Formulation, Process Optimization, and Biochemical Characterization of Cereal-Based Sweet Potato and Mulberry Instant Beverage

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Introduction
Instant beverage mix is referred to as a dry mix which can be blended with water or other liquid foods (i.e., milk) to prepare the instant beverage [1].Te hectic lifestyle of consumers promoted the consumption of instant beverage mixes due to their easy preparation and health benefts.Recently, due to the availability and increasing demand for "on-the-go" food and beverages, instant beverage mixes have received much demand across the developed countries [2].Many research studies showed that plant-based diets can prevent the type 2 diabetes [3].Te constituents (fbre, minerals, phytonutrients, and antioxidants) present in plant-based diets play a key role in preventing and treating type 2 diabetes as well as reducing the risk of other complications, such as overweight, abdominal obesity, high blood pressure, hyperlipidemia, and infammation.[4].According to the World Health Organization (WHO) and Food and Agriculture Organization (FAO) [5,6], diets rich in plant foods are not only efective in reducing healthrelated complications but also more environmentally sustainable than diets rich in animal products.
Instant foods such as ready-to-eat (RTE), ready-to-drink beverages (RTD), and semicooked or ready-to-cook breakfast and snack items are commonly prepared by using malted or unmalted cereals alone or by mixing with legumes, nuts, fruits and/or vegetables.Among cereals, rice is the second most important cereal in the world, consumed as a staple food by the Asian population, providing energy and other nutrients compared to other staple foods [7].Black rice is one of the healthiest foods and contains a variety of constituents including antioxidants, essential amino acids, minerals, and dietary fbre.Regular consumption of black rice can help in weight management and detoxifcation of the body as it gives a feeling of fullness which reduces food intake [8].Among all colored rice varieties, anthocyanin content is the highest (327.60 mg 100 g −1 ) in black rice, which is able to prevent the accumulation of bad cholesterol over the heart valves and also reduces the level of cholesterol in the blood.Tus, black rice results in controlling and preventing chronic diseases, such as coronary infarction, cancer, type 2 diabetes mellitus, allergies, and Alzheimer's disease [9,10].Many ready-to-serve (RTS) and RTE foods use white rice as the main ingredient; however, it can be replaced with black rice to develop a new food product with increased nutritional status [11].Among legumes, lentils have the highest content of functional components, such as total phenolic content, than other common legumes [12], which have potential health benefts as complementary medicinal foods, that exert antioxidant, hypolipidemic, cardioprotective, anti-infammatory, and anticancerous effects [13].Sweet potato four contains 2-3% of fbre [14].Potential benefts of soluble dietary fbre include lowering blood serum cholesterol levels, infuencing glucose metabolism, and thus regulating postprandial blood sugar levels and secretion of insulin in diabetic patients [15].Lund et al. [16] compared 28 varieties of fbre-rich test samples prepared from tropical fruits and vegetables for their capacity to bind cholesterol.On the other hand, the sweet potato-based test sample had the highest cholesterol-binding ability.Terefore, sweet potato genotypes with a high polyphenol content and used as a vegetable, tea, beverage breakfast food, food ingredient, and as a nutritional supplement can be developed to be used for the promotion of health [17].A natural antioxidant in black mulberry (Morus nigra) is the anthocyanin pigment which has excellent free radicals scavenging activity and leads to limit and cure the diseases caused by oxidative damage in human beings [18].
Te literature reported that black rice (Oryza sativa L.), sweet potato (Ipomoea batatas), lentil (Lens culinaris), and mulberry fruit (Morus nigra) have been used for the prevention and treatment of adult-onset diabetes via a specifc mechanism of insulin interaction [19].Te benefcial role of these functional food combinations provided us with the tool to think and design our study to develop an instant beverage mix (IBM) from functional ingredients of plant origin and to determine its efcacy on blood glucose levels of alloxan-induced diabetics [20].IBM thus might reveal the favorable therapeutic potential for the treatment of hyperglycemia and act as a functional beverage.
Tere is no research work available on the development of cereal-based instant beverages using the abovementioned functional ingredients.If properly processed, such a product would be novel and could fetch substantial revenue to the beverage processing industry.Apart from this, there is a need to enhance the postharvest processing of these underutilized grains by formulating value-added convenience and healthy products such as instant beverage mixes.Tis functional beverage meets the consumers' demand by supplying functional components.Tus, the present study was carried out to optimize the ratio of raw ingredients of a CIB, and the efects of the ingredient variations were observed on the organoleptic properties of it as well as the biochemical parameters of the fnal optimized CIB was also investigated for its therapeutic suitability.

