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The use of malolactic starter cultures, often offer no guarantee of microbiological success due to the chemical and physical factors (pH, ethanol, SO2, nutrient availability) that occur during the winemaking process. This study was born with the aim of improving the performance of the lactic acid bacteria used as a starter culture in the de-acidification of wines. Two commercial strains of Oenococcus oeni, were used. Was evaluated the effect of exogenous l-proline added during the bacterial growth, on the improvement of their survival in the presence of different ethanol concentrations and their ability to degrade l-malic acid in synthetic wine with the presence of 12% (v/v) and 13% (v/v) of ethanol. The results showed that l-proline improve ethanol tolerance and so the malolactic performances of O. oeni. This work represents an important strategy to ensure good vitality and improve the performance of the malolactic starter.

In industrial winery, the use of mixed starter cultures composed of Saccharomyces and non-Saccharomyces yeasts is an approach of growing importance for winemakers to enhance the sensory and complexity of the wine without compromising the quality. In this work, the oenological properties and enzymatic activities of 196 non-Saccharomyces yeasts, belonging to Hanseniaspora guilliermondii and Hanseniaspora uvarum species, were investigated. This screening has allowed the selection of the best non-Saccharomyces yeast strain and the use of vinification for white grape of Campania Vitis vinifera cv Fiano, in an industrial scale. The experimental fermentations were performed in four different batches: batch A (H. guilliermondii BF1 and S. cerevisiae 404 in sequential inoculum); batch B only inoculum of S. cerevisiae 404; batch C without inoculation (spontaneous fermentation); batch D with the inoculum of H. guilliermondii BF1 strain. The results of chemical and sensorial analyses have showed that the best wine comes from batch A. In conclusion, the use of H. guilliermondii BF1 with good oenological properties and a strong β-glucosidase activity allowed to improve the sensorial complexity of the wine. Selected non-Saccharomyces yeast could be applied profitably to winemaking to enhance the quality of the wine using new fermentation technologies, and could be used in industrial winery.

In this work was evaluated the effect of sequential inoculum of Hanseniaspora uvarum AS27 strain and a commercial Saccharomyces cerevisiae yeast on the physical–chemical and organoleptic features of Aglianico, a traditional red wine of Southern Italy. Four fermentation treatments on a pilot scale were performed. In fermentation treatment A, the alcoholic fermentation was spontaneously conducted by the indigenous yeasts present in grape must. In the fermentation treatments B and C were inoculated respectively S. cerevisiae FE and H. uvarum AS27 strains, as a single starter. The fermentation treatment D was initially inoculated with H. uvarum AS27, and S. cerevisiae strain was added after 72 h (sequential inoculation). Microbiological, physical–chemical parameters and sensory profiles of the wines have been defined. The results showed that the use of H. uvarum AS27, in sequential inoculum with S. cerevisiae FE, influenced the wine composition, enriching it in polyphenolic and volatile compounds. Further, the sensory evaluation showed that the use of H. uvarum AS27 strain, in co-culture with S. cerevisiae, gives the wine more pleasant characteristics. Therefore, the results have highlighted how the use of particular non-Saccharomyces yeasts can represent a biotechnological resource in red wine production.

Apilactobacillus kunkeei is an insect symbiont with documented beneficial effects on the health of honeybees. It belongs to fructophilic lactic acid bacteria (FLAB), a subgroup of lactic acid bacteria (LAB) notably recognized for their safe status. This fact, together with its recurrent isolation from hive products that are traditionally part of the human diet, suggests its possible safe use as human probiotic. Our data concerning three strains of A. kunkeei isolated from bee bread and honeybee gut highlighted several interesting features, such as the presence of beneficial enzymes (β-glucosidase, β-galactosidase and leucine arylamidase), the low antibiotic resistance, the ability to inhibit P. aeruginosa and, for one tested strain, E. faecalis, and an excellent viability in presence of high sugar concentrations, especially for one strain tested in sugar syrup stored at 4 °C for 30 d. This datum is particularly stimulating, since it demonstrates that selected strains of A. kunkeei can be used for the probiotication of fruit preparations, which are often used in the diet of hospitalized and immunocompromised patients. Finally, we tested for the first time the survival of strains belonging to the species A. kunkeei during simulated gastrointestinal transit, detecting a similar if not a better performance than that showed by Lacticaseibacillus rhamnosus GG, used as probiotic control in each trial.

