Glucose oxidase: A potential option to decrease the oxidative stress in stirred probiotic yogurt

The effect of the addition of glucose oxidase as a technological option todecrease the oxidative stress in a stirred yogurt during 15 days of refrigerated storage was investigated. A significant increase in the viable population of Bifidobacteria longum (6.9–8.7 log CFU g^?1) was observed in all yogurts added with glucose oxidase and glucose. Additionally, the yogurts containing glucose oxidase and glucose presented and caused higher pH, proteolysis rate, and contents of organic acids and aroma compounds, and this effect was comparable to the control sample (without glucose oxidase and glucose). Overall, the combined effect of the glucose oxidase system was important to reduce the oxygen content during storage of the probiotic yogurt. These findings suggest that glucose oxidase is a potential option to decrease the oxidative stress in probiotic yogurts.     

Effect of probiotic supplementation on laying hen diets on yield performance and serum and egg yolk cholesterol.

The effects of dietary supplementation of a commercial probiotic (BioPlus 2B) on daily feed consumption, egg yield, egg weight, specific gravity, body weight, feed conversion ratio, serum and egg yolk cholesterol, and serum trigylceride in layer hens were investigated. In 12 replicates, 480 27-week-old Brown-Nick layers were fed with diets containing 0, 250, 500 or 750 mg kg(-1) probiotic for 90 days. When compared with the controls, supplementation of 250, 500 and 750 mg kg(-1) probiotic increased egg production, but decreased the damaged egg ratio (p < 0.05), egg yolk cholesterol and serum cholesterol (p < 0.001) levels. In addition, serum triglyceride levels were reduced by using 500 and 750 mg kg(-1) probiotic supplementation (p < 0.001). Feed conversion ratios were positively affected by supplementation of 250 and 500 mg kg(-1) probiotic compared with controls (p < 0.05). There was no statistically significant difference between the control and all treatment groups on feed consumption, egg weight, specific gravity, body weight, and egg yolk weight.     

PARAFAC: Adjustment for modeling consumer study covering probiotic and conventional yogurt

The feasibility of the parallel factors model (PARAFAC) as a modeling tool for consumer sensory acceptance data of complex food matrices was investigated. The attributes of aroma, flavor, taste, texture, and overall liking of probiotic and conventional yogurts were evaluated by 120 consumers using a 9-point hedonic hybrid scale. Six yogurt samples were used: three were prototypes supplemented with a glucose oxidase/glucose system, which is a potential oxygen scavenging system, and the other three were commercial yogurts. The yogurts supplemented with the glucose oxidase/glucose system presented similar sensory acceptance towards commercial probiotic and conventional yogurts (p > 0.05), besides having garnered lower mean scores for the sensory attributes. Appearance and overall liking were the most significant attributes for the first two components of the PARAFAC model. Finally, the advantage of using this method to explore the intrinsic nature of consumer sensory data is discussed and compared to principal component analysis (PCA).     

Effects of partially replacing skimmed milk powder with dairy ingredients on rheology, sensory profiling, and microstructure of probiotic stirred-type yogurt during cold storage

This study aimed to evaluate the quality of stirred-type skim milk probiotic yogurt fortified by partially replacing skim milk powder (SMP) with whey protein concentrate (WPC) and sodium caseinate (Na-CN) during cold storage for 28 d compared with nonfortified yogurt. The rheological properties (as measured using dynamic oscillation) and sensory profiles of probiotic yogurts were greatly enhanced when SMP (i.e., 45%) was replaced with WPC and Na-CN. Higher values of mechanical parameters related to storage and loss modulus and consistent microstructure were found in the fortified yogurts. The acidification profile was not affected by supplementation of the solids in the milk base, and the viable counts of probiotic microbiota were high and satisfactory. These positive characteristics of probiotic yogurts were maintained until the end of the storage period. The microstructure of the fortified yogurt showed some differences compared with the nonfortified product, which were due to changes in chemical composition of the milk base in addition to the colloidal characteristics of the product.     

