Kefir micro-organisms: their role in grain assembly and health properties of fermented milk.

Kefir is a homemade viscous and slightly effervescent beverage obtained by milk fermentation with kefir grains, which are built up by a complex community of lactic acid and acetic acid bacteria and yeasts confined in a matrix of proteins and polysaccharides. The present review summarizes the role of kefir micro-organisms in grain assembly and in the beneficial properties attributed to kefir. The use of both culture-dependent and independent methods has made possible to determine the micro-organisms that constitute this ecosystem. Kefir consumption has been associated with a wide range of functional and probiotic properties that could be attributed to the micro-organisms present in kefir and/or to the metabolites synthetized by them during milk fermentation. In this context, the role of micro-organisms in kefir health promoting properties is discussed with particular attention to the contribution of yeast as well as bioactive metabolites such as lactic and acetic acid, exopolysaccharides and bioactive peptides. Even though many advances on the knowledge of this ancient fermented milk have been made, further studies are necessary to elucidate the complex nature of the kefir ecosystem.

Probiotic yeast Kluyveromyces marxianus CIDCA 8154 shows anti-inflammatory and anti-oxidative stress properties in in vivo models.

Inflammatory bowel diseases (IBDs) are complex affections with increasing incidence worldwide. Multiple factors are involved in the development and maintenance of the symptoms including enhanced oxidative stress in intestinal mucosa. The conventional therapeutic approaches for IBDs are based on the use anti-inflammatory drugs with important collateral effects and partial efficacy. In the present work we tested the anti-inflammatory capacity of Kluyveromyces marxianus CIDCA 8154 in different models. In vitro, we showed that the pretreatment of epithelial cells with the yeast reduce the levels of intracellular reactive oxygen species. Furthermore, in a murine model of trinitro benzene sulfonic acid-induced colitis, yeast-treated animals showed a reduced histopathological score (P<0.05) and lower levels of circulating interleukin 6 (P<0.05). The capacity to modulate oxidative stress in vivo was assessed using a Caenorhabditis elegans model. The yeast was able to protect the nematodes from oxidative stress by modulating the SKN-1 transcription factor trough the DAF-2 pathway. These results indicate that K. marxianus CIDCA 8154 could control the intestinal inflammation and cellular oxidative stress. Deciphering the mechanisms of action of different probiotics might be useful for the rational formulation of polymicrobial products containing microorganisms targeting different anti-inflammatory pathways.

Optimization the soda-AQ process for cellulose pulp production and energy content of black liquor from L. leucocephala K360.

A commercial variety of Leucaena leucocephala K360 was used for pulp production and papermaking employing the soda-anthraquinone process. Also, the chemical and energy contents of the resultant black liquors were determined to simultaneously optimize: pulp and paper production and energy generation. A process temperature of (185°C), an operating time of (120 min) and an active alkali concentration of (21%) provided sheets of paper with good strength (tensile index of 12.12 Nm/g, burst index of 0.38 kPa m(2)/g, tear index of 1.29 mN m(2)/g and a Kappa number of 20.5) and black liquor with a greater calorific value (14.1 MJ/kg) than that obtained with higher active alkali concentrations. However, reducing the active alkali concentration to a level in the low operation range led to less marked degradation of cellulose and allowed paper sheets with good properties to be obtained and energy to be optimally produced from the black liquor.

Biorefinery of olive pruning using various processes.

Biorefinery developed involve separation of olive pruning into two parts: main (OPM) (stems>1cm diameter), and residual (OPR) (stems<1cm diameter, and leaves). OPM was submitted to hydrothermal treatment, separating: a liquid fraction (HL), rich in products of hemicelluloses decomposition, and other solid (HS), rich in cellulose and lignin. HS is subject to pulping, resulting: a liquid fraction (HPL), rich in lignin, and other solid (HPS), rich in cellulose. Up to 42% of the polysaccharides from OPM were recovered in HL as valuable compounds. HPS can be used for the bioethanol production by saccharification and fermentation, reaching a bioethanol conversion of 90.6% of the theoretical value. In addition, HPS obtained paper with lower strength properties than those of paper obtained from OPM pulp directly. OPR provided 18.70 MkJ/t heating values, 1094-2234°C flame temperature, and 45-53°C dew point temperature, with a cost of the unit of heat (3.20 €/MkJ) much lower than fossil fuels fluids.