Apilactobacillus kunkeei is an insect symbiont with documented beneficial effects on the health of honeybees. It belongs to fructophilic lactic acid bacteria (FLAB), a subgroup of lactic acid bacteria (LAB) notably recognized for their safe status. This fact, together with its recurrent isolation from hive products that are traditionally part of the human diet, suggests its possible safe use as human probiotic. Our data concerning three strains of A. kunkeei isolated from bee bread and honeybee gut highlighted several interesting features, such as the presence of beneficial enzymes (&beta, glucosidase, &beta, galactosidase and leucine arylamidase), the low antibiotic resistance, the ability to inhibit P. aeruginosa and, for one tested strain, E. faecalis, and an excellent viability in presence of high sugar concentrations, especially for one strain tested in sugar syrup stored at 4 &deg, C for 30 d. This datum is particularly stimulating, since it demonstrates that selected strains of A. kunkeei can be used for the probiotication of fruit preparations, which are often used in the diet of hospitalized and immunocompromised patients. Finally, we tested for the first time the survival of strains belonging to the species A. kunkeei during simulated gastrointestinal transit, detecting a similar if not a better performance than that showed by Lacticaseibacillus rhamnosus GG, used as probiotic control in each trial.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a severe bacterial disease that affects larvae of honeybees. The present study evaluated, in vitro, antimicrobial activity of sixty-one Lactiplantibacillus plantarum strains, against P. larvae ATCC 9545. Five strains (P8, P25, P86, P95 and P100) that showed the greatest antagonism against P. larvae ATCC 9545 were selected for further physiological and biochemical characterizations. In particular, the hydrophobicity, auto-aggregation, exopolysaccharides production, osmotic tolerance, enzymatic activity and carbohydrate assimilation patterns were evaluated. The five L. plantarum selected strains showed suitable physical and biochemical properties for their use as probiotics in the honeybee diet. The selection and availability of new selected bacteria with good functional characteristics and with antagonistic activity against P. larvae opens up interesting perspectives for new biocontrol strategies of diseases such as AFB.

The use of malolactic starter cultures, often offer no guarantee of microbiological success due to the chemical and physical factors (pH, ethanol, SO(2), nutrient availability) that occur during the winemaking process. This study was born with the aim of improving the performance of the lactic acid bacteria used as a starter culture in the de-acidification of wines. Two commercial strains of Oenococcus oeni, were used. Was evaluated the effect of exogenous l-proline added during the bacterial growth, on the improvement of their survival in the presence of different ethanol concentrations and their ability to degrade l-malic acid in synthetic wine with the presence of 12% (v/v) and 13% (v/v) of ethanol. The results showed that l-proline improve ethanol tolerance and so the malolactic performances of O. oeni. This work represents an important strategy to ensure good vitality and improve the performance of the malolactic starter.

