The sensory evaluation indicated enhanced tenderness and juiciness without significant alteration of flavor, color, and overall acceptability of the cooked patties. Microstructure images of cooked patties indicated uniform/cohesive structures with small pore size of patties shaped with Osan. Obviously, good storability of the uncooked patties was reflected on the physiochemical, textural, color, and sensory evaluation of the cooked patties, which points to the benefit of using Osan in frozen patties and signifies possible use in the meat industry. The overall sensory acceptability scores were given to cooked patties containing Osan starch as well as the native starch, whereas 15% animal fat was favored too.Octenyl-succinylated corn starch (Osan) was used to improve the physicochemical properties of ground beef patties. The study involved incorporation of 5 and 15% Osan and storage for 30 or 60 days at −20 °C. The tested parameters included cooking loss, microstructure image, firmness, color, and sensory evaluation of the prepared patties. Along with Osan, native corn starch was used as control and considered the patties with added animal fat. The data showed that Osan reduced the cooking loss and dimensional shrinkage significantly ( Meat patties are considered the most popular ready to eat food, due to its desirable sensory and mouth feel. This qualifies its consumption to be considered as a good part of human diet in the past few decades, in addition to its nutritional value which includes essential amino acids, vitamins, and minerals . In procBecause of its poor water solubility and high retrogradation, native starches utilization is limited. This presented the need for physical, chemical, or enzymatic modifications of native starches so as to expand its utilization . Starch The objective of this work is to compare the quality characteristics of beef patties prepared with starch to those with animal fat. Therefore, ocetinyle succenylated and native corn starch as fat replacement in beef patties was utilized. This work includes the effect of storability at −20 °C on the quality characteristics of the patties .Lean beef meat and animal fat (Beef) were purchased from a central meat market . Ground lean meats were selected from animals of the same age, breed, and feeding protocol . While, fresh fat was obtained from Riyadh slaughterhouse . Corn starch was purchased from Middle East Food Solutions Company . Black paper, white paper, onion powder, garlic powder, were purchased from Panda Retail Company . Analytical grade reagents, HCL, 2-Octen-1-yl succinic anhydride, NaOH were purchased from Sigma-Aldrich Chemical Co. .g) for 10 min, washed twice with distill water and once with acetone. The product was dried at room temperature for two days, ground, sieved through 250 µm sieve and stored for further use. This product is considered as food grade according to the followed preparation method.Osan corn starch was synthesized via esterification according to the method of  with sli−1.The stretching vibrational mode of the functional group (Osan) on the modified starch was detected by FTIR spectrophotometer . One gram of dry sample was placed on the FT-IR cell and scanned between 4000 and 500 cmBeef patties were prepared and processed as described by Alejandre et al.  with sliMicrostructures of the obtained cooked patties was examined using JEOL-6360A SEM . Samples were cut into small pieces 5 × 5 × 1 mm, mounted on the pin stubs using copper tape before coating with gold using an automated sputter coater JFC-1600 Auto Fine Coater  for 5 min at 2.5 kV operation energy. Subsequently, four fields of each sample were spotted and the selected images were captured at magnifications ranging from 100× to 1000×.The cooking loss of patties was determined by weighing before and after cooking as recommended by Hollenbeck et al.  using thThe moisture contents of un-cooked and cooked patties were determined based on the AOAC Method 950.46 .The moisture retention, was determined as the amount of moisture retained in the cooked product per 100 g of raw sample. This value was calculated according to the following equation described by ,22, wherChange in beef patties’ diameter was determined before and after cooking using Digital Electric Caliper  by employing the following equations.The firmness of cooked beef patties was determined using a texture analyzer . Samples (60 mm diameter and 10 mm thickness) were pressed using aluminum cylinder probe  operated at 1 mm/s. Samples were compressed to 8 mm distance with 10% strain, where the needed force is expressed in Newton (N). The shear force corresponds to maximum peak force, expressed in Newton (N). The test was performed at room temperature (25 ± 1 °C).The pH was measured using a portable pH-meter  by injecting the probe in 25 g of meat patty and held for 10 s to obtain the pH value.The surface color characteristics of un-cooked and cooked patties were determined after the specified storage time  days. The measurements included, lightness (L*), redness (a*), and yellowness (b*), assessed using a portable colorimeter . Five color measurements were done, where each patty was separated into four quarters one measurement on the surface of each quarter was taken and the fifth was done in the middle.The sensory test was performed using a 9 points hedonic test (Affective Tests), which includes scale from 1 (dislike extremely) to 9 (like extremely) in a single session. This test is useful for evaluating the acceptance of new products. The sensory evaluation team included trained students and King Saud University staff average age between 22 and 60 years old. The panelists were trained to be able to evaluate the sensory properties of patties including overall product acceptability according to method of . After tp ≤ 0.05). The significance level of the analysis of variance (ANOVA) was applied to observe the differences. All measurements were done in triplicate.The statistical analysis was carried out using the Tukey HSD test  at (−1 were ascribed to (O-H) characteristics stretching vibration of amylose or amylopectin, while peaks around 2930 and 1645 cm−1 are attributed to C–H stretching and to the tightly bound water present in the starch, respectively [−1 was originated from the C–O stretching vibration of glucose monomer (Garcia and Grossmann 2014). Two new peaks emerged after modification. Evidently, the region between 1720 and 1570 cm−1 is considered a finger print for the main functional groups of the octenyl-succinylated (Osan) corn starch [−1 emerged after OSA modification was ascribed to the asymmetric stretching vibration of carboxylate RCOO–, whereas the other new peak at 1725 cm−1 was observed, which can be attributed to the characteristic C=O stretching vibration of an ester carbonyl group [FT-IR profile is shown in ectively . In addin starch . The newyl group .The gel network of patties with 5% addition of either fat or starch, was loose and irregular, because large holes emerged within the structure compared to the 15% . Nonethep ≤ 0.05) altered the cooking loss, moisture retention, thickness, diameter, and dimensional shrinkage of the patties. These parameters were compared to the raw patties. The data presented here are in agreement with the literature reports on the effectivity of corn starch to retain moisture of the cooked bologna [The cooking properties of the patties prepared with Osan-corn starch, native corn, and control are presented in  bologna ,32,33. T bologna . The fir bologna . The pH  bologna  reported bologna  reported bologna ,39,40,41The effect of storage on cooking loss, firmness, and other cooking properties is presented in p ≤ 0.05). The addition of 15% of either starches significantly improved the juiciness, flavor and, the overall acceptability of the patties, whereas the tenderness and the color were slightly improved more with Osan starch. After storage, the sensory evaluation showed superior performance of Osan over the control and the native starch, whereas longer storage appeared to have negative effect on the parameter of samples containing native starch  images of the samples confirmed the rise of intermolecular interaction between the proteins and the Osan starch, which resulted in small pores on the surface of the patties.