Raw Materials.
Black rice (Oryza sativa L.) and black mulberry (Morus nigra) were procured from the counter of the Agricultural Technology Information Centre (ATIC) and the sericulture farm of Assam Agricultural University (AAU), Jorhat, respectively.Both lentils (Lens culinaris) and pale yellow-feshed sweet potato (Ipomoea batatas) were purchased from the weekly market of Jorhat town, Assam, India.Soy milk (Life Health Foods, Jorhat, Assam, India) and sucralose with 100 purity (Brand: ProFoods Nutrition, Jaldhara and Co. Jorhat, Assam) were taken as an alternative to sugar in CIB preparation.

Pretreatment of Raw Materials and Its Processing.
Black rice was frst cleaned and washed, followed by soaking in water for half an hour.Te rice sample was steamed for 45-50 min.and dried using a hot air oven at 60 °C for 8 h (Castro Engineering Pvt. Ltd., Howrah, India).Finally, rice was ground into four using paddy dehuller (Paddy Dehusker, Osaw Industrial Product Pvt. Ltd., Assam, India) and sieved with 150 mess size.For germinated lentil four, lentil seeds were frst washed, followed by soaking in water by portioning into a cup and adding water (lentil: water � 1 : 2, w/w) for 24 h, and covering with a muslin cloth.After that, legume seeds were washed and allowed to germinate in a muslin cloth at 20 °C for 5 days.Germinated samples were steamed for 20 min (a ratio of 1 : 1.5, w/w of lentil and water) and fnally, dried at 40 °C for 72 h.Te germs or sprouts were separated by rubbing.After dehulling, legumes were ground into four using a paddy dehuller machine and sieved with 150 mesh sieves [21].Sweet potato was processed according to the method given by Hernandez-Aguilar et al. [22].In brief, yellow fashed sweet potato was peeled of, washed, and dabbed in a muslin cloth to remove surface water.Tereafter, the washed sweet potato was uniformly sliced in 10 × 10 × 5 mm size.Sweet potato slices were oven-dried at 2 Journal of Food Quality 60 °C for 10 h and ground into four using an electric grinder.Te four was screened with a 100-mesh sieve [22].Fresh ripe black mulberries were sorted and dried at 55 °C for 30 h using a cabinet dryer (XDL315 Dry Cabinet, Hibex India Pvt. Ltd., Assam, India).Te dried fruit sample was ground into powder using an electric grinder and sieved with a 100-mesh sieve size.All the four and powders were separately packed in high-density polyethylene (HDPE) virgin airtight containers and stored at room temperature.

Experimental Design of Optimization.
Te selection of the diferent ingredients for the development of CIB was based on preliminary experiments.Te range of fours (independent variables) comprising of black rice (C1: 40-70%), germinated lentil (C2: 10-20%), sweet potato (C3: 10-20%), and mulberry (C4: 10-20%) was taken and experimental formulations were designed using a D-optimal mixture design.Te efect of formulations was investigated on responses such as appearance, color, taste, texture, favor, consistency, and overall acceptability, as shown in Table 1.A total of 20 experiments were performed for the optimization of raw material ratio in CIB, including 4 replicates to obtain an estimate of pure error, as shown in Table 1.Te accuracy of ft and the signifcance of linear and quadratic models were performed by Design-Expert ® Version 13 (Stat-Ease, Inc., Minneapolis, MN, USA).

Preparation of CIB.
Te raw ingredients (30 g) were taken from each formulation (20 experiments) and added to 100 ml of warm soy milk containing 2 g of sucralose.
Tereafter, it was stirred well for proper mixing.Te organoleptic evaluation was conducted on the prepared instant beverage (CIB) of each formulation.It was carried out with respect to the following responses such as appearance, color, taste, texture, favor, consistency, and overall acceptability as shown in Table 1.

