Effect of gums on viability and β‐galactosidase activity of Lactobacillus spp. in milk drink during refrigerated storage

The viability and β‐galactosidase activity of four Lactobacillus strains in milk drink containing gums during 28 days of refrigerated storage at 4 °C were assessed. The population of Lactobacillus rhamnosus GGB101 and Lactobacillus rhamnosus GGB103 were maintained, whereas the population of Lactobacillus reuteri DSM20016 and Lactobacillus reuteri SD2112 significantly decreased. The recommended level of 6 log CFU g^?1 was exceeded for all tested trains throughout storage. The highest viable number of Lactobacillus rhamnosus GGB103 (8.76 ± 0.03 log CFU mL^?1) was obtained in the product containing carrageenan–maltodextrin. The addition of guar–locust bean–carrageenan led to 20‐fold increase in the level of β‐galactosidase activity for L. rhamnosus GGB101 (1208 ± 2.12 Miller units mL^?1) compared to the control (61 ± 2.83 Miller units mL^?1). Our results suggested that gums could be added to milk to improve viability and enhance β‐galactosidase activity of Lactobacillus.     

Extraction of water-soluble xylan from wheat bran and utilization of enzymatically produced xylooligosaccharides by Lactobacillus, Bifidobacterium and Weissella spp.

Xylan was extracted from wheat bran after heat pretreatment in water using either an autoclave or a microwave oven. Xylooligosaccharides (XOS) were produced from the xylan using the thermostable xylanase RmXyn10A and the potential prebiotic properties of XOS were studied in vitro with different human gut bacteria: Lactobacillus brevis (DSMZ 1269), Bifidobacterium adolescentis (ATCC 15703) and two strains of recently isolated lactic acid bacteria from the species pair Weissella cibaria/confusa. The highest yield of (arabino)xylan with the heat pretreatment was obtained at 185 °C for 10 min. Higher temperature led to fewer arabinose substitutions present on the backbone which in turn resulted in a slightly more efficient enzymatic hydrolysis by RmXyn10A.     

Viability and α‐ and β‐galactosidase activity of Bifidobacterium breve and Lactobacillus reuteri in yoghurt products supplemented with shiitake mushroom extract during refrigerated storage

This study examined the impact of shiitake mushroom extract (ME) on the viability and α/β enzyme activity of Bifidobacterium breve ATCC 15701 and Lactobacillus reuteri DSM20016 in yoghurt during refrigerated storage. The presence of ME significantly enhanced the viability of both strains. L. reuteri DSM20016 exhibited better stability in yoghurt than B. breve ATCC 15701. L. reuteri DSM20016 showed the highest viability in yoghurt samples containing 4% ME, followed by B. breve ATCC 15701, and then control samples. Results suggest that ME can be used as a natural additive to dairy products to improve the growth and viability of L. reuteri DSM20016 and B. breve ATCC 15701 strains.     

Aglycone production by Lactobacillus rhamnosus CRL981 during soymilk fermentation

Lactobacillus rhamnosus CRL981 showed the highest levels of β-glucosidase and was selected to characterize this enzyme system, among 63 strains of different Lactobacillus species. The maximum activity was obtained at pH 6.4 and 42 °C. The enzyme showed weak resistance to thermal inactivation maintaining only 20% of the initial activity when it was exposed at 50 °C for 5 min. It also, showed stability when stored at 4 °C for 60 days. Afterwards, L. rhamnosus was evaluated for hydrolysis of isoflavones to aglycones, cell population, residual sugars and organic acid produced during fermentation on soymilk (37 °C for 24 h). Higher viable counts were obtained after 12 h of fermentation (8.85 log CFU ml⁻¹) followed by a drop of pH and an increase of acidity during fermentation due the production of organic acids. L. rhamnosus CRL981 was able to proliferate in soymilk and produce a high β-glucosidase activity achieving a complete hydrolysis of glucoside isoflavones after 12 h of fermentation. The present study indicates that L. rhamnosus CRL981 could be used in the development of different aglycone-rich functional soy beverages.     

