Meat industries are eager to find natural low-cost additives for improving the health benefits and shelf life of meat products. The present study elucidated the effect of four different fruit peel powders, namely lemon, orange, grapefruit, and banana (1% each), on the oxidative stability, microbial quality, physicochemical properties, and sensory attributes of chicken patties during 3 months of storage at −18 °C. The total phenolics and flavonoids as well as the antioxidant activity of the fruit peel powders were analyzed. The lemon peel powder contained the highest bioactive substance  and had the highest free radical scavenging activity (90%). The fruit peel powders used, especially the banana peel powder, induced an increase in protein (22.18 g/100 g) and a decrease in fat (10.52 g/100 g) content. Furthermore, all the fruit peel powders exhibited significant antioxidant and antibacterial activities compared with the control samples. The sensory attributes were improved in all treated groups, especially in the lemon peel powder-treated patties. Consequently, the obtained results support the application of fruit peel powders, as natural sources of antioxidants with antibacterial effects, as health-promoting functional additives during the manufacturing of meat products. Chicken meat and its derivatives are one of the most widely consumed food products throughout the world. This may be due to the fact that this meat is not subjected to any cultural or religious limitations. Additionally, its consumption has been increasing rapidly due to its healthy and nutritional image. In this regard, the amount of proteins, essential amino acids, and vitamins are well presented along with a relatively low fat and cholesterol content . HoweverThe oxidative degradation could be successfully reduced by using synthetic antioxidants, including butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA). Nevertheless, the use of these compounds is limited due to their toxicity and damaging effects at the DNA level . CurrentIt has been previously demonstrated that some citrus peel extracts, such as lemon, orange, and grapefruit, as well as banana peel possess good antioxidant and antimicrobial properties due to their phenolic acid and flavonoid contents ,10. FurtFresh lemons, oranges, grapefruits, and bananas were acquired from a local retail market. After washing, the fruit peels were removed using a sharp kitchen knife in the case of the citrus fruits, and by hand in the case of the banana, while the edible portions were separated. The resulting peels were cut into small pieces using a sharp knife, dried in an air oven drier  at 50 °C for 48 h, and then minced to a fine powder with a kitchen grinder. The powders were passed through a 100 mesh sieve, packed in polyethylene bags, and subsequently stored at 20 °C until further use.The deboned chicken meat (~15 kg of thigh and breast) provided by a poultry processing plant was trimmed of all visible fat and connective tissue and then stored in frozen conditions (−18 °C) for the next day. Common salt and starch were purchased from a local market in Giza, Egypt, while seasoning mix and sodium tripolyphosphate were purchased from Loba Chemie .A simple traditional formulation was used to prepare a base batter for the chicken patties as follows: 80% chicken breast and thigh , 7.5% plant oil, 1.8% sodium chloride, 0.3% sodium tripolyphosphate, 0.3% seasoning mix, 5% water, and 5% starch. From this base batter, four formulations were prepared by addition of 1% of each fruit peel powder , besides a fifth formulation that was kept as a control with no fruit peel powders added.The chicken patty formulations were processed at three independent occasions. For each occasion, the cooled chicken breast and thigh meat were ground together using a plate grinder . The minced chicken meat was mixed with salt, water, polyphosphates, oil, seasonings, and starch. The obtained mixture was divided into five groups comprising the first, second, third, and fourth groups that were treated with 1% of lemon, orange, grapefruit, and banana peel powders, respectively, and a 5th group that constituted the control, without any treatment. Subsequently, the mixture of each formulation was homogenized for 3 min by hand using sterile gloves, and was then formed into 1 cm thick discs weighing 75 g using a manual patty former obtained from Fac Affettatrici . The chicken patties were packaged in plastic films and kept at −40 °C for 30 min. Next, the samples were placed in plastic containers and stored under frozen conditions (−18 °C) for three months. The samples were enrolled in triplicates from each formulation for analysis on the second day (time zero) from storage and then monthly.The total phenolic content of the analyzed samples were measured using the Folin–Ciocalteau reagent, as has been previously described by Singleton et al. . In ordeThe determination of the total flavonoid content of the fruit peel powders was achieved using the aluminum chloride colorimetric method, according to the protocol described by Zhishen et al. . One milThe determination of the antioxidant activity of the fruit peel powder was carried out according to the procedure described by Brand-Williams et al.  based onThe compositional analysis of chicken patties from the different groups was carried out following the methods recommended by the Association of Official Analytical Chemists (AOAC) . Thus, wStaphylococcus aureus colonies were counted on double sets of the inoculated Baird-Parker agar plates  after an incubation period of 48 h at 37 °C. The Enterobacteriaceae counts were determined using violet red bile glucose agar  after an incubation period of 24 h at 37 °C.The bacteriological examination of the chicken patties from different groups was performed on the second day after processing, and then monthly during frozen storage. The enumeration of the aerobic plate count (APC) was achieved after a 48 h incubation of the inoculated standard plate count agar plates  at 32 °C; the presence of psychrotrophic bacteria was determined after a 7 day incubation of the standard plate count agar plates at 7 °C. Presumptive The pH, TVBN, and TBA values of the chicken patties from different groups were measured after processing and each month during frozen storage. A digital pH meter  was used for the pH value measurements after the homogenization of 5 g of sample with 20 mL DW for 10 s and the calibration of the pH meter using pH 7.0 and 4.0 buffer solutions . The macThe color of chicken patties from different groups was measured after processing and monthly during frozen storage using a Chroma Meter  with an illuminant D65, 11 mm aperture size, and 10° observer, according to the method described by Shin et al. . The metThe sensory analysis of the chicken patties was carried out after processing and each month during storage (−18 °C), according to the recommended guidelines from the American Meat Science Association (AMSA) . For senp value ≤ 0.05 was considered significant.A triplicate measurement was applied for each of the tested samples. The statistical analysis of the obtained data was carried out using the IBM SPSS statistics software for Windows, version 27.0 . A one-way analysis of variance (ANOVA) was used for the determination of differences between the means of the values of the recorded measurements. In addition, multiple comparisons of the means were achieved using the post hoc  test. A p < 0.05) different, and were greatest in the lemon peel powder, followed by the orange peel powder, grapefruit peel powder, and banana peel powder  changes in the moisture and ash content, as compared with the control samples. Nonetheless, the protein content was significantly (p < 0.05) higher in banana peel powder-treated chicken patties, along with an insignificant (p > 0.05) increase in lemon, orange, or grapefruit peel powder-treated samples. On the other hand, a significant (p < 0.05) reduction in the fat content was noticed in all chicken patties treated with the different fruit peel powders, especially in those treated with the powder of banana peel.The compositional analysis of the chicken patties treated with different fruit peel powders is presented in Our findings are in accordance with those of Mahmoud et al. , who repIn another study, Chappalwar et al.  found thS. aureus, and Enterobacteriaceae counts were significantly (p < 0.05) lower in all fruit peel powder-treated chicken patties when compared with their counterpart untreated samples at the beginning of examination, on the second day of processing, and throughout the 3-month storage period at −18 °C  in lemon, orange, and grapefruit peel powder-treated samples at time zero of the examination and during the entire frozen storage period (S. aureus and Enterobacteriaceae counts were situated under the detectable limit (<2 log10 CFU/g) in banana peel powder-treated samples during the first month of frozen (−18 °C) storage, yet their recovery was observed during the examinations at the second and third months of storage (p < 0.05) increased during the storage time, especially in the control untreated samples, while such an increase was slow and quite less obvious in the fruit peel powder-treated samples  in the samples treated with lemon, orange, and grapefruit peel powders, and insignificantly (p > 0.05) in the samples treated with banana peel powder during frozen storage, as compared with the samples from the control group. However, the TBA and TVBN values were significantly (p < 0.05) decreased during storage in all the fruit peel powder-treated samples. Moreover, with the increase in storage time, all deterioration criteria were significantly (p < 0.05) increased in control samples and insignificantly (p > 0.05) increased in all the fruit peel powder-treated samples. Furthermore, among all treatment groups, the lemon peel powder-treated samples exhibited the lowest pH, TBA, and TVBN values, followed by the orange and grapefruit peel powder-treated samples, and lastly the banana peel powder-treated samples.The registered pH, TVBN, and TBA values of the chicken patties with the incorporation of different fruit peel powders during frozen (−18 °C) storage for a period of 3 months are illustrated in Our results are in harmony with those of Mahmoud et al. , who conThe reduction in pH values of citrus fruit peel-treated chicken patties may be related to their higher organic acid content, including citric, oxalic, malic, and malonic acids . AdditioThe antioxidant activity of fruit peels has been attributed to their vitamin C, vitamin E, flavonoid, phenolic, and carotenoid contents . Phenolip < 0.05) decrease in the L* value in all fruit peel powder-treated chicken patties, especially those treated with banana peel powder, when compared with control untreated patties at time zero of examination and throughout the storage period (p < 0.05) increases in a* and b* values, as compared with the control values (p > 0.05) increase in a* values and a significant (p < 0.05) decrease in b* values during the entire storage period (p < 0.05) increased, and the reverse pattern occurred for a*and b* values with the increase in the storage period; this effect was more pronounced in the control sample and less noticeable in all the fruit peel powder-treated samples  increases in appearance, flavor, tenderness, and juiciness scores, compared with the control samples  increases in flavor, tenderness, and juiciness scores, when compared with the control patties; however, they demonstrated an insignificant (p > 0.05) increase in appearance score  increased at time zero of examination, and throughout the storage period, while in banana peel powder-treated samples it showed a significant (p < 0.05) increase only at the second and third months of the storage  peel powder-treated chicken patties was attributed to the presence of aromatic compounds, which are produced as metabolites during the usage of organic acids  or to thHealthy meat products without any synthetic additives are a main target for the consumer. Moreover, the prudent use of wasted fruit by-products assists in environmental protection and increases industrial profitability. Therefore, our study concluded that the incorporation of orange, lemon, grapefruit, or banana peel powders (1%) into chicken patties could improve their microbial quality, oxidative stability, and sensory attributes, and such improvements were more pronounced in the lemon peel powder-treated samples. Moreover, among the four fruit peel powders added, the total phenolic and flavonoid contents, together with the DPPH% activity, were significantly higher in the lemon peel powder. Additionally, these fruit peel powders may provide benefits for both meat processors and consumers by increasing the nutritional value of the product and lowering the formulation cost. Based on the promising results obtained in the current investigation, future studies focusing on the examination of the effect of frozen storage on phenicol and flavonoid contents as well as the antioxidant activity of other fruit peel powders containing bioactive compounds in chicken meat patties or in other meat-derived products are recommended.