Organoleptic Evaluation.
After the preparation of the CIB of each formulation, organoleptic evaluation was conducted in the Department of Food Science and Nutrition, Assam Agricultural University, Jorhat, (India).Organoleptic evaluation of the fresh instant was conducted by a total number of 15 semitrained sensory panelists comprising fve males and ten females.Te coded CIB samples assigned with three-digit numbers were randomly presented to the trained panelists.All the panelists were asked to rinse their oral cavity with lukewarm water for palate cleansing in between each sample at 20 °C and RH of 62%.Te organoleptic evaluation was performed using a 9-point hedonic scale ranging from 1 to 9, where 1 depicted "dislike extremely" and 9 depicted "like extremely" [23].

Optimization Using D-Optimal Design.
Te ratio of raw ingredients/materials was optimized using organoleptic response values on the basis of better-set goals which were maximum for each response (appearance, color, taste, texture, favor, consistency, and overall acceptability).
Te criterion of the set goal for optimization was determined according to Pérez-Báez et al. [24].Te lower and upper limits for black rice four (C1: 40-70%), germinated lentil four (C2: 10-20%), sweet potato four (C3: 10-20%), and mulberry powder (C4: 10-20%) were fxed on the basis of our preliminary trials.For carrying out the optimum solution of multiple responses, the individual goals were combined into an overall composite function called the desirability function [25].Te desirability of optimized CIB was found to be closer to one.Contour plots for all responses showed better results with respect to the used ingredient levels.Moreover, optimized CIBs were further subjected to proximate, free radical scavenging activity, and biochemical analysis (in vivo).

Proximate Analysis of Optimized CIB.
Standard AOAC analytical methods [26] were used to analyse the proximate composition i.e., moisture, protein, fat, fbre, and ash of optimized CIB.Te carbohydrate content was determined by the diferential method and the energy value was determined by multiplying the proportion of protein, fat, and carbohydrate with its respective physiological energy value.

Determination of Free Radical Scavenging Activity of
Optimized CIB.Free radical scavenging activity was estimated using the DPPH method given by Vani et al. [27].
Analysis was carried out in the analytical laboratory of the Department of Food Science and Nutrition and Department of Food Science and Technology, Assam Agricultural University, Jorhat, Assam.

Biochemical Analysis of Optimized CIB
2.9.1.In Vivo Glycemic Index (GI).Twelve adult healthy male Wistar rats (150-250 g) from Chakraborty Enterprises, Kolkata were used to determine the glycemic index.All the animals were reared in the Department of Pharmacology and Toxicology, College of Veterinary Sciences, Assam Agricultural University, Khanapara, Assam.Te rats were housed in polypropylene cages (fve rats per cage).Te rats were incubated in a controlled condition with a 12 h light and dark cycle for acclimatization.Deionized distilled water was ofered ad libitum.Te animals were divided into two groups, namely, group A (control group) and group B (optimized CIB as a feed).Each group consists of 10 animals.Te control group (group A) was fed with rat ration along with standard glucose and group B was fed with optimized CIB.Te guidelines of the Institutional Animal Ethics Committee had been followed during animal experimentation (Approval no.770/GO/Re/S/03/CPCSEAFVSc/AAU/ IAEC/18-19/709 dated 28.12.2018).
(1) Glucose Tolerance Test (GTT).GTT was performed using blood samples taken from overnight fasting animals.Fasting blood sugar was taken from Group A before administration of 0.15 g of standard glucose solution (dissolved in distilled water).Blood glucose was determined after feeding at the interval of 15, 30, 45, 60, 90, and 120 mins.Te same method was performed with Group B (feed supplied as optimized CIB) after determining their fasting blood sugar level according to the method proposed by Tannoun [28].
A blood sample was collected from the experimental animals by the tail-tipping method.Blood glucose was determined by using the Accu-Check Roche blood glucose meter (Active blood glucometer kit, Pillbox Pharmacy, Assam, India).
Te glycemic index (GI) for each diet was determined by calculation of the incremental area under two hours of blood glucose response or curve (iAUC) for each diet and compared with iAUC for glucose solution standard according to the method of Jenkins [29]  Te rats that had a value above 200 mg/dl were considered as diabetic.
(1) Determination of Blood Glucose Level.Blood samples were collected from the experimental animals on days 0, 2, 7, 14, and 21 by retro-orbital puncture and centrifuged at 3000 rpm for 20 min.Serum was collected in a microcentrifuge and glucose level was determined using a commercially available assay kit with an auto analyser.Te standard laboratory method was used for blood glucose estimation by using a spectrophotometer (iCE 3000 series, Systronics, India).