Lactobacillus pentosus dominates spontaneous fermentation of Italian table olives

Culture-dependent and -independent approaches were applied to identify the bacterial species involved in Italian table olive fermentation. Bacterial identification showed that Lactobacillus pentosus was the dominant species although the presence of Lactobacillus plantarum, Lactobacillus casei, Enterococcus durans, Lactobacillus fermentum and Lactobacillus helveticus was observed. Rep-PCR allowed to obtain strain-specific profiles and to establish a correlation with table olive environment. PCR-DGGE (Denaturing Gradient Gel Electrophoresis) confirmed the heterogeneity of bacterial community structure in fermented table olives as well as the prevalence of L. pentosus. The strains were characterized on the basis of technological properties (NaCl tolerance, β-glucosidase activity and the ability to grow in synthetic brine and in presence of 1 g/100 mL oleuropein). L. pentosus showed a high capacity of adaptation to the different conditions characterizing the olive ecosystem. This species showed the highest percentage of strains able to grow in presence of 10 g/100 mL NaCl, oleuropein and in the synthetic brine. Moreover, all the strains belonging to L. pentosus and L. plantarum species showed a β-glucosidase activity. This study allowed both to identify the main species and strains associated to Italian table olives and to obtain a lactic acid bacteria collection to apply as starter culture in the process of olive fermentation.     

Growth and survival of Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus GR-1 in yogurt for use as a functional food

Both Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus GR-1 are considered probiotic agents with therapeutic properties. To prepare mother cultures for these organism bacteria, four formulations were made with milk (1% fat) with 0.33% yeast extract (T1); 0.4% inulin (T2); 0.33% yeast extract and 0.4% inulin (T3); and one with no additives (T4). The media were inoculated with 1% probiotic cultures and incubated anaerobically at 37 °C overnight. Low-fat (1%) probiotic yogurts were made. Survival of L. reuteri RC-14 and L. rhamnosus GR-1 was monitored after 1, 7, 14, 21, and 28 days of storage at 4 °C. In all treatments, L. rhamnosus GR-1 survived significantly better (P < 0.05) than L. reuteri RC-14. Survival was highest in media T1 and T3. This study shows that yogurt has the potential to deliver probiotic bacteria to consumers, with L. rhamnosus GR-1 providing excellent shelf life.Industrial relevanceThis study is of relevance to food industry because it deals with the effectiveness of dairy products as a good-vehicle for delivering probiotic microorganisms to consumers. The fermentation of milk into yogurt has gained widespread consumer acceptance in North America and its consumption has increased significantly over the past few years. The normal yogurt cultures, Lactobacillus delbreukii sub-species bulgaricus and Streptococcus thermophilus, are not bile resistant or acid tolerant and thus cannot survive in the intestinal tract, although they may help to lessen the symptoms of lactose intolerance. Various strains of lactic acid bacteria are considered probiotics. Two of the most documented probiotic strains, Lactobacillus reuteri (formerly fermentum) RC-14 and Lactobacillus rhamnosus GR-1 can colonize the intestine and vagina and reduce recurrences of bacterial vaginosis, yeast vaginitis and urinary tract infections. They are bile resistant and survive passage through the human gastrointestinal tract without induction of systemic immune or inflammatory responses. There is no published information on the growth and survival of L. rhamnosus GR-1 and L. reuteri RC-14 in yogurt. The incorporation of L. rhamnosus GR-1 and L. reuteri RC-14 in yogurt is an innovative idea. This research developed a new probiotic yogurt with sufficient viable counts of L. rhamnosus GR-1 accompanied by L. reuteri RC-14. The use of probiotic bacteria, especially those with proven therapeutic effects, in dairy products has attracted a lot of attention from dairy industry and health/wellness industry, and this type of product can provide a bridge between the two industries.     

Comparison of inactivation pathways of thermal or high pressure inactivated Lactobacillus rhamnosus ATCC 53103 by flow cytometry analysis

The effect of thermal and pressure treatments on Lactobacillus rhamnosus ATCC 53103 was evaluated by flow cytometric analysis in conjunction to standard cultivation techniques. A double staining technique with fluorochromes carboxyfluorescein diacetate (cFDA) and propidium iodide (PI) revealed that depending on temperature regime used heat-killed cells had different fluorescence behaviors. Cells killed at 60 °C were not stained at all whereas heat treatment at 75 °C resulted in a single population entirely labelled by PI. These findings indicated that thermal-induced cell death was achievable with or without membrane degradation. Hydrostatic pressures beyond 400 MPa inactivated L. rhamnosus ATCC 53103 in a different way. It was observed that the irreversible damage of the membrane-bound transport systems could be largely accounted for the cause of high pressure-induced cell death.     

