In Vitro Assessment of Bio-Functional Properties from Lactiplantibacillus plantarum Strains.

In recent years, alongside the conventional screening procedures for the evaluation of probiotics for human usage, the pharmaceutical and food industries have encouraged scientific research towards the selection of new probiotic bacterial strains with particular functional features. Therefore, this study intended to explore novel functional properties of five Lactiplantibacillus plantarum strains isolated from bee bread. Specifically, antioxidant, antimicrobial and ß-glucosidase activities, exopolysaccharides (EPS) production and the ability to synthesize γ-aminobutyric acid (GABA) were evaluated. The results demonstrated that the investigated L. plantarum strains were effective in inhibiting the growth of some human opportunistic pathogens in vitro (Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Enterococcus faecalis and Staphylococcus aureus). Moreover, the evaluation of antioxidant and ß-glucosidase activity and of EPS and GABA production, revealed a different behavior among the strains, testifying how these properties are strongly strain-dependent. This suggests that a careful selection within a given species is important in order to identify appropriate strains for specific biotechnological applications. The results highlighted that the five strains of L. plantarum are promising candidates for application as dietary supplements in the human diet and as microbial cultures in specific food productions.

Chemical Characterization and Antibiofilm Activities of Bulbs and Leaves of Two Aglione (Allium ampeloprasum var. holmense Asch. et Graebn.) Landraces Grown in Southern Italy.

The present study was carried out to determine some biochemical characteristics, in particular the total polyphenol content and the free radical scavenging activity, of the extracts recovered from bulbs and aerial parts (these last often considered as by-products) of two landraces of A. ampeloprasum var. holmense cultivated in Southern Italy. For the first time, the capacity of the extracts of these landraces to inhibit the formation of biofilm of different Gram-negative and Gram-positive bacteria and to affect the metabolism of the cells present within the bacterial biofilm was evaluated. All extracts exhibited an amount of total polyphenols not lower than 2.86 mg/g of dried product and revealed a noteworthy antioxidant activity, with EC50 values not exceeding 4.95 mg. In both cases, the aerial parts extracts were more effective than the bulb extracts, which also showed a minor amount of total polyphenols. The extracts inhibited mainly the adhesive capability of Pseudomonas aeruginosa and Staphylococcus aureus, by 95.78% and 85.01%, respectively. The extracts demonstrated to inhibit also the metabolism of the bacterial cells reaching levels up to 90%. Finally, as assessed by the assays performed on the 24-h preformed biofilms, all the extracts were also capable to cause a reduction in bacterial biomass and to affect their metabolism.

Effect of exogenous proline on the ethanolic tolerance and malolactic performance of Oenococcus oeni.

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.

Polyphenols, Antioxidant, Antibacterial, and Biofilm Inhibitory Activities of Peel and Pulp of Citrus medica L., Citrus bergamia, and Citrus medica cv. Salò Cultivated in Southern Italy.

The aim of this paper was to study the polyphenols of peel and pulp of three Citrus taxa-Citrus medica, Citrus bergamia, and Citrus medica cv. Salò-cultivated in the Cosenza province, Southern Italy, and to evaluate their antioxidant and antibacterial activity, performed against Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, and Pectobacterium carotovorum. Furthermore, we assessed the inhibitory effect of the extracts on bacterial capacity to form biofilm, and on the metabolic activity of the cells present therein. The results indicated that such extracts could find new potential applications in the field of natural antioxidant and anti-bacterial agents in pharmaceutics, agriculture, and food fields.

Alpha-amylase, α-glucosidase and lipase inhibiting activities of polyphenol-rich extracts from six common bean cultivars of Southern Italy, before and after cooking.

