Comparative studies on the antioxidant properties and polyphenolic content of wine from different growing regions and vintages,a pilot study to investigate chemical markers for climate change

Many health benefits of wine result from specific polyphenolic compounds. Factors such as climate, CO₂ levels and region are known to affect polyphenolic compounds in wine; therefore a pilot study was conducted to focus on the Australian climate which has shifted from El Niño to La Niña. This research paper presents the influence of climate conditions and growing regions on the in vitro and ex vivo antioxidant capacity of red and white wine and the profile and concentration of polyphenols in these wines from the 2008 and 2009 vintages. The ORAC and polyphenolic data show that warmer climate wines had lower in vitro antioxidant capacity values but retained good bioavailability based on data from the RBC ex vivo assay compared to cool climate wines. Based on this pilot study, further research is being conducted at the National Measurement Institute, Australia (NMIA) with the goal of determining more polyphenolic compounds which appear to be affected by climate conditions.     

Separation of an effective fraction from turmeric against Streptococcus mutans biofilms by the comparison of curcuminoid content and anti-acidogenic activity

Turmeric has long been used as a colouring and flavouring agent for foods. Curcuminoids are the main component of turmeric and have a range of pharmacological activities. In this study, a fraction that could show anti-biofilm activity was separated from turmeric, based on a comparison of curcuminoid content and anti-acidogenic activity against Streptococcus mutans, and the effects of the separated fraction and curcuminoids on the adherence ability of S. mutans and the physiological ability of S. mutans biofilms were examined at sub-minimum inhibitory concentration (MIC) levels. The separated fraction and curcuminoids had inhibitory effects on the sucrose-dependant adherence of S. mutans to saliva-coated hydroxyapatite (sHA) discs and the acidogenicity and aciduricity of S. mutans biofilms. These results suggest that the separated turmeric fraction and its components, curcuminoids, may be useful for controlling dental biofilms and subsequent dental caries formation.     

The inhibitory effect of Plectranthus barbatus and Plectranthus ecklonii leaves on the viability,glucosyltransferase activity and biofilm formation of Streptococcus sobrinus and Streptococcus mutans

Aqueous extracts of Plectranthus barbatus and Plectranthus ecklonii are traditionally used as anti-inflammatory and anti-fungal agents. The effect of these extracts and of its main component, rosmarinic acid, on the viability of the cariogenic bacteria, Streptococcus sobrinus and Streptococcus mutans, was determined by MIC and MBC. The influence of these extracts on the biofilm formation as well as on the inhibition of glucosyltransferase enzyme, produced by these species, was also analysed. Aqueous extracts of P. barbatus and P. ecklonii were stronger inhibitors than rosmarinic acid. MIC values of 3.8 and 4.7 mg/ml for S. sobrinus and 2.9 and 5.0 for S. mutans were obtained, while rosmarinic acid presented MIC values of 8.4 and 7.3 mg/ml. P. barbartus, P. ecklonii and rosmarinic acid presented MBC values of 9.5, 9.0 and 12.0 mg/ml for S. sobrinus, and 9.5, 10.0 and 12.5 mg/ml for S. mutans. The inhibition of biofilm formation by P. barbatus, P. ecklonii and rosmarinic acid presented IC₅₀ values of, respectively, 0.6, 1.0 and 3.1 mg/ml for S. sobrinus and 1.4 and 2.7 and 1.3 mg/ml for S. mutans. The glucosyltransferase inhibition activity by theses extracts and rosmarinic acid was calculated and IC₅₀ values presented were, respectively, 1.1, ca 1.2 and 2.1 mg/ml for S. sobrinus and 3.1, 1.6 and 3.9 mg/ml for S. mutans were obtained. These extracts may be useful in the prevention of dental carie.     