β-Glucosidase Activity of Lactobacilli for Biotransformation of Soy Isoflavones

Thirty isolates of lactobacilli were screened for β-glucosidase production and isoflavones biotransformation. The isolates exhibited enzyme activities in the range of 0.14–3.31 IU. Five highest enzyme producers were assessed for isoflavones biotransformation potential in soymilk that varied greatly among the isolates with an increase of 2–3 fold in genistein and 6–14 fold in daidzein. The biochemical and molecular identification classified the isolates as Lactobacillus rhamnosus. Among isolates, L. rhamnosus D13 was the highest enzyme producer, while L. rhamnosus D7 was the highest isoflavone aglycone producer. Our results are distinct because of no proportional relation between enzyme activity and isoflavones biotransformation. The study further confirms that the degree of biotransformation of isoflavones is characteristic of an individual strain.     

Fluidized bed microencapsulation of Lactobacillus reuteri with sweet whey and shellac for improved acid resistance and in-vitro gastro-intestinal survival

This study aimed at developing a micro-encapsulation process for Lactobacillus reuteri DSM 20016 with sweet whey powder and dietary shellac by a two-step fluidized bed granulation and top spray coating technique to enhance resistance to acid conditions and to increase bacterial survival under gastro-intestinal conditions. L. reuteri DSM 20016 was a top-spray fluid bed granulated at 50 °C with sweet whey and different thermo-protectants followed by top spray coating with an aqueous shellac solution. Moisture content and particle size of granules measured by infrared moisture balance and laser diffraction were appropriate for the encapsulation of the bacteria. Bacterial survival rates were evaluated by pour plate counting. Viabilities after granulation were 43 ± 4.5%, after 15 min shellac coating 22.69 ± 2.41% and 14.86 ± 1.29% after 30 min coating time. Endpoint bacterial count after 28 d storage at 4 °C remained equal for coated and uncoated bacteria. After incubation in gastric acid and intestinal conditions the final survival rate of coated L. reuteri DSM 20016 with 76.74 ± 24.36% was higher compared to free cells with 17.74 ± 10.51% (p = 0.0388).To conclude fluidized bed technology with a combination of sweet whey and shellac as encapsulation material offers distinct acid resistance for L. reuteri DSM 20016 and enables improved survival during gastro-intestinal transit. Hence the usage of shellac and sweet whey powder as encapsulation material improves the effectiveness of probiotic food applications.     

Production of exopolysaccharides by Lactobacillus and Bifidobacterium strains of human origin, and metabolic activity of the producing bacteria in milk

This work reports on the physicochemical characterization of 21 exopolysaccharides (EPS) produced by Lactobacillus and Bifidobacterium strains isolated from human intestinal microbiota, as well as the growth and metabolic activity of the EPS-producing strains in milk. The strains belong to the species Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus vaginalis, Bifidobacterium animalis, Bifidobacterium longum, and Bifidobacterium pseudocatenulatum. The molar mass distribution of EPS fractions showed 2 peaks of different sizes, which is a feature shared with some EPS from bacteria of food origin. In general, we detected an association between the EPS size distribution and the EPS-producing species, although because of the low numbers of human bacterial EPS tested, we could not conclusively establish a correlation. The main monosaccharide components of the EPS under study were glucose, galactose, and rhamnose, which are the same as those found in food polymers; however, the rhamnose and glucose ratios was generally higher than the galactose ratio in our human bacterial EPS. All EPS-producing strains were able to grow and acidify milk; most lactobacilli produced lactic acid as the main metabolite. The lactic acid-to-acetic acid ratio in bifidobacteria was 0.7, close to the theoretical ratio, indicating that the EPS-producing strains did not produce an excessive amount of acetic acid, which could adversely affect the sensory properties of fermented milks. With respect to their viscosity-intensifying ability, L. plantarum H2 and L. rhamnosus E41 and E43R were able to increase the viscosity of stirred, fermented milks to a similar extent as the EPS-producing Streptococcus thermophilus strain used as a positive control. Therefore, these human EPS-producing bacteria could be used as adjuncts in mixed cultures for the formulation of functional foods if probiotic characteristics could be demonstrated. This is the first article reporting the physicochemical characteristics of EPS isolated from human intestinal microbiota.     