Anthocyanins are antioxidants used as natural colorants and are beneficial to human health. Anthocyanins contribute to reactive oxygen species detoxification and sustain plant growth and development under different environmental stresses. They are phenolic compounds that are broadly distributed in nature and are responsible for a wide range of attractive coloration in many plant organs. Anthocyanins are found in various parts of plants such as flowers, leaves, stems, shoots, and grains. Considering their nutritional and health attributes, anthocyanin-enriched rice or pigmented rice cultivars are a possible alternative to reduce malnutrition around the globe. Anthocyanin biosynthesis and storage in rice are complex processes in which several structural and regulatory genes are involved. In recent years, significant progress has been achieved in the molecular and genetic mechanism of anthocyanins, and their synthesis is of great interest to researchers and the scientific community. However, limited studies have reported anthocyanin synthesis, transportation, and environmental conditions that can hinder anthocyanin production in rice. Rice is a staple food around the globe, and further research on anthocyanin in rice warrants more attention. In this review, metabolic and pre-biotic activities, the underlying transportation, and storage mechanisms of anthocyanins in rice are discussed in detail. This review provides potential information for the food industry and clues for rice breeding and genetic engineering of rice. Anthocyanins are water-soluble plant pigments, responsible for a wide range of attractive colors in leaves, fruits, grains, and flowers , with moAnthocyanins have been found in different rice plant parts and most interestingly in the rice caryopsis, which leads to attractive coloration . The colThe type of anthocyanin detected in black rice caryopses varies depending on the cultivar and the extraction method . Some meUntil now, about eighteen major types of anthocyanins have been found in rice. Among those types, four are most frequently reported, namely: cyanidin-3-glucoside (C3G), peonidin-3-glucoside (P3G), cyanidin-3-rutinoside (C3R), and cyanidin-3-galactoside . AnthocyIncreasing interest in health-promoting food has significantly generated a substantial market for potential nutritionally valuable rice . Subsequ, cinnamate 4-hydroxylase (C4H), and 4-coumarate Coenzyme A ligase (4CL), forms another substance called 4-coumaroyl CoA. Under the control of chalcone synthase (CHS), 4-coumaroyl-CoA and 3x malonyl-CoA will react to form chalcone, which is the first committed step of the flavonoid pathway [Continuous research progress has been made in pigmented plants, highlighting the pathway of anthocyanin synthesis in rice . The pat pathway  and has  pathway . With th pathway . s-transferase (GST), and other specific transporters localized in the vacuolar membrane [Thereafter, the oxidation of leucoanthocyanidins catalyzed by Leucoanthocyanidin oxidase/Anthocyanin Synthase (LDOX/ANS) leads to the formation of anthocyanidins, namely, delphinidins, pelargonidins, and cyanidins  which armembrane ,59.PAL, C4H, 4CL, CHS, CHI, and F3H, F 3′H, and F 3′5′H. The second set is more specific to anthocyanin biosynthesis, including genes such as DFR, ANS, and UFGT. All the structural genes encoding the enzymes involved in anthocyanin synthesis in rice have been deciphered , which is located in the cytoplasm and is associated with the ER [There is no evidence that anthocyanin is transported out of the synthesizing cells. However, trafficking and accumulation are known to be intracellular. Based on the model described by Grotewold and Davies  and the h the ER ,79. It aWe schematically present two models to explain the transport mechanisms of anthocyanin in rice. In the LT model, GST carries mainly glycosylated anthocyanin. These anthocyanins penetrate the vacuole through ABC. In the VT model, the MATE transporter carries mainly acylated anthocyanins, which are transported in the PVC. The anthocyanin is stored as AVI and diffuses inside the vacuole. OsMRP15 is an orthologous gene of maize anthocyanin transporter ZmMRP3 [OsMRP15 co-expresses with OsC1 and OsB1 transcription factors (TFs) in different tissues and organs except the embryo to promote a membrane-bound transporter protein OsMRP15 required for vacuole uptake of anthocyanin in rice [OsMRP15 resulted in green leaves, whereas its wild type had purple leaves [OsGSTU, a homolog of the maize Bronze2 [OsMRP15 and OsGSTU may play a central role in anthocyanin accumulation by facilitating the transport and sequestration of anthocyanin in the vacuole of pigmented rice.In rice, two anthocyanin transporters have been characterized . As a mer ZmMRP3 . OsMRP15 in rice . The Clue leaves . Likewis Bronze2  mutation Bronze2 . Thus, OFurthermore, it is believed that besides GST and ABC transporters, MATE plays a role in rice anthocyanin transport and accumulation. The MATE transporters are distinct from any other known multidrug resistance protein transporters because of the 12 predicted transmembrane I helices and serve as transporters in the accumulation and sequestration of the flavonoid and secondary metabolites in the vacuole ,85. The six rice MATE proteins  and five characterized transporters of various glycosides  involved in the transport of anthocyanin into vacuole were classified in the same sub-cluster . ThereafO. sativa). The broad range of biological functions for the bHLH partners in the MBW complex implies that they are not the determinants of anthocyanin in different cell types. Contrary to the arguments mentioned above, the Kala4 gene, which is the rearrangement of the promoter region of OsB2 (a bHLH protein gene), is expressed ectopically in the pericarp of the black rice and activates the transcription of anthocyanin biosynthetic genes [DFR) and anthocyanin synthase (ANS) [OsPAL, OsCHS, OsCHI, OsF3H, OsDFR, OsANS/LDOX, and OsUFGT/Os3GT) and OsADR function as a simple unit activated by a complex [In most cases, the ternary complex MYB-bHLH-WD40 is a player in activating the anthocyanin biosynthesis structural genes in plants. In this complex, the MYB TFs belong to subgroup 6 of the R2R3-MYB family and carry a highly conserved signature motif. The MYB proteins of the MBW complex bind to DNA and are believed to be the main components of the MBW complex that activates the target genes. The activity of MYB in controlling anthocyanin biosynthesis is specific. Usually, rice has multiple copies  of this gene, which confer anthocyanin production in different patterns or cell types. It is the activity of the MYB TFs that normally determines the amount of anthocyanin production by specific cells, and so differences in rice color intensity and pattern are usually attributable to differences in the expression of the MYB TFs in the MBW complex. The basic helix-loop-helix (bHLH) partners in the MBW complex may have many independent functions and are not present exclusively in the cells in which anthocyanin is synthesized. As such, their functions are not only limited to the regulation of anthocyanin biosynthesis but also to the regulation of proanthocyanidin biosynthesis, as well as the control of vacuolar Ph and seed coat morphology. In plants, bHLH proteins belong to multigenic families , encompaic genes . This deic genes . The dicse (ANS) . In dicose (ANS) . In a mo complex . Some coOsBBX14 and OsHY5 TFs were reported to be localized in the nucleus with transcriptional activation activity that triggered anthocyanin biosynthesis regulatory genes OsC1 and OsB2 [OsBBX14 is a functional region for transcriptional activation. The physical interaction of the basic leucine zipper (Bzip) domain of OsHY5 with the second B-box of OsBBX14 induced OsC1 expression. High expression of OsBBX14 is further worth noting, associated with the high transcript level of OsHY5 in the pigmentation of black rice seeds [OsBBX14 and OsHY5 could act in a coordinated or separate way to directly activate regulatory genes OsC1 and OsB2. The expression of OsBBX14 increased significantly and gradually with seed maturation from 15 DAF to 30 DAF [OsBBX14 is surprisingly involved in photo-morphogenesis, hypocotyl length, and chlorophyll accumulation [OsCHS, OsCHI, OsF3H) for anthocyanin biosynthesis [The possible regulation of the MBW complex has been deciphered. Two novel regulators and OsB2 . The C-tce seeds . This ino 30 DAF . In addimulation . This coynthesis .2 populations derived from the crosses between black rice and white rice. The segregation ratio was 9:3:4 for the three phenotypes, black pericarp, brown pericarp, and white pericarp [Pb synonym Prp-b) and purple pericarp p . The product of Pb has been revealed to be responsible for pigment deposition in the pericarp of brown grain, whereas Pp amplifies the accumulation of pigmentation leading to purple grain. The copy number of the Pp allele determines the intensity of the purple coloration, and the Pp allele is incompletely dominant to the recessive pp allele [Pb and Pp, results in the purple coloration of rice pericarp, while the presence of Pb and the absence of Pp result in brown coloration. In addition, Pb is epistatic to Pp and pp of rice pericarp; the absence of Pp results in white coloration. These two dominant genes, Pb and Pp, are mapped to chromosomes 4 and 1, respectively [Pb locus harbors two genes named Ra and bhlh16. The former is a homolog of the Myc TF Lc gene regulating anthocyanin biosynthesis in maize, and the latter is a homolog of the TT8 gene, which is an Myc TF gene controlling proanthocyanidin synthesis in the pericarp of Arabidopsis thaliana [Ra and OsB1 have been reported to have some common functions in anthocyanin synthesis [Ra gene in all purple pericarp compared to white pericarp was suggested to be the origin of purple pericarp [Kala1, Kala3, and Kala4 [Kala1 between the SSR markers RM 7405 and RM 7419 on chromosome 1, Kala3 between the SSR markers RM15191 and RM 3400 on chromosome 3, and Kala4 between SSR markers RM 1354 and RM 7210 on chromosome 4 [Kala 4 region harbored three alleles, namely, Os04g0557200, Os04g0557500, and Os04g0557800, coding for bHLH proteins [Os04g0557500 plays a key role in anthocyanin pigmentation in rice pericarp. Thereby, this allele was believed to be Kala4. Os04g0557500 has the same function as OsB2, whereas Os04g0557800 corresponds to OsB1 previously identified in Chr 4 at a purple leaf locus W) with a rearrangement in the promoter region could induce the black pericarp coloration [Kala1 includes the Rd locus coding for the DFR enzyme, which is regulated by a member of the Kala4 locus for anthocyanin synthesis. Thus, Rd may be synonymous with Pp, which is believed to be Kala1. It is likely that Kala3 is synonymous with MYB3 [The rice caryopsis consists of the pericarp, testa, embryo, and endosperm. Since the anthocyanin is mainly deposited in the pericarp of the caryopsis, genetic study of the