Common beans (Phaseolus vulgaris) are a good source of nutrients and phenolic compounds with versatile health benefits. Polyphenol-rich extracts of six ecotypes of P. vulgaris were analysed to determine their phenolic profiles and assayed in vitro for inhibitory effects on digestive enzymes relevant to carbohydrates and lipids metabolism. The extracts inhibited enzyme activities in a dose-dependent manner. IC 50 values ranged from 69 ± 1.9 to 126 ± 3.2 µg/mL and from 107.01 ± 4.5 to 184.20 ± 5.7 µg/mL, before and after cooking, for α-amylase, from 39.3 ± 4.4 to 74.13 ± 6.9 µg/mL and from 51 ± 7.7 to 122.1 ± 5.2 µg/mL for α-glucosidase and from 63.11 ± 7.5 to 103.2 ± 5.9 µg/mL and from 92.0 ± 6.3 to 128.5 ± 7.4 µg/mL for lipase. Results suggest encouraging their consumption, being natural sources of enzyme inhibitors important for type-2 diabetes and obesity prevention/control. Well-monitored in vivo studies would help to establish their beneficial effects, making them worthwhile of further consideration as functional foods.

Sequential inoculum of Hanseniaspora guilliermondii and Saccharomyces cerevisiae for winemaking Campanino on an industrial scale.

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.

Evolution of free amino acids during ripening of Caciocavallo cheeses made with different milks.

The evolution of free amino acids (FAA) in Caciocavallo cheeses, made with cow milk (CC) and cow milk mixed with ewe (CE) and goat (CG) milk, was studied throughout ripening. In all Caciocavallo cheeses produced, the total free amino acid (TFAA) content increased during ripening. In general, the highest TFAA content was found in cow cheeses, and the lowest in CG cheeses, whereas CE cheeses ranged over an intermediate level. In all the analyzed samples, during ripening, the content of the individual FAA increased with the exception of arginine. Tyrosine and histidine were found only in CE samples from the middle to the end of ripening. The major FAA found throughout the whole ripening period, in all types of cheese, were leucine, phenylalanine, lysine, valine, asparagine, γ-aminobutyric acid, and ornithine. The TFAA and several AA showed significant differences in ripening time, whereas tyrosine and histidine showed significant differences in kinds of milk.

Laurus nobilis: Composition of Essential Oil and Its Biological Activities.

Laurus nobilis is native to the southern Mediterranean region and cultivated mainly in Europe and the USA as an ornamental and medicinal plant. The chemical composition of the essential oil (EO) from leaves of L. nobilis, collected in Southern Italy, was studied by GC and GC-MS. In all, 55 compounds were identified, accounting for 91.6% of the total oil. 1,8-Cineole (31.9%), sabinene (12.2%), and linalool (10.2%) were the main components. Antimicrobial and antifungal activities of EO and 1,8-cineole were determined in vitro. The cytotoxicity of the EO was evaluated against SH-SY5Y cell line, as well as the influence of the EO on the expression of adenylate cyclase 1 (ADCY1), suggesting possible oil effects on the Central Nervous System.

Persistence of bacterial indicators and zoonotic pathogens in contaminated cattle wastes.

BACKGROUND: Manure can provide a favourable environment for pathogens' survival. De-contamination may be conducted by extended storage, until field conditions are suitable for application to land as source of agricultural nutrients. RESULTS: The hygienic evaluation of manure and slurry coming from a plant that collects cattle livestock wastes from a big slaughterhouse was carried out. Samples were even collected from spillages in the area around the plant. Microbial analyses highlighted the massive presence of faecal indicators in all samples: mean counts of Escherichia coli and enterococci were always above EU limits for marketable processed manure products. Cultures referable to the genus Brucella spp. were recorded in two samples of fresh manure but not in the aged ones. Conventional isolation techniques failed to detect members of the Mycobacterium genus, while by means of IS900 and F57 PCR real-time system on DNA directly extracted from environmental samples, the pathogen was detected in all cases. CONCLUSIONS: Thoughtful design of manure storage infrastructure is critical to prevent spills and over-topping of an open structure. The documented overload situation seems to lay the basis for an ongoing environmental contamination by enteric organisms and opportunistic pathogens circuit faecal-oral route. Moreover, the type of wastes analysed during this study, namely a mixture of fresh cattle manure, bedding and rumen content, needs a longer storage period or, alternatively, of specific chemical, biological or thermal treatments for stabilization.

