Optimization of Fermentation Time and Temperature to Enhance Phenolic Content and Antioxidant Activity of Traditional Tempe as a Functional Food

Optimization of Fermentation Time and Temperature to Enhance Phenolic Content and Antioxidant Activity of Traditional Tempe as a Functional Food | 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 Optimization of Fermentation Time and Temperature to Enhance Phenolic Content and Antioxidant Activity of Traditional Tempe as a Functional Food A.D. murtado, Suyatno Suyatno This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8715857/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 Tempe is a traditional Indonesian fermented soybean product with growing potential as a functional food due to its bioactive compounds. This study aimed to evaluate the effects of fermentation time and temperature on total phenolic content and antioxidant activity of tempe and to determine optimal fermentation conditions. Tempe samples were fermented for 24, 36, and 48 h at two different temperatures (28 and 32°C). Total phenolic content was determined using the Folin–Ciocalteu method, while antioxidant activity was measured by the DPPH radical scavenging assay. The results demonstrated that both fermentation time and temperature significantly affected total phenolic content and antioxidant activity. The highest phenolic content (11.14 mg GAE/g) and antioxidant activity (62.1% DPPH inhibition) were obtained at 36 h fermentation at 32°C. Prolonged fermentation (48 h) led to a decrease in both parameters. A positive relationship was observed between total phenolic content and antioxidant activity, indicating that phenolic compounds play a major role in the antioxidant potential of tempe. These findings suggest that optimization of fermentation conditions can enhance the functional properties of traditional tempe. Tempe fermentation phenolic compounds antioxidant activity functional food Figures Figure 1 Figure 2 INTRODUCTION Fermented foods have attracted increasing scientific and consumer interest due to their nutritional value and health-promoting properties. Fermentation has been shown to enhance the bioavailability of nutrients and generate bioactive compounds with antioxidant, anti-inflammatory, and metabolic health benefits (Marco et al., 2017 ; Pimentel et al., 2021 ). Among fermented foods, soybean-based products are particularly notable because soybeans are rich sources of phenolic compounds, isoflavones, and high-quality proteins that can be transformed into functional components through microbial fermentation (Shahidi & Ambigaipalan, 2015 ). Tempe is a traditional Indonesian fermented soybean product produced through solid-state fermentation using Rhizopus spp. During tempe fermentation, microbial enzymes such as β-glucosidase and protease play a critical role in modifying soybean constituents, particularly through the conversion of isoflavone glycosides into aglycone forms with higher bioavailability and biological activity (Nout & Kiers, 2005 ; Kuligowski et al., 2017 ). Isoflavone aglycones and other phenolic compounds generated during fermentation have been widely associated with enhanced antioxidant activity and potential health benefits (Granato et al., 2018; Xiao et al., 2015 ). Several studies have reported that fermentation significantly increases total phenolic content and antioxidant activity in tempe and other fermented soybean products. For instance, Chang et al. ( 2009 ) demonstrated that antioxidant activity of tempe increased with fermentation time up to an optimal point, after which prolonged fermentation led to a decrease in antioxidant capacity. Similarly, Kuligowski et al. ( 2017 ) observed that phenolic content and antioxidant activity of soybeans increased during tempe fermentation due to enzymatic hydrolysis of isoflavone conjugates. These findings highlight the importance of controlling fermentation parameters to maximize the functional properties of tempe. Fermentation conditions, particularly time and temperature, are critical factors influencing microbial growth, enzymatic activity, and the formation of bioactive compounds. Temperature affects the metabolic activity of Rhizopus spp. and the rate of enzymatic reactions, while fermentation time determines the extent of biochemical transformations occurring within the substrate (Polanowska et al., 2020 ; Rashad et al., 2011 ). Suboptimal fermentation conditions may limit the release of phenolic compounds, whereas