Food Proteins Research Articles

Dual-protein system composed of soy protein and β-lactoglobulin: effect of transglutaminase-mediated crosslinking on its allergenicity and conformation.

The escalating global prevalence of food allergies has intensified the need for hypoallergenic food products. Transglutaminase (TGase)-mediated crosslinking has garnered significant attention for its potential to reduce the allergenicity of food proteins. This study aimed to investigate the effects of TGase crosslinking on the potential allergenicity and conformational changes in a dual-protein system composed of β-lactoglobulin (β-LG) and soy protein isolate (SPI) at varying mass ratios (10:0, 7:3, 5:5, 3:7 and 0:10 (w/w)). TGase preferentially crosslinked the 7S and 11S subunits of soy protein, rather than β-LG. Crosslinking treatment reduced the allergenic potential of both soy protein and β-LG, with the degree of reduction depending on the protein ratio. The β-LG5:SPI5 and β-LG7:SPI3 mixtures showed the most significant reduction in antibody reactivity towards soy protein and β-LG, respectively. Additionally, TGase-mediated crosslinking significantly reduced the binding capacity of all dual-protein samples to serum immunoglobulin E from allergic patients, compared to the control group (P < 0.05). The allergenicity reduction was accompanied by structural modifications, including a decrease in β-sheet content, an increase in β-turn and random coil structures, enhanced ultraviolet absorption and intrinsic fluorescence, reduced free sulfhydryl levels and altered intermolecular forces. These changes suggest that TGase-induced crosslinking may disrupt or mask allergenic epitopes, thus lowering allergenicity. TGase crosslinking effectively reduced the allergenic potential of the dual-protein system by inducing structural changes. These findings underscore the potential of TGase in developing hypoallergenic dual-protein food products and provide a scientific foundation for future research and application in the food industry. © 2025 Society of Chemical Industry.

Protein extraction technologies for foxtail, pearl, and proso millets along with their structural and techno-functional properties: a review

Abstract As the world’s population grows, so does the demand for protein, and the growing population is actively seeking alternative protein sources that are affordable and available locally. Millet, a resilient crop with a remarkable ability to adapt to unfavourable conditions, has emerged as a potential alternative in recent years. Millet contains protein levels ranging from 7% to 21%, depending on the variety, growing condition, maturity of seeds, and species. This review looks closely into the current protein extraction technologies used on three popular millets: pearl, foxtail, and proso millet. Aside from the extraction technologies, this review provides a thorough critical analysis of structural and techno-functional properties of millet proteins, namely, solubility, emulsifying activity, water and oil holding capacity, and foaming ability, which are important attributes impacting the potential use of millet proteins in food and beverages. Thus, the insights gained from this review provide a strong foundation for future research and development on pearl, foxtail, and proso millet as an ingredient in a wide range of gluten-free food products, meat analogue formulations, and nutraceuticals.

Wheat2035: Integrating Pan-omics and Advanced Biotechnology for Future Wheat Design.

Wheat (Triticum aestivum L.) production is vital for global food security, providing energy and protein to millions of people worldwide. Recent advancements in wheat research have led to significant increases in production, fueled by technological and scientific innovation. Here, we summarize the major advancements in wheat research, particularly the integration of biotechnologies and a deeper understanding of wheat biology. The shift from multi-omics to pan-omics approaches in wheat research has greatly enhanced our understanding of the complex genome, genomic variations, and regulatory networks to decode complex traits. We also outline key scientific questions, potential research directions, and technological strategies for improving wheat over the next decade. Since global wheat production is expected to increase by 60% in 2050, continued innovation and collaboration are crucial. Integrating biotechnologies and a deeper understanding of wheat biology will be essential in addressing future challenges in wheat production, ensuring sustainable practices and improved productivity.

Feasibility, acceptability, and preliminary impact of "Supper Heroes", a family-based sustainable diet intervention.