Petroleum-based plastics are associated with environmental pollution problems owing to their non-biodegradable and toxic properties. In this context, renewable and biodegradable bioplastics possess great potential to replace petroleum-based plastics in mitigating these environmental issues. Fabrication of bioplastic films involves a delicate mixture of the film-forming agent, plasticizer and suitable solvent. The role of the plasticizer is to improve film flexibility, whereas the filler serves as a reinforcement medium. In recent years, much research attention has been shifted toward devising diverse methods for enhancing the performance of bioplastics, particularly in the utilization of environmentally benign nanoparticles to displace the conventional hazardous chemicals. Along this line, this paper presents the emergence of nanofillers and plasticizers utilized in bioplastic fabrication with a focus on starch-based bioplastics. This review paper not only highlights the influencing factors that affect the optical, mechanical and barrier properties of bioplastics, but also revolves around the proposed mechanism of starch-based bioplastic formation, which has rarely been reviewed in the current literature. To complete the review, prospects and challenges in bioplastic fabrication are also highlighted in order to align with the concept of the circular bioplastic economy and the United Nations’ Sustainable Development Goals. Fossil-fuel-derived plastics have vast applications ranging from food packaging, bottle drinks, furniture, household appliances and toys to medical equipment and even construction materials ,2. The hConsidering their non-biodegradability, for petroleum-based plastic wastes, recycling, landfill and incineration must be resorted to for their disposal . It has Bioplastics can be defined as biobased polymers derived from renewable resources or which are biodegradable and/or compostable naturally by microorganisms ,12. WithApart from the generation of plastic waste, food waste generation was also alarming in Malaysia as it contributed up to 70% of the national municipal solid waste . There iAs compared with petroleum-based plastics, bioplastics present three main disadvantages: the processing window, the performance and the cost. The narrow processing window, poor gas and water barrier properties, unbalanced mechanical properties, low softening temperature and weak resistivity of plastics have collectively limited their broader applications. Through substantial research work, the above limitations could be overcome by the introduction of nanofillers ,25. MeanThe present article reviews the main classifications of bioplastics according to the feedstocks, the critical roles of emerging nanofillers and the selection of environmentally benign plasticizers in enhancing the properties of starch-based bioplastics. This is followed by an elucidation of the formation mechanism of starch-based bioplastics to provide a deeper insight into their properties down to the molecular level. The importance of the effects of different synthesizing parameters in the solvent-casting technique on the optical, mechanical and barrier properties of bioplastics is also discussed. Challenges and suggestions for future research are highlighted. These state-of-the-art reviews are still rarely available in the current literature. Therefore, this review could bridge the gap of the relevant scientific information required to transform the bioplastics industry into a more sustainable pathway in line with the circular economy.Currently, bioplastics have been developed in three different generations based on the types of feedstock used for the fabrication. The classification of bioplastics with relevant examples is depicted in The feedstocks of the first-generation bioplastics are usually comprised of edible food crops or carbohydrate-rich plants such as corn or sugarcane. This is the most efficient feedstock since it requires the least land area to produce the highest yield based on highly matured plantation technology. Notable examples include corn, wheat, sugarcane, potato, sugar beet and rice. Starch-based bioplastics and PLA are examples of first-generation bioplastics derived from first-generation feedstocks.Starch is a type of carbohydrate comprised of carbon, hydrogen and oxygen with a mole ratio of 6:10:5 . It contNative starch is challenging to use due to its brittleness, low mechanical properties and poor film forming capacity, as previously described. Plasticization and blending of starch with other polymers are often adopted to overcome these shortcomings . In the In a bioplastic composite, starch acts as the matrix and plasticizer and filler as the plasticizing and reinforcement agents, respectively. The formation of starch-based bioplastics is related to starch gelatinization, whereby starch thickens in the presence of water after breaking down the intermolecular bonds of starch molecules. This allows the hydrogen bonding sites to engage with more water molecules. The starch granules dissolve irreversibly in water due to the plasticizer. Gelatinization simply turns a colloidal system from a temporary suspension to a permanent suspension, involving processes such as starch granule swelling, crystal or double helical melting and amylose leaching.At the initial stage, water molecules enter the amorphous amylose region of starch granules, causing them to swell and expand. They are unable to enter the crystalline amylopectin region at ambient temperature. With an increase in the temperature, the additional heat energy melts the crystalline amylopectin region  and dissOne of the well-known bioplastics is PLA. PLA is a transparent plastic that possesses similar characteristics to common petrochemical-based plastics, such as polyethylene and polypropylene . A wide The fabrication of PLA constitutes a multi-step process. The process begins with the fermentation of hydrolysed sugar from starch to produce lactic acid monomers, and ends with the polymerization of lactic acid to produce PLA . Lactic The first-generation feedstocks are generally rich in carbohydrate and are normally consumed by humans and animals. Continuous processing of the feedstock into bioplastic creates undesirable competition with food and animal feed supply ,39,40. TIn the fabrication of second-generation bioplastics, the feedstocks utilized are non-food crops such as lignocellulosic biomass. Lignocellulosic biomass derived from plants such as wood, straw, grass and bagasse is the most abundant biomass resource on earth . One of Cellulose is the most abundant natural resource on earth as it can be obtained from various living species such as plants , animals , algae , bacteria and even amoebas . CelluloThere are two major challenges in forming film materials directly from lignocellulosic biomass: its high immiscibility with water and the large amount of non-film forming fractions (lignin and hemicellulose) . SeveralThe most popular feedstocks used in third-generation bioplastic fabrication are algae and seaweed. The feedstocks can be cultivated naturally and they grow into enormous amounts in a short time span . They doPHAs are polymers with desirable properties; they are mainly produced by microorganisms (cyanobacteria) and blue-green algae in the presence of excess carbon sources and a limited supply of essential nutrients such as nitrogen and oxygen ,49. PHA Gracilariaceae and Gelidiaceae. It consists of two major components: agarose and agaropectin [Gracilaria comprised of more than 150 species distributed worldwide [Gracilaria changii is in high demand for applications in the cosmetic, pharmaceutical and food industries [The other type of third-generation bioplastic is based on agar. Agar is a polysaccharide produced by several families of red seaweed (Rhodophyceae) such as ropectin . This tyropectin . Being torldwide . Becausedustries .Agar extracted from seaweed has great potential to be used as raw material for bioplastic film fabrication. As demonstrated in the study conducted by Hii et al. , agar exBioplastics reinforced with filler enhance the mechanical properties of starch but reduce its hydrophilicity . In receNanoclays are also known as layered silicates . They arIn order to utilize film for food packaging, oxygen and moisture transport must be minimized to extend food shelf-life . In addi2. TPS film with hydrophilic nano-SiO2 had a lower retrogradation rate than that with hydrophobic nano-SiO2. During gelatinization of starch, the starch granules and crystalline structure were destroyed along with the breakdown of hydrogen bonds and the double helix structure. During short-term retrogradation, the amylose molecules started to crystallize and formed a crystal through a single helix structure. With longer retrogradation time, the amylopectin molecules gradually formed a crystal through a double helix structure. With the addition of nano-SiO2, the starch structure would be affected through interaction between O-H groups of starch and nano-SiO2. The addition of hydrophilic nano-SiO2 produced a V-type starch with a single helix structure. Formation of hydrogen bonds between O-H groups of hydrophilic nano-SiO2 and starch subsequently destructed the hydrogen bonds between the double helix structure of starch. In contrast, the double helix structure of starch remained with the addition of hydrophobic nano-SiO2, which was conducive to the regular arrangement of the double helix structure for crystallization. This indicated that the retrogradation degree of TPS was larger with hydrophobic nano-SiO2. Furthermore, the mobility of starch molecules was affected by the different surface properties of nano-SiO2. Hydrophilic nano-SiO2 has many O-H groups on its surface which could form hydrogen bonds with O-H groups of starch and reduce the mobility of starch molecules. On other hand, hydrophobic nano-SiO2 could not react with starch molecules as there is no O-H group on its surface. Therefore, TPS with hydrophobic nano-SiO2 had a higher retrogradation rate as a result of the faster movement of starch molecules during retrogradation.In the study by Liu et al. , retrogrCellulose is one of the examples of organic nanofillers. There are three types of cellulose in nanoscale: cellulose nanocrystals (CNC), cellulose nanofiber (CNF) and bacterial nanocellulose . CNC are2/g), high aspect ratio of ≥100, high crystallinity and high inherent rigidity [G. xylinus that was used as a reinforcement filler also had increased tensile strength and Young’s modulus up to 2 times and 2.1 times, respectively, as compared to that from non-reinforced bioplastic. Similar findings were also obtained in the study conducted by Noshirvani et al. [Similar to other fillers, CNCs can increase the stiffness of bioplastic based on their nanoscale dimension, low density, high surface area , electrical, magnetic, optical and thermal properties . CNT canHowever, the natural insolubility of CNT in water causes self-association and a strong aggregation tendency, giving rise to low adhesion with the hydrophilic polymer matrix such as the starch matrix . FunctioPlasticizers consist of polar and low-molecular-weight molecules that are capable of disrupting intermolecular bonds between polymer chains and forming new bonds with O-H groups on the glucosidic units of polysaccharide chains. This facilitates chain movements and produces a more flexible material with a lower glass transition temperature. Thus, starch with added plasticizer can be easily processed to form thermoplastic derivatives. Plasticizers such as water, glycerol and sorbitol are commonly used in bioplastic fabrication . HoweverVegetable oil is composed primarily of fatty acid and glycerol. It is highly biodegradable and renewable. Many edible vegetable oils such as palm oil, soybean oil and rapeseed oil have been utilized as plasticizers. This has raised