Variability in chemical and microbiological profiles of long-ripened Caciocavallo cheeses.

Five protected designation of origin (PDO) Caciocavallo Silano and 6 non-PDO Caciocavallo cheeses, ripened for 6mo and collected in the 5 Italian regions of the PDO area (Apulia, Basilicata, Calabria, Campania, and Molise, Italy), were studied to assess their physico-chemical (pH, acidity, moisture, fat, ash, protein, and free amino acid composition) and microbiological profiles. Analyses evidenced a certain fluctuation of previous parameters among samples regardless of the kind of cheese evaluated (PDO and non-PDO). The PCR-denaturing gradient gel electrophoresis analysis performed on the DNA directly extracted from cheeses gave different results, but a low number of bands was always observed. Only one band, corresponding to the species Streptococcus thermophilus, was detectable in 1 PDO and in 2 non-PDO cheese samples, whose free amino acid content was the lowest. Analyses were repeated on experimental Caciocavallo cheeses. Specifically, 2 productions were made, one mimicking the industrial technology (pasteurized milk and selected starter culture) and one the artisanal technology (raw milk and natural whey starter). Results obtained on experimental cheeses at 6mo of ripening showed that industrial samples had lower amounts of total free amino acids then the artisanal ones (1,188.2 vs. 7,523.67mg/100 g of dry matter). Moreover, the PCR-denaturing gradient gel electrophoresis analysis evidenced the sole presence of S. thermophilus in the case of the industrial technology. These data sustain the hypothesis that, out of 11 cheeses analyzed previously, 1 PDO Caciocavallo Silano and 2 non-PDO Caciocavallo cheeses were obtained with the industrial technology. These results could be of help in the discrimination of PDO products, taking into account that the PDO production regulation does not allow the milk pasteurization, nor the use of selected starters.

High resolution melting analysis (HRM) as a new tool for the identification of species belonging to the Lactobacillus casei group and comparison with species-specific PCRs and multiplex PCR.

The correct identification and characterisation of bacteria is essential for several reasons: the classification of lactic acid bacteria (LAB) has changed significantly over the years, and it is important to distinguish and define them correctly, according to the current nomenclature, avoiding problems in the interpretation of literature, as well as mislabelling when probiotic are used in food products. In this study, species-specific PCR and HRM (high-resolution melting) analysis were developed to identify strains belonging to the Lactobacillus casei group and to classify them into L. casei, Lactobacillus paracasei and Lactobacillus rhamnosus. HRM analysis confirmed to be a potent, simple, fast and economic tool for microbial identification. In particular, 201 strains, collected from International collections and attributed to the L. casei group, were examined using these techniques and the results were compared with consolidated molecular methods, already published. Seven of the tested strains don't belong to the L. casei group. Among the remaining 194 strains, 6 showed inconsistent results, leaving identification undetermined. All the applied techniques were congruent for the identification of the vast majority of the tested strains (188). Notably, for 46 of the strains, the identification differed from the previous attribution.

Optimization of water curing for the preservation of chestnuts (Castanea sativa Mill.) and evaluation of microbial dynamics during process.

Chestnuts are very perishable fruits, whose quality may be compromised during postharvest handling. Damage can be caused both by insects and fungi. Water curing, a commonly used postharvest method, is based on soaking fruits in water typically for about one week. Factors that affect effectiveness of water curing have only been explained partially. A decrease in pH, likely imputable to a light fermentation caused by lactic acid bacteria, may inhibit the growth of moulds. In this study a Lactobacillus pentosus strain was selected for its ability to inhibit fungi, and used as a starter culture during water curing. As second goal, a reduction of the environmental impact of the process was evaluated by using water that had been re-cycled from a previous curing treatment. Experiments were performed on pilot as well as on farm scale. In all trials, microbial dynamics were evaluated by means of a polyphasic approach including conventional and molecular-based analyses. According to results, the employment of an adjunct culture appears as a very promising opportunity. Even if no reduction in the duration of the process was achieved, waters exhibited a minor microbial complexity and fruits did not lose the natural lustre after the process.

