Enhancement of retrograded maize amylopectin in aged starch paste by screening Monascus strains under solid fermentation

Enhancement of retrograded maize amylopectin in aged starch paste by screening Monascus strains under solid fermentation | 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 Enhancement of retrograded maize amylopectin in aged starch paste by screening Monascus strains under solid fermentation Jiarui Yu, Zengfang Guo, Yuxian Lai, Yu Gou, Xijun Lian This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7928313/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Jan, 2026 Read the published version in Journal of Polymers and the Environment → Version 1 posted 13 You are reading this latest preprint version Abstract Retrograded starch (type 3 resistant starch), known for its laxative properties, has garnered significant research interest. Nevertheless, the inherently low levels of retrograded starch in aged starch paste restrict its practical applications. Monascus strains named as M1, M2, M3, M4, and M5 were isolated from various red mold rice produced in China and were employed to enhance contents of retrograded maize amylopectin in aged starch paste. The results demonstrated that optimal addition of aged starch and coxi seed was observed at a 1:1 ratio in a solid medium. The highest content of retrograded maize amylopectin in aged starch was achieved after 15 days of fermentation by strain M2 at 32 ℃, increasing from 39.5% to 73.1%, which represents an 85.1% enhancement. The results of FT-IR, 13C solid-state NMR, XRD and DSC revealed that the ω-gliadin at β-sheet and β-turn state served as a valuable nitrogen source for Monascus M2 growth. The maize amylopectin in the amorphous region were decomposed by Monascus . The crystals for retrograded maize amylopectin were characterized by diffraction angles at 2θ 19.1°. This study proposes a novel way to enhance the retrograded starch content in aged starch paste with no environmental pollution, thereby broadening the application potential of gluten. Monascus purpureus retrograded maize amylopectin contents aged starch paste nitrogen source Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Retrograded starches in aged starch paste are functional foods formed by reassociating disaggregated amylose and amylopectin chains in a gelatinized starch paste to form an ordered structure. This process makes them resistant to hydrolysis by amylase. It is well-known that resistant starches have many beneficial effects, including laxative effects [ 1 ], the alleviation of diabetes [ 2 ], the control of body weight [ 3 ], and the improvement of immunity [ 4 ]. However, only retrograded starch, not all aged starch paste, is resistant to hydrolysis by amylase, and conventional aged starch contains only about 20% retrograded starch [ 5 , 6 ]. To meet the recommended daily intake of 30 grams of dietary fiber, an adult must consume 150 grams of dry starch daily [ 7 ]. This is not a practical option. It is, therefore, of great importance to enhance the contents of retrograded starch in aged starch paste as to facilitate the commercialization of such functional foods. The primary purification approach is enzymatic hydrolysis. However, the glucose and maltose obtained through enzymatic hydrolysis are difficult to completely wash off, such washing also bring environmental pollution. Meanwhile, the laxative effect of the acquired high-purity starch in vivo is not as pronounced as expected. This may be due to the low molecular weight of the starch within the residual retrograded starch. These retrograded starches might be partially dissolved in the alkaline environment of the small intestine and hydrolyzed by enzymes. Furthermore, our research indicates that gliadin fractions can enhance the retrogradation of amylopectin [ 8 ]. The retrogradation rate of maize amylopectin has been observed to increase to more than 40%. The study of the laxative function of retrograded starch reveals that the resistance to decomposition of retrograded amylopectin is more robust than that of retrograded starch or retrograded amylose. Nevertheless, the retrograded amylopectin content in aged starch paste remains insufficient for industrial applications. Recent studies have demonstrated that Monascus can be cultivated in retrograded maize starch in winter outdoor air [ 9 ]. This process could enhance the contents of retrograded maize amylopectin in aged starch paste, which contains gliadin fractions. The reasons for selecting Monascus as the fermentation strain are that it possesses high activity of fermentation amylases and proteases, low acid production, a strong ability to resist the growth of miscellaneous bacteria, ease of industrial production, and has been utilized in the production of red yeast rice in China for thousands of years. Monascus purpureus is a tiny filamentous saprophytic fungus that produces a variety of enzymatic substances, including amylolytic enzymes [ 10 ], esterification enzymes [ 11 ], proteases [ 12 ] and others. The saccharifying enzyme, or α-1,4-glucan glucohydrolase, hydrolyzes α-1,4 and α-1,6 glycosidic linkages in starch to produce glucose, which is vital for Monascus growth [ 13 ]. Thus, Monascus purpureus can thrive on various substrates. Retrograded maize amylopectin containing gliadin exhibits high viscosity in water, impeding fermentation oxygen supply for Monascus . In contrast to rice, coix seed has a lower viscosity post-gelation, making the culture medium with retrograded maize amylopectin more permeable and favorable for Monascus growth. Moreover, coix seed is traditionally used in China to treat rheumatism, warts, chapped skin, and as an anti-helminthic and anti-inflammatory agent [ 14 ]. Retrograded starches in aged starch paste are functional foods that are formed by the reassociation of disaggregated amylose and amylopectin chains in a gelatinized starch paste to form an ordered structure. This paper aims to increase content of retrograded maize amylopectin containing gliadin fractions in aged starch paste by metabolizing non-retrograded amylopectin through the growth of different Monascus . At the same time, these surplus gliadin fractions could be utilized as a nitrogen source for solid fermentation. The best strain and optimum conditions for fermentation are determined, and a pattern for enhancing the contents of retrograded maize amylopectin in aged starch paste has been established based on the results of infrared spectroscopy (IR), 13 C solid-state nuclear magnetic resonance ( 13 C solid-state NMR), X-ray diffraction and differential thermal analysis (DSC). The findings in the paper demonstrate a novel way to enhance the contents of retrograded starch in aged starch paste. Materials and methods Materials The gluten (9.0% moisture, 7.0% lipids, 12.8% carbohydrates, 77.5% protein) was sourced from Henan Midaner Trading Co., Ltd (Xinzheng City, Henan Province, China). Alcohol was obtained from Henan Xinheyang Alcohol Co., Ltd (Mengzhou City, Henan Province, China). Maize starch was provided by Henan Enmiao Food Co., Ltd. (Beijing City, China); coix seed by Bozhou Wenshun Pharmaceutical Co., Ltd. (Bozhou City, China); Tween-80 by Tianjin Zhiyuan Chemical Reagent Co., Ltd. (Tianjin City, China). Soluble starch, maltose, peptone and agar were supplied by Tianjin Chemical Reagent Co., Ltd. (Tianjin City, China), while sodium chloride, hydrochloric acid, sodium hydroxide, low- temperature amylase, and iodine solution were form Tianjin Fengchuan Chemical Reagent Co., Ltd. (Tianjin City, China). Red mold rice was purchased from Diyuan Monascus Factory, (Gutian County, Fujian Province, 2023), Chengjiu Monascus Co., Ltd. (Gutian County, Fujian Province, 2023), and Shandong Zhonghui Biotechnology Co., Ltd. (Huimin County, Shandong Province, 2023) in China. Methods Isolation of gliadin fractions The isolation of gliadin fractions was performed in accordance with the methods described in the literature [ 15 , 16 ]. Gluten powder was dissolved in 65% ethanol at a ratio of 1 : 30 (g : mL) and continuously stirred in a water bath at 35 ℃ for 3 h. Following this, the solution was maintained at room temperature (15–28 ℃) for 3 h before being centrifuged at 4000×g for 5 min to obtain the first supernatant. The first supernatant was then subjected to coagulation and sedimentation at 4 ℃ for 24 h, yielding gliadin 1 (primarily ω-gliadin). The residual second supernatant was stored at -18 ℃ for 24 h and subsequently thawed at room temperature (15–28 ℃), resulting in the formation of a precipitate designated as gliadin 3 (primarily αβγ-gliadin). The preparation and purification of maize amylopectin Maize amylopectin was separated and purified according to reference with minor modifications [ 17 ]. Maize starch was blended with deionized water in a 1:5 ratio, and the solutions were heated to 70°C for 90 minutes to promote granule swelling. After cooling to room temperature (15 ~ 28 ℃), the solution was frozen for over 12 h. Upon thawing, the solutions were centrifugated (3500×g for 3 min) and mixed with a 0.5% NaCl solution (g/v). Following a 20-minute stirring, it was centrifuged again (3500×g for 3 minutes) to yield crude maize