Evaluation of Surface Characterization and Qualitative Parameters of Cottonseed Meal and Extracted Protein

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Kadam, Sayali S. Parab, Akansha Kasara This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4747495/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 The utilization of cottonseed meal and its extracted protein powder as valuable industrial products necessitates leveraging. The surface characteristics and qualitative parameters were evaluated by using FTIR, SEM, color and protein digestibility, lysine content, and microbial analysis respectively. CSPC and MW-CSPI had parallel β plated sheets and an accumulation of tiny particles due to soluble and insoluble pentosans, with changes resulting from the alkali salt protein extraction process. CSPC had superior in-vitro protein digestibility and microbiological analysis showed safe limits for bacterial and pathogenic bacterial count. Cottonseed protein, whether untreated or microwave pre-treated, can be used as an ingredient or supplement in various foods, except for lysine content as specified in Food Safety and Standards Regulations of 2011. Cottonseed protein FTIR SEM Protein Digestibility Lysine Content Figures Figure 1 Figure 2 Introduction Presently, a reliable supply of plant protein is currently more in need than ever. Cottonseed meal (CSM) is a highly neglected protein fountain, with 40–45% protein and a balanced profile of essential amino acids. The application of cottonseed as a protein-rich source in the majority of the underprivileged countries will prove to be a significant breakthrough in eliminating the widespread issue of malnutrition [ 1 ]. Based on recent studies, cottonseed meal (CSM) can be utilized as a promising protein resource to execute industrial ultimatum for a substandard raw material substitute to much more costly protein sources. However, the existence of anti-nutrients in the diet can have numerous contradicting impacts, including lethal results on animal advancement and reproductive livestock strength. If the gossypol content is reduced in CSM by using microwave aided treatment, safe consumption can be achieved [ 2 ]. Furthermore, dietary anti-nutritional variables can affect protein digestibility, amino acid bioavailability, and food protein quality. Recent research on the global diet's IAA balance revealed that impoverished, developing nations had low average lysine consumption. This suggests a significant danger of insufficient intake, particularly in susceptible categories like children and pregnant or nursing women. Protein supply quality is evaluated based on ability of protein to be digested, amino acid bio accessibility, IAA quantities and percentage and insignificant nitrogen. Availability varies by region/country based on balance sheets, surveys, and diet composition [ 3 ]. Mixed diets in underdeveloped nations have significantly lower protein digestibility and quality than those in developed nations [ 3 , 4 ]. Digestion affects the release of active molecules with higher or lower bioactivity than raw materials. Protein digestibility predicts protein availability during intestinal absorption [ 5 ]. More free amino acids are generated and oligopeptides have lower molecular weights (MW) as more peptide bonds are hydrolyzed [ 6 ]. The nutritional value of a protein is determined by the type and quantity of amino acids present within it. The body absorbs amino acids when protein breaks down into small peptides and free amino acids [ 7 ]. Heating and storing certain foods can alter proteins, reducing their nutritional value. This happens when sugars and lysine undergo a reaction called the Maillard reaction, found in dairy products, eggs, and cereals [ 8 ]. Lysine is changed into the physiologically inaccessible fructose or lactulose lysine (in dairy products) during the "early" Maillard reaction. Amino acids bond to form complex protein structures [ 9 ]. Proteins, which are the "second part of the genetic code" in living organisms, play essential roles in controlling metabolism, communication, transport, and structural integrity. To truly understand proteins, we need to learn the connection within their unique formation and functional characteristics. In addition to their chemical properties, proteins' functional features depend on a variety of structural traits [ 10 , 11 , 12 ]. FTIR spectroscopy is a quick and efficient method of identifying molecular composition by allocating all absorbance band to a particular functional category. It is an important aid for examine the biochemical features of biological variety, including proteins, cellular components, and tissues [ 13 ]. In SEM, the appearance is created accurately moving an intent electron beam over a solid specimen's surface [ 14 ]. Through secondary electron signal imaging, the sample surface morphology is examined using SEM. The study aimed to analyze the surface properties of CSM, MW-CSM, CSPC, and MW-CSPI. It also evaluated the quality of treated and untreated meals, and different protein samples. Material and Methods Untreated cottonseed meal, microwave pre-treated meal, and their extracted proteins were done using as per Kadam et al. [ 15 ]. Detailed process may be referenced as given in Kadam et al. published elsewhere [ 15 ]. Characterization of Protein Fourier transform infrared spectroscopy (FTIR) Fourier Transform Infrared Spectroscopy analyzed CSPC and MWCSPI with a Shimadzu IR Prestige-21® spectrophotometer equipped with diamond ATR attachment. The samples spectra averaged 49 scans from 4000 to 400 cm − 1 , with a resolution of 2 cm − 1 . Scanning electron microscopy (SEM) SEM (Philips XL30 SEM, Netherlands) was used to check the surface analysis of CSPC and MW-CSPI samples at 10 kV accelerating voltage and 500X magnification. The samples were coated with a gold-palladium combination, mounted on a holder, to improve conductivity and visibility under SEM. Color The computerized color matching system (X-rite color iMatch) was used to determine the color profile of CSPC and MW-CSPI. The values for lightness (L*), redness/greenness (a*), yellowness/blueness (b*), brightness/dullness (C*), and hue (h*) were used to calculate the total color difference (ΔE) of CSPC and MW-CSPI using Eq. ( 1 ). $$\:\varDelta\:E=\sqrt{\left({\left({L}^{*}-{L}_{0}\right)}^{2}+{\left({a}^{*}-{a}_{0}\right)}^{2}+{\left({b}^{*}-{b}_{0}\right)}^{2}\right)}$$ 1 Where \(\:{L}_{0}\) , \(\:{a}_{0}\) and \(\:{b}_{0}\) represents the white standard plate: \(\:\:{L}^{*}\) , \(\:{a}^{*}\) and \(\:{b}^{*}\) represents the sample. Qualitative analysis of protein Lysine Content The Galicia et al. method was used to examine the lysine content of CSM, MW-CSM, CSPC, and MW-CSPI. Papain (12.5 units/mg) was used to break down 100mg of lyophilized material in 5ml of water for 16 hours at 64°C [ 16 ]. After centrifugation at 2500 rpm for 5 minutes, a mixture was created by vortexing 1 ml of the solution with 0.5 ml of 0.05 M carbonates buffer (pH 9) and 0.5 ml of copper-phosphate suspension. The mixture was centrifuged at 2000 rpm for 5 min. Then, 1 ml of supernatant was combined with 0.1 ml of 2-chloro-3, 5 dinitropyridine reagent