Production of Stable Anthocyanin-Based Food Colorants from Hibiscus sabdariffa L. by Copigmentation with Different Protein Isolates

Production of Stable Anthocyanin-Based Food Colorants from Hibiscus sabdariffa L. by Copigmentation with Different Protein Isolates | 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 Production of Stable Anthocyanin-Based Food Colorants from Hibiscus sabdariffa L. by Copigmentation with Different Protein Isolates Vildan Eyiz, İsmail Tontul, Selman TÜRKER This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6401806/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 In this study, different proteins [pea protein isolate (PP), rice protein isolate (RP), gluten (GP) and whey protein isolate (WP)] were co-pigmented with Hibiscus sabdariffa L. anthocyanins and bathochromic shift, hyperchromic effect and thermal stability tests were performed on the samples obtained. The polymeric color percentage values, which are indicative of anthocyanin degradation during both processing and storage, showed that the samples WPH (%35.86) and RPH (%38.84) exhibited superior anthocyanin stability. The WPH samples provided the highest z values (112.78 K). Furthermore, the RPH samples demonstrated the highest Ea values (47.98 kJ/mol). The FTIR spectrums revealed the presence of prominent peaks at wavelengths of 3000–3500 cm − 1 (-OH tension) and 1600–1650 cm − 1 (C = O tension). The observed fluctuations suggest the presence of interactions between anthocyanins and proteins. Copigmentation process caused a significant reduction in fluorescence intensity. This suggests that there is strong interaction between proteins and anthocyanins. It was observed that hibiscus extracts copigmented with protein isolates, particularly whey and rice protein isolates, were able to increase anthocyanin stability. protein-anthocyanin interaction color stability colorants protein isolates Figures Figure 1 Figure 2 Figure 3 1. Introduction Colorants represent a class of additives of paramount importance in the food industry, given that the color of a foodstuff has a considerable effect on its visual appeal. The classification of food colorants can be divided into two broad categories: synthetic and natural. Synthetic colorants (tartrazine, Allura red, indigo carmine, etc.) exhibit remarkable stability when exposed to various environmental factors such as temperature, pH, light and oxygen 1 . However, synthetic colorants have been associated with various health concerns, including toxic effects, allergic reactions, and behavioral and neurological side effects 2 . Consequently, there is an increasing interest in the development of chemically stable food colorants derived from natural sources as an alternative to synthetic colorants. Anthocyanins are compounds belonging to the flavonoid group that are frequently found in fruits, vegetables, grains and other plants. These compounds have been shown to possess antioxidant, antiviral and anti-aging properties. In addition, anthocyanins have been shown to have beneficial effects on health, including a reduced risk of cancer, inflammation 3 , 4 , cardiovascular diseases, and diabetes 5 and a reduced obesity risk 6 . Despite the benefits, the low stability of anthocyanins when exposed to food processing and environmental factors limits their use in food formulations 7 . Consequently, it is imperative to implement some precautionary measures to enhance the stability of anthocyanins. Copigmentation has emerged as a promising technique for enhancing the stability of anthocyanins when subjected to conditions such as elevated temperatures, exposure to light, and the presence of oxygen 8 . It is estimated that there are more than ten thousand compounds that have the potential to be utilized as copigments 9 . Typically, copigments are characterized by the presence of π conjugated systems, which facilitate the formation of π − π interactions. Additionally, these compounds often possess hydrogen bond donor/acceptor groups such as hydroxyl (OH) and carbonyl (C = O) 10 . Proteins have emerged as a promising copigments to enhance the stability of anthocyanins. The interaction between proteins and anthocyanins is complex and occurs through a variety of chemical interactions, including hydrophobic and hydrophilic interactions, van der Waals forces, and hydrogen bonding 11 . Several studies showed that whey proteins exhibit enhanced copigmentation capabilities due to their high affinity for anthocyanins 12 – 14 . Chen, Guan, Zeng, Wang, Qin, Chen and He 15 used whey protein and phenolic compounds as copigmentation agents for mulberry anthocyanins in their studies and demonstrated that WPI was efficacious in mitigating thermal-induced color and anthocyanin loss. Recent studies have explored the use of plant-based proteins as anthocyanin copigments 16 . In a study, the effects of β-cyclodextrin, whey protein, and soy protein on anthocyanin degradation in purple-fleshed sweet potato anthocyanin extracts were investigated in model beverage systems. It was reported that the incorporation of whey protein and soy protein resulted in an increase in the thermal stability of the extracts 17 . In another study, the effect of three amino acids (L-phenylalanine, L-tyrosine, and L-tryptophan) and a peptide (poly-L-lysine) on the color of anthocyanins in model beverages during storage at 40°C and under light for seven days was investigated. The results showed that the incorporation of amino acids improved color stability, mainly through the formation of hydrogen bonds. Furthermore, Chung, Rojanasasithara, Mutilangi and McClements 18 reported that L-tryptophan had a significant impact on the color stability of purple carrot anthocyanins. In a separate study, copigmentation with soy protein isolate enhanced the stability of anthocyanins in purple corn. 19 The present study is an investigation into the copigmentation effect of different plant proteins on Hibiscus sabdariffa L. extract. Hibiscus extract is characterized by a deep red pigmentation, attributable to the presence of anthocyanins, thus establishing its prevalence as a colorant 20 . The anthocyanin content of hibiscus has been documented to range from 1.7–2.5% of dry weight 21 . The main anthocyanins in hibiscus have been identified as delphinidin-3-sambubioside and cyanidin-3-sambubioside 22 . The copigmentation effect of three different plant proteins (pea protein isolate, rice protein isolate, and gluten) on Hibiscus sabdariffa L. extract was examined comparatively to whey protein isolates. To this end, thermal stability tests were performed on samples and kinetic calculations were made to determine the most suitable copigmentation agent. 2. Material and Methods 2.1. Material Dried Hibiscus sabdariffa L. calices, obtained from a local herbalist, was used as an anthocyanin source. Rice protein isolate (Naturiga Natural Foods, Istanbul, Türkiye), pea protein isolate (Proteinocean, Ankara, Türkiye), gluten (Vatan enzymes, Istanbul, Türkiye) and whey protein isolate (PASP) (Hardline Nutrition, Kocaeli, Türkiye) were utilized as copigmentation agents. All chemicals used in the analyses were of reagent grade and obtained from Merck (Germany). 2.2. Extraction of anthocyanins from Hibiscus Sabdariffa The extraction of anthocyanins from hibiscus was carried out by modifying the methodology performed by Abou-Arab, Abu-Salem and Abou-Arab 23 . The hibiscus was pulverized and converted into a powdered form. The powdered sample was then combined with distilled water in a 1:10 ratio at a temperature of 90°C. The mixture was then subjected to agitation in a mechanical mixer (DLAB-OS20-S, China) at 600 RPM for 15 minutes at 90 o C. Subsequently, the sample was subjected to agitation (100 rpm) in a water bath maintained at 24°C for 24 hours. Finally, the extract was filtered to remove hibiscus pulps and stored at 4ºC until copigmentation experiments. 2.3. Copigmentation of anthocyanin extract with protein isolates The copigmentation conditions of hibiscus anthocyanins and protein isolates were determined by preliminary experiments in accordance with the methodologies outlined by Li, Wang, Zhang, Yu and Chen 24 and Ma and Jing 25 . A combined solution of hibiscus extract and protein isolates (10 mg/mL in distilled water) were prepared at a ratio of 1:5. Subsequently, the pH value of the mixture was adjusted to 6 using a sodium hydroxide solution (1 M). The copigmentation was carried out by means of a 30-minute mixing procedure in a rotator (Isolab Laborgerate, Germany). Then, the pH value of the solution was adjusted to 3 using HCl (1 M). At this stage, four different copigmented samples were formed with different protein isolates and a control sample using distilled water in lieu of the protein solution. Initially, the bathochromic shift and hyperchromic effects of the samples obtained were determined. Subsequently, a thermal stability test was conducted, followed by analyses of monomeric anthocyanin and polymeric color, as well as FTIR and fluorescence spectroscopy. 