black rice caryopsis pigmentation mainly focuses on the pericarp. The pigmentation of the pericarp has been widely studied, and the results showed three phenotypes of the Fpericarp ,99. The ectively ,99. The thaliana . Ra and ynthesis ,100,101,nd Kala4 . Quantitmosome 4 . The Kalproteins . Subsequus (PlW) . Since Ka4 locus . This stpericarp . Therefon leaves , while Kloration . The chrith MYB3 .With the advent of modern molecular techniques, e.g., QTL mapping, next-generation sequencing, and microarray, researchers have more and more tools to elucidate the pigmentation of colored rice. QTL identification technology was used to locate the QTLs underlying anthocyanins in rice, detecting that the seven QTLs for anthocyanin are located on chromosomes 1, 3, 7, and 10 .O. sativa microarray was used to evaluate the expression of genes after the heading stage. Eighty-two TFs genes classified into 12 groups were found to be associated with anthocyanin. Besides, the comparisons between the white and black cultivars revealed that twelve hypothetical unknown genes were involved in anthocyanin biosynthesis [To better understand the anthocyanin gene expression in black rice, the 135K ynthesis . Furtherynthesis ,102.Two kinds of genes are involved in rice anthocyanin biosynthesis, the structural genes encoding the enzymes in the biosynthesis pathway, and the regulatory genes encoding TFs. The regulatory genes control the expression levels of structural genes by the action of the TF, influencing the intensity and pattern of anthocyanin biosynthesis. In black rice, two classes of regulatory genes, namely, the R/B gene family encoding Myc bHLH proteins and the C1/Pl gene family encoding Myb-type R2R3 proteins, have been reported to control the accumulation and deposition pattern of anthocyanin ,103. SomOsCHS, OsCHI, OsF3H, OsDFR, OsANS/LDOX, and OsUFGT/Os3GT) function as a single unit activated by a complex MBW [Kala4/OsB2 gene located on chromosome 4 [OsWD40 is located on chromosome 2 and has been cloned and characterized [OsC1 has been revealed as a potential activator of DFR and ANS involved in the anthocyanin biosynthesis in other plant organs, mainly hull pigmentation [OsB2 in a tissue-specific manner [The structural genes  insertion in exon 7 of OSB1, which along with a 1 bp deletion of a guanine nucleotide in exon 8, results in a threonine for methionine substitution at position 64, resulting in a white grain phenotype [osb2 allele of OsB2 had a single-base insertion causing a premature stop codon, which resulted in the loss of function of the gene [A mutation in regulatory genes may lead to changes in pigmented rice. The henotype . Likewisthe gene . The MYBthe gene ,109,110.This overview summarizes the TFs involved in anthocyanin pigmentation and their interaction to regulate anthocyanin synthesis in black rice. In this process OsBBX14, a B-box BBX TF, interacts with OsHY5 to form a complex that triggers OsC1, OsB1, and OsB2 TFs. These TFs engage with other MYB and bHLH TFs to form different types of MBW (complex I & II), which activate the expression of structural genes for the formation of anthocyanin. OsB1-OsW40-OsC1 will form complex I, which regulates anthocyanin synthesis mainly in leaves and other vegetative parts, whereas, Kala4-OsW40-OsC1 binds to complex II that regulates anthocyanin mainly in the pericarp of seed . The antRecent high-throughput sequencing technologies include transcriptome analysis, genome re-sequencing, fine mapping, and cloning, unraveling the main candidate networks directly involved in the coordination of anthocyanin formation in rice. Coordinating sets of genes and TFs are responsible for phenotypic diversity in pigmented rice. Several authors endeavored to associate variant genes with the variation of phenotype in pigmented rice. Thereafter, this has been extensively studied using different advanced molecular biology techniques, and the basic genes and QTLs for anthocyanin expression of several rice parts have been found .Anthocyanin is a source of pharmaceuticals, food additives, flavors, and industrial biochemical components ,17. An iC1/R-S regulatory genes using the promoter of a rice prolamin gene. The result showed the expression of the gene in the endosperm and revealed numerous kinds of flavonoid compounds including anthocyanin endosperm [OsB2 after treatment with methyl jasmonate (MeJA) and 2,6 dichloro-isonicotinic (DCINA), resulting in the accumulation of anthocyanin in rice. Subsequently, chemical treatment of transgenic plants increased the activity of OsB2 and OsANS, leading to a high accumulation of anthocyanin in rice [Agrobacterium tumefaciens. The result showed that transgenic rice up-regulated the expression of structural genes, both EBG (F3H) and LBG , concomitantly [The transgenic approach is a powerful tool to investigate regulatory genes and genetic engineering in plants . The antndosperm . Anthocyndosperm  used a tndosperm  induced  in rice . Similar in rice  cloned tmitantly . This remitantly  developeEnvironmental conditions are crucial in rice production and can affect the productivity of black rice ,125,126.Anthocyanin structure is essential to maintain its stability. Highly hydroxylated anthocyanin is less stable than acylated and methylated anthocyanin. The larger the hydroxyl group, the bluer the color becomes ,129. TheCHS, F3H, DFR, ANS, and AN5 of the Heugjinju black rice cultivar was 200–500-fold lower for plants grown under 21 °C compared to those grown under 27 °C. Thus, the growing altitude may impact the anthocyanin content and color density of pigmented rice; an appropriate altitude for a cultivar can help to enhance the accumulation of anthocyanin. A black rice variety G60 increased its anthocyanin content about 2-fold when grown at an altitude of 1360 m as compared to when grown at an altitude of 76 m. Consequently, the color of the caryopsis was darker at high altitude compared to low altitude. This might be attributed to the temperature effect that was different between the two altitudes [2O2, resulting in the degradation of anthocyanin [Anthocyanin biosynthesis is mostly affected by temperature. Suitable temperature increases anthocyanin synthesis while extremely high or low temperature reduces the accumulation of anthocyanin and induces degradation of pigments ,134,135.ltitudes . Thermalhocyanin . Thus, uRuby1, a key activator of anthocyanin biosynthesis, was light-inducible [Melastoma malabathricus and found that light exposure positively regulated the accumulation of anthocyanin whereas shade or a dark environment repressed it. Higher accumulation of anthocyanin was observed with moderate light intensity (301–600 lx). The cultures exposed to 10 days of continuous darkness showed the lowest pigment content, while the cultures exposed to 10 days of continuous irradiance showed the highest pigment content [It is well known that light is a great stimulus for plant growth, and light intensity positively correlates with the level of phenolic compounds such as anthocyanin ,141. Altnducible  highligh content . Moreove content . This coOsPAL, OsCHS, OsDFR, and OsANS, were more influenced during salt stress and correlated with the anthocyanin variation in the leaf. The higher the total anthocyanin in leaves (deep purple leaf color) the less the plant is affected (growth parameter is less affected). This indicates that higher anthocyanin in the leaf can significantly reduce salt stress in a plant [Salt stress is among the most severe environmental stresses that cause the accumulation of ROS damage in plants ,145. Ant a plant .OsPAL, OsANS, OsDFR, OsF3H, and OsCHS [2+ acts as a co-enzyme regulator in anthocyanin biosynthesis that could sustain a high level of anthocyanin in the rice pericarp. Later on, Yamuangmorn and colleagues [Nutrients are essential for plant growth in all stages of plant development. Some minerals such as calcium, magnesium, iron, manganese, and copper can form a complex with anthocyanin in plant cells . It has nd OsCHS . This rend OsCHS , who replleagues  reportedlleagues . These eTo ensure food and nutritional security, rice has been considered the best staple food. The world’s population is expected to reach 9.7 billion by 2050; food and energy demands should be a great challenge . New strOver the last decade, anthocyanin has been considered an attractive and rich functional nutrient in food and has attracted many researchers due to its antioxidant properties. Significant progress has been made to understand its potential health benefit; some key genes involved in the regulatory network and transcriptional factors have been reported, and biosynthesis pathways have been revealed. However, spatial–temporal anthocyanin accumulation and various phenotypes make the understanding of anthocyanin synthesis in pigmented rice more complex. In this regard, this review highlights numerous studies leading to different results useful for a better understanding of the synthesis mechanism and transportation of anthocyanin in pigmented rice for their better handling.About eighteen types of anthocyanins are found in the rice caryopsis, among them, cyanidin-3-glucoside (C3G) and peonidin-3-glucoside (P3G) are the most abundant. Anthocyanins are spatially–temporally expressed differently in rice parts, and biosynthesis involves the regulatory ternary complex R2R3-MYB, bHLH and W40 transcriptional factors, which deferentially activate structural genes for enzyme synthesis. They are synthesized on the surface of the endoplasmic reticulum of cells and are transported to the vacuole of a large range of cells and tissues of vegetative and generative organs at a high level through vesicular transport and ligandin transportation.However, this field needs to be deeply studied to identify the gaps in relevant mechanisms, enabling the enhancement of anthocyanin in rice. For instance, future investigations should address the functional characterization of the genes involved in the storage mechanism of anthocyanin in rice and gene regulation mechanisms encoding for anthocyanin transporters. Powerful tools such as gene-editing tools, e.g., CRISPR/Cas9, can be utilized to modulate transcriptional factors to enhance anthocyanin in rice endosperm. Besides, the conserved regulatory mechanism in the expression of a gene such as histone modification, non-coding RNA, and DNA methylation, could be used to highlight the biological processes of anthocyanin in rice. The environmental factors  that can influence the accumulation of anthocyanin could be addressed to promote anthocyanin production in rice. Whole-genome resequencing could accelerate and allow the discovery of novel allelic variants, with unknown genes subsequently used to develop markers. The development of good quality markers can be helpful to support marker-assisted selection for rapid breeding. Indeed, there are flourishing opportunities for breeders to develop productive pigmented varieties for their antioxidant properties.