Processing optimization of probiotic yogurt containing glucose oxidase using response surface methodology

Exposure to oxygen may induce a lack of functionality of probiotic dairy foods because the anaerobic metabolism of probiotic bacteria compromises during storage the maintenance of their viability to provide benefits to consumer health. Glucose oxidase can constitute a potential alternative to increase the survival of probiotic bacteria in yogurt because it consumes the oxygen permeating to the inside of the pot during storage, thus making it possible to avoid the use of chemical additives. This research aimed to optimize the processing of probiotic yogurt supplemented with glucose oxidase using response surface methodology and to determine the levels of glucose and glucose oxidase that minimize the concentration of dissolved oxygen and maximize the Bifidobacterium longum count by the desirability function. Response surface methodology mathematical models adequately described the process, with adjusted determination coefficients of 83% for the oxygen and 94% for the B. longum. Linear and quadratic effects of the glucose oxidase were reported for the oxygen model, whereas for the B. longum count model an influence of the glucose oxidase at the linear level was observed followed by the quadratic influence of glucose and quadratic effect of glucose oxidase. The desirability function indicated that 62.32 ppm of glucose oxidase and 4.35 ppm of glucose was the best combination of these components for optimization of probiotic yogurt processing. An additional validation experiment was performed and results showed acceptable error between the predicted and experimental results.     

Survival of free and encapsulated probiotic bacteria and their effect on the sensory properties of yoghurt

The survival and effect of free and calcium-induced alginate-starch encapsulated probiotic bacteria (Lactobacillus acidophilus and Bifidobacterium lactis) on pH, exopolysaccharide production and influence on the sensory attributes of yogurt were studied over 7 weeks storage. Addition of probiotic bacteria (free or encapsulated) reduced acid development in yogurt during storage. Post-acidification in yogurt with encapsulated probiotic bacteria was slower compared to yogurt with free probiotic bacteria. More exopolysaccharides were observed in yogurts with probiotic cultures compared to those without probiotic cultures. The results showed that there was an increased survival of 2 and 1 log cell numbers of L. acidophilus and B. lactis, respectively due to protection of cells by microencapsulation. The addition of probiotic cultures either in the free or encapsulated states did not significantly affect appearance and colour, acidity, flavour and after taste of the yogurts over the storage period. There were, however, significant differences (P<0.05) in the texture (smoothness) of the yogurts. This study has shown that incorporation of free and encapsulated probiotic bacteria do not substantially alter the overall sensory characteristics of yogurts and microencapsulation helps to enhance the survival of probiotic bacteria in yogurts during storage.     

乳清分离蛋白纤维和典型抗氧化剂对罗伊氏乳杆菌TMW 1.656在常温贮藏胁迫下存活的影响

为研究新型纳米材料乳清分离蛋白纤维（whey protein isolate fibrils,WPIF）和典型抗氧化剂对益生菌常温贮藏氧胁迫的保护影响,采用乳清分离蛋白（whey protein isolate,WPI）和WPIF为壁材,添加不同质量浓度的典型抗氧化剂表没食子酸儿茶素没食子酸酯（epigallocatechin gallate,EGCG）和谷胱甘肽（glutathione,GSH）,采用喷雾干燥法制备干态菌粉。结果表明,在常温贮藏期间,益生菌的贮藏稳定性按照WPIF≈WPI>WPI+0.5 mg/mL EGCG>WPI+0.5 mg/mL GSH>WPI+5.0 mg/mL EGCG>WPI+5.0 mg/mL GSH的顺序下降。WPIF保护菌体的效果与WPI相差不大,其原因可能是高温使得纤维发生分解和聚集,从而导致没有发挥纤维优越的效果;抗氧化剂EGCG和GSH在抑菌实验和贮藏实验中都显著加速了益生菌的死亡,且具有质量浓度依赖性,这可能与其本身的抗菌活性或氧化产物的细胞毒性有关。     

Oxidative stress in probiotic Petit Suisse: Is the jabuticaba skin extract a potential option?

This research evaluated the action of jabuticaba skin extract (JSE) obtained through supercritical extraction compared with the antioxidants ascorbic acid, cysteine chloride, and glucose oxidase along probiotic Petit Suisse cheese processing. Physico-chemical analyses (pH, proteolysis, antioxidant activity, organic acids, and fatty acid profile) and microbiological (starter culture and probiotic culture counts) were carried out over 28 days of refrigerated storage. Addition of JSE on Petit Suisse was variable and dependent of the evaluated parameters with satisfactory findings in consumer test. However, the supplementation of probiotic Petit Suisse with JSE should be evaluated with care.     