The present study, considering for the first time microbiological concerns due to the use of lemon albedo as a fat replacer, aimed at the selection of an anti-Listeria strain to be used as protective culture in low-fat southern Italian fermented sausages. In fact, these kinds of products require appropriate bio-protective strategies to avoid risks due to Listeria monocytogenes. Sixty-seven Lactiplantibacillus plantarum strains isolated from diverse sources were screened for their antimicrobial activity and their interaction with starter strains (Latilactobacillus sakei 152 and Staphylococcus xylosus MVS9). Lactiplantibacillus plantarum Lpls100, highlighting both listericidal activity and the ability to promote Staphylococcus xylosus MVS9 growth, was used as a protective strain in low-fat fermented sausages prepared with lemon albedo as a fat replacer. The effect of the albedo and the protective strain on the fermentation process and the final quality was ascertained. Results highlighted that the use of the albedo did not affect the growth of starter strains and enhanced some quality features, such as fatty acid profiles and certain sensory attributes. However, the albedo also produced a slow decrease in water activity, compromising the microbial quality. The anti-Listeria strain, enhancing coagulase negative cocci growth and exerting antimicrobial activity, avoided the inconveniences caused by the use of the albedo. Moreover, the anti-Listeria effectiveness was assessed through a challenge test using a Listeria cocktail. The study revealed that Lactiplantibacillus plantarum Lpls100, regardless of the presence of the albedo, assures a prompt inhibition of Listeria spp. Therefore, its use could be an important contribution to the quality of low-fat fermented sausages.

In this study, for the first time, we examined some of the physico-chemical properties of the cell surface of Akkermansia muciniphila DSM 22959, comparing it with those of Lactobacillus rhamnosus GG&mdash, one of the most extensively studied probiotic microorganisms. In particular, hydrophobicity, auto-aggregation, co-aggregation, and biofilm formation were investigated. In addition, antibiotic susceptibility, co-culture, and antimicrobial activity of the two strains were compared. Hydrophobicity was evaluated using xylene and toluene, showing that A. muciniphila DSM 22959 possessed moderate hydrophobicity. A. muciniphila showed a faster and higher auto-aggregation ability than Lb. rhamnosus GG, but a lower aptitude in biofilm formation. In the co-aggregation test, the best performance was obtained by Lb. rhamnosus GG. Regarding the susceptibility to antibiotics, the differences between the two strains were remarkable, with A. muciniphila DSM 22959 showing resistance to half of the antibiotic tested. Interesting results were also obtained with regard to the stimulating effect of Lb. rhamnosus GG on the growth of A. muciniphila when co-cultured.

Biofilm life-style of Lactobacillus plantarum (L. plantarum) strains was evaluated in vitro as a new and suitable biotechnological strategy to assure L-malic acid conversion in wine stress conditions. Sixty-eight L. plantarum strains isolated from diverse sources were assessed for their ability to form biofilm in acid (pH 3.5 or 3.2) or in ethanol (12% or 14%) stress conditions. The effect of incubation times (24 and 72 h) on the biofilm formation was evaluated. The study highlighted that, regardless of isolation source and stress conditions, the ability to form biofilm was strain-dependent. Specifically, two clusters, formed by high and low biofilm producer strains, were identified. Among high producer strains, L. plantarum Lpls22 was chosen as the highest producer strain and cultivated in planktonic form or in biofilm using oak supports. Model wines at 12% of ethanol and pH 3.5 or 3.2 were used to assess planktonic and biofilm cells survival and to evaluate the effect of biofilm on L-malic acid conversion. For cells in planktonic form, a strong survival decay was detected. In contrast, cells in biofilm life-style showed high resistance, assuring a prompt and complete L-malic acid conversion.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a severe bacterial disease that affects larvae of honeybees. The present study evaluated, in vitro, antimicrobial activity of sixty‐one Lactiplantibacillus plantarum strains, against P. larvae ATCC 9545. Five strains (P8, P25, P86, P95 and P100) that showed the greatest antagonism against P. larvae ATCC 9545were selected for further physiological and biochemical characterizations. In&nbsp, particular, the hydrophobicity, auto‐aggregation, exopolysaccharides production, osmotic tolerance, enzymatic activity and carbohydrate assimilation patterns were evaluated. The five L. plantarum selected strains showed suitable physical and biochemical properties for their use as probiotics in the honeybee diet. The selection and availability of new selected bacteria with good functional characteristics and with antagonistic activity against P. larvae opens up interesting perspectives for new biocontrol strategies of diseases such as AFB.