amylopectin, which was washed multiple times with 0.5% NaCl until no blue color appeared upon iodine titration, indicating the removal of amylose. Residual NaCl was eliminated by washing with deionized water. Lipase (1500 U/g) was then added to hydrolyze lipids bound to maize amylopectin at 40 ℃ for 12 h. The pH was adjusted to 8.0 with 2.0 mol·L⁻¹ NaOH, and alkali protease (20,000 U/g) was introduced to hydrolyze proteins. The final maize amylopectin was obtained by centrifugation (at 3500 ×g for 10 min) followed by washing with deionized water to remove residual fatty acids and amino acids, resulting in purified maize amylopectin. The preparation of aged starch paste containing gliadin fractions Wet gliadin 1, gliadin 3, and maize amylopectin were mixed in a of 4 : 1 : 95 mass ratio and stirred at 50 ℃ for 10 min in a water bath. The mixtures were then autoclaved at 105 ℃ for 20 min and kept at 4 ℃ for 7 d to obtain aged starch paste containing gliadin fractions. Screening of Monascus strains Monascus strains named as M1, M2, M3, M4, and M5 were isolated from five red mold rice. M1 was sourced from Diyuan Monascus Factory, (Gutian County, Fujian Province, China, 2023), M2, M3, and M4 from Chengjiu Monascus Co., Ltd. (Gutian County, Fujian Province, China, 2023), and M5 from Shandong Zhonghui Biotechnology Co., Ltd. (Huimin County, Shandong Province, China, 2023). The samples (0.1 g) were dissolved in 6 mL of Tween 80 solvent and diluted with sterilized water to concentrations of 1/10, 1/100, 1/1000, 1/10000, and 1/100000. These Monascus samples (0.1 mL) were then inoculated onto the surface of the plate culture medium in sterilized Petri dishes and incubated at 32 ℃ for 4 days. Well-grown Monascus mycelium was scraped and cultured on a medium composed of 5 g soluble starch, 4 g maltose, 3 g peptone, 2 g agar and 86 g deionized water (sterilized at 121 ℃ for 30 minutes), using an inoculation ring. The suspected Monascus strains were separated and purified on the culture medium through 3 to 5 iterations until single colonies appeared. The strains were incubated at 32 ℃ for 4 days and subsequently sub-cultured in test tube slants over five generations to yield strains suitable for further experimentation. M2 was genetically identified as Monascus purpureus by the General Microbiology Center of China Microbial Culture Preservation and Management Committee (CGMCC). Solid-state fermentation Solid-state fermentation was carried out according to the reference with specific modifications [ 18 ]. The growth conditions for Monascus purpureus were initially assessed through a pre-experiment. Soaked coix seeds and retrograded maize amylopectin were dried at 80 ℃ for 6 h before being mixed with sterile deionized water. After drying, a high-speed grinder crushed the retrograded maize amylopectin through a 100-mesh sieve. The solid culture medium was prepared with soaked coix seeds, retrograded maize amylopectin and sterile deionized water in a mass ratio of 3:3 :4 (g/g/v), and sterilized at 110 ℃ for 40 min. After cooling, 2 ml of deionized water was added to the slant culture medium to dissolve the strains, which were then transferred into a watering can. Finally, the Monascus strains were sprayed onto the solid culture medium in triangular flasks and incubated at 32 ℃ in a thermostat. Determination of retrogradation rate The retrogradation rate of maize amylopectin was determined using the method from reference [ 19 ]. The solid culture medium was dried in an oven at 60 ℃ until a constant weight was achieved, then pulverized into powder. Powder samples (m 1 ) were mixed with deionized water and low-temperature α-amylase for enzymatic hydrolysis of non-retrograded maize amylopectin at 45 ℃ for 2 h. Subsequently, the solutions were centrifuged at 3500 ×g for 10 min. The precipitate was dried in an oven at 60 ℃ until a constant weight was reached and weighed to obtain a mass of m 2 . Retrogradation rate = (m 2 /m 1 ) × 100% FT-IR spectrum analysis Samples weighing 0.8 mg were homogeneously mixed with 150 mg of spectroscopic-grade potassium bromide (KBr) and subsequently compressed into pellets under controlled conditions. Spectroscopic data were acquired using a Fourier-transform infrared spectrometer (Perkin-Elmer, Buckinghamshire, UK). The samples were scanned over the wavenumber range of 4000 to 400 cm − 1 with a nominal resolution of 4 cm − 1 . 13 C solid-state NMR spectroscopy The samples were placed in a sealed pencil-type (5-mm) zirconia rotor and analyzed using a JEOL ECZ600R 600 MHz spectrometer, featuring a resonance frequency of 150.87 kHz , corresponding to a 90° pulse width of 2.4 µs. A 4 mm double resonance HX CP/MAS (cross polarization/magic angle rotation) probe was employed, with the magic angle rotation (MAS) speed automatically controlled between 9–12 kHz . The temperature range for the test was 28–120 ℃. X-ray diffraction (XRD) analysis X-ray diffraction (XRD) patterns of the samples were obtained using a D/MAX-2500 diffractometer (Rigaku, Japan) with copper as the target material. The operating voltage and current were set to 40 kV and 40 mA, respectively. The angular velocity ranged from 0 o to 60 o with a step size of 0.02 o . Differential scanning calorimetry (DSC) analysis Samples weighing 5.0 mg were sealed in aluminum pans and stored at 4 ℃ overnight. After an equilibrium period of 1 h at room temperature (15 ~ 28 ℃), the samples were heated from 25 ℃ to 235 ℃ at a rate of 10 ℃ per minute using a differential scanning calorimeter (DSC404C, Netzsch Instruments NA LLC; Burlington, MA). The peak temperature ( Tp ) and enthalpy of melting ( ΔH ) were calculated using a universal analysis program. Statistical analysis Data were presented as mean ± standard deviation from triplicate measurements. Statistical significance was assessed using a two-sample ANOVA t-test, conducted with the Microsoft Excel software and IBM SPSS 27.0 software. Results and discussion Enhancement of retrograded maize amylopectin contents by different Monascus strains The colonial morphology of different Monascus strains with distinct growth characteristics on PDA medium in Fig. 1 suggested preliminary classification into different species. Obvious aerial mycelium was observed in all strains after two days of growth. With the exception of M2, all other strains exhibited significant pigment secretion after three days of growth. Unlike other Monascus , the mycelium at both the center and edge of the colonies of M2 and M4 appeared white, suggesting that the growth process of these two strains primarily involves the secretion of extracellular pigments. Notably, the strains M2, M3, and M4 were sourced from red yeast rice produced by the same company (Chengjiu Monascus Co., Ltd). Such results indicated that the production process of red yeast rice in enterprises involved the co-fermentation of various strains of Monascus , resulting in a complex and integrated product. M3 had been identified as Monascus purpureus by us [ 20 ] and M2 might also be Monascus purpureus based on the analysis of the mycelial growth morphology [ 21 ]. The effects of fermentation on the contents of retrograded maize amylopectin-containing gliadin in aged starch paste by various Monascus strains were presented in Table 1 . The results indicated a significant increase in contents of retrograded maize amylopectin in aged starch paste with extended fermentation times. Notably, the greatest increase occurred on 6th day, where the content rose from 39.5% in the control group to over 52.0% in experimental group. At this stage, the sensory evaluation indicated that bitterness levels remained relatively low. The M2 strain, after 15 days of fermentation, achieved the highest content of 73.1%, representing an 85.1% increase compared to the control. This content exceeded ones (55%) achieved with debranching enzymes combined with moist heat treatment [ 22 ]. This suggested that strain M2 was particularly effective in enhancing the contents of retrograded maize amylopectin in aged starch paste. The mechanism behind this enhancement will be discussed in relation to FT-IR, 13 C solid-state NMR, XRD, and DSC results. Figure 1 The colonial morphology of different Monascus strains on PDA medium Table 1 The effects of fermentation on the contents of retrograded maize amylopectin containing gliadin in aged starch paste by different Monascus strains (%) Fermentation times (days) Monascus strains M1 M2 M3 M4 M5 0 39.5 ± 0.0 f 39.5 ± 0.0 e 39.5 ± 0.0 d 39.5 ± 0.0 d 39.5 ± 0.0 f 3 41.9 ± 0.0 e 44.0 ± 0.9 d 39.9 ± 0.6 d 39.9 ± 0.002 d 42.9 ± 0 e 6 54.8 ± 0.1 d 62.2 ± 0.1 c 52.9 ± 0.5 c 63.1 ± 0.008 c 55.7 ± 0.2 d 9 62.1 ± 0.3 c 69.3 ± 0.2 b 66.5 ± 0.3 b 64.6 ± 0.002 b 62.9 ± 0.1 c 12 69.0 ± 0.6 b 70.6 ± 0.0 b 68.4 ± 0.3 a 65.2 ± 0.003 b 68.8 ± 0.3 b 15 72.1 ± 0.6 a 73.1 ± 0.6 a 69.2 ± 0.5 a 69.9 ± 0.002 a 72.5 ± 0.1 a Note: Different alphabets in a column indicate significant differences at P < 0.05 FT-IR spectra of retrograded maize amylopectin before and after fermentation by M2 The FT-IR spectra of aged starch paste before and after fermentation by M2 at different times was showed in Fig. 2 . The bands at 3431.2/3407.6/3408.6 cm -1 corresponded to the