Evaluation and Comparison of Sphenostylis stenocarpa Bean and Wheat Flours: Functional attributes, Antioxidant property, FAME analysis, FTIR, SDS-PAGE and ELISA Validation of the Gluten Content Produced Biscuits

Evaluation and Comparison of Sphenostylis stenocarpa Bean and Wheat Flours: Functional attributes, Antioxidant property, FAME analysis, FTIR, SDS-PAGE and ELISA Validation of the Gluten Content Produced Biscuits | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation and Comparison of Sphenostylis stenocarpa Bean and Wheat Flours: Functional attributes, Antioxidant property, FAME analysis, FTIR, SDS-PAGE and ELISA Validation of the Gluten Content Produced Biscuits S. A. Shodehinde, D. Indrani, P. Prabhasankar, K. Shwetha This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6407277/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and Objective This study of 100% Sphenostylis stenocarpa bean (ssB) flour with incorporation of glyceryl mono stearate (GMS), sodium stearoyl-2-lactylate (SSL) and without additive (W) for the production of Gluten Free (GF) biscuits in comparison to wheat flour biscuit as control was carried out. Materials and Methods : The functional properties of flours, texture, proximate analysis, physico-sensory, biofunctional activities [total phenol content and DPPH (1,1-diphenyl-2-picryl-hydrazyl-hydrate) radical scavenging ability], fatty acid methyl esters (FAME) analysis, fourier transform infrared spectrometry (FTIR), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme linked immunosorbent assay (ELISA) validation of the gluten content were determined in the produced biscuits. Results : Among the composite blends, ssB + GMS had the highest functional properties. The obtained values for sensory evaluation, proximate analysis and hardness in ssB + GMS biscuit were also closer to that of control biscuit. The control biscuit showed lower values of total phenol content and DPPH radical scavenging ability compared to the GF biscuits. However, there was no significant difference (p < 0.05) in the values observed in ssB + GMS and ssB + SSL biscuits. The FAME and FTIR analyses revealed the presence of omega-3, omega-6, omega-9 and similar IR spectra in all the GF biscuits. SDS-PAGE showed a more distinct similar band in GF biscuits in comparison to control biscuit that was not visible to the eye. ELISA evaluation confirmed < 20 ppm content of gluten present in the GF biscuit with the highest sensory score (ssB + GMS). Conclusion : Thus, it can be concluded that Sphenostylis stenocarpa bean flour can be utilized 100% to produce satisfactory and nutritious gluten free biscuits. Sphenostylis stenocarpa Gluten fatty acids antioxidant sensory analysis biscuit Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION ‘Gluten free’ (GF) is a collocation that is identified with the reduction or elimination of wheat, barley, rye and oats is becoming one of the most public used terms among celiac patients and non-celiac individuals [ 1 ] . Celiac disease (CD) is a chronic disease that affects the gastrointestinal system with characteristic atrophy of the small intestinal mucosa on exposure to gluten in food. The genetically predisposed people are affected by the action of human leukocyte antigen (HLA) tagged as genetic determinant factor for celiac autoimmunity [ 2 ] . The number of sufferers of this ailment is put at 1% of the world population. The deleterious effect of CD also causes injury to the skin, liver, joints, brain, heart, and other organs. The centerpiece of celiac disease treatment lies in the consumption of gluten free diets as they have been proven to improve clinical symptoms, forestalls the incidence of malignancies [ 3 ] and other autoimmune disorders such as type 1diabetes and inflammatory bowel [ 4 ] . Recent research work has shown that GF food products differ in their nutrient content and technological characteristics in comparison to gluten-containing food products. Apart from CD patients some consumers who are not suffering from CD do express their interest in gluten-free products as a lifestyle thereby increasing the demand for GF diets [ 5 ] . In the light of this, the current trend in the bakery requires the addition of different flours but most formulations involve the use of carbohydrate-rich but protein-poor contents from tapioca, rice, corn and potato that leads to lower protein content in GF foods [ 6 ] . In recent years, diverse approach has also been taken by researchers to add value to GF products by combining protein rich contents cereals with other non-gluten sources such as dairy products, prebiotics and additives in order to produce baked products with sensory characteristics that compare with those made from wheat flour [ 7 ] . Another area yet to be fully explored is the use of underutilized beans such as Sphenostylis stenocarpa also known as African yam bean. Sphenostylis stenocarpa is a leguminous plant that is native to Africa. Earlier researches have revealed its protein content as it compared with many conventional beans such cashew nut, bambara groundnut, pigeon pea, lima bean, groundnuts and cowpea. It is also rich in phenolic compounds with antioxidant properties [ 8 ] . It is one of the classified underutilized beans, long forgotten and almost going into extinction due to its hard-to-cook nature, beany flavour and bitter taste [ 9 ] . There are speculations about the impossibility of producing GF bakery products (for example biscuits) from legumes 100% with high acceptability due to its low dough forming properties except with the incorporation of other non-wheat flours such as maize starch, maize flour, rice flour and additives [ 10 ] or reducing legume incorporation to formulations by adding legume proteins just for enhancement of the quality of GF biscuits as experimented in some Italian produced biscuits [ 11 ] . In general, leguminous crop such as Sphenostylis stenocarpa is a good source of complex carbohydrate, protein and fibre [ 9 ] . Their use in bakery to address food for celiac patients will require the use of additives such as emulsifiers in order to keep the structure of dough during baking to eventually produce food with improved sensory attributes that will compete with gluten-containing food products. The chemical structures of emulsifiers such as glycerol monostearate (GMS) and Sodium stearoyl-2-lactylate (SSL) contain both hydrophilic and hydrophobic moieties. Their amphiphilic property elevates the stability of a thermodynamically unstable system in dough thus facilitating the possibility of dynamic interaction between starch and protein [ 12 ] . Biscuit is the one of the universally appealing ready-to-eat snack items that is regularly consumed by all age groups. Its popularity is associated with the low cost, varied crispy taste, convenience and longer shelf compared to other baked foods [ 13 ] . GF biscuit, once produced, is expected to possess some considerable quality parameters and can be verified with the use of some classical analytical methods that are time effective. Among these are fourier transform infrared spectrometry (FTIR) spectroscopy, fatty acid methyl esters (FAME) analysis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The FT-IR spectroscopy qualifies and quantifies the macro-components of food and intensity of absorption IR that is associated with the molecular content of their identified chemical group (functional group) in the range 400–4000 cm [ 14 ] . FAME analysis is majorly conducted on gas chromatography-mass spectrometry (GC/MS) to qualify and quantify the saturated and unsaturated fatty acids [ 15 ] . SDS-PAGE is widely used for the identification of protein profile through the separation of proteins according to their molecular weight and is compared to molecular weight markers used [ 16 ] . All these classical analytical techniques are put in place to ensure the safety of food for consumption as this is paramount for the maintenance of a healthy lifestyle. Nevertheless, in order to maintain standard requirements for declaration of gluten content in GF products, a worldwide Codex Standard on Food Labelling has recently been established to forestall danger of unknown gluten consumption and to also ensure a sufficient declaration of gluten content and additives in food [ 17 ] . On the other hand, food products can be contaminated by gluten containing cereals at any stage of production. Enzyme linked immunosorbent assay ( ELISA) test kit is one of the recently developed methods of validating the presence of gluten in food products [ 18 ] . So far, there is still dearth of comprehensive data about the use of various analytical methods in the measurement of GF biscuit quality from Sphenostylis stenocarpa . This present study therefore sought to carry out the following objectives: To utilize wheat flour and Sphenostylis stenocarpa bean flour as raw materials To prepare blends of Sphenostylis stenocarpa bean flour (100%) incorporated with different emulsifiers To prepare blend wheat flour blend To determine the functional properties of composite flour blends in comparison to wheat flour blend To produce GF biscuits from Sphenostylis stenocarpa bean flour (100%) and control biscuits from wheat flour To determine proximate composition and quality parameters of GF biscuits produced using FTIR, FAME analysis and SDS-PAGE (protein profile pattern) in comparison to control biscuit. To validate the gluten content of the most accepted GF biscuit using ELISA kit in comparison to control biscuit. Methods Sample collection and preparation Sphenostylis stenocarpa bean seed was purchased from Osele market, Ikare Akoko, Ondo State. Nigeria [7.5248 ºN, 5.7669 ºE]. The bean seed was identified and authenticated with designated Voucher Number- 257 as that of Sphenostylis stenocarpa (Hochst ex A.Rich) in the herbarium of Plant Science and Biotechnology, Adekunle Ajasin University, Akungba Akoko, Ondo State. Nigeria. The bean was soaked at room temperature until the coat was soft, sun dried and then milled into flour in a hammer mill 3,100 (Perten Instruments AB, Huddinge, Sweden) and sifted through a 250-µm sieve and was stored until further use. Hydrogenated fat (Bunge India Pvt Ltd., Mumbai, India), water, salt (common food grade sodium chloride) and sugar were also used. Additives namely sodium stearoyl-2-lactylate (SSL), glyceryl monostearate (GMS) were procured from PD Navkar Bangalore, India and conventional wheat flour was procured from the market. Preparation of blends Individual preparation of blends was carried out for wheat flour (WF) and 100% Sphenostylis stenocarpa bean flour with different emulsifiers. The preparations were given in the Table 1 below. Functional properties Functional properties such as water absorption capacity, swelling power, emulsion capacity and emulsion stability for the prepared blends are described below: Water absorption capacity (WAC) Water absorption capacity (WAC) was determined according to the method of Sosulski et al. [ 19 ] . Sample (1 g) was mixed with 10 ml of distilled water kept at ambient temperature for 30 mins and centrifuged at 2,000xg for 10 mins. Water absorption capacity was expressed in percent of the water absorbed per gram of sample. Swelling capacity (SC) The swelling capacity was determined by the method described by Okaka and Potter [ 20 ] . A graduated cylinder of 100 mL calibration was filled with the sample to 10 mL mark. The distilled water was added to give a total volume of 50 mL. The top of the graduated cylinder was tightly covered and mixed by inverting the cylinder. The suspension was inverted again after 2 mins and left to stand for a further 8 mins. The volume occupied by the sample was taken after the 8th min. Emulsion capacity and emulsion stability The emulsion capacity (EC) and emulsion stability (ES) were determined by the method of Yasumatsu [ 21 ] with slight modifications. The emulsion (1 g sample, 10 mL distilled water, 10 mL Sunflower oil) was prepared in a calibrated centrifuge tube. The emulsion was centrifuged at 2,0009g for 5 mins. The ratio of the height of the emulsion layer to total layer of the mixture was calculated and expressed in percent. The stability of emulsion was determined according to the method of Neto et al. [ 22 ] the method is as follows heating the emulsion at 85 ºC for 30 mins before centrifuging at 2,0009g for 5 mins. Preparation of biscuits The method used for the biscuit production is as described by Oyewole et al. [ 23 ] with slight modifications. Each sample blend given in Table 1 was mixed for 40 mins until good textured, slightly firm dough was obtained. The dough was kneaded on a flat clean stainless metal table for 4 mins. The mixture was manually rolled into sheets and cut into shapes using biscuits cutter. The dough was baked in an oven at 150 ºC for 20 mins to attain pale brown colour. The biscuits produced were removed from the oven and allowed to assume room temperature. They were later packed and stored at room temperature for further analysis. Proximate analysis Determinations of proximate compositions of biscuits were carried out using standard methods. Moisture, fat, ash, protein, fibre, protein and carbohydrate contents were estimated using AACC approved methods [ 24 ] . Instrumental color measurement Color of biscuit was measured using Hunter color measurement (Color measuring Labscan XE system, USA). Color readings were expressed by Hunter values for L*, a* and b*. L* values measure black to white (0–100), a* values measure red (+ ve) to green (-ve) and b* values measure yellow (+ ve) to blue (-ve). Physical properties of GF biscuits The diameter and thickness of five biscuits measured by placing edge to edge and by stacking one above the other. To obtain the average, measurements were made by rearranging and re-stacking biscuits. The average weight of five biscuits was recorded. Spread ratio was calculated as the average diameter/thickness [ 25 ] . Sensory evaluation The sensory evaluation was carried out by twenty panellists of age between 25 to 55 years, including twelve male and 8 females, who have