Lactose triggers biofilm formation by Streptococcus mutans

The most cariogenic bacterium, Streptococcus mutans, often adopts a sessile lifestyle in response to carbohydrates. Biofilm formation represents one of the most successful strategies for survival by S. mutans in a dental environment. This study reports induced biofilm formation by S. mutans in the presence of lactose, the primary sugar in milk. Importantly, no major difference was observed in S. mutans growth in the presence of different concentrations of lactose in growth medium, indicating that lactose has a specific effect on biofilm formation. Moreover, extracellular polysaccharides produced by S. mutans in response to lactose were found to be different from the polysaccharides produced in the presence of sucrose. It is further reported that several biofilm-related genes of S. mutans were significantly up-regulated in response to lactose. These results lead to the conclusion that lactose may promote biofilm formation by S. mutans, the most important bacterium involved in dental diseases.     

Species,roasting degree and decaffeination influence the antibacterial activity of coffee against Streptococcus mutans

Coffee beverage has been associated with antibacterial activity against Streptococcus mutans, a cariogenic bacterium. This study aimed at identifying natural compounds in coffee that contribute to such activity and investigate the influence of species, roasting and decaffeination on it. Coffee chemical compounds and aqueous extracts of green and roasted regular and decaffeinated Coffea arabica and Coffea canephora beans were tested. MIC, biofilm inhibition and biofilm reduction results were correlated with the concentration of coffee compounds in the extracts. 5-Caffeoylquinic acid, trigonelline and caffeic acid solutions showed bacteriostatic activity (MIC = 0.8 mg/mL). Lighter and regular extracts showed higher inhibitory activity than darker and decaffeinated extracts, with an inverse correlation between bacterial colony-forming units and roasting degree. Only regular C. canephora extracts showed biofilm formation inhibition. The joint effect of chlorogenic acids, trigonelline and caffeine or other compounds removed by decaffeination seems to be one of the causes for coffee antibacterial activity against S. mutans.     

Attachment strength and biofilm forming ability of Bacillus cereus on green-leafy vegetables: cabbage and lettuce

The present study was designed to investigate the ability of six Bacillus cereus strains to attach and form biofilm on cabbage and lettuce surfaces. These six strains were; a reference strain DSMZ 345 and five biofilm-producing strains (aquatic strains; TUB8, TUB30, TUB31, TUB32 and TUB33) isolated from drinking-water distribution network. Hydrophobicity, biofilm formation ability, attachment strength (S_R) of spores and vegetative cells of the six B. cereus strains were also determined. Due to their high hydrophobicity, spores of all strains had high ability to attach polystyrene and did not affect by dilution of tryptone soy broth (TSB, 1:20 v/v) in the in vitro experiment. Significant (p < 0.05) enhancement in vitro biofilm formation by vegetative cells of B. cereus was recorded in the diluted TSB. The highest biofilm formation on cabbage and lettuce surfaces was obtained by spores and vegetative cells of all tested strains on the 4^th hour of the incubation period. These populations were significantly (p < 0.05) increased by elongating incubation time from 4 h to 24 h except DSMZ 345 and TUB8. Biofilm formation behavior obtained by B. cereus spores and vegetative cells on the polystyrene surface was different compared with that recorded on produce surface. The S_R of both spores and vegetative cells of the studied strains to the lettuce surface was higher than that of the cabbage surface. The hydrophobicity, biofilm formation and S_R of spores and vegetative cells of the biofilm-producing strains were higher than that of the reference strain DSMZ 345. Scanning electron microscopy (SEM) exposed random distribution of cells either on the surface or cut edge, without clear obvious affinity for the surface structures. Increasing in the presence of large clusters of cells on leaf surfaces was demonstrated after 4 and 24 h.In conclusion, use of aquatic environmental isolates is more useful for studying biofilm formation than the reference strain. Lettuce surface supported the attachment of B. cereus spores and vegetative cells compared with the cabbage surface. Further investigations are required to improve our knowledge of biofilm formation mechanisms by the human pathogenic microorganisms, especially by using the environmental and clinical isolates. To ensure safety level of green-leafy vegetables, biofilm formation after harvest should be considered as critical control point during handling of these vegetables.     