2.4. Bathochromic shift and hypochromic effect Hyperchromic effect (the absorbance value at λ max ) and bathochromic shift (a shift of wavelength of λ max ) were determined according to the method of Malaj, De Simone, Quartarolo and Russo 26 . 2.6. Thermal stability test The samples were subjected to thermal stability testing, with temperatures ranging from 80 to 100°C for a duration of up to 120 minutes. Sampling was conducted at predetermined intervals, and the samples were chilled to room temperature. Subsequently, each solution was then centrifuged at 10,000 rpm for 30 minutes. The polymeric color values and total monomeric anthocyanin concentration of supernatants were determined. The data obtained from these tests were then used to calculate the kinetic parameters. The second-order reaction kinetics (determined according to kinetic calculations) (Eq. 1 .) and the Arrhenius equation (Eq. 2)were employed to calculate the thermal degradation kinetic parameters of samples by Simonin 27 . $$\:\frac{1}{C}\:-\:\frac{1}{C0}=\:kt$$ 1 C = Anthocyanin content (time t) C 0 = Anthocyanin content (time zero) k = rate constant [(mg.L) −1 .min − 1 ] t = Time (min.) A = Arrhenius constant E a = Activation energy (cal/mol − 1 ) k = rate constant [(mg.L) −1 .min − 1 ] R = ideal gas constant (1.987 cal/mol − 1 ) T: Temperature (K) 2.7. Total monomeric anthocyanin content The total anthocyanin content was determined by employing the pH differential method. The total anthocyanin content of the extracts was expressed as milligrams of cyanidin-3-glucoside equivalent (C3G) per 100 grams of dry matter of the sample 28 . 2.8. Polymeric color The determination of color intensity and polymeric color values was conducted by first diluting the samples with a KCl solution (0.025 M) or distilled water. 2.8 mL of each diluted sample was transferred into two separate cuvettes. To one of the cuvettes, 0.2 mL of K 2 S 2 O 5 (20%, w/v) was added, while 0.2 mL of distilled water was added to the other. Both cuvettes were kept in the dark for 15 min. Finally, the absorbances ​​of both cuvettes were measured against distilled water at 420 nm (A 420 ), λ max (A 518 ) and 700 (A 700 ) nm. Polymeric color (Eq. 3), color intensity (Eq. 4) and polymeric color ratio (Eq. 5) was calculated according to Giusti and Wrolstad 29 . Polymeric color = (A 420 -A 700 ) + (A 518 -A 700 ) x dilution factor (3) Color intensity = (A 420 -A 700 ) + (A 518 -A 700 ) x dilution factor (4) Polymeric color ratio (%) = (Polymeric color / Color intensity) x 100 (5) 2.9. Fourier transform infrared spectroscopy (FTIR) Fourier Transform Infrared Spectrometry analysis was determined with Thermo Scientific – Nicolet iS20 (CA, USA). Spectroscopic readings were collected within the 4000 − 600 cm - 1 wavelength range, with a resolution of 2 cm - 1 and a scanning speed of 1 cm/s for each spectrum, and the spectrum graph was obtained. 2.10. Fluorescence spectroscopy Fluorescence spectrometer measurements were performed with an Agilent Cary Eclipse instrument (Agilent, Germany). Emission spectra were recorded between 300 and 400 nm with an excitation wavelength of 280 nm (slit width 5 nm) in a 1.0 cm quartz cuvette. 2.11. Statistical analyses The data were subjected to analysis of variance, and the appropriate mean separations were calculated using Duncan's Multiple Comparison Test. Statistical analyses were performed using the SAS software program (SAS Institute, Cary, NC, USA). 3. Results and discussion 3.1. Bathochromic shift and hypochromic effect Copigmentation refers to the hydrophobically oriented association of an anthocyanin chromophore with the planar electronically saturated part of the copigment. This phenomenon can be attributed to van der Waals interactions and hydrophobic effects in aqueous media that result in the 'π–π' stacking of anthocyanin and copigment molecules. This association leads to an increase in absorbance within the visible range (hyperchromic effect, ΔA) and a shift of λ max wavelengths (bathochromic effect, Δλ max ) 30 . The magnitude of the copigmentation effect is influenced by several factors, including the type and chemical structure of the copigment. This can be quantified through the use of hyperchromic effect and bathochromic shift parameters 31 . The hyperchromic effect generally signifies a shift in the hydration balance from the colorless hydrated hemiketal form to the colored flavylium form. Conversely, the bathochromic shift is indicative of the formation of electrostatic interactions between the pigment and the copigment 32 . The bathochromic shift and hyperchromic effect results for hibiscus samples copigmented with PP, RP, GP, and WP are presented in Table 1 . As illustrated in the table, a reduction in both bathochromic shift (with the exception of BPH sample) and the hyperchromic effect was observed in samples copigmented with proteins. This finding indicates that protein isolates do not effectively function as copigmentation agents. In general, while there was an improvement in the bathochromic shift and hyperchromic effects of anthocyanin pigments copigmented with protein isolates was observed in previous studies 33 , 34 , a decrease was noted in the present study. It is hypothesized that this is due to interference of proteins with the color of the hibiscus extract. In their studies, Chen, Gao, Liao, Zou, Yan and Li 33 copigmented mulberry anthocyanins with whey protein isolate and a whey protein isolate-phenolic complex. They reported that the use of protein isolates alone resulted in the weakening of anthocyanin color and that the resulting mixture was unstable and prone to precipitation at high temperatures. Table 1 Bathochromic shift and hyperchromic effect results of copigmented samples Sample ƛ max (Bathochromic shift, wavelength (nm)) Δ λ max (nm) ABS (Hyperchromic effect, A) ΔA (%) Control 519 - 1.964 - PPH 519 0 1.149 -41.50 RPH 514 -5 1.098 -44.09 GPH 518 -1 1.019 -48.12 WPH 518 -1 1.448 -26.27 PPH: Hibiscus extract copigmented with pea protein isolate, RPH: Hibiscus extract copigmented with rice protein isolate, GPH: Hibiscus extract copigmented with gluten, WPH: Hibiscus extract copigmented with whey protein isolate 3.2. Polymeric color The term “polymeric color percentage” is defined as the ratio of polymeric color to color intensity. It is used as a measure of the percentage of color of a sample that can be attributed to polymerized compounds. Available evidence indicates that an increase in polymeric color is proportional to a loss of anthocyanin 35 – 37 . The change in polymeric color values during the heat treatment of the control extract is illustrated in Fig. 1 . An increase in the percentage of polymeric color was observed in correlation with both an increase in the temperature of the heat treatment and an extension of its duration. During the heat treatment, it was observed that polymeric color values ranged from 33.22–54.34%. A comparative analysis of the polymeric color values according to the protein type revealed that the polymeric color belonged to the GPH, RPH, PPH, and WPH samples, respectively (Table 2 ). Consequently, it can therefore be concluded that the color was effectively preserved in hibiscus extracts copigmented with whey protein isolate. According to the extant literature, previous studies have also demonstrated the efficacy of whey proteins in inhibiting the degradation of anthocyanins 8 , 12 , 14 . Conversely, the samples copigmented with gluten exhibited the highest polymeric color value. This phenomenon is hypothesized to be attributable to the low solubility of gluten, which leads to the turbidity of the sample 38 . An increase in the polymeric color percentage was observed in response to an increase in temperature and an extension in heating duration. This phenomenon is hypothesized to be attributable to the promotion of the formation of chalcones, an intermediate product of anthocyanin degradation, at elevated temperatures 39 . Chalcones possess reactive properties, which enable them to combine with other components. This combination results in an increase in polymeric color 36 . Table 2 Polymeric color contents of copigmented samples Variation sources Polymeric color (%) Protein type PPH 38.84 ± 8.30c RPH 41.83 ± 10.79b GPH 47.86 ± 5.73a WPH 35.86 ± 5.47d Temperature ( o C) 80 40.01 ± 8.66b 90 40.21 ± 9.04b 100 43.08 ± 9.07a Heating time (min) 0 32.47 ± 7.70d 10 38.85 ± 9.80c 30 39.76 ± 5.17c 60 44.51 ± 6.54b 120 49.91 ± 6.70a Mean ± std error, different letters in same column shows significant difference (P < 0.05). PPH: Copigmented with pea protein, RPH: Copigmented with rice protein, GPH: Copigmented with gluten protein, WPH: Copigmented with whey protein. 