Ecological linkages between biotechnologically relevant autochthonous microorganisms and phenolic compounds in sugar apple fruit (Annona squamosa L.).

Our study investigated the potential of Annona squamosa (L.) fruit as a reservoir of yeasts and lactic acid bacteria having biotechnological implications, and phenolics capable of modifying the ecology of microbial consortia. Only a single species of lactic acid bacteria (Enterococcus faecalis) was identified, while Annona fruit seemed to be a preferred niche for yeasts (Saccharomyces cerevisiae, Hanseniaspora uvarum), which were differentially distributed in the fruit. In order to identify ecological implications for inherent phenolics, the antimicrobial potential of water- and methanol/water-soluble extracts from peel and pulp was studied. Pulp extracts did not show any antimicrobial activity against the microbial indicators, while some Gram-positive bacteria (Staphylococcus aureus, Staphylococcus saprophyticus, Listeria monocytogenes, Bacillus megaterium) were susceptible to peel extracts. Among lactic acid bacteria used as indicators, only Lactococcus lactis and Weissella cibaria were inhibited. The chemical profiling of methanol/water-soluble phenolics from Annona peel reported a full panel of 41 phenolics, mainly procyanidins and catechin derivatives. The antimicrobial activity was associated to specific compounds (procyanidin dimer type B [isomer 1], rutin [isomer 2], catechin diglucopyranoside), in addition to unidentified catechin derivatives. E. faecalis, which was detected in the epiphytic microbiota, was well adapted to the phenolics from the peel. Peel phenolics had a growth-promoting effect toward the autochthonous yeasts S. cerevisiae and H. uvarum.

Bacterial cellulose production by Novacetimonas hansenii MSCL 1646 on apple juice.

Biomaterials and biopolymers, such as bacterial cellulose (BC), are becoming increasingly important as sustainable materials with a wide range of potential applications. However, BC industrial production is associated with several difficulties such as low BC production yields and high production costs; therefore, cheap alternative growth media, e.g. apple juice are being studied intensively. The aim of this study is to evaluate BC synthesis under static conditions on apple juice medium (AJM). The optimal concentration of apple juice in unsupplemented AJM for Novacetimonas hansenii MSCL 1646 was shown by its dilution 1:6 with water, which resulted in 0.89 ± 0.01 g/L of dry BC weight after 10 cultivation days. Low BC synthesis can be associated with insufficient N concentration in apple juice; therefore, different organic and inorganic N sources were evaluated in combination with AJM, and beef extract (5 g/L) was found to be the most suitable. Further, AJM optimisation experiment showed the optimal apple juice and beef extract concentrations as 1:2 and 15 g/L respectively, which resulted in 17.27 ± 0.07 g/L of dry BC weight, which is significantly higher than in standard Hestrin-Schramm (HS) medium (4.07 ± 0.02 g/L). Analysis of mechanical and physical properties showed that use of AJM results in changes in BC properties compared with the standard HS medium. Results of the study indicate that apple juice is an effective and cheap C source that in combination with appropriate N source leads to high BC synthesis and makes it suitable for industrial BC production. KEY POINTS: • Low quality apples can be used as raw material for BC production; • Beef extract improves BC synthesis in apple juice medium; • Use of apple juice and beef extract affect mechanical properties of BC.

Diversity and dynamics of bacterial and fungal communities in cider for distillation.

Bacterial and fungal population dynamics in cider for distillation have so far been explored by culture-dependant methods. Cider for distillation can be produced by the spontaneous fermentation of apples that do not undergo any intervention during the process. In this study, cider microbiomes extracted from six tanks containing ciders for distillation from four producers in Normandy were characterized at three main stages of the fermentation process: fermentation Initiation (I), end of the alcoholic Fermentation (F) and end of the Maturation period (M). Cider samples were subjected to Illumina MiSeq sequencing (rRNA 16S V1-V3 and ITS1 region targeting) to determine bacterial and fungal communities. Yeasts (YGC), Zymomonas (mZPP) and lactic acid bacteria selective media (mMRS, mMLO, mPSM) were also used to collect 807 isolates. Alcoholic levels, glycerol, sugar content (glucose, fructose and sucrose), pH, total and volatile acidity, nitrogen, malic and lactic acid contents were determined at all sampling points. Alpha diversity indexes show significant differences (p < 0.05) in microbial populations between I, F and M. Fungal communities were characterized by microorganisms from the environment and phytopathogens at I followed by the association of yearsts with alcoholic fermentation like Saccharomyces and non-Saccharomyces yeasts (Hanseniaspora, Candida). A maturation period for cider leads to an increase of the Dekkera/Brettanomyces population, which is responsible for off-flavors in cider for all producers. Among bacterial communities, the genera community associated to malolactic fermentation (Lactobacillus sp., Leuconostoc sp., Oenococcus sp.) was the most abundant at F and M. Acetic acid bacteria such as Acetobacter sp., Komagataeibacter sp. and Gluconobacter sp. were also detected during the process. Significant differences (p < 0.05) were found in fungal and bacterial populations between the four producers and during the fermentation process. The development of microorganisms associated with cider spoilage such as Zymomonas mobilis, Lactobacillus collinoides or Brettanomyces/Dekkera sp. was anticipated by a metagenomic approach. The monitoring of microbial diversity via high throughput sequencing combined with physical-chemical analysis is an interesting approach to improve the fermentation performance of cider for distillation and therefore, the quality of Calvados.

