Acrosiphonia orientalis, Caulerpa scalpelliformis, Ulva fasciata, Ulva lactuca; six brown—Iyengaria stellata, Lobophora variegate, Padina boergesenii, Sargassum linearifolium, Spatoglossum asperum, Stoechospermum marginatum; and five red—Amphiroa anceps, Grateloupia indica, Halymenia porphyriformis, Scinaia carnosa, Solieria chordalis). All seaweeds were analyzed for the proximate composition, including moisture content, ash content, total sugar content, total proteins, total lipids, crude fiber, carotenoid content, total chlorophyll content, proline, iodine content, nitrogen-free extract, total phenolic content and total flavonoid content. Green seaweeds showed higher nutritional proximate composition, followed by brown and red seaweeds. Among the different seaweeds, Ulva, Caulerpa, Sargassum, Spatoglossum and Amphiroa showed high nutritional proximate composition compared to other seaweeds. High cation scavenging, free radical scavenging and total reducing activities were observed for Acrosiphonia, Caulerpa, Ulva, Sargassum, Spatoglossum and Iyengaria. It was also observed that 15 tropical seaweeds contained negligible amounts of antinutritional compounds, including tannic acid, phytic acid, saponins, alkaloids and terpenoids. Nutritionally, green and brown seaweeds provided higher sources of energy  compared to red seaweeds . Additionally, this study also confirmed that tropical seaweeds improved the oxidative stability of food oils and, therefore, might be recommended as natural antioxidant additives. The overall results confirm that tropical seaweeds are potential sources of nutrition and antioxidants and may be explored as functional food, dietary supplementation or animal feed. Additionally, they may also be explored as food supplements for fortifying food products, as food toppings or for garnishing and seasoning foods. However, a human or animal toxicity analysis is required before any conclusive recommendation for daily food or feed intake can be made.Foods enriched with nutritional compounds and biological activities, especially antioxidants, are considered healthier for human and/or animal consumption. Seaweeds are rich sources of biologically active metabolites and are used as functional foods. In this study, proximate compositions, physicobiochemical characteristics and oil oxidative stability were analyzed for 15 abundant tropical seaweeds (four green— Seaweeds have been traditional foods in many cultures since ancient periods, as well as being used as fertilizer, condiments and different phycocolloids, including agar, carrageenan and alginates . The demThe world aquaculture economy amounted to USD 275 billion in 2019, and seaweeds accounted for 5.4% USD 14.85 billion) . Seaweed5 billionMore than 10,000 seaweed species have been reported globally and broadly classified into three taxonomic groups: green ; brown (about 2000 species under Ochrophyta/Phaeophyta); and red (approximately 7200 species under Rhodophyta). However, only 27 different seaweed species were cultivated in 2019 ,13. MostConsumption of seaweeds in Western diets was previously limited to coastal communities and artisanal practices, but now it has gained wider consumer attention. The current surge of attention is fueled by awareness of the bioactive components of seaweed, which show possible benefits in nutraceutical applications  and as lGlobally used edible oils contain high concentrations of unsaturated fatty acids, mainly polyunsaturated fatty acids, which are more susceptible to oxidation. Exposure of oils to high temperature and light can cause oxidation and amplify the peroxide value, causing the oils to become unpalatable . Oil oxiAcrosiphonia spp., Caulerpa spp., Grateloupia spp., Padina spp., Sargassum spp., Spatoglossum spp. and Ulva spp. are considerd edible seaweeds [Previously, we demonstrated the antioxidant and nutraceutical potential of abundantly available tropical seaweeds from the Saurashtra coast of the Arabian Sea and their application as dietary supplements or functional foods ,25,26. Aseaweeds ,27. TherAcrosiphonia orientalis (J.Agardh) P.C.Silva (ACR), Caulerpa scalpelliformis f. dwarkensis Børgesen (CAU), Ulva fasciata Delile (UF) and Ulva lactuca Linnaeus (UL); six brown—Iyengaria stellata (Børgesen) Børgesen (IYN), Lobophora variegata (J.V.Lamouroux) Womersley ex E.C. Oliveira (LOB), Padina boergesenii Allender & Kraft (PAD), Sargassum linearifolium (Turner) C. Agardh (SAR), Spatoglossum asperum J. Agardh (SPA) and Stoechospermum marginatum (J.V. Lamouroux) J. Agardh (STO); and five red—Amphiroa anceps (Lamarck) Decaisne (AMP), Grateloupia indica Børgesen (GRA), Halymenia porphyriformis P.G. Parkinson (HAL), Scinaia carnosa (Kützing) J. Agardh (SCI) and Solieria chordalis (C. Agardh) J. Agardh (SOL)) were collected from their natural habitat , following the phenol sulfuric acid method ,31.W0) were digested with 150 mL sulfuric acid (0.128 M) at 100 °C for 30 min. Digested mixtures were filtered and washed twice using 25 mL hot distilled water. Washed samples were further treated with 150 mL potassium hydroxide (0.223 M), filtered, washed twice with 25 mL hot distilled water and dried. Finally, samples were washed with acetone and incubated for 2 h in an oven at 100 °C. Samples were cooled and weighed (W1). The residue was ashed at 550 °C for 3 h in a muffle furnace. The ashed residue was desiccated, cooled and weighed (W2). Crude fiber was calculated in percent using the following equation [To determine crude fiber content , seaweedequation :Percent v/v) and incubated at room temperature for 24 h in the dark. The incubated mixture was filtered, and the crude extract was washed with 20% of its volume in 0.9% NaCl solution and vigorously mixed for phase separation. The lower phase containing the total lipids was transferred to a pre-weighed beaker (emptyW), which was kept on a hot plate until complete removal of the solution; finally, the weight of the beaker with the residue (residueW) was estimated. Total lipid content was estimated by subtracting with the weight of the empty beaker (emptyW) from the weight of the beaker containing the residue (residueW) [Total lipid content was estimated using the chloroform–methanol method ,34. Abouresidue) ,34.The nitrogen-free extract (NFE) of the seaweed was calculated based on the percent weight difference of crude fiber, crude protein, crude ash and crude lipid, as per the following equation :NFE%=100Proline content was determined based on the method described by Bates et al.  using nig and 4 °C, and the supernatant was collected. Absorbance (Abs) was recorded at 664.5, 664, 647 and 461 nm, and total chlorophyll and carotenoid contents were determined using the following equations:Total chlorophyll and carotenoid contents were determined based on the methods described by Inskeep and Bloom  and Cham2CO3 solution (600 µL) were added. The solution was mixed thoroughly and incubated at 40 °C for 30 min in a dark place. Incubated samples were centrifuged at 9000× g for 2 min at room temperature. The supernatant was used to measure the total phenolic content by recording the absorbance at 765 nm [−1 Dw) of seaweed sample [Total phenolic content was determined based on the method described by Singleton and Rossi  and optit 765 nm . TPC wasd sample .−1 Dw) of seaweed sample [Total flavonoid content (TFC) was determined by following the colorimetric method . About 1d sample .3:HCl:H2O2; 1:2:3) solution. The mixture was vortexed, filtered and subjected to acid digestion on a hot plate until the brown fumes gently disappeared. Distilled water was added to the acid-digested mixture until it became colorless. The sample was filtered and the volume made up with deionized water, and then it was subjected to inductive coupled plasma mass spectrometry analysis [Inductively coupled plasma mass spectrometry analysis was performed to determine the mineral composition . About 1analysis .−1 and the eluent was 20 mM NaOH.The ion chromatography (IC) method was used to estimate iodine content ,43. To dThe seaweed sample (200 mg dried power) was vigorously mixed with 20 mL distilled water. The volume was measured (before swelling), and it was kept for 24 h at room temperature (25 °C) . The samg for 25 min at room temperature, and the residue was collected and weighed. The WHC was calculated using the following equation and expressed as grams of water held per gram dry weight of the sample [To determine the water-holding capacity (WHC), the dry seaweed sample (200 mg dried power) was vigorously mixed with 20 mL of distilled water in pre-weighed tubes and kept for 24 h at room temperature (25 °C) with continuous shaking. The mixture was centrifuged at 3000× e sample :WHC  was expressed as grams of oil held per gram dry weight of the sample [−1.Corn oil (10 g) was mixed with 3 g of sample in a pre-weighed centrifugation tube to determine the oil-holding capacity of the seaweed samples . The mixe sample . The denv/v) to ensure appropriate dispersion in oil. Simultaneously, a set of experiments were performed with the widely used synthetic antioxidant BHT instead of seaweed samples. The mixture was incubated in ultrasonic water bath for 10 min and then subjected to evaporation under a vacuum. The sample or the BHT was added to crude peanut oil at a concentration of 500 ppm. Refined oil, without the addition of any antioxidant, was used as a control. The oil-mix samples  were kept at 60 °C with continuous shaking (100 rpm) for different time ranges (0–16 days). Lipid oxidation was analyzed by measuring different indicators , including peroxide value, p-anisidine value, thiobarbituric acid-reactive substance (TBARS) value and conjugated dienes, as described in To determine the effectiveness of seaweed against lipid oxidation, the oxidative stability of oil under accelerated conditions was analyzed . The sea2 kg−1 (mEq O2 kg−1) and calculated using the following formula:V is the volume (in mL) of sodium thiosulfate consumed in the titration, N (0.01) is the normality of the sodium thiosulfate solution and W is the weight (0.5 g) of the sample.For the measurement of the peroxide value (PV), 500 mg of the oil-mix sample was added to 10 mL of trichloromethane, followed by the addition of 15 mL acetic acid and 1 mL saturated aqueous solution of potassium iodide. Samples were slightly agitated for 1 min and kept in the dark for 5 min. About 75 mL distilled water was added and vigorously mixed. The mixture was titrated against sodium thiosulfate (0.01 N) until the yellow color disappeared . The conAV) was determined according to an IUPAC-approved method [w/v) for 10 min to produce a colored complex [AV was calculated as follows:bE is the absorbance of the oil solution (before reaction), aE is the absorbance of the oil–anisidine reaction solution and W is the weight of the sample (in grams).The p-anisidine value  and K0 is the absorptivity in relation to acid or ester groups. In this case, b, c and K0 were 12.776 cm, 0.5 g L–1 and 0.03, respectively.To determine the conjugated dienes (CDs) value, the oil-mix sample was diluted with hexane in a 25 mL volumetric flask. The absorbance of the diluted sample was measured at 233 nm, and the CDs value, expressed as a percentage of the conjugated dienoic acid, was calculated as follows :CD=0.84×TOTOX) value was determined with the following equation:The total oxidation (TBARS) value estimation, 200 mg of the sample was mixed with 25 mL 1-butanol in a volumetric flask. Then, 5 mL was transferred to another tube, and 5 mL freshly prepared TBA reagent (200 mg TBA in 100 mL butanol) was added and heated at 95 °C in a water bath for 120 min. A pink color developed, the mixture was allowed to cool at room temperature and the absorbance was measured at 532 nm [TBARS values were calculated with the equation given below:For thiobarbituric acid-reactive substance  with potassium persulfate (2.45 mM) for 12–16 h in the dark . These AFor scavenging activity, DPPH solution was diluted with methanol until an absorbance 0.98 ± 0.02 at 517 nm was obtained . Differe4 was used as a standard, and absorbance was read at 593 nm. The ferric reducing antioxidant power (FRAP) assay reagent was prepared from 10 mmol L−1 tripyridyltriazine (TPTZ), 40 mmol L−1 hydrochloric acid, 20 mmol L−1 ferric chloride and 0.3 mol L−1 acetate buffer (pH 3.6). Different amounts of seaweeds (1–5 mg) or FeSO4 (used as standard) were mixed with a FRAP assay reagent (1 mL) and incubated for 10 min at 37 °C. Absorbance was measured at 593 nm, and total reducing capacity was determined over the linear range of the standard curve [The reducing power of the seaweed sample was determined according to their capacity to reduce ferric ions . FeSO4 wrd curve . 