expertise in sensory evaluation of bakery products. The panelists were trained in four sessions involving 2 hr of training in each session. Four samples of biscuits in four replicates were evaluated by each panelist following a score card consisting of various quality parameters like appearance, aroma, taste, crispy, colour and overall quality score (OQS). The OQS (max 50) was taken as the total score of all the five quality parameters. Evaluation of hardness of biscuits Biscuit hardness was determined using a Texture Profile Analyzer (TPA) according to AACC [ 26 ] . Biscuit hardness was determined by a universal testing machine (Brookfield Engineering Lab. Inc., Middleboro, MA 02346 − 1031, USA). A 25-mm diameter cylindrical probe was used in a TPA at 2 mm/s speed. Hardness was calculated from TPA graphic in Newton (N). Fatty acids determination A portion of the homogenized samples (about 10 g) was extracted with a methanol/chloroform mixture according to Folch method [ 27 ] . The lipid extract was converted into fatty acid methyl esters (FAME) by treatment with 0.01 M sodium hydroxide in methanol at 60–65 ºC, for 30 min at room temperature, followed by collection of the FAMEs dissolved in hexane (analytical grade from Sigma Aldrich, Milano, Italy). GC–MS Analysis The analysis of the standard (FAME) and samples was carried out using gas chromatography with a Shimadzu model GC-17A gas chromatograph equipped with a flame-ionization detector and a 30-metre capillary column of 0.22 mm, that is, with a film thickness of 0.25 µm. The internal standard (IS) used was heptadecanoate. The column temperature was programmed to increase from 60 to 230°C and the injector and detector ports were set at 225°C and 250°C respectively Fourier Transform Infrared (FTIR) Spectroscopy The 2000 System Perkin Elmer instrument operated by PEGRAMS software running on Windows 95 platform was used to register FT-IR spectra. The transmission technique was applied to conduct 25 scans for each of the studied flours in the spectral range of 4000–370 cm − 1. KBr matrix pellets were prepared by mixing 300 mg of KBr with 1 mg of sample in laboratory ball mill. Then mixture was pressed in laboratory press with press 10 tones. Ready pellet was placed in measuring holder-dedicated accessory of System 2000 spectrometer and placed in measuring chamber. Average spectrum was considered final. The resolution was 4 cm − 1 and the shift velocity 2 cm s − 1 . DTGS (deuterated triglycine sulphate) detector is a part of used spectrometer. Extraction of phenolic compounds The antioxidant components of biscuits samples were extracted following the method according to Li et al. [ 28 ] . One gram of sample was mixed with 10 mL 1 N HCl/95% ethanol (v/v, 15/85) solvent in an amber bottle. The mixture was vortexed. Then, extraction was performed in a temperature-controlled (65°C) water bath shaker (VWR International, Radnor, PA, USA) at a constant speed for 80 min. The resulting mixture was centrifuged at 7800× g (10,000 rpm at 5°C for 15 mins). The supernatant was collected and stored in the dark at − 20°C until their use for total phenol content and antioxidant property. Determination of total phenol content Total phenolic content of each sample extract was determined using the Folin–Ciocalteu method as adopted by Shodehinde and Oboh [ 29 ] . Briefly, a 10-fold dilution of Folin–Ciocalteu reagent was prepared just prior to use. Then, 2.5 mL of freshly diluted Folin–Ciocalteu reagent was used to oxidize 0.2 mL sample extracts. After allowing the mixture to equilibrate for 5 mins, the reaction was then neutralized with 2.0 mL sodium carbonate solution (7.5%) for 40 mins at 45°C. The absorbance of the resulting solution was measured at 765 nm (JENWAY 6305). The total phenolic content of samples was expressed as mg/g GAE. Determination of antioxidant activity DPPH Radical Scavenging Assay (RSA) of biscuits was evaluated according to the method adopted by Shodehinde and Oboh [ 30 ] . The percentage of DPPH inhibition was calculated according to the equation below: (%) inhibition = \(\:\frac{\text{A}\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\text{A}\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}{\text{A}\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}\) \(\:X\) \(\:100\) SDS PAGE (Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis) Defatted biscuit samples were analyzed by Electrophoresis. SDS-PAGE was carried out as per the method adopted by Prabhasankar [ 31 ] . 12% acrylamide gel was used to separate the protein fractions of GF biscuits. Gel was stained with coomassie brilliant blue R250. Enzyme linked immunosorbent assay (ELISA ) Gluten analysis was performed using the RIDASCREENR7001Gliadin ELISA (R Biopharm, Germany). Extraction procedure described in the kit insert using Cocktail (R Biopharm, Germany) was followed for the analysis. Each homogenized sample (0.25 g) was weighed into a 15 mL centrifuge tube, 2.5 mL of cocktail solution was added, and the tubes were mixed well followed by incubation for 40 mins at 50°C. Samples were brought to room temperature and added 7.5 mL of 80% ethanol and incubated for 60 mins. On a shaker followed by centrifugation at room temperature for 10 mins at 2,500 g (Eppendorf, 5810R). Supernatant (80 µL) was removed and diluted with 920 µL of sample diluent and 100 µL of this solution was used in the assay. Standard and samples were added in wells on the plate and allowed to incubate for 30 mins at room temperature followed by three steps washing. At this point substrate and chromogen were added to each well and allowed to react for 30 mins followed by addition of stop reagent. The absorbance was read at 450 nm and data was analyzed to determine gluten concentration. The gliadins concentration in µg/kg (ppb) was read from the calibration curve, and further multiplied by dilution factor, then multiplied by 2 in order to obtain the gluten concentration. However, the composite biscuit with the highest overall acceptability was assayed alongside the control biscuit for comparison of gluten level present. Statistical analysis The data pertaining to chemical and nutritional characteristics were expressed as mean ± standard deviation. Sensory mean scores were analyzed using Duncan’s new MRT with different experiment groups appropriate to the completely randomized design with three replicates each, as described by Steel and Torrie [ 32 ] . The significant level was established at p < 0.05. Results and Discussion Table 1: Formulation for the preparation of blends WF; wheat flour, ssBW; Sphenostylis stenocarpa bean flour (without additive), ssB + GMS; Sphenostylis stenocarpa bean flour and glycerol mono stearate, ssB + SSL; Sphenostylis stenocarpa bean flour and sodium stearoyl lactylate. Table 2: Functional properties of flour blends WF; wheat flour, ssBW; Sphenostylis stenocarpa bean flour (without additive), ssB + GMS; Sphenostylis stenocarpa bean flour and glycerol mono stearate, ssB + SSL; Sphenostylis stenocarpa bean flour and sodium stearoyl lactylate. WAC; water absorption capacity, SC; swelling capacity, EC; emulsion capacity, ES; emulsion stability. Values with the same superscript along the same row are not significantly (p < 0.05) different. Values are means of triplicates ± standard deviation Table 5 : Validation of gluten content in biscuits using ELISA ELISA; Enzyme linked immunosorbent assay (ELISA), ssB+GMS; Sphenostylis stenocarpa bean flour plus glycerol mono stearate. Values with the different superscript along the same column are significantly (p<0.05) different. Values are means of three replicates ± standard deviation. The cross examination of functional properties is very vital in processing as it guarantees the quality of products with good quality. The results of functional properties of flour blends of the present study showed significant difference (p < 0.05) and are presented in Table 2 . The result of water absorption capacity (WAC) for WF (96.30%) is lower than that of ssBW (121.20%). Incorporation of additives influenced the WAC as observed in ssB + GMS (130.97%) with the highest WAC was followed by ssB + SSL (127.68%). The reason for the increase in the bean flour blends could be linked to the protein compositions as hydrophilic components interact by hydrogen bonding. WAC has been described as a function of baking quality as it impacts consistency and gives body to food [ 33 ] . High resistance to swelling power is one of the unique properties that have been reported about legumes [ 34 ] . The swelling capacity (SC) values for the flour blends are displayed in Table 2 . The SC value reported for ssBW (3.58 g/g) was slightly lower than that of WF (3.96 g/g). Again, there was an observed significant increase (p < 0.05) in SC of ssB + GMS (4.35 g/g) and ssB + SSL (5.67 g/g) which could be attributed to the presence of incorporated additives. Beans are leguminous plants that are also rich in starch and the determination of SC explains the extent at which the forces within the starch granules associate in flour. However, higher reinforcement of the starch granules in beans has been reported to be responsible for display of low level of SC [ 34 ] . Addition of emulsifiers significantly improved the SC of the flour blends as this also influenced the obtained WAC values. The action of the emulsifiers facilitates an increase in water absorption capacity in flour and to bind to macromolecules in a condition that is similar to that of sucrose [ 35 ] . Table 2 also depicted the emulsion capacity (EC) values which ranged from 33.25 to 42.71. There was significant difference (p < 0.05) in the reported EC values for the bean flour blends. However, the least and the highest values were reported for WF (33.25%) and ssB + GMS (42.71%) respectively. This result is in line with the work of Prajapati et al. [ 36 ] in which the emulsion capacity was higher in fat soya flour in comparison to wheat flour. However, the blends with additives had higher EC values in comparison to ssBW (without additive). EC is a functional parameter that exhibits the ability of protein to bind to fat and is also a means of measuring the maximum amount of oil that is emulsified by protein in a given amount of flour [ 35 ] . The resultant increase of emulsion stability (Table 2 ) in the bean flour blends with additive and may be attributed to the boiling of emulsion as this could have contributed to the melting of protein-lipid complex. The interaction of protein and emulsifier has been shown to build up structure of dough in a way similar to that of gluten protein. The measurement of EC and ES are considered in the maintenance of thick consistency in biscuits baking [ 36 ] . The reported values for all the composite flours exhibited a good capacity of emulsion activity that will be useful for baking processes like the regular WF. The proximate analysis of biscuits is presented in Table 3 . The result of the moisture content ranged from 3.10 to 8.67%. The GF biscuits had significantly (p < 0.05) higher moisture content than that of control biscuit that was produced from wheat flour. The presented varied moisture contents in biscuit samples are in line with the information earlier provided by different producers that (a) typical moisture content of biscuit after baking falls below 10%. (b) During baking, variations come to play as a result of differences in their thickness and weight that occur during forming and shaping [ 37 ] . However, the lower moisture content of biscuits is an important indicator of longer shelf life [ 38 ] . The values of protein content of the biscuits ranged from 10.61–20.91% (Table 3 ). There was significant difference (p < 0.05) in the protein content of the control and GF biscuits. All the GF biscuits exhibited higher protein values than control biscuits. However, ssBW had the highest protein value while control biscuit had the least. This finding supports the general fact that beans contain more protein than cereals [ 39 ] . The fat content of the biscuit samples as shown in Table 3 ranged from 10.55–14.35%. As observed in this finding, there was significant difference (p < 0.05) in fat content values as ssB + GMS had the highest value (14.35%) while ssBW had the least (10.55%). The fat content of biscuits in this present work was lower than the values recorded for some biscuits in earlier research [ 40 ] . Fat is a known flavour retainer that improves the sensory qualities of baked products. The condition of low-fat content in food products promote low energy value to prevent lipid peroxidation [ 41 ] . The ash content of biscuit samples ranged from 0.84–2.47% with high significant difference (p < 0.05) among them. The GF biscuits displayed higher ash contents than the control biscuit. Ash content is one of the parameters recognized for measuring the functional properties of food samples [ 39 ] . In addition, the estimation of ash content in food materials serves as an index of mineral components of food that is considered as an inorganic residue that remains after removing water and inorganic matter with heat in the presence of oxidizing agent [ 42 ] . The fiber content of biscuit samples ranged from 1.95–4.67% and was shown to be significantly (p < 0.05) higher in GF biscuits than control biscuit. This present finding is in line with the work of Hama-Ba et al. [ 43 ] in which lower fibre content was recorded in biscuits produced from wheat flour than those produced from non-wheat flour. The carbohydrate content of biscuit samples ranged from 48.74–71.77%. Again, the significant difference (p < 0.05) in carbohydrate content showed that GF biscuits (ssBW, ssB + GMS and ssB + SSL) at 51.46, 48.74 and 50.95% respectively had lower values than that of control biscuit (71.77%). This finding also supports the work of Oghbaei and Prakash [ 44 ] in which conventional wheat flour was reported to be concentrated in starch as a result of the removal of bran and germ during processing. The physical properties of the biscuit are presented in Table 3 . The weight of the biscuit samples ranged from 6.08 to 7.13 g. There was