N-H and O-H stretching vibration [ 8 ]. The observed lower wavenumber shifts in Fig. 2 a-c indicated enhanced hydrogen bonding among maize amylopectin among amylopectin molecules and between amylopectin and gliadin [ 23 ], likely due to the metabolism of the small molecular starch and protein that disrupt hydrogen bond formation among the larger macromolecules during fermentation. A weak band was observed at 1735.6 cm -1 in Fig. 1a, which indicated the presence of lipids in a solid medium [ 24 ]. The intensification of the signal in Fig. 1b and the disappearance of the band in Fig. 1c indicated that these lipids, initially bound to protein in coix seeds, were fully metabolized during fermentation. They potentially aided Monascus growth. The strong vibrations at 1640.2/1654.1/1647.9 cm -1 and 1535.1/1538.0/1541.3 cm -1 were attributed to amide I and II of gliadin fractions [ 25 ]. The changes in infrared absorption intensities during fermentation indicated that gliadin fractions in aged starch paste, possessing specific secondary structures, were beneficial for the growth of Monascus . Note a. Fermented for 0 day; b. Fermented for 6 days; c. Fermented for 15 days The results in Table 2 showed that the gliadin fractions with β-sheet conformation in aged starch paste were reduced during fermentation, providing a nitrogen source that supported Monascus growth. Table 2 Secondary structure and thermal properties of the medium at different fermentation stages Samples α-helix (%) β-sheet (%) β-turn (%) random coil (%) T p (℃) ΔH (J/g) Day 0 19.57 43.96 16.13 20.34 107.16 ± 0.50 a 173.04 ± 0.34 a Day 6 21.67 43.79 11.92 22.62 97.06 ± 0.30 c 115.30 ± 1.99 b Day 15 22.07 42.49 12.56 22.88 100.70 ± 0.13 b 118.06 ± 0.65 b Note: Different alphabets in a column indicate significant differences at P < 0.05 Based on the secondary structure of gliadin fractions in reference [ 15 ], the reduced gliadin should be ω-gliadin. The fact that M2 could grow on a medium with ω-gliadin as the sole nutrient source also supported the speculation (Data not shown). The results in Fig. S1 demonstrated that the additive amounts of aged starch paste in medium significantly influenced Monascus growth, with optimal effects observed at a 1:1 ratio of the aged starch and coix seed. Both higher and lower additive amount diminished this facilitative effect, suggesting that an appropriate amount of ω-gliadin enhanced Monascus M2 growth. The results in Fig. S2 revealed that the ω-gliadin more significantly promoted the growth of M2, and the red color of the culture medium manifested conspicuously after just three days of fermentation, indicating a much faster growth rate compared to existing literature [ 18 ](Jiang et al., 2024). Additionally, the results in Table 2 also described that the enhanced intensities of amide I and II in Fig. 2 b and c corresponded to the increase of α-helix and random coil among gliadin fractions. The bands at 1023.0/1023.5 cm − 1 (Fig. 1a/b) and ~ 1081/1040.4 cm − 1 (Fig. 2 c) corresponded to the amorphous and crystallization regions (in inside and outside of granule) of maize amylopectin, respectively [ 26 ]. The presence of the band at 1041.5 cm − 1 in Fig. 2 c conformed the findings in Table 1 , indicating that Monascus fermentation greatly enhanced the contents of retrograded maize amylopectin in aged starch paste. The mechanism by which Monascus fermentation enhanced the content of retrograded starch in aged maize amylopectin involved the decomposition and metabolism of amorphous maize amylopectin that did not contribute to retrogradation in the aged starch. 13 C NMR spectra of aged starch paste before and after fermentation by M2 The 13 C NMR spectra of aged starch paste before and after fermentation by M2 was displayed in Fig. 3 . According to the literature [ 15 , 27 – 29 ], the resonances of gliadin in Fig. 3 were assigned as follows: 174.7/174.8/174.6 ppm (Q δ of Gln), 128.8/128.9 ppm (Yε of Tyr), 103.2/103.5/102.9 ppm (C1 of maize amylopectin and starch in coxi seed), 82.5 ppm (C4 of maize amylopectin and starch in coxi seed), 72.7/72.5 ppm (C2,3,5 of maize amylopectin and starch in coxi seed), 62.2/61.9 ppm (C6 of maize amylopectin and starch in coxi seed), 30.3/30.1 ppm (Q γ of Gln, P β of Pro), 25.7/25.4 ppm (P γ of Pro, L γ of Leu). When gliadin interacted with maize amylopectin in Fig. 3 a, the nuclear magnetic resonance (NMR) signals of all chemical bonds in gliadin, except for the amide bond, were shielded. As the molecular weight of amylopectin decreased or its crystalline regions increased during fermentation in Fig. 3 b and c, the resonance of all carbon atoms shifted toward the high field. This finding offered a reliable and effective NMR-based screening approach to distinguish structural changes in amylopectin molecules. The C4 nuclear magnetic resonance (NMR) signal at 82.5 ppm in Fig. 3 a corresponded to the starch located in the amorphous region [ 29 ]. The disappearance of this signal in Fig. 3 b and c strongly supported the catabolism of maize amylopectin in the amorphous region during the fermentation process by Monascus , thereby validating the accuracy of the infrared spectroscopy analysis in Fig. 2 . The heightened intensities of resonances for Q γ / P β in Fig. 3 b and c suggested that they were primarily embedded at β-sheet and β-turn became more exposed as Monascus hydrolyzed the former during fermentation. This indicated that gliadin fractions at β-sheet and β-turn state served as a valuable nitrogen source for Monascus growth, which were crucial for regulating Monascus solid fermentation. Note a. Fermented for 0 day; b. Fermented for 6 days; c. Fermented for 15 days XRD patterns of aged starch paste fermented by M2 for varying durations The X-ray diffraction patterns of aged starch paste fermented by M2 for varying durations were shown in Fig. 4 . The diffraction angles for aged starch pastes in Fig. 4 a differed from the reference values for gliadin [ 8 ] or retrograded maize amylopectin (2θ at ~ 17°, 20°, 22°) [ 19 ]. The characteristic diffraction angles of aged starch in Fig. 4 a for 0-day fermentation were observed at 2θ 15.0°, 17.1°, 19.4°, and 22.1°, which might be from coxi seed granules (2θ at ~ 15°, 17°, 18°, and 23°) [ 30 ]. After 6 days of fermentation, the diffraction pattern shifted to angles at 2θ 18.5° and 21.3° in Fig. 4 b. By 15 days of fermentation, the angles further changed to 2θ 8.5° and 19.1° (Fig. 4 c). These linear diffractomes arose from the noise signals generated by trace amounts of small-molecule sugars, proteins, and metal salts present in the culture medium. The crystallinity of the aged starch pastes firstly decreased from 12.24% to 11.26% and then increased to 20.22% during fermentation. Based on the analysis of the above results, it could be inferred that the crystalline amylopectin in coix seed starch as well as not-retrograded amylopectin in the culture medium had been metabolized by Monascus during fermentation. Consequently, the residual starch forms a novel crystal structure. The thermal property of the medium containing aged starch paste and coix seeds, as shown in Table 2 , also supported this hypothesis. The peak temperature (T p ) and the transition enthalpy ( ΔH ) reflected the presence of retrogradation crystals in the samples [ 31 ]. Initially, the T p and ΔH of the original medium were 107.165 ℃ and 173.04 J·g -1 , respectively. After 6 days of fermentation, these values changed to 97.06 ℃ and 115.305 J·g -1 , respectively, indicating the metabolism of crystallized amylopectin in coxi seed granules corresponding to X-ray diffraction angles of 2θ 15.0° and 17.1°. After 15 days of fermentation, T p and ΔH of the medium underwent a transform, reaching values of 100.70℃ and 118.06 J·g -1 , respectively. This slight increase suggested the formation of a more ordered structure of maize amylopectin in the medium. Whether this structure was a consequence of Monascus fermentation or molecular recombination needed further study. Note a. Fermented for 0 day; b. Fermented for 6 days; c. Fermented for 15 days Conclusions Monascus fermentation (M2) significantly enhances the contents of retrograded maize amylopectin in aged starch paste. Strain M2 exhibited the ability to secrete extracellular pigments. ω-gliadin in the β-sheet and β-turn conformations in aged starch paste serves as a valuable nitrogen source for the growth of Monascus M2 during fermentation. The infrared absorption peaks at approximately 1081 cm − 1 and 1040.4 cm − 1 correspond to the crystallization regions in coxi seed granules and retrograded maize amylopectin, respectively. Retrograded maize amylopectin crystals are characterized by diffraction angles at 2θ of 19.1°. The mechanism underlying the enhancement of retrograded starch content in aged maize amylopectin by Monascus fermentation involves the decomposition of amorphous maize amylopectin. The method of enhancing retrograded resistant starch in gelatinized starch through Monascus fermentation represents a novel, cost-effective, efficient, and environmentally sustainable technology. Declarations Competing interest The authors have declared no conflict of interest. Author Contribution Jiarui Yu and Zengfang Guo contribute this work equally. Jiarui Yu: Methodology, Data curation, Writing draft, Zengfang Guo: Methodology, Formal analysis, Data curation, Yuxian Lai: Methodology, Yu Gou, Methodology, Formal analysis, Xijin Lian: Conceptualization, Funding acquisition, Project administration, Supervision, Writing - review & editing. Acknowledgements This work is supported by National Natural Science Foundation of China (No. 31571834); Natural Science Foundation of Tianjin Municipality (No. 22JCYBJC00130). Data availability statement The data that support the findings of this study are available on reasonable request from the corresponding author. References Maki KC, Sanders LM, Reeves MS et al (2009) Beneficial effects of resistant starch on laxation in healthy adults. 