Background Sparkling wines produced by traditional method owe their characteristics to secondary fermentation and maturation that occur during a slow ageing in bottles. Yeast autolysis plays an important role during the sparkling wine aging. Using a combination of killer and sensitive yeasts is possible to accelerate yeast autolysis and reduce maturing time. Methods killer and sensitive Saccharomyces cerevisiae strains, separately and in co-cultures, were inoculated in base wine and bottled on pilot-plant scale. Commercial Saccaromyces bayanus strain was also investigated. Protein free amino acid and polysaccharides contents and sensory analysis were determined on the wine samples at 3, 6 and 9 months of aging. Yeast autolysis that occurs during the production of sparkling wines, obtained with co-cultures of killer and sensitive strains, has influenced free amino acids, total protein and polysaccharides content after 3 months aging time: sparkling wines, produced without the use of these yeasts, have reached the same results only after 9 months aging time. Results These results demonstrate that killer and sensitive yeasts in co-culture can accelerate the onset of autolysis in enological conditions, and has a positive effect on the quality of the aroma and flavor of sparkling wine. Conclusion This paper offers an interesting biotechnological method to reduce production time of sparkling wine with economical benefits for the producers. We revised all patents relating to sparkling wine considering only those of interest for our study.

Lactic acid bacteria (LAB) are an important group of honeybee gut microbiota. These bacteria are involved in food digestion, stimulate the immune system, and may antagonize undesirable microorganisms in the gastrointestinal tract. Lactobacillus kunkeei is a fructophilic lactic acid bacterium (FLAB) most frequently found in the gastrointestinal tracts of honeybees. Ascosphaera apis is an important pathogenic fungus of honeybee larvae, it can colonize the intestine, especially in conditions of nutritional or environmental stress that cause microbial dysbiosis. In this work, some functional properties of nine selected L. kunkeei strains were evaluated. The study focused on the antifungal activity of these strains against A. apis DSM 3116, using different matrices: cell lysate, broth culture, cell-free supernatant, and cell pellet. The cell lysate showed the highest antifungal activity. Moreover, the strains were shown to possess good cell-surface properties (hydrophobicity, auto-aggregation, and biofilm production) and a good resistance to high sugar concentrations. These L. kunkeei strains were demonstrated to be functional for use in &ldquo, probiotic syrup&rdquo, useful to restore the symbiotic communities of the intestine in case of dysbiosis and to exert a prophylactic action against A. apis.

Malolactic fermentation (MLF) is a biological process that, in addition to deacidifying, also improves biological stability and changes the chemical and sensorial characteristics of wines. However, multiple biotic and abiotic factors, present in must and wine, make the onset and completion of MLF by indigenous malolactic bacteria or added commercial starters difficult. This work illustrates the metabolic and fermentative dynamics in winemaking Fiano wine, using a commercial starter of Saccharomyces cerevisiae and the selected strain Lactobacillus plantarum M10. In particular, an inoculum of malolactic starter was assessed at the beginning of alcoholic fermentation (early co-inoculum), at half alcoholic fermentation (late co-inoculum), and post alcoholic fermentation (sequential inoculum). The malolactic starter, before its use, was pre-adapted in sub-optimal growth conditions (pH 5.0). In sequential inoculum of the Lb. plantarum M10, even in a wine with high acidity, has confirmed its good technological and enzymatic characteristics, completing the MLF and enriching the wine with desirable volatile compounds.

