Genetic improvement of non-conventional Torulaspora delbrueckii for traditional sparkling winemaking by mixing for eventual hybridization with Saccharomyces cerevisiae.

Non-conventional yeasts such as Torulaspora delbrueckii (Td) have been proposed for sparkling winemaking. Unfortunately, this yeast has poor efficiency in completing wine fermentation as compared to Saccharomyces cerevisiae (Sc). New mutants with increased resistance to SO2, ethanol, and high CO2 pressure were previously isolated from spore clones of Td. Although these mutants showed improved capability for base wine fermentation, there is still room for genetic improvement of Td yeasts until the fermentative capacity of Sc is achieved. As an alternative approach, yeast mixture for eventual hybridization of Td with Sc was assayed in this study. The new yeast mixture clones (Sc-mixed Td) showed an intermediate phenotype between both parent yeasts for some relevant biotechnological properties, such as resistance to SO2, ethanol, copper, high CO2 pressure, and high temperature, as well as flocculation potential. These properties varied depending on the specific Sc-mixed Td clone. Several mixture clones showed improved capability for base wine fermentation as compared to the Td parent strain, approaching the fermentation capability of the Sc parent strain. The organoleptic quality of sparkling wine was also improved by using some mixture clones and this improved quality coincided with an increased amount of acetate and ethyl esters. The genetic stability of some Sc-mixed Td clones was good enough for commercial yeast production and winery applications.

Evaluating the effect of using non-Saccharomyces on Oenococcus oeni and wine malolactic fermentation.

Interest in using non-Saccharomyces yeasts in winemaking has increased in recent years due to their ability to improve wine quality. However, little information has been published regarding the possible effect on malolactic fermentation (MLF), carried out mostly by Oenococcus oeni. The aim of this paper is therefore to evaluate the effect of the most representative non-Saccharomyces species on O. oeni and wine MLF. Different strains of Torulaspora delbrueckii, Metschnikowia pulcherrima, Hanseniaspora uvarum, Hanseniaspora vineae and Starmerella bacillaris (syn. Candida zemplinina) were used in sequential alcoholic fermentation with Saccharomyces cerevisiae. The resulting wines were inoculated with four O. oeni strains. The action of non-Saccharomyces affected the final wine composition and the later role of O. oeni. Some of its strains could not perform MLF in H. uvarum wine due to high SO2 concentrations. In some cases, MLF was inhibited in wines inoculated with S. bacillaris. All the H. uvarum and H. vineae strains notably increased acetic acid concentrations, thus threatening wine quality. The best conditions for MLF were provided by some T. delbruecckii and M. pulcherrima strains, which showed increased concentrations of mannoproteins - compounds described as MLF activators -, no production of SO2, and low consumption of L-malic acid. In conclusion, non-Saccharomyces yeasts have diverse effects on O. oeni and MLF depending on the species, with T. delbrueckii and M. pulcherrima being those that showed the best compatibility with MLF development.

Effects of Different Stress Parameters on Growth and on Oleuropein-Degrading Abilities of Lactiplantibacillus plantarum Strains Selected as Tailored Starter Cultures for Naturally Table Olives.

The use of ß-glucosidase positive strains, as tailored-starter cultures for table olives fermentation, is a useful biotechnological tool applied to accelerate the debittering process. Nowadays, strains belonging to Lactiplantibacillus plantarum species are selected for their high versatility and tolerance to stress conditions. The present study investigated the effect of different stress factors (pH, temperature and NaCl) on growth and on oleuropein-degrading abilities of selected L. plantarum strains. In addition, the presence of the beta-glucosidase gene was investigated by applying a PCR based approach. Results revealed that, overall, the performances of the tested strains appeared to be robust toward the different stressors. However, the temperature of 16 °C significantly affected the growth performance of the strains both singularly and in combination with other stressing factors since it prolongs the latency phase and reduces the maximum growth rate of strains. Similarly, the oleuropein degradation was mainly affected by the low temperature, especially in presence of low salt content. Despite all strains displayed the ability to reduce the oleuropein content, the beta-glucosidase gene was detected in five out of the nine selected strains, demonstrating that the ability to hydrolyze the oleuropein is not closely related to the presence of beta-glucosidase. Data of the present study suggest that is extremely important to test the technological performances of strains at process conditions in order to achieve a good selection of tailored starter cultures for table olives.

