Simulated gastrointestinal digestion of polylactic acid (PLA) biodegradable microplastics and their interaction with the gut microbiota.

The accumulation of microplastics (MPs) in the environment as well as their presence in foods and humans highlight the urgent need for studies on the effects of these particles on humans. Polylactic acid (PLA) is the most widely used bioplastic in the food industry and medical field. Despite its biodegradability, biocompatibility, and "Generally Recognized As Safe" (GRAS) status, recent animal model studies have shown that PLA MPs can alter the intestinal microbiota; however, to date, no studies have been reported on the possible gut and health consequences of its intake by humans. This work simulates the ingestion of a realistic daily amount of PLA MPs and their pass through the gastrointestinal tract by combining the INFOGEST method and the gastrointestinal simgi® model to evaluate possible effects on the human colonic microbiota composition (16S rRNA gene sequencing analysis) and metabolic functionality (lactic acid and short-chain fatty acids (SCFA) production). Although PLA MPs did not clearly alter the microbial community homeostasis, increased Bifidobacterium levels tended to increase in presence of millimetric PLA particles. Furthermore, shifts detected at the functional level suggest an alteration of microbial metabolism, and a possible biotransformation of PLA by the human microbial colonic community. Raman spectroscopy and field emission scanning electron microscopy (FESEM) characterization revealed morphological changes on the PLA MPs after the gastric phase of the digestion, and the adhesion of organic matter as well as a microbial biofilm, with surface biodegradation, after the intestinal and colonic phases. With this evidence and the emerging use of bioplastics, understanding their impact on humans and potential biodegradation through gastrointestinal digestion and the human microbiota merits critical investigation.

Neuroprotective Effects of Selected Microbial-Derived Phenolic Metabolites and Aroma Compounds from Wine in Human SH-SY5Y Neuroblastoma Cells and Their Putative Mechanisms of Action.

Moderate wine consumption has shown the potential to delay the onset of neurodegenerative diseases. This study investigates the molecular mechanisms underlying the protective effects of wine-derived phenolic and aroma compounds in a neuroinflammation model based on SIN-1 stress-induced injury in SH-SY5Y neuroblastoma cells. Cell pretreatment with microbial metabolites found in blood after wine consumption, 3,4-dihydroxyphenylacetic (3,4-DHPA), 3-hydroxyphenylacetic acids and salicylic ß-d-O-glucuronide, at physiologically concentrations (0.1-10 µM) resulted in increased cell viability versus SIN-1 control group (p < 0.05). Results also showed significant decreases in mitogen-activated protein kinase (MAPK) p38 and ERK1/2 activation as well as in downstream pro-apoptotic caspase-3 activity by some of the studied compounds. Moreover, pretreatment with p38, MEK, and ERK1/2-specific inhibitors, which have a phenolic-like structure, also resulted in an increase on cell survival and a reduction on caspase-3 activity levels. Overall, these results contribute with new evidences related to the neuroprotective actions of wine, pointing out that wine-derived human metabolites and aroma compounds may be effective at protecting neuroblastoma cells from nitrosative stress injury by inhibiting neuronal MAPK p38 and ERK1/2, as well as downstream caspase 3 activity.

Antibacterial activity of hen egg white lysozyme modified by heat and enzymatic treatments against oenological lactic acid bacteria and acetic acid bacteria.

The antimicrobial activity of heat-denatured and hydrolyzed hen egg white lysozyme against oenological lactic acid and acetic acid bacteria was investigated. The lysozyme was denatured by heating, and native and heat-denatured lysozymes were hydrolyzed by pepsin. The lytic activity against Micrococcus lysodeikticus of heat-denatured lysozyme decreased with the temperature of the heat treatment, whereas the hydrolyzed lysozyme had no enzymatic activity. Heat-denatured and hydrolyzed lysozyme preparations showed antimicrobial activity against acetic acid bacteria. Lysozyme heated at 90°C exerted potent activity against Acetobacter aceti CIAL-106 and Gluconobacter oxydans CIAL-107 with concentrations required to obtain 50% inhibition of growth (IC50) of 0.089 and 0.013 mg/ml, respectively. This preparation also demonstrated activity against Lactobacillus casei CIAL-52 and Oenococcus oeni CIAL-91 (IC50, 1.37 and 0.45 mg/ml, respectively). The two hydrolysates from native and heat-denatured lysozyme were active against O. oeni CIAL-96 (IC50, 2.77 and 0.3 mg/ml, respectively). The results obtained suggest that thermal and enzymatic treatments increase the antibacterial spectrum of hen egg white lysozyme in relation to oenological microorganisms.