Evaluation of the fermentative capacity of an indigenous Hanseniaspora sp. strain isolated from Lebanese apples for cider production.

The present work studied the fermentative potential and carbon metabolism of an indigenous yeast isolated from Lebanese apples for cider production. The indigenous yeast strain was isolated from a spontaneous fermented juice of the Lebanese apple variety 'Ace spur'. The sequencing of the Internal Transcribed Spacer (ITS) domain of rRNA identified the isolated yeast strain as a member of the Hanseniaspora genus. These results suggest an intragenomic ITS sequence heterogeneity in the isolated yeast strain specifically in its ITS1 domain. The different investigations on the yeast carbon metabolism revealed that the isolated yeast is 'Crabtree positive' and can produce and accumulate ethanol from the first hours of fermentation. Thus, our findings highlight the possibility of using the isolated indigenous Hanseniaspora strain as a sole fermentative agent during cider production.

Pulsed electric field-assisted fermentation of Hanseniaspora sp. yeast isolated from Lebanese apples.

Hanseniaspora sp. yeast was stimulated using pulsed electric field (PEF) during the different fermentation phases. The impact of PEF parameters on the growth rate and substrate consumption was studied. The PEF intensities chosen for this study were mainly in the range of 72-285 V cm-1. A PEF treatment chamber was designed for this study with a ratio of 1:50 between the volume of the fermenter and the volume of the chamber. It allows the recycling of the culture medium using a peristaltic pump, and the yeast treatment by PEF during the fermentation. The continuous circulation of the medium allows avoiding the increase of the temperature inside the fermenter, the cell aggregation, as well as the agitation and the scale-up issues that are associated with the PEF treatment of the entire volume in batch mode. The maximal yeast growth rate was obtained using an electric field strength of 285 V cm-1 applied during both Lag and early exponential phase, and Log phase. This observation was accompanied by a faster consumption of glucose in the medium during the fermentation. Besides, the sensitivity of Hanseniaspora sp. yeast to PEF treatment was more pronounced during the Lag and early exponential phase than the Log phase. The results obtained exposed the great benefit of stimulating Hanseniaspora sp. yeast using moderate PEF as it reduces the fermentation time along with increasing the biomass concentration.

Ultrasound-assisted fermentation for cider production from Lebanese apples.

The present work studies the impact of low-intensity ultrasound (US) on Hanseniaspora sp. yeast fermentations. The effect of pulse duration and growth phase on US application was first evaluated using a synthetic medium. The optimal conditions were then applied to apple juice US-assisted fermentation. An US treatment chamber was first designed to allow the recycling of the culture medium. The optimal US pulse duration on the yeast growth rate was of 0.5 s followed by 6 s rest period, and during 6 h of both Lag and Log phases. These US parameters led to a faster consumption of glucose in the medium during the fermentation, compared to the untreated culture. The impact of US was also depending on the growth phase, showing higher sensitivity of the yeast to US during the Lag phase rather than the Log phase. US-assisted fermentation of apple juice showed a significant increase in biomass growth and glucose consumption, along with a significant decrease in the ethanol yield. The fastest growth kinetic (by 52%), and the highest ethanol reduction (by 0.55% (v, v)) were obtained for the treatment during the first 12 h of fermentation, thereby, the stationary phase was reached faster, and the maximum biomass growth rate was 10 folds higher compared to the untreated culture. The results obtained in this study demonstrated the promising efficiency of US-assisted fermentation in stimulating the biomass growth and reducing the ethanol content in alcoholic beverages.