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07:36:10","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":97141,"visible":true,"origin":"","legend":"","description":"","filename":"8d40f84087244cf5ac0dc5d65f5f45b71structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7928313/v1/62889516aef1f96d0df7494c.xml"},{"id":96086251,"identity":"97ff10b4-cb12-4b0d-b648-db1ae54bf426","added_by":"auto","created_at":"2025-11-17 12:34:15","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":104329,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7928313/v1/baf5afd5e81512e50b655b56.html"},{"id":96247599,"identity":"4897b24b-5004-4df9-9df4-154c6c540b9e","added_by":"auto","created_at":"2025-11-19 07:27:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":126439,"visible":true,"origin":"","legend":"\u003cp\u003eThe colonial morphology of different \u003cem\u003eMonascus\u003c/em\u003e strains on PDA medium\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7928313/v1/c091104dd80dc6d4dfd3f8a9.jpg"},{"id":96086237,"identity":"1e921aab-66a5-4eec-8d89-0b8686006e6d","added_by":"auto","created_at":"2025-11-17 12:34:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":68054,"visible":true,"origin":"","legend":"\u003cp\u003eThe FT-IR spectra of aged starch paste fermented by \u003cem\u003eMonascus\u003c/em\u003e \u003cem\u003epurpureus\u003c/em\u003e for varying durations\u003c/p\u003e\n\u003cp\u003eNote: a. Fermented for 0 day; b. Fermented for 6 days; c. Fermented for 15 days\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7928313/v1/1348e506d9b03a5d075502ef.jpg"},{"id":96086242,"identity":"085309cc-a8ca-47ee-8619-d700ecc0ee2d","added_by":"auto","created_at":"2025-11-17 12:34:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72672,"visible":true,"origin":"","legend":"\u003cp\u003eThe \u003csup\u003e13\u003c/sup\u003eC solid-state NMR spectra of retrograded maize amylopectin containing gliadin fractions fermented by M2 for varying durations\u003c/p\u003e\n\u003cp\u003eNote: a. Fermented for 0 day; b. Fermented for 6 days; c. Fermented for 15 days\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7928313/v1/f2f760eecf56b3345a8a9468.jpg"},{"id":96086238,"identity":"dea2e62d-e4d2-470a-b194-6cc96839100c","added_by":"auto","created_at":"2025-11-17 12:34:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":69156,"visible":true,"origin":"","legend":"\u003cp\u003eThe XRD patterns of retrograded maize amylopectin containing gliadin fractions fermented by \u003cem\u003eMonascus purpureus\u003c/em\u003e for varying durations.\u003c/p\u003e\n\u003cp\u003eNote: a. Fermented for 0 day; b. Fermented for 6 days; c. Fermented for 15 days\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7928313/v1/6655910c467c6a1fb5bd6456.jpg"},{"id":100614514,"identity":"a0cc31f7-da5b-47be-8726-d80c80f89283","added_by":"auto","created_at":"2026-01-19 17:21:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1255885,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7928313/v1/29cd0c5c-beb6-4b4e-bdbe-3774e27cc2da.pdf"},{"id":96248056,"identity":"a69aeb11-5179-4b1c-b246-ddb44dc2a260","added_by":"auto","created_at":"2025-11-19 07:27:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1659410,"visible":true,"origin":"","legend":"","description":"","filename":"Supfig.docx","url":"https://assets-eu.researchsquare.com/files/rs-7928313/v1/18c5439ae16dbd7f47978079.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancement of retrograded maize amylopectin in aged starch paste by screening Monascus strains under solid fermentation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRetrograded starches in aged starch paste are functional foods formed by reassociating disaggregated amylose and amylopectin chains in a gelatinized starch paste to form an ordered structure. This process makes them resistant to hydrolysis by amylase. It is well-known that resistant starches have many beneficial effects, including laxative effects [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], the alleviation of diabetes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], the control of body weight [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and the improvement of immunity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, only retrograded starch, not all aged starch paste, is resistant to hydrolysis by amylase, and conventional aged starch contains only about 20% retrograded starch [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. To meet the recommended daily intake of 30 grams of dietary fiber, an adult must consume 150 grams of dry starch daily [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This is not a practical option. It is, therefore, of great importance to enhance the contents of retrograded starch in aged starch paste as to facilitate the commercialization of such functional foods. The primary purification approach is enzymatic hydrolysis. However, the glucose and maltose obtained through enzymatic hydrolysis are difficult to completely wash off, such washing also bring environmental pollution. Meanwhile, the laxative effect of the acquired high-purity starch in vivo is not as pronounced as expected. This may be due to the low molecular weight of the starch within the residual retrograded starch. These retrograded starches might be partially dissolved in the alkaline environment of the small intestine and hydrolyzed by enzymes. Furthermore, our research indicates that gliadin fractions can enhance the retrogradation of amylopectin [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The retrogradation rate of maize amylopectin has been observed to increase to more than 40%. The study of the laxative function of retrograded starch reveals that the resistance to decomposition of retrograded amylopectin is more robust than that of retrograded starch or retrograded amylose. Nevertheless, the retrograded amylopectin content in aged starch paste remains insufficient for industrial applications. Recent studies have demonstrated that \u003cem\u003eMonascus\u003c/em\u003e can be cultivated in retrograded maize starch in winter outdoor air [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This process could enhance the contents of retrograded maize amylopectin in aged starch paste, which contains gliadin fractions. The reasons for selecting \u003cem\u003eMonascus\u003c/em\u003e as the fermentation strain are that it possesses high activity of fermentation amylases and proteases, low acid production, a strong ability to resist the growth of miscellaneous bacteria, ease of industrial production, and has been utilized in the production of red yeast rice in China for thousands of years.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMonascus purpureus\u003c/em\u003e is a tiny filamentous saprophytic fungus that produces a variety of enzymatic substances, including amylolytic enzymes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], esterification enzymes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], proteases [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and others. The saccharifying enzyme, or α-1,4-glucan glucohydrolase, hydrolyzes α-1,4 and α-1,6 glycosidic linkages in starch to produce glucose, which is vital for \u003cem\u003eMonascus\u003c/em\u003e growth [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Thus, \u003cem\u003eMonascus purpureus\u003c/em\u003e can thrive on various substrates. Retrograded maize amylopectin containing gliadin exhibits high viscosity in water, impeding fermentation oxygen supply for \u003cem\u003eMonascus\u003c/em\u003e. In contrast to rice, coix seed has a lower viscosity post-gelation, making the culture medium with retrograded maize amylopectin more permeable and favorable for \u003cem\u003eMonascus\u003c/em\u003e growth. Moreover, coix seed is traditionally used in China to treat rheumatism, warts, chapped skin, and as an anti-helminthic and anti-inflammatory agent [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRetrograded starches in aged starch paste are functional foods that are formed by the reassociation of disaggregated amylose and amylopectin chains in a gelatinized starch paste to form an ordered structure. This paper aims to increase content of retrograded maize amylopectin containing gliadin fractions in aged starch paste by metabolizing non-retrograded amylopectin through the growth of different \u003cem\u003eMonascus\u003c/em\u003e. At the same time, these surplus gliadin fractions could be utilized as a nitrogen source for solid fermentation. The best strain and optimum conditions for fermentation are determined, and a pattern for enhancing the contents of retrograded maize amylopectin in aged starch paste has been established based on the results of infrared spectroscopy (IR), \u003csup\u003e13\u003c/sup\u003eC solid-state nuclear magnetic resonance (\u003csup\u003e13\u003c/sup\u003eC solid-state NMR), X-ray diffraction and differential thermal analysis (DSC). The findings in the paper demonstrate a novel way to enhance the contents of retrograded starch in aged starch paste.