significant difference (p < 0.05) between the weight of the GF biscuits and control biscuit. Looking at the result, ssBW biscuit had moderately increased weight than the control biscuit while ssB + GMS and ssB + SSL displayed an increase in the weight. This present finding run contrary to the work of Zucco et al. [ 45 ] in which the addition of surfactants affected the weight of cookies supplemented with pulse by decreasing the density. The evaluated thickness of the biscuit samples ranged from 7.00 mm to 7.60 mm (Table 3 ). The thickness value for control biscuit (7.00 mm) was significantly different (p < 0.05) from the GF biscuits (ssB + GMS, ssB + SSL and ssBW) at 7.20 < 7.40 < 7.60 respectively. The recorded higher thickness values in GF biscuits in this present study did not follow the trend of the earlier reported work of Manorah and Rao [ 46 ] in which decreased density influenced reduction in the thickness of biscuits on addition of surfactants. Diameter and spread ratio are important parameters that determine the quality of flour in relation to its ability to rise during biscuit making [ 39 ] . The spread ratio is calculated as the ratio of diameter to thickness [ 25 ] . The higher the spread ratio value in biscuits, the higher they are appreciated [ 47 ] . In this present study, there was significant difference (p < 0.05) in the spread ratio and diameter values of the GF and control biscuits (Table 3 ). The GF biscuits had higher values than that of control biscuit. However, ssB + GMS exhibited the highest values of the parameters. According to Hutchinson et al. [ 48 ] the incorporation of emulsifiers into cookies had been reported as a monitor of spread ratio as it impacts diameter and thickness of biscuits for better textural characteristics. To corroborate this finding, Kissell and Yamazaki [ 49 ] are of the opinion that the incorporation of surfactants into biscuit dough contributes to an effective decrease in dough viscosity by extending the availability of water during the expansion process to increase of biscuit dough during baking thus, increasing the spread ratio. The measurement of hardness in GF and control biscuits as shown in Table 3 ranged between 10.67 and 23.34 N. The evaluated hardness was significantly (p < 0.05) higher in ssBW (GF biscuit without additive) in comparison to others while control biscuit had the least value. The reduced level of hardness in ssB + GMS and ssB + SSL is in line with the opinion of Tsen et al. [ 50 ] that emulsifiers improve the spread and softness of biscuits. The color measurement is an important parameter that appeals to the consumers’ priority and are presented in Table 3 . The colour measurement addresses tri-stimulus attributes L*, a* and b* values [ 51 ] . The GF biscuits (ssBW, ssB + GMS and ssB + SSL) exhibited significantly (p < 0.05) decreased lightness in the reported ‘L*’ values (50.26, 51.38 and 51.08 respectively) in comparison to control biscuit (61.33) that tends towards brightness. Also, there was no significant difference (p < 0.05) in the reported values of a* and b* in all the GF biscuits in comparison to control biscuit. According to earlier report, the colour of processed product should still be as close as possible to the raw material [ 52 ] . The characteristic darker colour in GF biscuits in this present study can also be associated with the negative correlation of protein to lightness while maillard reactions and caramelization also played their roles that lead to the browning of biscuits during baking [ 53 ] . In relation to the additves, the incorporation of emulsifiers did not display any considerable changes in colour parameters of ssB + GMS and ssB + SSL. Similar work had been reported by Shimray et al. [ 54 ] in which there were no significant change in the color parameter of finger millet incorporated biscuits with the addition of emulsifiers. The results of sensory attributes of biscuits as revealed in Table 3 showed significantly (p < 0.05) different values in the evaluation of appearance, aroma, taste, crispy and overall acceptability. As revealed in Table 3 , the appearance of control biscuit in comparison to GF biscuits had the highest score of 10 followed by ssB + GMS (8.5), ssB + SSL (7) and ssBW (6.5). The usual preference given to control biscuit could be attributed to the smoothness of the wheat flour it was produced from. The emphasis on the importance of appearance in food assessment directly influence its acceptability [ 55 ] . In agreement with the work of Shimray et al. [ 54 ] , the addition of emulsifiers however improved the sensory characteristics of the GF biscuits in terms of surface character (Fig. 1 ). The aroma of the biscuit samples ranged from 7.00 to 9.00. The control biscuit had the highest score and was significantly different (p < 0.05) from GF biscuits. There was no significant difference (p < 0.05) in the evaluation of the beany aroma perceived in GF biscuits. Stenostylis stenocarpa naturally has beany flavour that will also be perceived in its products [ 56 ] . The taste of the biscuit samples evaluated ranged from 6.00 to 9.50. The result showed that control biscuit was the most preferred (9.50) which differed significantly (p < 0.05) with respect to GF biscuits. However, ssB + GMS (8.50) was more acceptable among the GF biscuits which was followed by ssB + SSL (8.00) while ssBW (6.00) had the least value. Similar results were also displayed in relation to crispiness of the sample biscuits. There was significant difference (p < 0.05) in the evaluation of the panelists which ranged from 6.00–9.50. The control biscuit had the highest value in comparison to GF biscuits. The evaluation of crispiness/taste in ssB + GMS and ssB + SSL biscuits were more of improved characteristics than that of ssBW biscuit. The assessment of the panelists indicated that the incorporation of surfactants improved the taste and crispiness of GF biscuits thereby influencing the increase in their level of acceptability. With respect to overall acceptability, control biscuit made from wheat flour was the most accepted followed by ssB + GMS which had the most satisfactory sensory attributes from among the GF biscuits. The order of preference by the panelists goes from control > ssB + GMS > ssB + SSL > ssBW at the rated values of 38.00 > 33.00 > 30.00 > 25.50 respectively. Using FAME analysis, the identified fatty acids are classed into saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) and are presented in Table 4 . Their chromatographic representations are also shown in Fig. 2 A-D. Saturated fatty acids were identified in all biscuits and the evaluated percentage ranged from 55.40–60.31%. There was no significant difference (p < 0.05) in the reported value for control (57.42) and ssB + GMS (57.28%). ssBW had the least content (55.40%) while ssB + SSL had the highest reported value (60.31%). Palmitic acid, myristic acid and stearic acid are the most prevalent SFAs in human diet with palmitic acid considered the highest in value [ 15 ] . According to Mensink [ 57 ] , SFAs such as myristic acid and palmitic acid have been reported as the most important dietary risk factors in coronary heart disease (CHD) while stearic acid on the other hand plays no hazardous role as it could be converted to oleic acid (a MUFA). The evaluated SFA value in this present study of the GF biscuits is at a close range with average value of SFA earlier reported in Sphenostylis stenocarpa bean seeds. The contributory role of Sphenostylis stenocarpa towards the CHD is considered low as it’s not an oil seed in itself [ 58 ] . Oleic acid also known as omega-9 fatty acid is considered one of the healthy sources of dietary fat that prevents oxidative rancidity in food. The present study identified oleic acid (C18:1) as the major monounsaturated fatty acids in all the biscuit samples. There was no significant difference (p < 0.05) in the quantity of higher values reported for ssB + GMS (29.54%) and ssB + SSL (29.73%) in comparison to the control biscuit with the least value (26.58%). Linoleic acid (omega-6) and α-Linolenic (omega-3) are essential fatty acids that can only be ingested by human through diet due to the absence of specific enzymes that synthesizes PUFAs with C3 and C6 on its methyl end. Their presence has been linked to health promoting activities as they influence the concentration of lipoproteins to prevent aging and other degenerative disease therefore, considering them as functional food and nutraceuticals [ 59 ] . The result of the polyunsaturated fatty acids of the present biscuit samples showed that Linoleic acid (C18:2 n-6) was identified in all the biscuit samples. However, there was no significant difference (p < 0.05) in the quantity of higher values reported for ssBW biscuit (16.75%) and control biscuit (16.25%), followed by ssB + GMS biscuit (12.90%) while ssB + SSL biscuit had the least value (10.66%). The other identified PUFA is α-Linolenic and was more of higher value in ssB + GMS biscuit (0.69%) than control biscuit (0.45%). FTIR is one the techniques used for the identification of structural and molecular features of phytocompounds in plants. It’s a sensitive method that reveals the functional groups residing in plants which are determined based on peak values with the aid of IR region within the range of 400-4000cm − 1 [ 14 ] . The present FTIR spectroscopy results reveal the presence of various functional groups of chemical compounds as the most important absorption peaks in the IR spectra which were depicted in two zones that range between 3276 − 1742 cm − 1 and 1634 − 525 cm − 1 in all the biscuit samples (Fig. 3 A-D). The displayed instant wide band identified at < 3300 cm − 1 are assigned to the H-bonded and O-H stretching vibration that are majorly generated by macromolecules such as proteins, carbohydrates and phenols. The characteristic bands also identified between 3000 and 2800 cm − 1 are assigned to symmetric and asymmetric stretching of -CH (CH 2 ) and -OH that are generated by lipids and phenols respectively [ 60 ] . The displayed characteristic bands that were identified between 2300 − 1900 cm − 1 are attributed to the presence of multiple bonding that are generated by nitrile compounds. The functional groups such as carbonyl (C = O) and carboxyl (C = C) were identified at the absorption peaks that ranged between < 1800 and 1600 cm − 1 which are known to be generated by ketone and carboxylic acid respectively [ 61 ] . The mid IR region (1750-1000cm-1) displayed large numbers of sharp peaks that yielded broad and overlapped bands that indicates the rich chemical components of carbohydrates, proteins and lipids. Interestingly, it could be observed that the spectra of ssB + GMS (Fig. 3 C) and ssB + SSL (Fig. 3 D) showed higher proportion of signals in comparison to ssBW (Fig. 3 B). According to Stehfest et al. [ 62 ] , some IR absorption peaks within this region have also been attributed to the presence of protein molecules that contain amide (I) and amide (II). This present work also agrees with the earlier work of Hemmalakshmi et al . [ 61 ] that characterized the IR spectra which peaks between 1500 − 600 to reveal the presence of aromatic rings (C = C-C) and amide group C-N which are identified as the fingerprint region. The presence of low-molecular weight carbohydrates and monosaccharides are also indicated in this region [ 63 ] . The present result of higher absorption band as revealed in ssB + GMS and ssB + SSL could be attributed to the contributory role of emulsifiers which is in line with the work of Chung and Tsen [ 50 ] to increase the interface activity between water and the non-aqueous phases of legume. Nevertheless, there are some buried absorption bands in GF biscuits thus revealing a slightly different features from control biscuit (Fig. 3 A). This present study is hereby revealing the presence of various functional molecules such as carboxyl, carbonyl, hydroxyl, amide and nitrile groups to be the major constituents of polyphenols in the understudied legume which do not exist alone but are mostly conjugated with one or more sugar moieties. Earlier and recent research interest in phenolic compounds has been elated for their potential health benefits. According to Shodehinde and Oboh [ 29 ] , the presence of one or more hydroxyl groups and conjugation of side chains to the aromatic rings in phenolic compounds is an indication of antioxidant potentials with inhibitory capacity against free radical generation to maintain the stability of a compound. Earlier work of Diblan et al . [ 64 ] has characterized and differentiated different legumes extracts by different solvents using FTIR spectra to correlate their total phenolic contents. Of note is the fact that the present GF biscuits revealed almost identical FTIR spectra. The similarities in the IR spectroscopy values reported for the GF biscuits follow the phenomenon that is in line with the work of Kemsley et al. [ 65 ] which states that similar active components of close IR absorbance values can be recorded in species of the same genus. Nevertheless, the emulsifiers (GMS and SSL) were observed to show no negative influence on the IR readings in their respective incorporation in GF biscuits. The produced GF biscuits are phenolic- rich hence, can be considered as functional foods. The phenolic acids are compounds with powerful actions of antioxidant that are derived from natural sources. Under experimental conditions, the evaluation of total phenolic content in plants is always linked majorly to their electron donation and metal ion scavenging ability [ 29 ], [ 30 ] . The evaluated total phenol contents of control and GF biscuits are presented in Fig. 4 . There was significant difference (p < 0.05) in the result values which ranged from 3.35–6.59 (mg/g GAE). There was no significant difference (p < 0.05) in the values reported for the GF biscuits. However, control biscuit had lowest value of total phenol content (3.35 mg/g GAE). This result however is in line with the experimental work of Walaa et al. [ 66 ] in which legume product displayed higher phenolic content than wheat product. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging ability (RSA) is one of the methods adopted to carry out an investigation on the antioxidant property of natural products. The resultant effect is measured by the ability to reduce the purple colour of DPPH (a stable diamagnetic molecule) to almost yellow colour at its maximum optical density (OD) 515 nm. There was significant difference (p < 0.05) in the recorded value of DPPH radical scavenging ability of the biscuits which ranged from 20.54% − 59.62% (Fig. 4 ). The result also showed that the control biscuit had the least value of DPPH radical scavenging ability (20.54%) while there was no significant difference (p < 0.05) in ssB + GMS and ssB + SSL DPPH radical scavenging ability values. Again, this finding agrees with the work of Walaa et al. [ 66 ] in which higher DPPH RSA was reported in legume with the higher total phenol content. SDS-PAGE is a valuable simple and informative method that is widely used for the separation of proteins according to their molecular weight and is compared to molecular weight markers used [ 16 ] . The characterization of biological objects thus reveals information of protein constituents. The result of SDS-PAGE protein profile patterns of control and GF biscuits in comparison to the molecular weight markers is revealed Fig. 5 . The GF biscuits displayed the same clear protein regions which were more intense while the band for the control biscuit was hardly visible to the eyes. The intensity of the bands in GF biscuits is an indication of protein abundance as evident in the percentage of protein (Table 3 ). This result also agrees with the fact that higher percentage of protein is profound in legumes than cereals [ 39 ] . Gluten content determination was carried out on GF biscuit for validation through the use of ELISA method (Table 5 ). Cocktail method was adopted as it had also been used by Spaenij-Dekking et al. [ 67 ] and it’s expressed in ppm. With differed significance (p < 0.05), the gluten content in the control biscuit was 75.20 ppm while the preferred GF biscuit with the most acceptable sensory attributes in this present study (ssB + GMS) had 2.01 ppm gluten content. The detected quantity of gluten in the GF biscuit was less than 20 ppm and is considered to be within the permissible limits. The amount of detected gluten content in the validated ssB + GMS biscuit might have occurred as a result of contamination during processing. Conclusion Sphenostylis stenocarpa is an economical crop with high nutritional values reported to its credit. Various analyses have been carried out to substantiate its possible use as supplement in wheat flour but with poor sensory attributes which were improved with the inclusion of emulsifiers to produce GF biscuits of satisfactory attributes that will compete with wheat flour biscuit. The outcome of these findings however suggests that the utilization of African yam bean as an underutilized crop can open ways for further diversification to the production of baked GF diets with higher nutritional properties. Declarations Acknowledgements The Authors are grateful Tertiary Education Trust fund (TETFund), Nigeria, Government of India and Director, CSIR-CFTRI, for extending facilities. Authors’ contributions SAS designed the project and was involved in the bench work, KS was involved in the laboratory works, PP and DI supervised the project and did the internal review of the manuscript. All authors have read and approved the submission of this manuscript. Funding This research was funded by DBT-TWAS (Award number: 3240300000). Availability of data and materials All data are available upon request. Consent for publication Not applicable. Competing interest None declared. Author(s) details 1 Department of Biochemistry, Faculty of Science, Adekunle Ajasin University, Akungba Akoko. Nigeria. 2 Flour Milling, Baking and Confectionery Technology Department, CSIR-Central Food Technological Research Institute, Mysore 570 020, India. References Pinto-Sanchez MI, Verdu EF (2018) Non-celiac gluten or wheat sensitivity: It’s complicated. Neurogastroenterol Motil 30:13392 Sciurti M, Fornaroli F, Gaiani F, Bonaguri C, Leandro G, Di Mario F, De' Angelis GL (2018) Genetic susceptibility and celiac disease: what role do HLA haplotypes play? Acta Bio-Med 89(9-S):17-21 Kelly CP; Bai JC; Liu E, Leffler DA (2015) Advances in diagnosis and management of celiac disease. 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As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6407277","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":440356954,"identity":"d78dd477-bfc1-4cf4-8a4e-1474ddb4e4ab","order_by":0,"name":"S. A. 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Prabhasankar","email":"","orcid":"","institution":"CSIR-Central Food Technological Research Institute","correspondingAuthor":false,"prefix":"","firstName":"P.","middleName":"","lastName":"Prabhasankar","suffix":""},{"id":440356957,"identity":"c3ffddb3-8ef5-43c1-9f85-05ad53453983","order_by":3,"name":"K. Shwetha","email":"","orcid":"","institution":"CSIR-Central Food Technological Research Institute","correspondingAuthor":false,"prefix":"","firstName":"K.","middleName":"","lastName":"Shwetha","suffix":""}],"badges":[],"createdAt":"2025-04-09 02:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6407277/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6407277/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80280482,"identity":"d7f6b139-ca4b-41e3-a2d6-19379b8789d8","added_by":"auto","created_at":"2025-04-10 05:41:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106388,"visible":true,"origin":"","legend":"\u003cp\u003ePhoto of biscuits.\u003c/p\u003e\n\u003cp\u003essBW; \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean (without additive), ssB+GMS; \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003ebean flour and glycerol mono stearate, ssB+SSL; \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003ebean flour and sodium stearoyl lactylate biscuits.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6407277/v1/7dc89edee0811360997a2aca.png"},{"id":80280487,"identity":"dca3c353-3887-4df8-8856-103bb6af8a22","added_by":"auto","created_at":"2025-04-10 05:41:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":529597,"visible":true,"origin":"","legend":"\u003cp\u003eChromatograms of identified fatty acid components in biscuits.\u003c/p\u003e\n\u003cp\u003eA; control, B; ssBW (\u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour without additive), C; ssB+GMS (\u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour and glycerol mono stearate), D; ssB+SSL (\u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour and sodium stearoyl lactylate). IS; internal standard (heptadecanoate).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6407277/v1/ac398cd1d7008154d3762581.png"},{"id":80280488,"identity":"0bbf7995-e5c0-4524-8e10-ebfa2a48c596","added_by":"auto","created_at":"2025-04-10 05:41:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":517811,"visible":true,"origin":"","legend":"\u003cp\u003eChromatograms of IR spectra for biscuits.\u003c/p\u003e\n\u003cp\u003eA; control, B; ssBW (\u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour without additive), C; ssB+GMS (\u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour and glycerol mono stearate), D; ssB+SSL (\u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour and sodium stearoyl lactylate).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6407277/v1/55c504868648bf1c373429ed.png"},{"id":80280486,"identity":"c53037f4-eaf0-4049-84e0-74ef892928e8","added_by":"auto","created_at":"2025-04-10 05:41:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":126122,"visible":true,"origin":"","legend":"\u003cp\u003eTotal phenol content and DPPH radical scavenging ability of biscuits\u003c/p\u003e\n\u003cp\u003econtrol, ssBW; \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour (without additive), ssB+GMS; \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour and glycerol mono stearate and ssB+SSL; \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour and sodium stearoyl lactylate biscuits.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6407277/v1/d81f5a9779887e2b865ec324.png"},{"id":80280490,"identity":"92d2b76e-e9d4-40e3-b54d-d013d3ca7876","added_by":"auto","created_at":"2025-04-10 05:41:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":181646,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE protein profile pattern of biscuits.\u003c/p\u003e\n\u003cp\u003eA; control, B; ssBW (\u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour without additive), C; ssB+GMS (\u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour and glycerol mono stearate), D; ssB+SSL (\u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour and sodium stearoyl lactylate).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6407277/v1/b7009a35068e3df5bd92d584.png"},{"id":80303456,"identity":"d1bb1777-463d-4830-a5c1-55c4de98dbc7","added_by":"auto","created_at":"2025-04-10 09:47:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2784466,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6407277/v1/abc1553c-a5fe-4cd5-8b1c-b90e0d827fc5.pdf"},{"id":80280483,"identity":"e5323183-b8f0-47ca-bac8-a4afd80b7013","added_by":"auto","created_at":"2025-04-10 05:41:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26515,"visible":true,"origin":"","legend":"","description":"","filename":"Table3and4.docx","url":"https://assets-eu.researchsquare.com/files/rs-6407277/v1/11ff34f21cad82ae1ae27e02.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation and Comparison of Sphenostylis stenocarpa Bean and Wheat Flours: Functional attributes, Antioxidant property, FAME analysis, FTIR, SDS-PAGE and ELISA Validation of the Gluten Content Produced Biscuits","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003e\u0026lsquo;Gluten free\u0026rsquo; (GF) is a collocation that is identified with the reduction or elimination of wheat, barley, rye and oats is becoming one of the most public used terms among celiac patients and non-celiac individuals \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Celiac disease (CD) is a chronic disease that affects the gastrointestinal system with characteristic atrophy of the small intestinal mucosa on exposure to gluten in food.\u003c/p\u003e \u003cp\u003eThe genetically predisposed people are affected by the action of human leukocyte antigen (HLA) tagged as genetic determinant factor for celiac autoimmunity \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The number of sufferers of this ailment is put at 1% of the world population. The deleterious effect of CD also causes injury to the skin, liver, joints, brain, heart, and other organs. The centerpiece of celiac disease treatment lies in the consumption of gluten free diets as they have been proven to improve clinical symptoms, forestalls the incidence of malignancies \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e and other autoimmune disorders such as type 1diabetes and inflammatory bowel \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent research work has shown that GF food products differ in their nutrient content and technological characteristics in comparison to gluten-containing food products. Apart from CD patients some consumers who are not suffering from CD do express their interest in gluten-free products as a lifestyle thereby increasing the demand for GF diets \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the light of this, the current trend in the bakery requires the addition of different flours but most formulations involve the use of carbohydrate-rich but protein-poor contents from tapioca, rice, corn and potato that leads to lower protein content in GF foods \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. In recent years, diverse approach has also been taken by researchers to add value to GF products by combining protein rich contents cereals with other non-gluten sources such as dairy products, prebiotics and additives in order to produce baked products with sensory characteristics that compare with those made from wheat flour \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAnother area yet to be fully explored is the use of underutilized beans such as \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e also known as African yam bean. \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e is a leguminous plant that is native to Africa. Earlier researches have revealed its protein content as it compared with many conventional beans such cashew nut, bambara groundnut, pigeon pea, lima bean, groundnuts and cowpea. It is also rich in phenolic compounds with antioxidant properties \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. It is one of the classified underutilized beans, long forgotten and almost going into extinction due to its hard-to-cook nature, beany flavour and bitter taste \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. There are speculations about the impossibility of producing GF bakery products (for example biscuits) from legumes 100% with high acceptability due to its low dough forming properties except with the incorporation of other non-wheat flours such as maize starch, maize flour, rice flour and additives \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e or reducing legume incorporation to formulations by adding legume proteins just for enhancement of the quality of GF biscuits as experimented in some Italian produced biscuits \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn general, leguminous crop such as \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e is a good source of complex carbohydrate, protein and fibre \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Their use in bakery to address food for celiac patients will require the use of additives such as emulsifiers in order to keep the structure of dough during baking to eventually produce food with improved sensory attributes that will compete with gluten-containing food products. The chemical structures of emulsifiers such as glycerol monostearate (GMS) and Sodium stearoyl-2-lactylate (SSL) contain both hydrophilic and hydrophobic moieties. Their amphiphilic property elevates the stability of a thermodynamically unstable system in dough thus facilitating the possibility of dynamic interaction between starch and protein \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBiscuit is the one of the universally appealing ready-to-eat snack items that is regularly consumed by all age groups. Its popularity is associated with the low cost, varied crispy taste, convenience and longer shelf compared to other baked foods \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. GF biscuit, once produced, is expected to possess some considerable quality parameters and can be verified with the use of some classical analytical methods that are time effective. Among these are fourier transform infrared spectrometry (FTIR) spectroscopy, fatty acid methyl esters (FAME) analysis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The FT-IR spectroscopy qualifies and quantifies the macro-components of food and intensity of absorption IR that is associated with the molecular content of their identified chemical group (functional group) in the range 400\u0026ndash;4000 cm \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. FAME analysis is majorly conducted on gas chromatography-mass spectrometry (GC/MS) to qualify and quantify the saturated and unsaturated fatty acids \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. SDS-PAGE is widely used for the identification of protein profile through the separation of proteins according to their molecular weight and is compared to molecular weight markers used \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. All these classical analytical techniques are put in place to ensure the safety of food for consumption as this is paramount for the maintenance of a healthy lifestyle.