and incubated at room temperature for 2 hours with occasional agitation every 30 minutes. After that, the tubes were filled with 5 ml of 1.2 N HCl and 5ml of ethyl acetate, and vortexed it. The tubes were covered and gently inverted ten times (twice) to mix the contents. Finally, a micropipette was used to separate the top phase, and the lysine content was calculated based on the residual solution's reading (absorbance) at 390 nm. $$\:\text{L}\text{y}\text{s}\text{i}\text{n}\text{e}\:\text{C}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t}\:\left(\text{\%}\right)=\:\frac{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\times\:\text{H}\text{y}\text{d}\text{r}\text{o}\text{l}\text{y}\text{s}\text{i}\text{s}\:\text{v}\text{o}\text{l}\text{u}\text{m}\text{e}}{\text{S}\text{l}\text{o}\text{p}\text{e}\:\times\:\text{S}\text{a}\text{m}\text{p}\text{l}\text{e}\:\text{W}\text{e}\text{i}\text{g}\text{h}\text{t}\:\left(\text{m}\text{g}\right)}\times\:100$$ 2 In vitro protein digestibility (IVPD) assay The method was developed by Akeson & Stahmann used to test in vitro protein digestibility of CSPC and MW-CSPI with modifications [ 17 ]. 15 ml of 0.1M HCL containing 1.5 mg/ml pepsin was used to suspend 250 mg of each sample and incubated for 3 hours at 37°C. Pepsin hydrolysis was stopped using 7.5 ml of 0.5 M NaOH. Pancreatic digestion was initiated by adding 10 ml of 0.2 M phosphate buffer (pH 8), 10 mg of pancreatin, and 1 ml of 0.005 M sodium azide. The mixture was kept at 37°C for the entire night. After pancreatic hydrolysis, 1 mL of 10 g/100 mL trichloroacetic acid was added. The solution was then centrifuged at 500g for 20 minutes. Sample was duplicated and supernatant collected. Total protein content estimated using Kjeldahl AOAC method based on nitrogen content. Microbial analysis CSM, MW-CSM, CSPC, and MW-CSPI (10 g) powders were blended with distilled water (90 ml) and agitated for 30 minutes in an Erlenmeyer flask. The suspension was infused in 9 ml water blanks until the infusion factor was 10 − 5 . 100 mg of each dilution were plated in duplicates on nutritional agar, MacConkey's agar, and modified brilliant green agar base to enumerate bacteria. Nutrient agar and MacConkey, Brilliant green agar plates were cultured for two days at 30°C and 37°C, respectively. Colonies were noted and counted according to He et al . [ 18 ]. Statistical analysis The ANOVA was performed on duplicated data to determine significant differences between samples (p < 0.05). Results and Discussion Fourier transform infrared spectroscopy (FTIR) ATR-FTIR Spectroscopy is a method used to obtain detailed information on protein secondary structure in both solidified and liquid forms by examining how infrared light interacts with matter. The sample absorbs infrared light rays according to the molecular vibration of the matter, with several vibrational bands arising from different functional groups like carbonyl group, amide group etc [ 19 ]. Protein concentrates demonstrate five characteristic bands, three of which are amide bands, with the Amide I band in 1600–1700 cm − 1 region strongly absorbing infrared light due to amide C = O stretching vibrations. The Amide I band in CSPC and MW-CSPI absorbs the most infrared light at 1637.56 and 1627.92 cm − 1 respectively, indicating different secondary configurations of protein structures (Fig. 1 ). These structures configurations of protein such as α-helix, β plated sheet–parallel and antiparallel, β-turn and unordered expose distinctive frequencies and intensities due to variations in hydrogen bonding [ 19 , 20 , 21 ]. The sharpest peak is obtained at 1637.56 cm − 1 and 1627.92 cm − 1 for CSPC and MW-CSPI showing a secondary structure mainly composed of parallel β plated sheets [ 22 , 23 ]. Other peak values at 1637.56 cm − 1 for CSPC and 1627.92 cm − 1 for MW-CSPI attributed to intermolecular β sheet structure [ 23 ]. The absence of the Amide VII band is shown in the absence of 476.42 cm − 1 in CSPC. The peak values obtained closely match the secondary structure of cotton seed protein isolate, as concluded by Ma et al. and Kumar et al. [ 1 , 24 ]. Similarities were found in the FTIR curves of soybean protein and cottonseed protein (1, 10, 24]. Scanning electron microscopy (SEM) The surface characteristics of CSPC and MW-CSPI were analyzed by SEM. CSPC had wrinkled surfaces with feather and permeable structures, likely due to insoluble polysaccharide components such as lignin, cellulose, hemicellulose and fibers present after protein extraction (Fig. 2 ) [ 25 , 26 ]. Protein particles had small flat areas with a wide range of sizes and shapes. Both CSPC and MW-CSPI showed a spongy, light structure with many pores, and appeared to be more or less spherical but with very uneven surfaces. Several studies indicate that CSPC and MW-CSPC residues have similar structures to isolated wheat protein and arabinoxylans (hemicelluloses), respectively [ 27 ]. CSPC and MW-CSPI showed spongy structures with many pores, formed by agglomerations of small elements. The microwaved cottonseed protein (MW-CSPI) particles have crystalline features with tight surfaces, likely due to the loosening of protein microstructure and fragmentation and formation of cavities resulting from microwave treatment, which may involve the reduction of amide residue proteins and the conversion of the amide group into the carboxyl group (Fig. 2 ) [ 26 ]. The changes in microstructure are caused by the eradication method and absorption of alkali [ 1 , 10 , 26 ]. Color profile Color quality is crucial for food acceptability and is a key factor. CSPC (L* = 65.35, a*= 5.13, b*= 19.08) and MW-CSPI (L* = 64.24, a*= 6.77, b*= 23.22) were lighter in color than sunflower protein isolates obtained by gamma irradiation (L* = 57.93 ± 61.2, a*= 1.8 ± 2.4, b*= 11.1 ± 12.8) [ 28 ], ultrasound-assisted extraction of sunflower protein isolates (L* = 48.08, a*= 1.36, b*= 5.53) [ 29 ], soybean protein isolates made from extrusion with varying screw speeds (L* =51.06–53.46, a*= − 4.02 - -5.16, b*= 11.08–13.25) [ 30 ] and slightly darker than bean protein isolate gel (L* =73.55–75.78, a*= 0.83–0.04, b*= 12.95 − 15.25) [ 31 ]. CSPC and MW-CSPI showed no significant color differences. No remarkable changes in color were observed. Lysine content Lysine is an essential amino acid that plays a crucial role in maintaining proper bodily functions. The lysine content of CSM, MW-CSM, CSPC and MW-CSPI were 1.534 ± 0.05%, 1.043 ± 0.128%, 1.863 ± 0.008% and 0.560 ± 0.001%, respectively. Due to heat treatment, lysine content in MW-CSM decreased compared to CSM. The gossypol may bind by heat and moisture, causing a reduction in the available lysine to about 0.06% [ 32 ]. Various drying methods affect lysine availability, as gossypol can bond with lysine and create indigestible Maillard linkages under extreme heat, reducing its nutritional value [ 33 , 34 ]. Lysine is also sensitive to Maillard reactions. Reduced lysine availability in cottonseed meals is caused by gossypol's interaction with protein amino groups, hindering their ability to absorb lysine [ 35 ]. Therefore, the primary cause for reduced lysine availability in CSM and MW-CSM was due to higher