Leuconostoc performance in soy-based fermentations - Survival, acidification, sugar metabolism, and flavor comparisons.

Leuconostoc spp. is often regarded as the flavor producer, responsible for the production of acetoin and diacetyl in dairy cheese. In this study, we investigate seven plant-derived Leuconostoc strains, covering four species, in their potential as a lyophilized starter culture for flavor production in fermented soy-based cheese alternatives. We show that the process of lyophilization of Leuconostoc can be feasible using a soy-based lyoprotectant, with survivability up to 63% during long term storage. Furthermore, the storage in this media improves the subsequent growth in a soy-based substrate in a strain specific manner. The utilization of individual raffinose family oligosaccharides was strain dependent, with Leuconostoc pseudomesenteroides NFICC99 being the best consumer. Furthermore, we show that all investigated strains were able to produce a range of volatile flavor compounds found in dairy cheese products, as well as remove certain dairy off-flavors from the soy-based substrate like hexanal and 2-pentylfuran. Also here, NFICC99 was strain producing most cheese-related volatile flavor compounds, followed by Leuconostoc mesenteroides NFICC319. These findings provide initial insights into the development of Leuconostoc as a potential starter culture for plant-based dairy alternatives, as well as a promising approach for generation of stable, lyophilized cultures.

A multi-phase approach to select new wine yeast strains with enhanced fermentative fitness and glutathione production.

The genetic improvement of winemaking yeasts is a virtually infinite process, as the design of new strains must always cope with varied and ever-evolving production contexts. Good wine yeasts must feature both good primary traits, which are related to the overall fermentative fitness of the strain, and secondary traits, which provide accessory features augmenting its technological value. In this context, the superiority of "blind," genetic improvement techniques, as those based on the direct selection of the desired phenotype without prior knowledge of the genotype, was widely proven. Blind techniques such as adaptive evolution strategies were implemented for the enhancement of many traits of interest in the winemaking field. However, these strategies usually focus on single traits: this possibly leads to genetic tradeoff phenomena, where the selection of enhanced secondary traits might lead to sub-optimal primary fermentation traits. To circumvent this phenomenon, we applied a multi-step and strongly directed genetic improvement strategy aimed at combining a strong fermentative aptitude (primary trait) with an enhanced production of glutathione (secondary trait). We exploited the random genetic recombination associated to a library of 69 monosporic clones of strain UMCC 855 (Saccharomyces cerevisiae) to search for new candidates possessing both traits. This was achieved by consecutively applying three directional selective criteria: molybdate resistance (1), fermentative aptitude (2), and glutathione production (3). The strategy brought to the selection of strain 21T2-D58, which produces a high concentration of glutathione, comparable to that of other glutathione high-producers, still with a much greater fermentative aptitude.

Oxidative fermentations and exopolysaccharides production by acetic acid bacteria: a mini review.

Acetic acid bacteria are versatile organisms converting a number of carbon sources into biomolecules of industrial interest. Such properties, together with the need to limit chemical syntheses in favor of more sustainable biological processes, make acetic acid bacteria appropriate organisms for food, chemical, medical, pharmaceutical and engineering applications. At current, well-established bioprocesses by acetic acid bacteria are those derived from the oxidative pathways that lead to organic acids, ketones and sugar derivates. Whereas emerging applications include biopolymers, such as bacterial cellulose and fructans, which are getting an increasing interest for the biotechnological industry. However, considering the industrial demand of high performing bioprocesses, the production yield of metabolites obtained by acetic acid bacteria, is still not satisfying. In this paper we review the major acetic acid bacteria industrial applications, considering the current status of bioprocesses. We will also describe new biotechnological advances in order to optimize the industrial production, offering also an overview on future directions.

Evolved Saccharomyces cerevisiae wine strains with enhanced glutathione production obtained by an evolution-based strategy.