50) was calculated via linear regression graph analysis of the dose–response curve for each bioactivity.The half-maximal effective concentration (ECg for 20 min. Supernatant was collected and the volume was topped up to 1000 mL. The extracted sample (1 mL) was further diluted by adding 75 mL distilled water. Folin–Denis reagent (5 mL) and sodium carbonate (10 mL) were added to the diluted sample and the volume was topped up to 100 mL using distilled water. The sample was incubated for 30 min and absorbance was recorded at 700 nm. Tannic acid was used as a standard (10–100 µg). The tannin content (µg g−1) of the sample was calculated with a regression graph for the tannic acid solution [Tannin was estimated based on the method described by Saxena et al. . Powderesolution .g and the supernatant was collected by filtering through Whatman filter paper into a tube that contained pre-weighed NaCl (1 g). The solution was vigorously mixed to dissolve the salt and kept on a shaker platform for 20 min at 300 rpm. The sample was kept idle to allow it to settle for 60 min at 4 °C. The sample was centrifuged at 10 °C and 3000× g for 20 min, the supernatant was collected and 1 mL of this supernatant was diluted with deionized water to 25 mL. The diluted sample (3 mL) was mixed with the Wade reagent (1 mL), vortexed thoroughly and centrifuged for 10 min at 10 °C (3000× g). Absorbance was recorded at 500 nm and phytic acid content was calculated with the regression curve for the standard [Phytic acid was estimated based on the method described by Gao et al. . First, standard .−1 to 10−5 were produced from this filtered solution, vigorously mixed and incubated at room temperature for 15 min. Saponin was determined from the presence of persistent foam [To determine the saponins, 100 mg of powdered seaweed was mixed with distilled water (20 mL) and incubated in boiling water bath for 5 min. The sample was cooled to room temperature and filtered through a nylon membrane, and the volume was brought up to 100 mL with distilled water. Various dilutions ranging from 10ent foam .v/v) H2SO4 (10 mL) followed by incubation in a boiling water bath for 2 min. The mixture was filtered with Whatman paper and 1 mL of filtrate was mixed with 40 mL of Dragendorff reagent. The presence of alkaloids was determined from the appearance of an orange-red precipitate [Seaweed samples (1 g) were treated with 1% (cipitate .2SO4 (3 mL) were added to the extract (5 mL). The presence of terpenoids in seaweed samples was confirmed by the appearance of a reddish-brown color [Terpenoids were extracted from seaweeds by adding 1 g of sample to the 10 mL of methanol followed by proper mixing. Further chloroform (2 mL) and concentrated Hwn color .p < 0.05.Data are shown as means ± SE. ANOVA and Tukey’s honest significant difference (HSD) were applied for statistical analysis. Statistical differences are expressed with different letters at a significance Proximate composition analyses included estimation of moisture content, ash content, total sugar content, total protein content, total lipids, crude fiber, carotenoid content, total chlorophyll content, proline, iodine content, nitrogen-free extract, total phenolic content and total flavonoid content . Among tAmong all 15 tropical seaweeds, the 3 seaweeds UL (56 ± 2%), SAR (53 ± 3%) and SPA (49 ± 0.5%) contained the highest total sugar content, while IYN (9 ± 1%) and AMP (9 ± 1%) contained the lowest total sugar content. Most seaweeds contained total sugars in the range of 15–30%. Total protein content was observed in the range of 6–7% in green seaweeds, 3–7% in brown (except STO) and 2–4% in red seaweeds (except GRA). However, the highest content (10 ± 1%) was detected in the brown seaweed STO, while the lowest (2 ± 0.1%) was observed in the red seaweed SCI. Low lipid content < 5% was found in all seaweeds except the brown seaweeds IYN and SPA; both had about 7% total lipid content. −1). Similar to the fiber content, the highest carotenoid content was found in green seaweeds (0.5–11 mg L−1), followed by brown (0.5–3 mg L−1), while the lowest carotenoid content was found in red seaweeds (0.2–0.7 mg L−1). The highest total chlorophyll was found in the red seaweed GRA (41 ± 0.35 mg L−1), followed by the brown seaweed SAR (34 ± 0.27 mg L−1) and green seaweed UL (14 ± 0.11 mg L−1). Other seaweeds contained total chlorophyll in the range of 1.5–13 mg L−1. The highest fiber content was detected in green seaweeds (11–27%), followed by brown seaweeds , while the lowest content of crude fiber was found in red seaweeds (2–5%). The green seaweed ACR not only contained the highest fiber content (27 ± 0.5%) but also the highest carotenoid content (11 ± 0.01 mg LUlva spp. (14–17 µg mg−1), followed by the brown seaweed STO (15 ± 0.5 µg mg−1), red seaweed SOL (14 ± 1 µg mg−1) and brown seaweed LOB (13 ± 0.3 µg mg−1). Other seaweeds contained proline in the range of 2–8 µg mg−1. The lowest iodine content (1–5 µg g−1) was noticed in red seaweeds, while brown seaweeds contained the highest iodine content, followed by green seaweeds. The highest iodine content was found in the brown seaweed LOB (60 ± 6 µg g−1), followed by SAR (41 ± 2 µg g−1), SPA (33 ± 2 µg g−1) and STO (30 ± 0.5 µg g−1). Among green seaweeds, UL had the highest iodine content of 30 µg g−1, while others were in the range of 5–15 µg g−1. Most seaweeds contained 40–60% nitrogen-free extract (NFE), except AMP and IYN, which contained the lowest NFE contents of 21–25%, while HAL contained the highest at 75 ± 8%, followed by UF (66 ± 4%). The highest proline content was detected in the green seaweed −1), followed by the brown seaweeds SAR and IYN (61 ± 1 mg g−1), SPA (47 ± 0.5 mg g−1) and STO (42 ± 0.5 mg g−1). All other seaweeds contained TPC in the range of 10–30 mg g−1. The highest total flavonoid content (TFC) was detected in the brown seaweed SPA (290 ± 4 mg g−1), followed by the green seaweeds ACR (277 ± 3 mg g−1), SAR (200 ± 18 mg g−1) and PAD (156 ± 10 mg g−1). Most seaweeds contained TFC in the range of 25–75 mg g−1.The highest phenolic content (TPC) was measured in the green seaweed ACR  and SOL . Green seaweeds contained 3000–9000 mg per 100 g K content, while brown seaweeds contained 5300–16,000 mg per 100 g K content. Potassium content of the brown seaweed SPA  was comparable to that of the red seaweed SOL. The red seaweed AMP contained the lowest K content (860 ± 90 mg per 100 g) among all seaweeds. Similarly, the highest Mg content was detected in red seaweeds (1400–5000 mg per 100 g), followed by green  and brown  seaweeds. The highest Mg content was detected in the red seaweed AMP (5000 ± 50 mg per 100 g), followed by the green seaweeds UF and UL. The magnesium content of the brown seaweed PAD and red seaweed SCI were comparable, while the lowest Mg content was detected in the green seaweed CAU (800 ± 100 mg per 100 g), followed by the brown seaweed SAR (900 ± 75 mg per 100 g). The maximum Ca content was detected in the red seaweed AMP (3500 ± 50 mg per 100 g); however, other red seaweeds contained lower ranges (45–85 mg per 100 g) of Ca content. The highest range of Ca content was detected in brown seaweeds (160–820 mg per 100 g), followed by green seaweeds (50–270 mg per 100 g).The highest concentration of iron (36 ± 10 mg per 100 g) was found in the red seaweed AMP, followed by the brown seaweed IYN (6 ± 0.1 mg per 100 g) and green seaweed ACR (2 ± 0.01 mg per 100 g); however, all other seaweeds contained iron in the range of 0.1–1.0 mg per 100 g dry weight. Similarly, Zn and Mn contents were detected in all seaweeds in the range of 0.1–3.0 mg per 100 g (except PAD) and 0.2–4.5 mg per 100 g , respectively. Most of the elements were detected in traces and mostly in brown and red seaweeds; however, none were found in green seaweed, except Co in ACR. −1 dry weight (DW) was observed for the red seaweed HAL, followed by GRA (7 ± 1 mL g−1) and SOL (6 ± 0.5 mL g−1), respectively. All other seaweeds showed WSCs in the range of 1–5 mL g−1 DW. The water- and oil-holding capacities were observed to be almost similar in all seaweeds and in ranges of 6–8 and 7–9 g g−1 DW, respectively. Seaweeds were characterized in terms of the water-swelling capacity (WSC) and water- and oil-holding capacities . The higThe oxidative stability of peanut oil resulting from different seaweeds and BHT (standard synthetic antioxidant) was studied for various accelerated storage condition times (0–15 days) by determining the peroxide value (PV), p-anisidine value (AV), conjugated dienes (CDs), total oxidation (TOTOX) and thiobarbituric acid-reactive substances (TBARS). 2 kg−1) at the 15th day for all seaweeds (2 kg−1), followed by the brown seaweed SAR (224 mEq O2 kg−1). For all other seaweeds, PV values ranged from 270 to 400 mEq O2 kg−1 (except HAL: 472 mEq O2 kg−1), while a 568 mEq O2 kg−1 PV was noticed for oil kept without any supplementation (control). Similarly, lower PA values were found  was determined for oil supplemented with the red seaweed AMP, followed by the green seaweed UF (0.32 mg kg−1) and ACR (0.33 mg kg−1), at day 15 of the accelerated storage condition. All other seaweeds showed 0.34–0.36 mg kg−1 TBARS, lower than the standard BHT (0.39 mg kg−1) and the control (0.45 mg kg−1).The CDs , TOTOX  and TBARThe cation scavenging, free radical scavenging and total reducing activities of seaweed powders (freeze-dried) were determined by using ABTS and DPPH radicals and ferric ions, respectively, on the basis of the concentration gradient .50 dose, or lowest antioxidant activity, was observed for CAU and Hal, followed by SOL.The green seaweed ACR and brown seaweeds SAR and IYN showed more than 50% of the total antioxidant  at their lowest concentration (<1 mg) . Approxi50 dose, was found for HAL , followed by AMP  and SOL . The lowest EC50 dose was found for UL (1 mg mL−1), followed by SAR (1 mg mL−1), ACR (2 mg mL−1), STO (3 mg mL−1) and CAU (4 mg mL−1)  scavenging activity , ACR shomg mL−1) .50 dose was detected for the green seaweed ACR (1.5 mg mL−1), followed by the brown seaweeds SAR, SPA and STO (3–4 mg mL−1)  . Other stivities .−1) in seven seaweeds, with the lowest concentration of 1.5 ± 0.5 µg g−1 in LOB and the highest of 6 ± 0.5 µg g−1 in STO. Phytic acid was also detected in negligible amounts in seven seaweeds, with the highest concentration of 24 ± 0.5 µg g−1 in SCI, followed by CAU (12 ± 0.5 µg g−1), whereas SAR, SPA, STO and SOL contained phytic acid in low concentrations (2–7 µg g−1). Other antinutritional compounds, including saponins, alkaloids and terpenoids, were not detected in any of the seaweeds  [Gelidiella acerosa and Sargassum wightii collected from the intertidal region of the Gulf of Mannar (India). High ash content was observed in marine macroalgae in comparison to terrestrial plants, in which the ash content ranges from 5 to 10%, except some vegetables, such as spinach [Enteromorpha clathrata, Gracilaria corticata and Sargassum linearifolium from the Red Sea coast near Massawa, Eritrea [Moisture content affects food stability and is a key factor for the preservation of various products . The moi6 to 9%) . In this6 to 9%)  for Geli spinach ,57. A co spinach . In the  Eritrea . A low r Eritrea . A high  Eritrea .Ulva lactuca (56%), which is widely considered an edible seaweed.The metabolic processes of all organisms use carbohydrates as one of the most important components. Carbohydrates are also a source of energy for respiration and other vital mechanisms. Total sugar content in seaweeds varies based on multiple factors, such as environmental conditions, species, location, etc. In this study, it ranged from almost 9 to 55% of the dry weight, which was in concordance with previous reports of 4 to 75% ,62. CompProtein content depends on seaweed species, geographical area and environmental conditions. Previously, protein content was measured as 5–15% of dry weight in brown seaweeds and 10–30% of dry weight in green and red seaweeds . In thisThe lipid content of seaweeds is generally found to be lower than 5% of dry weight, and in this study, similar results of between 0.5 and 7% of dry weight were estimated . SimilarUtilization of foods that contain high fiber protects individuals from various chronic diseases, such as colon cancer . In the −1 to 11 mg L−1, which was a little higher than the previously reported data [−1, and it is the main pigment in seaweeds providing color. The NFE ranged from 21% to 75%, which was similar to previous reports [Several Spanish seaweeds have shown new chlorophyll derivatives that help unravel the role of these chlorophyll derivatives in food composition and bioactive compound databases . Previouted data . The chl reports .