levels of gossypol compared to CSPC and MW-CSPI. Various drying techniques also impact the availability of lysine [ 34 ]. The lysine content in CSPC and MW-CSPI was also below the established standard of 3.16 g/100 g according to FSSR, version IV [ 36 ]. Furthermore, recovering additional lysine from CSPC and MW-CSPI was difficult due to the scarcity of lysine in CSM and MW-CSM. In vitro protein digestibility (IVPD) assay The composition of an amino acid and its digestibility determine its nature of protein. The amount and ease of digestion of vital amino acids in protein are the main factors that decide its quality. In vitro protein digestibility of CSPC was 87.728 ± 0.22 whereas MW-CSPI had a protein digestibility of 84.168 ± 2.58. CSPC is more digestible than MW-CSPI, possibly due to microwave pre-heat treatment of cottonseed meal [ 37 , 38 ]. This particular treatment might cause a reduction in the availability of amino acids and the digestibility of protein. During the processing of protein, heat treatments are applied for various reasons such as sterilization or pasteurization, to improve flavour and texture, to prepare concentrated protein products, or to deactivate anti-nutritional factors [ 4 , 39 ]. However, heat treatments may lead to chemical changes in proteins, such as Maillard reactions between reducing sugars and lysine that can reduce their nutritional values and make their residues biologically unavailable [ 35 ]. Anti-nutritional factors and microwave pre-heat treatment were found to affect the lysine content and protein digestibility of basic and pre-treated meal and protein samples. Microbial analysis Microbial analysis was conducted on CSM, MW-CSM, CSPC, and MW-CSPI powders. The total bacterial count was 1425 cfu/g for CSM and 25 cfu/g for CSPC. MW-CSM and MW-CSPI had no bacterial count. No Salmonella or coliform bacteria were found in CSM, MW-CSM, CSPC and MW-CSPI powder. The total bacterial count, total coliform count, and total Salmonella bacterial count were all confirmed to be within acceptable limits as set by food control authorities for microbiological quality parameters (Table 1 ). CSPC and MW-CSPI deemed safe for food supplements with 1295 cfu/g total bacterial count and no Salmonella or coliform bacteria [ 1 ]. According to the microbiological study, no pathogenic bacteria such as Coliform and Salmonella were found. Table 1 Standards of cottonseed meal and their protein samples according to FSSR 2011, to be used as food supplement. Parameters Standards as per FSSR 2011 Quality of samples according to current study CSM MW-CSM CSPC MW-CSPI Lysine Content 3.6 g per 100 g or more of crude protein 1.534 ± 0.058 0.982 ± 0.174 1.863 ± 0.008 0.56 ± 0.001 Total Bacterial Count Less than 50,000 per g 1425 cfu/g 0 25 cfu/g 0 Coliform Bacteria Less than 10 per g Nil Nil Nil Nil Salmonella Bacteria Should be nil in 25 g Nil Nil Nil Nil Correlation between cottonseed protein structure and functionality It is essential to recognize the correlation between protein formation and functional properties to ensure top-notch food quality and flavor. Microwave pre-treatment was observed to decrease the solubility of microwaved treated cottonseed protein isolates, but it increased foaming and emulsifying properties slightly when compared to untreated cottonseed protein concentrate [ 15 ]. The expected consequence could be caused by the formation of free radicals as an outcome of kinetic energy absorption throughout microwave treatment which results in the unfolding or refolding of secondary and tertiary structures [ 40 , 41 ]. Protein structure changes can affect important functional properties in food processing [ 40 ]. Increasing solubility boosts viscosity, foaming, and emulsification per MWCSPI [ 41 ]. According to Khan et al ., microwaved-treated rice bran protein isolates had little effect on protein solubility, but exposing more hydrophobic surfaces could enhance foaming and emulsification [ 42 ]. In Ma et al . study, the solubility features of CSPC/MW-CSPI were correlated with protein solubility and diffusion of FC and FS [ 24 ]. Protein solubility was inversely proportional to emulsification due to heat treatment. Ghribi et al. found that convective drying at 40°C resulted in low solubility but higher EAI and ESI than freeze-drying and convective drying at 50°C [ 43 ]. Conclusion The study evaluated CSPC and MW-CSPI through various analytical techniques including FTIR, SEM, color analysis, protein digestibility, lysine content, and microbial analysis to assess their surface characteristics and qualitative parameters. Both CSPC and MW-CSPI exhibited parallel β plated sheets and an accumulation of tiny particles due to soluble and insoluble pentosans, with changes resulting from the alkali salt protein extraction process. CSPC demonstrated superior in-vitro protein digestibility, and the microbiological analysis revealed safe limits for bacterial and pathogenic bacterial count. Research findings suggest that microwave treatment alters the surface characteristics and protein properties of CSPI. Cottonseed can be used in food as a constituent or supplement, except for lysine content regulated by Food Safety and Standards Regulations, 2011. Abbreviations FTIR Fourier transform infrared spectroscopy SEM Scanning electron microscopic CSPC Cottonseed protein concentrate MW-CSPI Microwave pre-treated protein isolates CSM Cottonseed meal IAA Indispensable amino acids MW-CSM Microwave pre-treated cottonseed meal HCL Hydrochloric Acid NaOH Sodium Hydroxide Declarations Funding The authors express their gratitude to the Department of Science and Technology (DST), Government of India, New Delhi, for funding the project with File No: DST/TDT/Agro-06/2019 (G) & (C) Dated 05–02–2021. The authors would like to express their gratitude to the Director of ICAR-CIRCOT, Mumbai for extending infrastructural support. Declaration of competing interest The authors affirm that they have no conflicts of interest and highly recommend their work for publication in your esteemed journal. With their extensive research and dedication to the subject matter, their article is sure to be a valuable addition to your publication. Author Contribution Author 1: Funding, Planning, Experiment Design, Guiding and Monitoring, Data analysis and editing.Authors 2 and 3: Execution of experiments, data analysis, preparing draft, corrections and modifications. Acknowledgement The authors express their gratitude to the Department of Science and Technology (DST), Government of India, New Delhi, for funding the project with File No: DST/TDT/Agro-06/2019 (G) & (C) Dated 05–02–2021. The authors would like to express their gratitude to the Director of ICAR-CIRCOT, Mumbai for extending infrastructural support. Data availability All data generated or analyzed during this study is included in this published article. 