Lactobacillus plantarum IFPL935 impacts colonic metabolism in a simulator of the human gut microbiota during feeding with red wine polyphenols.

The colonic microbiota plays an important role in the bioavailibility of dietary polyphenols. This work has evaluated the impact on the gut microbiota of long-term feeding with both a red wine polyphenolic extract and the flavan-3-ol metabolizer strain Lactobacillus plantarum IFPL935. The study was conducted in the dynamic Simulator of the Human Intestinal Microbial Ecosystem (SHIME). The feeding of the gut microbiota model with red wine polyphenols caused an initial decrease in the counts of total bacteria in the ascending colon (AC), with Bacteroides, Clostridium coccoides/Eubacterium rectale and Bifidobacterium being the most affected bacterial groups. The bacterial counts recovered to initial numbers faster than the overall microbial fermentation and proteolysis, which seemed to be longer affected by polyphenols. Addition of L. plantarum IFPL935 helped to promptly recover total counts, Lactobacillus and Enterobacteriaceae and led to an increase in lactic acid formation in the AC vessel at the start of the polyphenol treatment as well as butyric acid in the transverse (TC) and descending (DC) vessels after 5 days. Moreover, L. plantarum IFPL935 favoured the conversion in the DC vessel of monomeric flavan-3-ols and their intermediate metabolites into phenylpropionic acids and in particular 3-(3'-hydroxyphenyl)propionic acid. The results open the possibilities of using L. plantarum IFPL935 as a food ingredient for helping individuals showing a low polyphenol-fermenting metabotype to increase their colonic microbial capacities of metabolizing dietary polyphenols.

Antibacterial activity of wine phenolic compounds and oenological extracts against potential respiratory pathogens.

AIMS: To investigate the effect of seven wine phenolic compounds and six oenological phenolic extracts on the growth of pathogenic bacteria associated with respiratory diseases (Pseudomonas aeruginosa, Staphylococcus aureus, Moraxella catarrhalis, Enterococcus faecalis, Streptococcus sp Group F, Streptococcus agalactiae and Streptococcus pneumoniae). METHODS AND RESULTS: Antimicrobial activity was determined using a microdilution method and quantified as IC(50) . Mor. catarrhalis was the most susceptible specie to phenolic compounds and extracts. Gallic acid and ethyl gallate were the compounds that showed the greatest antimicrobial activity. Regarding phenolic extracts, GSE (grape seed extract) and GSE-O (oligomeric-rich fraction from GSE) were the ones that displayed the strongest antimicrobial effects. CONCLUSIONS: Results highlight the antimicrobial properties of wine phenolic compounds and oenological extracts against potential respiratory pathogens. The antimicrobial activity of wine phenolic compounds was influenced by the type of phenolic compounds. Gram-negative bacteria were more susceptible than Gram-positive bacteria to the action of phenolic compounds and extracts; however, the effect was species-dependent. SIGNIFICANCE AND IMPACT OF STUDY: The ability to inhibit the growth of respiratory pathogenic bacteria as shown by several wine phenolic compounds and oenological extracts warrants further investigations to explore the use of grape and wine preparations in oral hygiene.

Degradation of biogenic amines by vineyard ecosystem fungi. Potential use in winemaking.

AIMS: To evaluate the ability of grapevine ecosystem fungi to degrade histamine, tyramine and putrescine in synthetic medium and in wines. METHODS AND RESULTS: Grapevine and vineyard soil fungi were isolated from four locations of Spain and were subsequently identified by PCR. A total of 44 fungi were evaluated for in vitro amine degradation in a microfermentation system. Amine degradation by fungi was assayed by reversed-phase (RP)-HPLC. All fungi were able to degrade at least two different primary amines. Species of Pencillium citrinum, Alternaria sp., Phoma sp., Ulocladium chartarum and Epicoccum nigrum were found to exhibit the highest capacity for amine degradation. In a second experiment, cell-free supernatants of P. citrinum CIAL-274,760 (CECT 20782) grown in yeast carbon base with histamine, tyramine or putrescine, were tested for their ability to degrade amines in three different wines (red, white and synthetic). The highest levels of biogenic amine degradation were obtained with histamine-induced enzymatic extract. CONCLUSION: The study highlighted the ability of grapevine ecosystem fungi to degrade biogenic amines and their potential application for biogenic amines removal in wine. SIGNIFICANCE AND IMPACT OF STUDY: The fungi extracts described in this study may be useful in winemaking to reduce the biogenic amines content of wines, thereby preventing the possible adverse effects on health in sensitive individuals and the trade and export of wine.