\u003c/p\u003e \u003cp\u003eNevertheless, in order to maintain standard requirements for declaration of gluten content in GF products, a worldwide Codex Standard on Food Labelling has recently been established to forestall danger of unknown gluten consumption and to also ensure a sufficient declaration of gluten content and additives in food \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. On the other hand, food products can be contaminated by gluten containing cereals at any stage of production. Enzyme linked immunosorbent assay \u003cb\u003e(\u003c/b\u003eELISA) test kit is one of the recently developed methods of validating the presence of gluten in food products \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. So far, there is still dearth of comprehensive data about the use of various analytical methods in the measurement of GF biscuit quality from \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e. This present study therefore sought to carry out the following objectives:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo utilize wheat flour and \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour as raw materials\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo prepare blends of \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour (100%) incorporated with different emulsifiers\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo prepare blend wheat flour blend\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo determine the functional properties of composite flour blends in comparison to wheat flour blend\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo produce GF biscuits from \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour (100%) and control biscuits from wheat flour\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo determine proximate composition and quality parameters of GF biscuits produced using FTIR, FAME analysis and SDS-PAGE (protein profile pattern) in comparison to control biscuit.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo validate the gluten content of the most accepted GF biscuit using ELISA kit in comparison to control biscuit.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection and preparation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean seed was purchased from Osele market, Ikare Akoko, Ondo State. Nigeria [7.5248 \u0026ordm;N, 5.7669 \u0026ordm;E]. The bean seed was identified and authenticated with designated Voucher Number- 257 as that of \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e (Hochst ex A.Rich) in the herbarium of Plant Science and Biotechnology, Adekunle Ajasin University, Akungba Akoko, Ondo State. Nigeria.\u003c/p\u003e \u003cp\u003eThe bean was soaked at room temperature until the coat was soft, sun dried and then milled into flour in a hammer mill 3,100 (Perten Instruments AB, Huddinge, Sweden) and sifted through a 250-\u0026micro;m sieve and was stored until further use. Hydrogenated fat (Bunge India Pvt Ltd., Mumbai, India), water, salt (common food grade sodium chloride) and sugar were also used. Additives namely sodium stearoyl-2-lactylate (SSL), glyceryl monostearate (GMS) were procured from PD Navkar Bangalore, India and conventional wheat flour was procured from the market.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of blends\u003c/h3\u003e\n\u003cp\u003eIndividual preparation of blends was carried out for wheat flour (WF) and 100% \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour with different emulsifiers. The preparations were given in the Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e below.\u003c/p\u003e\n\u003ch3\u003eFunctional properties\u003c/h3\u003e\n\u003cp\u003eFunctional properties such as water absorption capacity, swelling power, emulsion capacity and emulsion stability for the prepared blends are described below:\u003c/p\u003e\n\u003ch3\u003eWater absorption capacity (WAC)\u003c/h3\u003e\n\u003cp\u003eWater absorption capacity (WAC) was determined according to the method of Sosulski et al. \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Sample (1 g) was mixed with 10 ml of distilled water kept at ambient temperature for 30 mins and centrifuged at 2,000xg for 10 mins. Water absorption capacity was expressed in percent of the water absorbed per gram of sample.\u003c/p\u003e\n\u003ch3\u003eSwelling capacity (SC)\u003c/h3\u003e\n\u003cp\u003eThe swelling capacity was determined by the method described by Okaka and Potter \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. A graduated cylinder of 100 mL calibration was filled with the sample to 10 mL mark. The distilled water was added to give a total volume of 50 mL. The top of the graduated cylinder was tightly covered and mixed by inverting the cylinder. The suspension was inverted again after 2 mins and left to stand for a further 8 mins. The volume occupied by the sample was taken after the 8th min.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEmulsion capacity and emulsion stability\u003c/h2\u003e \u003cp\u003eThe emulsion capacity (EC) and emulsion stability (ES) were determined by the method of Yasumatsu \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e with slight modifications. The emulsion (1 g sample, 10 mL distilled water, 10 mL Sunflower oil) was prepared in a calibrated centrifuge tube. The emulsion was centrifuged at 2,0009g for 5 mins. The ratio of the height of the emulsion layer to total layer of the mixture was calculated and expressed in percent. The stability of emulsion was determined according to the method of Neto \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e the method is as follows heating the emulsion at 85 \u0026ordm;C for 30 mins before centrifuging at 2,0009g for 5 mins.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of biscuits\u003c/h3\u003e\n\u003cp\u003eThe method used for the biscuit production is as described by Oyewole et al. \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e with slight modifications. Each sample blend given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e was mixed for 40 mins until good textured, slightly firm dough was obtained. The dough was kneaded on a flat clean stainless metal table for 4 mins. The mixture was manually rolled into sheets and cut into shapes using biscuits cutter. The dough was baked in an oven at 150 \u0026ordm;C for 20 mins to attain pale brown colour. The biscuits produced were removed from the oven and allowed to assume room temperature. They were later packed and stored at room temperature for further analysis.\u003c/p\u003e\n\u003ch3\u003eProximate analysis\u003c/h3\u003e\n\u003cp\u003eDeterminations of proximate compositions of biscuits were carried out using standard methods. Moisture, fat, ash, protein, fibre, protein and carbohydrate contents were estimated using AACC approved methods \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eInstrumental color measurement\u003c/h2\u003e \u003cp\u003eColor of biscuit was measured using Hunter color measurement (Color measuring Labscan XE system, USA). Color readings were expressed by Hunter values for L*, a* and b*. L* values measure black to white (0\u0026ndash;100), a* values measure red (+\u0026thinsp;ve) to green (-ve) and b* values measure yellow (+\u0026thinsp;ve) to blue (-ve).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhysical properties of GF biscuits\u003c/h2\u003e \u003cp\u003eThe diameter and thickness of five biscuits measured by placing edge to edge and by stacking one above the other. To obtain the average, measurements were made by rearranging and re-stacking biscuits. The average weight of five biscuits was recorded. Spread ratio was calculated as the average diameter/thickness \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSensory evaluation\u003c/h2\u003e \u003cp\u003eThe sensory evaluation was carried out by twenty panellists of age between 25 to 55 years, including twelve male and 8 females, who have expertise in sensory evaluation of bakery products. The panelists were trained in four sessions involving 2 hr of training in each session. Four samples of biscuits in four replicates were evaluated by each panelist following a score card consisting of various quality parameters like appearance, aroma, taste, crispy, colour and overall quality score (OQS). The OQS (max 50) was taken as the total score of all the five quality parameters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of hardness of biscuits\u003c/h2\u003e \u003cp\u003eBiscuit hardness was determined using a Texture Profile Analyzer (TPA) according to AACC \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Biscuit hardness was determined by a universal testing machine (Brookfield Engineering Lab. Inc., Middleboro, MA 02346\u0026thinsp;\u0026minus;\u0026thinsp;1031, USA). A 25-mm diameter cylindrical probe was used in a TPA at 2 mm/s speed. Hardness was calculated from TPA graphic in Newton (N).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFatty acids determination\u003c/h2\u003e \u003cp\u003eA portion of the homogenized samples (about 10 g) was extracted with a methanol/chloroform mixture according to Folch method \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. The lipid extract was converted into fatty acid methyl esters (FAME) by treatment with 0.01 M sodium hydroxide in methanol at 60\u0026ndash;65 \u0026ordm;C, for 30 min at room temperature, followed by collection of the FAMEs dissolved in hexane (analytical grade from Sigma Aldrich, Milano, Italy).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGC\u0026ndash;MS Analysis\u003c/h2\u003e \u003cp\u003eThe analysis of the standard (FAME) and samples was carried out using gas chromatography with a Shimadzu model GC-17A gas chromatograph equipped with a flame-ionization detector and a 30-metre capillary column of 0.22 mm, that is, with a film thickness of 0.25 \u0026micro;m. The internal standard (IS) used was heptadecanoate. The column temperature was programmed to increase from 60 to 230\u0026deg;C and the injector and detector ports were set at 225\u0026deg;C and 250\u0026deg;C respectively\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFourier Transform Infrared (FTIR) Spectroscopy\u003c/h2\u003e \u003cp\u003eThe 2000 System Perkin Elmer instrument operated by PEGRAMS software running on Windows 95 platform was used to register FT-IR spectra. The transmission technique was applied to conduct 25 scans for each of the studied flours in the spectral range of 4000\u0026ndash;370 cm\u0026thinsp;\u0026minus;\u0026thinsp;1. KBr matrix pellets were prepared by mixing 300 mg of KBr with 1 mg of sample in laboratory ball mill. Then mixture was pressed in laboratory press with press 10 tones. Ready pellet was placed in measuring holder-dedicated accessory of System 2000 spectrometer and placed in measuring chamber. Average spectrum was considered final. The resolution was 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the shift velocity 2 cm s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. DTGS (deuterated triglycine sulphate) detector is a part of used spectrometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eExtraction of phenolic compounds\u003c/h2\u003e \u003cp\u003eThe antioxidant components of biscuits samples were extracted following the method according to Li \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. One gram of sample was mixed with 10 mL 1 N HCl/95% ethanol (v/v, 15/85) solvent in an amber bottle. The mixture was vortexed. Then, extraction was performed in a temperature-controlled (65\u0026deg;C) water bath shaker (VWR International, Radnor, PA, USA) at a constant speed for 80 min. The resulting mixture was centrifuged at 7800\u0026times; g (10,000 rpm at 5\u0026deg;C for 15 mins). The supernatant was collected and stored in the dark at \u0026minus;\u0026thinsp;20\u0026deg;C until their use for total phenol content and antioxidant property.