Objective: Coronavirus disease 19 is a well-established cause of rare arterial thrombosis. Nevertheless, the exact mechanism of arterial thrombosis remains to be elucidated. We herein report the case of a large floating thrombus of the aortic arch, its surgical management and histological analysis.Case: A 65-year-old patient presented to the emergency department with a suspected stroke. He was non-smoker, but presented cardiovascular risk factors, namely hypertension, type 2 diabetes and hyperlipidaemia. A computed tomography of the aorta revealed a large floating thrombus of the aortic arch, at the base of the brachiocephalic trunk, suspected to be the etiology of stroke. Therapeutic anticoagulation was immediately started. The decision was made to perform an open aortic replacement surgery because of the symptomatic thromboembolic event with recent cerebral infarction and the potential harmfulness of the thrombus due to its size. A mobile thrombus was observed at the base of the brachiocephalic trunk by echocardiography. It was attached to a small area of the upper aortic wall and had an irregular surface. Histology revealed a platelet-rich thrombus lying on an aortic atherosclerotic plaque without pronounced inflammation. No plaque ulceration was present but endothelial cell desquamation was observed consistent with plaque erosion.Conclusion: In our case, there was a thrombus lying on an atherosclerotic plaque with intact thick fibrous cap, but associated with a plaque erosion mechanism. The thrombus formation appeared more likely to relate to a very localized endothelial injury. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the worldwide pandemic coronavirus disease 19 (COVID-19), a systemic disorder presenting typically with fever and respiratory symptoms. However, vascular manifestations, in the form of thrombosis, were rapidly reported. The first Chinese series found significant biological changes in coagulation biomarkers, associated with endothelial damage . ArteriaA 65-year-old patient presented to the emergency department with a suspected stroke. He was a non-smoker, but presented cardiovascular risk factors, namely hypertension, type 2 diabetes and hyperlipidaemia. His usual prescription included valsartan 160 mg, amlodipine 10 mg, spironolactone 25 mg, atenolol 50 mg, chlortalidone 12.5 mg, gliclazide 60 mg, metformin 2,000 mg, vildagliptin 100 mg, and atorvastatin 10 mg per day. Due to his resistant hypertension, the patient had previously participated to a clinical trial testing a carotid baroreceptor stimulation device  that he was still carrying.The patient reported unusual and fluctuating headaches 4 days earlier with a climax within the last 48 hours immediately followed by a sensory and motor loss in his left arm. He fully recovered from those neurological deficits and later went to the emergency department, off-delay for a potential thrombolysis. The clinical examination did not reveal any sensory or motor neurological deficit, the ECG was normal with sinus rhythm and the cerebral computed tomography (CT) scan was normal. MRI could not be performed due to presence of the carotid baroreceptor stimulation device. Although confirmation between a transient ischaemic attack and a stroke could not be proven, the diagnosis of stroke was retained due to the prolonged deficit and the normality of the early CT scan. The patient was immediately admitted and treated with aspirin. Biologically, sodium and potassium levels were normal, creatinine 112 μmol/L, C-reactive protein was elevated at 13.4 mg/L, hemoglobin 156 g/L, platelets 292 G/L, leucocytes 6.2 G/L. There was no obvious coagulation disorder, with a prothrombin time at 13.2 s (control at 13.2 s), an activated partial thromboplastin time at 29 s (control at 30 s), and a fibrinogen at 3.4 g/L. The investigations were completed at Day 2 with carotid and intracranial Duplex ultrasound, reporting the absence of significant stenosis of the carotid, vertebral or intracranial arteries. A CT-angiogram of the aorta revealed a large floating thrombus at the base of the brachiocephalic trunk , measuriWe report the case of a mobile thrombus of the initial part of the aortic arch, most likely responsible for a cerebral infarction associated and potentially subsequent to a slightly symptomatic SARS-CoV-2 infection. Pathology analysis showed the presence of a large thrombus attached to an extensive eroded atherosclerotic plaque. Cases of arterial thrombosis linked to COVID-19 seem to be favored by an underlying atherosclerosis, affecting patients at high cardiovascular risk, particularly those with high blood pressure . ArteriaThe original contributions presented in the study are included in the article/Ethical approval was not provided for this study on human participants because Case report. The patients/participants provided their written informed consent to participate in this study.GG, AD, and TM wrote the document. RE performed the histological analysis. MA, AB, and PA performed the aortic surgery. NG and DS performed the biological analysis. All authors interpreted the data, drafted and revised the manuscript, and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study examined the association between physical activity (PA) and quality of life (QOL) in Korean patients with inflammatory bowel disease (IBD).We enrolled 158 patients with IBD (81 men and 47 women). PA levels were assessed using the International PA questionnaire. Using self-reported frequency (day) and duration (h) of physical activities, the patients were categorized into 3 groups based on their total metabolic equivalent (MET-h/wk) values: least, moderate, and most active. The QOL of patients with IBD was assessed using the inflammatory bowel disease questionnaire (IBDQ), the Medical Outcomes Study 36-Item Short Form Version 2 (SF36v2), the EuroQOL five dimensions questionnaire (EQ5D), and the EuroQOL visual analog scale (EQ-VAS).P < .001 and P = .004 respectively). In addition, depending on the type of PA, the amount of leisure activity was associated with higher IBDQ , physical function of SF36v2 , EQ5D , and EQ-VAS  scores. The frequency of sweat-inducing exercise showed an inverse correlation with IBDQ , physical function of SF36v2 , EQ5D , and EQ-VAS  scores.Of 158 patients, 62, 73, and 23 patients with Crohn disease, ulcerative colitis, and intestinal Behçet disease, respectively, were included. The mean age was 45.96 ± 17.58 years, and 97 (61.4%) patients were men. Higher PA levels correlated with higher EQ5D and EQ-VAS scores (Increased PA levels were associated with improved QOL in patients with IBD. More leisure activity and non-sweat-inducing exercise were associated with improved QOL in patients with IBD. Thus, patients with IBD are frequently affected by depressive syndromes.–12 Patients with IBD generally have higher levels of daily stress and lower QOL compared with healthy subjects and even patients with other chronic diseases.,14Gastrointestinal and extra-intestinal discomfort negatively affect the quality of life (QOL) of patients with IBD.–18 Therefore, strategies, including complementary pharmacological approaches and psychosocial support, are commonly used by patients with IBD.–21IBD has no cure despite multimodal medical treatments, including immune-modulating drugs and anti-tumor necrosis factor antibodies. Drug therapy aims at altering the course of disease, reducing its’ symptoms, and improving health-related QOL (HRQOL).–25 However, PA as a therapeutic option for IBD has not been studied sufficiently. Information on the effects of regular PA on disease activity, inflammation, and QOL is limited. Similar to other chronic diseases, muscle function, peak power, and peak oxygen uptake are reduced in patients with IBD.,28 Furthermore, to date, no negative side effects of moderate exercise on the physical condition of patients with IBD have been observed. These data suggest that PA may be a safe method for patients with IBD in order to minimize the side effects of IBD and improve HRQOL.Regular physical activity (PA) has become an important complementary treatment strategy in several chronic diseases, including coronary heart disease, metabolic syndrome, heart failure, breast cancer, and depression.Therefore, the present study focused on examining the association between PA and QOL in patients with IBD in Korea.22.1The protocols of the research were approved by the institutional review board of Severance Hospital (4–2014–0364). Self-administered questionnaires that measure PA and HRQOL were used. The protocol was first explained to patients with IBD, and informed consent was provided by the patients who agreed to participate before receiving and answering the questionnaires. The data of 158 patients with IBD  between March 3, 2013 and December 31, 2013 who submitted questionnaires were included in this study. The questionnaires were first distributed at an IBD workshop that was held at Severance Hospital on March 3, 2013. The questionnaires (n = 111) were submitted to 1 of 8 research staff who were familiar with the questionnaire and had previously been trained to help explain the questionnaires to patients with IBD. Additional questionnaires (n = 47) were distributed by health care providers at Severance Hospital who had previously been briefed on the purpose of this study and had agreed to play an active role in the distribution of the questionnaires.2.2 The questionnaire has 2 items regarding leisure time physical activity: frequency of mild , moderate (not exhausting), strenuous (heart beats rapidly) exercise of at least 15-minutes or more per week, and the frequency of participation in sweat-inducing activities during a