Feasibility and application of liquid-liquid extraction combined with gas chromatography-mass spectrometry for the analysis of phenolic acids from grape polyphenols degraded by human faecal microbiota.

In this study the feasibility of a LLE-GC-EI-MS method for the analysis of 43 phenolic acids belonging to different chemical structure families which have been described in the literature as microbial-derived metabolites after consumption of dietary polyphenols was proved. In addition, the method was applied for the characterisation of phenolic metabolites resulting from the incubation, in anaerobic conditions, of a commercial grape seed extract (GSE) and their corresponding flavan-3-ol monomeric (GSE-M) and oligomeric (GSE-O) fractions with human faeces from healthy volunteers (n=3). The method showed average values of repeatability and reproducibility of 5.0% and 6.3%, respectively, adequate and low detection (1.8-30.8 µg L(-1)) and quantification limits (6.0-102.8 µg L(-1)) and good recovery values (95%, as average value). A total of 27 phenolic acids were identified in the faecal solutions after incubation with the grape seed extracts. In general, faecal samples incubated with GSE and GSE-M (monomeric fraction) yield a higher formation of phenolic acids compared to the samples incubated with the oligomer fraction (GSE-O).

Analytical performance of three commonly used extraction methods for the gas chromatography-mass spectrometry analysis of wine volatile compounds.

The analytical performance of three extraction procedures based on cold liquid-liquid extraction using dicloromethane (LLE), solid phase extraction (SPE) using a styrene-divinylbenzene copolymer and headspace solid phase microextraction (SPME) using a carboxen-polydimethylsiloxane coated fibre has been evaluated based on the analysis of 30 representative wine volatile compounds. From the comparison of the three procedures, LLE and SPE showed very good linearity covering a wide range of concentrations of wine volatile compounds, low detection limits, high recovery for most of the volatile compounds under study and higher sensitivity compared to the headspace-SPME procedure. The latter showed in general, poor recovery for polar volatile compounds. Despite some drawbacks associated with the LLE and SPE procedures such as the more tedious sampling treatment and the use of organic solvents, the analytical performance of both procedures showed that they are more adequate for the analysis of wine volatiles.

Inactivation of oenological lactic acid bacteria (Lactobacillus hilgardii and Pediococcus pentosaceus) by wine phenolic compounds.

AIMS: To investigate the inactivation properties of different classes of phenolic compounds present in wine against two wine isolates of Lactobacillus hilgardii and Pediococcus pentosaceus, and to explore their inactivation mechanism. METHODS AND RESULTS: After a first screening of the inactivation potency of 21 phenolic compounds (hydroxybenzoic and hydroxycinnamic acids, phenolic alcohols, stilbenes, flavan-3-ols and flavonols) at specific concentrations, the survival parameters (MIC and MBC) of the most active compounds were determined. For the L. hilgardii strain, the flavonols morin and kaempferol showed the strongest inactivation (MIC values of one and 5 mg l(-1), and MBC values of 7.5 and 50 mg l(-1), respectively). For the P. pentosaceus strain, flavonols also showed the strongest inactivation effects, with MIC values between one and 10 mg l(-1) and MBC values between 7.5 and 300 mg l(-1). Observations by epifluorescence and scanning electron microscopy revealed that the phenolics damaged the cell membrane and promoted the subsequent release of the cytoplasm material into the medium. CONCLUSIONS: The antibacterial activity of wine phenolics against L. hilgardii and P. pentosaceus was dependent on the phenolic compound tested, and led not only to bacteria inactivation, but also to the cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: New information about the inactivation properties of wine lactic acid bacteria by phenolic compounds is presented. It opens up a new area of study for selecting/obtaining wine phenolic preparations with potential applications as a natural alternative to SO(2) in winemaking.

Evolution of red wine anthocyanins during malolactic fermentation, postfermentative treatments and ageing with lees.