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of total phenol content\u003c/h2\u003e \u003cp\u003eTotal phenolic content of each sample extract was determined using the Folin\u0026ndash;Ciocalteu method as adopted by Shodehinde and Oboh \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Briefly, a 10-fold dilution of Folin\u0026ndash;Ciocalteu reagent was prepared just prior to use. Then, 2.5 mL of freshly diluted Folin\u0026ndash;Ciocalteu reagent was used to oxidize 0.2 mL sample extracts. After allowing the mixture to equilibrate for 5 mins, the reaction was then neutralized with 2.0 mL sodium carbonate solution (7.5%) for 40 mins at 45\u0026deg;C. The absorbance of the resulting solution was measured at 765 nm (JENWAY 6305). The total phenolic content of samples was expressed as mg/g GAE.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of antioxidant activity\u003c/h2\u003e \u003cp\u003eDPPH Radical Scavenging Assay (RSA) of biscuits was evaluated according to the method adopted by Shodehinde and Oboh \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. The percentage of DPPH inhibition was calculated according to the equation below:\u003c/p\u003e \u003cp\u003e(%) inhibition = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{A}\\text{C}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}-\\text{A}\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}{\\text{A}\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}\\)\u003c/span\u003e\u003c/span\u003e \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:X\\)\u003c/span\u003e\u003c/span\u003e \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSDS PAGE (Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis)\u003c/h2\u003e \u003cp\u003eDefatted biscuit samples were analyzed by Electrophoresis. SDS-PAGE was carried out as per the method adopted by Prabhasankar \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. 12% acrylamide gel was used to separate the protein fractions of GF biscuits. Gel was stained with coomassie brilliant blue R250.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEnzyme linked immunosorbent assay (ELISA\u003c/b\u003e)\u003c/p\u003e \u003cp\u003eGluten analysis was performed using the RIDASCREENR7001Gliadin ELISA (R Biopharm, Germany). Extraction procedure described in the kit insert using Cocktail (R Biopharm, Germany) was followed for the analysis. Each homogenized sample (0.25 g) was weighed into a 15 mL centrifuge tube, 2.5 mL of cocktail solution was added, and the tubes were mixed well followed by incubation for 40 mins at 50\u0026deg;C. Samples were brought to room temperature and added 7.5 mL of 80% ethanol and incubated for 60 mins. On a shaker followed by centrifugation at room temperature for 10 mins at 2,500 g (Eppendorf, 5810R). Supernatant (80 \u0026micro;L) was removed and diluted with 920 \u0026micro;L of sample diluent and 100 \u0026micro;L of this solution was used in the assay. Standard and samples were added in wells on the plate and allowed to incubate for 30 mins at room temperature followed by three steps washing. At this point substrate and chromogen were added to each well and allowed to react for 30 mins followed by addition of stop reagent. The absorbance was read at 450 nm and data was analyzed to determine gluten concentration. The gliadins concentration in \u0026micro;g/kg (ppb) was read from the calibration curve, and further multiplied by dilution factor, then multiplied by 2 in order to obtain the gluten concentration. However, the composite biscuit with the highest overall acceptability was assayed alongside the control biscuit for comparison of gluten level present.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data pertaining to chemical and nutritional characteristics were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Sensory mean scores were analyzed using Duncan\u0026rsquo;s new MRT with different experiment groups appropriate to the completely randomized design with three replicates each, as described by Steel and Torrie \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. The significant level was established at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003eTable 1:\u0026nbsp;\u003c/strong\u003eFormulation for the preparation of blends\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003eWF; wheat flour, ssBW; \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour (without additive), ssB\u0026thinsp;+\u0026thinsp;GMS; \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour and glycerol mono stearate, ssB\u0026thinsp;+\u0026thinsp;SSL; \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour and sodium stearoyl lactylate.\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2:\u0026nbsp;\u003c/strong\u003eFunctional properties of flour blends\u003c/p\u003e\n\n\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img174426288569.png\"\u003e\u003cbr\u003e\u003c/div\u003e\n\u003cdiv align=\"left\" class=\"colspec\"\u003eWF; wheat flour, ssBW; \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour (without additive), ssB\u0026thinsp;+\u0026thinsp;GMS; \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour and glycerol mono stearate, ssB\u0026thinsp;+\u0026thinsp;SSL; \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour and sodium stearoyl lactylate. WAC; water absorption capacity, SC; swelling capacity, EC; emulsion capacity, ES; emulsion stability. Values with the same superscript along the same row are not significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) different. Values are means of triplicates\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e: Validation of gluten content in biscuits using ELISA\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1744262885.png\"\u003e\u003c/p\u003e\n\n\u003cdiv align=\"left\" class=\"colspec\"\u003eELISA; Enzyme linked immunosorbent assay (ELISA), ssB+GMS; \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour plus glycerol mono stearate. Values with the different superscript along the same column are significantly (p\u0026lt;0.05) different. Values are means of three replicates \u0026plusmn; standard deviation.\u003c/div\u003e\n\u003cp\u003eThe cross examination of functional properties is very vital in processing as it guarantees the quality of products with good quality. The results of functional properties of flour blends of the present study showed significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and are presented in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. The result of water absorption capacity (WAC) for WF (96.30%) is lower than that of ssBW (121.20%). Incorporation of additives influenced the WAC as observed in ssB\u0026thinsp;+\u0026thinsp;GMS (130.97%) with the highest WAC was followed by ssB\u0026thinsp;+\u0026thinsp;SSL (127.68%). The reason for the increase in the bean flour blends could be linked to the protein compositions as hydrophilic components interact by hydrogen bonding. WAC has been described as a function of baking quality as it impacts consistency and gives body to food \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eHigh resistance to swelling power is one of the unique properties that have been reported about legumes \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. The swelling capacity (SC) values for the flour blends are displayed in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. The SC value reported for ssBW (3.58 g/g) was slightly lower than that of WF (3.96 g/g). Again, there was an observed significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in SC of ssB\u0026thinsp;+\u0026thinsp;GMS (4.35 g/g) and ssB\u0026thinsp;+\u0026thinsp;SSL (5.67 g/g) which could be attributed to the presence of incorporated additives. Beans are leguminous plants that are also rich in starch and the determination of SC explains the extent at which the forces within the starch granules associate in flour. However, higher reinforcement of the starch granules in beans has been reported to be responsible for display of low level of SC \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Addition of emulsifiers significantly improved the SC of the flour blends as this also influenced the obtained WAC values. The action of the emulsifiers facilitates an increase in water absorption capacity in flour and to bind to macromolecules in a condition that is similar to that of sucrose \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e also depicted the emulsion capacity (EC) values which ranged from 33.25 to 42.71. There was significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the reported EC values for the bean flour blends. However, the least and the highest values were reported for WF (33.25%) and ssB\u0026thinsp;+\u0026thinsp;GMS (42.71%) respectively. This result is in line with the work of Prajapati \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e in which the emulsion capacity was higher in fat soya flour in comparison to wheat flour. However, the blends with additives had higher EC values in comparison to ssBW (without additive). EC is a functional parameter that exhibits the ability of protein to bind to fat and is also a means of measuring the maximum amount of oil that is emulsified by protein in a given amount of flour \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe resultant increase of emulsion stability (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) in the bean flour blends with additive and may be attributed to the boiling of emulsion as this could have contributed to the melting of protein-lipid complex. The interaction of protein and emulsifier has been shown to build up structure of dough in a way similar to that of gluten protein. The measurement of EC and ES are considered in the maintenance of thick consistency in biscuits baking \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. The reported values for all the composite flours exhibited a good capacity of emulsion activity that will be useful for baking processes like the regular WF.\u003c/p\u003e\n\u003cp\u003eThe proximate analysis of biscuits is presented in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The result of the moisture content ranged from 3.10 to 8.67%. The GF biscuits had significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) higher moisture content than that of control biscuit that was produced from wheat flour. The presented varied moisture contents in biscuit samples are in line with the information earlier provided by different producers that (a) typical moisture content of biscuit after baking falls below 10%. (b) During baking, variations come to play as a result of differences in their thickness and weight that occur during forming and shaping \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. However, the lower moisture content of biscuits is an important indicator of longer shelf life \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe values of protein content of the biscuits ranged from 10.61\u0026ndash;20.91% (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). There was significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the protein content of the control and GF biscuits. All the GF biscuits exhibited higher protein values than control biscuits. However, ssBW had the highest protein value while control biscuit had the least. This finding supports the general fact that beans contain more protein than cereals \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe fat content of the biscuit samples as shown in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e ranged from 10.55\u0026ndash;14.35%. As observed in this finding, there was significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in fat content values as ssB\u0026thinsp;+\u0026thinsp;GMS had the highest value (14.35%) while ssBW had the least (10.55%). The fat content of biscuits in this present work was lower than the values recorded for some biscuits in earlier research \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Fat is a known flavour retainer that improves the sensory qualities of baked products. The condition of low-fat content in food products promote low energy value to prevent lipid peroxidation \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe ash content of biscuit samples ranged from 0.84\u0026ndash;2.47% with high significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) among them. The GF biscuits displayed higher ash contents than the control biscuit. Ash content is one of the parameters recognized for measuring the functional properties of food samples \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. In addition, the estimation of ash content in food materials serves as an index of mineral components of food that is considered as an inorganic residue that remains after removing water and inorganic matter with heat in the presence of oxidizing agent \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe fiber content of biscuit samples ranged from 1.95\u0026ndash;4.67% and was shown to be significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) higher in GF biscuits than control biscuit. This present finding is in line with the work of Hama-Ba \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e in which lower fibre content was recorded in biscuits produced from wheat flour than those produced from non-wheat flour.\u003c/p\u003e\n\u003cp\u003eThe carbohydrate content of biscuit samples ranged from 48.74\u0026ndash;71.77%. Again, the significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in carbohydrate content showed that GF biscuits (ssBW, ssB\u0026thinsp;+\u0026thinsp;GMS and ssB\u0026thinsp;+\u0026thinsp;SSL) at 51.46, 48.74 and 50.95% respectively had lower values than that of control biscuit (71.77%). This finding also supports the work of Oghbaei and Prakash \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e in which conventional wheat flour was reported to be concentrated in starch as a result of the removal of bran and germ during processing.