3.3. Total monomeric anthocyanin content In order to determine the effect of copigmentation on the anthocyanin content of the hibiscus extract, the samples were subjected to a thermal stability test, and kinetic calculations were performed. The degradation rate constants (k), correlation coefficients (R²), z values and activation energy (Ea) of anthocyanin degradation were calculated. The degradation of total monomeric anthocyanins in the samples during heat treatment found to follow a second order kinetic model, consistent with findings reported in other studies 40 – 42 . This behavior is attributed to the reaction of electropositive aglycones (cyanidin + 287, delphinidin + 303, pelargonidin + 271 and peonidin + 301) in accordance with the second-order kinetics 43 – 45 . The degradation rate constants (k) of the samples at different temperatures ranged from 0.22×10 − 3 to 0.59×10 − 3 (mg/L) −1 .min − 1 , with the rate of anthocyanin degradation increasing as temperature rose (Table 3 ). Similarly, Kanha, Surawang, Pitchakarn and Laokuldilok 46 reported an increase in degradation rate constants with elevated temperature when black rice extract subjected to copigmentation and encapsulation. A study on anthocyanin degradation in blueberry juice also demonstrated a rise in k values with increasing temperature, emphasizing the accelerating effect of temperature on degradation 47 . Furthermore, Kechinski, Guimarães, Noreña, Tessaro and Marczak 48 observed a similar trend in blueberry juice, with k values ranging from 0.064 to 2.254 x 10 − 3 (mg/L) −1 .min − 1 under heat treatment between 40–80 ºC. These findings are consistent with the results of current study. The activation energy (Ea) values ​​for the degradation of hibiscus anthocyanins copigmented with different protein isolates were calculated from the slope of the Arrhenius plot and ranged between 22.39 and 47.98 kJ/mol. These values align with those reported in the literature, including 41.1–58.0 kJ/mol 49 , 35–125 kJ/mol 50 and 49.16–77.77 kJ/mol 51 . Among the samples, the highest Ea value was observed in the RPH sample, while the lowest was in the WPH sample. The Ea of the extracts copigmented with PPH and GPH were comparable to those of the control. A higher activation energy value indicates that a greater amount of energy is required for the degradation reaction to occur, suggesting increased stability of the anthocyanins 52 . The z value indicates the temperature increase required to reduce the decimal disintegration time of a compound by a factor of 10, while maintaining a constant thermal conditions. A lower z value reflects greater sensitivity of the reaction to temperature. In this study, z values ranged from 52.65 to 112.78 K, with the lowest observed in the RP sample and the highest in the WPH sample. Typically, higher z values are associated with increased thermal resistance 46 . Previous studies on anthocyanin degradation kinetics have reported z values between 34 ºC and 57 ºC 53 , 54 . In contrast, the higher z values observed in our study suggest reduced thermal sensitivity, likely due to the stabilizing effect of copigmentation. Table 3 Kinetic parameters for the anthocyanin degradation of copigmented hibiscus extracts Sample Temperature ( o C) k*10 3 (mg/L) −1 min − 1 z value (K) E a (kJ/mol) R 2 Control 80 0.25 96.22 26.13 90 0.29 0.94 100 0.40 PPH 80 0.22 93.63 27.07 90 0.33 0.91 100 0.37 RPH 80 0.23 52.65 47.98 90 0.37 0.99 100 0.55 GPH 80 0.35 89.89 27.94 90 0.41 0.91 100 0.59 WPH 80 0.34 112.78 22.30 90 0.43 0.99 100 0.51 Control: Product using pure water instead of copigmentation agent. PPH: Hibiscus extract copigmented with pea protein isolate. RPH: Hibiscus extract copigmented with rice protein isolate. GPH: Hibiscus extract copigmented with gluten. WPH: Hibiscus extract copigmented with whey protein isolate 3.4. FTIR Fourier transform infrared spectroscopy (FTIR) is a technique used to obtain the infrared absorption or emission spectrum of a substance. FTIR shows whether hydrogen bond interactions exist in anthocyanin-protein complexes and how these interactions change the spectroscopic properties of anthocyanins and proteins 55 . The formation of copigmentation complexes is evidenced by FTIR measurements with spectra recorded in the range of 4000 − 400 cm⁻¹ (Fig. 1 ). Notable spectral changes upon copigmentation were observed, particularly in the range of 3300–3400 cm⁻¹ range, indicating hydrogen bond formation 56 . A broader in the 3200–3300 cm⁻¹ range suggest the presence of –OH groups and –NH₂, as previously reported 57 . The observed wavelength shift is attributed to -OH stresses between anthocyanin and the copigment 14 , 33 , 58 . Samples subjected to copigmentation exhibited higher wavelength in the 3000–3400 cm⁻¹ range compared to natural protein isolates, suggesting a reduction in the β-sheet content of the protein structure. It is hypothesized that the addition of anthocyanins disrupts the hydrogen bonds within the β-sheet structures of both natural and denatured proteins, allowing the anthocyanins to be retained by the altered protein matrix 59 . The samples also exhibited peaks within the 1630–1650 cm⁻¹ range (Fig. 1 ). Peaks observed between 1600 and 1680 cm⁻¹ indicate the presence of strong unconjugated C = C vibrations 60 . This region is significant for examining conformational changes in protein structures. The vibrational spectra of proteins are characterized by two main regions: amide I (1600–1700 cm⁻¹) and amide II (1480–1600 cm⁻¹). The amide I region, corresponding to the C = O stretching, is especially valuable for characterizing the secondary structure of proteins, while the amide II region corresponds to the NH/CH stretching. Within the amide I region, specific secondary structures are identified, including intermolecular β-sheet (1610–1625 cm⁻¹), native β-sheet (1625–1635 cm⁻¹), random coil/α-helix (1635–1665 cm⁻¹), β-turn (1665–1690 cm⁻¹) and antiparallel amyloid β-sheets (1690–1705 cm⁻¹) 61 . 3.5. Fluorescence spectroscopy Fluorescence spectroscopy is a spectrometric technique used to study molecular interactions by analyzing the excitation of electrons in covalent bonds through infrared radiation emitted by the instrument 62 . As shown in Fig. 2 , the fluorescence intensity of copigmented samples is significantly lower compared to that of protein isolates alone, indicating that copigmentation substantially quenches the fluorescence intensity of proteins. This quenching effect suggests a strong interaction between proteins and anthocyanins 63 . While the fluorescence intensity of native proteins was ranged between 100–1000 au, the fluorescence intensity of copigmented samples decreased drastically to between 3 and 5 au. The presence of aromatic groups in the anthocyanin molecules renders their natural fluorescence very weak, particularly around 320 nm. In contrast, proteins, exhibit intrinsic fluorescence due to the fluorescence-producing capabilities of tryptophan, tyrosine and phenylalanine residues at specific excitation wavelengths 64 . The observed decrease in fluorescence intensity in this study suggests an increase in the stability of the anthocyanin-protein complex, likely due to interactions between anthocyanins and proteins, which may alter the molecular conformation of the proteins 65 . For instance, in soy protein isolate-anthocyanin complexes, the decrease in fluorescence intensity was attributed to strong interactions between soy protein isolate and anthocyanins, leading to structural changes in the polypeptide chain and its subsequent 66 . Similarly, Khalifa, Nie, Ge, Li and Li 14 demonstrated copigmentation of mulberry anthocyanins with whey proteins reduced fluorescence intensity. While whey proteins exhibited maximum fluorescence absorption at 340 nm with an intensity of 2800 a.u., copigmentation reduced this intensity to 2200 a.u., further highlighting the interaction-induced structural changes in the protein-anthocyanin complexes. 