a tremendous concern as the use of edible oil, as plasticizer feedstock tends to incur food supply competition leading to a food security threat. Moreover, environmental issues such as deforestation will likely occur if a massive propagation of plants producing edible oil is to take place . To reso2O2. Unsaturated fatty acids are the primary target during the epoxidation of vegetable oils due to the higher amount of carbon–carbon double bonds [Jatropha oil, which consists of high unsaturated fatty acid, including oleic (C18:1) and linoleic (C18:2) acids, is suitable to be converted into epoxidized Jatropha oil before being used as plasticizers in bioplastics fabrication. A typical epoxidation reaction of Jatropha oil is presented in Epoxidized vegetable oils are often used as plasticizers instead of pure vegetable oils. The epoxidized vegetable oils have greater molecular weight and a bulkier structure than the pure vegetable oils which have helped to resist migration by minimizing volatility in the polymeric matrix . Epoxidale bonds . For exaVegetable oil can also be used as an anti-microbial agent. In the algae biofilm synthesized by Othman et al. , cinnamo+ acted as electron acceptor centres while Cl− as electron donor centred. Upon interaction, the intermolecular hydrogen bonds were cleaved, resulting in the dissolution of cellulose with BMIMCl [Ionic liquids (ILs) are organic salts of bulky organic cation and a smaller organic or inorganic anion, with a melting point below the boiling point of water at 100 °C ,98. Due h BMIMCl . It was h BMIMCl . It has Even though ILs have always been considered as green solvents, uncertainty about their environmental impacts had also been raised . For insDeep eutectic solvents are mixtures of two or more compounds which consist of hydrogen bond acceptors  and hydrogen bond donors . The bond acceptors and the bond donors react with each other through strong hydrogen bond interactions to produce DES with a lower melting point and vapour pressure but higher thermal stability than their individual components ,104. TheDES has been employed to replace the traditional imidiazolium-based ILs. Although exhibiting similar performances as conventional IL, DES is comparatively cheaper, safer and easier to prepare in large amounts via a simple mixing process ,94. BecaThe important optical properties of bioplastic film usually refer to its colour and opacity. The opacity of the film indicates the amount of visible wavelength that can pass through the film . BesidesBioplastic often encounters fragility issues due to its high intermolecular forces, which render it very rigid. With the addition of plasticizer, the moisture content of the starch solution is enhanced, and thus the starch granule can move more freely. The elasticity and flexibility of bioplastic is enhanced through the weakening of the internal hydrogen bonds among polymer chains and increasing the molecular spacing ,107. TheIncrement of the plasticizer loading during the fabrication of bioplastic increases the elongation at break of bioplastic. This relationship was proven based on the bioplastic synthesized by Ginting et al. . The eloE), chromaticity parameters, a* (red-green) and b* (yellow-blue)) and a decrement in the lightness (L*). Similar trends were noted in other chitosan films incorporating different additives such as tea polyphenols [The addition of suitable fillers can affect the optical properties of bioplastic. When Almeida et al.  preparedyphenols  or cinnayphenols . Moreoveyphenols  believedyphenols .Unlike plasticizer, the addition of filler could improve tensile strength and Young’s modulus while reducing elongation at break. Filler promotes the formation of strong hydrogen bonds between O-H groups on the interface of both the filler and starch matrix. The formation of the stronger molecular bonding increased the tensile strength and Young’s modulus of bioplastic. Conversely, elongation at break of bioplastic decreased as intermolecular bonding distance was reduced . Strong Modification of property in bioplastic was reported in the work of Agustin et al.  using CN2O, O2 or CO2 [It is worth noting that the density of bioplastic has a large impact on its mechanical properties. A denser bioplastic usually possesses higher tensile strength, which improves its mechanical properties. It is essential to ascertain the density of bioplastic so that the most appropriate packaging and efficiency can be selected . In the 2 or CO2 .Minimal moisture uptake of film is favourable, which indicates that the film is water-resistant . Starch-max) decreased from 323.75 °C to 277.26 °C as the CNC loading increased from 0 wt.% to 15 wt.% [The thermal stability of bioplastic film, which can be characterized using TGA, is an indicator of its durability at high temperature applications . A decli 15 wt.% . This wa 15 wt.% .The bioplastic synthesized by Anggraini et al.  was also−2∙day−1 from 114 g∙mm∙m−2∙day−1 with the addition of 5 wt.% chitin nanocrystals in the TPS film. Interestingly, with the same amount of 10 wt.% chitin nanofibers, the WVTR value was on par with the pure TPS film. A further increase of the nanofibers to 20 wt.% would surpass the WVTR value of the pure TPS film. The findings recommended low loading of chitin nanocrystal and nanofiber contents as they could create a more tortuous path to block water molecules from passing through. Otherwise, the presence of excessive contents of chitin nanocrystal and nanofiber would increase the WVTR. With regards to the OP of pure TPS film, its value decreased by roughly 30% and 25% with the addition of 10 wt.% chitin nanocrystal and nanofiber, respectively. Incorporating filler into TPS film has the advantage of imparting the antimicrobial properties against fungi and bacteria. This was proven by the growth inhibition of the A. niger exerted by chitin nanocrystal- and nanofiber-reinforced TPS film. At filler loading at 20 wt.%, chitin nanofiber-reinforced TPS film exhibited better inhibition effect compared to the chitin nanocrystal, i.e., fungal growth inhibition , 96% vs. 89%. Unlike the antimicrobial property, the inhibition rate was not affected by the concentration of chitin nano-materials. When chitin nanocrystals of 10 wt.% and 20 wt.% were incorporated into TPS film, there was only a slight FGI difference of 2% . Furthermore, the mechanism demonstrated by the antimicrobial property of chitin was not conclusive and is still under intense research [In a study on TPS film conducted by Salaberria et al. , the watresearch .Chitosan is commonly incorporated into starch-based bioplastics as a co-polymer or filler to reduce the water sensitivity as well as enhance the mechanical and barrier properties of film . In addiThe processing temperature at 90 °C achieved the optimum mechanical properties for bioplastic film as determined by Ginting et al. . Higher It was also discovered that increasing the processing temperature of the bioplastic solution beyond the optimum range deteriorates its tensile strength due to the weakening of the intermolecular bonds in starch chains. Excessive heating would break the glycosidic bonds (bonds between monomers) in amylose . This waFor elongation at break, it increases with the increasing processing temperature of the bioplastic solution. As previously discussed, the addition of sorbitol increased the elongation at break of bioplastic . The incAs mentioned previously in In addition, the amount of lipids in the starch interferes with the opacity of the film. An increase in starch concentration in the film increases the lipid concentration. Meanwhile, the lipid particles could scatter the visible light through the film, causing it to be more opaque . Potato Pseudomonas aeruginosa. The film which contained higher starch content had a colour change in the media after 10 days of incubation and was completely biodegraded after 30 days. Owing to the presence of glucosidic bonds in the amylose and amylopectin units, bioplastic film with greater starch content could be biodegraded easily through the hydrolysis mechanism.The biodegradability of bioplastic was evaluated by Hasan and Rahmayani , who incBased on the review presented, nanofiller has a demonstrated ability to enhance the mechanical, optical and barrier properties of bioplastics. However, the scientific literature reporting on the utilization of nanofillers in starch-based bioplastics is still limited. Considering the knowledge gaps waiting to be bridged by the fundamental studies in dealing with the reaction mechanism of nanofiller on starch-based bioplastics, more in-depth and related studies should be carried out. As for the preparation of nanofiller, the process remains complex and not economically feasible. These have restricted the potential of reinforced starch-based bioplastics in replacing conventional petroleum-based plastics . As discUniform dispersion of materials is desired when nanofiller is incorporated in bioplastic as its effect can be manifested in its physical and mechanical properties. Nano-sized particles tend to agglomerate with each other, and this problem can be resolved with the aid of ultrasound in dispersing nanomaterials in the polymer . HoweverThe leaching of plasticizer during storage or end-user applications is also a major concern which could restrict the utilization of plasticizers in essential sectors such as the medical, pharmaceutical and food packaging industries. Plasticizer has comparatively low molecular weight (300–600 g/mol), which could potentially migrate from packaging materials into packaged food, thereby becoming indirect “food additives” . Ideal pWhen using starch-based bioplastics as food packaging materials, their hydrophilic nature exhibits a low water vapour barrier, which is responsible for poor processing ability, high brittleness, vulnerability to degradation, limited long-term stability and poor mechanical properties . In addiThe biodegradable and green starch-based bioplastic is highly desirable to replace the conventional petroleum-based plastic due to its sustainability and environmental friendliness together with its readily available starch sources from food waste. The optical properties of starch-based bioplastics are affected by the amylose and amylopectin contents in starch. Lower amylose or higher amylopectin content resulted in more transparent film. On the other hand, more lipid content in starch can cause the film to become more opaque. The addition of environmentally benign nanofillers in bioplastic fabrication can overcome two main inadequacies of starch-based bioplastics, namely, high water affinity and poor mechanical properties. Filler-incorporated bioplastic has an enhanced tensile strength and Young’s modulus due to the large surface area and aspect ratio, which increase the hydrogen bonding between filler–amylose–amylopectin molecular chains. Moreover, barrier properties can be improved by forcing the permeable molecules to follow a more tortuous path which will impede their diffusion. The addition of plasticizer weakens the structure of bioplastic and decreases the tensile strength while increasing the elongation at break of bioplastic. From the collated findings, it can be deduced that tensile strength is closely related to the added fillers, while elongation at break is associated with the addition of plasticizer. Overall, utilizing agricultural or food waste for the production of starch-based bioplastic can maximize their values and reduce the accumulation of hazardous petroleum-based plastic wastes. Commercialization of starch-based bioplastic is highly plausible if it can strike a balance among its mechanical properties, production cost, toxicity and an acceptable level of biodegradability. Therefore, a synergistic relationship between the addition of fillers and plasticizers is crucial to establish in future research studies.