Extracting value from Eucalyptus wood before kraft pulping: effects of hemicelluloses solubilization on pulp properties.

Eucalyptus globulus wood samples were subjected to autohydrolysis for extracting hemicelluloses, and the resulting solids were assayed as substrates for kraft pulping and further Totally Chlorine Free (TCF) bleaching. The susceptibility of treated solids to kraft processing was assessed under selected experiments covering the optimum experimental range. In order to establish a basis for comparison, samples of untreated wood were also subjected to kraft delignification. The best kraft pulps obtained from autohydrolyzed solids were subjected to an optimized TCF bleaching sequence involving double alkaline oxygen and pressurized H(2)O(2) processing, and characterized using standard methods. The suitability of the final product obtained by autohydrolysis-kraft delignification-TCF bleaching for specific purposes is discussed on the basis of the experimental results.

Inhibitory activity of cheese whey fermented with kefir grains.

We investigated the chemical and microbiological compositions of three types of whey to be used for kefir fermentation as well as the inhibitory capacity of their subsequent fermentation products against 100 Salmonella sp. and 100 Escherichia coli pathogenic isolates. All the wheys after fermentation with 10% (wt/vol) kefir grains showed inhibition against all 200 isolates. The content of lactic acid bacteria in fermented whey ranged from 1.04 × 10(7) to 1.17 × 10(7) CFU/ml and the level of yeasts from 2.05 × 10(6) to 4.23 × 10(6) CFU/ml. The main changes in the chemical composition during fermentation were a decrease in lactose content by 41 to 48% along with a corresponding lactic acid production to a final level of 0.84 to 1.20% of the total reaction products. The MIC was a 30% dilution of the fermentation products for most of the isolates, while the MBC varied between 40 and 70%, depending on the isolate. The pathogenic isolates Salmonella enterica serovar Enteritidis 2713 and E. coli 2710 in the fermented whey lost their viability after 2 to 7 h of incubation. When pathogens were deliberately inoculated into whey before fermentation, the CFU were reduced by 2 log cycles for E. coli and 4 log cycles for Salmonella sp. after 24 h of incubation. The inhibition was mainly related to lactic acid production. This work demonstrated the possibility of using kefir grains to ferment an industrial by-product in order to obtain a natural acidic preparation with strong bacterial inhibitory properties that also contains potentially probiotic microorganisms.

Hydrothermal treatment and ethanol pulping of sunflower stalks.

The influence of temperature in the hydrothermal treatment of sunflower stalks on the composition of the liquid fraction obtained was examined. The remaining solid fraction was subjected to ethanol pulping in order to obtain pulp that was used to produce paper sheets. The pulp was characterized in terms of yield, kappa index, viscosity, and cellulose, hemicellulose and lignin contents; and the paper sheets in terms of breaking length, stretch, burst index and tear index. Hydrothermal treatment of the raw material at 190 degrees C provided a liquid phase with maximal hemicellulose-derived oligomers and monosaccharide (glucose, xylose and arabinose) contents (26.9 and 4.2 g/L, respectively). Pulping the solid fraction obtained by hydrothermal treatment at 180 degrees C, with 70% ethanol at a liquid/solid ratio of 8:1 at 170 degrees C for 120 min provided pulp with properties on a par with those of soda pulp from the sunflower stalks, namely: 36.3% yield, 69.1% cellulose, 12.6% hemicellulose, 18.2% lignin and 551 ml/g viscosity. Also, paper sheets obtained from the ethanol pulp were similar in breaking length (3.8 km), stretch (1.23%), burst index (1.15 kN/g) and tear index (2.04 m Nm(2)/g) to those provided by soda pulp.