–) during the alkali digestion, allowing the determination of the total iodine concentration in matrices rich in fat or carbohydrates. Iodine concentration is normally high in seaweeds compared to terrestrial plants (<1 µg g−1) [−1, and the recommended daily intake (RDI) level for an adult is 150 µg. Brown seaweeds showed higher iodine content compared to the other seaweeds and previous reports. Overall, brown seaweeds contained high iodine compared to green and red seaweeds. Based on the iodine content, it can be recommended that consumption of seaweeds should be kept within the RDI limit of 150 µg iodine per day.The decomposition technique is used to determine iodine content. In this technique, potentially interfering organic compounds are destroyed through incineration in the presence of KOH, which is a strong alkali that uses air as an oxidant. In seaweeds, iodine is found in both organic and inorganic forms . All org µg g−1) . The wat µg g−1) . In thisG. corticata and G. edulis showed 4.03 ± 0.39 g g−1 and 4.09 ± 0.32 g g−1 WHCs, respectively, which were higher than those of AMP and SCI but lower than the other red seaweeds in the present study [U. intestinalis showed a higher WHC (9.54 ± 0.02 g g−1) than the green seaweeds in the present study [Previously, a positive impact was reported for seaweed supplementation on food emulsion stability . This prnt study . The grent study . The watG. fisheri and G. tenuistipitata, which showed 1.83 ± 0.08 g g−1 and 2.12 ± 0.03 g g−1, respectively [−1 [−1 [The hydrophobicity of proteins plays an important role in the absorbance of oil ,76. In tectively . The OHCvely [−1 , which i [−1 [−1 , much loMinerals are chemical elements that are crucial for normal metabolic functioning. Definite concentrations of a wide range of minerals are required by the human body to function properly, and human health is also affected by these minerals . The daiUlva species including U. fasciata and U. lactuca, showed positive correlations with the antioxidant activity and thus negatively correlated with their EC50 doses [Kappaphycus alvarezii were studied for their bioactivity. It was found that gaint (commonly known as white seaweed) had the highest phenolic and flavonoid contents, which also showed positive correlations with the antioxidant activity [50 doses. The present study is similar to the previous reports, in which phenolic and flavonoid contents showed strong positive correlations with antioxidant activities [U. lactuca and Sargassum wightii  showed a direct relation with their antioxidant activities; these results support the strong correlation between bioactive compounds and antioxidant activity [50 doses [Polyphenol and flavonoids can donate hydrogen atoms to free radicals, resulting in the inactivation of the free radicals and transforming them into chain-breaking antioxidants . It is e50 doses . Three vactivity . In the tivities . In a practivity . Our preactivity , and the50 doses ,26,49.In an oxidation reaction, oxygen reacts with a substance, which results in the synthesis of free radicals and other dangerous chemical compounds. Generally, oxidation is more sensitive in unsaturated fats, whereas saturated fats are comparatively resistant to oxidation. In polyunsaturated fats, the number of unstable double bonds is high and, therefore, fats are susceptible to oxidation. These fats can be oxidized by exposure to sunlight, air or moisture and even by simple heating. Peanut oil contains high amounts of polyunsaturated fatty acids, and the oil is commonly used at high temperatures to fry food items and is susceptible to oxidation. Peanut oil oxidation generates free radicals that may contribute to many diseases, such as cancer, heart diseases and premature aging ,88,89.Macrocystis pyrifera and Ecklonia radiate) and two red (Porphyra sp. and Champia sp.)—had the ability to reduce the oxidation of fish oil [Bifurcaria bifurcate, Ascophyllum nodosum and Fucus vesiculosus, significantly reduced the oxidation of canola oil, more so than the synthetic antioxidant BHT [Many natural antioxidants are useful for the reduction of lipid oxidation in lipid-enriched foods and increase their lifespan. Seaweed extracts have shown reductions in the oxidation of oil ,91. In afish oil . Extractdant BHT . In the Addition of the synthetic antioxidant BHT reduced oxidation by about 18%, while supplementation with ACR, SAR and IYN reduced oil oxidation by about 70%, 60% and 50%, respectively. However, with supplementation with other seaweeds, 30–50% reductions were noticed, except HAL, which reduced oil oxidation by about 17–18% (similarly to BHT). The seaweeds ACR, SAR and IYN contained high phenolic and flavonoid contents and, therefore, showed high oxidation-inhibition activities compared to the other seaweeds. The results confirmed that seaweeds can be used to improve the biological value and oxidative stability of food oils, especially peanut oil.The absence of alkaloids, terpenoids and saponins, and the presence of negligible or trace amounts of phytic acid and tannic acid, confirmed that the studied seaweeds have potential to be explored as nutritional food supplements. Saponins affect health and metabolism because of the inhibitory activity of a number of digestive enzymes, including chymotrypsin, lipase, glucosidase, trypsin and amylase, and inhibition of these enzymes results in health disorders related to indigestion ,94,95,96Nutritionally, 155–230 calories per 100 g of dry seaweed were estimated for green seaweeds, 105–300 calories per 100 g for brown seaweed and 80–165 calories per 100 g for red seaweeds. The highest calorie value of about 300 calories per 100 g was observed for SPA, followed by SAR , STO , UF  and CAU . Red seaweed showed a low calorie intake value compared to the other seaweeds. Low-calorie seaweeds may also be explored in food science and technology to develop low-calorie food or food items.Ulva and Caulerpa; the brown seaweeds Spatoglossum asperum, Sargassum linearifolium and Padina boergesenii; and the red seaweed Scinaia carnosa are good sources of energy. The study also confirmed the absence of antinutritional compounds and toxic mineral elements in the studied tropical seaweeds. The present study recommends green seaweeds for overall nutrition, followed by brown and red seaweeds. Overall, tropical seaweeds have high nutritional potential and may deserve further exploration in relation to dietary supplementation and food/feed consumption purposes; however, a detailed, prolonged toxicity analysis with a human and/or animal model system is needed before any conclusive recommendations can be made regarding regular daily consumption in food/feed. Furthermore, they may also be explored as key ingredients in food toppings and for garnishing or seasoning food or food products.The antioxidant activities of the studied seaweeds confirmed their potential for application as natural antioxidants to improve the biological value of food and the oxidative stability of food oils, especially peanut oil. Proximate composition analyses of the abundant tropical seaweeds confirmed that the green and brown seaweeds contained more calories per 100 g of dry seaweed. The green seaweeds


Esr1-deficient mouse strain and analyzed LCN2 expression in reproductive  and non-reproductive tissues  of both sexes. Tissues collected from adult wild-type (WT) and Esr1-deficient animals were analyzed by immunohistochemistry, Western blot analysis, and RT-qPCR for Lcn2 expression. In non-reproductive tissues, only minor genotype- or sex-specific differences in LCN2 expression were detected. In contrast, significant differences in LCN2 expression were observed in reproductive tissues. Particularly, there was a strong increase in LCN2 in Esr1-deficient ovaries when compared to WTs. In summary, we found an inverse correlation between the presence of ERα and the expression of LCN2 in testes and ovaries. Our results provide an important basis to better understand LCN2 regulation in the context of hormones and in health and disease.Estrogen receptor alpha (ERα) is widely expressed in reproductive organs, but also in non-reproductive tissues of females and males. There is evidence that lipocalin 2 (LCN2), which has diverse immunological and metabolic functions, is regulated by ERα in adipose tissue. However, in many other tissues, the impact of ERα on LCN2 expression has not been studied yet. Therefore, we used an  Despite increased consideration of sex as a biological variable in preclinical studies, sex bias in favor of males widely persists ,2,3. HowEsr1 and Esr2 genes [Estrogens, especially 17β-estradiol (E2), form the main class of female sex hormones. They are primarily synthesized in the ovaries, and to a lesser extent in adipose, testis, and adrenal tissues ,7. Neverr2 genes ,8. In adr2 genes ,8, whichr2 genes .Lcn2 promotor contains an ERE [ERα is expressed primarily in the liver, bone, mammary gland, uterus, ovary, and vagina . ERα sigs an ERE . This ans an ERE .Lcn2 encodes for lipocalin 2 (LCN2), also known as neutrophil gelatinase-associated lipocalin (NGAL), and 24p3 lipocalin. LCN2 is a 25-kDa glycoprotein that has been linked to an increasingly broad range of immunological and metabolic functions [unctions ,14,15. Cunctions ,16,17,18unctions . Immunolunctions . LCN2 isunctions ,21. Furtunctions ,22. In sThe function of LCN2 to transport the reproductive hormone precursor cholesterol suggests a link to the endocrine system ,24. ThisLcn2-deficient (−/−)Lcn2 mice develop a more severe phenotype of fructose-induced steatosis compared to males [In males, LCN2 possibly affects overall fertility, while in females, an immunological function is discussed ,28. In fto males . Furtherto males . Regardito males ,30,33. SEsr1 knockout mouse strain generated in 2003 [Esr1-deficient animals because LCN2 levels strongly increase in reproductive tissues when ERα is absent.To date, differences in LCN2 levels associated with ERα expression have not been systematically investigated yet. Therefore, the present study focused on different reproductive  and non-reproductive  organs in which LCN2, ERα, or both proteins are known to have profound effects. We examined the expression of these proteins in the tissues of male and female wild-type (WT) animals and compared them with the expression in tissues of a conventional  in 2003 . The obtEsr1−/−) mice are widely used in research to study aspects of estrogen receptors and their functions [Esr1 in the used mouse model (B6N(Cg)-tm4.2KskEsr1/J), DNA was isolated from ear punches and subjected to genotyping. In our genotyping protocol, the WT allele produced an amplicon of 287 bp in size with the chosen primer combination, while DNA taken from Esr1-depleted mice resulted in the amplification of a 550 bp fragment, which is due to the insertion of a floxed TK-neo selection cassette  and areal expression sites  analysis was used to compare the hepatic mRNA expression of nd males . AlthougEsr1-depleted tissue sections and IgG negative controls remained negative for ERα with low background staining. In addition, Western blot analysis of ERα confirmed staining results . ERα protein expression could not be detected in kidney tissue, while LCN2 expression in male kidneys showed strong