Genetic Improvement of Torulaspora delbrueckii for Wine Fermentation: Eliminating Recessive Growth-Retarding Alleles and Obtaining New Mutants Resistant to SO2, Ethanol, and High CO2 Pressure.

The use of Torulaspora delbrueckii has been repeatedly proposed to improve a wine's organoleptic quality. This yeast has lower efficiency in completing wine fermentation than Saccharomyces cerevisiae since it has less fermentation capability and greater sensitivity to SO2, ethanol, and CO2 pressure. Therefore, the completion of fermentation is not guaranteed when must or wine is single-inoculated with T. delbrueckii. To solve this problem, new strains of T. delbrueckii with enhanced resistance to winemaking conditions were obtained. A genetic study of four wine T. delbrueckii strains was carried out. Spore clones free of possible recessive growth-retarding alleles were obtained from these yeasts. These spore clones were used to successively isolate mutants resistant to SO2, then those resistant to ethanol, and finally those resistant to high CO2 pressure. Most of these mutants showed better capability for base wine fermentation than the parental strain, and some of them approached the fermentation capability of S. cerevisiae. The genetic stability of the new mutants was good enough to be used in industrial-level production in commercial wineries. Moreover, their ability to ferment sparkling wine could be further improved by the continuous addition of oxygen in the culture adaptation stage prior to base wine inoculation.

The use of multifunctional yeast-lactobacilli starter cultures improves fermentation performance of Spanish-style green table olives.

In this work, Lactobacillus pentosus LPG1, Lactobacillus pentosus Lp13, Lactobacillus plantarum Lpl15, and Wickerhanomyces anomalous Y12, all of them previously isolated from fermented table olive biofilms, were used (alone or in combination) as multifunctional starters for Manzanilla Spanish-style green table olive fermentations. Their performances were evaluated through the changes in the key physico-chemical and microbiological parameters, correlation between AI-2 production and biofilm formation, inoculum imposition, metataxonomic analysis and sensory characteristics of the finished products. Inoculation only with lactic acid bacteria (LAB) strains led to higher titratable acidities and lower pH values than the spontaneous fermentation (non-inoculated control), mainly during the first steps of processing. However, the sequential inoculation of the yeast and then the combination of the 3 LAB strains showed the most favourable evolution. LPG1 strain and, particularly Lp13, were excellent biofilms former and showed the major imposition on the fruit epidermis, as corroborated by rep-PCR analysis. Production of AI-2 was lower in the treatment inoculated exclusively with yeast Y12 but had the highest presence in the sequential yeast-LAB inoculum, with its maximum concentration and maximum LAB population on fruits (19th days) strongly related. Metataxonomic analysis of the biofilms at the end of the fermentation revealed, in addition to Lactobacillus, high proportions of sequences from genera Marinilactobacillus, Alkalibacterium, Halolactobacillus, and low levels of Halomonas and Aerococcus. Compositional data analysis of the omics data revealed that Lpl15 was scarcely efficient for controlling the spontaneous microbiota since its treatment presented the highest proportions of Aerococcus genus. Finally, the sensory analysis showed similar characteristics for the treatment inoculated with LPG1 and the spontaneous process, with olives inoculated with the yeast (alone or in combination with Lactobacillus strains) showing attractive scores. Then, inoculation of Spanish-style table olive fermentations with a sequential yeast and LAB combination could be an advisable practice.

Probiotic potential of a Lactobacillus rhamnosus cheese isolate and its effect on the fecal microbiota of healthy volunteers.