Microstructural, textural, and sensory characteristics of probiotic yogurts fortified with sodium calcium caseinate or whey protein concentrate

The influence of milk protein-based ingredients on the textural characteristics, sensory properties, and microstructure of probiotic yogurt during a refrigerated storage period of 28 d was studied. Milk was fortified with 2% (wt/vol) skim milk powder as control, 2% (wt/vol) sodium calcium caseinate (SCaCN), 2% (wt/vol) whey protein concentrate (WPC) or a blend of 1% (wt/vol) SCaCN and 1% (wt/vol) WPC. A commercial yogurt starter culture and Bifidobacterium lactis Bb12 as probiotic bacteria were used for the production. The fortification with SCaCN improved the firmness and adhesiveness. Higher values of viscosity were also obtained in probiotic yogurts with SCaCN during storage. However, WPC enhanced water-holding capacity more than the caseinate. Addition of SCaCN resulted in a coarse, smooth, and more compact protein network; however, WPC gave finer and bunched structures in the scanning electron microscopy micrographs. The use of SCaCN decreased texture scores in probiotic yogurt; probably due to the lower water-holding capacity and higher syneresis values in the caseinate-added yogurt sample. Therefore, the textural characteristics of probiotic yogurts improved depending on the ingredient variety.     

Effects of source on bioavailability of selenium,antioxidant status,and performance in lactating dairy cows during oxidative stress-inducing conditions

In the current study, we used heat stress (HS) as an oxidative stress model to examine the effects of hydroxy-selenomethionine (HMSeBA), an organic selenium source, on selenium's bioavailability, antioxidant status, and performance when fed to dairy cows. Eight mid-lactation Holstein dairy cows (141 ± 27 d in milk, 35.3 ± 2.8 kg of milk/d, parity 2 or 3) were individually housed in environmental chambers and randomly assigned to 1 of 2 treatments: inorganic Se supplementation (sodium selenite; SS; 0.3 mg of Se/kg of dry matter; n = 4) or HMSeBA supplementation (0.3 mg of Se/kg of dry matter; n = 4). The trial was divided into 3 continuous periods: a covariate period (9 d), a thermal neutral (TN) period (28 d), and a HS period (9 d). During the covariate and TN periods, all cows were housed in TN conditions (20°C, 55% humidity). During HS, all cows were exposed to cyclical HS conditions (32–36°C, 40% humidity). All cows were fed SS during the covariate period, and dietary treatments were implemented during the TN and HS periods. During HS, cows fed HMSeBA had increased Se concentrations in serum and milk, and total Se milk-to-serum concentration ratio compared with SS controls. Superoxide dismutase activity did not differ between Se sources, but we noted a treatment by day interaction in glutathione peroxidase activity as HS progressively reduced it in SS controls, whereas it was maintained in HMSeBA cows. Supplementation with HMSeBA increased total antioxidant capacity and decreased malondialdehyde, hydrogen peroxide, and nitric oxide serum concentrations compared with SS-fed controls. We found no treatment effects on rectal temperature, respiratory rate, or dry matter intake. Supplementing HMSeBA tended to increase milk yield and decrease milk fat percentage. No other milk composition parameters differed between treatments. We observed no treatment effects detected on blood biochemistry, except for a lower alanine aminotransferase activity in HMSeBA-fed cows. These results demonstrate that HMSeBA supplementation decreases some parameters of HS-induced oxidative stress.     

Probiotic viability and storage stability of yogurts and fermented milks prepared with several mixtures of lactic acid bacteria