Whey fermented by Enterococcus faecalis M157 exhibits antiinflammatory and antibiofilm activities against oral pathogenic bacteria

The aim of the present study was to investigate the antiinflammatory and antibiofilm effects of whey fermented by Enterococcus faecalis M157 (M157-W) against oral pathogenic bacteria. The M157-W significantly inhibited IL-1β, IL-6, and nitric oxide induced by the lipopolysaccharide of Porphyromonas gingivalis in RAW 264.7 cells. The M157-W also inhibited the production of IL-1β and IL-8 in human periodontal ligament cells. Treatment with M157-W suppressed the phosphorylation of mitogen-activated protein kinases as well as the activation of nuclear factor-κB in RAW 264.7 cells stimulated by P. gingivalis lipopolysaccharide. Furthermore, M157-W dose-dependently inhibited Streptococcus mutans biofilm, whereas unfermented whey did not inhibit the biofilm. Treatment with M157-W significantly suppressed gtfB, gtfC, and gtfD gene expression in S. mutans compared with the control (0 μg/mL), indicating that M157-W inhibits S. mutans biofilm formation by reducing the synthesis of extracellular polymeric substances. Collectively, these results suggest that M157-W has antiinflammatory and antibiofilm activities against oral pathogenic bacteria.     

Effect of lysozyme on "flor" velum yeasts in the biological aging of sherry wines

Biological aging is a key step in the production of Sherry wine classified as “fine”. During this stage, a film of yeast referred to as “flor velum” covers the surface of the wine and substantially alters its characteristics. Other microorganisms may coexist with flor yeasts, such as lactic acid bacteria and non-Saccharomyces yeasts, whose growth may be favored under certain conditions, causing organoleptic deviations and deterioration of the wine. To prevent the development of lactic bacteria, lysozyme usage has been introduced. Lysozyme is a hydrolytic enzyme with muramidase activity that can lyse gram-positive bacteria; its use in winemaking was approved by the OIV in 1997 (resolution OENO 10/97). Thus far, the use of lysozyme during the production of Sherry wines is not widespread despite its effectiveness in controlling lactic acid bacteria. However, there have been no studies on the effect of lysozyme on flor velum. The aim of this study was to determine the influence of lysozyme on yeast growth and the formation, development and metabolism of flor velum during the biological aging process of Sherry wine. The results indicate that lysozyme does not affect the flor yeast during the fermentative stage or biofilm stage. However, if yeast inoculation is carried out under submerged culture conditions during biological aging, low doses of lysozyme (≥12.5 g/hL) affect cell multiplication and the membrane hydrophobicity of the yeast, inhibiting their aggregation and flotation and the subsequent development of flor velum. Thus, the yeast inoculation protocol and the methodology used for the addition of lysozyme influence velum development, its metabolism and the wine characteristics.     

Biofilm formation by Campylobacter jejuni in controlled mixed-microbial populations

This study was to screen the ability of biofilm formation by Campylobacter jejuni strains found in New Zealand, and investigate the biofilm growth of C. jejuni in a controlled mixed-microbial population that includes five different bacteria. The ability of C. jejuni to form a biofilm in monoculture and mixed-microbial populations was measured in a laboratory assay using a microtiter plate screening assay. The optical density of the biofilm and cell growth from mixed-microbial populations was converted to a Biofilm Formation Index (BFI). This index was used to standardize the biofilm formation in the mixed-microbial populations. High BFI was observed for Enterococcus faecalis (2.30) and Staphylococcus simulans (3.75) when they were grown with C. jejuni multilocus sequence type ST-474: a dominant poultry and human-associated type in New Zealand. C. jejuni cells were recovered from most of the biofilms containing E. faecalis and/or S. simulans. These results suggest that E. faecalis and S. simulans may play a role in biofilm formation in the poultry environment as both of these microorganisms are found in poultry processing environments and were able to form a biofilm in association with C. jejuni under microaerobic conditions. Understanding the relationships among C. jejuni, E. faecalis and S. simulans in poultry processing plants and farms may help in the design of strategies to reduce the reservoir of contamination of these bacteria and reduce the incidence of campylobacteriosis.     