Raw milk from vending machines: Effects of boiling, microwave treatment, and refrigeration on microbiological quality.

In Italy, the sale of raw milk from vending machines has been allowed since 2004. Boiling treatment before its use is mandatory for the consumer, because the raw milk could be an important source of foodborne pathogens. This study fits into this context with the aim to evaluate the microbiological quality of 30 raw milk samples periodically collected (March 2013 to July 2013) from 3 vending machines located in Molise, a region of southern Italy. Milk samples were stored for 72 h at 4 °C and then subjected to different treatments, such as boiling and microwaving, to simulate domestic handling. The results show that all the raw milk samples examined immediately after their collection were affected by high microbial loads, with values very close to or even greater than those acceptable by Italian law. The microbial populations increased during refrigeration, reaching after 72 h values of about 8.0 log cfu/mL for Pseudomonas spp., 6.5 log cfu/mL for yeasts, and up to 4.0 log cfu/mL for Enterobacteriaceae. Boiling treatment, applied after 72 h to refrigerated milk samples, caused complete decontamination, but negatively affected the nutritional quality of the milk, as demonstrated by a drastic reduction of whey proteins. The microwave treatment at 900 W for 75 s produced microbiological decontamination similar to that of boiling, preserving the content in whey proteins of milk. The microbiological characteristics of raw milk observed in this study fully justify the obligation to boil the raw milk from vending machines before consumption. However, this study also showed that domestic boiling causes a drastic reduction in the nutritional value of milk. Microwave treatment could represent a good alternative to boiling, on the condition that the process variables are standardized for safe domestic application.

Innovative Caciocavallo cheeses made from a mixture of cow milk with ewe or goat milk.

This study assessed and compared the physicochemical, microbiological, and sensorial characteristics of Caciocavallo cheeses, made from cow milk and a mixture of cow with ewe or goat milk, during ripening. Different cheese-making trials were carried out on an industrial scale following the standard procedure of pasta filata cheeses, with some modifications. The percentage of the different added milk to cow milk influenced compositional and nutritional characteristics of the innovative products, leading to a satisfactory microbiological and sensorial quality.

Ability of synbiotic encapsulated Saccharomyces cerevisiae boulardii to grow in berry juice and to survive under simulated gastrointestinal conditions.

The probiotic yeast, Saccharomyces cerevisiae boulardii, was microencapsulated in a mixture of alginate-inulin-xanthan gum, and its ability to grow in berry juice and survive 4 weeks of storage at 4 °C was determined. Exposure of the yeast in these forms to artificial gastrointestinal conditions was also assessed. Encapsulation significantly enhanced cell viability after fermentation and storage compared with the free yeast (7.59 log10 colony forming units/ml versus 6.98 log10 colony forming units/ml, respectively) and protected it from exposure to a simulated gastrointestinal transit after 4 weeks of storage. Conversely, a dramatic loss of viability was exhibited by free yeast after 4 weeks of storage, and viability values closer to zero (0.23 log10 cfu/ml) were found after the simulated gastrointestinal treatment. Microcapsules were capable of absorbing a certain amount of polyphenols and anthocyanins. This work, based on use of microencapsulated probiotic yeasts, might represent the starting point for the development of new functional foods or functional ingredients. Microcapsules were capable to absorb, from berry juice, a certain amount of anthocyanins which, maintaining their native form after the in vitro gastrointestinal transit, might in vivo therein be transformed into other, simpler molecules, with beneficial effect on microflora and human health too.

Eruca sativa might influence the growth, survival under simulated gastrointestinal conditions and some biological features of Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus rhamnosus strains.

The growth and viability of three Lactobacillus strains, Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus rhamnosus, after their passage through simulated gastric and pancreatic juices were studied as a function of their presence in the growth medium of rocket salad (Eruca sativa). The presence of E. sativa affected some of the biological properties of the strains. For example, L. acidophilus and L. plantarum worked more efficiently in the presence of E. sativa, increasing not only the antioxidant activity of the medium, but also their own antioxidant power and antimicrobial activity; L. rhamnosus was not affected in the same manner. Overall, the presence of vegetables might help to boost, in specific cases, some of the characteristics of lactobacilli, including antioxidant and antimicrobial power.