The primary objective of this research was to evaluate the feasibility and acceptability of "Supper Heroes", a superhero-themed mobile health (mHealth) intervention designed to reduce food waste and increase plant-based protein food intake among families with children aged 9-14 in Ontario, Canada. A single arm, pre-post mixed methods design was used to evaluate the intervention. The 4-month mHealth intervention included 7 online modules with infographics, videos, and activities to help families reduce their food waste and eat more plant-based protein foods. Feasibility was assessed using website analytics to monitor intervention completion and attrition. Acceptability was assessed at post-intervention using online surveys of parents and children, and in-depth interviews with parents. 23 out of 30 families (77%) were retained from pre-to post-intervention; 33 parents (22 mothers, 10 fathers; and 1 parent who did not disclose their gender) completed the pre-intervention and post-intervention surveys and 33 children (16 girls; 15 boys; 1 child who did not disclose their gender; and 1 non-binary child) completed the post-intervention surveys. Most parents (95% of mothers and 100% of fathers) and children (88%) reported that they were satisfied or highly satisfied with the intervention. Overall, the intervention was feasible and well-accepted by parents and children. A randomized, controlled trial among a larger sample is recommended to further evaluate intervention impacts. These results can inform future research and public health interventions aimed at promoting sustainable diets.

Feasibility and preliminary effects of the mindful healthy family project among rural families

ABSTRACT Background/purpose: Rural adults and children are at higher risk for overweight and obesity. However, there are relatively few lifestyle modification programs available for these high-risk families, mainly because of the difficulty in reaching them. This mindfulness-based motivational interviewing (MM-based-MI) pilot aimed to improve parents’ healthy eating index (HEI), collective family efficacy, family satisfaction, perceived stress, and depressive symptoms as well as parent–child dyads’ eating patterns, physical activity (PA), and body mass index (BMI). Methods: This randomized controlled trial (RCT) was conducted in the Midwestern US to examine the feasibility (enrollment, attendance, and attrition), acceptability, and preliminary effects of an MM-based-MI intervention that contained nine sessions of health coaching (1-on-1), while the active-control included nine emailed health handouts sent over an 18-week period. Results: A total of 46 parents (29 intervention, 17 control; Mage = 38.5 years, 85% female) participated. The enrollment rate, intervention attendance rate, and attrition rate were 12.6%, 79.8%, and 23.9% respectively. Although not statistically significant, compared to the control, the MM-based-MI intervention showed positive effects on improving parents’ HEI in terms of increasing total HEI score (Cohen’s d = 0.43), vegetable intake (d = 0.41), greens/beans (d = 0.59), protein food (d = 0.82), and self-efficacy in exercise (d = 0.21), as well as decreasing total calories (Kcal, d = −0.58), added sugar (d = −0.50), and depressive symptoms (d = −0.42), while controlling for marital status. Controlling for age, sex, and marital status, intervention children had greater improvement in increasing fiber (d = 0.75) and protein (d = 0.72) intake compared to the active-control group. Moreover, parents in both groups reported improvement (small to large effects) in ↑mindful eating, ↑collective family efficacy, ↑family satisfaction, and ↓perceived stress over time. Conclusions: Despite some limitations (small sample size, virtual at home measurement), our results support the feasibility, acceptability, and preliminary effects of this Mindful Healthy Family program on potentially mitigating some obesogenic behaviors among rural parent–child dyads. Trial registration: ClinicalTrials.gov identifier: NCT05324969.

Характеристика белкового комплекса сортов сои селекции ВНИИМК

The biochemical composition of soybean seeds determines the use of soybeans as a source of food and feed proteins. In this study, the protein complex of seeds of soybean varieties bred at V.S. Pustovoit All-Russian Research Institute of Oil Crops was evaluat-ed. The mass fraction of protein was determined by the Kjeldahl method on a VELP Scientifica device (Italy). Amino-acid composition was determined by high-performance liquid chromatography on a Sevco ARM-1000 amino-acid analyzer (Russia). Standard methods were used in the work. The ratio of amino-acid composition of soybean protein by functional groups as well as the protein content in soybean varieties bred at V.S. Pustovoit All-Russian Research Institute of Oil Crops (39.35–42.93%) were determined. The wholesomeness of soybean protein was estimated on the basis of the content of essential amino ac-ids in relation to the FAO/WHO reference protein. During the research it was determined that all essential amino acids have score more than 100%. The biological value of the soybean samples was calculated and ranged from 71.20 to 79.10 %. The ratio of differences in amino acid composition varied from 20.90 to 28.80%. The utility coefficient of the amino acid composition of soybean seed protein differed slightly among varieties (0.68-0.72 fractions of unit). The best ratio of qualitative characteristics of the protein complex was observed in the variety Puma.