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003eThe gluten (9.0% moisture, 7.0% lipids, 12.8% carbohydrates, 77.5% protein) was sourced from Henan Midaner Trading Co., Ltd (Xinzheng City, Henan Province, China). Alcohol was obtained from Henan Xinheyang Alcohol Co., Ltd (Mengzhou City, Henan Province, China). Maize starch was provided by Henan Enmiao Food Co., Ltd. (Beijing City, China); coix seed by Bozhou Wenshun Pharmaceutical Co., Ltd. (Bozhou City, China); Tween-80 by Tianjin Zhiyuan Chemical Reagent Co., Ltd. (Tianjin City, China). Soluble starch, maltose, peptone and agar were supplied by Tianjin Chemical Reagent Co., Ltd. (Tianjin City, China), while sodium chloride, hydrochloric acid, sodium hydroxide, low- temperature amylase, and iodine solution were form Tianjin Fengchuan Chemical Reagent Co., Ltd. (Tianjin City, China). Red mold rice was purchased from Diyuan \u003cem\u003eMonascus\u003c/em\u003e Factory, (Gutian County, Fujian Province, 2023), Chengjiu \u003cem\u003eMonascus\u003c/em\u003e Co., Ltd. (Gutian County, Fujian Province, 2023), and Shandong Zhonghui Biotechnology Co., Ltd. (Huimin County, Shandong Province, 2023) in China.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eIsolation of gliadin fractions\u003c/h2\u003e\u003cp\u003eThe isolation of gliadin fractions was performed in accordance with the methods described in the literature [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Gluten powder was dissolved in 65% ethanol at a ratio of 1 : 30 (g : mL) and continuously stirred in a water bath at 35 ℃ for 3 h. Following this, the solution was maintained at room temperature (15\u0026ndash;28 ℃) for 3 h before being centrifuged at 4000\u0026times;g for 5 min to obtain the first supernatant. The first supernatant was then subjected to coagulation and sedimentation at 4 ℃ for 24 h, yielding gliadin 1 (primarily ω-gliadin). The residual second supernatant was stored at -18 ℃ for 24 h and subsequently thawed at room temperature (15\u0026ndash;28 ℃), resulting in the formation of a precipitate designated as gliadin 3 (primarily αβγ-gliadin).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eThe preparation and purification of maize amylopectin\u003c/h3\u003e\n\u003cp\u003eMaize amylopectin was separated and purified according to reference with minor modifications [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Maize starch was blended with deionized water in a 1:5 ratio, and the solutions were heated to 70\u0026deg;C for 90 minutes to promote granule swelling. After cooling to room temperature (15\u0026thinsp;~\u0026thinsp;28 ℃), the solution was frozen for over 12 h. Upon thawing, the solutions were centrifugated (3500\u0026times;g for 3 min) and mixed with a 0.5% NaCl solution (g/v). Following a 20-minute stirring, it was centrifuged again (3500\u0026times;g for 3 minutes) to yield crude maize amylopectin, which was washed multiple times with 0.5% NaCl until no blue color appeared upon iodine titration, indicating the removal of amylose. Residual NaCl was eliminated by washing with deionized water. Lipase (1500 U/g) was then added to hydrolyze lipids bound to maize amylopectin at 40 ℃ for 12 h. The pH was adjusted to 8.0 with 2.0 mol\u0026middot;L⁻\u0026sup1; NaOH, and alkali protease (20,000 U/g) was introduced to hydrolyze proteins. The final maize amylopectin was obtained by centrifugation (at 3500\u003cem\u003e\u0026times;g\u003c/em\u003e for 10 min) followed by washing with deionized water to remove residual fatty acids and amino acids, resulting in purified maize amylopectin.\u003c/p\u003e\n\u003ch3\u003eThe preparation of aged starch paste containing gliadin fractions\u003c/h3\u003e\n\u003cp\u003eWet gliadin 1, gliadin 3, and maize amylopectin were mixed in a of 4 : 1 : 95 mass ratio and stirred at 50 ℃ for 10 min in a water bath. The mixtures were then autoclaved at 105 ℃ for 20 min and kept at 4 ℃ for 7 d to obtain aged starch paste containing gliadin fractions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eScreening of\u003c/b\u003e \u003cb\u003eMonascus\u003c/b\u003e \u003cb\u003estrains\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eMonascus\u003c/em\u003e strains named as M1, M2, M3, M4, and M5 were isolated from five red mold rice. M1 was sourced from Diyuan \u003cem\u003eMonascus\u003c/em\u003e Factory, (Gutian County, Fujian Province, China, 2023), M2, M3, and M4 from Chengjiu \u003cem\u003eMonascus\u003c/em\u003e Co., Ltd. (Gutian County, Fujian Province, China, 2023), and M5 from Shandong Zhonghui Biotechnology Co., Ltd. (Huimin County, Shandong Province, China, 2023). The samples (0.1 g) were dissolved in 6 mL of Tween 80 solvent and diluted with sterilized water to concentrations of 1/10, 1/100, 1/1000, 1/10000, and 1/100000. These \u003cem\u003eMonascus\u003c/em\u003e samples (0.1 mL) were then inoculated onto the surface of the plate culture medium in sterilized Petri dishes and incubated at 32 ℃ for 4 days. Well-grown \u003cem\u003eMonascus\u003c/em\u003e mycelium was scraped and cultured on a medium composed of 5 g soluble starch, 4 g maltose, 3 g peptone, 2 g agar and 86 g deionized water (sterilized at 121 ℃ for 30 minutes), using an inoculation ring. The suspected \u003cem\u003eMonascus\u003c/em\u003e strains were separated and purified on the culture medium through 3 to 5 iterations until single colonies appeared. The strains were incubated at 32 ℃ for 4 days and subsequently sub-cultured in test tube slants over five generations to yield strains suitable for further experimentation. M2 was genetically identified as \u003cem\u003eMonascus purpureus\u003c/em\u003e by the General Microbiology Center of China Microbial Culture Preservation and Management Committee (CGMCC).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSolid-state fermentation\u003c/h2\u003e\u003cp\u003eSolid-state fermentation was carried out according to the reference with specific modifications [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The growth conditions for \u003cem\u003eMonascus purpureus\u003c/em\u003e were initially assessed through a pre-experiment. Soaked coix seeds and retrograded maize amylopectin were dried at 80 ℃ for 6 h before being mixed with sterile deionized water. After drying, a high-speed grinder crushed the retrograded maize amylopectin through a 100-mesh sieve. The solid culture medium was prepared with soaked coix seeds, retrograded maize amylopectin and sterile deionized water in a mass ratio of 3:3 :4 (g/g/v), and sterilized at 110 ℃ for 40 min. After cooling, 2 ml of deionized water was added to the slant culture medium to dissolve the strains, which were then transferred into a watering can. Finally, the \u003cem\u003eMonascus\u003c/em\u003e strains were sprayed onto the solid culture medium in triangular flasks and incubated at 32 ℃ in a thermostat.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDetermination of retrogradation rate\u003c/h3\u003e\n\u003cp\u003eThe retrogradation rate of maize amylopectin was determined using the method from reference [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The solid culture medium was dried in an oven at 60 ℃ until a constant weight was achieved, then pulverized into powder. Powder samples (m\u003csub\u003e1\u003c/sub\u003e) were mixed with deionized water and low-temperature α-amylase for enzymatic hydrolysis of non-retrograded maize amylopectin at 45 ℃ for 2 h. Subsequently, the solutions were centrifuged at 3500\u003cem\u003e\u0026times;g\u003c/em\u003e for 10 min. The precipitate was dried in an oven at 60 ℃ until a constant weight was reached and weighed to obtain a mass of m\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eRetrogradation rate = (m\u003csub\u003e2\u003c/sub\u003e/m\u003csub\u003e1\u003c/sub\u003e) \u0026times; 100%\u003c/p\u003e\n\u003ch3\u003eFT-IR spectrum analysis\u003c/h3\u003e\n\u003cp\u003eSamples weighing 0.8 mg were homogeneously mixed with 150 mg of spectroscopic-grade potassium bromide (KBr) and subsequently compressed into pellets under controlled conditions. Spectroscopic data were acquired using a Fourier-transform infrared spectrometer (Perkin-Elmer, Buckinghamshire, UK). The samples were scanned over the wavenumber range of 4000 to 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a nominal resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003csup\u003e\u003cb\u003e13\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC solid-state NMR spectroscopy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe samples were placed in a sealed pencil-type (5-mm) zirconia rotor and analyzed using a JEOL ECZ600R 600 MHz spectrometer, featuring a resonance frequency of 150.87 \u003cem\u003ekHz\u003c/em\u003e, corresponding to a \u003cem\u003e90\u0026deg;\u003c/em\u003e pulse width of \u003cem\u003e2.4\u003c/em\u003e \u0026micro;s. A 4 mm double resonance HX CP/MAS (cross polarization/magic angle rotation) probe was employed, with the magic angle rotation (MAS) speed automatically controlled between 9\u0026ndash;12 \u003cem\u003ekHz\u003c/em\u003e. The temperature range for the test was 28\u0026ndash;120 ℃.