A comparative study was conducted on nine batches of wine, from the same initial wine, subjected to malolactic fermentation and ageing in barrels, under different technological conditions: Malolactic fermentation in barrel or in tank, with or without wine clarification, ageing with or without lees and stirring or no stirring of the lees. Samples were taken of the initial wine, of the wine at the end of malolactic fermentation, of the wines after clarifying treatments, and after 3, 6, 9, 12 and 14 months of ageing in the barrel, making a total of 48 wines. As a result of the anthocyanin analysis of all the wines studied, a total of 21 different anthocyanin compounds were detected, which can be classified into four groups: simple glucosides, acetyl glucosides, cinnamoyl glucosides and pyroanthocyanins. During MLF, it was shown that the effect of the container used seems to be more important than the metabolic activity of the bacteria responsible for the process. From application of the LSD test, significant differences were found in the concentrations of all the anthocyanin compounds identified due to ageing time and significant differences were also revealed for most anthocyanin compounds in relation to the manufacturing method, especially the presence or absence of lees.

Biogenic amine production by lactic acid bacteria isolated from cider.

AIMS: To study the occurrence of histidine, tyrosine and ornithine decarboxylase activity in lactic acid bacteria (LAB) isolated from natural ciders and to examine their potential to produce detrimental levels of biogenic amines. METHODS AND RESULTS: The presence of biogenic amines in a decarboxylase synthetic broth and in cider was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). Among the 54 LAB strains tested, six (five lactobacilli and one oenococci) were biogenic amine producers in both media. Histamine and tyramine were the amines formed by the LAB strains investigated. Lactobacillus diolivorans were the most intensive histamine producers. This species together with Lactobacillus collinoides and Oenococcus oeni also seemed to produce tyramine. No ability to form histamine, tyramine or putrescine by Pediococus parvulus was observed, although it is a known biogenic amine producer in wines and beers. CONCLUSIONS: This study demonstrated that LAB microbiota growing in ciders had the ability to produce biogenic amines, particularly histamine and tyramine, and suggests that this capability might be strain-dependent rather than being related to a particular bacterial species. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of biogenic amines by food micro-organisms has continued to be the focus of intensive study because of their potential toxicity. The main goal was to identify the microbial species capable of producing these compounds in order to control their presence and metabolic activity in foods.

Contribution of malolactic fermentation by Oenococcus oeni and Lactobacillus plantarum to the changes in the nonanthocyanin polyphenolic composition of red wine.

The changes in the nonanthocyanin phenolic composition during red wine malolactic fermentation carried out spontaneously and by four different starter cultures of the species Oenococcus oeni and Lactobacillus plantarum were examined to determine whether differences in nonanthocyanin polyphenolic compounds could be attributed to the lactic acid bacteria (LAB) strain that performs this important step of the wine-making process. The polyphenolic compounds were analyzed by high-performance liquid chromatography with photodiode array detection and HPLC with electrospray ionization-mass spectrometry detection. The malolactic cultures selected for this study were indigenous wine LAB strains from the A.O.C. Rioja (Spain). Results showed different malolactic behaviors in relation to wine phenolic compositions for O. oeni and L. plantarum, and also, a diversity was found within each group. The hydroxycinnamic acids and their derivatives, the flavonols and their glycosides, the flavanol monomers and oligomers, and trans-resveratrol and its glucoside were the main compounds modified by the different LAB. The wild LAB population exerted a greater impact in the wine content of some of these phenolic compounds than the inoculated selected monocultures of this study.

Biogenic amines in natural ciders.

Biogenic amines play an important physiological role in mammals, and high amounts of some exogenous amines in human diet may contribute to a wide variety of toxic effects. These amines are commonly found in many foodstuffs, particularly in fermented products such as cheese, meat products, beer, wine, and ciders. Here, the level of biogenic amines in some natural ciders was examined. Twenty-four samples of cider purchased from commercial sources were analyzed by reverse-phase high-performance liquid chromatography and fluorescence detection after precolumn derivatization with o-phthaldialdehyde. Amine levels were variable, ranging from not detected to 23 mg/liter. The average level of total biogenic amines in ciders was 5.94 +/- 8.42 mg/liter. Putrescine, histamine, and tyramine were the prevailing amines being present in 50.0, 37.5, and 33.3% of the ciders studied; very small amounts of ethylamine and phenylethylamine were observed in only one sample. Other cider parameters were analyzed to determine whether they affect the biogenic amine content in ciders, and the results were evaluated by applying cluster analysis and principal component analysis. Ciders that showed lower glycerol contents and higher amounts of 1,3-propanediol had much higher levels of histamine, tyramine, and putrescine, suggesting a high activity of lactic acid bacteria during cider making and thus the need for effective control of lactic acid bacteria.