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LWT, 73, 675–682. https://doi.org/10.1016/j.lwt.2016.07.011 Damodaran S., 2007. Amino acids, peptides, and proteins, In: Damodaran S., Parkin K. L., Fennema O. R. (4th Eds), Fennema’s Food Chemistry , pp. 217–330. Marcel Dekker Inc., New York. Food Safety and Standards Authority of India. Food Safety and Standards (food products standards and additives) regulation, part III section 4 (2011), 368–369. Gilani, G. S., Xiao, C. W. & Cockell, K. A. (2012). Impact of anti-nutritional factors in food proteins on the digestibility of protein and the bioavailability of amino acids and on protein quality. British Journal of Nutrition, 108 (S2), S315-S332. https://doi.org/10.1017/S0007114512002371 Olakanmi, S., Barbhuiya, R. I., Wroblewski, C., Ramalingam, S., Wang, J., Nair, G. R. & Singh, A. (2023). Effect of microwave and conventional heat treatment on trypsin inhibitor activity and in vitro digestibility of edamame milk protein. Food Bioengineering, 2, 114–126. https://doi.org/10.1002/fbe2.12050 Schwass, D. E. & Finley, J. W. (1984). Heat and alkaline damage to proteins: racemization and lysinoalanine formation. Journal of Agricultural and Food Chemistry, 32(6), 1377–1382. https://doi.org/10.1021/jf00126a040 Han, Z., Cai, M. J., Cheng, J. H. & Sun, D. W. (2018). Effects of electric fields and electromagnetic wave on food protein structure and functionality: A review. Trends in food science & technology, 75, 1–9. https://doi.org/10.1016/j.tifs.2018.02.017 Subasi, B. G., Yildirim-Elikoğlu, S., Altay, İ., Jafarpour, A., Casanova, F., Mohammadifar, M. A. & Capanoglu, E. (2022). Influence of non-thermal microwave radiation on emulsifying properties of sunflower protein. Food Chemistry, 372, 131275. https://doi.org/10.1016/j.foodchem.2021.131275 Khan, S. H., Butt, M. S., Sharif, M. K., Sameen, A., Mumtaz, S. & Sultan, M. T. (2011). Functional properties of protein isolates extracted from stabilized rice bran by microwave, dry heat, and parboiling. Journal of Agricultural and Food Chemistry, 59(6), 2416–2420. https://doi.org/10.1021/jf104177x Ghribi, A. M., Gafsi, I. M., Blecker, C., Danthine, S., Attia, H. & Besbes, S. (2015). Effect of drying methods on physico-chemical and functional properties of chickpea protein concentrates. Journal of Food Engineering, 165, 179–188. https://doi.org/10.1016/j.jfoodeng.2015.06.021 Captions of Table Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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-4747495","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":342882184,"identity":"a62dc8d3-dd17-4883-93cd-24093d8add36","order_by":0,"name":"Dattatreya M. Kadam","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYDCCA2wMDDxAxMbDfADIlZAhWosMHw9bAkgLD9FabOR4eAxAfMJa+K4dS/zwpmYb0GFnPr+6UWPBw8B++OgGfFokb6cdlpxz7DYPG2/vNuucY0CH8aSl3cCnxeB2eoM0DxtQCz/vNuMcNqAWCR4zQlqaf/P8A2nheWac848oLWnHpHnbQA7rYX6c20aEFqBf0izn9gG18BwzY87tkwAyCPiF73aa8Y03327by/ckP/6c861Ojp/98DG8WpABmwSYJFY5CDB/IEX1KBgFo2AUjBwAAGWeRcwzKwHQAAAAAElFTkSuQmCC","orcid":"","institution":"ICAR-Central Institute for Research on Cotton Technology (ICAR-CIRCOT)","correspondingAuthor":true,"prefix":"","firstName":"Dattatreya","middleName":"M.","lastName":"Kadam","suffix":""},{"id":342882185,"identity":"95dc2ebf-bfce-4d68-82fc-cb8945886579","order_by":1,"name":"Sayali S. Parab","email":"","orcid":"","institution":"ICAR-Central Institute for Research on Cotton Technology (ICAR-CIRCOT)","correspondingAuthor":false,"prefix":"","firstName":"Sayali","middleName":"S.","lastName":"Parab","suffix":""},{"id":342882186,"identity":"012f2665-d632-4667-a0bd-590c48099987","order_by":2,"name":"Akansha Kasara","email":"","orcid":"","institution":"ICAR-Central Institute for Research on Cotton Technology (ICAR-CIRCOT)","correspondingAuthor":false,"prefix":"","firstName":"Akansha","middleName":"","lastName":"Kasara","suffix":""}],"badges":[],"createdAt":"2024-07-16 06:30:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4747495/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4747495/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62951050,"identity":"e3674249-0caf-4960-94b4-fc38d7c6206c","added_by":"auto","created_at":"2024-08-21 11:20:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":14208,"visible":true,"origin":"","legend":"\u003cp\u003eFourier Transform infrared spectroscopy profile of CSPC and MW- CSPI.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4747495/v1/3244c30f433408e965ddb45a.png"},{"id":62951049,"identity":"e0cb715d-82ff-47f0-b65d-a5224edf27a4","added_by":"auto","created_at":"2024-08-21 11:20:17","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":899994,"visible":true,"origin":"","legend":"\u003cp\u003eScanning Electron Microscopic of Cottonseed meal protein concentrate (CSPC) (A) and Microwave treated cottonseed meal protein concentrate (MW- CSPI) (B).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4747495/v1/63d38baaa00351b9c05481c6.jpeg"},{"id":81608598,"identity":"3d6400eb-ba31-422a-a6c8-8c06d786c13e","added_by":"auto","created_at":"2025-04-29 06:31:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1744844,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4747495/v1/e30aca0d-00c5-48ff-8720-f002e56252b6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of Surface Characterization and Qualitative Parameters of Cottonseed Meal and Extracted Protein","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePresently, a reliable supply of plant protein is currently more in need than ever. Cottonseed meal (CSM) is a highly neglected protein fountain, with 40\u0026ndash;45% protein and a balanced profile of essential amino acids. The application of cottonseed as a protein-rich source in the majority of the underprivileged countries will prove to be a significant breakthrough in eliminating the widespread issue of malnutrition [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Based on recent studies, cottonseed meal (CSM) can be utilized as a promising protein resource to execute industrial ultimatum for a substandard raw material substitute to much more costly protein sources. However, the existence of anti-nutrients in the diet can have numerous contradicting impacts, including lethal results on animal advancement and reproductive livestock strength. If the gossypol content is reduced in CSM by using microwave aided treatment, safe consumption can be achieved [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Furthermore, dietary anti-nutritional variables can affect protein digestibility, amino acid bioavailability, and food protein quality. Recent research on the global diet's IAA balance revealed that impoverished, developing nations had low average lysine consumption. This suggests a significant danger of insufficient intake, particularly in susceptible categories like children and pregnant or nursing women.