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eX-ray diffraction (XRD) analysis\u003c/h2\u003e\u003cp\u003eX-ray diffraction (XRD) patterns of the samples were obtained using a D/MAX-2500 diffractometer (Rigaku, Japan) with copper as the target material. The operating voltage and current were set to 40 kV and 40 mA, respectively. The angular velocity ranged from \u003cem\u003e0\u003c/em\u003e \u003csup\u003e\u003cem\u003eo\u003c/em\u003e\u003c/sup\u003e to \u003cem\u003e60\u003c/em\u003e \u003csup\u003e\u003cem\u003eo\u003c/em\u003e\u003c/sup\u003e with a step size of \u003cem\u003e0.02\u003c/em\u003e \u003csup\u003e\u003cem\u003eo\u003c/em\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eDifferential scanning calorimetry (DSC) analysis\u003c/h2\u003e\u003cp\u003eSamples weighing 5.0 mg were sealed in aluminum pans and stored at 4 ℃ overnight. After an equilibrium period of 1 h at room temperature (15\u0026thinsp;~\u0026thinsp;28 ℃), the samples were heated from 25 ℃ to 235 ℃ at a rate of 10 ℃ per minute using a differential scanning calorimeter (DSC404C, Netzsch Instruments NA LLC; Burlington, MA). The peak temperature (\u003cem\u003eTp\u003c/em\u003e) and enthalpy of melting (\u003cem\u003eΔH\u003c/em\u003e) were calculated using a universal analysis program.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation from triplicate measurements. Statistical significance was assessed using a two-sample ANOVA t-test, conducted with the Microsoft Excel software and IBM SPSS 27.0 software.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cb\u003eEnhancement of retrograded maize amylopectin contents by different\u003c/b\u003e \u003cb\u003eMonascus\u003c/b\u003e \u003cb\u003estrains\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe colonial morphology of different \u003cem\u003eMonascus\u003c/em\u003e strains with distinct growth characteristics on PDA medium in Fig.\u0026nbsp;1 suggested preliminary classification into different species. Obvious aerial mycelium was observed in all strains after two days of growth. With the exception of M2, all other strains exhibited significant pigment secretion after three days of growth. Unlike other \u003cem\u003eMonascus\u003c/em\u003e, the mycelium at both the center and edge of the colonies of M2 and M4 appeared white, suggesting that the growth process of these two strains primarily involves the secretion of extracellular pigments. Notably, the strains M2, M3, and M4 were sourced from red yeast rice produced by the same company (Chengjiu \u003cem\u003eMonascus\u003c/em\u003e Co., Ltd). Such results indicated that the production process of red yeast rice in enterprises involved the co-fermentation of various strains of \u003cem\u003eMonascus\u003c/em\u003e, resulting in a complex and integrated product. M3 had been identified as \u003cem\u003eMonascus purpureus\u003c/em\u003e by us [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and M2 might also be \u003cem\u003eMonascus purpureus\u003c/em\u003e based on the analysis of the mycelial growth morphology [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe effects of fermentation on the contents of retrograded maize amylopectin-containing gliadin in aged starch paste by various \u003cem\u003eMonascus\u003c/em\u003e strains were presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The results indicated a significant increase in contents of retrograded maize amylopectin in aged starch paste with extended fermentation times. Notably, the greatest increase occurred on 6th day, where the content rose from 39.5% in the control group to over 52.0% in experimental group. At this stage, the sensory evaluation indicated that bitterness levels remained relatively low. The M2 strain, after 15 days of fermentation, achieved the highest content of 73.1%, representing an 85.1% increase compared to the control. This content exceeded ones (55%) achieved with debranching enzymes combined with moist heat treatment [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This suggested that strain M2 was particularly effective in enhancing the contents of retrograded maize amylopectin in aged starch paste. The mechanism behind this enhancement will be discussed in relation to FT-IR, \u003csup\u003e13\u003c/sup\u003eC solid-state NMR, XRD, and DSC results.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;1 The colonial morphology of different \u003cem\u003eMonascus\u003c/em\u003e strains on PDA medium\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\u003eThe effects of fermentation on the contents of retrograded maize amylopectin containing gliadin in aged starch paste by different \u003cem\u003eMonascus\u003c/em\u003e strains (%)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFermentation times (days)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003e\u003cem\u003eMonascus\u003c/em\u003e strains\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eM3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eM4\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eM5\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e39.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e39.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e39.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e39.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e39.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e41.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e44.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e39.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e39.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e42.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e54.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e62.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e52.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e63.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e55.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e62.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e69.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e66.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e64.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e62.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e69.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e70.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e68.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e65.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e68.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e72.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e73.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e69.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e69.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e72.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eNote: Different alphabets in a column indicate significant differences at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eFT-IR spectra of retrograded maize amylopectin before and after fermentation by M2\u003c/h2\u003e\u003cp\u003eThe FT-IR spectra of aged starch paste before and after fermentation by M2 at different times was showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The bands at 3431.2/3407.6/3408.6 cm\u003csup\u003e-1\u003c/sup\u003e corresponded to the N-H and O-H stretching vibration [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The observed lower wavenumber shifts in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c indicated enhanced hydrogen bonding among maize amylopectin among amylopectin molecules and between amylopectin and gliadin [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], likely due to the metabolism of the small molecular starch and protein that disrupt hydrogen bond formation among the larger macromolecules during fermentation. A weak band was observed at 1735.6 cm\u003csup\u003e-1\u003c/sup\u003e in Fig.\u0026nbsp;1a, which indicated the presence of lipids in a solid medium [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The intensification of the signal in Fig.\u0026nbsp;1b and the disappearance of the band in Fig.