typical week. In addition, the mean duration of each exercise intensity was assessed in order to calculate metabolic equivalent hours per week (MET-h/wk). Patients were then categorized into 3 groups based on their total metabolic equivalent (MET-h/wk) values: least, moderate, and most active.The exercise participation of patients with IBD was assessed using the Godin leisure-time exercise questionnaire.2.3The QOL of patients with IBD was assessed using the inflammatory bowel disease questionnaire (IBDQ), the Medical Outcomes Study 36-Item Short Form Version 2 (SF-36v2), the EuroQOL 5 dimensions questionnaire (EQ5D), and the EuroQOL visual analog scale (EQ-VAS).The IBDQ is a validated questionnaire that assesses disease-related dysfunction in patients with IBD. This questionnaire consists of 32 questions covering 4 QOL dimensions . Up to 7 points can be given to each question, 224 being the highest achievable score , and higher values indicate a better HRQOL.The SF-36v2  is a generic instrument for measuring HRQOL. The SF-36v2 uses norm-based scoring wherein the scale and component summary scores have a mean and standard deviation (SD) of 50 and 10, respectively, in the US general population. It consists of 36 questions that measure functional health and well-being from the patient's point of view and has been found to be valid and reliable after extensive psychometric evaluations. Responses to items are computed into an 8-domain profile of the scores: physical functioning, role-physical, bodily pain, general health, vitality, social functioning, role-emotional, and mental health (MH). In addition, SF36v2 physical and MH scores  are generated. In this study, the higher values indicate a better HRQOL. The descriptive system is composed of 5 dimensions . Each dimension has 5 levels: no, slight, moderate, severe, and extreme problems. The respondent is asked to indicate his/her health state by checking (or placing a cross) the box against the most appropriate statement in each of the 5 dimensions. The digits for 5 dimensions can be combined in a 5-digit number describing the respondent's health state. The index scores are based on general population valuation surveys that used time trade-off methods in various countries. In this study, we used the Korean value set, and higher values  indicate a better HRQOL.The EQ5D is one of the most commonly used generic questionnaires to measure HRQOL. The conceptual basis of the EQ-5D is a holistic view of health, which includes the medical definition, as well as the fundamental importance of independent physical, emotional, and social functioning. In this study, the higher values  indicate a better HRQOL.The EQ-VAS derives information regarding the patients’ subjective health perception, scored on a visual analog scale with endpoints labeled “the best health you can imagine” and “the worst health you can imagine.”2.4The questionnaires that were used were forward-translated to Korean, and back-translated separately, by experts in the field and compared afterward. The translated questionnaires were subsequently examined by an expert panel for face validation before being used in this study.2.5P < .05.All statistical analyses were performed using SPSS 21 . Continuous and categorical data were presented as mean (SD) and frequency (percentage), respectively. For the univariate analyses, in the comparison of the 3 groups by PA, a one-way analysis of variance (ANOVA) followed by a post-hoc Bonferroni test was used to analyze the relationship between the PA level and the HRQOL. The correlation between leisure activity and the HRQOL was analyzed with a κ test. The results were considered statistically significant if 33.1We enrolled 158 patients between March 3, 2013 and December 31, 2013. Of 158 patients, 62, 73, and 23 patients with CD, UC, and intestinal BD, respectively, were included. The baseline characteristics of 158 patients are described in Table 3.2Exercise participation of patients based on the type of IBD can be seen in Table 3.3P = .026). In the SF-36v2, physical functioning and the SF-36v2_PH domains scored higher in the most active group than in the least active group . The same trend could also be seen for the EQ5D and the EQ-VAS scores .All patients were divided into 3 groups based on their PA, and the mean ± standard deviation of the questionnaires is presented in Table 3.4κ = 0.212, P = .018), physical function of the SF36v2 , the EQ5D , and the EQ-VAS  scores. In contrast, the frequency of sweat-inducing exercise showed an inverse correlation with the IBDQ , physical function of the SF36v2 , the EQ5D , and the EQ-VAS  scores. Sweat-inducing exercise was associated with better general health .The Godin leisure-time exercise questionnaire as a PA measurement includes both leisure activity and sweat-inducing exercise. The correlation between the PA type and the HRQOL questionnaires is presented in Table 4More recently, PA has been generally regarded as beneficial for patients with IBD as it may counteract some IBD-specific complications by improving the immunological response, psychological health, nutritional status, bone mineral density, and reverse the decrease of muscle mass and strength. Therefore, there have been specific efforts to confirm these favorable effects and to clarify the pathophysiology of PA in IBD.–38 Those studies also showed that moderate-intensity exercise is well tolerated by patients with IBD who are in remission. Ploeger et al tested the effect of moderate-intensity continuous exercise and high-intensity intermittent exercise in youth with CD. The authors concluded that such patients can engage in distinctly different types of exercise without significant exacerbations of the disease. Although previous research suggested that exercise may be beneficial for patients with IBD, the results of this study suggested that the opposite effect could also be possible regarding the HRQOL. Therefore, further studies are necessary to evaluate the effectiveness of various types of exercise, as well as the duration and intensity of an exercise program to determine an optimal PA level for patients with IBD.Most studies have examined the beneficial effects of exercise in terms of QOL and general fitness. Studies on sedentary patients with inactive or mildly active CD have shown that moderate exercise, such as walking or yoga, lead to significant improvements in the measures of the quality of life and levels of stress.This study has several limitations. First, this study was a cross-sectional study; hence, a causal relationship between PA and HRQOL cannot be determined. Second, the number of patients in each IBD type  was small; hence, the relationship between PA and HRQOL in the subgroup of each IBD type could not be analyzed. Finally, because the majority of questionnaires were collected from patients attending an IBD workshop, IBD disease activity at the time of the survey was mostly clinically remission to mild. This could mean that the participants of this study may not accurately represent the general IBD population in Korea. Nevertheless, in patients with IBD, in a state of high disease activity, the measurement of PA or QOL itself might be meaningless, and intensive treatment is usually performed in hospitalization. Therefore, as the subjects of this study, dealing with PA and QOL, it is considered appropriate to target the patients with well controlled disease.This study is the first study to examine the association of PA and QOL in patients with IBD in Korea, and to the best of our knowledge, in East Asia. The results of this study clearly show that increased PA levels are associated with better QOL in patients with IBD. Moreover, the results of this study also suggest that the impact of PA on QOL may be determined by the type, and, possibly, the intensity, of PA. More specifically, more leisure activities were associated with better QOL; however, sweat-inducing exercise had an inverse association with QOL. This result warrants more research. These findings also demonstrate a need for the establishment of appropriate exercise regime for patients with IBD.In conclusion, patients with IBD generally have higher levels of daily stress and lower QOL when compared with healthy persons. Regular PA has become an important complementary treatment strategy in several chronic diseases, including coronary heart disease, metabolic syndrome, heart failure, breast cancer, and depression. However, PA as a therapeutic option for IBD has not been studied sufficiently. Particularly, there has been no study regarding the effects of PA on IBD patients in East Asia. Our results illustrate that increased levels of PA are associated with a better quality of life in patients with IBD, even though a causal relationship between PA and health related QOL cannot be determined. Moreover, the results of this study also suggest that the impact of PA on QOL may be determined by the type, and possibly, the intensity, of PA. In this study, more leisure activities were associated with a better QOL; however, sweat-inducing exercise had an inverse association with QOL. Therefore, more research is essential in order to establish the appropriate exercise regime for the improvement of QOL in patients with IBD.Conceptualization: Jae Hee Cheon, Tae Il Kim, Won Ho Kim, Soo Jung Park.Data curation: Bun Kim, Eun Hye Kim, Soo Jung Park.Formal analysis: Bun Kim, Jisuk Chae, Hyuk In Yang.Investigation: Justin Y. Jeon, Soo Jung Park.Methodology: Jisuk Chae, Hyuk In Yang, Justin Y. Jeon.Project administration: Justin Y. Jeon, Soo Jung Park.Supervision: Jae Hee Cheon, Tae Il Kim, Won Ho Kim, Justin Y. Jeon, Soo Jung Park.Writing – original draft: Bun Kim, Eun Hye Kim.