Lactose hydrolyzing enzymes in Lactobacillus acidophilus strains

Five Lactobacillus acidophilus strains were examined for lactose hydrolyzing enzymes under several growth conditions. All have both β-galactosidase (β-gal) and phospho-β-galactosidase (P-β-gal) activities. Strains W, ATCC 4356 and NCDO 1748 had different amounts of β-gal activity, but all had 100-fold or more higher than P-β-gal activity. In contrast, in strains ATCC 4962 and ATCC 19992, activities of both enzymes were similar. The β-gal synthesis in strains W, 4356 and 1748 was induced by lactose and galactose, while in strains 4962 and 19992 it appeared not to be inducible. At 45°C in the presence of lactose synthesis of only β-gal occurred in strains 4356, 1748 and W. These differences in lactose hydrolyzing enzymes between the strains could be used, along with other characteristics, for taxonomic differentiation between L. acidophilus strains. Studies with L. acidophilus W indicated that cells have maximum β-gal activity at their early stationary phase of growth and when grown in the presence of an inducer and in the absence of oxgall. Selection of strains, type of carbohydrate in growth medium, growth temperature, culture age, and the absence of oxgall during cell growth should be considered in producing L. acidophilus cultures containing high β-gal activity for use as a dietary adjunct by lactose-intolerant individuals and in the manufacture of acidophilus-fermented dairy products.     

Bioconversion of anthocyanin glycosides by Bifidobacteria and Lactobacillus

Eight strains of Lactobacillus plantarum, 6 strains of Lactobacillus casei, Lactobacillus acidophilus LA-5, and Bifidobacterium lactis BB-12 were screened for β-glucosidase activity. We then proceeded to investigate the enzymatic potential of selected strains for bioconversion of delphinidin and malvidin glycosides to their metabolites. L. plantarum and L. casei strains showed the highest cell-envelope associated β-glucosidase activity. Intracellular β-glucosidase activity from B. lactis BB-12 was up to 287-fold higher than that of the other strains. The L. acidophilus strain showed low β-glucosidase activity, both, intra and extracellularly. No aglycons were detected in bacterial extract reactions with anthocyanin glycosides. Delphinidin-3-glucoside underwent chemical degradation to form mainly gallic acid, although delphinidin-3-glucoside degradation due to B. lactis BB-12 and enzymatic activity towards chemically-formed metabolites due to L. casei LC-01 were observed. Incubation of malvidin-3-glucoside with B. lactis BB-12, L. plantarum IFPL722, and L. casei LC-01 cell-free extracts led to different patterns of gallic, homogentisic and syringic acid formation.     

Application of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and L. helveticus for sourdough bread making

The application of Kluyveromyces marxianus (IFO 288), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842) and Lactobacillus helveticus (ATCC 15009) as starter cultures for sourdough bread making was examined. Production of lactic and acetic acids, bread rising, volatile composition, shelf-life and organoleptic quality of the sourdough breads were evaluated. The amount of starter culture added to the flour, the dough fermentation temperature and the amount of sourdough used were examined in order to optimise the bread making process. The use of mixed cultures led to higher total titratable acidities and lactic acid concentrations compared to traditionally made breads. Highest acidity (3.41 g lactic acid/kg of bread) and highest resistance to mould spoilage were observed when bread was made using 50% sourdough containing 1% K. marxianus and 4% L. delbrueckii ssp. bulgaricus. The use of these cultures also improved the aroma of sourdough breads, as shown by sensory evaluations and as revealed by GC–MS analysis.     

