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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Probiotic properties are highly strain-dependent but rarely studied in enological lactic acid bacteria (LAB). In this study, the probiotic features of 11 strains of Lactobacillus spp., Pediococcus spp., and Oenococcus oeni, including saliva and acid resistance, bile tolerance and exopolysaccharides' production, were investigated. The assays included two probiotic reference strains (Lactobacillus plantarum CLC 17 and Lactobacillus fermentum CECT5716). The Lactobacillus and Pediococcus strains showed high resistance to lysozyme (>80% resistance to 100 mg/L of lysozyme under conditions simulating the in vivo dilution by saliva) and were capable of surviving at low pH values (pH 1.8) and bile salts, suggesting good adaptation of the wine strains to gastrointestinal conditions. The ability of the strains to adhere to the intestinal mucosa and the inhibition of the adhesion of Escherichia coli to human intestinal cells were also evaluated. Adhesion levels of enological LAB to Caco-2 cells varied from 0.37% to 12.2%, depending on the strain. In particular, Pediococcus pentosaceus CIAL-86 showed a high percentage of adhesion to intestinal cells (>12%), even higher than that shown by the probiotic reference strains, and a high anti-adhesion activity against E. coli CIAL-153 (>30%), all of which support this wine LAB strain as a potential probiotic., (Published by Elsevier Ltd.)

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.

Molecular techniques have been applied to study the evolution of wine-associated lactic acid bacteria from red wines produced in the absence and presence of antimicrobial phenolic extracts, eucalyptus leaves and almond skins, and to genetically characterize representative Oenococcus oeni strains. Monitoring microbial populations by PCR-DGGE targeting the rpoB gene revealed that O. oeni was, as expected, the species responsible for malolactic fermentation (MLF). Representative strains from both extract-treated and not-treated wines were isolated and all were identified as O. oeni species, by 16S rRNA sequencing. Typing of isolated O. oeni strains based on the mutation of the rpoB gene suggested a more favorable adaptation of L strains (n = 63) than H strains (n = 3) to MLF. Moreover, PFGE analysis of the isolated O. oeni strains revealed 27 different genetic profiles, which indicates a rich biodiversity of indigenous O. oeni species in the winery. Finally, a higher number of genetic markers were shown in the genome of strains from control wines than strains from wines elaborated with phenolic extracts. These results provide a basis for further investigation of the molecular and evolutionary mechanisms leading to the prevalence of O. oeni in wines treated with polyphenols as inhibitor compounds., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Fermentation extracts from fungi isolated from vineyard ecosystems were tested for antimicrobial activities against a set of test microorganisms, including five food-borne pathogens (Staphylococcus aureus EP167, Acinetobacter baumannii (clinically isolated), Pseudomonas aeruginosa PAO1, Escherichia coli O157:H7 (CECT 5947) and Candida albicans MY1055) and two probiotic bacteria (Lactobacillus plantarum LCH17 and Lactobacillus brevis LCH23). A total of 182 fungi was grown in eight different media, and the fermentation extracts were screened for antimicrobial activity. A total of 71 fungi produced extracts active against at least one pathogenic microorganism, but not against any probiotic bacteria. The Gram-positive bacterium S. aureus EP167 was more susceptible to antimicrobial fungi broth extracts than Gram-negative bacteria and pathogenic fungi. Identification of active fungi based on internal transcribed spacer rRNA sequence analysis revealed that species in the orders Pleosporales, Hypocreales and Xylariales dominated. Differences in antimicrobial selectivity were observed among isolates from the same species. Some compounds present in the active extracts were tentatively identified by liquid chromatography-mass spectrometry. Antimicrobial metabolites produced by vineyard ecosystem fungi may potentially limit colonization and spoilage of food products by food-borne pathogens, with minimal effect on probiotic bacteria., (Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Phenolic acids (benzoic, phenylacetic and phenylpropionic acids) are the most abundant phenolic structures found in fecal water. As an approach towards the exploration of their action in the gut, this paper reports the antimicrobial activity of thirteen phenolic acids towards Escherichia coli, Lactobacillus spp., Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The growth of E. coli ATCC 25922 was inhibited by only four of the phenolic acids tested at a concentration of 1000 microg/mL, whereas pathogenic E. coli O157:H7 (CECT 5947) was susceptible to ten of them. The genetically manipulated E. coli lpxC/tolC strain was highly susceptible to phenolic acids. The growth of lactobacilli (Lactobacillus paraplantarum LCH7, Lactobacillus plantarum LCH17, Lactobacillus fermentum LPH1, L. fermentum CECT 5716, Lactobacillus brevis LCH23, and Lactobacillus coryniformis CECT 5711) and pathogens (S. aureus EP167 and C. albicans MY1055) was also inhibited by phenolic acids, but to varying extents. Only P. aeruginosa PAO1 was not susceptible to any of the phenolic compounds tested. Structure-activity relationships of phenolic acids and some of their diet precursors [(+)-catechin and (-)-epicatechin] were established, based on multivariate analysis of microbial activities. The antimicrobial properties of phenolic acids reported in this paper might be relevant in vivo., (2010 Elsevier Masson SAS. All rights reserved.)