Protective action of Lactobacillus kefir carrying S-layer protein against Salmonella enterica serovar Enteritidis.

Eight Lactobacillus kefir strains isolated from different kefir grains were tested for their ability to antagonize Salmonella enterica serovar Enteritidis (Salmonella enteritidis) interaction with epithelial cells. L. kefir surface properties such as autoaggregation and coaggregation with Salmonella and adhesion to Caco-2/TC-7 cells were evaluated. L. kefir strains showed significantly different adhesion capacities, six strains were able to autoaggregate and four strains coaggregated with Salmonella. Coincubation of Salmonella with coaggregating L. kefir strains significantly decreased its capacity to adhere to and to invade Caco-2/TC-7 cells. This was not observed with non coaggregating L. kefir strains. Spent culture supernatants of L. kefir contain significant amounts of S-layer proteins. Salmonella pretreated with spent culture supernatants (pH 4.5-4.7) from all tested L. kefir strains showed a significant decrease in association and invasion to Caco-2/TC-7 cells. Artificially acidified MRS containing lactic acid to a final concentration and pH equivalent to lactobacilli spent culture supernatants did not show any protective action. Pretreatment of this pathogen with spent culture supernatants reduced microvilli disorganization produced by Salmonella. In addition, Salmonella pretreated with S-layer proteins extracted from coaggregating and non coaggregating L. kefir strains were unable to invade Caco-2/TC-7 cells. After treatment, L. kefir S-layer protein was detected associated with Salmonella, suggesting a protective role of this protein on association and invasion.

Refining of autohydrolysis liquors for manufacturing xylooligosaccharides: evaluation of operational strategies.

When xylan-containing lignocellulosic materials are subjected to the action of heat in an aqueous medium (autohydrolysis reaction) under suitable operational conditions, the heterocyclic ether bonds of the polymeric chain are split to give xylooligosaccharides (XO) and a variety of other products derived from hemicelluloses, lignin and extractives. For product applications requiring high-purity XO, subsequent purification stages have to be implemented. Various strategies have been evaluated for refining Eucalyptus wood autohydrolysis liquors, including the two-stage reaction, precipitation and solvent extraction of freeze-dried, dewaxed liquors. Data on the degree of xylooligosaccharide recovery as well as on the composition of the isolates (measured in terms of hemicellulose-derived products and non-saccharide products) are provided for the various operational procedures assayed.

Hydrothermal and pulp processing of Eucalyptus.

Hydrothermal processing of Eucalyptus wood was performed at operation temperature of 181 degrees C, processing time or 37.5 min and solid water ratio of 1/6 to ensure a maximum loss of xylan recuperation with minimum cellulose fibre degradation. Under those conditions, the loss of xylan was 22% less than that achieved with the conditions 196 degrees C, 50.6 min and 1/8 (solid/water). IN In addition, an experimental design was used to study the influence of process variables: temperature (145-175 degrees C), pulping time (40-120 min) and ethanol concentration (40-70% weight concentration), on the properties of pulps (yield, kappa number, viscosity, cellulose, xylan, lignin acetyl groups contents and brightness) and paper sheets (stretch index, burst index and tear index) obtained from the solid fraction after hydrothermal treatment of Eucalyptus globulus. Pulps with acceptably high physical and chemical properties can be obtained operating at 175 degrees C for 90 min with 55% ethanol concentration.

Manufacture of xylose-based fermentation media from corncobs by posthydrolysis of autohydrolysis liquors.

Milled corncob samples were mixed with water and heated to obtain a liquid phase containing oligosaccharides, sugars, and acetic acid as main reaction products (autohydrolysis reaction). To hydrolyze the sugar oligomers to the correspondent monomers, sulfuric acid was added to the autohydrolysis liquors to reach 0.5-2 wt% of solution, and the reaction media were heated at 101.5-135 degrees C. With this operational procedure, sugar solutions suitable as fermentation media (containing xylose as the major component) were obtained. The kinetics of the posthydrolysis step was characterized on the basis of experimental data concerning the time courses of the concentrations of xylooligosaccharides, xylose, furfural, and acetic acid. The concentrations of other reaction byproducts (glucose or arabinose) were also measured.

