Alcoholic fermentation drives the selection of Oenococcus oeni strains in wine but not in cider.

Oenococcus oeni is the predominant lactic acid bacteria species in wine and cider, where it performs the malolactic fermentation (MLF). The O. oeni strains analyzed to date form four major genetic lineages named phylogroups A, B, C and D. Most of the strains isolated from wine, cider, or kombucha belong to phylogroups A, B + C, and D, respectively, although B and C strains were also detected in wine. This study was performed to better understand the distribution of the phylogroups in wine and cider. Their population dynamics were determined by qPCR all through wine and cider productions, and the behavior of the strains was analyzed in synthetic wines and ciders. Phylogroups A, B and C were all represented in grape must and throughout the alcoholic fermentation, but on the transition to MLF, only phylogroup A remained at high levels in all wine productions. In the case of cider, phylogroups A, B and C were detected in stable levels during the process. When they were tested in synthetic wine and cider, all phylogroups performed MLF, but with different survival rates depending on the ethanol content. In this sense, ethanol and fermentation kinetics are the main agent that drives the selection of phylogroup A strains in wine, while B and C strains dominates in cider containing less ethanol.

Microbiome Status of Cider-Apples, from Orchard to Processing, with a Special Focus on Penicillium expansum Occurrence and Patulin Contamination.

Patulin is a secondary metabolite produced primarily by the fungus Penicillium expansum, responsible for the blue mold disease on apples. It is found in apple products including apple cider when apple juice is added after fermentation. In the present study, two hundred and twenty-five cider-apples of the variety "Bedan", cultivated in Brittany in France, were sampled from the orchard during harvesting until the storage step, right before processing. The patulin analysis on these samples reported a low contamination at the orchard and a significantly higher-level of contamination in the cider-apples starting from the transporting bin. The percentage of positive samples increased from 6% to 47% after 12 h in the harvesting bin before transporting and reached 95% after 24 h of transporting, decreasing then to 69% at the end of the storage. Penicillium expansum was quantified on the surface of apples using real-time PCR and was observed to be mostly consistent between the harvest and post-harvest steps. It was detected on average, on the surface of 85% of all sampled apples with a mean value around 2.35 × 106Penicillium expansum DNA/g of apple. Moreover, the changes in the fungal and bacterial epiphytic microbiota in the different steps were studied using a metabarcoding approach. The alpha and beta diversity analysis revealed the presence of unique and more diverse bacterial and fungal communities on the surface of apples picked from the orchard compared to the rest of the sampling steps. Potential indigenous biological control agents were identified on the surface of sampled apples. Future perspective includes developing actions of prevention and control of the contamination by Penicillium expansum during the harvest and along the various critical post-harvest stages before transformation in a sustainable development concern.

Implications of Lactobacillus collinoides and Brettanomyces/Dekkera anomala in phenolic off-flavour defects of ciders.

Different Lactobacillus collinoides and Brettanomyces/Dekkera anomala cider strains were studied for their ability to produce volatile phenols in synthetic medium. All strains were able to produce 4-ethylcatechol (4-EC), 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) from caffeic, p-coumaric and ferulic acids, respectively. Interestingly, D. anomala and L. collinoides were also able to produce 4-EC, 4-EP and 4-EG in cider conditions. The quantities of ethylphenols produced by these two species were similar in both tested ciders. The impact of precursor quantities was studied and it showed that the addition of caffeic and p-coumaric acids in ciders allowed for higher 4-EC and 4-EP production by D. anomala and L. collinoides. In parallel, D. anomala and L. collinoides strains were isolated from a phenolic off-flavour defective bottled cider after ethylphenol production hence confirming the implication of these two species in this cider spoilage. Finally, detection thresholds of the main ethylphenols were determined in ciders by orthonasal and retronasal sampling. The 4-EC and 4-EP detection thresholds (close to 20-25mg/l and 1.5-2.0mg/l, respectively) were matrix dependant.

Screening of representative cider yeasts and bacteria for volatile phenol-production ability.

Representative cider microorganisms (47 yeast strains and 16 bacterial strains) were studied for their ability to produce volatile phenols in a synthetic medium simulating cider conditions and supplemented with the necessary precursors. The various strains were tested for cinnamoyl esterase activity and only Lactobacillus collinoides were able to hydrolyse chlorogenic acid. Phenolic acid decarboxylase (PAD) activities were observed for 6 yeasts and 4 bacterial species allowing them to produce vinylphenols from hydroxycinnamic acids. On the other hand, 4 bacterial species exhibited phenolic acid reductase (PAR) activities leading to the formation of hydroxyphenylpropionic acids. Brettanomyces/Dekkera anomala and L. collinoides were able to produce 4-ethylcatechol (4-EC) and 4-ethylphenol (4-EP) from caffeic and p-coumaric acid, respectively, indicating that both species exhibit PAD and vinylphenol reductase (VPR) activities. In the experimental conditions used, the production of ethylphenols by L. collinoides was faster than the one observed for D. anomala.

Biogenic amines content in Spanish and French natural ciders: application of qPCR for quantitative detection of biogenic amine-producers.

Biogenic amines (BA) are low molecular weight nitrogenous bases commonly found in fermented foods and beverages and their consumption can induce undesirable reactions. In this work, the BA content in natural cider from Spain and France was determined. Samples from commercially available cider or obtained during the elaboration process were analyzed. A different profile and BA concentration was observed depending on cider origin. qPCR tools developed for the quantitative detection of BA producers from cheese and wine were tested in the cider samples. A good connection between the BA content and the presence of BA-producing microorganisms was observed. Based on these tools, BA-producing bacteria were isolated from the analyzed cider samples, including new potential histamine- and putrescine-producing Lactobacillus paracollinoides strains.