Plasmodium. Various antigenic proteins of Plasmodium are considered as the major targets for the development of an effective vaccine. The aim of the current study was a comprehensive analysis of the experimentally validated epitopes of Plasmodium obtained from various immunoassays.Malaria is a life-threatening disease caused by protozoan parasite of genus Plasmodium species epitopes were prefetched from Immune Epitope Database (IEDB). Species specific classification of available epitopes was done for both human and murine malaria parasites. Further, these T cell and B cell epitopes along with MHC I/II binders of different Plasmodium species were examined to find out their capability to induce IFN-γ and IL-10 using IFNepitope and IL-10 Pred, respectively.Plasmodium species. Further, the attempt was made to analyse the immune reactivity of these epitopes for their ability to induce cytokines namely IFN-γ and IL-10. Total, 2775 epitopes were predicted to possess IFN-γ inducing ability, whereas 1275 epitopes were found to be involved in the induction of IL-10.The species-specific classification of 6874 unique epitopes resulted in the selection of predominant human and murine Plasmodium epitopes and associated proteins as a potential approach to design and develop an epitope-based vaccine. Moreover, the results highlight the epitope-based immunization in malaria to induce a protective immune response.This study facilitates the assessment of The online version contains supplementary material available at 10.1186/s12866-022-02480-7. Plasmodium is a serious life-threatening infectious disease and mainly transmitted by female mosquito vector. According to recent World Malaria report (2021), there were an estimated 241 million cases of malaria around the world with approx. 0.4 million estimated deaths [Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae Plasmodium ovale and Plasmodium knowlesi Several recent reports suggest that Plasmodium knowlesi has an ability to infect humans along with simian malaria [P. falciparum malaria parasite is accountable for most of the malaria-related demises with prevalence in the African content while P. vivax which has wider geographical range and considered to be less malignant than falciparum. Moreover, rodent malaria parasites such as Plasmodium berghei, Plasmodium yoelii, Plasmodium chabaudi and Plasmodium vinckei with different genetic background are being studied globally to understand immunology and other physiology of malaria [Malaria, caused by the protozoan parasite of genus eport 202, there w malaria .Plasmodium species are known to share a similar basic life cycle. There is a clinically silent phase of infection in the liver while exponential growth of parasite occurs in blood stage [Plasmodium parasite has developed several escape mechanisms by modifying its genome over the period of time. Therefore, due to multiple complexities involved at both host and parasite end, clinical representation of malaria may range from asymptomatic, uncomplicated to severe malaria. Additionally, different stages of the parasite life cycle are known to express a variety of surface proteins that activate different defence mechanisms of the host [Despite this species diversity, all od stage . With ththe host , 6. Thisthe host . Till dathe host . Moreovethe host , 10.Plasmodium species-based epitopes and their ability to influence immune system. It is well believed that host immune response during Plasmodium infection is very complicated and requires interaction of parasite with RBCs during blood stage [Whether human or non-human host, vaccines are known to activate immune system involving various immune mechanisms. In the present study, we have made a systematic attempt to understand the od stage . This blod stage . Howeverod stage , 14. ManPlasmodium epitopes identified from different stages of parasite life cycle and subsequently these epitopes were characterised for their ability to induce the production of two selected cytokines, namely IFN-γ and IL-10. The balance of these cytokines known to determine the outcome of Plasmodium infection and thus it is important to investigate their underlying mechanism of action during infection. Interferon-γ, a key Th1-derived inflammatory cytokine is responsible for killing of intracellular parasites, however, overproduction may lead to tissue pathology [Plasmodium infection [+ T cells become the major producer of IFN- γ which further activates cytotoxic T cells and performs a vital role in the cytotoxic killing of malaria parasite infected cells [In the light of the above discussion, this study was designed to assess various athology , 16. On athology . IFN-γ, nfection . IFN-γ infection . Once thed cells . Additioed cells . These aed cells . Varioused cells –24. Thered cells .+ and CD8+ T cells along with macrophages, B cells, NK cells, dendritic cells etc.[+Tbet+CD4+ regulatory T cells are considered an important source of IL-10 during malaria pathogenesis [P. falciparum infection [γ induced inhibited secretion of TNF-α, IL-1β, IL-6, IL-8, G-CSF, and GM-CSF [Similarly, interleukin-10 (IL-10) is an important anti-inflammatory cytokine reported to have key role in infection by limiting the immune response to pathogen and thereby preventing the host. At first, it was described to be produced by Th2 cells to inhibit Th1 response. Now a days it is well known to be produced by various subsets of CD4ells etc.. Moreoveogenesis . The rologenesis . Additionfection . IL-10 id GM-CSF . Moreoved GM-CSF –33.Plasmodium (ID: 5820) genus in the IEDB database  resulted in 6874 unique epitopes that are positive or experimentally validated and comprising of both linear and discontinuous sequences. These epitopes are defined as peptides fragments that are immunogenic in nature and capable of producing a specific T cell or B cell immune response. Next, we retrieved all epitope sequences derived from a total of 14,816 immunoassays comprising of 3947 T cell assay, 8893 B cell assays and 1976 MHC ligand assays from approximately 800 studies. Further, only uniquely represented epitopes from all three assays were considered to determine their species-specific distribution by removing the repetitive epitope sequences.Plasmodium genus obtained from T cell, B cell and MHC ligand assay were considered. As result, it was observed that these experimentally validated epitopes mainly belong to 9 different species of Plasmodium namely P. falciparum, P. vivax, P. berghei, P. yoelii, P. chabaudi, P. brasilianum, P. cynomolgi, P. knowlesi and P. fragile  freely available to predict and design the epitopes having ability to induce release of interferon-γ cytokine. In current study B cell epitopes, T cell epitopes and MHC I/II binders of four most predominant species of Plasmodium were analysed for their IFN-γ inducing ability using IFNepitope server , rifin, circumsporozoite (CS) protein, Merozoite Surface Protein 1 (MSP1), Erythrocyte membrane protein 1(PfEMP1). Stevor is known to contribute to parasite-mediated pathology through rosetting, in so doing protects released merozoites from immune detection [P. falciparum blood-stage malaria vaccine candidates [P. vivax proteins which dominated for IFN- γ induction. Similarly, from T cell epitopes, for P. falciparum, 622 epitopes were IFN-γ inducing out of 885 epitopes, and 86 out of 107 epitopes were found to have IFN-γ inducing ability for P. vivax  and duffy receptor are top proteins of P. vivax with the highest number of IFN- γ inducing epitopes obtained from MHC binding assays  and circumsporozoite protein precursor of P. yoelii. Also, epitopes available from MHC binding assays were checked for IFN-γ prediction. It was found that among 55 epitopes of P. berghei, 29 were positive for IFN-γ prediction and 9 out of 23 were found to have IFN-γ inducing ability for P. yoelii. Major proteins to which these epitopes from MHC binding assay belongs are L-lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, CS protein, circumsporozoite protein precursor and sporozoite surface protein 2.In addition, IFN-γ inducing ability in murine malaria parasites namely https://webs.iiitd.edu.in/raghava/il10pred/) web server, for determination of IL-10 inducer and IL-10 non-inducer antigenic peptides. B cell, T cells and MHC I/II binders of four major species of Plasmodium were checked for IL-10 inducing ability using IL-10 pred  protein. Similarly, for P. berghei T cell epitopes, 14 out of 82 epitopes were identified with IL-10 inducing ability and 13 out of 60 epitopes from P. yoelii were positive for IL-10 induction. Most of these T cell epitopes are represented by CS protein, circumsporozoite protein precursor, merozoite surface protein 1. Along with T cell and B cell epitopes, epitopes available from MHC binding assays were also subjected for predicting IL-10 inducing ability. It was found that among 55 epitopes of P. berghei, 6 were positive for IL-10 prediction and 2 from a total of 23 epitopes were found to have IL-10 inducing ability for P. yoelii. These epitopes with IL-10 inducing ability were representation of three major proteins like circumsporozoite protein precursor of P. yoelii and L-lactate dehydrogenase and ATP synthase beta subunit of P. berghei.We also analysed IL-10 inducing epitopes in murine malaria parasites namely Plasmodium falciparum, Plasmodium vivax, Plasmodium berghei, Plasmodium yoelii were included for next analysis. These experimentally validated epitopes were cross-checked for their presence in epitopes predicted with IFN-epitope and IL-10 Pred. Details of these epitopes is provided in Supplementary file In addition to prediction of IFN-γ and IL-10 inducing ability of epitopes by IFNepitope and IL-10 Pred respectively, we also assessed the experimentally validated IFN-γ and IL-10 inducing epitopes available from T cell assays in the IEDB database. Epitopes from four major species including human and murine malaria parasites namely P. falciparum . Moreover, it was noticed that most of these epitopes belongs to proteins like circumsporozoite (CS) protein, Merozoite Surface Protein 1 (MSP1) of Plasmodium falciparum.It was observed that almost all experimentally