\u003c/p\u003e \u003cp\u003eProtein supply quality is evaluated based on ability of protein to be digested, amino acid bio accessibility, IAA quantities and percentage and insignificant nitrogen. Availability varies by region/country based on balance sheets, surveys, and diet composition [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Mixed diets in underdeveloped nations have significantly lower protein digestibility and quality than those in developed nations [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Digestion affects the release of active molecules with higher or lower bioactivity than raw materials. Protein digestibility predicts protein availability during intestinal absorption [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. More free amino acids are generated and oligopeptides have lower molecular weights (MW) as more peptide bonds are hydrolyzed [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The nutritional value of a protein is determined by the type and quantity of amino acids present within it. The body absorbs amino acids when protein breaks down into small peptides and free amino acids [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Heating and storing certain foods can alter proteins, reducing their nutritional value. This happens when sugars and lysine undergo a reaction called the Maillard reaction, found in dairy products, eggs, and cereals [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Lysine is changed into the physiologically inaccessible fructose or lactulose lysine (in dairy products) during the \"early\" Maillard reaction. Amino acids bond to form complex protein structures [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProteins, which are the \"second part of the genetic code\" in living organisms, play essential roles in controlling metabolism, communication, transport, and structural integrity. To truly understand proteins, we need to learn the connection within their unique formation and functional characteristics. In addition to their chemical properties, proteins' functional features depend on a variety of structural traits [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. FTIR spectroscopy is a quick and efficient method of identifying molecular composition by allocating all absorbance band to a particular functional category. It is an important aid for examine the biochemical features of biological variety, including proteins, cellular components, and tissues [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In SEM, the appearance is created accurately moving an intent electron beam over a solid specimen's surface [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Through secondary electron signal imaging, the sample surface morphology is examined using SEM. The study aimed to analyze the surface properties of CSM, MW-CSM, CSPC, and MW-CSPI. It also evaluated the quality of treated and untreated meals, and different protein samples.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003eUntreated cottonseed meal, microwave pre-treated meal, and their extracted proteins were done using as per Kadam et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Detailed process may be referenced as given in Kadam et al. published elsewhere [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eCharacterization of Protein\u003c/h3\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eFourier transform infrared spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eFourier Transform Infrared Spectroscopy analyzed CSPC and MWCSPI with a Shimadzu IR Prestige-21\u0026reg; spectrophotometer equipped with diamond ATR attachment. The samples spectra averaged 49 scans from 4000 to 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with a resolution of 2 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eScanning electron microscopy (SEM)\u003c/h2\u003e \u003cp\u003eSEM (Philips XL30 SEM, Netherlands) was used to check the surface analysis of CSPC and MW-CSPI samples at 10 kV accelerating voltage and 500X magnification. The samples were coated with a gold-palladium combination, mounted on a holder, to improve conductivity and visibility under SEM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eColor\u003c/h2\u003e \u003cp\u003eThe computerized color matching system (X-rite color iMatch) was used to determine the color profile of CSPC and MW-CSPI. The values for lightness (L*), redness/greenness (a*), yellowness/blueness (b*), brightness/dullness (C*), and hue (h*) were used to calculate the total color difference (ΔE) of CSPC and MW-CSPI using Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\varDelta\\:E=\\sqrt{\\left({\\left({L}^{*}-{L}_{0}\\right)}^{2}+{\\left({a}^{*}-{a}_{0}\\right)}^{2}+{\\left({b}^{*}-{b}_{0}\\right)}^{2}\\right)}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{L}_{0}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{a}_{0}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{b}_{0}\\)\u003c/span\u003e\u003c/span\u003e represents the white standard plate:\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:{L}^{*}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{a}^{*}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{b}^{*}\\)\u003c/span\u003e\u003c/span\u003e represents the sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eQualitative analysis of protein\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eLysine Content\u003c/h2\u003e \u003cp\u003eThe Galicia et al. method was used to examine the lysine content of CSM, MW-CSM, CSPC, and MW-CSPI. Papain (12.5 units/mg) was used to break down 100mg of lyophilized material in 5ml of water for 16 hours at 64\u0026deg;C [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. After centrifugation at 2500 rpm for 5 minutes, a mixture was created by vortexing 1 ml of the solution with 0.5 ml of 0.05 M carbonates buffer (pH 9) and 0.5 ml of copper-phosphate suspension. The mixture was centrifuged at 2000 rpm for 5 min. Then, 1 ml of supernatant was combined with 0.1 ml of 2-chloro-3, 5 dinitropyridine reagent and incubated at room temperature for 2 hours with occasional agitation every 30 minutes. After that, the tubes were filled with 5 ml of 1.2 N HCl and 5ml of ethyl acetate, and vortexed it. The tubes were covered and gently inverted ten times (twice) to mix the contents. Finally, a micropipette was used to separate the top phase, and the lysine content was calculated based on the residual solution's reading (absorbance) at 390 nm.\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:\\text{L}\\text{y}\\text{s}\\text{i}\\text{n}\\text{e}\\:\\text{C}\\text{o}\\text{n}\\text{t}\\text{e}\\text{n}\\text{t}\\:\\left(\\text{\\%}\\right)=\\:\\frac{\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\times\\:\\text{H}\\text{y}\\text{d}\\text{r}\\text{o}\\text{l}\\text{y}\\text{s}\\text{i}\\text{s}\\:\\text{v}\\text{o}\\text{l}\\text{u}\\text{m}\\text{e}}{\\text{S}\\text{l}\\text{o}\\text{p}\\text{e}\\:\\times\\:\\text{S}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}\\:\\text{W}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t}\\:\\left(\\text{m}\\text{g}\\right)}\\times\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro protein digestibility (IVPD) assay\u003c/h2\u003e \u003cp\u003eThe method was developed by Akeson \u0026amp; Stahmann used to test in vitro protein digestibility of CSPC and MW-CSPI with modifications [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. 