Theuse of nanostructured fluids (NSFs), that is, micellar solutionsand microemulsions, in art conservation is often associated with cleaningpurposes as the removal of polymeric coatings and/or soil from artisticsurfaces. In both cases, the use of NSFs grants significant improvementsover the use of traditional cleaning techniques that employ neat unconfinedorganic solvents, water, or aqueous solutions. The study of the natureand properties of surfactants present in NSF formulations is importantto boost the effectiveness of these systems in applicative contextsand in the search of innovative and highly performing amphiphiles.This work reports on the methoxy-pentadeca(oxyethylene) dodecanoate(MPD) surfactant in two different NSFs, whose utilization in conservationof cultural heritage is new. Its effectiveness is compared to theconventional nonionic amphiphiles used in conservation practice, aspentadeca(oxyethylene) dodecyl ether, for the cleaning of poly(ethylmethacrylate/methyl acrylate) 70:30, p(EMA/MA), and artificially soiledsurfaces. The mechanism, through which NSFs interact with polymericcoatings or soiled surfaces, was investigated by confocal laser scanningmicroscopy, fluorescence correlation spectroscopy, photographic observation,contact angle, surface tension measurements, and small-angle X-rayscattering. The results highlighted the superior MPD’s performance,both in inducing polymer removal and in detaching the soil from coatedsurfaces. At the microscale, the cleaning involves dewetting-likeprocesses, where the polymer or the soil oily phase is detached fromthe surface and coalesce into separated droplets. This can be accountedby considering the different surface tensions and the different adsorptionmechanisms of MPD with respect to ordinary nonionic surfactants (likelydue to the methyl capping of the polar head chain and to the presenceof the ester group between the hydrophilic and hydrophobic parts ofthe MPD surfactant molecule), showing how a tiny change in the surfactantarchitecture can lead to important differences in the cleaning capacity.Overall, this paper provides a detailed description of the mechanismand the kinetics involved in the NSFs cleaning process, opening newperspectives on simple formulations that are able to target at a specificsubstance to be removed. This is of utmost importance in the conservationof irreplaceable works of art. Nowadays, they are an important part of the paletteof the methodologies commonly used by conservators for the cleaningof works of art.8 NSFs used in art conservation are mostly related to two main cleaningissues: the removal of polymeric coatings11  and the removal of soil14 . This lastrepresents the most common of the interventions on artworks. In bothcases, the use of NSFs grants significant improvements over the useof traditional cleaning methods, that is, the use of neat unconfinedorganic solvents, water, or aqueous solutions. The synergistic actionof organic solvents and surfactants allows excellent cleaning performances,combined with a safe and controlled application. In fact, in a genericNSF, the organic solvent is confined in the water continuous phaseand its amount is reduced to a few percentages, drastically loweringboth the environmental impact of the methodology and the health riskfor operators. Moreover, compared to unconfined organic solvents,NSFs are particularly effective for the removal of (hydrophobic) polymericcoatings. Different from organic solvents, which are chosen to dissolvea given polymer, NSFs are usually selected to be non-solvents forthe polymer, in order to swell the film and detach it from the substratesurface through, depending on the polymer nature, a dewetting process.18 Dewetting is a well-known physical phenomenon defined as the spontaneouswithdrawal of a film of fluid  from a surface and subsequent rearrangementin the form of separated droplets.24 The dewetting process of polymeric coatings from artistic surfacesinduced by NSFs grants that polymer macromolecules are not spreadinto the work of art, as it would happen with neat unconfined organicsolvents, resulting in an effective and controlled cleaning action.The nature of the organic solvents included in the NSF has a majorrole in the dewetting of polymers from solid surfaces, as they areselected to increase the mobility of polymer chains by swelling thefilm. Moreover, the surfactant nature is crucial to kinetically favorthis process. In fact, the surfactant, lowering the polymer/solidinterfacial tension, energetically favors the detachment of the filmfrom the solid surface, and it was shown that a partial detachmentof the polymer from the surface represents the first step of dewettingprocesses.18 Thus, amphiphile-based systems having low interfacialtension may be particularly effective as dewetting agents. Most recently,it was also observed that surfactants too have a role in increasingpolymer chains mobility, making them the key components in NSFs forpolymer removal.18Nanostructured fluids (NSFs), such as micellar solutions and microemulsions,have been proposed as innovative cleaning systems in the field ofconservation of cultural heritage, see Chelazzi et al.,28 or technologically more advanced solutions, such as highly retentivesemi-interpenetrated or twin-chain polymer chemical gels, which grantthe safest and most controllable cleaning action.30 Among the chemicals used for soil removal,surfactants certainly play a major role, and in particular when theyare formulated as micellar solutions or microemulsions constitutethe most effective tools available to conservators. Thus, nature andproperties of surfactants are important to boost the effectivenessof NSFs in applicative contexts, and the search for innovative andhighly performing amphiphiles is one of the main goals in the fieldof conservation of Cultural Heritage and in many practical applicationsin cosmetics, detergency, and so forth.In many cases, works ofart do not present polymeric coatings,but their visual aspect is compromised by the presence of soil/grimeat the surface. Soil is composed of a variety of usually low molecularweight substances that accumulate on the surface of works of art asa result of ageing, unsuitable storage, or detrimental practices,from previous conservations, and so forth. A wide choice of cleaningmethodologies is employed for soil removal, according to the specificneeds of the given conservation case, spanning from the use of mechanicalmethods, to pure water, to the use of aqueous solutions of pH buffers,chelating agents, or surfactants, which may be applied by means ofbrushes, cotton swabs, poultices, thickeners, physical gels,35 which is sometimes present in commercial detergents,38 but its cleaning mechanism is poorly understood and its utilizationin conservation of cultural heritage is completely new, to the bestof our knowledge. In particular, the effectiveness of MPD-based NSFswas studied and compared to the commonly employed PDE  dodecyl ether)-based NSFs.In order to quantify the mechanism of action and the effectivenessof the cleaning systems, the MPD- and PDE-based NSFs were formulatedto solve two conservative challenges: (i) polymer coatings removaland (ii) soil removal. In the first case, the removal of poly(ethylmethacrylate/methyl acrylate) 70:30, p(EMA/MA), commercially knownas Paraloid B72, was studied on model systems as polymer-coated glassslides. The interaction mechanism between the polymer film and thecleaning fluid was investigated by means of confocal laser scanningmicroscopy (CLSM), fluorescence correlation spectroscopy (FCS), dynamiclight scattering (DLS), and small-angle X-ray scattering (SAXS). ParaloidB72 is one of the most used polymers in conservation of cultural heritage,42 and it was widely used for a variety of different purposes and ondifferent substrates. MPD- and PDE-based NSFs have been tested forsoil removal from glass and polystyrene substrates coated with anartificial soil, prepared following standard procedures availablein the literature,43 and characterizedby means of visual and photographic observation, CLSM investigation,contact angle, surface tension measurements, and SAXS. Overall, MPD-NSFswere found to be superior over NSFs based on conventional nonionicsurfactants.This work reports onthe use of a relatively innovative surfactant,a methoxy-pentadeca(oxyethylene) dodecanoate (MPD),22.111(C=O)EO15–CH3, MPD ,C12EO15, PDE ,dodecyl dimethyl amine oxide ,sodium dodecyl sulfate , propylenecarbonate , 2-butanone , 2-butanol , ethylacetate, and thefluorescent probes used for CLSM experiments, i.e., rhodamine 110chloride, Nile red, coumarin 6 ,and Bodipy 558/568 C12 -4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid) (Thermo Fisher) were usedwithout further purification. Water was purified with a MilliporeMilli-Q gradient system (resistivity >18 MΩ cm). Carbon black,iron oxide (ochre), silica, kaolin, gelatin powder, Japanese paper(9.6 g/m2), poly(ethyl methacrylate/methyl acrylate) [p(EMA/MA)],Paraloid B72, pellets, and cellulose powder  were purchased from Zecchi, Florence. Soluble starch,cement, olive oil, mineral oil, and white spirit were commerciallyavailable and thus purchased in non-specialized stores.C2.217 (see Tables S1 and S2). Besides MPD andPDE, an anionic (SDS) and a zwitterionic/cationic (DDAO) surfactantwere used as reference amphiphiles.44 Forsoil removal experiments, micellar solutions of MPD and PDE were used,at two different surfactant concentrations, that is, 1 and 5% w/w.The experimentsreported in this study involved different NSFs. In particular, forthe experiments on polymer  removal, four differentformulations were selected, by combining the two surfactants MPD andPDE with two different organic solvents, PC and MEK, both partly misciblewith water, and used as reference solvents in previous studies2.343 and detailed in Supporting Information Table S3.The artificial soilmixture was prepared according to the standard formulation availablein the literature2.42.4.1For CLSM experiments on ParaloidB72/NSFs interaction, polymerfilms of about 2 μm thickness were prepared by spin-coatingabout 200 μL of a 10% w/w p(EMA/MA) solution in EtAc on coverglasses. The polymer films were stained with coumarin 6,co-dissolved with the polymer solution.2.4.22 frosted glass slides were coatedby drop-casting a 10% w/w p(EMA/MA), Paraloid B72, solution in EtAc,which was let drying until constant weight was reached. The averagefinal amount of p(EMA/MA) on each glass slide was about 80 mg.Weighed 5 × 5 cm2.4.3–6 M, previously dissolvedin white spirit. The samples were let completely drying for at leasta week and then used for the experiments.For CLSMexperiments on soil/NSF interaction, glass slides werecoated by drop-casting 150 μL of the artificial soil dispersionstained with Nile red 102.4.42 frosted glass andpolystyrene slides were coated by drop-casting 1 mL of the artificialsoil dispersion. The samples were let drying until constant weightwas reached, and the final “dry” weight of the soilcoating was about 2–3 mg/cm2, on average.Weighed 5 × 5 cm2.4.5ParaloidB72 films were labeled by dissolving the hydrophobic dyecoumarin 6 in the 10% w/w p(EMA/MA) solution in EtAc, to a final concentrationof 1 mM ca. 2 μm thick films were prepared on glass slides throughthe same spin-coating procedure reported for CLSM experiments. FCSallows the tracking of fluorescent-labeled species diffusing in solution.Thus, the