validated epitopes positive for IFN-γ inducing ability from IEDB database are similar epitopes predicted with software. Very few epitopes among four species from IEDB database that are experimentally positive for IFN-γ and were not predicted by software due to long epitope length are also listed in Table SP. falciparum (53 out of 68 epitopes) and P. vivax (12 out of 68 epitopes). Only 3 epitopes belonging to murine malaria parasite P. berghei are positive for IL-10 induction. Further observations indicated that most of these epitopes belongs to merozoite surface protein 1 (MSP 1), thrombospondin-related anonymous protein (TRAP) and circumsporozoite (CS) protein of Plasmodium falciparum.Similarly, all experimentally validated epitopes with IL-10 inducing ability form IEDB database were found same as predicted by IL-10 pred except one epitope that belong to merozoite surface protein 3 (MSP3). Moreover, we observed that number of epitopes predicted by software are more than number of epitopes experimentally validated. Only 68 epitopes from IEDB database that are positive for IL-10 induction are known as compared to 358 epitopes predicted by IL-10 Pred. Additionally, we observed that most of these experimentally validated epitopes belongs to Plasmodium are considered as the major targets for the development of an effective vaccine [in-vivo and in-vitro assays, a critical step required in the development of epitope-driven vaccines to determine the immune response post-immunization.Various antigenic proteins specific to different stages of  vaccine . MoreovePlasmodium epitopes in current study provided us evidence about predominant murine and human malaria parasites. Additionally, number of epitopes in each species explored so far were recorded. Although, it is also important to mention that the predominant status of a given Plasmodium epitope in the immune epitope data is not the true representation as many genetic and experimental factors may attribute to immunodominance of an epitope [Plasmodium whether human or non-human. It is an important vaccine candidate and has been investigated throughout the world in various studies for its efficacy [Plasmodium protein essential for sporozoite motility and liver cell invasion [P. falciparum liver-stage antigen 1 (LSA-1) exact function is not clear to date, however, it is thought to have a role in liver schizogony and release of merozoites [P. falciparum and P. vivax [P. vivax is an analogue of MSP-1 from P. falciparum [P. falciparum protein, ring-infected erythrocyte surface antigen (RESA) located at the membrane of erythrocytes infected with ring-stage parasites, is also a major vaccine candidate against P. falciparum and has been studied widely [Plasmodium infection [Plasmodium species were identified. It was apprehended that most of these epitopes belong to parasite proteins like CS protein, stevor, merozoite surface protein 1 and erythrocyte membrane protein 1 (EMP 1) from both liver and blood stages of parasite indicating a broader immune response. IL-10 concentration has been interrelated with parasite density implying that IL-10 has an important immunoregulatory role, pronounced in both experimental and human models of malaria [P. falciparum natural infections endorses the protective role of IL-10 [Species specific classification of  epitope . Further epitope  was obseefficacy . Thromboinvasion . Thereforozoites . It is wrozoites  with abirozoites . Similarrozoites . VariousP. vivax –49. Morelciparum . Many mulciparum , 51 and lciparum . Anotherd widely . A largenfection , 54 provnfection , 56. Alsnfection . Multiplnfection , 59. The malaria , 60–62. of IL-10 , 64 and Plasmodium were predicted to possess IL-10 inducing potential. Several studies have demonstrated the protective role of IFN-γ and IL-10 against a number of plasmodial antigenic proteins like LSA-1, MSP-1, CS protein, erythrocyte membrane protein (EMP) etc. [+ T cells in acute malaria infection with malaria parasites [Conclusively, it was observed that 50% of the epitopes from each species hold IFN-γ inducing ability. Similarly, approx. 26% epitopes of unique epitopes from each species of MP) etc. , 65–68. arasites . This anPlasmodium species. It also helps in identification of epitopes and associated antigens known to express at different stages of the parasite life cycle. The current study also provides a comprehensive as well stringent analysis of Plasmodium epitopes and proteins associated with their ability to modulate immune response through induction of cytokines like IL-10 and IFN-γ. Gaining insights into the epitopes and their ability to induce some important cytokines paves the ways for assessment of Plasmodium proteins as candidate for design and development of an epitope-based vaccine in future. The vaccine development based on synthetic peptides is already proved as successful strategy in many infectious diseases and is well established in cancer field. In malaria, the efforts made in current study may enhance vaccine development and understanding the pathogenesis caused by malaria parasite. However, it is also possible that there may be differences in sequence of some proteins when host is different. Therefore, to have a comprehensive overview of Plasmodium epitopes and their ability to modulate immune response, we included both murine and human Plasmodium species in the current study.This comprehensive analysis provides a wider depiction of malaria-related epitope data of both murine and human Plasmodium parasite and are in accordance with its promising candidate for malaria vaccine development. RTS,S/AS01  vaccine developed for malaria is well known first human malaria vaccine based on Circumsporozoite (CS) protein. Progress with RTS,S represents a historic milestone, but its partial efficacy leaves room for further improvement. Meanwhile, novel malaria vaccine candidate clinical development has continued apace, being new R21/Matrix-M under trial is again a circumsporozoite protein-based vaccine [These observations in the present study suggested that circumsporozoite protein predominates along with other proteins and is exclusively expressed in all species of  vaccine . AdditioPlasmodium species were prefetched from IEDB using Plasmodium (ID: 5820) by considering all three search parameters available such as T and B cell epitopes along with MHC binders. Also, the MHC restriction pane was selected for any MHC available and all host parameter was selected to include all available host whether human, or non-human. Data prefetched with above mentioned search criteria included epitopes from different species such as Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium knowlesi, Plasmodium berghei, Plasmodium chabaudi, Plasmodium yoelii, Plasmodium brasilianum, Plasmodium cynomolgi. This information was further processed for preliminary analysis.The Immune Epitope Database and Analysis Resource  is a freely available data resource funded by the National Institute for Allergy and Infectious Diseases (NIAID). It is a repository of experimental data on antibody and T cell epitopes with context of four broad disease categories: Infectious diseases, allergy, autoimmunity and transplantation. It also offers multiple tools for epitopes prediction and analysis . EpitopePlasmodium whether belong to human or animal (murine/ non-human primates). Next, each species was checked for the availability of all antigenic proteins known so far. Further, unique antigens or parent proteins along with their prevalence in each Plasmodium species were recorded for further analysis.Initially all three files encompassing experimentally validated epitopes from T, B and MHC ligand assays were downloaded including detailed information about epitope sequences, epitope ID, parent species and parent protein of epitopes. These epitopes were subjected to classification for each available species of Plasmodium species were used individually for fasta files generation. Further, these individual species fasta files were processed to find out their capability to induce two major cytokines (IFN-γ and IL-10).Peptide sequences are considered as a key input for prediction of their ability to induce different cytokines. First, all non-linear, discontinuous peptides and other non-peptidic sequences were excluded for further analysis. Next, the peptide sequences which were reported more than once in literature were considered as one during fasta file generation by removing identical sequences. Moreover, peptide sequences of more than 29 amino acids long were excluded for further study due to their incompatibility with epitope prediction software. Next to streamline further analysis, T cell and B cell epitopes along with MHC I/II binders of different P. falciparum and P. vivax that are responsible for human malaria were subjected to publicly available web based servers namely IFNepitope [P. berghei and P. vivax were also examined for their potential to induce IL-10 and IFN-γ using the same web-based servers.Epitopes belonging to the two most important parasite species Nepitope  and IL10Nepitope , to findPlasmodium epitopes for each species. Search model used for prediction was SVM (support vector machine) motif hybrid which provides results after combining the SVM based model with motifs contributing to IFN-γ induction. The model performs better compared with SVM model and motif based model separately. Similarly, IL-10 Pred a web server was used, which is freely available that allows users to discover, design and mapping of peptides with capability to induce IL-10 cytokine. Plasmodium epitopes for determining their IL-10 inducing ability were subjected IL-10 pred with SVM based prediction model. In case of IL-10 Pred we used the default cut off values as recommended by developers. Here in addition to predicted values of IL-10 induction, we can also get the different physiochemical parameters assigned based on amino acid index database.IFNepitope server allows users to predict the IFN-γ inducing epitopes from a set of peptide sequences or library of peptides. Therefore, this server was used to predict IFN-γ inducing Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.