excessive fermentation can lead to degradation or oxidation of bioactive molecules, resulting in reduced antioxidant activity (Chang et al., 2009 ; Xiao et al., 2015 ). Despite growing evidence on the functional properties of tempe, studies examining the combined effects of fermentation time and temperature on both total phenolic content and antioxidant activity remain limited. Moreover, few studies have systematically evaluated the relationship between phenolic content and antioxidant activity under controlled fermentation conditions. Therefore, this study aimed to investigate the effects of fermentation time and temperature on total phenolic content and antioxidant activity of tempe and to determine optimal fermentation conditions for enhancing its functional properties. Understanding these relationships is essential to support the transformation of traditional tempe into a scientifically validated functional food. MATERIALS AND METHODS 1 Materials Soybeans were obtained from a local supplier. Commercial tempe starter containing Rhizopus spp. was used for fermentation. All chemicals used for analysis, including Folin–Ciocalteu reagent, gallic acid, DPPH (2,2-diphenyl-1-picrylhydrazyl), and methanol, were of analytical grade. 2. Methods 2.1. Tempe Fermentation Soybeans were soaked, dehulled, cooked, and inoculated with tempe starter. Fermentation was conducted at two temperatures (28 and 32 °C) for 24, 36, and 48 h. Each treatment was performed in triplicate. 2.2. Determination of Total Phenolic Content Total phenolic content was determined using the Folin–Ciocalteu method and expressed as mg gallic acid equivalents per gram of sample (mg GAE/g). 2.3. Determination of Antioxidant Activity Antioxidant activity was measured using the DPPH radical scavenging assay and expressed as percentage of DPPH inhibition. 2.4. Statistical Analysis Data were analyzed using analysis of variance (ANOVA) at a significance level of p < 0.05 RESULTS AND DISCUSSION 1. Effect of Fermentation Time and Temperature on Total Phenolic Content The results demonstrated that fermentation time and temperature significantly affected the total phenolic content (TPC) of tempe. TPC increased from 24 to 36 h of fermentation and subsequently decreased at 48 h. The highest TPC value was observed at 36 h fermentation at 32 °C (11.14 mg GAE/g), indicating an optimal fermentation condition for phenolic compound accumulation. The increase in TPC during fermentation is closely associated with the enzymatic activity of Rhizopus spp., particularly β-glucosidase, which hydrolyzes isoflavone glycosides into their aglycone forms with higher solubility and bioactivity (Kuligowski et al., 2017; Rashad et al., 2011). Isoflavone aglycones such as genistein and daidzein are major contributors to the phenolic fraction in fermented soybean products (Xiao et al., 2015). Fermentation temperature played a crucial role in modulating phenolic formation. Samples fermented at 32 °C consistently demonstrated higher TPC compared to those fermented at 28 °C at the same fermentation time. This finding is consistent with Polanowska et al. (2020), who reported that fermentation temperatures close to the optimal growth temperature of Rhizopus enhanced the release and formation of bioactive compounds. Similarly, Nout and Kiers (2005) emphasized that controlled fermentation conditions are essential to maximize the functional quality of tempe. The decline in TPC observed after 48 h of fermentation suggests that prolonged fermentation may lead to degradation or further metabolism of phenolic compounds. Chang et al. (2009) reported that excessive fermentation time can result in oxidative degradation or structural modification of antioxidant compounds, leading to reduced measurable phenolics. Similar trends have been reported in other fermented soybean products (Xiao et al., 2015; Rashad et al., 2011). (Figure 1, blue) 2. Effect of Fermentation Time and Temperature on Antioxidant Activity (DPPH Assay) Antioxidant activity measured by the DPPH radical scavenging assay followed a pattern similar to that of TPC. Antioxidant activity increased with fermentation time, reaching a maximum at 36 h at 32 °C (62.1% inhibition), and declined thereafter at 48 h. The enhancement of antioxidant activity during fermentation is primarily attributed to the increased concentration of phenolic compounds and isoflavone aglycones, which act as effective hydrogen or electron donors capable of neutralizing DPPH radicals (Shahidi & Ambigaipalan, 2015; Chang et al., 2009). Phenolic compounds are widely recognized as key contributors to the antioxidant capacity of functional foods (Granato et al., 2018). In addition to phenolic compounds, fermentation-induced proteolysis generates bioactive peptides with antioxidant properties. Jakubczyk et al. (2020) demonstrated that peptides released during soybean fermentation significantly contribute to radical scavenging activity. Therefore, the elevated antioxidant activity observed at 36 h fermentation may result from the combined effects of phenolic compounds and antioxidant peptides. The decrease in antioxidant activity at 48 h fermentation indicates that prolonged fermentation may reduce antioxidant effectiveness. Kuligowski et al. (2017) and Rashad et al. (2011) suggested that extended fermentation could cause degradation of phenolic compounds or alteration of antioxidant structures, reducing their radical scavenging capacity. (Figure 1, yellow) 3. Relationship Between Total Phenolic Content and Antioxidant Activity A positive relationship was observed between total phenolic content and antioxidant activity, indicating that higher phenolic levels were associated with increased DPPH radical scavenging activity. This relationship supports the role of phenolic compounds as the main contributors to the antioxidant potential of fermented tempe. The strong association between phenolic content and antioxidant activity has been widely reported in fermented soybean-based foods (Kuligowski et al., 2017; Shahidi & Ambigaipalan, 2015). Granato et al. (2018) further emphasized that phenolic-rich functional foods exhibit enhanced antioxidant properties and potential health benefits. The fermentation condition of 36 h at 32 °C can therefore be considered optimal in this study, as it simultaneously maximized total phenolic content and antioxidant activity. These findings align with the concept of functional food development, where process optimization is essential to enhance the nutritional and health-promoting properties of traditional foods (Marco et al., 2017; Pimentel et al., 2021). Declarations Author Contribution AD wrote the manuscript and I prepared the drawings. References Astuti, M., Meliala, A., Dalais, F. S., & Wahlqvist, M. L. (2000). Tempe, a nutritious and healthy food from Indonesia. Asia Pacific Journal of Clinical Nutrition, 9(4), 322–325. Chang, C. T., Hsu, C. K., Chou, S. T., Chen, Y. C., Huang, F. S., & Chung, Y. C. (2009). Effect of fermentation time on the antioxidant activities of tempe. Journal of Food Science, 74(8), C655–C660. Granato, D., Barba, F. J., Bursać Kovačević, D., Lorenzo, J. M., & Cruz, A. G. (2020). Putative health benefits of phenolic compounds in functional foods: A review. Food Chemistry, 312, 126091. Kuligowski, M., Pawłowska, K., Jasińska-Kuligowska, I., & Nowak, J. (2017). Isoflavone composition, polyphenols content and antioxidant activity of soybean seeds during tempe fermentation. Food Chemistry, 222, 109–116. Marco, M. L., et al. (2017).Health benefits of fermented foods: microbiota and beyond. Nature Reviews Gastroenterology & Hepatology, 14, 196–208. Nout, M. J. R., & Kiers, J. L. (2005).Tempe fermentation, innovation and functionality: Update into the third millennium. Journal of Applied Microbiology, 98(4), 789–805. Pimentel, T. C., et al. (2021).Fermented foods and health-promoting properties. Food Research International, 140, 110067. Polanowska, K., Grygier, A., Kuligowski, M., Rudzinska, M., & Nowak, J. (2020). Effect of tempe fermentation by different strains of Rhizopus on nutritional and functional properties of soybeans. Journal of Food Composition and Analysis, 92, 103534. Rashad, M. M., et al. (2011).Biochemical changes during soybean fermentation by Rhizopus. Food Chemistry, 124(2), 685–692. Shahidi, F., & Ambigaipalan, P. (2015).Phenolics and polyphenolics in foods: Antioxidant activity. Journal of Functional Foods, 18, 820–897. Xiao, Y., et al. (2015).Enhancement of antioxidant capacity of soybeans during fermentation. Journal of Functional Foods, 14, 594–602. 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. 