Statistical Analysis.
In the present study, a D-optimal mixture design was applied to design the experiments and to analyse the data of organoleptic observations.All experiments were performed independently with at least three determinations.Mean values ± standard deviations for all TS1 to TS20, test formulations 1 to 20 based on the D-optimal mixture design. 4 Journal of Food Quality quantitative parameters were calculated using Microsoft Excel ® 2016 (Microsoft Co., Ltd., Washington, USA).Paired "t" test, analysis of variance, and F-test were used to compare the efect of a specifc treatment, the ratio of between-group variability to within-group variability, and the equality of the diferent treatments on the time of the interval using IBM ® SPSS ® Statistics version 22.0 (New York, USA).A proba- bility value ≤ 0.05 was considered to be signifcant.

Efect of Ingredients Ratio on Organoleptic Properties of CIB.
Te role of the diferent raw ingredients on organoleptic properties was determined by the second-order polynomial model which examined the efect of the signifcant diference between the independent variable (linear efect) and combined variable (interactive efect) on the organoleptic responses of the developed products.Te linear and interactive efects of all the ingredients on the individual organoleptic parameters of the formulated CIB are shown in Table 3.
Te independent variables showed a signifcant (p < 0.05) efect on the organoleptic properties of the product.Organoleptic evaluation of the 20 test samples obtained through a D-optimal mixture design revealed that the appearance of CIB formulation ranged from 6.6 to 7.9 in diferent test samples.Appearance and color of the product were linearly and signifcantly (p < 0.05) improved by pulse, fruit, and vegetable.Te texture was linearly and signifcantly (p < 0.05) improved by cereal, pulse, and fruit, while consistency was linearly and signifcantly (p < 0.05) improved by pulse and vegetable.Moreover, the texture ranged from 6.7 to 8.2.Te favor, taste, and overall acceptability of the product were linearly and signifcantly (p < 0.05) improved by all the independent variables.
Te interactive efect between pulse and fruit and fruit and vegetable signifcantly (p < 0.01) improved the appearance.Similarly, the interactive efect between cereal and pulse, pulse and fruit, as well as fruit and vegetable significantly improved the color.It ranged from 6.5 to 7.9.Te efect between cereal and pulse, pulse and fruit, as well as fruit and vegetable signifcantly improved the favor (6.6 to 7.7) and consistency (6.6 to 7.8) of the product.Te interactive efect between cereal and pulse, cereal and fruit, pulse and fruit, pulse and vegetable, as well as fruit and vegetable signifcantly (p < 0.01) improved the taste of the product.Similarly, the interactive efect between cereal and fruit as well as pulse and fruit signifcantly (p < 0.01) improved the texture of the CIB.All the independent combined variables (interactive efect) signifcantly (p < 0.05) raised the overall acceptability of the product.Te organoleptic responses including appearance, color, favor, taste, texture, and consistency of CIB together contributed to 89% overall acceptability of the product.Sensory evaluation indicated that the overall acceptability of the diferent test samples ranged from 6.7 to 7.6 as shown in Table 1.Te 2D representation of the D-optimal mixture design in relation to the independent variables involved in the organoleptic properties of CIB is shown in Figures 1(a)-1(n).