Prunus dulcis, Pistachio vera, and Juglans regia during the winter months are indeed associated with high yield, while high levels of the NSC in late summer often correlate with low yield. An evaluation of monthly NSC level importance on yield revealed that for P. dulcis high levels in February were a good predictor of yield and that low levels throughout summer were associated with high yield. In P. vera, high levels of NSC in December were best predictors of yield. J. regia exhibited peculiar patterns; while high pre-budbreak reserves were associated with high yields they only played a minor role in explaining crop, the most important months for predicting yields were June and July. Results suggest that NSC levels can serve as good predictors of orchard yield potential and should be monitored to inform orchard management.Successful yield in orchards is the culmination of a series of events that start with plants entering dormancy with adequate energy reserves . These NSC are responsible for the maintenance of activities during dormancy and extending onto the period of activeness. Using multi-year yield information and monthly NSC content in twigs, we show that high levels of carbohydrate in  The nonstructural carbohydrates (NSC) reserve pool in trees, mostly consisting of soluble sugars and starch, constitutes the primary resource-supply for energetic disparities4. Short-term variability typically results from the day/night cycle wherein metabolic processes continuously draw energy from NSC reserves which are all the while replenished by the daily photosynthetic activity5. Long-term variability can result from either periods of drought, forcing plants to keep stomata closed (days–weeks), or from seasonal shifts like dormancy when photosynthetic activity is relatively absent (summer–winter). Thus, the survival of perennial species depends on their ability to accumulate adequate NSC reserves to meet resource demands during any period in which photosynthetic output is absent or lacking.Perennials are characterized by their persistence across seasonal cycles, including recurring periods of dormancy and activity, that may be intermittently punctuated by biotic and abiotic disturbances. As such, they must accommodate both short and long-term fluctuations in energy supply and demand3 and is of major importance in terms of energy supply for flower development and during the initial phases of vegetative bud growth in species like Prunus dulcis, Pistacia vera, and Juglans regia6. Additionally, there is increasing evidence suggesting that conversions between NSC forms, soluble sugars and starches, may potentially serve as a ‘dormancy clock’6. Hence, the timing and synchrony of bloom is strongly impacted by disturbances especially to the NSC accumulation and dispersion that flank dormancy8.Furthermore, not only are sufficient NSC reserves critical for supplementing energy deficiencies but a growing body of evidence suggests that whole-tree NSC reserve levels/status play an important role in sustaining and synchronizing certain phenological progressions. NSC reserves, specifically those present when approaching the end of the season/dormancy, can affect all aspects of tree physiology including effective bloom, spring growth, and ultimately yield. The level of NSC in twigs, in particular, seems to be the most variable yet the most indicative of whole tree storage status9. The amount of reserves needed to maintain dormancy and a healthy growth resumption (bloom/leafing) can be variable and not easily predicted. The build-up of pre-dormancy reserves may be subject to changing abiotic and biotic conditions, growth, and reproductive activity during the active season3 especially in alternate bearing species like P. vera. Additionally, not only is the NSC reservoir contingent upon the active season but the length and conditions of the dormant period itself can also vary from year-to-year, further affecting the amount of reserves readily available to sustain phenological transitions. The unpredictable nature of the local climate combined with the selection for yield maximization most likely enforce the need to store more NSC reserves in domesticated plants than what is required for the average dormancy period in most undomesticated perennials11. While the accumulation of reserves is often seen as a byproduct of an excess of carbohydrates, mounting evidence suggests that it may actually be a sink that actively competes with growth and reproduction rather than merely a passive process13. To their undomesticated counterparts, this ‘excess’ might provide a competitive advantage in which long-term survival is promoted over current vegetative growth and reproductive capacity (yield). However, in domesticated fruit and nut species this may instead shift to promoting short-term gains in reproductive capacity in lieu of long-term NSC reserve formation. This, in turn, potentially makes selected varieties potentially more susceptible to the negative impacts of unexpected changes in dormancy conditions and reduces their resilience to additional stresses. Finally, as a healthy and synchronous bloom is a prerequisite for pollination and fruit set, any changes to NSC content and its forms as affected by weather, biological stress or management can result in significant yield variation. Furthermore, since NSC levels and their form can affect a range of physiological activities, it is important to ask if and when NSC content has the greatest impact on tree productivity and if it is always better to assure high NSC content to generate high yields. Therefore, to answer these questions, we used multi-year observations of NSC content in twigs of P. dulcis, P. vera, and J. regia and combined them with reported yields for over 300 orchards located across the Central Valley, CA, USA.Surprisingly, despite the importance of NSC activity in providing protection against adverse weather conditions through dormancy and in influencing the timing and synchrony of bloom, little attention has been given to how trees physiologically prepare for this quintessential period of quiescencePrunus dulcis (Mill. D.A Webb), Pistacia vera L. and Juglans regia L.  using a ball grinder . To analyze for soluble sugar and starch content, we used the previously described protocol14 with modifications to use smaller sample sizes15. Specifically, 25 mg of powder per sample was placed in 1.5 mL tubes. Tubes were then treated with 1 mL of sodium acetate buffer , vortexed, and incubated in a 70 °C water bath for 15 min and centrifuged . 50 µL of supernatant was extracted and diluted in ultra-pure (UP) water  and vortexed. Soluble sugar content was quantified from diluted supernatant tubes using an anthrone/sulfuric acid colorizing reagent (0.1% (m:v) in 98% sulfuric acid) and reading absorbance at 620 nm in a spectrophotometer.Specific details of sample collection and handling were described previouslyg). Tubes were then centrifuged and 50 µL of supernatant was extracted and diluted in 1 mL of ultra-pure (UP) water  and vortexed. Total soluble sugar content was analyzed using the same method described above. Starch content was determined by subtracting the original from the post-digestion soluble sugar content. All samples were plated onto 96-well plates. To account for any procedural variability , each plate contained a glucose standard curve (4 wells per plate) and wood/bark standard tissue samples with known soluble sugar and starch contents (4 wells of each per plate) that were concurrently undergoing all steps in the same chemical analysis. The wood/bark standard tissue is a sample from a homogenous mix of several thousand ground samples leftover from a 2016 preliminary part of the study1.The remaining centrifuged tubes containing the pellet and buffer were used for starch quantification. To extract the starch, the tubes were boiled at 100 °C for 10 min to allow starch gelatinization, and let sit for 20 min at room temperature (22 °C). Once cooled, 100 µL of amyglucosidase  and 100 µL amylase  were added to the tubes and incubated for 4 h at 37 °C in a rotating incubator. Samples were then centrifuged  was used to estimate the slope parameter . To determine the most important months, for each carbohydrate type, in predicting the observed yields we used the Random Forest Regressor in PyCaret . The native function ‘feature importances’, in which ‘months’ were assigned as features, was used to indicate which months were the most important predictor of yield.To calculate the coefficient of correlation (r) between yield and carbohydrate content for each of the 12 months preceding harvest (September till August) we used the ‘cor’ function using the Pearson method (R-core). A linear model Yield = ß1 but are also characterized by high variation within each month of the year in all three species . Due to the seasonal variation in NSC content, calculation of the coefficient of correlation between NSC content and yield was performed separately for each month. In general, the analysis revealed that the coefficients of correlation between NSC concentrations and yield were positive and significant  during mid-winter (January and February) in all three species  was characterized by the presence of multiple periods of significant negative correlations between NSC content and yield; during the active period (April to July), highly significant negative correlations before harvest (August), and following harvest in September. Soluble sugar concentration in the bark was only weakly correlated with yield, while total NSC concentration and NSC concentration in wood was significantly correlated in 5 or 6 months during the year preceding harvest  was characterized by significant positive correlations between concentrations of NSC and yield over the period from post-harvest till the end of dormancy (September till March). Specifically, starch content, in wood and bark, was the main driver behind these positive correlations. During the active period spanning from April till July, NSC contents were not significantly correlated with the current-year yield. A shift to negative correlations occurred in August (before harvest) when total NSC, NSC in bark, and starch in bark showed negative correlations with yield. Interestingly, starch content in wood and bark as well as the total content of NSC was most correlated with yield across all months, while soluble sugar concentration in wood remained uniformly non-correlated to yield through the entire season  were the least correlated with yield. Only during the late dormancy period (February–March), the content of NSC and their forms were positively correlated with yield. Unlike in both other species, no significant negative correlations between NSC and yield were observed in J. regia  till harvest (August), NSC concentrations in twigs not only show seasonal variationes Table , 3, 4. Mest Fig. . P. veraP. dulcis, NSC content in February was the most important positive feature contributing to yield, while NSC concentration in August was the most important negative feature in predicting yield  was independently analyzed and the relative importance of each month’s content as the predictor of yield was determined using the Random Forest Regressor algorithm  NSC concentration was positively correlated with yield, while the summer (active period) NSC concentration was negatively correlated with yield. Thus, more is not always better. In fact, the apparent exhaustion of NSC just prior to harvest can be linked to high yield, in which case, less is better. The presence of such correlations underlines the importance of monitoring NSC reserves for enhancing yield success.The two main goals of the presented work were 1) to test for the presence of correlations between NSC concentration in twigs of nut trees and orchards’ yields and (2) to determine the months at which carbohydrate concentration were the best predictors of realized yield. In general, several correlations were significant . Out of all the months however, December NSC concentration was the most important positive predictor of yield, this was true in all of its studied forms and locations . NSC concentration was only negatively correlated with yield in the short period preceding fruit maturation (August). The low levels of August NSC associated with high yields, suggest that reserve exhaustion during this period was correlated with a high accumulation of nut biomass. This pattern may reflect sink dominance of fruit over reserve formation. However, if high yield results in the depletion of NSC concentrations to the extent that they cannot be replenished prior to senescence (high levels of NSC are required in December to assure high yield), then this may lead to a reduction in the following year’s yield and ultimately explain the alternate bearing habit seen in P. vera16. If true, breeding objectives aiming to reduce alternate bearing in P. vera18 may benefit from selecting varieties that show a strong NSC recovery pattern in the fall.The inverse relationship between yield and summer NSC suggests that yield comes at the expense of NSC reserve formation. Nevertheless, the positive correlation in the fall and winter requires that NSC reserves be replenished during the short postharvest period, prior to senescence, to assure adequate reserves for bloom. If NSC reserves are not replenished, a lower yield may be expected and may help