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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-8715857","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":581574904,"identity":"133ab4c2-f387-4b2a-8f67-6daecff4c569","order_by":0,"name":"A.D. murtado","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYNACNhjDwIaBQYKwesYGJC1pJGthOExYi+6M3OcPfpTZMPD3nzH8XFFwPrF/dvPBBww1NtG4tJjdSDds7DmXxiBxI8dY8ozB7cQZd44lGzAcS8ttwKkljbGBtw3onhs8BpINQC0NN3LMJBgbDuPV0vgXqEX+/Bnjnw0G5xLnE6OlGWSLwYEcM6AtBxI3ENRy5hnjbJlzaTyGN9LKLBsMko033khLNkjA55fjaQwf35TZyMmdP7z5ZsMfO9l5N5IPPvhQY4NTCwzwMDBwGIAYjmCVCQSUQwH7AxBpT5ziUTAKRsEoGEkAAEOyXryXpgYpAAAAAElFTkSuQmCC","orcid":"","institution":"Muhammadiyah university of Palembang","correspondingAuthor":true,"prefix":"","firstName":"A.D.","middleName":"","lastName":"murtado","suffix":""},{"id":581574908,"identity":"9950375a-17e6-4eaa-bfb6-653ee1d1212c","order_by":1,"name":"Suyatno Suyatno","email":"","orcid":"","institution":"Muhammadiyah university of Palembang","correspondingAuthor":false,"prefix":"","firstName":"Suyatno","middleName":"","lastName":"Suyatno","suffix":""}],"badges":[],"createdAt":"2026-01-28 03:38:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8715857/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8715857/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101490714,"identity":"e7c09f60-5edc-4247-988a-c4d7e7e2a5e4","added_by":"auto","created_at":"2026-01-30 10:00:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":46195,"visible":true,"origin":"","legend":"\u003cp\u003eTotal Phenolic and Antioxidant Activity of Tempe\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8715857/v1/410eaa9978c7945e92c098a4.png"},{"id":101490716,"identity":"9884d7c2-9ded-4705-8ae6-16f29884e854","added_by":"auto","created_at":"2026-01-30 10:00:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":28125,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship Between Total Phenolic Content and Antioxidant Activit\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8715857/v1/1d4977a16f211c80594faba8.png"},{"id":102403989,"identity":"c591df21-b33d-4c45-bc8c-7d1210befd2e","added_by":"auto","created_at":"2026-02-11 10:51:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":455366,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8715857/v1/c75af907-cb84-4899-b961-1f66950e2e2f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optimization of Fermentation Time and Temperature to Enhance Phenolic Content and Antioxidant Activity of Traditional Tempe as a Functional Food","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eFermented foods have attracted increasing scientific and consumer interest due to their nutritional value and health-promoting properties. Fermentation has been shown to enhance the bioavailability of nutrients and generate bioactive compounds with antioxidant, anti-inflammatory, and metabolic health benefits (Marco et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Pimentel et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Among fermented foods, soybean-based products are particularly notable because soybeans are rich sources of phenolic compounds, isoflavones, and high-quality proteins that can be transformed into functional components through microbial fermentation (Shahidi \u0026amp; Ambigaipalan, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTempe is a traditional Indonesian fermented soybean product produced through solid-state fermentation using Rhizopus spp. During tempe fermentation, microbial enzymes such as β-glucosidase and protease play a critical role in modifying soybean constituents, particularly through the conversion of isoflavone glycosides into aglycone forms with higher bioavailability and biological activity (Nout \u0026amp; Kiers, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Kuligowski et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Isoflavone aglycones and other phenolic compounds generated during fermentation have been widely associated with enhanced antioxidant activity and potential health benefits (Granato et al., 2018; Xiao et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral studies have reported that fermentation significantly increases total phenolic content and antioxidant activity in tempe and other fermented soybean products. For instance, Chang et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) demonstrated that antioxidant activity of tempe increased with fermentation time up to an optimal point, after which prolonged fermentation led to a decrease in antioxidant capacity. Similarly, Kuligowski et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) observed that phenolic content and antioxidant activity of soybeans increased during tempe fermentation due to enzymatic hydrolysis of isoflavone conjugates. These findings highlight the importance of controlling fermentation parameters to maximize the functional properties of tempe.