\u003c/p\u003e\n\u003cp\u003eThe physical properties of the biscuit are presented in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The weight of the biscuit samples ranged from 6.08 to 7.13 g. There was significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between the weight of the GF biscuits and control biscuit. Looking at the result, ssBW biscuit had moderately increased weight than the control biscuit while ssB\u0026thinsp;+\u0026thinsp;GMS and ssB\u0026thinsp;+\u0026thinsp;SSL displayed an increase in the weight. This present finding run contrary to the work of Zucco \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e in which the addition of surfactants affected the weight of cookies supplemented with pulse by decreasing the density.\u003c/p\u003e\n\u003cp\u003eThe evaluated thickness of the biscuit samples ranged from 7.00 mm to 7.60 mm (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The thickness value for control biscuit (7.00 mm) was significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) from the GF biscuits (ssB\u0026thinsp;+\u0026thinsp;GMS, ssB\u0026thinsp;+\u0026thinsp;SSL and ssBW) at 7.20\u0026thinsp;\u0026lt;\u0026thinsp;7.40\u0026thinsp;\u0026lt;\u0026thinsp;7.60 respectively. The recorded higher thickness values in GF biscuits in this present study did not follow the trend of the earlier reported work of Manorah and Rao \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e in which decreased density influenced reduction in the thickness of biscuits on addition of surfactants.\u003c/p\u003e\n\u003cp\u003eDiameter and spread ratio are important parameters that determine the quality of flour in relation to its ability to rise during biscuit making \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. The spread ratio is calculated as the ratio of diameter to thickness \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. The higher the spread ratio value in biscuits, the higher they are appreciated \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. In this present study, there was significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the spread ratio and diameter values of the GF and control biscuits (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The GF biscuits had higher values than that of control biscuit. However, ssB\u0026thinsp;+\u0026thinsp;GMS exhibited the highest values of the parameters. According to Hutchinson \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e the incorporation of emulsifiers into cookies had been reported as a monitor of spread ratio as it impacts diameter and thickness of biscuits for better textural characteristics. To corroborate this finding, Kissell and Yamazaki \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e are of the opinion that the incorporation of surfactants into biscuit dough contributes to an effective decrease in dough viscosity by extending the availability of water during the expansion process to increase of biscuit dough during baking thus, increasing the spread ratio.\u003c/p\u003e\n\u003cp\u003eThe measurement of hardness in GF and control biscuits as shown in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e ranged between 10.67 and 23.34 N. The evaluated hardness was significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) higher in ssBW (GF biscuit without additive) in comparison to others while control biscuit had the least value. The reduced level of hardness in ssB\u0026thinsp;+\u0026thinsp;GMS and ssB\u0026thinsp;+\u0026thinsp;SSL is in line with the opinion of Tsen \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e that emulsifiers improve the spread and softness of biscuits.\u003c/p\u003e\n\u003cp\u003eThe color measurement is an important parameter that appeals to the consumers\u0026rsquo; priority and are presented in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The colour measurement addresses tri-stimulus attributes L*, a* and b* values \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. The GF biscuits (ssBW, ssB\u0026thinsp;+\u0026thinsp;GMS and ssB\u0026thinsp;+\u0026thinsp;SSL) exhibited significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decreased lightness in the reported \u0026lsquo;L*\u0026rsquo; values (50.26, 51.38 and 51.08 respectively) in comparison to control biscuit (61.33) that tends towards brightness. Also, there was no significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the reported values of a* and b* in all the GF biscuits in comparison to control biscuit. According to earlier report, the colour of processed product should still be as close as possible to the raw material \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. The characteristic darker colour in GF biscuits in this present study can also be associated with the negative correlation of protein to lightness while maillard reactions and caramelization also played their roles that lead to the browning of biscuits during baking \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. In relation to the additves, the incorporation of emulsifiers did not display any considerable changes in colour parameters of ssB\u0026thinsp;+\u0026thinsp;GMS and ssB\u0026thinsp;+\u0026thinsp;SSL. Similar work had been reported by Shimray \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e in which there were no significant change in the color parameter of finger millet incorporated biscuits with the addition of emulsifiers.\u003c/p\u003e\n\u003cp\u003eThe results of sensory attributes of biscuits as revealed in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e showed significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) different values in the evaluation of appearance, aroma, taste, crispy and overall acceptability. As revealed in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the appearance of control biscuit in comparison to GF biscuits had the highest score of 10 followed by ssB\u0026thinsp;+\u0026thinsp;GMS (8.5), ssB\u0026thinsp;+\u0026thinsp;SSL (7) and ssBW (6.5). The usual preference given to control biscuit could be attributed to the smoothness of the wheat flour it was produced from. The emphasis on the importance of appearance in food assessment directly influence its acceptability \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. In agreement with the work of Shimray \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e, the addition of emulsifiers however improved the sensory characteristics of the GF biscuits in terms of surface character (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe aroma of the biscuit samples ranged from 7.00 to 9.00. The control biscuit had the highest score and was significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) from GF biscuits. There was no significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the evaluation of the beany aroma perceived in GF biscuits. \u003cem\u003eStenostylis stenocarpa\u003c/em\u003e naturally has beany flavour that will also be perceived in its products \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe taste of the biscuit samples evaluated ranged from 6.00 to 9.50. The result showed that control biscuit was the most preferred (9.50) which differed significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with respect to GF biscuits. However, ssB\u0026thinsp;+\u0026thinsp;GMS (8.50) was more acceptable among the GF biscuits which was followed by ssB\u0026thinsp;+\u0026thinsp;SSL (8.00) while ssBW (6.00) had the least value. Similar results were also displayed in relation to crispiness of the sample biscuits. There was significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the evaluation of the panelists which ranged from 6.00\u0026ndash;9.50. The control biscuit had the highest value in comparison to GF biscuits. The evaluation of crispiness/taste in ssB\u0026thinsp;+\u0026thinsp;GMS and ssB\u0026thinsp;+\u0026thinsp;SSL biscuits were more of improved characteristics than that of ssBW biscuit. The assessment of the panelists indicated that the incorporation of surfactants improved the taste and crispiness of GF biscuits thereby influencing the increase in their level of acceptability.\u003c/p\u003e\n\u003cp\u003eWith respect to overall acceptability, control biscuit made from wheat flour was the most accepted followed by ssB\u0026thinsp;+\u0026thinsp;GMS which had the most satisfactory sensory attributes from among the GF biscuits. The order of preference by the panelists goes from control\u0026thinsp;\u0026gt;\u0026thinsp;ssB\u0026thinsp;+\u0026thinsp;GMS\u0026thinsp;\u0026gt;\u0026thinsp;ssB\u0026thinsp;+\u0026thinsp;SSL\u0026thinsp;\u0026gt;\u0026thinsp;ssBW at the rated values of 38.00\u0026thinsp;\u0026gt;\u0026thinsp;33.00\u0026thinsp;\u0026gt;\u0026thinsp;30.00\u0026thinsp;\u0026gt;\u0026thinsp;25.50 respectively.\u003c/p\u003e\n\u003cp\u003eUsing FAME analysis, the identified fatty acids are classed into saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) and are presented in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. Their chromatographic representations are also shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-D. Saturated fatty acids were identified in all biscuits and the evaluated percentage ranged from 55.40\u0026ndash;60.31%. There was no significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the reported value for control (57.42) and ssB\u0026thinsp;+\u0026thinsp;GMS (57.28%). ssBW had the least content (55.40%) while ssB\u0026thinsp;+\u0026thinsp;SSL had the highest reported value (60.31%). Palmitic acid, myristic acid and stearic acid are the most prevalent SFAs in human diet with palmitic acid considered the highest in value \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. According to Mensink \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e, SFAs such as myristic acid and palmitic acid have been reported as the most important dietary risk factors in coronary heart disease (CHD) while stearic acid on the other hand plays no hazardous role as it could be converted to oleic acid (a MUFA). The evaluated SFA value in this present study of the GF biscuits is at a close range with average value of SFA earlier reported in \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean seeds. The contributory role of \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e towards the CHD is considered low as it\u0026rsquo;s not an oil seed in itself \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOleic acid also known as omega-9 fatty acid is considered one of the healthy sources of dietary fat that prevents oxidative rancidity in food. The present study identified oleic acid (C18:1) as the major monounsaturated fatty acids in all the biscuit samples. There was no significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the quantity of higher values reported for ssB\u0026thinsp;+\u0026thinsp;GMS (29.54%) and ssB\u0026thinsp;+\u0026thinsp;SSL (29.73%) in comparison to the control biscuit with the least value (26.58%).\u003c/p\u003e\n\u003cp\u003eLinoleic acid (omega-6) and \u0026alpha;-Linolenic (omega-3) are essential fatty acids that can only be ingested by human through diet due to the absence of specific enzymes that synthesizes PUFAs with C3 and C6 on its methyl end. Their presence has been linked to health promoting activities as they influence the concentration of lipoproteins to prevent aging and other degenerative disease therefore, considering them as functional food and nutraceuticals \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e. The result of the polyunsaturated fatty acids of the present biscuit samples showed that Linoleic acid (C18:2 n-6) was identified in all the biscuit samples. However, there was no significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the quantity of higher values reported for ssBW biscuit (16.75%) and control biscuit (16.25%), followed by ssB\u0026thinsp;+\u0026thinsp;GMS biscuit (12.90%) while ssB\u0026thinsp;+\u0026thinsp;SSL biscuit had the least value (10.66%). The other identified PUFA is \u0026alpha;-Linolenic and was more of higher value in ssB\u0026thinsp;+\u0026thinsp;GMS biscuit (0.69%) than control biscuit (0.45%).\u003c/p\u003e\n\u003cp\u003eFTIR is one the techniques used for the identification of structural and molecular features of phytocompounds in plants. It\u0026rsquo;s a sensitive method that reveals the functional groups residing in plants which are determined based on peak values with the aid of IR region within the range of 400-4000cm\u003csup\u003e\u0026minus;\u0026thinsp;1 [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The present FTIR spectroscopy results reveal the presence of various functional groups of chemical compounds as the most important absorption peaks in the IR spectra which were depicted in two zones that range between 3276\u0026thinsp;\u0026minus;\u0026thinsp;1742 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1634\u0026thinsp;\u0026minus;\u0026thinsp;525 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in all the biscuit samples (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-D).\u003c/p\u003e\n\u003cp\u003eThe displayed instant wide band identified at \u0026lt;\u0026thinsp;3300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are assigned to the H-bonded and O-H stretching vibration that are majorly generated by macromolecules such as proteins, carbohydrates and phenols. The characteristic bands also identified between 3000 and 2800 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are assigned to symmetric and asymmetric stretching of -CH (CH\u003csub\u003e2\u003c/sub\u003e) and -OH that are generated by lipids and phenols respectively \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe displayed characteristic bands that were identified between 2300\u0026thinsp;\u0026minus;\u0026thinsp;1900 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are attributed to the presence of multiple bonding that are generated by nitrile compounds. The functional groups such as carbonyl (C\u0026thinsp;=\u0026thinsp;O) and carboxyl (C\u0026thinsp;=\u0026thinsp;C) were identified at the absorption peaks that ranged between \u0026lt;\u0026thinsp;1800 and 1600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e which are known to be generated by ketone and carboxylic acid respectively \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/sup\u003e. The mid IR region (1750-1000cm-1) displayed large numbers of sharp peaks that yielded broad and overlapped bands that indicates the rich chemical components of carbohydrates, proteins and lipids. Interestingly, it could be observed that the spectra of ssB\u0026thinsp;+\u0026thinsp;GMS (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC) and ssB\u0026thinsp;+\u0026thinsp;SSL (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD) showed higher proportion of signals in comparison to ssBW (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). According to Stehfest \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/sup\u003e, some IR absorption peaks within this region have also been attributed to the presence of protein molecules that contain amide (I) and amide (II). This present work also agrees with the earlier work of Hemmalakshmi \u003cem\u003eet al\u003c/em\u003e. \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/sup\u003e that characterized the IR spectra which peaks between 1500\u0026thinsp;\u0026minus;\u0026thinsp;600 to reveal the presence of aromatic rings (C\u0026thinsp;=\u0026thinsp;C-C) and amide group C-N which are identified as the fingerprint region.