Formation of biogenic amines throughout the industrial manufacture of red wine.

Changes in biogenic amines (histamine, methylamine, ethylamine, tyramine, phenylethylamine, putrescine, and cadaverine) were monitored during the industrial manufacture of 55 batches of red wine. The origin of these amines in relation to must, alcoholic fermentation, malolactic fermentation, sulfur dioxide addition, and wine aging and the interactions between amines and their corresponding amino acids and pH were statistically evaluated in samples from the same batches throughout the elaboration process. Some amines can be produced in the grape or the musts (e.g., putrescine, cadaverine, and phenylethylamine) or can be formed by yeast during alcoholic fermentation (e.g., ethylamine and phenylethylamine), although quantitatively only very low concentrations are reached in these stages (less than 3 mg/liter). Malolactic fermentation was the main mechanism of biogenic amine formation, especially of histamine, tyramine, and putrescine. During this stage, the increase in these amines was accompanied by a significant decline in their amino acid precursors. Significant correlations between biogenic amine formation and the disappearance of their corresponding amino acids were observed, which clearly supports the hypothesis that malolactic bacteria are responsible for accumulation of these amines in wines. No increase in the concentration of biogenic amines was observed after SO2 addition and during wine aging, indicating that sulfur dioxide prevents amine formation in subsequent stages.

Wine volatile and amino acid composition after malolactic fermentation: effect of Oenococcus oeni and Lactobacillus plantarum starter cultures.

Red wine amino acids and volatile compounds were analyzed before and after malolactic fermentation carried out by four different starter cultures of the species Oenococcus oeni and Lactobacillus plantarum. The purpose of this study was to determine whether differences can be attributed to the lactic acid bacteria strain used in this important step of the wine-making process. The malolactic cultures selected for this study were indigenous wine lactic acid bacteria strains. The data were evaluated using different multivariate analysis techniques. Results showed different malolactic behaviors for O. oeni and L. plantarum and significant metabolic differences between both species. A degree of diversity was found within each lactic acid bacteria group, since wines presented specific characteristics depending on the lactic acid bacteria strain used. In all cases, malolactic fermentation seemed to modify the amino acid and volatile composition of the wine.

Assessment of the native electrophoretic analysis of total grape must proteins for the characterization of Vitis vinifera L. cultivars.

The findings of an ampelographic analysis of vines belonging to a Germoplasm Bank were compared to the results of native electrophoresis of the total proteins in their musts. Cluster analysis of the data from the morphological description produced correct groupings, in terms of variety, for all samples. When cluster analysis was performed on the electrophoretic data, 10 of the 11 musts studied were grouped correctly. Electrophoresis was also performed on 30 musts made from a mixture of grapes from large vineyards. In the cluster analysis of the electrophoretic data on the proteins of the 41 musts studied, all the musts are grouped correctly in terms of variety. Electrophoretic analysis of proteins is a simple technique that can be used routinely, provides complementary information to morphological analysis for varietal characterization of vines, and in the majority of cases, makes it possible to ascertain the grape variety from which musts originate.

Capillary isoelectric focusing of erythropoietin glycoforms and its comparison with flat-bed isoelectric focusing and capillary zone electrophoresis.

The influence of several operation conditions on separation of recombinant human erythropoietin glycoforms by capillary isoelectric focusing (cIEF) is explored. From this study it is deduced that in order to separate several glycoforms of erythropoietin, urea has to be added to sample, which should not be completely depleted of the excipients used in its formulation. On-line desalting does not provide separation enhancement for samples with high content of salt. Better resolution is obtained using a mixture of a broad and a narrow pH-range carrier ampholytes than with either one used separately. Under the experimental conditions, focusing voltages of 25 kV improve separation compared to lower and higher electric fields. Focusing times shorter than the time necessary for electric current to reach a minimum provide similar separations than longer focusing times at which a minimum value of the current has already been achieved. The optimized method allows the separation and quantitation in 12 min of at least seven bands containing glycoforms of recombinant erythropoietin with apparent isoelectric points in the range 3.78-4.69. Compared to flat-bed isoelectric focusing, cIEF provides better separation of bands of glycoforms in a shorter time, and allows quantitative determination. Capillary zone electrophoresis (CZE) gives rise to resolution of erythropoietin glycoforms similar to that obtained by cIEF. Although CZE requires a longer analysis time, its reproducibility in terms of peak area of glycoforms is better than in cIEF.