Chemical composition, sensorial properties, and aroma-active compounds of ciders fermented with Hanseniaspora osmophila and Torulaspora quercuum in co- and sequential fermentations.

In this study, apple juice was fermented using Hanseniaspora osmophila X25-5 in pure culture as well as mixed culture with Torulaspora quercuum X24-4, which was inoculated simultaneously or sequentially. H. osmophila inhibited the growth of T. quercuum, while T. quercuum had little effect on the growth of H. osmophila. The simultaneous fermentation consumed relatively more sugar and resulted in the highest ethanol content. The production of organic acids varied depending on the yeast species employed and inoculation modality. Esters and alcohols were the main volatile families produced during fermentation, while ethyl esters and terpenes contributed most to the temperate fruity aroma. Gas chromatography-olfactometry (GC-O) showed that 3-methyl-1-butanol, ethyl 2-methylbutanoate, phenylethyl alcohol, ß-phenethyl acetate, and ß-damascenone were the most potent odorants in all samples. This study suggested that simultaneous fermentation with H. osmophila and T. quercuum might represent a novel strategy for the future production of cider.

Fourier Transform Near-Infrared Spectroscopy and Chemometrics To Predict Zygosacchromyces rouxii in Apple and Kiwi Fruit Juices.

This study investigated the capability of near-infrared spectroscopy (NIRS) to predict the concentration of Zygosaccharomyces rouxii in apple and kiwi fruit juices. The yeast was inoculated in fresh kiwi fruit juice ( n = 68), reconstituted kiwi juice ( n = 85), and reconstituted apple juice ( n = 64), followed by NIR spectra collection and plate counting. A principal component analysis indicated direct orthogonal signal correction preprocessing was suitable to separate spectral samples. Parameter optimization algorithms increased the performance of support vector machine regression models developed in a single variety juice system and a multiple variety juice system. Single variety juice models achieved accurate prediction of Z. rouxii concentrations, with the limit of quantification at 3 to 15 CFU/mL ( R2 = 0.997 to 0.999), and the method was also feasible for Hanseniaspora uvarum and Candida tropicalis. The best multiple variety juice model obtained had a limit of quantification of 237 CFU/mL ( R2 = 0.961) for Z. rouxii. A Bland-Altman analysis indicated good agreement between the support vector machine regression model and the plate counting method. It suggests that NIRS can be a high-throughput method for prediction of Z. rouxii counts in kiwi fruit and apple juices.

Yeast species diversity in apple juice for cider production evidenced by culture-based method.

Identification of yeasts isolated from apple juices of two cider houses (one located in a plain area and one in an alpine area) was carried out by culture-based method. Wallerstein Laboratory Nutrient Agar was used as medium for isolation and preliminary yeasts identification. A total of 20 species of yeasts belonging to ten different genera were identified using both BLAST algorithm for pairwise sequence comparison and phylogenetic approaches. A wide variety of non-Saccharomyces species was found. Interestingly, Candida railenensis, Candida cylindracea, Hanseniaspora meyeri, Hanseniaspora pseudoguilliermondii, and Metschnikowia sinensis were recovered for the first time in the yeast community of an apple environment. Phylogenetic analysis revealed a better resolution in identifying Metschnikowia and Moesziomyces isolates than comparative analysis using the GenBank or YeastIP gene databases. This study provides important data on yeast microbiota of apple juice and evidenced differences between two geographical cider production areas in terms of species composition.

Distribution of apple and blackcurrant microbiota in Lithuania and the Czech Republic.

The microbial assemblies on the surface of plants correlate with specific climatic features, suggesting a direct link between environmental conditions and microbial inhabitation patterns. At the same time however, microbial communities demonstrate distinct profiles depending on the plant species and region of origin. In this study, we report Next Generation Sequencing-based metagenomic analysis of microbial communities associated with apple and blackcurrant fruits harvested from Lithuania and the Czech Republic. Differences in the taxonomic composition of eukaryotic and prokaryotic microorganisms were observed between plant types. Our results revealed limited geographic differentiation between the bacterial and fungal communities associated with apples. In contrast, blackcurrant berries harvested from different regions demonstrated high diversity in both bacterial and fungal microbiota structures. Among fungal and bacterial microorganisms, we identified both potentially beneficial (Cryptococcus, Hanseniaspora, Massilia, Rhodotorula, Sphingomonas) and phytopathogenic microorganisms (Cladosporium, Pantoea, Phoma, Pseudomonas, Septoria, Taphrina) indicating their important roles in ecological and evolutionary processes.