The present study describes an in vitro characterization of strains of lactic acid bacteria, focusing on physiological characters of probiotic interest, and a subsequent placebo-controlled, crossover administration trial, with a cohort of healthy volunteers. The strains of lactic acid bacteria were previously isolated from a fermented food (ripened cheese) and several ones resulted to have promising probiotic characteristics. Based on comprehensive evaluation of the data obtained, one strain was chosen and supplemented in a fermented milk. The fermented milk was then used in the administration trial with the goal of assessing its effect on the composition of the intestinal microbiota, as reflected in the feces. The fermented milk, with or without probiotic, had an effect on the intestinal microbiota and significant inter-individual differences were observed in response to the intervention. A common trend was observed related to two important populations of the human gut microbiota; a reduction in the relative abundance of Bacteroides and increase in the abundance of Prevotella in subjects during treatment compared to baseline were registered.

Effect of Sequential Inoculum of Beta-Glucosidase Positive and Probiotic Strains on Brine Fermentation to Obtain Low Salt Sicilian Table Olives.

In the present study, the ß-glucosidase positive strain Lactobacillus plantarum F3. 3 was used as starter during the fermentation of Sicilian table olives (Nocellara Etnea cultivar) at two different salt concentrations (5 and 8%), in order to accelerate the debittering process. The latter was monitored through the increase of hydroxytyrosol compound. In addition, the potential probiotic Lactobacillus paracasei N24 strain was added after 60 days of fermentation. Un-inoculated brine samples at 5 and 8% of salt were used as control. The fermentation was monitored till 120 days through physico-chemical and microbiological analyses. In addition, volatile organic compounds and sensorial analyses were performed during the process and at the end of the fermentation, respectively. Lactic acid bacteria and yeasts were, in depth, studied by molecular methods and the occurrence of the potential probiotic N24 strain in the final products was determined. Results highlighted that inoculated brines exhibited a higher acidification and debittering rate than control ones. In addition, inoculated brines at 5% of salt exhibited higher polyphenols (hydoxytyrosol, tyrosol, and verbascoside) content compared to samples at 8% of NaCl, suggesting a stronger oleuropeinolytic activity of the starter at low salt concentration. Lactobacilli and yeasts dominated during the fermentation process, with the highest occurrence of L. plantarum and Wickerhamomyces anomalus, respectively. Moreover, the potential probiotic L. paracasei N24 strain was able to survive in the final product. Hence, the sequential inoculum of beta-glucosidase positive and potential probiotic strains could be proposed as a suitable technology to produce low salt Sicilian table olives.

Reduction of the Bitter Taste in Packaged Natural Black Manzanilla Olives by Zinc Chloride.

The work assays the use of various concentrations of ZnCl2 (0.0-0.1%, w/v) in packaged natural black Manzanilla table olives. The transformations were followed for 4 months. The presence of Zn modified the leaching of total sugars (sucrose, glucose, fructose, and mannitol) into the brine, which decreased as the ZnCl2 content increased. Over the study, sucrose and glucose were exhausted while fructose, although consumed, left some final residues and the use of mannitol was limited. Titratable acidity was always gradually formed causing the subsequent pH decrease, which stabilized at ≈3.5. Acetic and mainly lactic acid were also formed during the assay, reaching the highest level of lactic acid in the 0.050% ZnCl2 treatment, followed by the Control. The acids were formed by lactic acid bacteria (LAB) (Lactobacillus pentosus, 39%, and Lactobacillus plantarum, 61%). However, the most outstanding Zn effect was found on the olive sensory characteristics: its presence markedly reduced the bitter notes, increased the overall appreciation, and the treatment containing 0.075% ZnCl2 had the highest scores in hardness, crunchiness, and overall appreciation. Therefore, the addition of ZnCl2 into packaged natural table olives may lead to healthy products with desirable sensory characteristics which, in turn, could promote consumption.