In winemaking, the application of glutathione (GSH) has been the subject of ever-growing interest because of its important role in limiting must and wine oxidation and in protecting various aromatic compounds. Glutathione concentration in wine is highly variable, involving as it does several factors from must, through alcoholic fermentation, to yeast strain activity. Consequently, the development of new wine yeast strains able to improve flavor stability is in great demand. To generate evolved Saccharomyces cerevisiae strains with enhanced GSH production, we have applied an evolution-based strategy that combines the sexual recombination of spores with the application of molybdate, which is toxic for the cells at high concentration, as specific selective pressure. Eight molybdate-resistant strains were selected and further screened for GSH production in synthetic grape must and in microvinification assay. By this nongenetically modified strategy, we obtained two evolved strains, Mo21T2-5 and Mo21T2-12, both able to enhance GSH content in wine with an increase of 100% and 36%, respectively, compared with the parental strain 21T2, and 120% and 50% compared with initial GSH content in the must.

A Microbial Co-Culturing System for Producing Cellulose-Hyaluronic Acid Composites.

In this study, a co-culture system combining bacterial cellulose (BC) producers and hyaluronic acid (HA) producers was developed for four different combinations. AAB of the genus Komagataeibacter sp. and LAB of the Lactocaseibacillus genus were used to produce BC and HA, respectively. Fourier-transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction were used to investigate changes in BC-HA composites chemical and morphological structure. Water absorption, uptake, and antibacterial properties were also tested. Outcomes highlighted a higher bacterial cellulose yield and the incorporation of hyaluronic acid into the composite. The presence of hyaluronic acid increased fiber dimension-nearly doubled for some combinations-which led to a decreased crystallinity of the composites. Different results were observed based on the BC producer and HA producer combination. However, water holding capacity (WHC) in all the samples improved with the presence of HA, while water uptake worsened. A thymol-enriched BC-HA composite showed high antibacterial activity against Escherichia coli DSM 30083T and Staphylococcus aureus DSM 20231T. Results could contribute to opening new applications in the cosmetics or pharmaceutical fields.

Acetobacter pasteurianus strain AB0220: cultivability and phenotypic stability over 9 years of preservation.

Acetobacter species are members of the α-subclass of Proteobacteria, which harbors a large number of bacteria recalcitrant to cultivation. Strain AB0220 was isolated from a superficial acetification system and preserved for 9 years by short and long time methods. Under short time preservation it was estimated that 540.54 number of generations occurred, whereas in long time preservation conditions the number of generations was 17.40. Ethanol oxidation to acetic acid was stable and confirmed, as well as acetate assimilation during long time preservation. Cultivability checks showed persistence of phenotypic traits (growth on ethanol and methanol, growth on different carbon sources and cellulose production) over the extended preservation time. 16S rRNA gene sequences analysis showed 100 % of similarity with A. pasteurianus (Accession number GQ240636). Stability of subcultures related to the culture age and subcultures frequency, tested by ERIC/PCR, confirmed the suitability of long term preservation at least over a period of 9 years.

Candidate Acetic Acid Bacteria Strains for Levan Production.

In this study, twelve strains of acetic acid bacteria (AAB) belonging to five different genera were tested for their ability to produce levan, at 70 and 250 g/L of sucrose concentration, respectively. The fructan produced by the bacterial strains was characterized as levan by NMR spectroscopy. Most of the strains produced levan, highlighting intra- and inter-species variability. High yield was observed for Neoasaia chiangmaiensis NBRC 101099 T, Kozakia baliensis DSM 14400 T and Gluconobacter cerinus DSM 9533 T at 70 g/L of sucrose. A 12-fold increase was observed for N. chiangmaiensis NBRC 101099 T at 250 g/L of sucrose concentration. Levan production was found to be affected by glucose accumulation and pH reduction, especially in Ko. baliensis DSM 14400 T. All the Gluconobacter strains showed a negative correlation with the increase in sucrose concentration. Among strains of Komagataeibacter genus, no clear effect of sucrose on levan yield was found. Results obtained in this study highlighted the differences in levan yield among AAB strains and showed interdependence between culture conditions, carbon source utilization, and time of incubation. On the contrary, the levan yield was not always related to the sucrose concentration.