variations in both genotypes  and STAR  showed positive staining signals in interstitial segments in both WT and Esr1-deficient males . STAR, which facilitates the rapid movement of cholesterol from the outer to the inner mitochondrial membrane, is highly expressed in Leydig cells ,46,47. Lnt males . Strikinnt males C. In addd to WTs D, demonsEstrogen, the major female reproductive hormone, is associated with a broad range of functions, such as affecting gene expression in reproductive organs . Serum eEsr1-deficient uteri, underlining a connection between ERα and LCN2 in uterine tissue.In our study, female mice were histologically assigned to the most likely stage of the estrous cycle by analyzing H & E staining of vagina, uterus, and ovary tissues . For thiEsr1-deficient ovaries exhibited haemorrhagic cystic follicles, as previously described , integrin αM (Itgam), myeloperoxidase (Mpo), and interleukin 1β (Il1b) through RT-qPCR. Tnf and Itgam expression levels were significantly increased in Esr1 knockouts compared to WT ovaries, while the mRNA expression levels of Mpo and Il1b tended to be elevated in knockouts compared to WTs  knockout mouse model [Esr1-deficient mice, Fshb knockouts exhibited normal E2 serum levels; conversely, Esr1 knockouts were associated with normal FSH levels [Interestingly, Burns and colleagues demonstrated a similar LCN2 expression pattern in a follicle-stimulating hormone (FSH) β -tm4.2KskEsr1/J) mice were generated as described by Korach and colleagues [®  and stored at −80 °C. In this study, n = 8 male and n = 6 female Esr1-deficient mice were included, as well as n = 8 male and n = 9 female WT mice.lleagues  and obtaw/v) sodium dodecyl sulphate (SDS) in H2O supplemented with proteinase K (0.1 mg/mL) and incubated at 55 °C overnight under shaking. The next day, samples were centrifuged for 10 min at 15,000× g at room temperature (RT) and the supernatant was supplemented with isopropanol (1:1). After incubation for 10 min at RT, samples were centrifuged . Pellets were washed with 70% ethanol, centrifuged , and finally resuspended in H2O. For Esr1 genotyping, the DNA was used as a template in a standard genotyping PCR according to the protocol recommended by The Jackson Laboratory [2O, primers (primer 1 (22055): CTG CCA AGG AGA CTC GCT AC (WT forward); primer 2 (22056): CCA CTT CTC CTG GGA GTC TG (common); primer 3 (22057): ATC CCA TGT GCT TGA GTG GT (MUT forward)), and dNTP mix. The amplification was conducted as follows: 30 cycles at 95 °C for 5 min, 95 °C for 1 min, 55 °C for 1 min, 72 °C for 3 min, and 72 °C for 10 min. Amplification was achieved on a T3000 Thermocycler , and fragments were separated in 1.6% agarose gels supplemented with ethidium bromide for 40 min (90 V) using 1× TAE  as a running buffer. Results were visualized in a GEL iX20 imager . Amplicon sizes were 287 bp  and ~550 bp , respectively.Genotyping was performed with DNA samples prepared from ear punches stored at −20 °C after biopsy collection. In brief, tissues were digested in lysis buffer containing 100 mM Tris-HCl, 200 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), and 0.2% (boratory . Brieflyw/v) NP-40, 0.1% (w/v) SDS, and 0.5% (w/v) sodium deoxycholate supplemented with the CompleteTM mixture of phosphatase inhibitors  and stored at −80 °C. Tissue samples were homogenized in an MM400 mixer mill  as previously described [N-morpholino) ethanesulfonic acid (MES) as a running buffer. For analyzing serum samples, 1 µL of serum was loaded into each lane. Proteins were transferred to Protran® Western blotting membranes  in NuPAGE buffer containing 10% methanol. Equal loading and successful protein transfer was visualized by Ponceau S staining. Subsequently, unspecific binding sites were blocked by 5% (w/v) non-fat milk powder in Tris-buffered saline supplemented with 0.1% Tween 20 (TBS-T). Membranes were incubated with primary antibodies (w/v) non-fat milk powder in TBS-T. Incubation was conducted on a shaker at 4 °C overnight. For visualization of protein signals, horseradish–peroxidase-conjugated secondary antibodies were used in combination with the SuperSignal chemiluminescent substrate . Membranes were re-probed with either glyceraldehyde-3-phophate dehydrogenase (GAPDH)  or vinculin (in kidneys) [Isolated tissue was resuspended on ice in RIPA buffer containing 20 mM Tris-HCl (pH 7.2), 150 nM NaCl, 2% (escribed . The DC tibodies  that werkidneys)  to demonkidneys) .2O and stored at −20 °C as previously described [TM qPCR SuperMix . Consecutive cycle conditions were initial denaturation at 95 °C for 10 min, followed by amplification for 40 cycles at 90 °C for 15 s and 60 °C for 1 min. All samples were loaded in duplicate. mRNA expression was normalized to either the expression of β-actin  or ribosomal protein S6 . Final mRNA quantities were calculated with the 2−ΔΔCT method [Dissected tissue samples were snap-frozen in liquid nitrogen and stored at −80 °C until further processing. Tissue was re-suspended in RNA lysis buffer with DTT and homogenized in an MM400mixer mill  as described previously . RNA extescribed . For revT method .Fresh tissue samples were fixated for 24 h at 4 °C in 4% neutral buffered formaldehyde (stabilized with methanol), dehydrated, and embedded into paraffin. Subsequently, 3 µm thick sections were prepared, deparaffinized in xylene, and subjected to decreasingly graded ethanol for rehydration. Samples were stained with hematoxylin  for 12 min and were afterwards placed under running tap water for a further 10 min. Slides were incubated in Eosin at a pH of 4.5  for 30 s and dehydrated in increasingly graded ethanol and xylene. DPX was used as a mounting medium . The H & E stain was used for estrous cycle staging as previously described ,80,85. T1, #X0931, DAKO) were equally prepared and applied. On the next day, endogenous peroxidase was blocked for 15 min in a solution of 3% H2O2  prepared in H2O. An avidin/biotin-based peroxidase system  was applied to all slides in accordance with the manufacturer’s instructions and incubated in a wet chamber at RT for 1 h. Afterwards, secondary antibodies  and counterstained with methyl green  as previously described for nuclear localization of ERβ [Formalin-fixed and paraffin-embedded tissues were deparaffinized and prepared as described above. Heat-induced antigen retrieval was performed by placing the slides in citrate buffer  for 30 min in a steamer, followed by cooling on ice for 30 min. Subsequently, samples were washed in phosphate buffered saline (PBS) and PBS supplemented with 1% Tween 20 (PBS-T). For immunohistochemical detection of ERα and LCN2, endogenous biotin and avidin binding sites were blocked with a biotin blocking system . Furthermore, unspecific antibody binding sites were blocked at RT for 90 min in 5% normal serum from the source species of the secondary antibody  in PBS supplemented with 1% bovine serum albumin, 0.1% cold fish skin gelatine, 0.1% Triton X-100, and 0.05% Tween 20. Primary antibodies were diluted in blocking solution : #MA1-27107, Thermo Fisher Scientific, 1:50) and incubated on samples at 4 °C overnight. IgG negative controls . Control slides were treated equally except for incubation with H2O instead of a primary antibody. Stained slides, which gradually lose intensity after a couple of days, were stored in the dark at 4 °C for a short period of time prior to stable documentation  or negative controls  overnight at 4 °C. 5% normal donkey serum  was used in the blocking solution compare . Samplestion see .Images were acquired with a Nikon Eclipse E80i fluorescence microscope equipped with the NIS-element Vis software . Selected slides were further scanned with a NanoZoomer  and viewed with NDP.view2 software .t-test was employed, otherwise, a non-parametric Mann–Whitney Test was chosen. For data that could be influenced by two factors (sex and genotype), two-way analysis of variance (ANOVA) was performed . Different significance levels are indicated by asterisks: * p < 0.05, ** p < 0.01, *** p < 0.001.All calculations were performed using Excel v16 . Protein expression was quantified densitometrically using ImageJ (software version 1.52n) and plotted relative to the protein loading control. Statistical analysis was performed with GraphPad Prism v.8.0 . Gaussian distribution was determined with Shapiro–Wilk tests. If normality could be assumed, a Student’s ed i.e., D. Data aEsr1-deficient mouse strain. Possible tissue-specific changes in LCN2 were examined in both male and female animals to highlight whether there is a sex-specific difference in LCN2 expression. The data obtained in our study indicate that the expression of LCN2 in some non-reproductive and reproductive tissues is Esr1-dependent. While knockout of Esr1 resulted in significantly increased expression of LCN2 in the testes and ovaries, the female lungs, as a non-reproductive organ, showed significantly lower expression of LCN2. Moreover, in some non-reproductive organs, there were no changes in LCN2 expression due to Esr1 knockout, nor between the two sexes. Since ERα is not physiologically expressed in the lungs and kidney, it is most likely that the regulation of LCN2 is not primarily driven by ERα in these organs, but rather by systemic parameters. These differences suggest that the regulation of LCN2 is tightly controlled in each tissue. Further studies are now necessary to unravel the precise molecular pathways by which ERα impacts LCN2 expression in different tissues.The aim of this study was to investigate whether the absence of ERα alters the expression of LCN2 in various non-reproductive and reproductive organs using an


Adansonia. The disjointed tree thrives in arid or semi-arid regions, is native to the thorn woodlands of Africa, along tracks, and is associated with human-populated forest areas. It is considered indigenous to Central and West Africa and has been introduced to the Arabian Peninsula, South-East Asia, the Indian sub-continent, and the Caribbeans. Adansonia digitata is a multifunctional tree with a long lifespan of over 1000 years old. The leaves, roots, flowers, fruit pulp, seeds and barks are used for food, medicine, or other ethnic-practices. The utilisation level and distribution are significantly undermined by climate change and poor use practices. The data set offers insight into the distribution pattern and genetic diversity of Adansonia digitata across the savannah belt of Nigeria using the rbcL gene.The African baobab species belong to the family Malvaceae and the genus  Specifications Table•Adansonia digitata sequences across the Savannah belt of Nigeria using information from partial rbcL gene sequences, nucleotide polymorphism and amino acid composition.The data provides information on the distribution and genetic diversity of •Adansonia digitata, which can be adopted for the improvement of the species and germplasm bank for species conservation.The data identifies areas of high genetic diversity of •Adansonia digitata.The rbcL gene sequences can be employed by plant molecular taxonomists to trace the molecular phylogeny, evolution as well as sub-speciation of •This data presents information on the species' amino acid composition and codon usage bias.1A. digitata accessions deposited on NBCI GenBank. A. digitata, the number of segregating sites (S), within-group mean distance, nucleotide diversity (π), nucleotide diversity (SD), and average no of nucleotide differences (k). A. digitata amongst the accessions; A. digitata accessions. of A. digitata accessions.The study presents the nucleotide sequences of rbcL gene for the 39 22.1Adansonia digitata accessions (39) were collected during a nationwide expedition across 17 states and Nigeria's Federal Capital City (FCT) in 2016-2017 , Ibadan, Nigeria.Using the rbcL primers H1f - forward (CCACAAACAGAGACTAAAGC) and Fofana - Reverse (GTAAAATCAAGTCCACCGCG) 20.25 µL PCR cocktail mix was generated for each sample with the following 2.5 µL of 10x PCR buffer, 1.0 µL of 50 mM MgCl2, 1.5 µL of 5 pMol forward primer, 1.5 µL of 5 pMol reverse primer, 1.0 µL of DMSO (Dimethyl sulfoxide), 2.0 µL of 25 mM dNTPs, 0.06 µL of Taq, 2.0 µL of 100 ng/µL DNA (extract), and 13.44 µL of HPCR amplification of the DNA samples