15 ml of 0.1M HCL containing 1.5 mg/ml pepsin was used to suspend 250 mg of each sample and incubated for 3 hours at 37\u0026deg;C. Pepsin hydrolysis was stopped using 7.5 ml of 0.5 M NaOH. Pancreatic digestion was initiated by adding 10 ml of 0.2 M phosphate buffer (pH 8), 10 mg of pancreatin, and 1 ml of 0.005 M sodium azide. The mixture was kept at 37\u0026deg;C for the entire night. After pancreatic hydrolysis, 1 mL of 10 g/100 mL trichloroacetic acid was added. The solution was then centrifuged at 500g for 20 minutes. Sample was duplicated and supernatant collected. Total protein content estimated using Kjeldahl AOAC method based on nitrogen content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMicrobial analysis\u003c/h2\u003e \u003cp\u003eCSM, MW-CSM, CSPC, and MW-CSPI (10 g) powders were blended with distilled water (90 ml) and agitated for 30 minutes in an Erlenmeyer flask. The suspension was infused in 9 ml water blanks until the infusion factor was 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e. 100 mg of each dilution were plated in duplicates on nutritional agar, MacConkey's agar, and modified brilliant green agar base to enumerate bacteria. Nutrient agar and MacConkey, Brilliant green agar plates were cultured for two days at 30\u0026deg;C and 37\u0026deg;C, respectively. Colonies were noted and counted according to He \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe ANOVA was performed on duplicated data to determine significant differences between samples (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFourier transform infrared spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eATR-FTIR Spectroscopy is a method used to obtain detailed information on protein secondary structure in both solidified and liquid forms by examining how infrared light interacts with matter. The sample absorbs infrared light rays according to the molecular vibration of the matter, with several vibrational bands arising from different functional groups like carbonyl group, amide group etc [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Protein concentrates demonstrate five characteristic bands, three of which are amide bands, with the Amide I band in 1600\u0026ndash;1700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e region strongly absorbing infrared light due to amide C\u0026thinsp;=\u0026thinsp;O stretching vibrations. The Amide I band in CSPC and MW-CSPI absorbs the most infrared light at 1637.56 and 1627.92 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e respectively, indicating different secondary configurations of protein structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese structures configurations of protein such as α-helix, β plated sheet\u0026ndash;parallel and antiparallel, β-turn and unordered expose distinctive frequencies and intensities due to variations in hydrogen bonding [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The sharpest peak is obtained at 1637.56 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1627.92 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for CSPC and MW-CSPI showing a secondary structure mainly composed of parallel β plated sheets [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Other peak values at 1637.56 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for CSPC and 1627.92 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for MW-CSPI attributed to intermolecular β sheet structure [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The absence of the Amide VII band is shown in the absence of 476.42 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in CSPC. The peak values obtained closely match the secondary structure of cotton seed protein isolate, as concluded by Ma et al. and Kumar et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Similarities were found in the FTIR curves of soybean protein and cottonseed protein (1, 10, 24].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eScanning electron microscopy (SEM)\u003c/h2\u003e \u003cp\u003eThe surface characteristics of CSPC and MW-CSPI were analyzed by SEM. CSPC had wrinkled surfaces with feather and permeable structures, likely due to insoluble polysaccharide components such as lignin, cellulose, hemicellulose and fibers present after protein extraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Protein particles had small flat areas with a wide range of sizes and shapes. Both CSPC and MW-CSPI showed a spongy, light structure with many pores, and appeared to be more or less spherical but with very uneven surfaces. Several studies indicate that CSPC and MW-CSPC residues have similar structures to isolated wheat protein and arabinoxylans (hemicelluloses), respectively [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCSPC and MW-CSPI showed spongy structures with many pores, formed by agglomerations of small elements. The microwaved cottonseed protein (MW-CSPI) particles have crystalline features with tight surfaces, likely due to the loosening of protein microstructure and fragmentation and formation of cavities resulting from microwave treatment, which may involve the reduction of amide residue proteins and the conversion of the amide group into the carboxyl group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The changes in microstructure are caused by the eradication method and absorption of alkali [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eColor profile\u003c/h2\u003e \u003cp\u003eColor quality is crucial for food acceptability and is a key factor. CSPC (L* = 65.35, a*= 5.13, b*= 19.08) and MW-CSPI (L* = 64.24, a*= 6.77, b*= 23.22) were lighter in color than sunflower protein isolates obtained by gamma irradiation (L* = 57.93\u0026thinsp;\u0026plusmn;\u0026thinsp;61.2, a*= 1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4, b*= 11.1\u0026thinsp;\u0026plusmn;\u0026thinsp;12.8) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], ultrasound-assisted extraction of sunflower protein isolates (L* = 48.08, a*= 1.36, b*= 5.53) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], soybean protein isolates made from extrusion with varying screw speeds (L* =51.06\u0026ndash;53.46, a*= \u0026minus;\u0026thinsp;4.02 - -5.16, b*= 11.08\u0026ndash;13.25) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and slightly darker than bean protein isolate gel (L* =73.55\u0026ndash;75.78, a*= 0.83\u0026ndash;0.04, b*= 12.95 \u0026minus;\u0026thinsp;15.25) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. CSPC and MW-CSPI showed no significant color differences. No remarkable changes in color were observed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLysine content\u003c/h2\u003e \u003cp\u003eLysine is an essential amino acid that plays a crucial role in maintaining proper bodily functions. The lysine content of CSM, MW-CSM, CSPC and MW-CSPI were 1.534\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05%, 1.043\u0026thinsp;\u0026plusmn;\u0026thinsp;0.128%, 1.863\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008% and 0.560\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001%, respectively. Due to heat treatment, lysine content in MW-CSM decreased compared to CSM. The gossypol may bind by heat and moisture, causing a reduction in the available lysine to about 0.06% [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Various drying methods affect lysine availability, as gossypol can bond with lysine and create indigestible Maillard linkages under extreme heat, reducing its nutritional value [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Lysine is also sensitive to Maillard reactions.