Viability of Hanseniaspora uvarum yeast preserved by lyophilization and cryopreservation.

Hanseniaspora yeasts are known to produce volatile compounds that give fruity aromas in wine and fermented fruit. This study aimed to verify the feasibility of the Hanseniaspora uvarum strain that had been isolated and identified during a previous study and preserved by lyophilization and freezing at -80 °C (cryopreservation). This strain was assessed in relation to its macroscopic and microscopic morphology and for its ability to ferment apple must. After having been subjected to lyophilization and cryopreservation, viability was assessed in relation to these characteristics during 12 months of storage. The strain showed stable colonial features and its microscopic appearance was unchanged during all recoveries. The plate count results showed consistency in both processes. Regarding the fermentative capacity, the kinetic results showed 100% viability for the strain subjected to lyophilization, as well as for those preserved at -80 °C. These results demonstrate that the preservation methods used are compatible with the maintenance of the relevant characteristics of the strain for the period of evaluation of this study (12 months).

A yeast isolated from cashew apple juice and its ability to produce first- and second-generation ethanol.

The aim of this study was to isolate and identify an indigenous yeast from cashew apple juice (CAJ) and then use it in the production of first- and second-generation ethanol, using CAJ and the enzymatic hydrolysate of cashew apple bagasse (MCAB-OH), respectively. The isolated yeast was identified as belonging to the genus Hanseniaspora. Afterward, the effect of the medium initial pH on the production of ethanol from CAJ was evaluated in the range of 3.0 to 5.5, with its maximum ethanol production of 42 g L(-1) and Y P/S of 0.44 g g(-1) and 96 % efficiency. The effect of temperature (28-38 °C) on ethanol production was evaluated in a synthetic medium, and no difference in ethanol production in the temperature range evaluated (28-36 °C) was observed. At 32 °C, the yield, concentration, efficiency, and productivity of ethanol when using the CAJ medium were higher when compared to the results achieved for the synthetic medium. Regarding second-generation ethanol, the results showed that the yeast produced 24.37 g L(-1) of ethanol with an efficiency of 80.23 % and a productivity of 4.87 g L(-1) h(-1) at 5 h. Therefore, Hanseniaspora sp., isolated from CAJ, is a promising microorganism for the production of first- and second-generation ethanol.

Influence of fermentation with Hanseniaspora sp. yeast on the volatile profile of fermented apple.

This study aims to evaluate the aromatic profile of fermented apple, obtained by the action of strains of Hanseniaspora uvarum and Hanseniaspora guilliermondii using two methods of analysis: static headspace and solid phase microextraction (SPME). The results obtained confirm that the strains of the Hanseniaspora genus contributed positively to the aroma profile of fermented apple, producing considerable amounts of esters and higher alcohols. In comparing the methods of analysis of aromatic compounds using headspace and SPME, it was verified that by using the headspace method it was possible to capture amounts that were up to 16 times greater than the value of the volatile compounds obtained by SPME. However, when using SPME, 5 times more compounds were obtained than when using headspace. Even so, in the conditions of this study it was noted that headspace was more efficient in the extraction of the aromatics of fermented apple when taking into consideration the cost effectiveness of both methods.

Osmotic adaptation in halotolerant yeast, Debaryomyces nepalensis NCYC 3413: role of osmolytes and cation transport.

Debaryomyces nepalensis NCYC 3413, a food spoiling yeast isolated from rotten apple, has been previously demonstrated as halotolerant yeast. In the present study, we assessed its growth, change in cell size, and measured the intracellular polyol and cations (Na(+) or K(+)) accumulated during growth in the absence and presence of different concentrations of salts (NaCl and KCl). Cells could tolerate 2 M NaCl and KCl in defined medium. Scanning electron microscopic results showed linear decrease in mean cell diameter with increase in medium salinity. Cells accumulated high amounts of K(+) during growth at high concentrations of KCl. However, it accumulated low amounts of Na(+) and high amounts of K(+) when grown in the presence of NaCl. Cells grown in the absence of salt showed rapid influx of Na(+)/K(+) on incubation with high salt. On incubation with 2 M KCl, cells grown at 2 M NaCl showed an immediate efflux of Na(+) and rapid uptake of K(+) and vice versa. To withstand the salt stress, osmotic adjustment of intracellular cation was accompanied by intracellular accumulation of polyol (glycerol, arabitol, and sorbitol). Based on our result, we hypothesize that there exists a balanced efflux and synthesis of osmolytes when D. nepalensis was exposed to hypoosmotic and hyperosmotic stress conditions, respectively. Our findings suggest that D. nepalensis is an Na(+) excluder yeast and it has an efficient transport system for sodium extrusion.