Draft Genome Sequences of Six Lactobacillus pentosus Strains Isolated from Brines of Traditionally Fermented Spanish-Style Green Table Olives.

Here, we report the genome sequences of six Lactobacillus pentosus strains isolated from traditional noninoculated Spanish-style green table olive brines. The total genome sizes varied between 3.77 and 4.039 Mbp. These genome sequences will assist in revealing the genes responsible for both technological and probiotic properties of these strains.

Non-Saccharomyces in Wine: Effect Upon Oenococcus oeni and Malolactic Fermentation.

This work is a short review of the interactions between oenological yeasts and lactic acid bacteria (LAB), especially Oenococcus oeni, the main species carrying out the malolactic fermentation (MLF). The emphasis has been placed on non-Saccharomyces effects due to their recent increased interest in winemaking. Those interactions are variable, ranging from inhibitory, to neutral and stimulatory and are mediated by some known compounds, which will be discussed. One phenomena responsible of inhibitory interactions is the media exhaustion by yeasts, and particularly a decrease in L-malic acid by some non-Saccharomyces. Clearly ethanol is the main inhibitory compound of LAB produced by S. cerevisiae, but non-Saccharomyces can be used to decrease it. Sulfur dioxide and medium chain fatty acids (MCFAs) produced by yeasts can exhibit inhibitory effect upon LAB or even result lethal. Interestingly mixed fermentations with non-Saccharomyces present less MCFA concentration. Among organic acids derived as result of yeast metabolism, succinic acid seems to be the most related with MLF inhibition. Several protein factors produced by S. cerevisiae inhibiting O. oeni have been described, but they have not been studied in non-Saccharomyces. According to the stimulatory effects, the use of non-Saccharomyces can increase the concentration of favorable mediators such as citric acid, pyruvic acid, or other compounds derived of yeast autolysis such as peptides, glucans, or mannoproteins. The emergence of non-Saccharomyces in winemaking present a new scenario in which MLF has to take place. For this reason, new tools and approaches should be explored to better understand this new winemaking context.

Assessing the Challenges in the Application of Potential Probiotic Lactic Acid Bacteria in the Large-Scale Fermentation of Spanish-Style Table Olives.

This work studies the inoculation conditions for allowing the survival/predominance of a potential probiotic strain (Lactobacillus pentosus TOMC-LAB2) when used as a starter culture in large-scale fermentations of green Spanish-style olives. The study was performed in two successive seasons (2011/2012 and 2012/2013), using about 150 tons of olives. Inoculation immediately after brining (to prevent wild initial microbiota growth) followed by re-inoculation 24 h later (to improve competitiveness) was essential for inoculum predominance. Processing early in the season (September) showed a favorable effect on fermentation and strain predominance on olives (particularly when using acidified brines containing 25 L HCl/vessel) but caused the disappearance of the target strain from both brines and olives during the storage phase. On the contrary, processing in October slightly reduced the target strain predominance on olives (70-90%) but allowed longer survival. The type of inoculum used (laboratory vs. industry pre-adapted) never had significant effects. Thus, this investigation discloses key issues for the survival and predominance of starter cultures in large-scale industrial fermentations of green Spanish-style olives. Results can be of interest for producing probiotic table olives and open new research challenges on the causes of inoculum vanishing during the storage phase.

Transcriptomic and Proteomic Analysis of Oenococcus oeni Adaptation to Wine Stress Conditions.