4. Conclusion In the present study, pea protein isolate, rice protein isolate, gluten and whey protein isolate were employed as copigmentation agents, with Hibiscus sabdariffa extract was serving as the anthocyanin source. Monomeric anthocyanin losses observed during heat treatment were attributed to the formation of anthocyanin polymers, necessitating polymeric color analyses to assess the stability of copigmented anthocyanins. The polymeric color results revealed that the sample copigmented with whey protein isolate demonstrated the lowest polymeric color percentage, indicating superior preservation properties. Kinetic analysis of the total monomeric anthocyanin degradation showed that the copigmentation with rice protein isolate provided the highest Ea value, reflecting reduced reactivity. FTIR and fluorescence spectroscopy analyses provided evidence of interactions and the bond formations between proteins and anthocyanins. This study aims to enhance the stability of anthocyanins by copigmenting them with protein isolates, offering a pathway to develop more stable natural colorants. The findings contribute to a better understanding of anthocyanin-protein interaction and highlight the potential for expanding the application of anthocyanins in the food industry. However, the complexity of copigmentation and the incomplete understanding of structure-affinity relationships underscore the need for further research. Future studies should focus on systematically designing model copigmentation complexes and developing advanced analytical techniques to establish robust structure-affinity and structure-optical-property correlations. Declarations Conflict of interest The authors declared no conflict of interest. Funding The authors have not disclosed any funding. Author Contribution Vildan Eyiz: Writing – original draft, Investigation. Ismail Tontul: Writing – original draft, Supervision, Conceptualization. Selman Türker: Supervision, Conceptualization. Data availability Data will be made available on request. References M. Kucharska, J. 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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-6401806","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":459825792,"identity":"1e8636d9-aef4-4363-8349-c2a2cd79b226","order_by":0,"name":"Vildan Eyiz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYLCCDwwMCQw8YCYzEcrZGBgYZ6BoYSNCCzMPSVoM7jcfe2xTcS+Pv+fwswcMFdaJDfK9D/BrOcaWbpxzprhY4mybuQHDmfTEBjZ2A7xaJNt4zKRz2xISG84zmEkwth0GaiHgMrAWy38JifPPs3+TYPxHhBZ+NqAWxoaExA1ne4C2NBClJS3dsOdYQrHhmTNlEgnH0o3b2NLwa2FjPnzswY+ahDy5M+nbJD7UWMv2Mx/Dr4UBJR4SGIiISSLVjIJRMApGwYgGANdyPdzOGO3sAAAAAElFTkSuQmCC","orcid":"","institution":"Necmettin Erbakan University","correspondingAuthor":true,"prefix":"","firstName":"Vildan","middleName":"","lastName":"Eyiz","suffix":""},{"id":459825793,"identity":"1493c8d2-22bf-4f53-8cca-84a771a1ba97","order_by":1,"name":"İsmail Tontul","email":"","orcid":"","institution":"Necmettin Erbakan University","correspondingAuthor":false,"prefix":"","firstName":"İsmail","middleName":"","lastName":"Tontul","suffix":""},{"id":459825794,"identity":"78ced30d-9c54-43cc-a5d8-c930bb9c0cf3","order_by":2,"name":"Selman TÜRKER","email":"","orcid":"","institution":"Necmettin Erbakan University","correspondingAuthor":false,"prefix":"","firstName":"Selman","middleName":"","lastName":"TÜRKER","suffix":""}],"badges":[],"createdAt":"2025-04-08 09:38:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6401806/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6401806/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83286717,"identity":"4477d356-e023-4f1e-a21d-4e1a3d6f714e","added_by":"auto","created_at":"2025-05-22 11:44:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":29152,"visible":true,"origin":"","legend":"\u003cp\u003ePolymeric color values of control sample\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6401806/v1/726513dc8dc99f3c86bceeb8.png"},{"id":83286718,"identity":"d01f8a65-d633-4bda-b98f-8990d0f0f32f","added_by":"auto","created_at":"2025-05-22 11:44:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":134596,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 1.\u003c/strong\u003e FTIR spectra of hibiscus extract and four copigmentation complexes\u003c/p\u003e\n\u003cp\u003e(a) PPH: Hibiscus extract copigmented with pea protein isolate, PP: pea protein isolate, (b) RPH: Hibiscus extract copigmented with rice protein isolate, RP: Rice protein isolate, (c) GPH: Hibiscus extract copigmented with gluten protein, GP: Gluten protein, (d) WPH: Hibiscus extract copigmented with whey protein isolate, WP: Whey protein isolat\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6401806/v1/35d675f318a71a355ff1cba1.png"},{"id":83288288,"identity":"ab188b31-0ca5-4435-a6e8-7d5702c4425f","added_by":"auto","created_at":"2025-05-22 12:08:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":137506,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 2.\u003c/strong\u003eFluorescence spectra of hibiscus extract and four copigmentation complexes\u003c/p\u003e\n\u003cp\u003e(a) PPH: Hibiscus extract copigmented with pea protein isolate, PP: pea protein isolate, (b) RPH: Hibiscus extract copigmented with rice protein isolate, RP: Rice protein isolate, (c) GPH: Hibiscus extract copigmented with gluten protein, GP: Gluten protein, (d) WPH: Hibiscus extract copigmented with whey protein isolate, WP: Whey protein isolate\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6401806/v1/ad01d6671d1f341f78200c53.png"},{"id":87734573,"identity":"ffe95724-7f46-418d-9ec4-e50fee3d6d39","added_by":"auto","created_at":"2025-07-28 12:17:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1199180,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6401806/v1/e81a3c2f-341c-4267-b6d1-707e102ccdc9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Production of Stable Anthocyanin-Based Food Colorants from Hibiscus sabdariffa L. by Copigmentation with Different Protein Isolates","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eColorants represent a class of additives of paramount importance in the food industry, given that the color of a foodstuff has a considerable effect on its visual appeal. The classification of food colorants can be divided into two broad categories: synthetic and natural. Synthetic colorants (tartrazine, Allura red, indigo carmine, etc.) exhibit remarkable stability when exposed to various environmental factors such as temperature, pH, light and oxygen \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. However, synthetic colorants have been associated with various health concerns, including toxic effects, allergic reactions, and behavioral and neurological side effects \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Consequently, there is an increasing interest in the development of chemically stable food colorants derived from natural sources as an alternative to synthetic colorants.\u003c/p\u003e \u003cp\u003eAnthocyanins are compounds belonging to the flavonoid group that are frequently found in fruits, vegetables, grains and other plants. These compounds have been shown to possess antioxidant, antiviral and anti-aging properties. In addition, anthocyanins have been shown to have beneficial effects on health, including a reduced risk of cancer, inflammation \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, cardiovascular diseases, and diabetes \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e and a reduced obesity risk \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite the benefits, the low stability of anthocyanins when exposed to food processing and environmental factors limits their use in food formulations \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Consequently, it is imperative to implement some precautionary measures to enhance the stability of anthocyanins. Copigmentation has emerged as a promising technique for enhancing the stability of anthocyanins when subjected to conditions such as elevated temperatures, exposure to light, and the presence of oxygen \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. It is estimated that there are more than ten thousand compounds that have the potential to be utilized as copigments \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Typically, copigments are characterized by the presence of π conjugated systems, which facilitate the formation of π\u0026thinsp;\u0026minus;\u0026thinsp;π interactions. Additionally, these compounds often possess hydrogen bond donor/acceptor groups such as hydroxyl (OH) and carbonyl (C\u0026thinsp;=\u0026thinsp;O) \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eProteins have emerged as a promising copigments to enhance the stability of anthocyanins. The interaction between proteins and anthocyanins is complex and occurs through a variety of chemical interactions, including hydrophobic and hydrophilic interactions, van der Waals forces, and hydrogen bonding \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Several studies showed that whey proteins exhibit enhanced copigmentation capabilities due to their high affinity for anthocyanins \u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Chen, Guan, Zeng, Wang, Qin, Chen and He \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e used whey protein and phenolic compounds as copigmentation agents for mulberry anthocyanins in their studies and demonstrated that WPI was efficacious in mitigating thermal-induced color and anthocyanin loss. Recent studies have explored the use