microemulsion droplets in solution were labeled by dissolvingBodipy in the NSFs to a final concentration of 10 nM. Bodipy is anamphiphilic dye with absorption and emission spectra well separatedfrom the ones of coumarin 6.2.5The study of the polymer removalwas performed on frosted glass slides,prepared as reported in 2.6t = 0, 3, 6, and 24 h, the immersed samples were photographed andthe cleaning fluid in contact with the soil layer was sampled by takingsmall amounts of liquid, which was subsequently investigated by SAXSmeasurements, in order to follow the possible NSF structural evolutionduring the interaction with soil. After 24 h, samples were taken outfrom the NSFs and tilted with care, in order to check for the residualadhesion of the soil coating to the glass/polystyrene surface.Soiled glass and polystyrene slides were immersedfor 24 h in 40 mL of the following aqueous micellar solutions: MPD1%, MPD 5%, PDE 1%, PDE 5% (w/w). During the experiments, the sampleswere not subjected to any mechanical action. At 2.7Confocal Microscopyexperiments were performed on a Leica TCS SP8confocal microscope  equippedwith a 63× water immersion objective. Rhodamine 110 chlorideand coumarin 6 were excited with the 488 nm laser line of an argonlaser, while Nile red was excited with a DPSS solid state laser at561 nm. The emission of the dyes was acquired with two PMTs in therange 498–530 and 571–630 nm, respectively. CLSM experimentswere performed to monitor the interaction of the polymer films orsoil with different NSFs, as detailed in 2.7.1Unlabeledliquid phase (200 μL) were left in contact with the coumarin6-stained Paraloid B72-coated coverglass, and the morphological variationsof the polymeric film were monitored over time, up to 20 min.2.7.2Liquid phase(200 μL) labeled with rhodamine 110 chloride were left in contactwith the Nile red-stained soiled coverglass, and the morphologicalvariations of the soil coating were monitored over time, up to 10min.2.8Surfactantadsorption was indirectly evaluated by measuring the contact angleof 5 μL of Milli-Q water droplets on soiled glass slides witha Rame-Hart model 190 CA Goniometer. Three samples were analyzed,that is, pristine soil-coated glass slide and two soil-coated glassslides immersed for 1 min in a 1% w/w MPD and PDE solution, respectively.The equilibrium contact angle was measured in at least five differentareas, and the average value and standard deviation were evaluated.2.9Surfacetension values of MPD and PDE aqueous solutions were determined witha K100 Tensiometer . The surfacetension was measured at different concentrations by adding a concentratedstock solution of surfactant in water to a known volume of water (40mL). Surface tension measurements were carried out with a platinumplate, and for each concentration, the average of ten readings wastaken after attaining the equilibrium.2.102), and two position sensitive detectors (PSD-50M)consisting of 1024 channels with a width of 54 μm. The Kα radiation (λ = 1.542 Å) emittedby a Cu anode from the Oxford 50 W microfocus source with customizedFOX-3D single-bounce multilayer point focusing optics was used, while the Kβ line wasremoved using a multilayer filter. The voltage was generated by theGeniX system . The sample-to-detector distance was26.9 cm. The volume between the sample and the detector was kept undervacuum during the measurements to minimize the scattering from theatmosphere. The camera was calibrated in the small-angle region usingsilver behenate (d = 58.38 Å). Scattering curveswere obtained in the q-range between 0.008 and 0.5Å–1. The temperature control was set to 25°C. Samples were contained in 2 mm thick quartz capillary tubessealed with hot-melting glue. Scattering curves were corrected forthe empty capillary contribution considering the relative transmissionfactors. Desmearing of the SAXS curves was not necessary thanks tothe focusing system. The fitting model adopted is described in detailin the Supporting Information file.SAXSmeasurements were performed with a HECUS S3-MICRO SWAXS-camera, equippedwith a Hecus System 3 2D-point collimator .FCS measurementswere performed with a Leica TCS SP8 confocal microscope equipped with a PicoQuantFCS modulus . A water immersion objective63×/1.2 W (Zeiss) was used. The evolution of the structure ofthe fluorescent-labeled film during the interaction with the NSFswas followed by confocal imaging, exciting the green dye coumarin6 with the 488 nm laser line, and collecting the emitted signal witha PMT in the range 498–530 nm, as reported in the 2.1245 In the second case, the values of diffusioncoefficients were obtained as weighted average of the most recurrentcomponents (components accounting for less than 8% of the populationwere not considered). All data shown are the average of three repetitions,with relative standard deviations.DLS measurementswere performed on a Brookhaven Instruments apparatus (BI 9000AT correlatorand BI 200 SM goniometer) equipped with a EMI 9863B/350 photomultiplier.The 633 nm He-Ne laser was used to avoid light absorption by the Bodipylabeled systems. Measurements on the simple water-surfactant systemsand on the NFSs were carried out at 90 and 25 °C. The signalwas collected performing 8 min measurements, and the diffusion coefficientswere obtained either from a second-order cumulant analysis or by theweighted average of the values obtained by the CONTIN algorithm.3This study focuses on the mechanism and kineticsof MPD cleaningand detergent properties in the context of cultural heritage conservation.As reported above, two main conservative issues are the scope of thisstudy, that is, polymer removal and soil removal. Besides testingthe efficacy of proposed NSFs based on MPD surfactant, our aim wasto understand the interaction between this surfactant and the materialsto be removed because in art conservation the value of the works ofart impose very specific, controlled, and performing cleaning, withoutany possible damage to the original works. To this aim, all the experimentson MPD behavior and performances were compared to those obtained replacingMPD surfactant with its alcohol ethoxylate homologue, PDE.46 showedthat the physico-chemical behaviors of MPD and PDE are significantlydifferent, despite their similar molecular architecture. In particular,phase behavior in water and the polar head hydration are differentfor the two surfactants. This leads to a different excluded volumefor the micelles and different effective micellar volume fractionfor the two systems. These findings partly explain the experimentalevidence that MPD possesses better cleaning efficiency than PDE, especiallyin low mechanical conditions, that is, when micellar solutions arekept in contact with soiling materials without stirring. In particular,the removal of oleic acid from fabrics by PDE and MPD micellar solutionswas studied by means of several techniques, and it was found thatat the equilibrium state both surfactants have almost the same emulsificationand solubilization power toward oleic acid, while, before the systemis equilibrated, PDE is preferentially adsorbed onto oleic acid coatingsin the form of lamellar structures. On the contrary, MPD is less efficientlyadsorbed onto the soil surface and tends to solubilize the oil inthe hydrophobic core of micelles.47Recently,a thorough study by Sato et al.3.1S, which accounts for the energetic balance of the system. In thecase of a polymer film laid on a glass surface and immersed in a liquid, S is expressed as follows48LG is the interfacialtension between the glass and the liquid, γPG isthe interfacial tension between the glass and the polymer, and γPL is the interfacial tension between the polymer and the liquid.In total wetting regime (S > 0), films are alwaysstable and dewetting does not occur. On the other hand, when a fluid is only partlywetting (S < 0), films are unstable or metastableand dewetting is thermodynamically favored below a critical thickness hc, which for most substances is in the rangeof millimeters. Even in these last conditions, dewetting does notnecessarily take place. In fact, it can be inhibited by a kineticfactor, that is, an energy barrier has to be overcome in order toinduce the process. For thin films , this energy barrier is usually low and the film is unstable.This instability generates capillary waves through the film, and whentheir fluctuation exceeds the film thickness h, thefilm itself spontaneously breaks down into separated droplets accordingto a mechanism termed spinodal dewetting.19 Thick films (h > 100 nm), on the other hand,aremetastable. A 2 μm-thick hydrophobic polymer film laid on ahydrophilic surface, such as glass, as in the case of the experimentshere reported, is a good example of metastable system, where dewettingwould be thermodynamically favored but kinetically inhibited becauseof the low mobility of entangled macromolecular chains in the film.Whenever this mobility is enhanced, the film becomes unstable anddewetting occurs. Enhanced chain mobility can be induced essentiallyin two distinct ways: (i) the film is heated at a temperature higherthan its glass transition temperature, Tgz-axis (here not reported)shows that the dark areas are not holes that go through the wholefilm thickness; instead, they are liquid-filled cavities that formas the polymer is locally detached and lifted from the solid surface.The rims of polymer remaining onto the glass surface draw a characteristicshape termed Voronoi pattern or tessellation.19 As the cavities grow and coalesce, they become weaker and the filmeventually breaks with the nucleation of holes, according to a well-knownand described mechanism for the dewetting of thick films,19 and as the glass is exposed to the bulk liquidphase, the polymer withdraws from the surface in the form of thickrims, which again describe a Voronoi pattern but on a larger scale.Complete dewetting is reached when polymer rims are also disruptedand swollen polymer globular droplets form.The interaction process was monitoredat the polymer/glass interface. 2O/PC/MPD system is almost completely dewetted, whileat the same time, the film interacting with the H2O/PC/PDEsystem showed just a few 20–30 μm large holes in an otherwisecontinuous polymer film. The same trend could be observed in the MEK-basedsystems. 