explain the presence of alternate bearing at either the whole-tree or orchard level in the case of Prunus dulcis presents a slightly more complicated picture of carbohydrates’ impact on yield. NSC total, starch, and sugar concentrations in wood were positively correlated with yield in late fall and during dormancy (November through February) with February reserves being the most important positive feature associated with high yield. This would suggest that high NSC levels just prior to and during bloom are the most important prerequisite for achieving higher yields. Hence, a high NSC content during dormancy, achieved either by preservation and/or by the influx of sugars from more distal sources during bloom, to provide sufficient energy and structural material for flowers is the key element to yield success. This finding may also provide an interesting opportunity for P. dulcis breeding efforts, wherein selection could be informed by high NSC levels in February19. The sudden change in direction, from a positive relationship in February to a negative in March, most likely reflects the strong dependence on local twig reserves for sustaining a healthy bloom and promoting vegetative bud pushing. The ensuing negative correlation, characterized by a steep decline in NSC concentration beginning in March, continuing through the summer3 suggests that during the most active period, the reproductive NSC sink takes precedence over reserve formation. While reproductive prioritization outweighs reserve formation in both P. dulcis and P. vera, the persistent decline observed in P. dulcis comes in stark contrast to P. vera, where only a narrow time frame, the nut filling period, was negatively correlated with yield. This prolonged period of low NSC content and its associated negative correlation with yield in P. dulcis may be offset by the fact that while P. dulcis has the earliest harvest amongst the three species, its senescence occurs at the same time as in P. vera and J. regia. Thus, in effect allowing more time for the recovery of NSC and potentially avoiding a pronounced alternate bearing habit17.Juglans regia presents the most ambiguous pattern of carbohydrate impact on yield. Much like P. dulcis and P. vera, we also found the strongest positive correlations between NSC content and yield in the months just prior to budbreak. However, in contrast to the two-former species, J. regia is a wind-pollinated species with female flowers developing on new vegetative extension growth and thus relies on the concurrent development of both the vegetative and flowering structures. As a result, this magnifies the burden of bearing enough NSC reserves to initiate both their growths following dormancy. In walnut, this burden exceeds the storage-supply capacity within the twigs and it must therefore import NSC from distant sources to attain sufficient energy. Therefore, in preparation for bloom, J. regia may strongly depend on the redistribution of NSC, via the xylem, from the stem to twigs thus reducing its reliance on autumnal carbohydrate reserves. Indeed, in February and March, we observed that increases in twig NSC were most consistently in the woody conductive tissues20. Furthermore, given that flowering and vegetative growth occur simultaneously, photosynthetic independence promptly follows thereby quickly becoming the main energy source for supporting both their growths. Hence, the dependence on a distal energetic supply for growth initiation and then on current photosynthate for growth sustenance may explain the lack of significant correlations between twig NSC storage and yield. As a consequence, the antagonistic relationship between storage and reproductive sinks that is more apparent in P. vera and P. dulcis is diminished, making J. regia potentially less sensitive to twig reserve carbohydrate content. Interestingly, when analyzing monthly importance there were consecutive shifts; from a high NSC content in June, as the most important predictor of low crop, to a high NSC content in July, as the most important predictor of high yield. These months, in particular, coincide with the transitional phases that occur between growth and storage accumulation. In J. regia specifically, June marks the fastest growth rates and the lowest carbohydrates content while July is the moment at which growth slows but also the point of maximum reserve accumulation rates1. Therefore, in the context of phenology and yield, it is thought-provoking that the crux between the interplay of these months is captured as most important for predicting final yields. Such increasing importance on mid-summer carbohydrate concentrations further supports the notion that in J. regia, high crop is not dependent on the competition between storage and yield but rather on an overall high photosynthetic productivity especially at the end of summer. The low dependence of yield on autumnal NSC reserves can be expected from the fact that multi-year observations of NSC content on walnut tree twigs were relatively unaffected by seasonality compared to the two other studied species1. In addition, it is important to note that an impact of NSC reserves on yield is not always detected, for example in Olea europea L. (olive tree) no such impact has been reported21. However, such a relationship may be very difficult to detect in small, short term experimental studies due to high temporal and year-to-year variation in NSC content9.P. vera or February for P. dulcis, can help project yield and provide information for assessing irrigation and fertilization needs. As non-structural carbohydrate content represents a buffer between photosynthetic capacity and needs , knowledge on their dynamics may provide physiological insights to better understand the physiological status of trees. Sudden and unexpected changes to NSC concentrations may reflect orchard health issues. The introduction of NSC analysis to breeding may open new avenues in the search for high-yielding varieties. We can also expect that adding NSC content analysis to yield prediction models which consider environmental elements  as physiological attributes and encompass a range of abiotic and biotic stressors  will improve their performance.In all cases, only 1 or 2 months shared a high importance for crop prediction and such distribution of importance may suggest the practical implications in using carbohydrate analyses for orchard management and decision making. A simple NSC concentration analysis in twigs at specific months for example, December for


The extent of retrogradation strongly affects certain physical and cooking properties of rice starch (RS), which are important to consumers. In this study, oligomeric procyanidins from lotus seedpod (LSOPC) was prepared and used to investigate its inhibitory effect on RS retrogradation. Various structural changes of RS during retrogradation were characterized by differential scanning calorimetry, low field nuclear magnetic resonance, X-ray diffraction, scanning electron microscopy, and Fourier transform infrared spectroscopy. The results showed LSOPC could effectively retard both short- and long-term retrogradation of RS, and its inhibitory effect was dependent on the administered concentration of LSOPC. Molecule simulation revealed the interactions of RS and LSOPC, which indicated that the competition of hydrogen bonds between RS and LSOPC was the critical factor for anti-retrogradation. This inhibitory effect and mechanism of action of LSOPC could promote its applications in the field of starch anti-retrogradation. Rice is a common food and a basic staple for hundreds of millions of people . Starch Several methods have been found to retard or decrease starch retrogradation, such as various modifications and specially designed additives. Extrusion and microwaving are easy-to-operate physical ways, however, the effects of anti-retrogradation are limited –6. AcetyIn addition, increased awareness of food safety has led to the development of natural food additives. Natural polyphenols have attracted attention because their non-negligible inhibiting effect on starch retrogradation. Rutin, ferulic acid, epigallocatechin gallate and tannin have already been used in research on starch retrogradation –16. ProcIn this study, PC was prepared from lotus seedpod. The retrogradation of rice starch with and without PC was then investigated. Various structural changes of rice starch during retrogradation were characterized by differential scanning calorimetry (DSC), low field nuclear magnetic resonance (LF-NMR), X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and computer molecular simulation .Rice starch (RS) was purchased from Wuxi Jinnong Biotechnology Co., Ltd. . Lotus seedpods were bought from the local supermarket (Wu Zhi 2 hao).The method of Wu et al.  was usedonset), peak temperature (Tpeak) and end temperature (Tend) were obtained from the pasting curve. The pasting enthalpy (ΔHg) was analyzed by the calculation of peak area.A DSC 214  was used to measure the thermodynamic properties of RS with or without LSOPC. The sample (2 mg) was used, and the heating rate is programmed at 10°C/min from 20 to 100°C. After that, the onset temperature . The test conditions were as follows: the probe was P/0.5 type, the pre-test, test and post-test speed were all 1.0 mm/s, the trigger force was set to 5.0 g, and the shape variable was set to 40%. TPA software was used to analyze the final results.Gelatinized samples were stored at 4°C for 7 days. All samples were transferred to the NMR tubes and a NMI 20X NMR imaging analyzer  was used to determine the water distribution. The treatment conditions were as follows: the number of sample point was set to 1,024, the number of repeated scans was set to 8, and the relaxation attenuation time was set to 2,000 ms. CPMG pulse sequence was applied to measure the relaxation time.−1 and the final results were analyzed by omnic 8.0 software.The samples were analyzed by a Nexus 470 FT-IR spectrometer  according to the previous method  with somThe crystal structure of the freeze-dried powder sample (10 mg) was measured by a D8-Advance diffractometer . The scanning range (2 θ) was 5°-40° and the scanning speed was 2°/min. MDI jade 6.0 software was used to analyze the final results.About 2 mg of freeze-dried sample was fixed and sprayed gold. Then the sample was observed by a SU-8010 scanning electron microscope .All samples were cooled to room temperature after gelatinization. A DHR-3 rotational rheometer  was applied to determine the dynamic rheological properties of samples. The test conditions were as follows: the plate diameter was 40 mm, the gap was 0.5 mm, the temperature was set to 25°C, the scanning strain was set to 1%, and the frequency was 0.1–10 Hz. Finally, the spectra of storage modulus (G') and loss modulus (G”) were obtained.The structural models of RS and procyanidins were downloaded from the National Center for Biotechnology Information (NCBI) using PubChem SID: 135332954 and 374367001. ChemOffice 2010 was used to optimize these molecular structures. After that, Materials Studio 8.0 was used to simulate the interactions between RS and procyanidins.All data were presented as means ± standard deviation (means ± S.D.). Analyses of variance (ANOVA) were performed by SPSS 25.0. The graph was drawn by OriginPro 8.0.onset, Tpeak, Tend and ΔHg of RS with LSOPC trended downward, and the decline correlated positively to the amount of LSOPC added. This result indicated that RS with LSOPC could gelatinize at a lower temperature, which may be due to the hydroxyl group in procyanidins. The hydroxyl group, which has strong hydrophilicity, could have interacted with the side chain of the starch and then combined with its non-crystalline region. Therefore, the degree of crystallinity changed, and the energy required for starch gelatinization was reduced  corresponded to the binding water, which bound the matter most tightly; T22 (17–165 ms) matched the water that did not flow easily, which was between the binding water and the free water; and T23  corresponded to the free water, which possessed the highest degree of fluidity. 23, indicating the internal water was mainly free water. With the increase of LSOPC addition, the wave peak shifted to the left and finally located at T22, suggesting the water flow became harder. The results of LF-NMR showed procyanidins could increase the water holding capacity of RS, and subsequently hinder its recrystallization.The changes in water distribution of samples were determined by using LF-NMR analysis, and the corresponding spectra were shown in −1 for RS. Notably, the absorption peak did not change significantly after adding 3% LSOPC, but moved toward the direction of low wavenumber after adding 6 or 9% LSOPC. The band of 3,100–3,500 cm−1 in the infrared spectrum could reflect the hydrogen bond strength formed by intramolecular hydroxyl groups: the stronger the hydrogen bond strength, the larger the shift of the spectral band to the lower wavenumber , State Key Laboratory of Marine Resource Utilization in South China Sea (Hainan University) (No. MRUKF2021002), Key Laboratory of Food Nutrition and Functional Food of Hainan Province (No. KF202009), and the Collaborative Grant-in-Aid of the HBUT National 111 Center for Cellular Regulation and Molecular Pharmaceutics (No. XBTK-2020005).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.