A Lupin (Lupinus angustifolius) Protein Hydrolysate Decreases the Severity of Experimental Autoimmune Encephalomyelitis: A Preliminary Study.

Multiple sclerosis (MS) is a neurodegenerative disease, with inflammation and oxidative stress in the central nervous system being the main triggers. There are many drugs that reduce the clinical signs of MS, but none of them cure the disease. Food proteins have been shown to contain encrypted peptides that can be released after hydrolysis and exert numerous biological activities. Recently, we have demonstrated the anti-inflammatory and antioxidant activities of a lupin protein hydrolysate (LPH) both in vitro and in vivo. Therefore, the aim of this study was to evaluate whether LPH is capable of reducing the clinical signs of experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. EAE was induced in female C57BL/6N mice and they were treated intragastrically with LPH (100 mg/kg) or vehicle (control group) from day 0 (prophylactic approach) or from the onset of the disease (day 12 post-induction; therapeutic approach) and the clinical score of each mouse was recorded daily. Prophylactic treatment with LPH reduced the clinical score of the mice compared to the control group, as well as the maximum and cumulative scores, without changing the day of onset of the symptoms while the therapeutic intervention did not significantly improve the severity of the disease. For the first time, we demonstrated that prophylactic administration of LPH reduces the severity of MS, suggesting a potential nutraceutical or new functional foods in neuroinflammation. However, further studies are needed to confirm this nutritional effect in a clinical context.

Novel foods in the European framework: benefits and risks.

Given the rapidly increasing global demand for food, it is mandatory to consider new sources of nutrients, safe and sustainably produced protein foods to complement the current traditional and limited sources of protein in the human diet. In recent years, a wide range of nontraditional protein foods have been explored, prompting the European Union to legislate on how novel foods can be introduced and traded on the European market to ensure their safety. This review will illustrate the range of novel foods authorized in the EU and their potential impact on human health, highlighting the gaps, the potential risks, and the future research opportunities and perspectives.

Electrochemical Magnetic Immunoassay for the Determination of the Fish Allergen β-Parvalbumin.

β-parvalbumin (β-PV) is the primary fish allergen responsible for most allergic reactions in individuals sensitive to fish. To ensure food safety, a sandwich-based magnetic immunoassay was developed using maleimide-functionalized magnetic beads (NH-MBs). Specific anti-β-PV antibodies were immobilized on these MBs, and a screen-printed carbon electrode was employed as the electrochemical transducer. A linear concentration range from 10 to 1000 ng/mL, a limit of detection of 1.8 ng/mL, and a limit of quantification of 7.1 ng/mL were achieved. Nineteen commercial food samples were analyzed to assess the potential of the sensor for routine use in food quality control. Important factors such as protein source and food processing (e.g., boiling, grilling, and frying) and preservation (e.g., in oil, and vacuum) were evaluated. The validated results confer the usefulness of the assay for food quality control.

The Effects of Ultrasound on the Rehydration of Konjac Glucomannan/Soy Protein Isolate Gel and Simulation of Gas-Liquid Interface Evolution During the Rehydration Process.

Soy protein isolate (SPI) possesses potential gelling properties, making it suitable for gel-based applications. However, the gel network stability and mechanical properties of SPI are relatively poor and can be improved through modifications or by combining it with other polymers, such as Konjac Glucomannan (KGM). Combining SPI with KGM can overcome the poor gel network stability and mechanical properties of SPI, but it reduces the water-absorbing capacity of the gel network after drying, which affects the quality characteristics of plant-based protein rehydrated foods and limits the economic feasibility of soy protein foods. In this study, SPI and KGM are the main research objects. By using the alkali method to construct SPI/KGM dry gels with good gel properties, the influence of different ultrasonic powers on the rehydration kinetics and performance changes of SPI/KGM dry gels is examined. The speed and state of water entering the pores are simulated by constructing different pore-size capillary filling models, and the rehydration mechanism of the gel is elucidated. This study provides research ideas and a theoretical basis for the application of ultrasonic wave technology in the study of dry product rehydration performance.