Currently, the food industry wants to expand the range of probiotic yogurts but each probiotic bacteria offers different and specific health benefits. Little information exists on the influence of probiotic strains on physicochemical properties and sensory characteristics of yogurts and fermented milks. Six probiotic yogurts or fermented milks and 1 control yogurt were prepared, and we evaluated several physicochemical properties (pH, titratable acidity, texture, color, and syneresis), microbial viability of starter cultures (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) and probiotics (Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus reuteri) during fermentation and storage (35 d at 5°C), as well as sensory preference among them. Decreases in pH (0.17 to 0.50 units) and increases in titratable acidity (0.09 to 0.29%) were observed during storage. Only the yogurt with S. thermophilus, L. delbrueckii ssp. bulgaricus, and L. reuteri differed in firmness. No differences in adhesiveness were determined among the tested yogurts, fermented milks, and the control. Syneresis was in the range of 45 to 58%. No changes in color during storage were observed and no color differences were detected among the evaluated fermented milk products. Counts of S. thermophilus decreased from 1.8 to 3.5 log during storage. Counts of L. delbrueckii ssp. bulgaricus also decreased in probiotic yogurts and varied from 30 to 50% of initial population. Probiotic bacteria also lost viability throughout storage, although the 3 probiotic fermented milks maintained counts ≥10⁷ cfu/mL for 3 wk. Probiotic bacteria had variable viability in yogurts, maintaining counts of L. acidophilus ≥10⁷ cfu/mL for 35 d, of L. casei for 7 d, and of L. reuteri for 14 d. We found no significant sensory preference among the 6 probiotic yogurts and fermented milks or the control. However, the yogurt and fermented milk made with L. casei were better accepted. This study presents relevant information on physicochemical, sensory, and microbial properties of probiotic yogurts and fermented milks, which could guide the dairy industry in developing new probiotic products.     

Stability and viability of synbiotic microgels incorporated into liquid,Greek and frozen yogurts

The viability of Lactobacillus acidophilus when co-encapsulated with fructooligosaccharides in alginate–gelatin microgels, for incorporation into liquid, Greek, and frozen yogurts, during storage and in vitro-simulated digestion was studied. Liquid yogurt provided the highest viability for the encapsulated probiotics during storage, followed by frozen and Greek formulations when compared to free probiotics, highlighting the influence of microencapsulation, yogurt composition, and storage conditions. Addition of up to 20% of probiotic (AG) and symbiotic (AGF) microgels did not cause significant changes in the liquid and frozen yogurts’ apparent viscosity (η_ap); however, it decreased η_ap for the Greek yogurt, indicating that microgels can alter product acceptability in this case. Both AG and AGF microparticles improved viability of cells face to gastric conditions for liquid and frozen yogurts, delivering cells in the enteric stage. Summarizing, liquid yogurt was the most appropriate for probiotic viability during storage, while frozen yogurt presented better protection along digestibility.     

Study of α-, γ-, and δ-tocopherols in the oxidative stability of lard

Effects of α-, γ-, and δ-tocopherols at 0, 50, 100, 250, 500, and 1,000 ppm on the oxidative stability of lard during storage in the dark at 55°C for 7 days were studied by determining headspace oxygen content and peroxide value. The headspace oxygen content of the lard without tocopherol addition decreased from 21.1 to 14.4% and the peroxide value increased from 2.5 to 15.4 meq/kg lard for 7 days. The headspace oxygen depletion and the peroxide value of lard decreased by the addition of α-tocopherol at 50 ppm, but significantly increased at 250, 500, or 1,000 ppm. As the concentration of γ-tocopherol or δ-tocopherol increased, the headspace oxygen depletion and the peroxide value decreased. α-Tocopherol at 250 ppm acted as a prooxidant in the autoxidation of lard. The concentrations of γ- or δ-tocopherol to be applied to improve the autoxidative stability of lard may be 100–250 ppm.     

Effect of bovine lactoferrin addition to milk in yogurt manufacturing

The aim of this work was to study the effect of milk supplementation with lactoferrin of different iron saturation on the manufacturing and characteristics of yogurt. Bovine lactoferrin was added at concentrations of 0.5, 1, and 2 mg/mL in the holo (iron saturated) and apo (without iron) forms. Some physicochemical properties, such as pH, concentration of lactic acid, and texture of supplemented yogurts, were determined throughout the shelf-life period storage (28 d) at 4°C. We also evaluated the stability of lactoferrin in supplemented yogurt throughout the storage time. The supplementation of milk with bovine lactoferrin did not greatly affect the physical properties of the yogurt, though apo-lactoferrin slightly delayed the decrease of pH. This could be attributed to the partial inhibition observed on the growth of Streptococcus thermophilus. The integrity and immunoreactive concentration of lactoferrin, determined by Western blotting and noncompetitive ELISA, respectively, remained constant throughout the shelf life of yogurt.     