Optimization of Formulation and Verifcation.
Optimization of the independent variables was performed for the organoleptic responses based on better-set goals for each response, which were "in range" for all ingredients and "maximum" for all responses.Te results of the optimization process showed that a maximum desirability could be obtained at a level of 40% incorporation of cereals and 20% incorporation of pulses, fruits, and vegetables.At this optimal level, the values of the predicted responses were found to be appearance (7.90), color (7.90), favor (7.60), taste (8.00), texture (8.00), consistency (7.80), and overall acceptability (7.60).For verifcation of the optimized level of variables (40% of cereal, 20% of pulse, 20% of fruit, and 20% of vegetable), organoleptic evaluation was again performed at this level and observed values for each response were found to be closer to predicted values.Te variation was found to be 1% in all the organoleptic parameters.Tus, the ratio of raw ingredients with 40 : 20 : 20 : 20 (cereal: pulse: fruit: vegetable) was termed as "optimized CIB" formulation and later it was subjected to proximate, free radical scavenging activity and in vivo studies.4. Te moisture content of the optimized CIB was 7.55 ± 0.16 g/100 g, which was within the recommended limit given by the Food Safety and Standards Regulation of India, 2011 [30].Te protein content of the optimized CIB formulation was 9.71 ± 0.10 g/ 100 g.Te optimized CIB contained a range of crude protein levels, varying from 13.92g/100g to 22.12g/100g, attributable to the inclusion of germinated lentils [31].Tis could be related to the germination and malting process, in which lentil seeds absorb water by imbibition process to initiate Journal of Food Quality  (i)   Journal of Food Quality sprouting [32].Sprouting increases the crude protein content, reduces the phytate level of legumes, and promotes the activity of protease and phytase enzymes, which makes solubilization of phytates easier and releases soluble protein and minerals that increase protein content [33].Te fat content of optimized CIB was 4.73 ± 0.09 g/100 g, which was higher than the fat value (1 g/100 g) of the healthy drink mix available in the market.Tis could be due to the use of fatrich lentil four in optimized CIB formulation.A similar range of fat content from 3.75 g/100 g to 7.26 g/100 g was reported by Qiu et al. [34].Te total mineral content of optimized CIB was 1.08 g/100 g.Tis was in agreement with Santos et al. [35], who reported a total mineral content of 1.9 g in black rice four.Chen et al. [36] reported that black rice bran had a higher mineral content than other cereal brans.Te crude fbre content of CIB was 4.48 g/100 g.Cereal's outer bran layer is rich in crude fbre content.

Proximate of Optimized CIB. Te proximate composition of optimized CIB is shown in Table
According to the World Health Organization (WHO), dietary fbre promotes metabolism and prevents noncommunicable diseases [37].Te total carbohydrate content of the optimized CIB was 72.45 g/100 g and the energy value was 371.21 kcal/100 g as shown in Table 4. Similar energy content from 309 kcal/100 g to 350 kcal/100 g in diferent samples of sprouted legume-based composite diet was reported by Okorie et al. [38].Tus, an increased metabolic process and degradation of starch into simple sugar provide energy during the germination process [39].