Application of AprX from Pseudomonas paralactis for the improvement of the emulsifying properties of milk, plant and insect protein and estimation of their hydrolysate’s bitter potential

AbstractProtein properties can be modified by selective enzymatic hydrolysis. In this study, the alkaline metalloendopeptidase AprX (Serralysin; EC 3.4.24.40) from Pseudomonas paralactis was used for the tailored hydrolysis of different food proteins resulting in the production of protein hydrolysates with improved emulsifying properties. Sodium caseinate, wheat gluten and buffalo worm protein were used for AprX hydrolysis at 40 °C and pH 8 to cover a spectrum of different protein sources. A maximum degree of hydrolysis (DH) of 13.1 ± 0.2%, 14.2 ± 0.1% and 20.7 ± 0.1% was reached for sodium caseinate, wheat gluten and the worm protein, respectively. The corresponding hydrolysate properties were analyzed regarding their particle size, peptide composition, solubility, viscosity, surface hydrophobicity and interfacial tension. The emulsifying properties were investigated by the oil-droplet size, ζ-potential and stability of emulsions prepared from the hydrolysates. Using partially hydrolyzed sodium caseinate (DH = 10.6%) as an emulsifier lead to an eightfold increase of the emulsion stability (t1/2 = 180 ± 0 min) compared to unhydrolyzed sodium caseinate. The emulsion stability using wheat gluten hydrolysates (DH = 11.9%) was increased 30-fold (t1/2 = 45 ± 5 min). Simultaneously, the solubility of gluten was increased by 60%. Buffalo worm hydrolysates (DH = 14.6%) had a twofold (t1/2 = 85 ± 5 min) increased emulsion stability. In conclusion, AprX can be used to improve the solubility and emulsifying properties of food proteins at a relatively high DH.

Relationship between Fertility Diet Score Index Items and Ovulation in Women with Polycystic Ovary Syndrome: A Narrative Review

Polycystic Ovary Syndrome (PCOS) is a common endocrine disorder affecting women of reproductive age, characterized by ovarian dysfunction and a leading cause of infertility due to ovulatory issues. Lifestyle interventions, including dietary modifications, exercise, and weight management, are considered first-line therapies for women with PCOS; however, the optimal treatment remains unidentified. The Fertility Diet (FD), introduced in 2007, represents a dietary approach that may positively impact fertility by emphasizing specific micronutrients, dietary composition modifications, weight management, and increased physical activity. This narrative review aims to evaluate how various components of the Fertility Diet influence ovulation and overall fertility, assessed through a fertility diet score. The findings of this study suggest that adherence to the Fertility Diet, particularly higher intake of the monounsaturated to trans-fat ratio, and increased vegetable protein intake, may positively influence fertility outcomes in individuals with PCOS. In contrast, high consumption of animal protein and high glycemic load food may have adverse effects. However, the current evidence remains insufficient for definitive conclusions, warranting further interventional studies to explore this relationship.

Starch to protein ratio and food moisture content influence water balance and urine supersaturation in cats.

Two starch to protein ratios (high starch [HS], 25% starch and 36% protein; high protein [HP], 15% starch and 53% protein on DM basis) and two moisture contents (5%, dry kibbles; 80%, wet food) were compared in a 2 x 2 factorial arrangement totaling 4 diets. Each diet was evaluated in 9 cats, with 8 d of total collection of urine and feces. Results were subjected to an analysis of variance of the effects of starch to protein ratio, moisture content and their interactions (P<0.05). Urine density was lower and volume was higher in cats fed wet foods (P<0.01). Calcium (Ca) urine concentration was higher for dry and HP diets (P<0.05). The oxalate urine concentration was 60% higher for cats fed both HS formulations (dry and wet; P<0.05). The relative supersaturation (RSS) of urine for calcium oxalate was higher for dry foods and HS formulations (P<0.01), and for struvite, it was lower for both wet foods, and among the dry diets, it was lower for the HS than for the HP formulation (P<0.01). Foods with a high protein-to-starch ratio reduced urine oxalate and RSS for calcium oxalate in wet and dry diets, and wet foods reduced RSS for calcium oxalate and struvite.