\u003c/p\u003e \u003cp\u003eFermentation conditions, particularly time and temperature, are critical factors influencing microbial growth, enzymatic activity, and the formation of bioactive compounds. Temperature affects the metabolic activity of Rhizopus spp. and the rate of enzymatic reactions, while fermentation time determines the extent of biochemical transformations occurring within the substrate (Polanowska et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rashad et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Suboptimal fermentation conditions may limit the release of phenolic compounds, whereas excessive fermentation can lead to degradation or oxidation of bioactive molecules, resulting in reduced antioxidant activity (Chang et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Xiao et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite growing evidence on the functional properties of tempe, studies examining the combined effects of fermentation time and temperature on both total phenolic content and antioxidant activity remain limited. Moreover, few studies have systematically evaluated the relationship between phenolic content and antioxidant activity under controlled fermentation conditions. Therefore, this study aimed to investigate the effects of fermentation time and temperature on total phenolic content and antioxidant activity of tempe and to determine optimal fermentation conditions for enhancing its functional properties. Understanding these relationships is essential to support the transformation of traditional tempe into a scientifically validated functional food.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e1 Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSoybeans were obtained from a local supplier. Commercial tempe starter containing Rhizopus spp. was used for fermentation. All chemicals used for analysis, including Folin\u0026ndash;Ciocalteu reagent, gallic acid, DPPH (2,2-diphenyl-1-picrylhydrazyl), and methanol, were of analytical grade.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1. Tempe Fermentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSoybeans were soaked, dehulled, cooked, and inoculated with tempe starter. Fermentation was conducted at two temperatures (28 and 32 \u0026deg;C) for 24, 36, and 48 h. Each treatment was performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Determination of Total Phenolic Content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal phenolic content was determined using the Folin\u0026ndash;Ciocalteu method and expressed as mg gallic acid equivalents per gram of sample (mg GAE/g).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Determination of Antioxidant Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntioxidant activity was measured using the DPPH radical scavenging assay and expressed as percentage of DPPH inhibition.\u003cbr\u003e\u003cstrong\u003e2.4. Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were analyzed using analysis of variance (ANOVA) at a significance level of p \u0026lt; 0.05\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003e1. Effect of Fermentation Time and Temperature on Total Phenolic Content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results demonstrated that fermentation time and temperature significantly affected the total phenolic content (TPC) of tempe. TPC increased from 24 to 36 h of fermentation and subsequently decreased at 48 h. The highest TPC value was observed at 36 h fermentation at 32 \u0026deg;C (11.14 mg GAE/g), indicating an optimal fermentation condition for phenolic compound accumulation.\u003c/p\u003e\n\u003cp\u003eThe increase in TPC during fermentation is closely associated with the enzymatic activity of Rhizopus spp., particularly \u0026beta;-glucosidase, which hydrolyzes isoflavone glycosides into their aglycone forms with higher solubility and bioactivity (Kuligowski et al., 2017; Rashad et al., 2011). Isoflavone aglycones such as genistein and daidzein are major contributors to the phenolic fraction in fermented soybean products (Xiao et al., 2015).