\u003c/p\u003e \u003cp\u003eReduced lysine availability in cottonseed meals is caused by gossypol's interaction with protein amino groups, hindering their ability to absorb lysine [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Therefore, the primary cause for reduced lysine availability in CSM and MW-CSM was due to higher levels of gossypol compared to CSPC and MW-CSPI. Various drying techniques also impact the availability of lysine [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The lysine content in CSPC and MW-CSPI was also below the established standard of 3.16 g/100 g according to FSSR, version IV [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Furthermore, recovering additional lysine from CSPC and MW-CSPI was difficult due to the scarcity of lysine in CSM and MW-CSM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro protein digestibility (IVPD) assay\u003c/h2\u003e \u003cp\u003eThe composition of an amino acid and its digestibility determine its nature of protein. The amount and ease of digestion of vital amino acids in protein are the main factors that decide its quality. In vitro protein digestibility of CSPC was 87.728\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 whereas MW-CSPI had a protein digestibility of 84.168\u0026thinsp;\u0026plusmn;\u0026thinsp;2.58. CSPC is more digestible than MW-CSPI, possibly due to microwave pre-heat treatment of cottonseed meal [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This particular treatment might cause a reduction in the availability of amino acids and the digestibility of protein.\u003c/p\u003e \u003cp\u003eDuring the processing of protein, heat treatments are applied for various reasons such as sterilization or pasteurization, to improve flavour and texture, to prepare concentrated protein products, or to deactivate anti-nutritional factors [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, heat treatments may lead to chemical changes in proteins, such as Maillard reactions between reducing sugars and lysine that can reduce their nutritional values and make their residues biologically unavailable [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Anti-nutritional factors and microwave pre-heat treatment were found to affect the lysine content and protein digestibility of basic and pre-treated meal and protein samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMicrobial analysis\u003c/h2\u003e \u003cp\u003eMicrobial analysis was conducted on CSM, MW-CSM, CSPC, and MW-CSPI powders. The total bacterial count was 1425 cfu/g for CSM and 25 cfu/g for CSPC. MW-CSM and MW-CSPI had no bacterial count. No Salmonella or coliform bacteria were found in CSM, MW-CSM, CSPC and MW-CSPI powder. The total bacterial count, total coliform count, and total Salmonella bacterial count were all confirmed to be within acceptable limits as set by food control authorities for microbiological quality parameters (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). CSPC and MW-CSPI deemed safe for food supplements with 1295 cfu/g total bacterial count and no Salmonella or coliform bacteria [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. According to the microbiological study, no pathogenic bacteria such as Coliform and Salmonella were found.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStandards of cottonseed meal and their protein samples according to FSSR 2011, to be used as food supplement.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStandards as per FSSR 2011\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003eQuality of samples according to current study\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCSM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMW-CSM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCSPC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMW-CSPI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLysine Content\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.6 g per 100 g or more of crude protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.534\u0026thinsp;\u0026plusmn;\u0026thinsp;0.058\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.982\u0026thinsp;\u0026plusmn;\u0026thinsp;0.174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.863\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Bacterial Count\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLess than 50,000 per g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1425 cfu/g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25 cfu/g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColiform Bacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLess than 10 per g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalmonella Bacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShould be nil in 25 g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation between cottonseed protein structure and functionality\u003c/h2\u003e \u003cp\u003eIt is essential to recognize the correlation between protein formation and functional properties to ensure top-notch food quality and flavor. Microwave pre-treatment was observed to decrease the solubility of microwaved treated cottonseed protein isolates, but it increased foaming and emulsifying properties slightly when compared to untreated cottonseed protein concentrate [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The expected consequence could be caused by the formation of free radicals as an outcome of kinetic energy absorption throughout microwave treatment which results in the unfolding or refolding of secondary and tertiary structures [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Protein structure changes can affect important functional properties in food processing [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIncreasing solubility boosts viscosity, foaming, and emulsification per MWCSPI [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. According to Khan \u003cem\u003eet al\u003c/em\u003e., microwaved-treated rice bran protein isolates had little effect on protein solubility, but exposing more hydrophobic surfaces could enhance foaming and emulsification [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In Ma \u003cem\u003eet al\u003c/em\u003e. study, the solubility features of CSPC/MW-CSPI were correlated with protein solubility and diffusion of FC and FS [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Protein solubility was inversely proportional to emulsification due to heat treatment. Ghribi et al. found that convective drying at 40\u0026deg;C resulted in low solubility but higher EAI and ESI than freeze-drying and convective drying at 50\u0026deg;C [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe study evaluated CSPC and MW-CSPI through various analytical techniques including FTIR, SEM, color analysis, protein digestibility, lysine content, and microbial analysis to assess their surface characteristics and qualitative parameters. Both CSPC and MW-CSPI exhibited parallel β plated sheets and an accumulation of tiny particles due to soluble and insoluble pentosans, with changes resulting from the alkali salt protein extraction process. CSPC demonstrated superior in-vitro protein digestibility, and the microbiological analysis revealed safe limits for bacterial and pathogenic bacterial count. Research findings suggest that microwave treatment alters the surface characteristics and protein properties of CSPI. Cottonseed can be used in food as a constituent or supplement, except for lysine content regulated by Food Safety and Standards Regulations, 2011.