Free soluble carbohydrates of different wine samples were analyzed by GC-MS as their trimethylsilyloximes using a methylsilicone column. Besides alpha,alpha-trehalose, several beta-glucosylglucoses such as cellobiose, sophorose, laminaribiose and gentiobiose were the main disaccharides identified. With the exception of gentiobiose, these disaccharides are now reported for the first time in wine. Lactose (4-O-beta-D-galactopyranosyl-D-glucose), previously described in this product, was also tentatively identified. Several free glycosides: beta-ethyl-glucoside and seven glyceryl-glycosides (including glucosides and galactosides) were also identified for the first time in wine. On the contrary, disaccharides in grape juice were mainly constituted of fructose derivatives, including sucrose, and no glycosides were detected. Although the total amount of disaccharides was different in white wines (<50mg/L) from those in rosé and red wines (80-130 mg/L), the chromatographic profile was noticeably similar in all wine samples. The method here reported allows the identification of several carbohydrates which have not been previously detected in wines and could contribute to increase the understanding of enzymatic activity during winemaking.

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.

Biologically aged sherry-type wines are elaborated by the so-called 'criadera and solera' system, which essentially involves development of the yeast on the wine surface forming a film velum for several years. Lactic acid bacteria can also develop and contribute to sherry-type wine quality, although their presence and role in this enological process have received very little attention. In this study, lactic acid bacteria microbiota and the presence of biogenic amines were investigated throughout the manufacture and biological aging of 36 samples of sherry wines. Malolactic fermentation was found to mainly take place during the first stage of biological ageing. The incidence and populations of lactic acid bacteria in sherry wines were low. The diversity of bacterial species isolated from the wines was greater than previously reported and included species of Lactobacillus, with prevailing Lactobacillus hilgardii, Lactobacillus plantarum and Lactobacillus zeae and Leuconostoc mesenteroides. The biogenic amine-producing capacity of the isolates was also determined. Five strains were putrescine producers, while another strain was shown to produce tyramine and phenylethylamine, simultaneously. L. zeae was one of the predominant species in wines during the biological aging and seemed to be one of the main putrescine producers. The biogenic amine composition of the wines investigated was similar to that reported for other types of wines. Putrescine was the major amine, followed by cadaverine, histamine and tyramine. The amine contents detected were lower than those usually reported in red wines.

In this work, two different capillary electrophoresis-mass spectrometry (CE-MS) methods, namely, capillary electrophoresis-ion-trap mass spectrometry (CE-IT-MS) and capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF-MS), applied to analyze biogenic amines in wine samples are investigated. A group of five amines was selected as case study (namely, putrescine, cadaverine, histamine, phenylethylamine and tyramine) since they are the most frequently biogenic amines found in wines. The possibilities of both instruments in terms of sensitivity, selectivity and quantitation during the determination of the mentioned biogenic amines in wine samples was studied and their results corroborated by HPLC. After optimization of the analytical conditions, CE-IT-MS and CE-TOF-MS allowed the identification of biogenic amines in wines without any previous treatment except diluting 1:1 with water and filtering. Biogenic amines were determined in three red wines and one white wine showing, as expected, a higher concentration in red wines. Moreover, CE-IT-MS and CE-TOF-MS were compared regarding their capacity to detect other biogenic amines different to the selected ones in wine samples, showing CE-TOF-MS a much better capability (i.e., putrescine, cadaverine, histamine, phenylethylamine, tyramine, triptamine, spermidine, spermine, ethanolamine and isoamylamine were identified by CE-TOF-MS in a single analysis). Moreover, CE-TOF-MS allowed the quantitation of biogenic amines with limits of detection as low as 10 ng/mL, comparable to those obtained using HPLC with fluorescence detection, without any previous derivatization step and with analysis times fivefold faster (40 min by HPLC and 8 min by CE-TOF-MS).

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.

A central composite face design was used to study growth and tyramine production of two strains of lactic acid bacteria, Lactobacillus brevis CECT 4669 and Enterococcus faecium BIFI-58. The effects of five physicochemical factors (incubation temperature and time, environmental pH, added tyrosine concentration, and pyridoxal-5-phosphate (PLP) supplementation) on cell growth and tyramine production were analyzed under aerobic and anaerobic conditions. The parameters of the quadratic model for each response variable were estimated by multiple linear regression (MLR), and statistical analysis of the results led to the elucidation of mathematical models capable of predicting the behavior of the responses as a function of the main variables involved in the process. Incubation time was found to be the most important variable influencing growth in L. brevis, while pH showed the highest contribution in E. faecium. The production of tyramine was dependent on the added tyrosine concentration and incubation time. The proposed MLR model predicted the optimum conditions that gave maximum responses for L. brevis and E. faecium growth and tyramine production. In both strains, this model predicted that the anaerobic condition at acidic pH (4.4) in the presence of a high tyrosine concentration favors tyramine production.

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.

In a screening of primers, we have selected three pairs of primers for a multiplex PCR assay for the simultaneous detection of lactic acid bacteria (LAB) strains, which potentially produce histamine, tyramine, and putrescine on fermented foods. These primers were based on sequences from histidine, tyrosine, and ornithine decarboxylases from LAB. Under the optimized conditions, the assay yielded a 367-bp DNA fragment from histidine decarboxylases, a 924-bp fragment from tyrosine decarboxylases, and a 1,446-bp fragment from ornithine decarboxylases. When the DNAs of several target organisms were included in the same reaction, two or three corresponding amplicons of different sizes were observed. This assay was useful for the detection of amine-producing bacteria in control collection strains and in a LAB collection. No amplification was observed with DNA from nonproducing LAB strains. This article is the first describing a multiplex PCR approach for the simultaneous detection of potentially amine-producing LAB in foods. It can be easily incorporated into the routine screening for the accurate selection of starter LAB and in food control laboratories.

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.