Oenococcus oeni, the main lactic acid bacteria responsible for malolactic fermentation in wine, has to adapt to stressful conditions, such as low pH and high ethanol content. In this study, the changes in the transcriptome and the proteome of O. oeni PSU-1 during the adaptation period before MLF start have been studied. DNA microarrays were used for the transcriptomic analysis and two complementary proteomic techniques, 2-D DIGE and iTRAQ labeling were used to analyze the proteomic response. One of the most influenced functions in PSU-1 due to inoculation into wine-like medium (WLM) was translation, showing the over-expression of certain ribosomal genes and the corresponding proteins. Amino acid metabolism and transport was also altered and several peptidases were up regulated both at gene and protein level. Certain proteins involved in glutamine and glutamate metabolism showed an increased abundance revealing the key role of nitrogen uptake under stressful conditions. A strong transcriptional inhibition of carbohydrate metabolism related genes was observed. On the other hand, the transcriptional up-regulation of malate transport and citrate consumption was indicative of the use of L-malate and citrate associated to stress response and as an alternative energy source to sugar metabolism. Regarding the stress mechanisms, our results support the relevance of the thioredoxin and glutathione systems in the adaptation of O. oeni to wine related stress. Genes and proteins related to cell wall showed also significant changes indicating the relevance of the cell envelop as protective barrier to environmental stress. The differences found between transcriptomic and proteomic data suggested the relevance of post-transcriptional mechanisms and the complexity of the stress response in O. oeni adaptation. Further research should deepen into the metabolisms mostly altered due to wine conditions to elucidate the role of each mechanism in the O. oeni ability to develop MLF.

RT-PCR-DGGE Analysis to Elucidate the Dominant Bacterial Species of Industrial Spanish-Style Green Table Olive Fermentations.

This paper describes the dominant bacterial species metabolically active through the industrial production of Spanish-style Manzanilla and Gordal olives. For this purpose, samples (brines and fruits) obtained at 0, 15, and 90 fermentation days were analyzed by a culture-independent approach to determine viable cells by reverse transcription of RNA and further PCR-DGGE analysis, detecting at least 7 different species. Vibrio vulnificus, Lactobacillus plantarum group, and Lactobacillus parafarraginis were present in samples from both cultivars; Lactobacillus sanfranciscensis and Halolactobacillus halophilus were detected only in Gordal samples, while Staphylococcus sp. was exclusively found at the onset of Manzanilla fermentations. Physicochemical data showed a typical fermentation profile while scanning electron microscopy confirmed the in situ biofilm formation on the olive epidermis. Different Bacillus, Staphylococcus, and Enterococcus species, not detected during the fermentation process, were also found in the solid marine salt used by the industry for preparation of brines. Elucidation of these non-lactic acid bacteria species role during fermentation is then an appealingly challenge, particularly regarding safety issues.

Data on green Spanish-style Manzanilla table olives fermented in salt mixtures.

This article contains processed data related to the research published in "Fermentation in nutrient salt mixtures affects green Spanish-style Manzanilla table olives" [1]. It displays information on the salt substitution by other nutrient salts (potassium chloride and calcium chloride) during fermentation of green Spanish-style Manzanilla table olives to produce healthier products. Particularly, it studies the relationship between the different colour parameters (L*, a*, b* and C i), firmness, and sensory attributes (saltiness, bitterness, hardness, and fibrousness), and the composition of the initial brine in NaCl, KCl, and CaCl2. The composition of the brines affected the characteristics of the product. In general, the higher was the proportion of CaCl2 in the initial brines the better was the colour. Also, the presence of this salt mitigated the saltiness perception but increment those of bitterness, hardness, fibrousness, and crunchiness. Besides, most of the sensory attribute scores could successfully be predicted as a function of the Na, K, and Ca concentrations in the fermented olive flesh. The work allows the production of table olives with specific characteristics and predetermined mineral nutrient composition.

Fermentation in nutrient salt mixtures affects green Spanish-style Manzanilla table olive characteristics.

This work studies the effects of the substitution of NaCl with KCl and CaCl2 on the physicochemical, mineral and sensory profile of fermented green Spanish-style Manzanilla olives, using an enlarged centroid mixture design. An increasing presence of CaCl2 in the initial brines improved the colour index, L(∗), b(∗) values, and firmness. The Na in the olives decreased (linearly) while the levels of K and Ca increased (quadratic) as a function of the KCl and CaCl2 concentrations in the initial brines. CaCl2 also improved the retention of Zn and P in the flesh. PLS showed a strong relationship between Ca and bitterness, hardness, fibrousness, crunchiness and saltiness (negative) and allowed for the prediction of sensory attributes (except acid) from the mineral contents in the flesh. Most of the treatments could lead to new green Spanish-style Manzanilla olive presentations with reduced Na and healthier characteristics.

