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.

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.

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.

Short communication: Production of antihypertensive peptide HLPLP by enzymatic hydrolysis: optimization by response surface methodology.

This study evaluates the potential ability of proteolytic enzymes to release the antihypertensive peptide HLPLP, ß-casein f(134-138), from caseinate. Corolase PP (Röhm GmbH & Co. KG, Darmstadt, Germany) was found as the most appropriate enzyme to produce this peptide. The optimization of the main experimental variables involved in the process [concentration of Corolase PP, concentration of Peptidase 433P (Biocatalysts Ltd., Parc Nantgarw, UK), and the hydrolysis time on the HLPLP concentration, expressed as area of peak] were studied using a central composite face design. The optimum conditions to obtain the maximum concentration of HLPLP provided by the statistical program were a concentration of Corolase PP of 60 mg/g of protein and hydrolysis time of 24h. The use of the Peptidase 433P did not increase the amount of the active peptide. The obtained hydrolysate might be used as functional ingredient with antihypertensive properties.

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.

Hydrolysis under high hydrostatic pressure as a means to reduce the binding of beta-lactoglobulin to immunoglobulin E from human sera.

Cows' milk allergy is the most frequent food allergy in children, and beta-lactoglobulin (beta-Lg) is a major allergen. Milk-based hypoallergenic ingredients are manufactured by enzymatic hydrolysis, a process that could be improved by the application of high-pressure treatments. This study showed that the treatment of beta-Lg dissolved in buffer with chymotrypsin and trypsin under high pressure for relatively short times accelerated proteolysis by leading to a rapid removal of the intact protein. The rapid proteolysis of the beta-Lg substrate under pressure led to the production, in 20 min, of hydrolysates with lower immunoglobulin (Ig) G binding than those produced in 8 h (chymotrypsin) or 48 h (trypsin) at atmospheric pressure. However, those hydrolysates retained some residual IgE-binding properties that could be traced to the preferential release, during the initial stages of proteolysis, of peptides containing IgE epitopes, such as (Val-41-Lys-60), (Leu-149-Ile-162), and (Ser-21-Arg-40). The formation of these fragments was favored when proteolysis was conducted under high pressure due to the preferential hydrolysis of Arg-40 and Arg-148 by trypsin, and Tyr-42 and Leu-149 by chymotrypsin, all located at the dimer interface of beta-Lg or very close to it. Although our results do not support that trypsin and chymotrypsin under high pressure selectively address the allergenic regions of beta-Lg, it is possible to select the conditions that quickly produce hydrolysates with reduced potential allergenicity that could be used in hypoallergenic foods.

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.

Purification of lactulose from mixtures with lactose using pressurized liquid extraction with ethanol-water at different temperatures.

The viability of the purification of lactulose from a mixture with lactose [70:30 (w/w)] using pressurized liquid extraction (PLE) at 1500 psi for 30 min was studied. Different temperatures (from 40 to 130 degrees C) and proportions of ethanol:water (70:30, 80:20, 90:10, 95:5, and 100:0) as the extraction solvent were assayed. Lactose and lactulose were measured by gas chromatographic analysis as their trimethylsilyl derivatives. Data were fitted through multiple linear regressions to different quadratic models to describe both the extraction yield (in terms of mg of lactulose) and the purity of the lactulose extracted. The optimum extraction conditions provided by the model were as follows: extraction temperature, 40 degrees C; and solvent composition, 70:30 ethanol:water. PLE extraction under the optimized conditions was also applied to purify lactulose from lactose in a synthesis mixture. To our knowledge, this is the first time that PLE has been tested for extraction and purification of lactulose from its mixture with lactose; this technique showed several advantages over classical methods such as the short extraction time and the low solvent consumption.

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.

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.

Determination of the antihypertensive peptide LHLPLP in fermented milk by high-performance liquid chromatography-mass spectrometry.

Among different lactic acid bacteria isolated from raw milk, 4 Enterococcus faecalis strains have stood out as producers of fermented milk with potent antihypertensive activity. The peptide beta-casein f(133-138), LHLPLP, was identified as one of the major peptides responsible for the activity of these fermented milk products. A simple method was developed to quantify this peptide in fermented milk using high-performance liquid chromatography coupled in line with mass spectrometry. This procedure does not require any previous sample fractionation or extraction, and direct analysis of the water-soluble extract obtained from the fermented milk can be performed. Validation studies showed sufficient specificity, reproducibility, linearity, and recovery, demonstrating that this method can be used for the routine quantification of LHLPLP during the production of fermented milk products. The developed method was readily applied to quantify the peptide LHLPLP under different fermentation conditions and with different aromatized products.

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.

Nontraumatic lower extremity amputations in diabetic and non-diabetic subjects in Madrid, Spain.

The purpose of this study was to determine the incidence of non-traumatic lower extremity amputations (LEAs) in diabetic and non-diabetic subjects in Madrid, Spain, and their direct cost. All patients who underwent LEAs between the 1st of January 1994 and the 31st of December 1996, and who had lived in area 7 of the city (569,307 inhabitants) for at least the last 6 months, were identified through operating theatre records cross-checked with Vascular Surgery Department discharge records. In addition, the direct cost of LEAs per year was estimated, taking into account the length of the hospital stay, the period of rehabilitation in the outpatient clinic after discharge, and the use of artificial limbs and their maintenance. The incidence of LEAs was 1.6 (95% CI: 1.1-2.2) per 10(5) non-diabetic subjects and 46.1 (95% CI: 34.5-57.6) per 10(5) diabetic patients. Relative risk was 28. Total direct costs associated with LEAs per year were US$ 56,131 in the diabetic population and US$ 30,765 in the non-diabetic population. Thus, potential cost savings associated with excess amputations in the diabetic population was estimated at US$ 541,353 per year of US$ 94,736 per 10(5) inhabitants. It is concluded that the incidence of LEAs in both diabetic and non-diabetic populations in area 7 is the lowest reported in European countries. The potential cost savings per 10(5) inhabitants and per year is estimated at US$ 94,736.