of plant-based proteins as anthocyanin copigments \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In a study, the effects of β-cyclodextrin, whey protein, and soy protein on anthocyanin degradation in purple-fleshed sweet potato anthocyanin extracts were investigated in model beverage systems. It was reported that the incorporation of whey protein and soy protein resulted in an increase in the thermal stability of the extracts \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In another study, the effect of three amino acids (L-phenylalanine, L-tyrosine, and L-tryptophan) and a peptide (poly-L-lysine) on the color of anthocyanins in model beverages during storage at 40\u0026deg;C and under light for seven days was investigated. The results showed that the incorporation of amino acids improved color stability, mainly through the formation of hydrogen bonds. Furthermore, Chung, Rojanasasithara, Mutilangi and McClements \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e reported that L-tryptophan had a significant impact on the color stability of purple carrot anthocyanins. In a separate study, copigmentation with soy protein isolate enhanced the stability of anthocyanins in purple corn. \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe present study is an investigation into the copigmentation effect of different plant proteins on \u003cem\u003eHibiscus sabdariffa\u003c/em\u003e L. extract. Hibiscus extract is characterized by a deep red pigmentation, attributable to the presence of anthocyanins, thus establishing its prevalence as a colorant \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The anthocyanin content of hibiscus has been documented to range from 1.7\u0026ndash;2.5% of dry weight \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The main anthocyanins in hibiscus have been identified as delphinidin-3-sambubioside and cyanidin-3-sambubioside \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The copigmentation effect of three different plant proteins (pea protein isolate, rice protein isolate, and gluten) on \u003cem\u003eHibiscus sabdariffa\u003c/em\u003e L. extract was examined comparatively to whey protein isolates. To this end, thermal stability tests were performed on samples and kinetic calculations were made to determine the most suitable copigmentation agent.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Material\u003c/h2\u003e \u003cp\u003eDried \u003cem\u003eHibiscus sabdariffa\u003c/em\u003e L. calices, obtained from a local herbalist, was used as an anthocyanin source. Rice protein isolate (Naturiga Natural Foods, Istanbul, T\u0026uuml;rkiye), pea protein isolate (Proteinocean, Ankara, T\u0026uuml;rkiye), gluten (Vatan enzymes, Istanbul, T\u0026uuml;rkiye) and whey protein isolate (PASP) (Hardline Nutrition, Kocaeli, T\u0026uuml;rkiye) were utilized as copigmentation agents. All chemicals used in the analyses were of reagent grade and obtained from Merck (Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Extraction of anthocyanins from \u003cem\u003eHibiscus Sabdariffa\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe extraction of anthocyanins from hibiscus was carried out by modifying the methodology performed by Abou-Arab, Abu-Salem and Abou-Arab \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The hibiscus was pulverized and converted into a powdered form. The powdered sample was then combined with distilled water in a 1:10 ratio at a temperature of 90\u0026deg;C. The mixture was then subjected to agitation in a mechanical mixer (DLAB-OS20-S, China) at 600 RPM for 15 minutes at 90 \u003csup\u003eo\u003c/sup\u003eC. Subsequently, the sample was subjected to agitation (100 rpm) in a water bath maintained at 24\u0026deg;C for 24 hours. Finally, the extract was filtered to remove hibiscus pulps and stored at 4\u0026ordm;C until copigmentation experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Copigmentation of anthocyanin extract with protein isolates\u003c/h2\u003e \u003cp\u003eThe copigmentation conditions of hibiscus anthocyanins and protein isolates were determined by preliminary experiments in accordance with the methodologies outlined by Li, Wang, Zhang, Yu and Chen \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and Ma and Jing \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. A combined solution of hibiscus extract and protein isolates (10 mg/mL in distilled water) were prepared at a ratio of 1:5. Subsequently, the pH value of the mixture was adjusted to 6 using a sodium hydroxide solution (1 M). The copigmentation was carried out by means of a 30-minute mixing procedure in a rotator (Isolab Laborgerate, Germany). Then, the pH value of the solution was adjusted to 3 using HCl (1 M). At this stage, four different copigmented samples were formed with different protein isolates and a control sample using distilled water in lieu of the protein solution. Initially, the bathochromic shift and hyperchromic effects of the samples obtained were determined. Subsequently, a thermal stability test was conducted, followed by analyses of monomeric anthocyanin and polymeric color, as well as FTIR and fluorescence spectroscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Bathochromic shift and hypochromic effect\u003c/h2\u003e \u003cp\u003eHyperchromic effect (the absorbance value at \u003cem\u003eλ\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) and bathochromic shift (a shift of wavelength of \u003cem\u003eλ\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) were determined according to the method of Malaj, De Simone, Quartarolo and Russo \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Thermal stability test\u003c/h2\u003e \u003cp\u003eThe samples were subjected to thermal stability testing, with temperatures ranging from 80 to 100\u0026deg;C for a duration of up to 120 minutes. Sampling was conducted at predetermined intervals, and the samples were chilled to room temperature. Subsequently, each solution was then centrifuged at 10,000 rpm for 30 minutes. The polymeric color values and total monomeric anthocyanin concentration of supernatants were determined. The data obtained from these tests were then used to calculate the kinetic parameters. The second-order reaction kinetics (determined according to kinetic calculations) (Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.) and the Arrhenius equation (Eq.\u0026nbsp;2)were employed to calculate the thermal degradation kinetic parameters of samples by Simonin \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\frac{1}{C}\\:-\\:\\frac{1}{C0}=\\:kt$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eC\u0026thinsp;=\u0026thinsp;Anthocyanin content (time t)\u003c/p\u003e \u003cp\u003eC\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;Anthocyanin content (time zero)\u003c/p\u003e \u003cp\u003ek\u0026thinsp;=\u0026thinsp;rate constant [(mg.L)\u003csup\u003e\u0026minus;1\u003c/sup\u003e.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e]\u003c/p\u003e \u003cp\u003et\u0026thinsp;=\u0026thinsp;Time (min.)\u003c/p\u003e \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" height=\"28\" width=\"347\"\u003e\u003c/p\u003e\u003cp\u003eA\u0026thinsp;=\u0026thinsp;Arrhenius constant\u003c/p\u003e \u003cp\u003eE\u003csub\u003ea\u003c/sub\u003e = Activation energy (cal/mol \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003cp\u003ek\u0026thinsp;=\u0026thinsp;rate constant [(mg.L)\u003csup\u003e\u0026minus;1\u003c/sup\u003e.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e]\u003c/p\u003e \u003cp\u003eR\u0026thinsp;=\u0026thinsp;ideal gas constant (1.987 cal/mol \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003cp\u003eT: Temperature (K)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Total monomeric anthocyanin content\u003c/h2\u003e \u003cp\u003eThe total anthocyanin content was determined by employing the pH differential method. The total anthocyanin content of the extracts was expressed as milligrams of cyanidin-3-glucoside equivalent (C3G) per 100 grams of dry matter of the sample \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Polymeric color\u003c/h2\u003e \u003cp\u003eThe determination of color intensity and polymeric color values was conducted by first diluting the samples with a KCl solution (0.025 M) or distilled water. 2.8 mL of each diluted sample was transferred into two separate cuvettes. To one of the cuvettes, 0.2 mL of K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e (20%, w/v) was added, while 0.2 mL of distilled water was added to the other. Both cuvettes were kept in the dark for 15 min. Finally, the absorbances ​​of both cuvettes were measured against distilled water at 420 nm (A\u003csub\u003e420\u003c/sub\u003e), λ\u003csub\u003emax\u003c/sub\u003e (A\u003csub\u003e518\u003c/sub\u003e) and 700 (A\u003csub\u003e700\u003c/sub\u003e) nm. Polymeric color (Eq.