the toxic action of gamma-decalactone in microorganisms.