In this study, the effect of sequential inoculation with non-Saccharomyces (Hanseniaspora guilliermondii) and Saccharomyces cerevisiae yeast on the distinctive characteristics of the Campanino white wine was investigated. For this purpose, three independent winemaking experiments were carried out on an industrial scale (batches A, B and C). In detail, the first one was carried out using the sequential inoculation technique while the other two, using a S. cerevisiae single-strain starter or no inoculation representing the control batches. Microbiological and chemical parameters and sensorial profiles of the wines were defined. Interestingly, the results showed that when sequential cultures (H. guilliermondii in a sequential mixture with S. cerevisiae) were used, a better wine aroma and quality was observed. More specifically, the wine obtained by sequential inoculation showed lower acetic acid values and enhanced volatile profiles than the wine from the control batches. Finally, sensorial analysis confirmed that the sequential cultures led to an improvement in wine flavour. Therefore, results suggest that the sequential inoculation using non-Saccharomyces and Saccharomyces yeast represents a biotechnological practice that can improve the quality features of traditional white wine. It has been shown for the first time that on an industrial scale H. guilliermondii could be used in sequential inoculum with S. cerevisiae in making white Campanino wine., Research in our labs is funded by Grants AGL2015-64522-C2-R (Spanish Ministry of Economy and Competitiveness), ALIBIRD-CM 2013 S2013/ABI-2728 (Comunidad de Madrid) and within the framework of the project by Molise Region (Rural Development Programme 2007–2013; Measure 1.2.4. Cooperation for development of new products, processes and technologies in the agriculture and food sector and in forestry).

In this study, 23 samples of traditional wines produced in Southern Italy were subjected to microbiological analyses with the aim to identify and biotype the predominant species of lactic acid bacilli. For this purpose, a multiple approach, consisting in the application of both phenotypic (API 50CHL test) and biomolecular methods (polymerase chain reaction-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing) was used. The results showed that Lactobacillus plantarum was the predominant species, whereas Lb. brevis was detected in lower amount. In detail, out of 80 isolates 58 were ascribable to Lb. plantarum and 22 to Lb. brevis. Randomly amplified polymorphic DNA-polymerase chain reaction was used to highlight intraspecific variability among Lb. plantarum strains. Interestingly, the cluster analysis evidenced a relationship between different biotypes of Lb. plantarum and their origin, in terms of wine variety. Data acquired in this work show the possibility to obtain several malolactic fermentation starter cultures, composed by different Lb. plantarum biotypes, for their proper use in winemaking processes which are distinctive for each wine.

The aim of this work is to study alternative biotechnological procedures to prepare green table olives. Specifically, by working on laboratory-scale system and under controlled conditions, the technological potential of enzymes from olive leaf and/or Lactobacillus plantarum strain (TB38) isolated from wine were assayed on green olives in comparison with spontaneous fermentation and 3 % NaOH treatment. The fermentation process (microbial count, pH, ethyl alcohol and the lactic and acetic acids, in the cover liquid medium) and the changes of phenolic fraction (total phenols, oleuropein and hydroxytyrosol, in the olive fruits) were checked weekly for 30 days. In the first week, olive leaf enzymes showed to be very effective in degrading the oleuropein; conversely, their action showed a decline in the subsequent days. Also the TB38 LAB strain showed a good oleuropeinolytic activity and acidifying capacity. However, the presence of undissolved olive leaf solid residue inhibited clearly the metabolism of LAB.

The effect of some phenolic compounds recurrent in wines on technological features of Oenococcus oeni was studied in order to individuate those strains to be utilized as starter in the deacidification of aged red wines. For this purpose, the growth and the L-malic acid metabolism of 100 O. oeni strains, previously isolated from different wines, was assayed in a synthetic medium added with ethanol, malic acid and phenol carboxylic (gallic, caffeic, p-coumaric and ferulic) acids or flavonoids (catechin and quercetin) at different concentrations. Results evidenced a different sensitivity of strains to each assayed compound. All the compounds restrained or stimulated the growth of 57 and 11 strains respectively, while no effect was detected on 6 strains. The remaining 26 strains showed a different behaviour: all were restrained by ferulic acid and stimulated by gallic acid and catechin. As for caffeic acid, 17 out of 26 strains were restrained, while 9 strains were stimulated. The main result obtained in this study was the establishment of a relationship between the effect of phenolic compounds on the O. oeni growth and the behaviour of the malolactic fermentation. This study may enrich the selection criteria of strains for the deacidification of aged red wines.