\u003c/p\u003e\n\u003cp\u003eThe presence of low-molecular weight carbohydrates and monosaccharides are also indicated in this region \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/sup\u003e. The present result of higher absorption band as revealed in ssB\u0026thinsp;+\u0026thinsp;GMS and ssB\u0026thinsp;+\u0026thinsp;SSL could be attributed to the contributory role of emulsifiers which is in line with the work of Chung and Tsen \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e to increase the interface activity between water and the non-aqueous phases of legume. Nevertheless, there are some buried absorption bands in GF biscuits thus revealing a slightly different features from control biscuit (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003eThis present study is hereby revealing the presence of various functional molecules such as carboxyl, carbonyl, hydroxyl, amide and nitrile groups to be the major constituents of polyphenols in the understudied legume which do not exist alone but are mostly conjugated with one or more sugar moieties. Earlier and recent research interest in phenolic compounds has been elated for their potential health benefits. According to Shodehinde and Oboh \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, the presence of one or more hydroxyl groups and conjugation of side chains to the aromatic rings in phenolic compounds is an indication of antioxidant potentials with inhibitory capacity against free radical generation to maintain the stability of a compound.\u003c/p\u003e\n\u003cp\u003eEarlier work of Diblan \u003cem\u003eet al\u003c/em\u003e. \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/sup\u003e has characterized and differentiated different legumes extracts by different solvents using FTIR spectra to correlate their total phenolic contents. Of note is the fact that the present GF biscuits revealed almost identical FTIR spectra. The similarities in the IR spectroscopy values reported for the GF biscuits follow the phenomenon that is in line with the work of Kemsley \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e]\u003c/sup\u003e which states that similar active components of close IR absorbance values can be recorded in species of the same genus. Nevertheless, the emulsifiers (GMS and SSL) were observed to show no negative influence on the IR readings in their respective incorporation in GF biscuits. The produced GF biscuits are phenolic- rich hence, can be considered as functional foods.\u003c/p\u003e\n\u003cp\u003eThe phenolic acids are compounds with powerful actions of antioxidant that are derived from natural sources. Under experimental conditions, the evaluation of total phenolic content in plants is always linked majorly to their electron donation and metal ion scavenging ability \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e], [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. The evaluated total phenol contents of control and GF biscuits are presented in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. There was significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the result values which ranged from 3.35\u0026ndash;6.59 (mg/g GAE). There was no significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the values reported for the GF biscuits. However, control biscuit had lowest value of total phenol content (3.35 mg/g GAE). This result however is in line with the experimental work of Walaa \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e]\u003c/sup\u003e in which legume product displayed higher phenolic content than wheat product.\u003c/p\u003e\n\u003cp\u003eDPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging ability (RSA) is one of the methods adopted to carry out an investigation on the antioxidant property of natural products. The resultant effect is measured by the ability to reduce the purple colour of DPPH (a stable diamagnetic molecule) to almost yellow colour at its maximum optical density (OD) 515 nm. There was significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the recorded value of DPPH radical scavenging ability of the biscuits which ranged from 20.54% \u0026minus;\u0026thinsp;59.62% (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The result also showed that the control biscuit had the least value of DPPH radical scavenging ability (20.54%) while there was no significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in ssB\u0026thinsp;+\u0026thinsp;GMS and ssB\u0026thinsp;+\u0026thinsp;SSL DPPH radical scavenging ability values. Again, this finding agrees with the work of Walaa \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e]\u003c/sup\u003e in which higher DPPH RSA was reported in legume with the higher total phenol content.\u003c/p\u003e\n\u003cp\u003eSDS-PAGE is a valuable simple and informative method that is widely used for the separation of proteins according to their molecular weight and is compared to molecular weight markers used \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. The characterization of biological objects thus reveals information of protein constituents. The result of SDS-PAGE protein profile patterns of control and GF biscuits in comparison to the molecular weight markers is revealed Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. The GF biscuits displayed the same clear protein regions which were more intense while the band for the control biscuit was hardly visible to the eyes. The intensity of the bands in GF biscuits is an indication of protein abundance as evident in the percentage of protein (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). This result also agrees with the fact that higher percentage of protein is profound in legumes than cereals \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eGluten content determination was carried out on GF biscuit for validation through the use of ELISA method (Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Cocktail method was adopted as it had also been used by Spaenij-Dekking \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/sup\u003e and it\u0026rsquo;s expressed in ppm. With differed significance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), the gluten content in the control biscuit was 75.20 ppm while the preferred GF biscuit with the most acceptable sensory attributes in this present study (ssB\u0026thinsp;+\u0026thinsp;GMS) had 2.01 ppm gluten content. The detected quantity of gluten in the GF biscuit was less than 20 ppm and is considered to be within the permissible limits. The amount of detected gluten content in the validated ssB\u0026thinsp;+\u0026thinsp;GMS biscuit might have occurred as a result of contamination during processing.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e is an economical crop with high nutritional values reported to its credit. Various analyses have been carried out to substantiate its possible use as supplement in wheat flour but with poor sensory attributes which were improved with the inclusion of emulsifiers to produce GF biscuits of satisfactory attributes that will compete with wheat flour biscuit. The outcome of these findings however suggests that the utilization of African yam bean as an underutilized crop can open ways for further diversification to the production of baked GF diets with higher nutritional properties.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Authors are grateful Tertiary Education Trust fund (TETFund), Nigeria, Government of India and Director, CSIR-CFTRI, for extending facilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSAS designed the project and was involved in the bench work, KS was involved in the laboratory works, PP and DI supervised the project and did the internal review of the manuscript. All authors have read and approved the submission of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by DBT-TWAS (Award number: 3240300000).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone declared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor(s) details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Biochemistry, Faculty of Science, Adekunle Ajasin University, Akungba Akoko. Nigeria. \u003csup\u003e2\u003c/sup\u003eFlour Milling, Baking and Confectionery Technology Department, CSIR-Central Food Technological Research Institute, Mysore 570 020, India. \u0026nbsp; \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePinto-Sanchez MI, Verdu EF (2018) Non-celiac gluten or wheat sensitivity: It\u0026rsquo;s complicated. Neurogastroenterol Motil 30:13392\u003c/li\u003e\n\u003cli\u003eSciurti M, Fornaroli F, Gaiani F, Bonaguri C, Leandro G, Di Mario F, De\u0026apos; Angelis GL (2018) Genetic susceptibility and celiac disease: what role do HLA haplotypes play? Acta Bio-Med 89(9-S):17-21\u003c/li\u003e\n\u003cli\u003eKelly CP; Bai JC; Liu E, Leffler DA (2015) Advances in diagnosis and management of celiac disease. 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Food Health\u003cem\u003e \u003c/em\u003e4(2):80-88\u003c/li\u003e\n\u003cli\u003eKemsley EK, Belton PS, McCann MC, Ttofis S, Wilson R.H, Delgadillo I (1994). Spectroscopic method for the authentication of vegetable matter. Food Control. 5:241\u0026ndash;243\u003c/li\u003e\n\u003cli\u003eWalaa IAM, Ayman FK, Mosa ZM (2015) Phenolic compounds and antioxidants capacity of sweet lupine derivatives-wheat Flour mixtures and the effects on diabetic rats. IOSR J Environ Sci Toxicol Food Technol\u003cem\u003e \u003c/em\u003e9(5):61-69\u003c/li\u003e\n\u003cli\u003eSpaenij-Dekking L, Kooy Winkelaar Y, Van Veelen P, Drijfhout JW, Jonker H, Soest V (2005) Natural variation in toxicity of wheat: potential for selection of non-toxic varieties for celiac patients. Gastroenterology 129(3):797\u0026ndash;806\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 3 and 4","content":"\u003cp\u003eTable 3 and 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Sphenostylis stenocarpa, Gluten, fatty acids, antioxidant, sensory analysis, biscuit","lastPublishedDoi":"10.21203/rs.3.rs-6407277/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6407277/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground and Objective\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study of 100% \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean (ssB) flour with incorporation of glyceryl mono stearate (GMS), sodium stearoyl-2-lactylate (SSL) and without additive (W) for the production of Gluten Free (GF) biscuits in comparison to wheat flour biscuit as control was carried out.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods\u003c/strong\u003e: The functional properties of flours, texture, proximate analysis, physico-sensory, biofunctional activities [total phenol content and DPPH (1,1-diphenyl-2-picryl-hydrazyl-hydrate) radical scavenging ability], fatty acid methyl esters (FAME) analysis, fourier transform infrared spectrometry (FTIR), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme linked immunosorbent assay (ELISA) validation of the gluten content were determined in the produced biscuits.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Among the composite blends, ssB + GMS had the highest functional properties. The obtained values for sensory evaluation, proximate analysis and hardness in ssB + GMS biscuit were also closer to that of control biscuit. The control biscuit showed lower values of total phenol content and DPPH radical scavenging ability compared to the GF biscuits. However, there was no significant difference (p \u0026lt; 0.05) in the values observed in ssB + GMS and ssB + SSL biscuits. The FAME and FTIR analyses revealed the presence of omega-3, omega-6, omega-9 and similar IR spectra in all the GF biscuits. SDS-PAGE showed a more distinct similar band in GF biscuits in comparison to control biscuit that was not visible to the eye. ELISA evaluation confirmed \u0026lt; 20 ppm content of gluten present in the GF biscuit with the highest sensory score (ssB + GMS).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: Thus, it can be concluded that \u003cem\u003eSphenostylis stenocarpa\u003c/em\u003e bean flour can be utilized 100% to produce satisfactory and nutritious gluten free biscuits.\u003c/p\u003e","manuscriptTitle":"Evaluation and Comparison of Sphenostylis stenocarpa Bean and Wheat Flours: Functional attributes, Antioxidant property, FAME analysis, FTIR, SDS-PAGE and ELISA Validation of the Gluten Content Produced Biscuits","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 05:41:50","doi":"10.21203/rs.3.rs-6407277/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bff520b0-f3c0-4baa-b79b-e51560942359","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-10T09:38:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-10 05:41:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6407277","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6407277","identity":"rs-6407277","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}