\u0026nbsp;1c indicated that these lipids, initially bound to protein in coix seeds, were fully metabolized during fermentation. They potentially aided \u003cem\u003eMonascus\u003c/em\u003e growth. The strong vibrations at 1640.2/1654.1/1647.9 cm\u003csup\u003e-1\u003c/sup\u003e and 1535.1/1538.0/1541.3 cm\u003csup\u003e-1\u003c/sup\u003e were attributed to amide I and II of gliadin fractions [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The changes in infrared absorption intensities during fermentation indicated that gliadin fractions in aged starch paste, possessing specific secondary structures, were beneficial for the growth of \u003cem\u003eMonascus\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e\u003cp\u003ea. Fermented for 0 day; b. Fermented for 6 days; c. Fermented for 15 days\u003c/p\u003e\u003c/p\u003e\u003cp\u003eThe results in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e showed that the gliadin fractions with β-sheet conformation in aged starch paste were reduced during fermentation, providing a nitrogen source that supported \u003cem\u003eMonascus\u003c/em\u003e growth.\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\u003eSecondary structure and thermal properties of the medium at different fermentation stages\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\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=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSamples\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eα-helix\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eβ-sheet\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eβ-turn (%)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003erandom coil (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eT\u003csub\u003ep\u003c/sub\u003e(℃)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eΔH\u003c/em\u003e(J/g)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e19.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e43.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e20.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e107.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e173.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e21.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e43.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e22.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e97.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e115.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDay 15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e22.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e42.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e12.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e22.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e118.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: Different alphabets in a column indicate significant differences at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eBased on the secondary structure of gliadin fractions in reference [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], the reduced gliadin should be ω-gliadin. The fact that M2 could grow on a medium with ω-gliadin as the sole nutrient source also supported the speculation (Data not shown). The results in Fig. S1 demonstrated that the additive amounts of aged starch paste in medium significantly influenced \u003cem\u003eMonascus\u003c/em\u003e growth, with optimal effects observed at a 1:1 ratio of the aged starch and coix seed. Both higher and lower additive amount diminished this facilitative effect, suggesting that an appropriate amount of ω-gliadin enhanced \u003cem\u003eMonascus\u003c/em\u003e M2 growth. The results in Fig. S2 revealed that the ω-gliadin more significantly promoted the growth of M2, and the red color of the culture medium manifested conspicuously after just three days of fermentation, indicating a much faster growth rate compared to existing literature [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e](Jiang et al., 2024). Additionally, the results in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e also described that the enhanced intensities of amide I and II in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and c corresponded to the increase of α-helix and random coil among gliadin fractions. The bands at 1023.0/1023.5 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;1a/b) and ~\u0026thinsp;1081/1040.4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) corresponded to the amorphous and crystallization regions (in inside and outside of granule) of maize amylopectin, respectively [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The presence of the band at 1041.5 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ec conformed the findings in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, indicating that \u003cem\u003eMonascus\u003c/em\u003e fermentation greatly enhanced the contents of retrograded maize amylopectin in aged starch paste. The mechanism by which \u003cem\u003eMonascus\u003c/em\u003e fermentation enhanced the content of retrograded starch in aged maize amylopectin involved the decomposition and metabolism of amorphous maize amylopectin that did not contribute to retrogradation in the aged starch.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003e\u003cb\u003e13\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC NMR spectra of aged starch paste before and after fermentation by M2\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe \u003csup\u003e13\u003c/sup\u003eC NMR spectra of aged starch paste before and after fermentation by M2 was displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e. According to the literature [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], the resonances of gliadin in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e were assigned as follows: 174.7/174.8/174.6 ppm (Q\u003csub\u003eδ\u003c/sub\u003e of Gln), 128.8/128.9 ppm (Yε of Tyr), 103.2/103.5/102.9 ppm (C1 of maize amylopectin and starch in coxi seed), 82.5 ppm (C4 of maize amylopectin and starch in coxi seed), 72.7/72.5 ppm (C2,3,5 of maize amylopectin and starch in coxi seed), 62.2/61.9 ppm (C6 of maize amylopectin and starch in coxi seed), 30.3/30.1 ppm (Q\u003csub\u003eγ\u003c/sub\u003e of Gln, P\u003csub\u003eβ\u003c/sub\u003e of Pro), 25.7/25.4 ppm (P\u003csub\u003eγ\u003c/sub\u003e of Pro, L\u003csub\u003eγ\u003c/sub\u003e of Leu). When gliadin interacted with maize amylopectin in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, the nuclear magnetic resonance (NMR) signals of all chemical bonds in gliadin, except for the amide bond, were shielded. As the molecular weight of amylopectin decreased or its crystalline regions increased during fermentation in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and c, the resonance of all carbon atoms shifted toward the high field. This finding offered a reliable and effective NMR-based screening approach to distinguish structural changes in amylopectin molecules. The C4 nuclear magnetic resonance (NMR) signal at 82.5 ppm in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea corresponded to the starch located in the amorphous region [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The disappearance of this signal in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and c strongly supported the catabolism of maize amylopectin in the amorphous region during the fermentation process by \u003cem\u003eMonascus\u003c/em\u003e, thereby validating the accuracy of the infrared spectroscopy analysis in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The heightened intensities of resonances for Q\u003csub\u003eγ\u003c/sub\u003e / P\u003csub\u003eβ\u003c/sub\u003e in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and c suggested that they were primarily embedded at β-sheet and β-turn became more exposed as \u003cem\u003eMonascus\u003c/em\u003e hydrolyzed the former during fermentation. This indicated that gliadin fractions at β-sheet and β-turn state served as a valuable nitrogen source for \u003cem\u003eMonascus\u003c/em\u003e growth, which were crucial for regulating \u003cem\u003eMonascus\u003c/em\u003e solid fermentation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e\u003cp\u003ea. Fermented for 0 day; b. Fermented for 6 days; c. Fermented for 15 days\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eXRD patterns of aged starch paste fermented by M2 for varying durations\u003c/h2\u003e\u003cp\u003eThe X-ray diffraction patterns of aged starch paste fermented by M2 for varying durations were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The diffraction angles for aged starch pastes in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea differed from the reference values for gliadin [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] or retrograded maize amylopectin (2θ at ~\u0026thinsp;17\u0026deg;, 20\u0026deg;, 22\u0026deg;) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The characteristic diffraction angles of aged starch in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea for 0-day fermentation were observed at 2θ 15.0\u0026deg;, 17.1\u0026deg;, 19.4\u0026deg;, and 22.1\u0026deg;, which might be from coxi seed granules (2θ at ~\u0026thinsp;15\u0026deg;, 17\u0026deg;, 18\u0026deg;, and 23\u0026deg;) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. After 6 days of fermentation, the diffraction pattern shifted to angles at 2θ 18.5\u0026deg; and 21.3\u0026deg; in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb. By 15 days of fermentation, the angles further changed to 2θ 8.5\u0026deg; and 19.1\u0026deg; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). These linear diffractomes arose from the noise signals