The potential of biodetoxification activity as a probiotic property of Lactobacillus reuteri

Previous work on the metabolism of Lactobacillus reuteri ATCC 55730 anticipated a variability in the use of organic electron acceptors as a means to relieve metabolic redox problems. Therefore, investigations focusing on this unique metabolism of L. reuteri may reveal a basis for new probiotic properties. For instance, L. reuteri may use reactive aldehydes and ketones as electron acceptors to balance their redox metabolism, which opens the possibility to exploit this bacterium for in vivo bioreduction of deleterious compounds in the gastrointestinal tract (GIT). Herein we demonstrate that L. reuteri ATCC 55730 cultures on glucose are able to use furfural (1 g/L), and hydroxymethylfurfural (HMF) (0.5 g/L), as electron acceptors. The former enhances the growth rate by about 25% and biomass yield by 15%, whereas the latter is inhibitory. Furfural is stoichiometrically reduced to furfuryl alcohol by the culture. The conversion of furfural had no effect on the flux distribution between the simultaneously operating phosphoketolase and Embden-Meyerhof pathways, but initiated a flux to acetate production. In addition to furfural and HMF, cellular extracts showed potential to reoxidize NADH and/or NADPH with acrolein, crotonaldehyde, and diacetyl, indicating that conversion reactions take place intracellularly, however, utilization mechanisms for the latter compounds may not be present in this strain. The strain did not reduce other GIT-related reactive compounds, including acrylamide, glyoxal, and furan.     

A comprehensive approach to determine the probiotic potential of human-derived Lactobacillus for industrial use

Specific strains should only be regarded as probiotics if they fulfill certain safety, technological and functional criteria. The aim of this work was to study, from a comprehensive point of view (in vitro and in vivo tests), three Lactobacillus strains (Lactobacillus paracasei JP1, Lactobacillus rhamnosus 64 and Lactobacillus gasseri 37) isolated from feces of local newborns, determining some parameters of technological, biological and functional relevance. All strains were able to adequately grow in different economic culture media (cheese whey, buttermilk and milk), which were also suitable as cryoprotectants. As selective media, LP-MRS was more effective than B-MRS for the enumeration of all strains. The strains were resistant to different technological (frozen storage, high salt content) and biological (simulated gastrointestinal digestion after refrigerated storage in acidified milk, bile exposure) challenges. L. rhamnosus 64 and L. gasseri 37, in particular, were sensible to chloramphenicol, erythromycin, streptomycin, tetracycline and vancomycin, increased the phagocytic activity of peritoneal macrophage and induced the proliferation of IgA producing cells in small intestine when administered to mice. Even when clinical trails are still needed, both strains fulfilled the main criteria proposed by FAO/WHO to consider them as potential probiotics for the formulation of new foods.     

Evaluation of enzymic potential for biotransformation of isoflavone phytoestrogen in soymilk by Bifidobacterium animalis,Lactobacillus acidophilus and Lactobacillus casei

Three strains of Lactobacillus acidophilus, two of Lactobacillus casei and one of Bifidobacterium were screened for β-glucosidase activity using ρ-nitrophenyl-β-d-glucopyranoside as a substrate and their potential for the breakdown of isoflavone glucosides to the biologically active aglycones in soymilk. Isoflavones quantification with HPLC and β-glucosidase activity were performed after 0, 12, 24, 36, and 48 h of incubation in soymilk at 37 °C. All six micro-organisms produced β-glucosidase, which hydrolysed the predominant isoflavone β-glucosides. There was a significant increase and decrease (P < 0.05) in the concentration of isoflavone aglycones and glucosides, respectively, in fermented soymilk. Based on the concentration of isoflavones during peak β-glucosidase activity, the hydrolytic potential was calculated. L. acidophilus 4461 had the highest aglycone concentration of 76.9% after 24 h of incubation, up from 8% in unfermented soymilk (at 0 h). It also had the best isoflavone hydrolytic index of 2.01, signifying its importance in altering the biological activity of soymilk.     

Effects of different probiotic strains of Lactobacillus and Bifidobacterium on bacterial translocation and liver injury in an acute liver injury model