was performed on GeneAmp™ PCR System 9700  thermocycler; with initial denaturation at 94°C, 180 sec, 10 cycles; denaturation at 94°C, 15 sec; annealing at 65°C, 30 sec; extension at 72°C, 90 sec, 10 cycles; denaturation at 93°C, 15 sec; annealing at 55°C, 30 sec; extension at 72°C for 90 sec, 30 cycles; and final extension at 72°C, 300 sec. Amplicons were held at 10°C until use.2.4Amplified products were loaded on 1.5% agarose gel with 1kbplus GeneRuler DNA Ladders (Thermo Fisher Scientific®). Gel components ran at 100 volts for 1 hr. The resultant bands were visualised under a UV light trans-illuminator.2.5De novo (unrevised) forward and reverse reads assembly were performed to generate consensus sequences using the Geneious assembler (GENEIOUS Prime 2020.1). Where assembly of De novo was not feasible, the NCBI database mapped forward and reverse reads to the previously submitted rbcL gene sequence (GU981721.1). The reference sequence was trimmed using the sequencing primers before mapping the sequencing reads to the guide. Using the Geneious alignment tool, generated sequences were individually aligned with the reference rbcL sequence after assembly to check for sequencing errors that might result in translation errors. Cleaned lines were then submitted to GenBank using Bankit.2.6A. digitata accession sequences were aligned to generate consensus sequences using Geneious Basic A. digitata ranged from 245-555 bp for the study.Generated Population diversity indices were assessed using DnaSP 4.5 Adansonia digitata accessions sequences were determined using DnaSP 4.5. Codon usage indices were estimated using CodonW on Galaxy server .The codon usage frequency, amino acid and nucleotide compositions of each The field data presented in Conrad Asotie Omonhinmin: Conceptualization, Funding acquisition, Methodology, Supervision, Writing – review & editing; Panrot Peace Piwuna: Investigation, Writing – original draft; Miracle Omolara Aderanti: Investigation, Formal analysis, Writing – review & editing; Abimbola Odunayo Bolade: Investigation, Data Curation.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Fertilizer treatments showed a strong association with %N, δ15N, δ2H, and δ18O values while it did not impart any significant variation for the %C and δ13C values. Light intensity levels also showed a significant influence on the isotopic values of different rice fractions. The δ13C values showed a positive correlation with irradiance. The δ2H and δ15N values decreased with an increase in the irradiance. The light intensity levels did not show any significant change for δ18O values in rice fractions. Multivariate ANOVA showed a significant interaction effect of different factors  on the isotopic composition of rice fractions. It is concluded that all environmental and cultivation factors mentioned above significantly influenced the isotopic values and should be considered when addressing the authenticity and origin of rice. Furthermore, care should be taken when selecting rice fractions for traceability and authenticity studies since isotopic signatures vary considerably among different rice fractions.The effect of fertilizer supply and light intensity on the distribution of elemental contents (%C and %N) and light stable isotopes  in different rice fractions  of two hybrid rice cultivars (maintainer lines You-1B and Zhong-9B) were investigated. Significant variations were observed for  Oryza sativa L.) is a major cereal crop consumed by half of the global population and widely planted in Asia, Africa, and parts of America. In 2020, the total area under rice cultivation exceeded 195 million hectares. The quality characteristics of rice are mainly associated with its growing conditions, and recently the traceability of rice back to its growing origins has gained increasing interest from consumers, producers, and related industries since it is vulnerable to economic fraud [δ13C, δ15N, δ2H, and δ18O) in different rice fractions according to the differences in light intensity level, fertilizer type and concentration, and cultivar/variety would improve the validity of traceability methods for rice and its products.Rice , and cultivation practices have been shown to induce stable isotope fractionation in plants . Generalg period . Solar iuctivity . In addi enzymes . This inductance . Nitrogeductance .δ13C, δ15N, δ2H, and δ18O values in different rice fractions  in response to different fertilizer regimes and light intensity levels, and to identify how light shading and fertilizer application control isotopic ratios in rice. The results from this study will contribute more insight into the localized climatic, environmental, and farming effects on the isotopic composition of rice and will allow us to better predict rice origin and authenticity using stable isotope-based traceability models.Many studies have reported the limitation of stable isotopes in addressing the authenticity of agro-products when the samples are procured from close geographical locations or from a region where the same agricultural practices  are adopted . Therefo2 with 3 replicates for each treatment. Nitrogenous fertilizer was applied as 0 kg/ha (N0), 90 kg/ha (N6), and 180 kg/ha (N12), respectively, at different growing stages including basal planting, tillering, and heading stages which accounted for 50%, 30%, and 20% of the application, respectively. Phosphate fertilizers were used only as basal fertilizer, whereas potash was used as both basal and tillering fertilizers and the fertilizer proportion was N:P:K = 1:0.5:0.5, respectively. Field trials were carried out in 2018 at an experimental field at the China National Rice Research Institute (CNRRI) in Fuyang, Zhejiang province. Two commonly used hybrid rice maintainer lines , were studied. In total, three nitrogen treatments  with three light intensities  were arranged in a split plot design. The size of each plot was 10 mIn the case of N0, urea was not applied during the entire production period. In total, 450 kg/ha of super phosphate was applied as basal fertilizer and 150 kg/ha of potash as basal (75 kg/ha) and tillering stage (75 kg/ha), respectively. For N6 treatment, 195 kg/ha of urea was applied, including 97.5kg/ha as basal fertilizer, 58.5 kg/ha as tillering fertilizer (7 to 9 days after transplanting), and 39 kg/ha as heading fertilizer (30 days after transplanting), respectively. In the case of N12 treatment, a total of 390 kg/ha of urea was applied, of which 195 kg/ha was basal fertilizer, 117 kg/ha was tillering fertilizer, and 78 kg/ha was applied as heading fertilizer. The application rate of phosphate and potash fertilizers for both N6 and N12 treatments were the same as N0. For the light intensity investigation, natural sunlight treatment, LS-0 , and shaded treatments, LS-50 (50% shading) and LS-75 (75% shading) were applied. In the shaded treatments, plants were shaded with a shading screen/net. For LS-50, one layer was used and for LS-75 two layers of shading screen were applied over the top of the rice plants. δ13C and δ15N), dried powdered samples were weighed (4.5 to 5.5 mg) and packed into tin capsules (3 mm × 5 mm). The samples were combusted in an elemental analyzer  and the combustion gases were analyzed using an isotope ratio mass spectrometer (IRMS) . Sample combustion was carried out in a combustion furnace at 1150 °C and reduction in the N2Ox gases to N2 over copper wire occurred at 850 °C. An inert gas (He) with a flow rate of 230 mL/min was passed through a CentrION prior to mass spectrometry. Acetanilide  was used to calibrate elemental % C and % N. For δ13C and δ15N analysis, multipoint calibration was applied using reference standard materials including B2155 , IAEA-CH-6 , USGS40 , USGS64 , and IAEA-N-2 . The δ13C and δ15N values were measured relative to V-PDB and AIR, respectively. Rice grains (2 kg) from each plot were harvested at maturity and subsequently threshed by a hulling machine equipped with a rice polisher  to obtain different fractions including brown rice (BR), polished rice (PR), and rice husk (RH). All fractions were air-dried, then ground into a fine powder, and finally dried at 50 ± 2 °C for 24 h. The samples were stored in desiccators until further analysis. For the determination of elemental contents  and isotopes  and analyzed using EA  IRMS . Reference materials USGS54  and USGS55  were used to calibrate the δ2H and δ18O measurements. Samples and reference materials were freeze-dried at −60 °C for three days to remove all adsorbed water and subsequently equilibrated for five days in the laboratory and exposed to local atmospheric conditions prior to H and O analysis. Pyrolysis was performed at 1450 °C to convert organic H and O to gaseous H2 and CO, respectively, and finally the analytes were transferred into the IRMS for isotope determination. The δ2H and δ18O values were measured relative to Vienna Standard Mean Ocean Water (V-SMOW). All the samples were analyzed in triplicate. Reference materials were sourced from the International Atomic Energy Agency  and the United States Geological Survey . B2155 was supplied by Elemental Microanalysis . The analytical precision for δ13C, δ15N, δ2H, and δ18O was less than ±0.1‰, ±0.2‰, ±2‰, and 0.5‰, respectively. The delta values (δ) were calculated as follows:δE represents δ13C, δ15 N, δ2H, and δ18O whereas Rsample and Rstandard represent the 13C/12C, 15N/14N, 2H/1H, or 18O/16O ratios in samples and reference materials, respectively. For δ13C, δ15 N, δ2H, and δ18O of different rice fractions with multivariate analysis of variance. Light shading level, fertilizer type and concentration, and cultivars were considered as fixed variables. Differences among the treatments were evaluated using Duncan’s test at a significance level of 0.05. All analyses were performed using R software (version 3.0.3).The effect of light intensity, fertilizer type and concentration, cultivars, and their interaction were studied using %C, %N, δ13C, δ15N, δ2H, and δ18O values of different rice fractions, including polished rice (PR), brown rice (BR), and rice husk (RH). The results are summarized in δ13C, light shading and variety significantly contributed to all rice fractions. The interaction (vty × fertilizer)/(vty × light shading) effect also showed significant influence for PR and (vty × light shading) for RH. No interaction effect was observed for BR , shading level, and fertilizer (nitrogen) concentration on the carbon (%C), nitrogen (%N), d for BR b–d. δ15N values in RH, PR, and BR were significantly affected by fertilizer concentration, shading level, and interaction (vty × shading level) effects /(vty × light shading) effect was only observed for PR /(vty × light shading) effects for δ18O are shown in The  effects e–g. Signd for PR h and , PR (−28.3‰), and BR (−29.1‰) were significantly higher than Zhong-9B , respectively. The lower δ13C values of Zhong-9B rice suggest higher water use efficiency. Different factors are responsible for genetic variations in the δ13C values, including diffusive conductance, water use efficiency, stomatal activity, etc. [δ18O values, the two varieties also showed significant differences for PR and RH. Higher δ18O values of You-1B RH (23.7‰) indicate higher transpiration rates. Our results are consistent with previous findings where a similar trend was observed for δ18O variations among different rice cultivars [δ18O values are mainly due to the vegetative cycle of water [Differences in the %C, %N, O values . The %N ty, etc. . For theof water . δ2H values. You-1B BR (−88.6‰) and RH (−125.7‰) were significantly lower than Zhong-9B BR and RH (−84.7‰ and −117.0‰), respectively. This difference suggests that less water fractionation occurred in Zhong-9B rice. A previous study also reported a significant difference in the δ2H values among different rice cultivars [A different pattern was observed for the δ15N values also followed the same trend where Zhong-9B rice values were higher than You-1B, although the difference in traits between rice varieties was primarily reflected in RH. δ15N values of PR, BR, and RH in Zhong-9B and You-1B rice was  and , respectively. Our results are consistent with previous findings where significant differences in the δ15N values among different rice cultivars were reported [The reported .