Assessment of the lupin seed glucose-lowering protein intestinal absorption by using in vitro and ex vivo models

A lupin seed glycoprotein, termed γ-conglutin, has previously been found to display insulin-mimetic activity in myocyte models and reduce plasma glucose concentration when orally administered to both rats and humans. To envisage the possible metabolic fate of this bioactive protein, we used in vitro cell and ex vivo tissue models to monitor its transit through the intestinal barrier. Caco-2 cell monolayers and rat intestinal everted sacs were treated with purified γ-conglutin and the protein was immuno-assayed by chemi-luminescence-enhanced Western blotting. The in vitro approach showed that the intact protein can transit from the apical to the basolateral side of the cell monolayers. The unmodified lupin protein was also detected inside the intestinal everted sacs. Proper controls of cell monolayer and sac integrity ruled out the possibility of protein passive leakage.     

Determination of flavan-3-ols and trans-resveratrol in grapes and wine using HPLC with fluorescence detection

Concentrations of trans-resveratrol, catechin and epicatechin were analyzed in musts and wines produced from seven red and four white grape cultivars from various wine growing regions of Turkey. Phenolics were quantified using an HPLC method optimized for the separation of wine phenolics. Wine samples contained higher phenolics levels than the corresponding musts. With the exception of Semillion, white wines and musts contained lower concentrations of phenolics than red wines and musts. However, the white cultivar Semillion had the highest concentrations of catechin and epicatechin among all wine and must samples. Semillion wine catechin and epicatechin were 13.7 and 11.8 mg/L, respectively. The highest level of trans-resveratrol among the white cultivars was found in Narince wine (1.93 mg/L). Within the red wine and must cultivars, Bo?azkere, Öküzgozü, and Cabernet contained the highest concentrations of flavan-3-ols and trans-resveratrol. Catechin was the major phenolic in all wines and most musts. Epicatechin was the major phenolic in 6 of the 11 must samples, but none of the wine samples. trans-Resveratrol was generally found in lowest concentrations in both wines and musts.     

Biofilm-producing ability of Staphylococcus aureus isolates from Brazilian dairy farms

This study aimed to investigate the in silico biofilm production ability of Staphylococcus aureus strains isolated from milking parlor environments on dairy farms from São Paulo, Brazil. The Staph. aureus isolates were obtained from 849 samples collected on dairy farms, as follows: milk from individual cows with subclinical mastitis or history of the disease (n = 220); milk from bulk tank (n = 120); surfaces of milking machines and utensils (n = 389); and milk handlers (n = 120). Thirty-one Staph. aureus isolates were obtained and categorized as pulsotypes by pulsed-field gel electrophoresis and submitted to assays for biofilm formation on polystyrene, stainless steel, rubber, and silicone surfaces. Fourteen (45.2%) pulsotypes were considered producers of biofilm on the polystyrene microplate assay, whereas 13 (41.9%) and 12 (38.7%) pulsotypes were biofilm producers on stainless steel and rubber, respectively. None of the pulsotypes evaluated produced biofilms on silicone. Approximately 45% of Staph. aureus pulsotypes isolated from different sources on dairy farms showed the ability to produce biofilms in at least one assay, indicating possible persistence of this pathogen in the milking environment. The potential involvement of Staph. aureus in subclinical mastitis cases and its occurrence in milk for human consumption emphasize the need to improve hygiene practices to prevent biofilm formation on the farms studied.     