Development and validation of a plate technique for screening of microorganisms that produce volatile sulfur compounds.

Volatile sulfur compounds (VSCs) are of major importance for flavor development in foodstuffs such as cheeses. Such compounds originate from the amino acid l-methionine, which can be degraded to methanethiol (MTL), a common precursor to a variety of other VSCs. A plate assay based on double-layer petri dishes containing 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB), a chemical used for the estimation of free thiols, in the upper layer provides an easy and reliable detection method for thiol-producing, cheese-ripening microorganisms. MTL production was quantitated by measuring the yellow-orange color intensity resulting from reaction with DTNB. Using this method, 18 Geotrichum candidum strains isolated from cheeses were compared, and the color intensity was found to be correlated with the production of microbial VSCs as measured by gas chromatographic analysis.

Identification of trace volatile compounds in freshly distilled Calvados and Cognac using preparative separations coupled with gas chromatography-mass spectrometry.

Gas chromatography coupled with mass spectrometry (GC-MS) using both electron impact and chemical ionization detection modes led to the determination of the volatile composition of two samples of freshly distilled Cognac and two samples of freshly distilled Calvados. A total of 169 volatile compounds were directly identified in dichloromethane extracts obtained by liquid-liquid extraction. Trace compounds present in both spirits were characterized with the help of preparative separations. In a first step, groups of compounds were separated by preparative GC, and the fractions were analyzed on a polar stationary phase by GC-MS. In a second step, silica gel fractionation was used to separate them by polarity. In this study, 331 compounds, of which 162 can be considered as trace compounds, were characterized in both freshly distilled Cognac and Calvados. Of these, 39 are common to both spirits; 30 are specific to Cognac with numerous hexenyl esters and norisoprenoidic derivatives, whereas 93 are specific to Calvados with compounds such as unsaturated alcohols, phenolic derivatives, and unsaturated aldehydes.

Chemical and sensorial aroma characterization of freshly distilled Calvados. 1. Evaluation of quality and defects on the basis of key odorants by olfactometry and sensory analysis.

Eight freshly distilled samples of Calvados, a fermented and distilled apple juice, were analyzed by sensory evaluation and direct injection GC to determine the composition of higher alcohols, esters, and aldehydes. The composition determined by direct injection was tentatively related to sensory descriptors. Esters have a probable maximum level around 500 g/hl of pure alcohol (PA). This level also corresponds to the threshold of the main ester constituent, ethyl acetate. A high ratio of esters to ethyl acetate seems to be of prime importance for good quality. Total aldehydes, with a maximum level between 8 and 11 g/hl of PA and mainly comprising acetal (maximum between 5 and 9 g/hl of PA), were related to a "green" descriptor. Higher alcohols do not have a direct impact on quality, but other volatile compounds with a positive impact on flavor should probably be present at a high level. As overall quality was not well related to sensory quality, it was necessary to perform more a precise analysis to determine the key odorants. The Calvados samples were thus extracted using pentane. Gas chromatography, employing both a flame ionization detector and an olfactometry port, was used to analyze the obtained extracts. Seventy-one odors were detected and distributed according to Calvados quality determined by sensory evaluation. Nineteen odors common to all Calvados samples constituted the "skeleton" of the aroma. Twenty-eight odors were specific to a quality class: 6 for good quality, 4 for neutral, and 18 for defective. Twenty-four other odors had either too low an odor impact or no evident specificity.

Chemical and sensorial aroma characterization of freshly distilled Calvados. 2. Identification of volatile compounds and key odorants.

Eight samples of freshly distilled Calvados were extracted using pentane. Gas chromatography with either a mass spectrometer or flame ionization detector was used to determine the volatile compounds composition of the extracts. More than 120 molecules were identified in Calvados and then correlated with results obtained by olfactometric analysis in our earlier work [Guichard, H.; Lemesle, S.; Ledauphin, J.; Barillier, D.; Picoche, B. Chemical and Sensorial Aroma Characterization of Freshly Distilled Calvados. 1. Evaluation of Quality and Defects on the Basis of Key Odorants by Olfactometry and Sensory Analysis. J. Agric. Food Chem. 2002, 50, 424-432 (preceding paper in this issue)]. Of these, 16 of the 19 molecules that constitute the "aroma skeleton" were identified, including 5 esters, 2 ketones, 5 phenolic derivatives, 2 alcohols, and 2 carboxylic acids. Numerous compounds were also associated with odors found in part 1. These molecules can be considered as being responsible for the good quality of Calvados or, in contrast, for defects. Relative levels of some major olfactive compounds were also estimated and tentatively compared with olfactometric indices found in part 1. A good correlation was found in many cases. Two important markers of defects in Calvados were also identified. 3-Methylbut-2-en-1-ol leads to an "herbaceous" defect, and 1,1,3-triethoxypropane seems to give an "acrolein" defect in the product. "Floral" notes of the aroma of freshly distilled Calvados seem to be due to the presence of phenolic derivatives such as 2-phenylethanol and 2-phenylethyl acetate. Low-molecular-weight esters such as ethyl 2-methylpropanoate, ethyl 2-methylbutanoate, and 3-methylbutyl acetate give, in general, the "fruity" notes. However, the overall aroma of Calvados seems likely to be a subtle balance of various functionalized compounds.