17 Therefore, the interfacial tensionof both surfactants was measured over a wide concentration range,from well above the cmc of the surfactants to more than 100 timeslower . It was found thatthe surface tension of the MPD micellar solution, γMPD ≈ 34.5 N/m, is lower than that of PDE, γPDE ≈ 37.5 N/m, which is in agreement with a kinetically boosteddewetting process for this surfactant. In addition, it can be hypothesizedthat the presence of the methyl capping at the end of the polyoxyethylenechain of MPD confers to this surfactant an increased hydrophobicityand thus a higher capability of penetrating into the p(EMA/MA) film,with a consequent enhancement of polymer chains mobility. Overall,these factors account for the better performances of MPD over PDE.It was found thatthe two PC-based NSFs are able to completelydewet the polymer from the glass, while in the case of MEK-based NSFs,no complete dewetting was observed after 20 min of interaction see 1. IntereIn order to get a detailed picture of the polymer dewetting process,induced by MPD- and PDE-based NSFs, the diffusion and the evolutionof the droplets during the film/liquid interaction were investigatedby means of FCS and SAXS measurements. The main results of these experimentsare reported in 2O/PDE5% and H2O/MPD 5%) were labeled with Bodipy, as describedin the D values obtained by the cumulant analysis are reported in For confocal experiments, micellar solutions , obtained by FCS measurements have been analyzedconsidering two-components decays  and using as initial guessthe parameters obtained from DLS and SAXS data analysis. The diffusionof the Bodipy-labeled NSFs was measured through FCS, before and duringthe interaction with the polymer film, in different sample regions,as shown in The confocalanalysis of the cleaning process indicated that dewettingwas, as expected, the main process for the polymer removal. FCS wasused to shed light on the cleaning mechanism in the “dewetting-like”polymer removal. The autocorrelation functions, D, are reported in The results are shown in D2-bulk in the H2O/MEK/MPD system, whichdecreases after the interaction. However, the most remarkable featuresof these systems lie in the description of the diffusive behaviorof labeled species in the NSF confined into the polymer cavities thatform at the polymer/glass interface. The main result is that accordingto measured diffusion coefficients, NSF droplets are able to penetrateinside these cavities , suggestingthat the interaction with the NFS only slightly alters the polymerfilm permeability, and the polymer film acts as a sort of “molecularsieve”, where only smaller aggregates, probably swollen surfactantmicelles, are able to reach the liquid-filled cavities at the polymer/glassinterface.In the case of the H2O/MEK/MPD NSFshows an evolutionwith time, that is, the NSF confined into the cavities at the polymer/glassinterface eventually reaches the diffusion coefficient of bulk NSFlocated above the polymer film. The decrease of D2 values at t = 20 min seems to be mainlyascribable to the extraction and solubilization of low-molecular weightpolymer chains into micelles/microemulsion droplets.On the other hand, the HThe analysisof PC-based NSFs was more complicated in view of thefact that the polymer is completely and relatively quickly dewettedfrom the glass surface. It was not possible to perform any FCS measurementsinto the cavities that form at the polymer/glass interface, as theyevolved too fast. Conversely, it was possible to measure the diffusionof labeled species inside the liquid-filled cavities that were foundtrapped into the large droplets of swollen polymer , while the D1-cav value is probably an average offaster and slower diffusing species. In fact, before the rearrangementof the polymer in the form of large droplets, the NSF penetrates thefilm and is confined in the cavities at the glass/polymer interface,see panel D in The H2O/PC/MPD NSF significantlychanges during the interaction with the polymer. Slow-diffusing speciescan be found both in the cavities confined into dewetted polymer dropletsand in the liquid on top of the film. These data confirm that, asin the case of H2O/MEK/MPD, micelles/microemulsion dropletsare probably able to extract and dissolve some low-molecular polymerchains present inside the polymer film, and this effect is boostedby the co-presence of both the most effective solvent, PC, and surfactant,MPD.On the other side, the HSupporting Information) but different surfactant.Besides MPD and PDE, two additional surfactants  were selectedand used as reference.In conclusion, the different NSFs show different mechanismsthatdepend both on the organic solvent and surfactant forming the NSF.Considering the obtained results, we challenged the NSFs to the removalof Paraloid B72 polymer films to real cases, that is, thickness ofseveral μm. We compared and quantify the performances of fourdifferent NSFs having the same composition  is very complex, including differentmaterials, ranging from oils to more hydrophilic polymeric materials and to an inert mineral fraction composed of carbon black, iron oxide,kaolin, and silica. This makes the understanding of the interactionprocess between the NSFs and the soil coating more complex. Most likely,a synergistic combination of concomitant physical phenomena occurs;however, this system is closer to real cases.In the present study,glass and polystyrene slides were used as specimen for the experiments.They were coated with very thick layers (∼10–20 μm)of artificial soil; this amount of soil is not easily encounteredon real artworks surfaces, where the soil layer is usually less than1–2 μm thick. Therefore, these experimental conditionshave been chosen to amplify possible differences in the behavior ofdifferent NSFs involved with the cleaning process. Furthermore, thecomposition of the artificial soil used in the present work , meaning that the adhesion (or wetting) of the soil toglass is not particularly favored, that is, the soil has a poor affinityfor the glass slides surface, and in a few seconds, the oily phasepresent in the soil coalesces and rearranges itself in large droplets,recalling a dewetting-like process. This appears dark in the confocalimages at z coordinates close to the glass slidessurface. For both 5% MPD and PDE solutions, because of the presenceof the dissolved rhodamine 110, the aqueous phase is initially seenas green, and turns to bright yellow when the Nile red, present inthe soil layer, interacts (within 10 min of incubation) with rhodamine.At longer time of incubation, the oily phase of the soil is dark brownishbecause of the depletion in the fluorescent dye, which was initiallydispersed in the coating. At the end of the cleaning process, thebright yellow spots unevenly distributed are related to starch andgelatin particles that remain adherent on the glass.In order to follow the evolution of sample morphology at the glass/soilinterface, as reported for CLSM experiments on soil/NSFs interactionssee 2.7.2, boEven if the interpretation of the collected images is not straightforward,it is evident that surfactant concentration plays a major role indetermining a displacement of the soil coating, by detaching it fromthe glass surface. As observed for polymer/NSF interactions, soildetachment from the surface may be regarded as the first key stepof the removal process. The process observed for MPD 5% and PDE 5%is similar; however, on average, larger and more continuous soil detachmentareas were evidenced in samples incubated with 5% MPD.The effectivenessof MPD-based NSFs was also compared to PDE-basedNSFs on macroscopic soil removal experiments performed on both frostedglass and polystyrene slides. Four NSFs (40 mL) used in CLSM experimentswere left for 24 h in contact with the samples. The samples were monitoredat 0, 3, 6, and 24 h. To better clarify the MPD and PDE performances,the contact anglefor pure water on soil was measured. The contact angle at the water/soilinterface was 52 ± 8°. After the artificial soil immersionfor 1 min in the two 1% surfactant solutions, the contact angle was29 ± 1 and <10° for soil incubated with MPD and PDE,respectively. This, contrarily to what can be expected, results ina lower effectiveness of MPD surfactant, when a solubilization processis involved in the cleaning mechanism. SAXS measurements performedon the cleaning fluids samples at 0, 3, 6, and 24 h on glass and polystyreneslides, shed light on the different cleaning mechanism for the twosurfactants.Supporting Information). According to publisheddata on the effective volume fraction of these two surfactants inwater,46 it was assumed that the volumefraction of both MPD and PDE 5% w/w (at t = 0 h)is 0.2 with a 10% uncertainty on this value.46 It is worth noting that the shell thicknessis significantly high for both surfactants, having MPD micelles asmaller shell, in agreement with literature data,46 that report a lower hydration number for the polar headof MPD because of the methyl capping at the end of the polyoxyethylenechain. The results obtained for MPD and PDE micellar solutions incontact with the soil layer show a different behavior for the twosurfactants. For both surfactants, micelles’ size is almostconstant, while the volume fraction significantly changes. For the5% MPD solution, the volume fraction of scattering particles startsto increase after 6 h of interaction with the soil layer, and after24 h, it is about 80% larger than its original value. SAXS data showthat micelles do not grow indicating that the solubilization intothe micelles of hydrophobic components from the soil layer occurswith a subsequent reorganization of the micellar structure. In otherwords, the solubilization of soil leads to a higher number of micelleswith oil molecules replacing the surfactant, with the resulting effectof the presence of aggregates with similar size of the original micellesbut with a different number  70:30,p(EMA/MA), commercially known as Paraloid B72 from glass and polystyrenesurfaces, while aqueous micellar solutions of the two surfactantswere used for the cleaning of artificially soiled surfaces. The overallresults highlighted the better performance of MPD both for the polymerand the soil removal from coated surfaces. The interaction mechanismof NSFs for the removal of p(EMA/MA) polymer, observed at the micro-scalethrough CLSM imaging, involves a dewetting-like process. The polymeris detached from the surface and coalesces into separated dropletsas the liquid phase/solid surface interfacial area increases. ThePDE- and MPD-NSFs exhibit different mechanisms that depend both onthe organic solvent and surfactant because of the different surfacetensions and to the different adsorption/penetration of MPD onto/intothe polymer film, with respect to PDE. This is likely due to the methylcapping of the surfactant polar head and to the presence of the estergroup between the hydrophilic and hydrophobic moiety of the surfactantmolecule (PDE). FCS provided a more detailed picture of the cleaningprocess showing that the surfactants present in the NSFs are ableto penetrate through the Paraloid B72 film, that acts as a sort of“sieve”, and reach the polymer/solid surface interface,where liquid-filled cavities are formed. Moreover, CLSM experimentshighlighted better performances of MPD, if compared to PDE, also insoil removal. The mechanism involves a dewetting-like process, wherethe oily phase is detached from the glass or polystyrene substratesand coalesces into large droplets. Surfactant concentration was foundto be crucial to boost the interaction with the heterogeneous soil.1% surfactant solutions are less effective than 5%, even if micellesare present in both cases. Differently to PDE that adsorb on the soillayer surface it was found, for both glass and polystyrene substrates,that MPD micellar solutions solubilize soil. Both surfactants allowthe removal of soil and grime with different efficacy, no mechanicalaction, and with different times. The time necessary to perform thecleaning and the mechanical action in conservation are of uppermostimportance because long application times and mechanical action shouldbe avoided particularly in the case of fragile and delicate surfacesas those of works of art, which hardly tolerate mechanical stressesduring the cleaning operations. Overall, the results reported in thepresent work open up to new formulations for better-performing andsafer cleaning systems to be used by restorers for the conservationof cultural heritage or in other applications as detergency, cosmetics,and so forth.