δ13C, δ15N, δ2H, and δ18O values was measured and the results are shown in δ15N values among different rice fractions were significantly affected by nitrogen fertilizer application levels. The %N and δ15N values of different rice fractions under different fertilizer concentrations  followed different patterns of accumulation. δ15N value for N0 was significantly higher than N6 and N12 indicating that the synthetic urea fertilizer application level was negatively correlated with the δ15N values in different rice fractions  [δ15N signatures in plant tissues is generally complex since it can differ from the nutrient source as 15N fractionation occurs during plant physiological processes, mainly during nitrogen uptake, nitrate assimilation, and reduction, as well as during remobilization to the rice grain [δ15N values in the fertilized plant fractions during pre-anthesis reflect the direct N availability from the fertilizer applied. Discrimination against 15N occurs during the assimilation of inorganic nitrogen within the plant, and the enzymes responsible for this isotope discrimination include nitrate reductase and glutamine synthase [These results appear consistent with previous findings which suggest conventionally fertilized rice has lower tilizer) . The intce grain ,21. Lowesynthase . 15N in fertilized plants compared to non-fertilized plants [15N is marginal in non-fertilized rice ears  as compared to fertilized rice ears, probably because N limitation prevents discrimination and promotes a higher efficiency for N remobilization [15N discrimination. This fact suggests a decrease in the δ15N values for the rice grain in fertilized plants compared to non-fertilized plants. Discrimination within the plant is lower in the non-fertilized grains  because the ratio between the plant demand vs. available N is high [Higher N assimilation rates in fertilized plants enhance the uptake of inorganic fertilizer which results in the reduction in d plants . In addilization . The hig is high .δ2H values in PR and BR. The highest δ2H values in PR (−73.2‰) and BR (−83.2‰) were observed for the N12 treatment which indicates that the δ2H values increased with N fertilizer level. There is limited literature on this topic, which restricts the discussion of this mechanism, although a similar trend was observed for the δ2H values in a previous study, where it was reported that BS-fertilized (biogas slurry) rice had higher δ2H values than a control rice (without fertilizer) [δ18O values contrasted with δ2H values. There was no significant effect in the PR and BR grains, probably due to a weak or no effect on grain morphological parameters, and a lack of N effect on stomatal conductance and transpiration rates [Nitrogen fertilizer application had a significant effect on the tilizer) . The effon rates . δ18O values in RH. The mean δ18O values for N6 (21.3‰) and N12 (20.2‰) treatments were significantly lower than N0 treatments (22.9‰) suggesting that the δ18O values in RH decreased with increasing nitrogen fertilization. Lower δ18O values in RH than the rice grain may be associated with progressive enrichment in plant components from the root, to the stem, to the leaf, and finally to the grain, as well as physiological factors such as dehydration and plant tissue degradation that occurs during husk development [A significant difference was observed in the elopment .δ13C, δ15N, δ2H, and δ18O values of different rice fractions. The experiment was performed using two shading treatments (LS-0 and LS-50) for rice plants grown with fertilizer treatment N0 and N6, respectively  followed by LS-50 (2.0 ± 0.2%) and the lowest was observed in LS-0 (1.6 ± 0.1%), respectively. The δ15N values followed a unique pattern. It can be seen in δ15N signature among different rice fractions was significantly higher in LS-50 compared to LS-0 (non-shaded), but it decreased in LS-75. No significant difference was found in the δ15N values for LS-0 and LS-75. Further investigation is required to explore this mechanism since no literature was found that explained this phenomenon in rice. However, previous research has shown that the effect of maize kernel shading (40%) on %N at different stages showed a decrease in the %N content (up to 60%) compared to the control (ambient sunlight), which is not consistent with our study [Different shading treatments including LS-0, LS-50, and LS-75 were applied and the effect was observed on the %N, %C, ectively a, and thectively b. The shur study . This crδ13C values among different rice fractions also showed significant variations for different shading treatments. The %C values of LS-0 in different rice fractions including PR (−28.3‰), BR (−28.8‰), and RH (−30.5‰) were significantly higher than those of LS-50 , and LS-75 , respectively, indicating that at greater light intensity, the 13C isotopic fractionation is higher. In previous studies, a similar trend in δ13C values for citrus, grapefruit, and banana plants were reported under different shading treatments [δ13C values are positively correlated with irradiance and a similar trend of increasing δ13C values with increasing irradiance has been found in banana plants [δ13C values have been shown to be negatively correlated to internal CO2 concentration in plants during carbon uptake, so a decrease in δ13C values under shaded treatments indicates increased conductance during the time that the CO2 was fixed and/or a decreased photosynthetic rate [δ13C values in shaded treatments clearly indicated that shading disrupted the photosynthate metabolism, reducing the photosynthate accumulation in grains [The eatments ,28,29. Ttic rate . Another and RH −.5‰ were δ2H values also showed significant variations for PR and BR. In PR, the highest δ2H value (−65.3‰) was found in LS-75 followed by LS-50 (−69.3‰), and the lowest was observed in LS-0 (−77.0‰), respectively. The same trend was observed for BR and there were significant differences between LS 50/75 and LS-0. Soil water  is the main source of δ2H in plants, and the present results indicate that different light intensities affect the δ2H values of soil water, causing the δ2H signature of rice grains to vary through transpiration. No significant difference was observed for the δ18O values among the different rice fractions at different light intensities.The δ13C, δ15N, δ2H, and δ18O values of different rice fractions among different cultivars. Fertilizer application rates showed a strong association with %N, δ15N, δ2H, and δ18O values, although there was no significant variation in the %C and δ13C values. The light intensity levels also showed a significant influence on isotopic contents among different rice fractions. The δ13C values showed a positive correlation with irradiance. The δ2H and δ15N values decreased with an increase in the irradiance. It is concluded that all factors mentioned above significantly influence isotopic values and should be considered when addressing the authenticity of rice. Furthermore, care should be taken when selecting the rice fraction for future studies as isotopic signatures vary considerably among different rice fractions. The findings from this study will have a significant impact on understanding different climatic trends  and fertilizer effects on rice and allow better understanding of isotopic variability for different geographical regions, farming practices, and environmental conditions.In this study, the effect of cultivar type, fertilizer supply, and light intensity on the distribution of elemental contents and light stable isotopes  in different rice fractions were investigated. Significant variations were observed for


The arrangement of nanoscale building blocks into patternswithmicroscale periodicity is challenging to achieve via self-assemblyprocesses. Here, we report on the phase-transition-driven collectiveassembly of gold nanoparticles in a thermotropic liquid crystal. Atemperature-induced transition from the isotropic to the nematic phaseunder anchoring-driven planar alignment leads to the assembly of individualnanometer-sized particles into arrays of micrometer-sized agglomerates,whose size and characteristic spacing can be tuned by varying thecooling rate. Phase field simulations coupling the conserved and nonconservedorder parameters exhibit a similar evolution of the morphology asthe experimental observations. This fully reversible process offerscontrol over structural order on the microscopic level and is an interestingmodel system for the programmable and reconfigurable patterning ofnanocomposites with access to micrometer-sized periodicities. The presence of an isotropic–nematic coexistenceregion above a critical NP threshold enabled the formation of macroscopicNP-enriched networks.45 NP-based hollowmicrostructures were formed through a two-stage nematic nucleationprocess.47 Cooling of LC-NP composites also enabledthe spatial separation of quantum dot clusters; however, control overthe positional order was limited and only achieved by the introductionof macroscopic beads as nucleation points.49 While much progress has been made on colloid-liquid-crystal compositesover the past 20 years, it remains a challenge for such and otherself-assembling materials systems to spontaneously and reversiblyarrange nanoscale building blocks into patterns of microscale periodicitywith control over size and spatial arrangement.In principle,exploitation of liquid crystal phase transition processesoffers access to structure formation beyond the nanometer length scale.This was first achieved via the cooling of a colloid-liquid crystalcomposite comprised of 0.5 μm-sized poly(methyl methacrylate)particles and the thermotropic LC 4′-pentyl-biphenyl-4-carbonitrile(5CB) below its isotropic-to-nematic transition.In this work,we present how the temperature-induced isotropic-to-nematicphase transition of the thermotropic LC 4′-pentyl-biphenyl-4-carbonitrile(5CB) in an LC cell with planar alignment can drive a hierarchicalassembly of nanometer-sized gold particles into micrometer-sized agglomerates.This process is not only reversible but offers control over the characteristicsize and spacing of the resulting structures. We report on the dynamicsof this process by experiments and accompanying phase-field simulationsand correlate our findings to parameters such as the cooling rate,nanoparticle solubility, phase separation kinetics, and the propertiesof the nematic director field.50 First, oleylamine-protected NPs were synthesizedand subsequently functionalized by ligand exchange with a 60:40 mol% mixture of 1-hexanethiol (HT) and 4′-(12-mercaptododecyloxy)biphenyl-4-carbonitrile(MDD-CBO). This led to an effective ligand composition on the NP surfaceof 61:39 ± 3 mol % according to analysis by NMR spectroscopy.The Au NP size distribution was 4.7 ± 0.7 nm accordingto TEM analysis. Another batch of Au NPs with a size distributionof 6.0 ± 1.0 nm and a comparable ligand composition yieldedresults similar to those reported here. The NPs were then transferredto 5CB in the isotropic phase at a concentration of 5  wt %following an established protocol using dichloromethane as a volatilesolvent.51 Subsequently, the LC-NP compositewas infiltrated into an LC cell with a defined gap thickness of 20  μmand homogeneous surface alignment. Further details on the NP synthesis,cleaning procedure, solubility, and material characterization canbe found in the Supporting Information . Interestingly, the NPs showed superiorsolubility in isotropic 5CB compared to various common solvents, includingchloroform, chlorobenzene, and a 50/50 vol % mixture of acetonitrileand tetrahydrofuran .Au NPs were made in a modularapproach, which allowed independentcontrol over both the NP size and ligand composition on the surface.