The effect of ZnCl2 on green Spanish-style table olive packaging, a presentation style dependent behaviour.

BACKGROUND: Zinc chloride has been used previously as a preservative in directly brined olives with promising results. However, this is the first time that the effects of ZnCl2 addition (0-1 g L(-1) ) on green Spanish-style table olive (cv. Manzanilla) packaging has been studied. RESULTS: The presence of ZnCl2 affected the physico-chemical characteristics of the products; the presence of the Zn led to lower pH values (particularly just after packaging) and titratable and combined acidity values than the control but did not produce clear trends in the colour parameters. No Enterobacteriaceae were found in any of the treatments evaluated. At the highest ZnCl2 concentrations, the lactic acid bacteria were inhibited while, unexpectedly, its presence showed a lower effect than potassium sorbate against the yeast population. Regardless of the use of potassium sorbate or ZnCl2 , the packages had a reduced microbial biodiversity because only Lactobacillus pentosus and Pichia galeiformis were found at the end of the shelf life. With respect to organoleptic characteristics, the presentations containing ZnCl2 were not differentiated from the traditional product. CONCLUSION: Zinc chloride was less efficient than potassium sorbate as a yeast inhibitor in green Spanish-style olives, showing clear presentation style dependent behaviour for this property. Its presence produced significant changes in chemical parameters but scarcely affected colour or sensory characteristics.

Influence of yeasts on the oil quality indexes of table olives.

After moisture, fat is the major constituent of table olives. However, scarce studies have been carried out to determine the influence of microorganisms and type of processing on the modification of their quality indexes. The present survey studies the influence of lipolytic (Candida boidinii TOMC Y5 and Wickerhamomyces anomalus TOMC Y10) and nonlipolytic (Debaryomyces etchellsii TOMC Y9 and Pichia galeiformis TOMC Y27) yeasts on the oil quality indexes of Manzanilla and Hojiblanca green fruits processed as directly brined and lye-treated table olives. Overall, the inocula scarcely used available sugars, except the lipolytic C. boidinii strain in lye-treated olives. Acetic acid production was limited in all conditions, except for the D. etchellsii strain in directly brined Manzanilla fruits. Ethanol formation was also reduced, although the W. anomalus (in both types of elaboration) and the C. boidinii (in lye-treated olives) strains produced significantly higher proportions. Apparently, changes in the oil quality indexes of processed olives were not related to the presence of yeasts, and hence, could have been caused by the endogenous activity of the fruits. A principal component analysis using the microbiological, physicochemical, and oil quality data supported this hypothesis, grouping treatments according to olive variety and type of elaboration, while segregation due to yeast inocula was not observed.

Pigment, physicochemical, and microbiological changes related to the freshness of cracked table olives.

The changes in chloroplastic pigments, mineral nutrients, and characteristics related to freshness were studied during storage and packing of cracked seasoned olives. Cracking produced an initial loss in green pigments and color degradation. Later, storage caused a progressive degradation of chlorophylls and carotenoids, with a slower rate in refrigerated fruits (which preserved the greenish tones better), but after packing (and storage at room temperature), the differential effect disappeared and, at the end of the study, all olives showed similar pigment transformations, which were correlated with CIE a* and hue. Processing led to a Na content increase in olive flesh (and Ca and Zn, when added) but marked losses in the other mineral nutrients. Sodium metabisulfite and ZnCl2 promoted LAB growth while inhibiting yeast, thus enhancing product stability, and erythorbic acid caused yeast growth and firmness deterioration.