\u0026nbsp;3), color intensity (Eq.\u0026nbsp;4) and polymeric color ratio (Eq.\u0026nbsp;5) was calculated according to Giusti and Wrolstad \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePolymeric color = (A\u003csub\u003e420\u003c/sub\u003e-A\u003csub\u003e700\u003c/sub\u003e) + (A\u003csub\u003e518\u003c/sub\u003e-A\u003csub\u003e700\u003c/sub\u003e) x dilution factor (3)\u003c/p\u003e \u003cp\u003eColor intensity = (A\u003csub\u003e420\u003c/sub\u003e-A\u003csub\u003e700\u003c/sub\u003e) + (A\u003csub\u003e518\u003c/sub\u003e-A\u003csub\u003e700\u003c/sub\u003e) x dilution factor (4)\u003c/p\u003e \u003cp\u003ePolymeric color ratio (%) = (Polymeric color / Color intensity) x 100 (5)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Fourier transform infrared spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eFourier Transform Infrared Spectrometry analysis was determined with Thermo Scientific \u0026ndash; Nicolet iS20 (CA, USA). Spectroscopic readings were collected within the 4000\u0026thinsp;\u0026minus;\u0026thinsp;600 cm\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e wavelength range, with a resolution of 2 cm\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and a scanning speed of 1 cm/s for each spectrum, and the spectrum graph was obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Fluorescence spectroscopy\u003c/h2\u003e \u003cp\u003eFluorescence spectrometer measurements were performed with an Agilent Cary Eclipse instrument (Agilent, Germany). Emission spectra were recorded between 300 and 400 nm with an excitation wavelength of 280 nm (slit width 5 nm) in a 1.0 cm quartz cuvette.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Statistical analyses\u003c/h2\u003e \u003cp\u003eThe data were subjected to analysis of variance, and the appropriate mean separations were calculated using Duncan's Multiple Comparison Test. Statistical analyses were performed using the SAS software program (SAS Institute, Cary, NC, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Bathochromic shift and hypochromic effect\u003c/h2\u003e \u003cp\u003eCopigmentation refers to the hydrophobically oriented association of an anthocyanin chromophore with the planar electronically saturated part of the copigment. This phenomenon can be attributed to van der Waals interactions and hydrophobic effects in aqueous media that result in the 'π\u0026ndash;π' stacking of anthocyanin and copigment molecules. This association leads to an increase in absorbance within the visible range (hyperchromic effect, ΔA) and a shift of λ\u003csub\u003emax\u003c/sub\u003e wavelengths (bathochromic effect, Δλ\u003csub\u003emax\u003c/sub\u003e) \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The magnitude of the copigmentation effect is influenced by several factors, including the type and chemical structure of the copigment. This can be quantified through the use of hyperchromic effect and bathochromic shift parameters \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The hyperchromic effect generally signifies a shift in the hydration balance from the colorless hydrated hemiketal form to the colored flavylium form. Conversely, the bathochromic shift is indicative of the formation of electrostatic interactions between the pigment and the copigment \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe bathochromic shift and hyperchromic effect results for hibiscus samples copigmented with PP, RP, GP, and WP are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. As illustrated in the table, a reduction in both bathochromic shift (with the exception of BPH sample) and the hyperchromic effect was observed in samples copigmented with proteins. This finding indicates that protein isolates do not effectively function as copigmentation agents. In general, while there was an improvement in the bathochromic shift and hyperchromic effects of anthocyanin pigments copigmented with protein isolates was observed in previous studies \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, a decrease was noted in the present study. It is hypothesized that this is due to interference of proteins with the color of the hibiscus extract. In their studies, Chen, Gao, Liao, Zou, Yan and Li \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e copigmented mulberry anthocyanins with whey protein isolate and a whey protein isolate-phenolic complex. They reported that the use of protein isolates alone resulted in the weakening of anthocyanin color and that the resulting mixture was unstable and prone to precipitation at high temperatures.\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\u003eBathochromic shift and hyperchromic effect results of copigmented samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eƛ\u003csub\u003emax\u003c/sub\u003e (Bathochromic shift, wavelength (nm))\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eΔ λ\u003csub\u003emax\u003c/sub\u003e(nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eABS (Hyperchromic effect, A)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eΔA (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.964\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-41.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e514\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.098\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-44.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e518\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-48.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e518\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.448\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-26.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003ePPH: Hibiscus extract copigmented with pea protein isolate, RPH: Hibiscus extract copigmented with rice protein isolate, GPH: Hibiscus extract copigmented with gluten, WPH: Hibiscus extract copigmented with whey protein isolate\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Polymeric color\u003c/h2\u003e \u003cp\u003eThe term \u0026ldquo;polymeric color percentage\u0026rdquo; is defined as the ratio of polymeric color to color intensity. It is used as a measure of the percentage of color of a sample that can be attributed to polymerized compounds. Available evidence indicates that an increase in polymeric color is proportional to a loss of anthocyanin \u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe change in polymeric color values during the heat treatment of the control extract is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e. An increase in the percentage of polymeric color was observed in correlation with both an increase in the temperature of the heat treatment and an extension of its duration. During the heat treatment, it was observed that polymeric color values ranged from 33.22\u0026ndash;54.34%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA comparative analysis of the polymeric color values according to the protein type revealed that the polymeric color belonged to the GPH, RPH, PPH, and WPH samples, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Consequently, it can therefore be concluded that the color was effectively preserved in hibiscus extracts copigmented with whey protein isolate. According to the extant literature, previous studies have also demonstrated the efficacy of whey proteins in inhibiting the degradation of anthocyanins \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Conversely, the samples copigmented with gluten exhibited the highest polymeric color value. This phenomenon is hypothesized to be attributable to the low solubility of gluten, which leads to the turbidity of the sample \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAn increase in the polymeric color percentage was observed in response to an increase in temperature and an extension in heating duration. This phenomenon is hypothesized to be attributable to the promotion of the formation of chalcones, an intermediate product of anthocyanin degradation, at elevated temperatures \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Chalcones possess reactive properties, which enable them to combine with other components. This combination results in an increase in polymeric color \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePolymeric color contents of copigmented samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariation sources\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePolymeric color (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eProtein