Free Radical Scavenging Activity of Optimized CIB.
Te free radical scavenging activity of optimized CIB was 75.65%.Te free radical scavenging activity was observed to be signifcantly (p < 0.05) higher in CIB than in PIB.Tis could be due to the presence of black rice four as the main constituent which has a potent source of phytochemicals, comprising of anthocyanins, favones, favonoids, glycosides, carotenoids, and tocopherols.Te study also reported that the most abundant anthocyanin present in black rice extract is cyanidin 3-glucoside which prevents diseases associated with hyperlipidemia and hyperglycemia by regulating the hepatic lipogenic enzyme activities, inhibiting aglucosidase activity, and inducing pancreatic beta cells regeneration, thereby causing an increase in blood insulin level and reducing blood glucose level [40,41].
3.5.Glycemic Index of Optimized CIB.Group A animals fed with glucose standard had 75 mg/dl of initial fasting blood glucose level, followed by 92 mg/dl at 60 min and again decreased gradually to 77 mg/dl after 120 min.Group B animals fed with optimized CIB had 85 mg/dl of initial fasting blood glucose level, followed by 98 mg/dl at 60 min and again gradually decreased to 79 mg/dl after 120 min.In experimental groups, the peak blood glucose level was observed at 45 min and 60 min after consumption of optimized CIB, which signifcantly (p < 0.05) decreased within 120 min, indicating slow digestion and absorption of the optimized CIB (Figure 2).Comparatively and signifcantly (p < 0.05) minimum rise in blood glucose level was observed in group B fed with optimized CIB, and this may be due to the higher dietary fbre presence in optimized CIB whereas the glucose standard contains no dietary fbre.Several studies have reported that the soluble dietary fbre promotes the satiety feeling which decreases the quantity and frequency of food intake and exerts hypoglycemic efects as well as reduces the regulatory systems stress related to glucose homeostasis through the activation of endocrine L cells in the colon by their physiological ligands and short chain fatty acids.Tey promote proglucagon expression and GLP-1 secretion, thereby controlling insulin secretion and   Te glycemic index is an important parameter when considering diet as a treatment for metabolism-related disorders, such as adult-onset diabetes mellitus, an improvement in postprandial blood glucose concentration after having a meal [43].Te glycemic index of optimized CIB was calculated from their iAUC and iAUC of glucose standard.Te glycemic index of CIB was 37.70.Te studies reported that the low glycemic index of foods, such as black rice and germinated lentil seed, are rich in dietary fbres, proteins, and phytonutrients.Fibers and protein play crucial roles in producing short chain fatty acids (SCFAs) and amino acids (AAs).Tese SCFAs and AAs, once produced, insulin signaling and activate the insulin secretion channels.Consequently, a diet rich in dietary fber and amino acids helps in maintaining stable blood glucose levels [44].Several studies have reported that a water-soluble dietary fbre is low or resistant to digestion and absorption in the small intestine.It may be fermented by gut microfora in the large bowel or as such excreted through feces, thus maintaining the blood glucose levels [45].6. Tis might be due to the presence of crude fbre, which binds with water to form a viscous gel and passes through the small intestine.Tis is relatively unchanged until it reaches the colon, where it is fermented by the gut microfora and produces shortchain fatty acids, resulting in the decrease of serum-free fatty acids.Tis reduces blood glucose levels through competition in insulin-sensitive tissues, which is most benefcial for diabetic patients [46].Furthermore, the presence of phytonutrients in black rice and mulberry powder reduces the blood sugar level as it decreases the intracellular production of H 2 O 2 , resulting in the reduction of apoptosis and β-cells' destruction thus maintaining the insulin secretion in the blood [47,48].Franco San et al. [49] reported that anthocyanin compounds in functional foods help in re-growth and maintenance the function of β-cells.Tese cells are responsible for the synthesizing and secreting of insulin into the bloodstream, helping to control the blood glucose level during diabetic complications.Rathna et al. [50] reported that the whole grain black rice is digested slowly and is thereby important to slow releasing of glucose in the blood  as well as also promoting obesity reduction which is a main cause of type 2 diabetes mellitus.Several studies documented that zinc from sweet potato has the ability to raise adiponectin hormone levels in the blood that appears to play a crucial role in protecting against insulin resistance and minimizing the chance of developing adult-onset diabetes mellitus [51,52].

Conclusion
Te present study explored the utilization of diferent functional food matrix formulations to prepare cereal-based instant beverage that has a lower GI value of 37.70.Supplementation study found that the incorporation of optimized CIB in animal diets at 50%, 60%, and signifcantly decreased the level of glucose in blood up to 42.33 mg/dl, 50.34 mg/dl, and 52.50 mg/dl, respectively.Te fndings of this study provide evidence for the selection of suitable ingredients for the formulation of CIB that can manage and control the blood glucose and lipid profle, thereby exerting additional health benefts in the control and treatment of several noncommunicable diseases.Te technology for the production of instant beverage mixes with enhanced nutritional and functional properties can be transferred for commercialization to local food, health and wellness industries, and entrepreneurship.Tus, it can be accessible to people to have safe nutritious and convenient ready-to-serve food.Te obtained fndings will hopefully provide crucial supporting data for further study on other noncommunicable diseases.Te efect of a specifc amount of optimized CIB can also be investigated for liver diseases, CVDs, and obesity.

Figure 1 :
Figure 1: Efect of independent variables on sensory attributes of cereal-based instant beverage (CIB).Appearance (a and b), color (c and d), favor (e and f ), (g and h), texture (i and j), consistency (k and l), and overall acceptability (m and n).

Figure 2 :
Figure 2: Blood glucose response for rats fed with control and CIB.Data are expressed as the mean (n � 3).

Table 2 :
Proportion of diets for assessing the hypoglycemic efect of optimized CIB.

Table 3 :
Linear and interactive efect of each variable on organoleptic responses of CIB formulations.

Table 4 :
Proximate composition of optimized CIB.Te data of the blood glucose response were used to calculate the glucose response incremental area.Te iAUC value of the reference and sample are shown in Table5.Te mean iAUC of glucose standard and optimized CIB was 1132 and 427, respectively.Te iAUC values expressed that CIB had a lower level of glucose than orally given glucose.

Table 5 :
Mean incremental area under the curve (iAUC) and glycemic index for formulated CIB 1 .