Anti-Spoilage Activity and Exopolysaccharides Production by Selected Lactic Acid Bacteria.

In this study, eight lactic acid bacteria (LAB) strains, previously isolated from traditional and gluten-free sourdoughs, and selected for their potential in improving the sensory and rheological quality of bakery products, were screened against some common spoilage agents. The anti-mould activity was tested using strains of the species Fusarium graminearum, Aspergillus flavus, Penicillium paneum and Aspergillus niger. Regarding the antibacterial activity, it was assessed against four strains of the species Escherichia coli, Campylobacter jejuni, Salmonella typhimurium and Listeria monocytogenes. Furthermore, LAB strains were evaluated for their ability to produce exopolysaccharides, which are gaining considerable attention for their functional properties and applicability in different food industrial applications. A strain-specific behaviour against the moulds was observed. In particular, F. graminearum ITEM 5356 was completely inhibited by all the LAB strains. Regarding the antibacterial activity, the strains Leuconostoc citreum UMCC 3011, Lactiplantibacillus plantarum UMCC 2996, and Pediococcus pentosaceus UMCC 3010 showed wide activity against the tested pathogens. Moreover, all the LAB strains were able to produce exopolysaccharides, which were preliminarily characterized. The assessed features of the LAB strains allow us to consider them as promising candidates for single or multiple starter cultures for food fermentation processes.

Yeasts and Lactic Acid Bacteria for Panettone Production: An Assessment of Candidate Strains.

The recovery of yeasts and lactic acid bacteria (LAB) involved in sourdough fermentation is the first step in the selection of starters with suitable technological aptitude and capable of producing desired aromas and/or aromatic precursors. In this work, two sourdoughs samples (MA and MB) and the derived doughs (samples A and B) were collected from a bakery during artisanal Panettone manufacture. Yeasts and bacteria were isolated at different fermentation steps on selective agar media. A total of 77 isolates were obtained and characterized. Representative strains of yeasts and LAB were identified by sequencing the D1/D2 domain of the 26S rRNA and the 16S rRNA genes, respectively. Moreover, the volatile organic compounds (VOCs) produced in the collected samples were detected and correlated to the species found in the same samples. The results highlighted the occurrence of Kazachstania humilis in both samples A and B, while Saccharomyces cerevisiae strains were detected only in samples B. Among LAB, Fructilactobacillus sanfranciscensis was the main species detected in both sourdoughs. Furthermore, strains belonging to the species Lactiplantibacillus plantarum, Furfurilactobacillus rossiae, Lactobacillus parabuchneri, Leuconostoc citreum, and Leuconostoc mesenteroides were assessed in the dough samples.

A Mathematical Modeling of Freezing Process in the Batch Production of Ice Cream.

This study deals with the mathematical modeling of crystallization kinetics occurring during batch production of the ice cream. The temperature decrease was recorded in-situ through a computerized wireless system. A robust pattern-recognition algorithm of the experimental cooling curves was developed to determine the initial freezing point. The theoretical freezing point was used to calibrate the whole time-temperature profile. Finally, a modified Gompertz's function was used to describe the main steps of crystallization kinetics. Derivative analysis of the Gompertz's function allowed to determine the time-temperature physical markers of dynamic nucleation, ice crystal growth and air whipping. Composition and freezing properties were used as input variables in multivariate analysis to classification purposes of the ice cream mixtures as a function of their ability to produce high-quality ice cream. The numerical analysis of the whole cooling curve was used to build predictive models of the ice cream quality indices.

Succession of selected strains of Acetobacter pasteurianus and other acetic acid bacteria in traditional balsamic vinegar.