\u003c/p\u003e\n\u003cp\u003eFermentation temperature played a crucial role in modulating phenolic formation. Samples fermented at 32 \u0026deg;C consistently demonstrated higher TPC compared to those fermented at 28 \u0026deg;C at the same fermentation time. This finding is consistent with Polanowska et al. (2020), who reported that fermentation temperatures close to the optimal growth temperature of Rhizopus enhanced the release and formation of bioactive compounds. Similarly, Nout and Kiers (2005) emphasized that controlled fermentation conditions are essential to maximize the functional quality of tempe.\u003c/p\u003e\n\u003cp\u003eThe decline in TPC observed after 48 h of fermentation suggests that prolonged fermentation may lead to degradation or further metabolism of phenolic compounds. Chang et al. (2009) reported that excessive fermentation time can result in oxidative degradation or structural modification of antioxidant compounds, leading to reduced measurable phenolics. Similar trends have been reported in other fermented soybean products (Xiao et al., 2015; Rashad et al., 2011). (Figure 1, blue)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Effect of Fermentation Time and Temperature on Antioxidant Activity (DPPH Assay)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntioxidant activity measured by the DPPH radical scavenging assay followed a pattern similar to that of TPC. Antioxidant activity increased with fermentation time, reaching a maximum at 36 h at 32 \u0026deg;C (62.1% inhibition), and declined thereafter at 48 h.\u003c/p\u003e\n\u003cp\u003eThe enhancement of antioxidant activity during fermentation is primarily attributed to the increased concentration of phenolic compounds and isoflavone aglycones, which act as effective hydrogen or electron donors capable of neutralizing DPPH radicals (Shahidi \u0026amp; Ambigaipalan, 2015; Chang et al., 2009). Phenolic compounds are widely recognized as key contributors to the antioxidant capacity of functional foods (Granato et al., 2018).\u003c/p\u003e\n\u003cp\u003eIn addition to phenolic compounds, fermentation-induced proteolysis generates bioactive peptides with antioxidant properties. Jakubczyk et al. (2020) demonstrated that peptides released during soybean fermentation significantly contribute to radical scavenging activity. Therefore, the elevated antioxidant activity observed at 36 h fermentation may result from the combined effects of phenolic compounds and antioxidant peptides.\u003c/p\u003e\n\u003cp\u003eThe decrease in antioxidant activity at 48 h fermentation indicates that prolonged fermentation may reduce antioxidant effectiveness. Kuligowski et al. (2017) and Rashad et al. (2011) suggested that extended fermentation could cause degradation of phenolic compounds or alteration of antioxidant structures, reducing their radical scavenging capacity. (Figure 1, yellow)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Relationship Between Total Phenolic Content and Antioxidant Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA positive relationship was observed between total phenolic content and antioxidant activity, indicating that higher phenolic levels were associated with increased DPPH radical scavenging activity. This relationship supports the role of phenolic compounds as the main contributors to the antioxidant potential of fermented tempe.\u003c/p\u003e\n\u003cp\u003eThe strong association between phenolic content and antioxidant activity has been widely reported in fermented soybean-based foods (Kuligowski et al., 2017; Shahidi \u0026amp; Ambigaipalan, 2015). Granato et al. (2018) further emphasized that phenolic-rich functional foods exhibit enhanced antioxidant properties and potential health benefits.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe fermentation condition of 36 h at 32 \u0026deg;C can therefore be considered optimal in this study, as it simultaneously maximized total phenolic content and antioxidant activity. These findings align with the concept of functional food development, where process optimization is essential to enhance the nutritional and health-promoting properties of traditional foods (Marco et al., 2017; Pimentel et al., 2021).\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAD wrote the manuscript and I prepared the drawings.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAstuti, M., Meliala, A., Dalais, F. S., \u0026amp; Wahlqvist, M. L. (2000).\u003cbr\u003e\u0026nbsp;Tempe, a nutritious and healthy food from Indonesia. Asia Pacific Journal of Clinical Nutrition, 9(4), 322\u0026ndash;325.\u003c/li\u003e\n \u003cli\u003eChang, C. T., Hsu, C. K., Chou, S. T., Chen, Y. C., Huang, F. S., \u0026amp; Chung, Y. C. (2009).