–1 to 28 °C. Inthis experiment, the phase transition was observed to commence at34.3 °C, which is in line with earlier studies that reportedon the decrease of the phase transition temperature in the presenceof NPs.53 A series of images was then taken at this temperaturealongside microspectroscopic absorbance measurements by partial out-couplingof light. By analysis of the micrographs, the behavior observed uponcooling may be divided into two stages. In the initial stage withinthe first 60 s, a compartmentalization occurred into NP-depletedand NP-enriched regions that resembled a spinodal-type decomposition;over time, the characteristic continuous pattern evolved to yieldisolated NP agglomerates at the end of the process. Cross-polarizedoptical microscopy revealed that during the phase transition process,the regions, which appeared brighter under bright field illuminationin the initial decomposition phase, corresponded to nematic domainsaligned with the homogeneous orientation of the cell, while the darkerregions were nematic microdomains with random alignment . As shown inthe accompanying video of the isotropic–nematic transitionunder cross-polarization (see Supporting Video 1), the growth of the uniformly aligned nematic domains startedshortly after the isotropic–nematic transition, once the randomlyaligned domains formed at the transition became large enough to interactwith the boundaries of the cell. We hypothesize that at the isotropic–nematicphase transition, the NPs tended to accumulate in the isotropic phase,as was observed in other systems.48 Thesubsequent growth of large, uniformly aligned domains, energeticallyfavored by the cell alignment, promoted the segregation of NPs inthe interstitial regions. Here, the NPs became more and more concentratedand, consequently, the receding phase became more and more disordered.Because of the high solubility of the NPs and the mesogenic natureof their ligands, the NP-enriched phase still retained some birefringence,which suggests a nonzero order parameter.The dynamics of the LC phase transition and subsequent diffusionof NPs into the nematic phase are shown in max)decreased drastically from 1.46 to 0.90 in the newly formed alignednematic domains . The absorbance results are, thus,in line with a short-lived depletion of NPs in the planarly alignednematic domains followed by subsequent diffusion of NPs from the enrichedreceding random nematic to the aligned nematic phase. After reachingsteady state, the large majority of the NPs (93.8%) became dispersedin the aligned nematic phase with the residual fraction of NPs embeddedas agglomerates in the composite matrix. Importantly, the shape ofthe spectral absorbance remained similar after the phase transition,i.e., apart from the ordered formation of μm-sized agglomerates,we did not observe the aggregation of individual AuNPs in the alignednematic phase that would result in a spectral shift. We further emphasizeon the role of the homogeneous substrate, which imposes an in-planealignment of the mesogen director field. When cells without surfacealignment were used, cooling of the LC-NP composite led to the formationof NP-enriched and NP-depleted regions but these remained in coexistence, whichis in line with earlier findings.45The findings of in situ absorbance microspectroscopyare shownin  domains 1b, thus function 1d. BasedSupporting Figures S1 and S2). We find that while interdigitation of theligand shell generally facilitates solvation of the NP complex, thesolvation free energy depends on whether the medium is isotropic ornematic. In the nematic phase, solvation of an object requires distortingthe local order parameter, which introduces a free energy penaltyto the system. A solvation barrier as driving force for a phase transition-drivencollective assembly may be conceptualized as follows. Cooling of theLC-NP composite to the isotropic-to-nematic phase transition temperature Tiso–nem induces demixing and a short-termphase coexistence of NP-enriched regions with vanishing order parameterand NP-depleted aligned nematic regions. The buildup in NP concentrationin the receding disordered phase then forces additional NPs to slowlydiffuse into the aligned nematic phase until, eventually, micrometer-sizedagglomerates of residual NPs are retained.To examineunderlying effects, we developed a qualitative coarse-grainedmolecular dynamics model of a single NP complex with mixed ligandshell immersed in a host of nematogenic mesogens, which is discoursedin detail in the Supporting Information  was calculated. Rastering acquisitionwith a 5× objective allowed analysis of the entire active areaof the LC cell (10 × 10 mm2), which containedbetween 1500 and 60 000 agglomerates depending on the coolingrate.54 We refer to the Supporting Information for a comprehensive comparison of theRDF with the nearest neighbor distance (NND) approach, in particularon their robustness toward small positional deviations . As shown in In 2, was converted to a grayscale matrix. The information entropy canserve to evaluate the uniformity of a system, herein for the uniformityof pixel values.55 This was conducted followinga procedure introduced by our group recently, with results shown in 56 For a perfectly homogeneous system, each pixel value shouldbe equivalent, resulting in a maximum entropy per μm2 of 1. As the variation in absorbance for each pixel increases , the entropy is reduced. Dueto the presence of the glass spacer beads and other minor aberrationsin the LC cell and optical path, the upper limit may not be obtainedbut the reversibility over time can be tracked. Prior to the nematic-to-isotropicphase transition, the entropy per μm2 remained relativelyconstant at an average of 0.979 μm–2. At the transition onset, this dropped drastically to 0.828 μm–2. With diffusion of the AuNPs into the isotropic phase,the entropy per μm2 increased toward 1 followinga logistic trend, and, thus, demonstrating homogeneity.We want to emphasize boththe stability of the agglomerates uponformation and the reversibility of the process. Once formed, the NPagglomerates were found to be stable for months only to be redissolvedby heating to temperatures above the isotropic phase transition. Wedid not observe further coarsening of agglomerates or diffusion ofNPs in the composite matrix on the time scale of the observation.An analysis of the reversibility of agglomerate formation is shownin 57 The high solubility of the particles and their small size preventthem from jamming into soft solid structures. Instead, we discerneda dynamic, reversible separation between NP-enriched and NP-depletedregions, characterized by high and low LC order parameters, respectively,more similar to biomolecular condensates. An open question remains,whether this is a real equilibrium state or a long-lived kineticallytrapped state.It is interesting to notice that the nature ofthe steady statemay be rather different from the arrested aggregations observed inearly work with μm-sized particles.58 and further details of the method and the empiricalfree-energy function appear in the Supporting Information. We define the alignment order-parameter ŝ is the vector describing the specific orientationinduced by the substrate. Where η = 1 implies all domains ofmesogens are aligned by the substrate; η = 0 relates to thenonexistence of a substrate effect. For c = 1, thesystem is pure mesogen; c = 0 implies it is pureNP. Further details on the phase field simulations approach can befound in the Supporting Information. Asshown in A coarse-grained simulation the NP-mesogen system’smorphologicalevolution illustrates how an initially homogeneous system can undergoa transient spinodal decomposition followed by a breakup into isolatedNP-enriched regions as observed in 59 The outlined characteristics of structure formationby phase transition are significantly different to conventional spinodaldecomposition-based processes that exhibit a conserved order parameter,such as the demixing of polymer blends,61 sol–gel-polymer62 and fullerene-polymer composites,63 bicontinuous interfacially jammed emulsion gels,65 polyelectrolyte multilayers or the electrochemical or liquid metaldealloying.67 Beyond examples found in nature,69 some of the most successful synthetic approaches to arrest and controlphase separation and therefore the length scale of structure formationrely on mechanical forces to control the kinetics of demixing, e.g.,provided by gelation or cross-linking of polymer networks.71 The approach presented here starts with a homogeneous mixture thatis fully isotropic. Cooling induces demixing with NP-enriched nematicmicrodomains with random alignment and NP-depleted aligned nematicregions. The phase coexistence is only transient and thus, at theend of the process, the composite displays the aligned nematic phasewith a characteristic spacing and size of agglomerates depending onthe cooling rate.In general, it remains extremely difficult to spatiallycontrolmicrostructures formed by liquid–liquid phase separation processes.–1 were studied. Samples were cooled to 28 °Cand left to equilibrate for 2 h. Between cycles, the LC-NP compositewas heated to 50 °C for 2 h to erase the sample history,and the process was repeated multiple times to evidence full reversibility.The corresponding RDFs demonstrate a nonlinear decrease in interagglomeratespacing with increasing cooling rate. Below 1 °C min–1, a small increase in the cooling rate produced asignificant reduction in the average interagglomerate distance. Incontrast, higher cooling rates exhibited a much-diminished dependence.Likewise, the mean agglomerate size was found to correlate with thecooling rate as shown in Supporting Figure S17. Note that the increased noise for slower cooling rates is relatedto the significantly reduced population size considered in the RDF,because fewer agglomerates were formed and these were spaced furtherapart. We note that the herein described assembly process separatesthe agglomerates evenly over distances that are significantly largerthan the mean agglomerate sizes. We determined for spacing l and diameter d the dimensionless ratioto be in the range of 8 < l/d < 18. The domains did not agglomerate over time, and the formationof dimers was only a very rare event.The effect of the cooling protocol on theagglomerate orderingis shown in –1, 91.1% of the AuNPstransfer into the aligned nematic phase, leaving 8.9% in the residualagglomerates. Following the relative fraction of agglomerates by 2Dimage analysis, one obtains a densification factor of 11.2, whichwas found to be lower for higher cooling rates. However, in this approximation,the agglomerates were assumed to extend across the entire 20 μmthickness of the LC cell, which may from consideration of their cross-sectionalarea only be likely for cooling rates below 1 °C min–1. Hence, in a sample with an overall 5 wt %concentration, even within the NP agglomerates the LC mesogens retaineda volumetric majority, which may explain the reversibility of theprocess.Based on arguments of mass conservation and fromtracking the lossin absorbance for the aligned nematic phase as well as the cross-sectionalarea of the agglomerates, we calculated an densification factor ofNPs within the agglomerates. For a 5 wt % composite cooledat 0.1 °C min–1, the whole process requiredaround 100 min at 0.1 °C min–1. A comparison of the spinodal-type patterns before breakup of thepercolation for the receding NP-enriched phase is shown in Supporting Figure S18 for different cooling rates.The observed characteristic length scale mirrors the characteristicagglomerate spacing after completion of the isotropic-to-nematic phasetransition, demonstrating that the NP agglomerates are indeed a remnantof the initial phase separation and can therefore be controlled throughparameters that govern this process.72The dynamics of the assembly process for differentcooling rateswas further studied by time-lapse microscopy. Though the principlesof agglomerate formation were found to be similar to the observationsreported in Supporting Figure S19, reducingthe NP concentration to 2.5 wt % led to a decrease in themean agglomerate size and a less pronounced peak in the RDF. At concentrationsbelow 1.5 wt %, no μm-sized agglomerates were observedin the final aligned nematic composite, most likely due to the factthat the transient local increase in NP concentration in the recedingrandom nematic phase was eventually consumed by NP diffusion intothe aligned nematic phase. Note that doping of the LC with Au NPsalso led to a small decrease in the observed transition temperaturefrom 34.8 °C for 0 wt %  to 