Yeast species associated with the spontaneous fermentation of cider.

This paper reports the influence of cider-making technology (pneumatic and traditional pressing) on the dynamics of wild yeast populations. Yeast colonies isolated from apple juice before and throughout fermentation at a cider cellar of Asturias (Spain), during two consecutive years were studied. The yeast strains were identified by restriction fragment length polymorphism analysis of the 5.8S rRNA gene and the two flanking internal transcribed sequences (ITS). The musts obtained by pneumatic pressing were dominated by non-Saccharomyces yeasts (Hanseniaspora genus and Metschnikowia pulcherrima) whereas in the apple juices obtained by traditional pressing Saccharomyces together with non-Saccharomyces, were always present. The species Saccharomyces present were S. cerevisiae and S. bayanus. Apparently S. bayanus, was the predominant species at the beginning and the middle fermentation steps of the fermentation process, reaching a percentage of isolation between 33% and 41%, whereas S. cerevisiae took over the process in the final stages of fermentation. During the 2001 harvest, with independence of cider-making technology, the species Hanseniaspora valbyensis was always isolated at the end of fermentations.

Mapping of the apple scab-resistance gene Vb.

Apple scab, caused by the fungus Venturia inaequalis, is the major production constraint in temperate zones with humid springs. Normally, its control relies on frequent and regular fungicide applications. Because this control strategy has come under increasing criticism, major efforts are being directed toward the breeding of scab-resistant apple cultivars. Modern apple breeding programs include the use of molecular markers, making it possible to combine several different scab-resistance genes in 1 apple cultivar (pyramiding) and to speed up the breeding process. The apple scab-resistance gene Vb is derived from the Siberian crab apple 'Hansen's baccata #2', and is 1 of the 6 "historical" major apple scab-resistance genes (Vf, Va, Vr, Vbj, Vm, and Vb). Molecular markers have been published for all these genes, except Vr. In testcross experiments conducted in the 1960s, it was reported that Vb segregated independently from 3 other major resistance genes, including Vf. Recently, however, Vb and Vf have both been mapped on linkage group 1, a result that contrasts with the findings from former testcross experiments. In this study, simple sequence repeat (SSR) markers were used to identify the precise position of Vb in a cross of 'Golden Delicious' (vbvb) and 'Hansen's baccata #2' (Vbvb). A genome scanning approach, a fast method already used to map apple scab-resistance genes Vr2 and Vm, was used, and the Vb locus was identified on linkage group 12, between the SSR markers Hi02d05 and Hi07f01. This finding confirms the independent segregation of Vb from Vf. With the identification of SSR markers linked to Vb, another major apple scab-resistance gene has become available; breeders can use it to develop durable resistant cultivars with several different resistance genes.

The role of indigenous yeasts in traditional Irish cider fermentations.

AIMS: To study the role of the indigenous yeast flora in traditional Irish cider fermentations. METHODS AND RESULTS: Wallerstein laboratory nutrient agar supplemented with biotin, ferric ammonium citrate, calcium carbonate and ethanol was employed together with PCR-restriction fragment length polymorphism analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene in the identification of indigenous yeasts at the species level, from traditional Irish cider fermentations. By combining the molecular approach and the presumptive media it was possible to distinguish between a large number of yeast species, and to track them within cider fermentations. The Irish cider fermentation process can be divided into three sequential phases based on the predominant yeast type present. Kloeckera/Hanseniaspora uvarum type yeasts predominate in the initial 'fruit yeast phase'. Thereafter Saccharomyces cerevisiae type yeast dominate in the 'fermentation phase', where the alcoholic fermentation takes place. Finally the 'maturation phase' which follows, is dominated by Dekkera and Brettanomyces type yeasts. H. uvarum type yeast were found to have originated from the fruit. Brettanomyces type yeast could be traced back to the press house, and also to the fruit. The press house was identified as having high levels of S. cerevisiae type yeast. A strong link was noted between the temperature profile of the cider fermentations, which ranged from 22 to 35 degrees C and the yeast strain population dynamics. CONCLUSIONS: Many different indigenous yeast species were identified. The mycology of Irish cider fermentations appears to be very similar to that which has previously been reported in the wine industry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has allowed us to gain a better understanding of the role of indigenous yeast species in 'Natural' Irish cider fermentations.