Healthy eating patterns associated with reduced risk of inflammatory bowel disease by lowering low-grade inflammation: evidence from a large prospective cohort study

BackgroundLimited epidemiological evidence exists regarding the role of healthy eating patterns in reducing the risk of Crohn’s disease (CD) and ulcerative colitis (UC). This study aimed to investigate the association between adherence to four established healthy eating patterns and subsequent CD or UC risk, and further examined whether these associations are linked to anti-inflammatory mechanisms.MethodsWe conducted a prospective cohort study of 197,391 participants from the UK Biobank who completed at least one dietary questionnaire and were free from inflammatory bowel disease or cancer at baseline. Four dietary patterns were assessed, including Alternate Mediterranean Diet (AMED), Healthy Eating Index 2015 (HEI-2015), Healthful Plant-based Diet Index (HPDI), and EAT-Lancet. Cox proportional models with restricted cubic splines were applied to explore the associations. The potential role of low-grade inflammation in these associations was examined through mediation analysis.ResultsDuring 2,193,436 person-years follow-up, 260 CD and 601 UC cases were identified. Higher AMED and HEI-2015 scores were associated with a reduced risk of CD but no UC, with no evidence against nonlinearity. These associations remained consistent across multiple sensitive and subgroup analyses. For dietary components, the fruits and monounsaturated fatty acids: saturated fatty acids ratio in AMED, and total fruits, total protein foods and fatty acid in HEI-2015 were linked to a decreased CD risk. Both diets were also associated with lower plasma inflammation biomarkers. Mediation analysis indicated that 7.66% and 13.40% of the reductions in CD risk attributed to AMED and HEI-2015 diets, respectively, were mediated by low-grade inflammation scores.ConclusionsHigher adherence to AMED and HEI-2015 might significantly reduce CD risk, partly due to their anti-inflammatory properties.

Nutrition Promotion Provision of Balanced Nutritious Food to Optimize the Nutritional Status of Harapan Bunda Kindergarten Students, Muaro Jambi Regency

Optimal children’s nutritional status is an indicator of achieving child growth according to their age. Optimizing nutritional status is supported by providing nutrition intake to meet children's daily nutrition needs. Kindergarten students need nutritionally, healthy and safe food. Providing balanced nutritious food supplies for kindergarten students requires knowledge, attitudes and good practices from parents, as well as implementation of nutritionally balanced meal that are integrated into the kindergarten learning curriculum from the school. Harapan Bunda Kindergarten, which is located in Talang Belido Village, Sungai Gelam District, Muaro Jambi Regency, has never received nutrition promotion regarding the nutritious food for kindergarten students. The food supplies for kindergarten students brought by their parents also do not implement balanced diet. This community service activity aimed to organize nutritional promotion of balanced nutritious food to optimize the nutritional status of kindergarten students with the targets are teachers and parents. The results of community service activities showed that there is an increase in target’s knowledge before and after being given education which is not significantly different (Sig.&gt;0.05). It is necessary to get used to implementing balance diet on children's meal which contain carbohydrate foods, protein foods, vegetables, fruit, and drinking water consumption (1200-1500 ml per day)..

Assessment of transglutaminase catalyzed cross-linking on the potential allergenicity and conformation of heterologous protein polymers.

Transglutaminase (TGase)-mediated cross-linking has gained significant attention due to its potential to reduce the allergenicity of food proteins. This study investigates the effects of TGase cross-linking on allergenicity and conformational modifications in a dual-protein system comprising soy protein isolate (SPI) and β-lactoglobulin (β-LG). The results showed that TGase cross-linking effectively decreased the allergenic potential of both SPI and β-LG, with a more pronounced reduction observed in the allergenicity of soy protein in the dual-protein system. SDS-PAGE analysis revealed that the 7S and 11S subunits of soy protein were more easily cross-linked than β-LG. Secondary structure analysis indicated that TGase treatment disrupted β-sheet structures, increased the content of random coils, and enhanced protein flexibility. Ultraviolet absorption and intrinsic fluorescence analyses confirmed these structural alterations, with TGase treatment exposing additional aromatic amino acids. A reduction in free sulfhydryl groups and altered intermolecular forces further corroborated the occurrence of cross-linking. These findings suggest that TGase-mediated cross-linking effectively reduced the allergenicity of SPI and β-LG by modifying their conformations, offering potential strategies for the development of hypoallergenic dual-protein food products. PRACTICAL APPLICATION: This study has practical applications in the food industry to develop hypoallergenic food products, particularly those that combine soy and dairy proteins. By using TGase to cross-link these proteins, the allergenicity can be reduced, resulting in products that are safer for consumers with food allergies.