generated by trace amounts of small-molecule sugars, proteins, and metal salts present in the culture medium. The crystallinity of the aged starch pastes firstly decreased from 12.24% to 11.26% and then increased to 20.22% during fermentation. Based on the analysis of the above results, it could be inferred that the crystalline amylopectin in coix seed starch as well as not-retrograded amylopectin in the culture medium had been metabolized by \u003cem\u003eMonascus\u003c/em\u003e during fermentation. Consequently, the residual starch forms a novel crystal structure. The thermal property of the medium containing aged starch paste and coix seeds, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, also supported this hypothesis. The peak temperature (T\u003csub\u003ep\u003c/sub\u003e) and the transition enthalpy (\u003cem\u003eΔH\u003c/em\u003e) reflected the presence of retrogradation crystals in the samples [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Initially, the T\u003csub\u003ep\u003c/sub\u003e and \u003cem\u003eΔH\u003c/em\u003e of the original medium were 107.165 ℃ and 173.04 J\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e, respectively. After 6 days of fermentation, these values changed to 97.06 ℃ and 115.305 J\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e, respectively, indicating the metabolism of crystallized amylopectin in coxi seed granules corresponding to X-ray diffraction angles of 2θ 15.0\u0026deg; and 17.1\u0026deg;. After 15 days of fermentation, T\u003csub\u003ep\u003c/sub\u003e and \u003cem\u003eΔH\u003c/em\u003e of the medium underwent a transform, reaching values of 100.70℃ and 118.06 J\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e, respectively. This slight increase suggested the formation of a more ordered structure of maize amylopectin in the medium. Whether this structure was a consequence of \u003cem\u003eMonascus\u003c/em\u003e fermentation or molecular recombination needed further study.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e\u003cp\u003ea. Fermented for 0 day; b. Fermented for 6 days; c. Fermented for 15 days\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003e\u003cem\u003eMonascus\u003c/em\u003e fermentation (M2) significantly enhances the contents of retrograded maize amylopectin in aged starch paste. Strain M2 exhibited the ability to secrete extracellular pigments. ω-gliadin in the β-sheet and β-turn conformations in aged starch paste serves as a valuable nitrogen source for the growth of \u003cem\u003eMonascus\u003c/em\u003e M2 during fermentation. The infrared absorption peaks at approximately 1081 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1040.4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e correspond to the crystallization regions in coxi seed granules and retrograded maize amylopectin, respectively. Retrograded maize amylopectin crystals are characterized by diffraction angles at 2θ of 19.1\u0026deg;. The mechanism underlying the enhancement of retrograded starch content in aged maize amylopectin by \u003cem\u003eMonascus\u003c/em\u003e fermentation involves the decomposition of amorphous maize amylopectin. The method of enhancing retrograded resistant starch in gelatinized starch through \u003cem\u003eMonascus\u003c/em\u003e fermentation represents a novel, cost-effective, efficient, and environmentally sustainable technology.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interest\u003c/h2\u003e\u003cp\u003eThe authors have declared no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJiarui Yu and Zengfang Guo contribute this work equally. Jiarui Yu: Methodology, Data curation, Writing draft, Zengfang Guo: Methodology, Formal analysis, Data curation, Yuxian Lai: Methodology, Yu Gou, Methodology, Formal analysis, Xijin Lian: Conceptualization, Funding acquisition, Project administration, Supervision, Writing - review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThis work is supported by National Natural Science Foundation of China (No. 31571834); Natural Science Foundation of Tianjin Municipality (No. 22JCYBJC00130).\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available on reasonable request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMaki KC, Sanders LM, Reeves MS et al (2009) Beneficial effects of resistant starch on laxation in healthy adults. 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Food Chem 294:179\u0026ndash;186. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2019.05.077\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2019.05.077\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-polymers-and-the-environment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jooe","sideBox":"Learn more about [Journal of Polymers and the Environment](https://www.springer.com/journal/10924)","snPcode":"10924","submissionUrl":"https://submission.nature.com/new-submission/10924/3","title":"Journal of Polymers and the Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Monascus purpureus, retrograded maize amylopectin, contents, aged starch paste, nitrogen source","lastPublishedDoi":"10.21203/rs.3.rs-7928313/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7928313/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRetrograded starch (type 3 resistant starch), known for its laxative properties, has garnered significant research interest. Nevertheless, the inherently low levels of retrograded starch in aged starch paste restrict its practical applications. \u003cem\u003eMonascus\u003c/em\u003e strains named as M1, M2, M3, M4, and M5 were isolated from various red mold rice produced in China and were employed to enhance contents of retrograded maize amylopectin in aged starch paste. The results demonstrated that optimal addition of aged starch and coxi seed was observed at a 1:1 ratio in a solid medium. The highest content of retrograded maize amylopectin in aged starch was achieved after 15 days of fermentation by strain M2 at 32 ℃, increasing from 39.5% to 73.1%, which represents an 85.1% enhancement. The results of FT-IR, 13C solid-state NMR, XRD and DSC revealed that the ω-gliadin at β-sheet and β-turn state served as a valuable nitrogen source for \u003cem\u003eMonascus\u003c/em\u003e M2 growth. The maize amylopectin in the amorphous region were decomposed by \u003cem\u003eMonascus\u003c/em\u003e. The crystals for retrograded maize amylopectin were characterized by diffraction angles at 2θ 19.1\u0026deg;. This study proposes a novel way to enhance the retrograded starch content in aged starch paste with no environmental pollution, thereby broadening the application potential of gluten.\u003c/p\u003e","manuscriptTitle":"Enhancement of retrograded maize amylopectin in aged starch paste by screening Monascus strains under solid fermentation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-17 12:34:10","doi":"10.21203/rs.3.rs-7928313/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-20T10:40:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-16T07:51:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-12T14:40:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-11T06:48:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-10T17:33:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"302967954006424078431967641329641266549","date":"2025-11-06T08:55:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81375762845586033735403520876861619709","date":"2025-11-06T03:01:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120236954461357453587383864708812029703","date":"2025-11-05T16:06:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"284967448578470383509253164464755398988","date":"2025-11-05T15:46:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-05T14:39:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-24T01:06:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-24T01:05:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Polymers and the Environment","date":"2025-10-23T04:41:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-polymers-and-the-environment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jooe","sideBox":"Learn more about [Journal of Polymers and the Environment](https://www.springer.com/journal/10924)","snPcode":"10924","submissionUrl":"https://submission.nature.com/new-submission/10924/3","title":"Journal of Polymers and the Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f82ff5c5-3123-475f-8b63-1ecdf2711aad","owner":[],"postedDate":"November 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T16:45:03+00:00","versionOfRecord":{"articleIdentity":"rs-7928313","link":"https://doi.org/10.1007/s10924-026-03768-9","journal":{"identity":"journal-of-polymers-and-the-environment","isVorOnly":false,"title":"Journal of Polymers and the Environment"},"publishedOn":"2026-01-17 16:29:00","publishedOnDateReadable":"January 17th, 2026"},"versionCreatedAt":"2025-11-17 12:34:10","video":"","vorDoi":"10.1007/s10924-026-03768-9","vorDoiUrl":"https://doi.org/10.1007/s10924-026-03768-9","workflowStages":[]},"version":"v1","identity":"rs-7928313","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7928313","identity":"rs-7928313","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}