Biodegradable packaging prepared from starch is an alternative to fossil-based plastic packaging. However, the properties of starch packaging do not comply with the necessary physicochemical properties to preserve food. Hence, in a previous study, we reported the preparation of a composite polymer material based on starch-chitosan-pluronic F127 that was found to be an adequate alternative packaging material. In this study, we modified the physicochemical properties of this material by storing it for 16 months under ambient conditions. The results indicate that the incorporation of pluronic F127 in the blend polymer can help avoid the retrogradation of starch. Moreover, at higher concentrations of pluronic F127, wettability is reduced. Finally, after storage, the materials exhibited surface modification, which is related to a color change and an increase in solubility, as well as a slight increase in stiffness. However, their advantages, the environmental pollution resulting from their excessive use, slight or prolonged degradation, and inadequate waste disposal are critical. Studies have been focused on the development of polymeric materials of biological and biodegradable origin using biopolymers such as starch, cellulose, chitosan, and proteins that do not contribute to the accumulation of COl crisis ,2,3,4.Starch is an excellent choice for developing biodegradable packaging materials, owing to its abundance in nature and higher economical viability. As a nontoxic polymer, it can be safely used for food packaging applications; moreover, it does not impart flavor to the packaged food. Therefore, there is no risk of affecting the organoleptic properties of the packed foods .However, biodegradable packaging based on starch and other biopolymers, such as chitosan or gelatin, are susceptible to hydration and thus do not resist high relative humidity conditions, direct contact with liquid water, or foods with high moisture content ,7,8. TheStorage time and conditions also significantly affect the properties of starch-based packaging materials due to the retrogradation exhibited by starch. Starch is composed of amylose and amylopectin. Amylose is a molecule with a mainly linear structure, whereas amylopectin is a highly branched molecule. The amylose-to-amylopectin ratio in corn starch granules is reported to be approximately 25/75 . The proThe results obtained in a previous study were promising, suggesting this material, made using starch-chitosan-poloxamer, to be a viable alternative for producing packaging materials . HoweverThis study hypothesized that new poloxamer-containing starch-chitosan formulations could maintain their structural integrity for several months, significantly delaying the starch retrogradation process that typically occurs in short periods. Therefore, in this study, the thermal, structural, mechanical, and morphological behaviors of corn starch-chitosan and poloxamer F127 stored for zero and sixteen months at room temperature were evaluated using differential scanning calorimetry (DSC), X-ray, FTIR-attenuated total reflection (ATR), tensile stress, wettability by the contact angle, color, and scanning electron microscopy (SEM) techniques.The following biopolymers were used: corn starch from KMC ; shrimp shell chitosan ; pluronic F127 . Glacial acetic acid  and glycerol  were also used.w/w), chitosan 1% (w/w), and poloxamer . The polymer solutions were mixed in a ratio of 60:20:20, and glycerol was added as a plasticizer at 25% . Fifty milliliters of each polymer dispersion was poured into a plastic mold and dried at 60 °C  for four hours. The dried films were peeled off and stored in airtight storage bags for 16 months in the dark and at ambient conditions with a relative humidity (RH) of 32.73% and temperature of 25 °C.The methodology proposed by Fonseca-García, Jiménez-Regalado  was follL*, a*, and b* [L* = 91.76, a* = 2.14, and b* = −10.64.Color was determined using a portable spectrophotometer  based on the CIELab scale with the coordinates , and b* . The filThe contact angle was determined using the video goniometer  to determine the hydrophobicity of the films . A 5-µL To analyze the morphology of the packaging films, SEM  was performed with a voltage of 10 kV. The films were coated with a gold-palladium layer  .The vibrational modes of principal functional groups present in the packaging film were analyzed by FTIR spectrum using a Nicolet iS50 FTIR  by ATR technique .The XRD of biodegradable films was determined using an X-ray diffractometer  with a voltage of 35 kV, current of 25 mA, and Cu Kα radiation of 1.5406 Å over a Bragg angular range (2θ) of 5–35° .PT), melting temperatures (mT1 and m2T), and the melting enthalpy (ΔmH2). The analyses were performed at a temperature range of 25–300 °C under a nitrogen atmosphere (50 cm3 min−1) using a thermogravimetric analyzer , according to the standard ASTM D3418-15 [DSC was used to measure the gelatinization peak ; the bands in this region are sensitive to the gelation of starch because of their association with C–O stretching of the ring, linkages (C–O–C), and COH groups. Notably, the band at 1017 cm−1 is reported as sensitive to amorphous starch, which is constant in the spectra after 16 months of storage [−1, attenuated after 16 months of storage, can be assigned to the O-H stretching of the groups in amorphous amylose. Moreover, the molecule of water showed that the absorption bands at 3300 and 1646 cm−1 are associated with OH stretching and deformation vibrations, respectively [−1 was associated with C-H bond stretching, which did not show a modification in the four materials after 16 months [−1, which was associated with C-O stretching of C–O–C in glycosidic linkage; this result suggests the depolymerization of the starch molecule [ storage ,22,23. C storage  reportedectively . In addi6 months . Finallymolecule .To determine if there was retrogradation or modification in the conformation of the structural matrix of the biodegradable films, mT) of the neat corn starch film decreases with a significant increase in enthalpy (∆mH), indicating a gain in the ordering of the polymeric structures involved. In contrast, chitosan films exhibited an increase of 15 °C after 16 months of storage. The enthalpy values for this biopolymer decreased as a function of the evaluated time. Notably, the mixture of starch and chitosan (F127 0%) exhibited a thermal behavior in which starch transitions predominated. The incorporation of the poloxamer in the starch-chitosan matrix ensured gelatinization, with the absence of the band at ~62 °C . A decrease was observed in the melting temperature (56.1 °C) corresponding to neat F127. The increase in poloxamer content resulted in an increase in the melting temperature and a decrease in enthalpy after 16 months, with respect to its nonfunctionalized homologous (F127 0%). This allowed for demonstrating the plasticizing effect generated by the poloxamer due to the destabilization of the crystalline regions. The fusion of intra- and inter-molecular double helices and the partial recovery of the crystalline structure of amylopectin (The results of the thermal behavior of the biodegradable films are presented in lopectin  can be oThe WS of biodegradable materials is a crucial parameter because it is important that they can decompose in both terrestrial and aquatic environments after being discarded, where they can potentially contaminate the environment on several occasions. A significant increase in the water solubility capacity of the starch-chitosan-based materials was observed  at all pThe mechanical performance of corn starch and chitosan films with various poloxamer concentrations was significantly affected with storage time, as shown in Storage time can significantly modify the physicochemical, morphological, and structural properties of biodegradable films comprising starch, chitosan, and poloxamer. This information is important to ascertain the state of packaging materials of biodegradable origin when stored at room temperature. Based on physicochemical characterization, there is evidence suggesting that poloxamer avoids the retrogradation of cornstarch. The biodegradable films showed a slight modification in their surface morphology after 16 months, similar to erosion, and this phenomenon can modify the color, WS, mechanical behavior, and wettability of films. Lower melting temperatures and enthalpies in the poloxamer films, as well as storage for 16 months, indicate that the retrogradation of amylopectin only partially recovers the crystalline structure of the native starch. The use in the pharmaceutical, medical, and packaging industry of a biodegradable material such as the one developed in this study may be possible, owing to the favorable properties observed in its mechanical performance and acceptable solubility in liquid water, which can be sufficiently solubilized in a short period of time with the presence of the poloxamer.