Septic complications represent frequent causes of morbidity in liver diseases and following hepatic operations. Most infections are caused by the individual own intestinal microflora. The intestinal microflora composition is important in physiological and pathophysiological processes in the human gastrointestinal tract, but their influence on liver in different situations is unclear. We therefore studied the effect of different Lactobacillus strains and a Bifidobacterium strain on the extent of liver injury, bacterial translocation and intestinal microflora in an acute liver injury model.Sprague–Dawley rats were divided into five groups: acute liver injury control, acute liver injury+B. animalis NM2, acute liver injury+L. acidophilus NM1, acute liver injury+L. rhamnosus ATCC 53103, and acute liver injury+L. rhamnosus DSM 6594 and L. plantarum DSM 9843. The bacteria were administered rectally daily for 8 days. Liver injury was induced on the 8th day by intraperitoneal injection of -galactosamine (1.1 g/kg BW). Samples were collected 24 h after the liver injury. Liver enzymes and bilirubin serum levels, bacterial translocation (to arterial and portal blood, liver and mesenteric lymph nodes (MLNs)), and intestinal microflora were evaluated.L. acidophilus NM1; L. rhamnosus ATCC 53103, and L. rhamnosus DSM 6594+L. plantarum DSM 9843 decreased bacterial translocation compared to the liver injury control group. B. animalis NM2 increased bacterial translocation to the mesenteric lymph nodes. The levels of alanine aminotransferase (ALAT) were significantly lower in the L. acidophilus, L. rhamnosus ATCC 53103, L. rhamnosus DSM 6594+L. plantarum DSM 9843 groups compared to the liver injury group. The L. rhamnosus and L. rhamnosus+L. plantarum groups significantly reduced ALAT levels compared to the B. animalis group. All administered bacteria decreased the Enterobacteriaceae count in the cecum and colon.Administration of different lactobacilli and a Bifidobacterium strain in an acute liver injury rat model, has shown different effects on bacterial translocation and hepatocellular damage. L. acidophilus, L. rhamnosus, and L. rhamnosus+L. plantarum reduced bacterial translocation and hepatocellular damage. B. animalis NM2 increased bacterial translocation to the mesenteric lymph nodes and did not affect hepatocellular damage.     

Phenotypic and genotypic characterization of non-starter Lactobacillus species diversity in Indian Cheddar cheese

A total of 243 non-starter lactobacilli were isolated from 12 premium quality Indian Cheddar cheese samples ripened for different periods and in different plant conditions. They were classified up to species level using mainly sugar fermentation assay and PCR. Based upon phenotypes, a maximum of 46.50% were classified as Lactobacillus paracasei, followed by 34.98% isolates as Lactobacillus plantarum. Only 3.29% were classified as Lactobacillus rhamnosus and 4.12% as Lactobacillus delbrueckii species, while 22 (9.05%) isolates (of which 16 L. plantarum/Lactobacillus paraplantarum and 6 Lactobacillus delbrueckii ssp. lactis/Lactobacillus crispatus) could not be designated to a single species. One isolate of Lactobacillus coryniformis ssp. coryniformis was isolated for the first time from Cheddar cheese (0.41%) while 1.65% isolates remained unidentified. Mostly, the tentative characterization based on phenotype, could be confirmed by PCR targeting rRNA. Those isolate groups which could not be tested in PCR, or resembled with more than one species in their phenotypic traits, could be resolved by the BLAST homology analysis of the partial tuf gene sequences of few representative isolates.     

Capsular and slime-polysaccharide production by Lactobacillus rhamnosus JAAS8 isolated from Chinese sauerkraut: Potential application in fermented milk products

Exopolysaccharides (EPSs) produced by lactic acid bacteria (LAB) play an important role in the improvement of the physical properties of fermented dairy products. To find EPS-producing LAB strains with potential industrial applications, a Lactobacillus rhamnosus strain, L. rhamnosus JAAS8, that is capable of producing two forms of EPS when grown in MRS broth or semi-defined medium with glucose as a carbon source was isolated and identified from Chinese sauerkraut. The capsular-polysaccharide (CPS) present surrounding the bacterial surface of L. rhamnosus JAAS8 was observed by both optical microscopy and transmission electron microscopy. The slime-polysaccharide (SPS) present in the growth medium was produced mainly during the exponential growth phase, while the CPS was produced only in fermentation. Monosaccharide analysis of the purified polysaccharide samples showed that the CPS was composed of galactose and N-acetylglucosamine in a molar ratio of 5:1, and the SPS was composed of galactose, glucose and N-acetylglucosamine in a molar ratio of 4:1:1. The use of L. rhamnosus JAAS8 could be considered for potential applications in the dairy industry to improve the rheological properties of fermented milk products by increasing their water-holding capacity and viscosity.