Biodiversity of Lactobacillus plantarum from traditional Italian wines.

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.

Diversity of fungal communities on Cabernet and Aglianico grapes from vineyards located in Southern Italy.

Grape-associated microbial community is influenced by a combination of viticultural, climatic, pedological and anthropological factors, collectively known as terroir. Therefore, grapes of the same cultivar grown in different areas can be appreciated for their distinctive biogeographic characteristics. In our previous study, we showed that the phenotypic response of Aglianico and Cabernet grapevines from Molise and Sicily regions is significantly influenced by the prevailing pedoclimatic conditions, particularly soil physical properties. However, the scale at which microbial communities differ could be important in clarifying the concept of terroir, including whether it is linked to the grape variety present in a particular vineyard. To explore this further, in the research presented here, a comparative study on the fungal communities inhabiting the berry surfaces of Cabernet and Aglianico cultivars was conducted on different vineyards located in Southern Italy (Molise, Sicily and Campania regions, the first two of which had been involved in our previous study) by using high-throughput sequencing (HTS) and multivariate data analysis. The descriptive approach through relative abundance analysis showed the most abundant phyla (Ascomycota, Basidiomycota, and Chytridiomycota), families (Cladosporiaceae, Saccotheciaceae, Pleosporaceae, Saccharomycodaceae, Sporidiobolaceae, Didymellaceae, Filobasidiaceae, Bulleribasidiaceae, and Saccharomycetaceae) and genera (Cladosporium, Aureobasidium, Alternaria, Stemphylium and Filobasidium) detected on grape berries. The multivariate data analysis performed by using different packages (phyloseq, Vegan, mixOmics, microbiomeMarker and ggplot2) highlighted that the variable "vineyard location" significantly affect the fungal community, while the variable "grape variety" has no significant effect. Thus, some taxa are found to be part of specific vineyard ecosystems rather than specific grape varieties, giving additional information on the microbial contribution to wine quality, thanks to the presence of fermentative yeasts or, conversely, to the involvement in negative or detrimental roles, due to the presence of grape-deriving fungi implied in the spoilage of wine or in grapevine pathogenesis. In this connection, the main functions of core taxa fungi, whose role in the vineyard environment is still poorly understood, are also described.

Effect of chestnut extract and chestnut fiber on viability of potential probiotic Lactobacillus strains under gastrointestinal tract conditions.

The main challenge to probiotics, during their passage through the gastrointestinal tract, are the acidic gastric secretions of the stomach, and the bile salts released into the duodenum. The survival of the strains, in this phase, is strongly influenced by the food used for their delivery. This work is part of a project studying the development of novel food processes, based on the use of chestnuts from cultivar "Castagna di Montella". In detail, the effect of indigestible chestnut fiber and of chestnut extract on the viability of selected lactic acid bacteria strains was evaluated. Among 28 cultures, twelve strains were selected, on the basis of tolerance to low pH values and bile salts, and submitted to exposition to simulated gastric or bile juice in presence of chestnut extract with or without immobilization in chestnut fiber. The presence of chestnut extract proved to play a significant role on the gastric tolerance improvement of lactobacilli. The recorded protective effect could not be simply related to the starch or reducing sugars content. RP-HPLC demonstrated that in the chestnut flour, there are one or more hydrophobic peptides or oligopeptides, which specifically offer a marked resistance to simulated gastric juice, albeit present at low concentration. These beneficial effects proved to be dependent by the cultivar used to produce the flour.

Quorum sensing and phytochemicals.

Most infectious diseases are caused by bacteria, which proliferate within quorum sensing (QS)-mediated biofilms. Efforts to block QS in bacteria and disrupt biofilms have enabled the identification of bioactive molecules that are also produced by plants. This mini review primarily focuses on natural QS inhibitors, which display potential for treating bacterial infections and also enhance the safety of food supply.