\u003cbr\u003e\u0026nbsp;Effect of fermentation time on the antioxidant activities of tempe. Journal of Food Science, 74(8), C655\u0026ndash;C660.\u003c/li\u003e\n \u003cli\u003eGranato, D., Barba, F. J., Bursać Kovačević, D., Lorenzo, J. M., \u0026amp; Cruz, A. G. (2020).\u003cbr\u003e\u0026nbsp;Putative health benefits of phenolic compounds in functional foods: A review. Food Chemistry, 312, 126091.\u003c/li\u003e\n \u003cli\u003eKuligowski, M., Pawłowska, K., Jasińska-Kuligowska, I., \u0026amp; Nowak, J. (2017).\u003cbr\u003e\u0026nbsp;Isoflavone composition, polyphenols content and antioxidant activity of soybean seeds during tempe fermentation. Food Chemistry, 222, 109\u0026ndash;116.\u003c/li\u003e\n \u003cli\u003eMarco, M. L., et al. (2017).Health benefits of fermented foods: microbiota and beyond. Nature Reviews Gastroenterology \u0026amp; Hepatology, 14, 196\u0026ndash;208.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eNout, M. J. R., \u0026amp; Kiers, J. L. (2005).Tempe fermentation, innovation and functionality: Update into the third millennium. Journal of Applied Microbiology, 98(4), 789\u0026ndash;805.\u003c/li\u003e\n \u003cli\u003ePimentel, T. C., et al. (2021).Fermented foods and health-promoting properties. Food Research International, 140, 110067.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePolanowska, K., Grygier, A., Kuligowski, M., Rudzinska, M., \u0026amp; Nowak, J. (2020).\u003cbr\u003e\u0026nbsp;Effect of tempe fermentation by different strains of Rhizopus on nutritional and functional properties of soybeans. Journal of Food Composition and Analysis, 92, 103534.\u003c/li\u003e\n \u003cli\u003eRashad, M. M., et al. (2011).Biochemical changes during soybean fermentation by Rhizopus. Food Chemistry, 124(2), 685\u0026ndash;692.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eShahidi, F., \u0026amp; Ambigaipalan, P. (2015).Phenolics and polyphenolics in foods: Antioxidant activity. Journal of Functional Foods, 18, 820\u0026ndash;897.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eXiao, Y., et al. (2015).Enhancement of antioxidant capacity of soybeans during fermentation. Journal of Functional Foods, 14, 594\u0026ndash;602. \u003c/li\u003e\n\u003c/ol\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":"Tempe, fermentation, phenolic compounds, antioxidant activity, functional food","lastPublishedDoi":"10.21203/rs.3.rs-8715857/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8715857/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTempe is a traditional Indonesian fermented soybean product with growing potential as a functional food due to its bioactive compounds. This study aimed to evaluate the effects of fermentation time and temperature on total phenolic content and antioxidant activity of tempe and to determine optimal fermentation conditions. Tempe samples were fermented for 24, 36, and 48 h at two different temperatures (28 and 32\u0026deg;C). Total phenolic content was determined using the Folin\u0026ndash;Ciocalteu method, while antioxidant activity was measured by the DPPH radical scavenging assay. The results demonstrated that both fermentation time and temperature significantly affected total phenolic content and antioxidant activity. The highest phenolic content (11.14 mg GAE/g) and antioxidant activity (62.1% DPPH inhibition) were obtained at 36 h fermentation at 32\u0026deg;C. Prolonged fermentation (48 h) led to a decrease in both parameters. A positive relationship was observed between total phenolic content and antioxidant activity, indicating that phenolic compounds play a major role in the antioxidant potential of tempe. These findings suggest that optimization of fermentation conditions can enhance the functional properties of traditional tempe.\u003c/p\u003e","manuscriptTitle":"Optimization of Fermentation Time and Temperature to Enhance Phenolic Content and Antioxidant Activity of Traditional Tempe as a Functional Food","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-30 10:00:33","doi":"10.21203/rs.3.rs-8715857/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":"e45fbd54-cfb3-448e-99cc-21201a4e6675","owner":[],"postedDate":"January 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-03T02:09:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-30 10:00:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8715857","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8715857","identity":"rs-8715857","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}