The application of a selected Acetobacter pasteurianus strain for traditional balsamic vinegar production was assessed. Genomic DNA was extracted from biofilms after enrichment cultures on GYC medium (10% glucose, 1.0% yeast extract, 2.0% calcium carbonate) and used for PCR/denaturing gradient gel electrophoresis, 16S rRNA gene sequencing, and enterobacterial repetitive intergenic consensus/PCR sequencing. Results suggested that double-culture fermentation is suitable for traditional balsamic vinegar acetification.

Preservation, Characterization and Exploitation of Microbial Biodiversity: The Perspective of the Italian Network of Culture Collections.

Microorganisms represent most of the biodiversity of living organisms in every ecological habitat. They have profound effects on the functioning of any ecosystem, and therefore on the health of our planet and of human beings. Moreover, microorganisms are the main protagonists in food, medical and biotech industries, and have several environmental applications. Accordingly, the characterization and preservation of microbial biodiversity are essential not only for the maintenance of natural ecosystems but also for research purposes and biotechnological exploitation. In this context, culture collections (CCs) and microbial biological resource centres (mBRCs) are crucial for the safeguarding and circulation of biological resources, as well as for the progress of life sciences. This review deals with the expertise and services of CCs, in particular concerning preservation and characterization of microbial resources, by pointing to the advanced approaches applied to investigate a huge reservoir of microorganisms. Data sharing and web services as well as the tight interconnection between CCs and the biotechnological industry are highlighted. In addition, guidelines and regulations related to quality management systems (QMSs), biosafety and biosecurity issues are discussed according to the perspectives of CCs and mBRCs.

Genus-specific profile of acetic acid bacteria by 16S rDNA PCR-DGGE.

An effective method for grouping acetic acid bacteria (AAB) genera was defined and evaluated as a tool for preliminary screening of the major AAB species involved in vinegar production. Acetobacter, Gluconobacter, Gluconacetobacter, Asaia, Neoasaia, Saccharibacter, Frateuria and Kozakia AAB strains were screened on the basis of the 16S rDNA sequences using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique. The DGGE profile of all the strains tested, consisted of one single band of approximately 330 bp for each strain and allowed their clustering. The results obtained clearly reflected in silico phylogenetic analysis of the AAB species used in this study, in fact, the species with a higher 16S rDNA sequence homology showed a similar electrophoretic profile. In particular almost all the species belonging to the genus Gluconacetobacter showed a DGGE pattern nearly identical and well distinct from all the other AAB genera. Furthermore by PCR-DGGE it was possible to clearly group the species more frequently recovered from vinegar fermentation which are mainly distributed in the genera Acetobacter, Gluconobacter and Gluconacetobacter.

High-glutathione producing yeasts obtained by genetic improvement strategies: a focus on adaptive evolution approaches for novel wine strains.

Glutathione (GSH) is the most abundant non-protein thiol in living organisms. Due to its important antioxidant role, it is widely used in medicine, as a food additive, and in the cosmetic industry. Recently, GSH has received growing attention in winemaking because of its ability to control oxidative spoilage damage and to protect various aromatic compounds. Indeed, GSH concentration in wine is highly variable and several factors are involved in its regulation, ranging from grape must to yeast fermentation activity. This short review aims at highlighting the common genetic strategies, useful for obtaining wine yeasts with enhanced GSH production, paying particular attention to the adaptive evolution approaches. Moreover, other strategies, such as random mutagenesis, metabolic engineering and hybridization have been briefly reviewed with a stress on both their strengths and weaknesses in terms of actual feasibility and acceptance by wine consumers.

Genetic variation and expression changes associated with molybdate resistance from a glutathione producing wine strain of Saccharomyces cerevisiae.