34.3 °Cfor 5 wt %, which is in line with previous studies thatreported an NP-induced dilution effect.40 Further detailson the effect of NP concentration and applied cooling rate on theobserved phase transition temperature can be found in Supporting Figures S20 and S21.The concentration of NPs in the isotropic phase before coolingplayed an important role in the overall agglomerate formation. Asshown in x–y and polar coordinatesfor 0.5 and 5 °C min–1, respectively.Details of the applied methodology,73 theexperimental results for other cooling rates, as well as a simulationof the effect of disorder on the resulting 2D histograms can be foundin Supporting Figures S22 and S23.The mostimportant consequence of this process is the hierarchicalorganization of NPs into larger assemblies within a nematic composite.The RDF demonstrates positional order of agglomerates over the fullradial profile and does, therefore, not account for a possible anisotropydue the nematic phase and alignment along a common director. In –1 (–1), an anisotropy inthe depletion zone was not observed. Instead, an increased probabilityof finding agglomerates along the director field became evident, discernablein Y = 0 direction and in –1, the probability of finding another agglomerate alongthe director field was 9% higher than in any other direction. A similartrend was observed for cooling rates of 10 and 20 °Cmin–1.At 0.5 °C min–1 6a, i.e.,59 The observation of controlledand phase transition-driven reversible assembly of gold NPs takesinspiration from other forms of stimuli-directed self-assembly,74 where the stimuli may be a redox reaction, solventaddition, pH change, or light exposure.76 The presentedfindings of such a controlled assembly process may yield interestingopportunities toward programmable, switchable and reconfigurable dynamicphotonic materials for sensing, spatial light modulation and displayapplications.77 When applied to quantumdot materials, one may expect suitable properties toward reconfigurablelasers.78 Furthermore, in case the transitioncan be triggered by the system itself, e.g., by light absorption,this principle of structure formation may enable further explorationof active soft matter.80In summary, we report on the reversiblecollective assembly ofgold NPs in a thermotropic LC driven by the phase transition fromthe isotropic to the nematic phase under anchoring-driven planar alignment.Based on our observations, the phase dependent solubility, coolingtemperature and homogeneous alignment by the substrate play a decisiverole in the formation of hierarchically structured LC-NP compositeswith NP agglomerates of controllable size and characteristic spacing.While reduced solubility in the unidirectionally aligned nematic phaseresults in residual NPs to be expelled in agglomerates, their spacingdepends on the cooling rate that defines a characteristic length scalein the compartmentalization through the imposed transient decompositionprocess during phase transition. Once formed, the agglomerates werefound to be stable for months and the process remained reversibleby heating above the nematic-to-isotropic phase transition temperature.Phase field simulations, where the nematic ordering is determinedby an Allen–Cahn model and the NP concentration is governedby a Cahn–Hilliard model, exhibit a similar evolution of themicrostructural characteristics as the experimental findings. As amodel platform, this materials system provides insights into the controlover the size and spatial arrangement of hierarchical microstructuresfrom nanoscale building blocks formed by liquid–liquid phaseseparation.37The ligand synthesis was carried outfollowing an adapted literature procedure from Milette and co-workers.N,N-dimethylformamide (DMF) were purchased dry over molecular sievesfrom Acros Organics, and acetone was purchased dry from Sigma-Aldrich.All reagents were commercially obtained and used without further purification.4′-Hydroxy-4-biphenylcarbonitrile (99%) and hexamethyldisilthiane(97%) were purchased from ABCR, 1,12-dibromododecane (98%) was purchasedfrom TCI, potassium thioacetate (98%) was purchased from Alfa Aesar,and acetyl chloride (ACS reagent grade), anhydrous potassium carbonate(99%), and tetrabutylammonium fluoride (1 M solution in THF)were purchased from Acros Organics. TLC analyzes were performed onTLC plates from Merck (Silica gel 60 F254). UV-light (254 nm)or anisaldehyde staining was used for detection. Column chromatographywas conducted on Geduran silica gel Si 60 from Merck (40–60 μm).Unless otherwise noted, all reactionswere carried out in dried Schlenk glassware in an inert argon atmosphere.Chromatography solvents were purchased as reagent grade and distilledonce prior to use. For reactions, dichloromethane (DCM), methanol(MeOH), tetrahydrofuran (THF), and 4,and concentrated in vacuo. Column chromatography  afforded 4′-(12-bromododecyloxy)-4-biphenylcarbonitrile 1  as a colorless powder. 1H NMR : δ = 7.70–7.63 , 7.54–7.51 , 7.01–6.97, 4.01 , 3.41 , 1.89–1.77, 1.51–1.29 . 13C NMR : δ = 159.9, 145.4, 132.7, 131.4, 128.4, 127.2,119.3, 115.2, 110.2 ,68.3 (CH2OR), 34.2 (CH2Br), 33.0, 29.7, 29.7, 29.6, 29.6, 29.5, 29.4, 28.9,28.3, 26.2 (10 CH2).4′-Hydroxy-4-biphenylcarbonitrile  and 1,12-dibromododecane were dissolved in dry acetone (750 mL). Potassium carbonate was added, and the mixture was heatedto reflux for 10 h, after which 4′-hydroxy-4-biphenylcarbonitrilewas consumed according to TLC (DCM). After dilution with DCM (300 mL)the mixture was washed twice with 1 M HCl and once with saturatedNaCl solution. The organic phase was dried over MgSO1  was added to a dispersionof S-potas-sium thioacetate in DMF (30 mL) and the mixture was heated to 70 °Cfor 12 h. After dilution with DCM (150 mL) the mixturewas washed six times with 1 M HCl and once with saturated NaClsolution. The organic phase was dried over Na2SO4, and concentrated in vacuo. Column chromatography  afforded S-(12-(4′-(4-biphenylcarbonitrile)oxy)dodecyl)ethanethioate 2  asan off-white solid. 1H NMR : δ = 7.70–7.63 , 7.54–7.51 , 7.00–6.98, 4.00 , 2.86 , 2.32 , 1.81 , 1.60–1.52 , 1.50–1.43 , 1.35–1.28 . 13C NMR : δ = 196.2 (CH3COS), 160.0, 145.4,132.7, 131.4, 128.5, 127.2, 119.3, 115.2, 110.2 , 68.3 (CH2OR),30.8 (CH3COS), 29.7, 29.7, 29.6, 29.5,29.4, 29.3, 29.3, 29.0, 26.2 (11 CH2).4′-(12-Bromodo-decyloxy)-4-biphenylcarbonitrile S-(12-(4′-(4-Biphenyl-carbonitrile)oxy)dodecyl)ethanethioate 2  was dissolved in dry DCM(7 mL), and dry methanol (10 mL) was added. The mixturewas stirred at room temperature, acetyl chloride was added dropwise, and stirring was continued for 6 h. Afterdilution with DCM (30 mL) the mixture was washed once withsaturated NH4Cl solution and once with saturated NaCl solution.The organic phase was dried over Na2SO4, andconcentrated in vacuo. Column chromatography  afforded4′-(12-mercaptododecyloxy)-4-biphenylcarbonitrile 3  as an off-white solid.1  was dissolved in dry THF(100 mL), and the solution was cooled to 0 °C.Hexamethyldisilathiane  was addeddropwise to the solution, followed by dropwise addition of tetrabutylammoniumfluoride .The reaction was allowed to warm up to room temperature and stirredfor 2 h. After dilution with DCM (200 mL) the mixturewas washed twice with saturated NH4Cl solution and oncewith saturated NaCl solution. The organic phase was dried over Na2SO4, and concentrated in vacuo. Column chromatography afforded 4′-(12-mercaptododecyloxy)-4-biphenylcarbonitrile 3  as colorless solid. 1H NMR : δ = 7.70–7.63, 7.54–7.51 , 7.01–6.97 , 4.00, 2.52 , 1.81 , 1.64–1.57, 1.51–1.43  1.37–1.28 . 13C NMR : δ = 160.0, 145.4, 132.7, 131.4, 128.5, 127.2, 119.3,115.2, 110.2 , 68.3(CH2OR), 34.2, 29.7, 29.6, 29.5, 29.4,29.2, 28.5, 26.2, 24.8 (11 CH2).4′-(12-Bromododecyloxy)-4-biphenylcarbonitrile 50 A precursor solution containing10 ml n-octane , 10 mloleylamine  and 0.25 mmol HAuCl4. 3H2O  wasprepared and stirred under inert atmosphere at a temperature of 15°C, which was controlled with 0.1 K precision. Separately,0.25 mmol of the reducing agent t-butylamine borane was dissolved in a solvent mixture of 1 ml n-octane and 1 ml oleylamine. Subsequently, the reducing solutionwas injected quickly into the precursor solution and left stirringat for 1 h. The oleylamine-protected AuNPs were subsequently2× washed in ethanol  with a minimal amountof DCM  and subsequently redispersed in DCM.Ligand exchange was carried out by preparing a thiol solution containing15 ml DCM with 0.127 mmol of 1-hexanethiol and 0.085 mmol of MDD-CBO, i.e., a 60/40 mol % mixture. Subsequently,a solution with 25 mg oleylamine-protected AuNPs and 5 mlDCM was added and left stirring for 24 h at room temperature.AuNPs were cleaned by dispersing them in a 10/90 vol % mixtureof DCM and acetone  and subsequent precipitationby ultracentrifugation . This stepwas repeated 3 times. A mixture of 10/10/80 vol % tetrahydrofuran,acetonitrile , and acetone  was used for two subsequent cleaning cycles. We want to notethat usual protocols such as the repeated precipitation in a poorsolvent (here: acetone) and subsequent centrifugation  as well as cleaning by a Soxhlet extractor didnot result in an NMR signal that is dominated by surface-bound ligands.Oleylamine AuNPs were synthesizedas follows.51 In short, thiol-protected AuNPs were dissolved in DCM andmixed in the targeted wt % with the LC . The mixture was stirred then sonicated for 1 minbefore the volatile components were evaporated overnight at 60 °Cunder a stream of nitrogen (Eppendorf ThermoMixer C). Subsequently,the solution was transferred to a vacuum oven (Heraeus Vacutherm),which was set to 50 °C for 3 h to remove any remainingtraces of solvent. Once complete, the sample was sealed and storedin the thermomixer at 40 °C until use.AuNP-LC composites were fabricatedaccording to a protocol published by Qi and Hegmann.Subsequently,the AuNP-LC composite was infiltrated in a glass sandwich with definedgap thickness and surface functionalization. Cells with a homogeneousor homotropic surface alignment and 4–20  μmthicknesswere supplied by Instec Inc. Reference cells without surface alignmentwere built with precleaned microscope slides where an initially 25μm thick thermoplastic sealing film  served as spacer. Prior to infiltration, the cellwas placed on the temperature controlled stage and warmed to 40 °C.Once the cell had been heated, 10 μL of the AuNPLC compositewas taken from the vial and slowly deposited on the cell near theopening. Capillary action drew the composite into the cell. The cellwas left at 40 °C for at least 15 min to allow a uniformfilm to form inside.Optical microscopy was carried outin transmission on an Olympus BX61. The following objectives wereused: 5× , 10× , and20× . The samples were temperature controlledwith a Peltier-driven hot stage . The effective temperaturewithin the LC cell was determined by a calibration run.Spectroscopic absorption measurementswere carried out on the BX61 with a 20× magnifying objectiveand the in-built halogen lamp (100 W). The signal was collectedthrough a microspectroscopy port with a 200 μm fibre andan Ocean Optics QE 65000 spectrometer. This resulted in a collectionspot size of around 25  μm.1H nuclei and 100.62 MHzfor 13C nuclei or on a Bruker Avance 400 spectrometer witha BBIz 5 mm probe at a frequency of 400.13 MHz for 1H nuclei. Spectra were calibrated to the residual solventpeak of CDCl3 .81NMR experimentswere carried out at 297.2 K on a Bruker Avance III 400 spectrometerat frequencies of 400.13 MHz for 6 source that was operatedat 120 kV accelerating voltage. Size analysis was carried outusing imageJ.TEM was carriedout on a Philips/FEI CM12 with a LaB82c)and order (η) are understood to be time-dependent fields: Dc and Dη set the time scalesfor the diffusion of NP and the rate of ordering of the mesogens andare assumed to be constant in the simulations. Interfaces separatingtwo regions of differing compositions are associated with large |∇c| and the width of that interface scales with c isthe square-gradient coefficient), where fmax is maximum value of the free energy f in the interfacial region. The interfacial tension scalesas t and the other’s value at −Δt/2. A semi-implicit spectral method was employed.83The relevant equations are given by