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.216216216216218%\" valign=\"top\" style=\"width: 16.9951%;\"\u003e\n \u003cp\u003eFTIR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.27927927927928%\" valign=\"top\" style=\"width: 83.0049%;\"\u003e\n \u003cp\u003eFourier transform infrared spectroscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.216216216216218%\" valign=\"top\" style=\"width: 16.9951%;\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.27927927927928%\" valign=\"top\" style=\"width: 83.0049%;\"\u003e\n \u003cp\u003eScanning electron microscopic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.216216216216218%\" valign=\"top\" style=\"width: 16.9951%;\"\u003e\n \u003cp\u003eCSPC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.27927927927928%\" valign=\"top\" style=\"width: 83.0049%;\"\u003e\n \u003cp\u003eCottonseed protein concentrate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.216216216216218%\" valign=\"top\" style=\"width: 16.9951%;\"\u003e\n \u003cp\u003eMW-CSPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.27927927927928%\" valign=\"top\" style=\"width: 83.0049%;\"\u003e\n \u003cp\u003eMicrowave pre-treated protein isolates\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.216216216216218%\" valign=\"top\" style=\"width: 16.9951%;\"\u003e\n \u003cp\u003eCSM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.27927927927928%\" valign=\"top\" style=\"width: 83.0049%;\"\u003e\n \u003cp\u003eCottonseed meal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.216216216216218%\" valign=\"top\" style=\"width: 16.9951%;\"\u003e\n \u003cp\u003eIAA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.27927927927928%\" valign=\"top\" style=\"width: 83.0049%;\"\u003e\n \u003cp\u003eIndispensable amino acids\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.216216216216218%\" valign=\"top\" style=\"width: 16.9951%;\"\u003e\n \u003cp\u003eMW-CSM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.27927927927928%\" valign=\"top\" style=\"width: 83.0049%;\"\u003e\n \u003cp\u003eMicrowave pre-treated cottonseed meal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.216216216216218%\" valign=\"top\" style=\"width: 16.9951%;\"\u003e\n \u003cp\u003eHCL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.27927927927928%\" valign=\"top\" style=\"width: 83.0049%;\"\u003e\n \u003cp\u003eHydrochloric Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.216216216216218%\" valign=\"top\" style=\"width: 16.9951%;\"\u003e\n \u003cp\u003eNaOH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"79.27927927927928%\" valign=\"top\" style=\"width: 83.0049%;\"\u003e\n \u003cp\u003eSodium Hydroxide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors express their gratitude to the Department of Science and Technology (DST), Government of India, New Delhi, for funding the project with File No: DST/TDT/Agro-06/2019 (G) \u0026amp; (C) Dated 05\u0026ndash;02\u0026ndash;2021. The authors would like to express their gratitude to the Director of ICAR-CIRCOT, Mumbai for extending infrastructural support.\u003c/p\u003e \u003ch2\u003eDeclaration of competing interest\u003c/strong\u003e \u003cp\u003eThe authors affirm that they have no conflicts of interest and highly recommend their work for publication in your esteemed journal. With their extensive research and dedication to the subject matter, their article is sure to be a valuable addition to your publication.\u003c/p\u003e \u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor 1: Funding, Planning, Experiment Design, Guiding and Monitoring, Data analysis and editing.Authors 2 and 3: Execution of experiments, data analysis, preparing draft, corrections and modifications.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors express their gratitude to the Department of Science and Technology (DST), Government of India, New Delhi, for funding the project with File No: DST/TDT/Agro-06/2019 (G) \u0026amp; (C) Dated 05\u0026ndash;02\u0026ndash;2021. The authors would like to express their gratitude to the Director of ICAR-CIRCOT, Mumbai for extending infrastructural support.\u003c/p\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data generated or analyzed during this study is included in this published article.\u003c/p\u003e \u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKumar, M., Potkule, J., Patil, S., Saxena, S., Patil, P.G., Mageshwaran, V., Punia, S., Varghese, E., Mahapatra, A., Ashtaputre, N. \u0026amp; Charlene, D. S. (2021). Extraction of ultra-low gossypol protein from cottonseed: Characterization based on antioxidant activity, structural morphology and functional group analysis. 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Journal of Food Engineering, 165, 179\u0026ndash;188. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jfoodeng.2015.06.021\u003c/span\u003e\u003cspan address=\"10.1016/j.jfoodeng.2015.06.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaptions of Table\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Cottonseed protein, FTIR, SEM, Protein Digestibility, Lysine Content","lastPublishedDoi":"10.21203/rs.3.rs-4747495/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4747495/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe utilization of cottonseed meal and its extracted protein powder as valuable industrial products necessitates leveraging. The surface characteristics and qualitative parameters were evaluated by using FTIR, SEM, color and protein digestibility, lysine content, and microbial analysis respectively. CSPC and MW-CSPI had parallel β plated sheets and an accumulation of tiny particles due to soluble and insoluble pentosans, with changes resulting from the alkali salt protein extraction process. CSPC had superior in-vitro protein digestibility and microbiological analysis showed safe limits for bacterial and pathogenic bacterial count. Cottonseed protein, whether untreated or microwave pre-treated, can be used as an ingredient or supplement in various foods, except for lysine content as specified in Food Safety and Standards Regulations of 2011.\u003c/p\u003e","manuscriptTitle":"Evaluation of Surface Characterization and Qualitative Parameters of Cottonseed Meal and Extracted Protein","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-21 11:20:08","doi":"10.21203/rs.3.rs-4747495/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":"4b412231-9bbc-46df-a362-b72b5646bd1e","owner":[],"postedDate":"August 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-29T06:23:43+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-21 11:20:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4747495","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4747495","identity":"rs-4747495","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}