type\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.84\u0026thinsp;\u0026plusmn;\u0026thinsp;8.30c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41.83\u0026thinsp;\u0026plusmn;\u0026thinsp;10.79b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.86\u0026thinsp;\u0026plusmn;\u0026thinsp;5.73a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.86\u0026thinsp;\u0026plusmn;\u0026thinsp;5.47d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTemperature (\u003c/b\u003e\u003csup\u003e\u003cb\u003eo\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40.01\u0026thinsp;\u0026plusmn;\u0026thinsp;8.66b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40.21\u0026thinsp;\u0026plusmn;\u0026thinsp;9.04b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.08\u0026thinsp;\u0026plusmn;\u0026thinsp;9.07a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHeating time (min)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32.47\u0026thinsp;\u0026plusmn;\u0026thinsp;7.70d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.85\u0026thinsp;\u0026plusmn;\u0026thinsp;9.80c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39.76\u0026thinsp;\u0026plusmn;\u0026thinsp;5.17c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.51\u0026thinsp;\u0026plusmn;\u0026thinsp;6.54b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49.91\u0026thinsp;\u0026plusmn;\u0026thinsp;6.70a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;std error, different letters in same column shows significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). PPH: Copigmented with pea protein, RPH: Copigmented with rice protein, GPH: Copigmented with gluten protein, WPH: Copigmented with whey protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Total monomeric anthocyanin content\u003c/h2\u003e \u003cp\u003eIn order to determine the effect of copigmentation on the anthocyanin content of the hibiscus extract, the samples were subjected to a thermal stability test, and kinetic calculations were performed. The degradation rate constants (k), correlation coefficients (R\u0026sup2;), z values and activation energy (Ea) of anthocyanin degradation were calculated.\u003c/p\u003e \u003cp\u003eThe degradation of total monomeric anthocyanins in the samples during heat treatment found to follow a second order kinetic model, consistent with findings reported in other studies \u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. This behavior is attributed to the reaction of electropositive aglycones (cyanidin\u0026thinsp;+\u0026thinsp;287, delphinidin\u0026thinsp;+\u0026thinsp;303, pelargonidin\u0026thinsp;+\u0026thinsp;271 and peonidin\u0026thinsp;+\u0026thinsp;301) in accordance with the second-order kinetics \u003csup\u003e\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe degradation rate constants (k) of the samples at different temperatures ranged from 0.22\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e to 0.59\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (mg/L)\u003csup\u003e\u0026minus;1\u003c/sup\u003e.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with the rate of anthocyanin degradation increasing as temperature rose (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Similarly, Kanha, Surawang, Pitchakarn and Laokuldilok \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e reported an increase in degradation rate constants with elevated temperature when black rice extract subjected to copigmentation and encapsulation. A study on anthocyanin degradation in blueberry juice also demonstrated a rise in k values with increasing temperature, emphasizing the accelerating effect of temperature on degradation \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Furthermore, Kechinski, Guimar\u0026atilde;es, Nore\u0026ntilde;a, Tessaro and Marczak \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e observed a similar trend in blueberry juice, with k values ranging from 0.064 to 2.254 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (mg/L)\u003csup\u003e\u0026minus;1\u003c/sup\u003e.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under heat treatment between 40\u0026ndash;80 \u0026ordm;C. These findings are consistent with the results of current study.\u003c/p\u003e \u003cp\u003eThe activation energy (Ea) values ​​for the degradation of hibiscus anthocyanins copigmented with different protein isolates were calculated from the slope of the Arrhenius plot and ranged between 22.39 and 47.98 kJ/mol. These values align with those reported in the literature, including 41.1\u0026ndash;58.0 kJ/mol \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, 35\u0026ndash;125 kJ/mol \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e and 49.16\u0026ndash;77.77 kJ/mol \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Among the samples, the highest Ea value was observed in the RPH sample, while the lowest was in the WPH sample. The Ea of the extracts copigmented with PPH and GPH were comparable to those of the control. A higher activation energy value indicates that a greater amount of energy is required for the degradation reaction to occur, suggesting increased stability of the anthocyanins \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe z value indicates the temperature increase required to reduce the decimal disintegration time of a compound by a factor of 10, while maintaining a constant thermal conditions. A lower z value reflects greater sensitivity of the reaction to temperature. In this study, z values ranged from 52.65 to 112.78 K, with the lowest observed in the RP sample and the highest in the WPH sample. Typically, higher z values are associated with increased thermal resistance \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Previous studies on anthocyanin degradation kinetics have reported z values between 34 \u0026ordm;C and 57 \u0026ordm;C \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. In contrast, the higher z values observed in our study suggest reduced thermal sensitivity, likely due to the stabilizing effect of copigmentation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKinetic parameters for the anthocyanin degradation of copigmented hibiscus extracts\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=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTemperature (\u003csup\u003eo\u003c/sup\u003eC)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ek*10\u003csup\u003e3\u003c/sup\u003e (mg/L)\u003csup\u003e\u0026minus;1\u003c/sup\u003emin\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ez value (K)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eE\u003csub\u003ea\u003c/sub\u003e (kJ/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eR\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e96.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e26.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ePPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e93.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e27.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eRPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e52.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e47.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eGPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e89.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e27.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eWPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e112.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e22.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eControl: Product using pure water instead of copigmentation agent. PPH: Hibiscus extract copigmented with pea protein isolate. RPH: Hibiscus extract copigmented with rice protein isolate. GPH: Hibiscus extract copigmented with gluten. WPH: Hibiscus extract copigmented with whey protein isolate\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4. FTIR\u003c/h2\u003e \u003cp\u003eFourier transform infrared spectroscopy (FTIR) is a technique used to obtain the infrared absorption or emission spectrum of a substance. FTIR shows whether hydrogen bond interactions exist in anthocyanin-protein complexes and how these interactions change the spectroscopic properties of anthocyanins and proteins \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe formation of copigmentation complexes is evidenced by FTIR measurements with spectra recorded in the range of 4000\u0026thinsp;\u0026minus;\u0026thinsp;400 cm⁻\u0026sup1; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notable spectral changes upon copigmentation were observed, particularly in the range of 3300\u0026ndash;3400 cm⁻\u0026sup1; range, indicating hydrogen bond formation \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. A broader in the 3200\u0026ndash;3300 cm⁻\u0026sup1; range suggest the presence of \u0026ndash;OH groups and \u0026ndash;NH₂, as previously reported \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The observed wavelength shift is attributed to -OH stresses