Biochemical intervention of ergothioneine-rich edible mushroom (Flammulina velutipes) extract inhibits melanosis in crab (Chionoecetes japonicus)

We investigated the effects of decreasing phenoloxidase (PO) activity and prophenoloxidase (proPO) gene expression on the inhibition of postharvest melanosis formation in the red queen crab, Chionoecetes japonicus. The cDNA of proPO from hemocytes of C. japonicus was partially cloned and sequenced. Immersion of live crabs in a 1.0% ergothioneine (ESH)-rich mushroom extract (Flammulina velutipes; ME) solution resulted in significant inhibition of haemolymph PO activity and a reduction of the proPO gene expression in hemocytes that consequently controlled melanosis in the crabs during ice storage. Treatments with a 0.05% w/v sodium sulphite solution or a 0.05% w/v 4-hexyl-1,3-benzenediol solution had similar positive effects as the treatment with a 1.0% ME solution in vivo. In vitro experiments showed that authentic l-(+)-ESH inhibited PO activity and decreased proPO gene expression in crab hemocytes. Thus, the application of ESH-rich ME can be a novel alternative to synthetic melanosis-inhibiting agents to control postharvest melanosis in crabs.     

Short communication: Biofilm production characterization of mecA and mecC methicillin-resistant Staphylococcus aureus isolated from bovine milk in Great Britain

Staphylococcus aureus is an important cause of contagious intramammary infection in dairy cattle, and the ability to produce biofilm is considered to be an important virulence property in the pathogenesis of mastitis. The aim of this study was to characterize the biofilm formation capacity of methicillin-resistant Staph. aureus (MRSA), encoding mecA or mecC, isolated from bulk tank milk in Great Britain. For this purpose, 20 MRSA isolates were grown on microtiter plates to determine the biofilm production. Moreover, the spa-typing and the presence of the intercellular adhesion genes icaA and icaD were analyzed by PCR. All MRSA isolates tested belonged to 9 spa-types and were PCR-positive for the ica genes; 10 of them (50%) produced biofilm in the microtiter plate assay. This is also the first demonstration of biofilm production by mecC MRSA.     

Proanthocyanidin profile and antioxidant capacity of Brazilian Vitis vinifera red wines

The free flavan-3-ol and proanthocyanidin (PA) profile and the antioxidant capacity of wines Vitis vinifera L., 2006 and 2007 vintages, from the São Joaquim region, at southern Brazil, are reported here for the first time. Catechin and epicatechin were the two main monomers in the wine samples, followed by gallocatechin and epigallocatechin; and the PA B1 was the main dimer. The terminal units of the PAs were constituted mainly by catechin units, with the co-presence of epicatechin, gallocatechin, epigallocatechin and traces of epicatechin gallate. The epicatechin and epigallocatechin units were the main constituents of the extension units of PAs with the co-presence of catechin and epicatechin gallate. The values for the mean degree of polymerisation ranged from 4.9 to 9.8. The wine samples demonstrated effective scavenging activity against DPPH and ABTS radicals and against lipid peroxidation in vitro. A positive correlation existed between flavan-3-ol content and antioxidant capacity in vitro.     

Facultative anaerobic halophilic and alkaliphilic bacteria isolated from a natural smear ecosystem inhibit Listeria growth in early ripening stages

In vitro and in situ anti-listerial properties of 3 strains of Facultative Anaerobic Halophilic and Alkaliphilic (FAHA) species, i.e. Alkalibacterium kapii ALK 6, Marinilactibacillus psychrotolerans ALK 9 and Facklamia tabacinasalis ALK 1, were investigated. The 3 strains were isolated from a smear ecosystem originating from a commercial Raclette type cheese and exhibiting strong anti-listerial activity in situ on cheese surface.In a first step, strains were tested in vitro for production of antimicrobial compounds against Listeria innocua 81000-1 and Listeria ivanovii HPB 28. M. psychrotolerans ALK 9 inhibited both indicator strains in spot-on-the-lawn tests while A. kapii ALK 6 showed no inhibiting effect. F. tabacinasalis ALK 1 exerted an in vitro inhibition on L. ivanovii HPB 28, but induced the formation of dense ball-shaped microcolonies of L. innocua 81000-1 in the soft agar, a typical biofilm microstructure. The extent of the biofilm zone was enhanced when F. tabacinasalis ALK 1 and M. psychrotolerans ALK 9 were tested together.In a second step, different combinations of strains were applied on Raclette cheeses ripened at pilot scale and contaminated with 50 cfu/cm²L. innocua at day 7. A control flora of 6 strains, isolated from ecosystem F and corresponding to species commonly found on smear cheeses, was applied on control and test cheeses. In test cheeses, we investigated the impact on Listeria growth of the addition of the 3 FAHA strains, applied as single or mixed cultures. A 1-log inhibition was obtained at day 15 on cheeses treated with FAHA strains applied either as single or mixed cultures. This 1-log inhibition was correlated with the development of FAHA species that reached their maximal count at day 15.This study suggests that the development of FAHA species in early ripening likely contributes to the initial part of the in situ inhibition exerted by the complex cheese surface ecosystem investigated.     