Exposure to food proteins: from basic knowledge on the requirements to subgroup specificities.

Exposure to food proteins: from basic knowledge on the requirements to subgroup specificities.

Fostering Knowledge and Awareness about Healthy Nutrition through Science-based Educational Escape Games

AbstractThe prevalence of diseases stemming from poor nutrition emphasizes the importance of educating people about healthy eating habits. One approach to achieving this is through educational escape games, which embody the features of a situated learning environment. Utilized the situated learning theory as a theoretical and methodological framework, the goal of our study was to examine the role of science-based educational escape games in facilitating knowledge construction and awareness about healthy nutrition. The study was conducted in the setting of a science teacher preparation program, where 165 preservice science teachers were engaged in an escape game named Zombie Attack about proteins in food and the human body. The study applied the pretest-posttest design, in which quantitative and qualitative data were collected concurrently before and after game participation. The findings showed that the escape game experience had a positive effect on the participants’ knowledge gain associated with topics such as energy of macronutrients, protein percent daily value, and proteins in the body. With regards to awareness about healthy nutrition, the study identified five types: Health, Composition, Environment, Source, and Ethics, with a significant gain in all categories following the escape game experience. Overall, the study advocates the use of escape games as a method for fostering interactive learning of scientific concepts, encouraging collaborative problem-solving, and facilitating self-reflection activities.

Effect of Hydrolyzed Frozen Meat on Diet Palatability, Apparent Digestibility, Immune Response, Fecal Microbiota, and Metabolome in British Shorthair Cats

Frozen meat is an important source of protein in pet food, and has attracted much attention in recent years. In this study, we compared the effect of meat meal (MM), frozen meat (FM), and hydrolyzed frozen meat (HFM) as ingredients in extruded pet food on its palatability and apparent digestibility, as well as its effects on the immune response, fecal microbiota, and metabolome of British shorthair cats. A total of 24 British shorthair cats were allocated to the MM, FM, and HFM groups according to body weight and gender. The palatability test lasted 4 days and the feeding test lasted 45 days. The results showed that the FM and HFM diets had better palatability than the MM diet (p &lt; 0.05) and significantly improved dry matter and crude protein digestibility (p &lt; 0.05). The serum IL-10 level was significantly higher in the HFM group compared to the MM and FM groups (p &lt; 0.05). The serum IgM levels were also found to be significantly higher in the FM group compared to the MM and HFM groups (p &lt; 0.05). The blood urea nitrogen/creatinine ratio was significantly lower in the HFM and FM groups than in the MM group (p &lt; 0.05). Cats fed HFM had a higher abundance of fecal Actinobacteria and Bifidobacterium and a lower content of Bacteroidota (p &lt; 0.05). Furthermore, serum metabolomics analysis revealed that the tryptophan (Trp) metabolism and bile acid metabolism were affected by HFM. Overall, FM and HFM were better for the cat’s health than meat meal, but they also have some potential risks.

In vitro simulated digestion and Caco-2 cell absorption to explore potential of soy protein isolates as whey protein substitutes in canine and feline food.

Digestive properties and bioavailability of soy protein isolate and whey protein in canine and feline were evaluated using in vitro simulated digestion and cell absorption experiments. The amino acids and peptides in soybean protein isolate and whey protein after digestion were detected by high-performance liquid chromatography and liquid mass spectrometry. Gastrointestinal digestion rates of soy protein isolate and whey protein were analyzed by fitting digestive kinetics curves. The degree of absorption and utilization of amino acids and peptides from the digestive products was evaluated by establishing a Caco-2 cell model. The results demonstrated that soy protein isolate, like whey protein, could produce large amounts of free amino acids and peptides distributed 100-1500 Da. The gastrointestinal digestion speed of soy protein isolate was similar with whey protein. It is worth noting from the intestinal absorption of Caco-2 cell that more than 15 amino acids produced by soy protein isolate and whey protein could be absorbed, and a large number of peptides were also utilized by Caco-2 cell. The bioavailability of soy protein isolates and whey protein for dogs respectively reached 6.30% and 9.00%, and it reached 7.40% and 16.40% in cats, respectively. Soy isolate proteins can be digested and absorbed by pets like whey proteins. These findings may provide significant strategies and support for the application of plant-based proteins in pet foods.