Effects of Selenium and Methionine Supplementation of Breeder Hen Diets on Selenium Concentration and Oxidative Stability of Lipids in the Thigh Muscles of Progeny

ABSTRACT:  The effects of dietary supplementation to female chickens with selenium (Se) and methionine (Met) on the next generation were studied. Lang-shan breeding hens (450) were obtained at 52 wk of age and randomly allotted to 9 treatments; 5 replicates of each treatment were carried out. The breeders were fed a basal corn–soybean meal diet (0.13 mg Se/kg) supplemented with 0, 0.30, or 0.60 mg/kg Se from Sel-Plex and 0.32%, 0.40%, or 0.54% Met for the 30-d adapting period and 70-d experiment period. Se and glutathione (GSH) concentrations, glutathione peroxidase (GSH-Px) activity, and the oxidative stability of muscular lipids of 90-d progeny were determined by testing the TBARS values. When breeders received the highest levels of Met or Se, GSH-Px activity was decreased, the Se concentration and the oxidative stability of muscular lipids were increased with the supplementation of Se or Met. When breeder hens were given a Met-deficient diet, supplementing with Se decreased the Se deposition in progeny thigh. With regard to lipid oxidation, 0.3 mg/kg maternal dietary Se supplementation decreased the oxidative stability of muscle lipid and 0.6 mg/kg Se supplementation showed no difference from the control. When breeders were fed a Se-deficient diet, the GSH-Px activity was increased significantly and the oxidative stability of progeny muscles was decreased with the supplementation of Met. It was concluded that supplementation of the maternal diet with higher Se and Met can increase Se deposition in progeny muscle and lead to more effective protection against lipid oxidation in progeny thighs.     

Oxidative stability of yogurt with added lutein dye

This study evaluated the effect of adding lutein dye on the oxidative stability of yogurt during 35 d of refrigerated storage, in the presence and absence of light. Yogurts manufactured without and with the equivalent of 1.5 mg of lutein in 120 g of the final product were characterized for their total carotenoid and riboflavin contents, and the behaviors of both riboflavin and lutein were monitored during storage. A decrease in riboflavin content occurred, with concurrent appearance of its derived-oxidation products in the yogurts without added lutein and exposed to light during storage. The yogurts with added lutein dye showed constant lutein and riboflavin contents throughout storage both for the samples stored under light and for those stored in the dark. Yogurts (120 g) with the addition of 0.5, 1.5, and 2.5 mg of lutein dye were evaluated for their sensory acceptance, and the statistical analysis showed no differences between the samples for the attributes of aroma and flavor. These results indicate that the added lutein remained stable throughout the storage period and conferred protection for the riboflavin against photooxidation, preserving the quality of the yogurts.     

Survival of Lactobacillus acidophilus and Bifidobacterium animalis ssp. lactis in stirred fruit yogurts

The effect of commercial fruit preparations (mango, mixed berry, passion fruit and strawberry) on the viability of probiotic bacteria, Lactobacillus acidophilus LAFTI^® L10 and Bifidobacterium animalis ssp. lactis LAFTI^® B94 in stirred yogurts during storage (35 days) at refrigerated temperature (4 °C) was evaluated. The results showed that addition of either 5 or 10 g/100 g fruit preparations had no significant (p>0.05) effect on the viability of the two probiotic strains except on L. acidophilus LAFTI L10 yogurt with 10 g/100 g passion fruit or mixed berry. After the addition of fruit preparation, 96% of the yogurts incorporated with fruit preparation did not exhibit a greater loss in the viability of probiotic bacteria compared to plain yogurt during the storage period. A correlation between the post-storage pH in yogurts and the survival of probiotic bacteria was observed. All the yogurts, however, contained the recommended levels of (10⁶-10⁷ cfu/g) probiotic bacteria at the end of 35-day shelf life.     