Low concentration of ethylenediaminetetraacetic acid (EDTA) affects biofilm formation of Listeria monocytogenes by inhibiting its initial adherence

The distribution and survival of the food-borne pathogen Listeria monocytogenes is associated with its biofilm formation ability, which is affected by various environmental factors. Here we present the first evidence that EDTA at low concentration levels inhibits the biofilm formation of L. monocytogenes. This effect of EDTA is not caused by a general growth inhibition, as 0.1 mM EDTA efficiently reduced the biofilm formation of L. monocytogenes without affecting the planktonic growth. Adding 0.1 mM of EDTA at the starting time of biofilm formation had the strongest biofilm inhibitory effect, while the addition of EDTA after 8 h had no biofilm inhibitory effects. EDTA was shown to inhibit cell-to-surface interactions and cell-to-cell interactions, which at least partially contributed to the repressed initial adherence. The addition of sufficient amounts of cations to saturate EDTA did not restore the biofilm formation, indicating the biofilm inhibition was not due to the chelating properties of EDTA. The study suggests that EDTA functions in the early stage of biofilm process by affecting the initial adherence of L. monocytogenes cells onto abiotic surfaces.     

Antioxidant and immunomodulatory activity of selenium exopolysaccharide produced by Lactococcus lactis subsp. lactis

Exopolysaccharide (EPS) was isolated and purified from Lactococcus lactis subsp. Lactis culture broth. Selenium chloride oxide (SeCl₂O) was added to the EPS to synthesize selenium-exopolysaccharide (Se-EPS). The in vitro and in vivo antioxidant and in vivo immunomodulatory activity of EPS and Se-EPS were compared. EPS and Se-EPS scavenged superoxide anions and hydroxyl radicals. They also increased catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, while decreasing malondialdehyde (MDA) levels in serum and in the livers of mice. Se-EPS showed stronger in vitro and in vivo antioxidant activity than were shown by EPS. The in vivo immunoenhancement activity of EPS and Se-EPS induced by cyclophosphamide (CY) treatment in immunosuppressed mice was researched. EPS and Se-EPS treatments increased macrophage phagocytosis, spleen and thymus indices and haemolytic complement activity (HC₅₀). Se-EPS showed stronger immunomodulatory activity than did EPS.     

Identification and quantification of acetic acid bacteria in wine and vinegar by TaqMan–MGB probes

A Real-Time PCR (RT-PCR) assay was developed using TaqMan minor groove binder (MGB) probes for the specific detection and quantification of five acetic acid bacteria (AAB) species (Acetobacter pasteurianus, Acetobacter aceti, Gluconacetobacter hansenii, Gluconacetobacter europaeus and Gluconobacter oxydans) in wine and vinegar. The primers and probes, designed from the 16S rRNA gene, showed good specificity with the target AAB species. The technique was tested on AAB grown in glucose medium (GY) and inoculated samples of red wine and wine vinegar. Standard curves were constructed with the five target species in all these matrices. Quantification was linear over at least 5 log units using both serial dilution of purified DNA and cells. When this technique was tested in GY medium and inoculated matrices, at least 10²–10³ cells/ml were detected. To quantify low populations of AAB in microbiologically complex samples, a PCR enrichment including part of the 16S–23S rRNA gene ITS region was needed to increase the amount of target DNA compared to non-target DNA.