Estimation of the prevalence of diabetes mellitus diagnosed, and incidence of type 1 (insulin-dependent) diabetes mellitus in the Avila Health Care region of Spain.

We studied the prevalence of diabetes mellitus diagnosed in the Avila Health Care region, Spain, based on the prescribed daily dosage (PDD) of insulin (corrected in function of insulin loss (PDDc)) and sulphonureas, from a sample representative of prescribing physicians (A, n = 48) and pharmacies (B, n = 25) in our region. The amounts of the PDDcs of insulin sold during 1989 per 1000 inhabitants/day were 6.06 and 6.20, respectively for the two samples (A and B). The figures for PDDs of sulphonurea/1000 inhabitants/day were 16.25 and 19.16, respectively. We determined the proportion that diabetic patients on diet alone represented in relation to non-insulin treated diabetic patients who visited the only specialized clinic for diabetic patients in our province in 1989. These patients accounted for 40.83% of non-insulin treated diabetic patients. The patients being treated with biguanides or with a combination of anti-diabetic drugs accounted for less than 1% of those treated in the specialized clinic. Taking this into account, the prevalence of diabetes mellitus in Avila was 3.35% (C.I. 95%: 3.10-3.59), according to the sample A and 3.85% (C.I. 95%: 3.50-4.31) according to the sample B. In addition, we studied the hospital admission records during three consecutive years in order to find out the incidence of type 1 insulin-dependent diabetes mellitus (IDDM) below 15 years of age in the Avila Health Care region of Spain. All prescribing physicians that did not work in the Hospital (197 in the rural area and 18 in the urban area) were used as the secondary source for validation of case ascertainment.(ABSTRACT TRUNCATED AT 250 WORDS)

Behaviour modification in obese subjects with type 2 diabetes mellitus.

We have followed prospectively, 46 obese, type 2 diabetic patients for a 55-week period, in order to evaluate the efficiency of an educational programme based on behaviour modification to enhance weight loss and changes of other cardiovascular risk factors. No patient received pharmacological treatment during the study. At the end of the follow-up the patients obtained an average weight loss of 9.250 kg (range: 0.500-17.500 kg); the BMI was reduced from 34.2 +/- 0.8 kg/m2 to 30.6 +/- 1.1 kg/m2 (P less than 0.01); fasting serum glucose descended from 7.9 +/- 0.4 to 6.1 +/- 0.5 mM (P less than 0.05); SBP (systolic blood pressure) decreased from 145.7 +/- 3 to 126.4 +/- 5.1 mmHg (P less than 0.01); DBP (diastolic blood pressure) decreased from 83.5 +/- 2.5 to 65 +/- 2.6 mmHg (P less than 0.01); triglyceride levels were lowered from 164.5 +/- 12 to 109.7 +/- 10 mg/dl (P less than 0.01); HDL-cholesterol levels increased from 1.27 +/- 0.05 to 1.53 +/- 0.12 mM (P less than 0.01). Serum glucose 2 h after a 75 g glucose oral load decreased from 14.9 +/- 0.6 to 12.7 +/- 0.9 mM (P less than 0.05) on week 35 of follow-up. Twelve patients no longer presented a diabetic curve (8 normal oral glucose tolerance test (OGTT) curves, and 4 impaired glucose tolerance (IGT) curves). No significant changes in the parameters studied were obtained in the group of patients on conventional treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Polydimethylsiloxane solid-phase microextraction-gas chromatography method for the analysis of volatile compounds in wines. Its application to the characterization of varietal wines.

A study was made of the validity of the solid-phase microextraction method, using a polydimethylsiloxane coated fused-silica fiber, for the extraction-desorption of the minor volatile compounds from wine before their gas chromatographic analysis. The aspects considered were the influence of ethanol on extraction, repeatability, limits of detection, linearity and recovery of compounds. This method, together with the direct injection of the major volatile compounds, was applied to 16 varietal wines. The findings indicate that the method is a highly suitable technique for the analysis of wines and that the volatile composition of wines depends, at least partly, on the grapes with which they have been made.

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.

In vitro fermentation of a red wine extract by human gut microbiota: changes in microbial groups and formation of phenolic metabolites.

An in vitro batch culture fermentation experiment was conducted with fecal inocula from three healthy volunteers in the presence and absence of a red wine extract. Changes in main bacterial groups were determined by FISH during a 48 h fermentation period. The catabolism of main flavonoids (i.e., flavan-3-ols and anthocyanins) and the formation of a wide a range of phenolic microbial metabolites were determined by a targeted UPLC-PAD-ESI-TQ MS method. Statistical analysis revealed that catechol/pyrocatechol, as well as 4-hydroxy-5-(phenyl)-valeric, 3- and 4-hydroxyphenylacetic, phenylacetic, phenylpropionic, and benzoic acids, showed the greatest increases in concentration during fermentation, whereas 5-(3'-hydroxyphenyl)-γ-valerolactone, its open form 4-hydroxy-5-(3'-hydroxyphenyl)-valeric acid, and 3,4-dihydroxyphenylacetic acid represented the largest interindividual variations in the catabolism of red wine polyphenols. Despite these changes, microbial catabolism did not produce significant changes in the main bacterial groups detected, although a slight inhibition of the Clostridium histolyticum group was observed.

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).