Generation of xylose solutions from Eucalyptus globulus wood by autohydrolysis-posthydrolysis processes: posthydrolysis kinetics.

Eucalyptus wood samples were treated with water under selected operational conditions (autohydrolysis reaction) to obtain a liquid phase containing hemicellulose-decomposition products (mainly acetylated xylooligosaccharides, xylose and acetic acid). In a further acid-catalysed step (posthydrolysis reaction), xylooligosaccharides were converted into xylose, a carbon source for further fermentation. The kinetic pattern governing the posthydrolysis step was established by reacting xylooligosaccharide-containing liquors at 100.5 degrees C, 115 degrees C, 125 degrees C or 135 degrees C in media containing 0.5, 1.0, 1.5 or 2 wt% of catalyst (sulphuric acid). The time course of the concentrations of xylooligosaccharides, xylose, furfural and acetic acid were determined, and the results were interpreted by means of a kinetic model which allowed a close reproduction of the experimental data. Almost quantitative conversion of xylooligosaccharides into xylose was achieved under a variety of experimental conditions. The first-order, kinetic coefficient for xylooligosaccharide hydrolysis (k1, h(-1)) varied with both temperature (T, K) and molar sulphuric acid concentration (C) according to the equation In k1 = 36.66 + 1.00lnC - 108.0/(8.314T). The hydrolysis of acetyl groups followed a first-order kinetics. The corresponding kinetic coefficient (ka, h(-1) was correlated with the operational conditions by the equation Inka = 26.80+ 1.18 InC - 73.37/(8.314T).

Chemical and microbiological characterisation of kefir grains.

Chemical and microbiological composition of four Argentinean kefir grains from different sources as well as characteristics of the corresponding fermented milk were studied. Kefir grains CIDCA AGK1, AGK2 and AGK4 did not show significant differences in their chemical and microbiological composition. In contrast, protein and yeast content of AGK3 was higher than in the other grains. Although grain microflora comprised lactobacilli, lactococcus, acetic acid bacteria and yeast, we found an important difference regarding species. Lactococcus lactis subsp. lactis, Lactobacillus kefir, Lactobacillus plantarum, Acetobacter and Saccharomyces were present in all types of kefir grain. While Leuconostoc mesenteroides was only isolated from grains CIDCA AGK1 and Lactococcus lactis subsp. lactis biovar diacetylactis, Lactobacillus parakefir and Kluyveromyces marxianus were only isolated from CIDCA AGK2 grains. All grains produced acid products with pH between 3.5 and 4.0. The apparent viscosity of AGK1 fermented milk was greater than the product obtained with AGK4. All fermented milks had inhibitory power towards Escherichia coli but AGK1 and AGK2 supernatants were able to halt the bacterial growth for at least 25 h. Grain weight increment in AGK1, AGK2 and AGK3 during growth in milk did not show significant differences. Despite their fermenting activity, AGK4 grains did not increase their weight.

Inhibitory power of kefir: the role of organic acids.

Milk and MRS broth fermented with kefir grains from different households were examined for inhibitory activity toward gram-negative and gram-positive strains. Fermented milk obtained with 10 g per 100 ml of inoculum (final pH 3.32 to 4.25) and MRS broth fermented with 1 and 10 g per 100 ml of inocula (final pH 4.18 to 5.25) had inhibitory power demonstrated by spot test and agar well diffusion assay. This inhibitory effect could be assigned to the undissociated form of lactic and acetic acid produced during the fermentation process. Kefir supernatants inhibited the growth of Escherichia coli 3 in nutrient broth at 37 degrees C for 24 h. However, supernatants of yogurt or milk artificially acidified with lactic and acetic acids allowed the growth of E. coli 3 in the same conditions. A bacteriostatic effect of milk fermented with kefir grains over E. coli 3 was also demonstrated.