Glutathione (GSH) production during wine fermentation is a desirable trait as it can limit must and wine oxidation and protect various aromatic compounds. UMCC 2581 is a Saccharomyces cerevisiae wine strain with enhanced GSH content at the end of wine fermentation. This strain was previously derived by selection for molybdate resistance following a sexual cycle of UMCC 855 using an evolution-based strategy. In this study, we examined genetic and gene expression changes associated with the derivation of UMCC 2581. For genetic analysis we sporulated the diploid UMCC 855 parental strain and found four phenotype classes of segregants related to molybdate resistance, demonstrating the presence of segregating variation from the parental strain. Using bulk segregant analysis we mapped molybdate traits to two loci. By sequencing both the parental and evolved strain genomes we identified candidate mutations within the two regions as well as an extra copy of chromosome 1 in UMCC 2581. Combining the mapped loci with gene expression profiles of the evolved and parental strains we identified a number of candidate genes with genetic and/or gene expression changes that could underlie molybdate resistance and increased GSH levels. Our results provide insight into the genetic basis of GSH production relevant to winemaking and highlight the value of enhancing wine strains using existing variation present in wine strains.

Characterization of acetic acid bacteria in "traditional balsamic vinegar".

This study evaluated the glucose tolerance of acetic acid bacteria strains isolated from Traditional Balsamic Vinegar. The results showed that the greatest hurdle to acetic acid bacteria growth is the high sugar concentration, since the majority of the isolated strains are inhibited by 25% of glucose. Sugar tolerance is an important technological trait because Traditional Balsamic Vinegar is made with concentrated cooked must. On the contrary, ethanol concentration of the cooked and fermented must is less significant for acetic acid bacteria growth. A tentative identification of the isolated strains was done by 16S-23S-5S rDNA PCR/RFLP technique and the isolated strains were clustered: 32 strains belong to Gluconacetobacter xylinus group, two strains to Acetobacter pasteurianus group and one to Acetobacter aceti.

Application of denaturing gradient gel electrophoresis (DGGE) analysis to evaluate acetic acid bacteria in traditional balsamic vinegar.

Acetic acid bacteria (AAB) are fastidious micro-organisms to isolate and cultivate despite of the great number of growth media available. Moreover, conventional techniques used to study AAB populations are time consuming and not completely reliable. In this study, we tested the usefulness of the polymerase chain reaction-denaturing gradient gel electophoresis (PCR-DGGE) as a rapid and cost effective method for the screening of AAB in traditional balsamic vinegar (TBV). DGGE analysis was applied to 19 AAB strains isolated by agar plating from three different samples of TBV. DGGE was also used for the analysis of PCR products obtained from DNA extracted directly from the TBV samples. A tentative species identification was achieved comparing the PCR-DGGE patterns of the isolated strains and the TBV samples to those of 15 AAB reference strains. The results support that DGGE is functional to monitor vinegar's AAB population.

Limitations on the use of polymerase chain reaction--restriction fragment length polymorphism analysis of the rDNA NTS2 region for the taxonomic classification of the species Saccharomyces cerevisiae.

Different molecular techniques were tested to determine which was the most effective in the identification of Saccharomyces cerevisiae strains. In particular, polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) regions and the nontranscribed spacer 2 (NTS2) region, sequencing of the D1/D2 domain, and electrophoretic karyotyping were applied to 123 yeast strains isolated from different sourdoughs and tentatively attributed to the species S. cerevisiae. All of the strains tested showed an identical PCR-RFLP pattern for the ITS regions, an identical nucleotide sequence of the D1/D2 domain, and the typical electrophoretic karyo type of S. cerevisiae. In contrast, 14 out of the 123 strains tested showed some polymorphism with BanI restriction analysis of the NTS2 region. Our results indicate that while the sequencing of the D1/D2 domain, the PCR-RFLP analysis of the ITS regions, and the electrophoretic karyotype can be employed successfully to identify S. cerevisiae strains, PCR-RFLP analysis of the NTS2 region does not allow a consistent and accurate grouping for S. cerevisiae strains. The fact that the NTS2 region of a small number of strains (8.78% of the total strains tested) is different from that of the other S. cerevisiae strains confirms that molecular methods should always be tested on a great number of strains.