Effect of dietary α-tocopherol supplementation and gamma-irradiation on α-tocopherol retention and lipid oxidation in cooked minced chicken

The effects of dietary α-tocopherol supplementation and gamma-irradiation on α-tocopherol retention and lipid oxidation in cooked minced chicken during refrigerated storage were studied. Minced breast and thigh meat from broilers fed diets supplemented with 100, 200 or 400 mg α-tocopheryl acetate/kg feed was irradiated at 2.5 or 4.0kGy. Cooked irradiated and unirradiated meat was stored at 4 °C for 5 days. α-Tocopherol concentrations increased with increasing dietary supplementation. Concentrations decreased during storage, but retention was not affected by irradiation. Lipid stability was determined by measuring the formation of thiobarbituric acid-reacting substances (TBARS) and cholesterol oxidation products (COPs) during storage. TBARS and COPs increased during storage and were reduced by increasing levels of dietary α-tocopheryl acetate supplementation. Irradiation accelerated TBARS formation during storage, but this was prevented by supplementation with 200 mg α-tocopheryl acetate/kg feed. Irradiation tended to increase COPs during storage, although no consistent effects were observed. In general supplementation with over 400 mg α-tocopheryl acetate/kg feed may be required to control cholesterol oxidation in minced chicken. The results suggest that, overall, irradiation had little effect on lipid stability in α-tocopherol-supplemented meat following cooking and storage.     

Effects of dietary leucine and phenylalanine on pancreas development,enzyme activity,and relative gene expression in milk-fed Holstein dairy calves

This study aimed to investigate the effect of dietary supplementation with leucine and phenylalanine on pancreas development, enzyme activity, and related gene expression in male Holstein calves. Twenty male Holstein calves [1 d of age, 38 ± 3 kg of body weight (BW)] were randomly assigned to 1 of the following 4 treatment groups with 5 calves in each group: control, leucine supplementation (1.435 g/L of milk), phenylalanine supplementation (0.725 g/L of milk), and leucine and phenylalanine (1.435 + 0.725 g/L of milk). The diets were made isonitrogenous with the inclusion of alanine in each respective treatment. The feeding trial lasted for 8 wk, including 1 wk for adaption and 7 wk for the feeding experiment. Leucine tended to increase the concentration of total pancreatic protein (mg/kg of BW). Phenylalanine increased the concentrations of plasma insulin, cholecystokinin, and pancreatic DNA (mg/g) and the expression of trypsin gene but decreased the pancreatic protein:DNA ratio and tended to decrease the pancreas weight (g/kg of BW). No differences were observed in total pancreatic DNA (mg/pancreas and mg/kg of BW), pancreatic protein (mg/pancreas), or activities of α-amylase, trypsin, and lipase. The relative expression levels of the genes encoding α-amylase and lipase did not differ among the 4 groups. The supplementation of both leucine and phenylalanine showed an interaction on the pancreas weight (g and g/kg of BW) and a tendency of an interaction on the pancreatic protein concentration (mg/g of pancreas and mg/kg of BW) and the plasma glucose concentration. Leucine tended to increase the size of the pancreatic cells, whereas phenylalanine tended to increase the number of pancreatic cells. However, neither AA affected the activities of the pancreatic enzymes of the calves. These results indicate that leucine and phenylalanine supplementation in milk-fed Holstein calves differentially affect pancreatic growth and development.     

Hepatoprotective effects of lupeol and mango pulp extract of carcinogen induced alteration in Swiss albino mice

Lupeol, a triterpene present in mango and other fruits, is known to exhibit a number of pharmacological properties including antioxidant, antilithiatic, and antidiabetic effects. In the present study, chemopreventive properties of lupeol and mango pulp extract (MPE) were evaluated against 7,12-dimethylbenz(a)anthracene (DMBA) induced alteration in liver of Swiss albino mice. Lupeol (25 mg/kg body weight, bw) or 1 mL of 20% w/v aqueous MPE/mouse were daily given once for 1 wk after a single dose of DMBA (50 mg/kg bw). Lupeol/MPE supplementation effectively influenced the DMBA induced oxidative stress, characterized by restored antioxidant enzyme activities and decrease in lipid peroxidation. A reduction of apoptotic cell population in the hypodiploid region was observed in lupeol and MPE supplemented animals. The inhibition of apoptosis was preceded by decrease in reactive oxygen species (ROS) level and restoration of mitochondrial transmembrane potential followed by decreased DNA fragmentation. In DMBA treated animals, downregulation of antiapoptotic Bcl-2 and upregulation of proapoptotic Bax and Caspase 3 in mouse liver was observed. These alterations were restored by lupeol/MPE, indicating inhibition of apoptosis. Thus, lupeol/MPE was found to be effective in combating oxidative stress induced cellular injury of mouse liver by modulating cell-growth regulators.