between anthocyanin and the copigment \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Samples subjected to copigmentation exhibited higher wavelength in the 3000\u0026ndash;3400 cm⁻\u0026sup1; range compared to natural protein isolates, suggesting a reduction in the β-sheet content of the protein structure. It is hypothesized that the addition of anthocyanins disrupts the hydrogen bonds within the β-sheet structures of both natural and denatured proteins, allowing the anthocyanins to be retained by the altered protein matrix \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe samples also exhibited peaks within the 1630\u0026ndash;1650 cm⁻\u0026sup1; range (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Peaks observed between 1600 and 1680 cm⁻\u0026sup1; indicate the presence of strong unconjugated C\u0026thinsp;=\u0026thinsp;C vibrations \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. This region is significant for examining conformational changes in protein structures. The vibrational spectra of proteins are characterized by two main regions: amide I (1600\u0026ndash;1700 cm⁻\u0026sup1;) and amide II (1480\u0026ndash;1600 cm⁻\u0026sup1;). The amide I region, corresponding to the C\u0026thinsp;=\u0026thinsp;O stretching, is especially valuable for characterizing the secondary structure of proteins, while the amide II region corresponds to the NH/CH stretching. Within the amide I region, specific secondary structures are identified, including intermolecular β-sheet (1610\u0026ndash;1625 cm⁻\u0026sup1;), native β-sheet (1625\u0026ndash;1635 cm⁻\u0026sup1;), random coil/α-helix (1635\u0026ndash;1665 cm⁻\u0026sup1;), β-turn (1665\u0026ndash;1690 cm⁻\u0026sup1;) and antiparallel amyloid β-sheets (1690\u0026ndash;1705 cm⁻\u0026sup1;) \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Fluorescence spectroscopy\u003c/h2\u003e \u003cp\u003eFluorescence spectroscopy is a spectrometric technique used to study molecular interactions by analyzing the excitation of electrons in covalent bonds through infrared radiation emitted by the instrument \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the fluorescence intensity of copigmented samples is significantly lower compared to that of protein isolates alone, indicating that copigmentation substantially quenches the fluorescence intensity of proteins. This quenching effect suggests a strong interaction between proteins and anthocyanins \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. While the fluorescence intensity of native proteins was ranged between 100\u0026ndash;1000 au, the fluorescence intensity of copigmented samples decreased drastically to between 3 and 5 au. The presence of aromatic groups in the anthocyanin molecules renders their natural fluorescence very weak, particularly around 320 nm. In contrast, proteins, exhibit intrinsic fluorescence due to the fluorescence-producing capabilities of tryptophan, tyrosine and phenylalanine residues at specific excitation wavelengths \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. The observed decrease in fluorescence intensity in this study suggests an increase in the stability of the anthocyanin-protein complex, likely due to interactions between anthocyanins and proteins, which may alter the molecular conformation of the proteins\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. For instance, in soy protein isolate-anthocyanin complexes, the decrease in fluorescence intensity was attributed to strong interactions between soy protein isolate and anthocyanins, leading to structural changes in the polypeptide chain and its subsequent \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Similarly, Khalifa, Nie, Ge, Li and Li \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e demonstrated copigmentation of mulberry anthocyanins with whey proteins reduced fluorescence intensity. While whey proteins exhibited maximum fluorescence absorption at 340 nm with an intensity of 2800 a.u., copigmentation reduced this intensity to 2200 a.u., further highlighting the interaction-induced structural changes in the protein-anthocyanin complexes.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn the present study, pea protein isolate, rice protein isolate, gluten and whey protein isolate were employed as copigmentation agents, with \u003cem\u003eHibiscus sabdariffa\u003c/em\u003e extract was serving as the anthocyanin source. Monomeric anthocyanin losses observed during heat treatment were attributed to the formation of anthocyanin polymers, necessitating polymeric color analyses to assess the stability of copigmented anthocyanins. The polymeric color results revealed that the sample copigmented with whey protein isolate demonstrated the lowest polymeric color percentage, indicating superior preservation properties. Kinetic analysis of the total monomeric anthocyanin degradation showed that the copigmentation with rice protein isolate provided the highest Ea value, reflecting reduced reactivity. FTIR and fluorescence spectroscopy analyses provided evidence of interactions and the bond formations between proteins and anthocyanins. This study aims to enhance the stability of anthocyanins by copigmenting them with protein isolates, offering a pathway to develop more stable natural colorants. The findings contribute to a better understanding of anthocyanin-protein interaction and highlight the potential for expanding the application of anthocyanins in the food industry. However, the complexity of copigmentation and the incomplete understanding of structure-affinity relationships underscore the need for further research. Future studies should focus on systematically designing model copigmentation complexes and developing advanced analytical techniques to establish robust structure-affinity and structure-optical-property correlations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declared no conflict of interest.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors have not disclosed any funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eVildan Eyiz: Writing \u0026ndash; original draft, Investigation. Ismail Tontul: Writing \u0026ndash; original draft, Supervision, Conceptualization. Selman T\u0026uuml;rker: Supervision, Conceptualization.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eM. Kucharska, J. 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Jiang, Food Chem. \u003cb\u003e245\u003c/b\u003e, 871\u0026ndash;878 (2018)\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":"protein-anthocyanin interaction, color stability, colorants, protein isolates","lastPublishedDoi":"10.21203/rs.3.rs-6401806/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6401806/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, different proteins [pea protein isolate (PP), rice protein isolate (RP), gluten (GP) and whey protein isolate (WP)] were co-pigmented with \u003cem\u003eHibiscus sabdariffa\u003c/em\u003e L. anthocyanins and bathochromic shift, hyperchromic effect and thermal stability tests were performed on the samples obtained. The polymeric color percentage values, which are indicative of anthocyanin degradation during both processing and storage, showed that the samples WPH (%35.86) and RPH (%38.84) exhibited superior anthocyanin stability. The WPH samples provided the highest z values (112.78 K). Furthermore, the RPH samples demonstrated the highest Ea values (47.98 kJ/mol). The FTIR spectrums revealed the presence of prominent peaks at wavelengths of 3000\u0026ndash;3500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (-OH tension) and 1600\u0026ndash;1650 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O tension). The observed fluctuations suggest the presence of interactions between anthocyanins and proteins. Copigmentation process caused a significant reduction in fluorescence intensity. This suggests that there is strong interaction between proteins and anthocyanins. It was observed that hibiscus extracts copigmented with protein isolates, particularly whey and rice protein isolates, were able to increase anthocyanin stability.\u003c/p\u003e","manuscriptTitle":"Production of Stable Anthocyanin-Based Food Colorants from Hibiscus sabdariffa L. by Copigmentation with Different Protein Isolates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-22 11:44:39","doi":"10.21203/rs.3.rs-6401806/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":"9daf1256-d944-4174-b04d-a13bfac0ef7c","owner":[],"postedDate":"May 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-28T12:09:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-22 11:44:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6401806","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6401806","identity":"rs-6401806","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}