From Food Waste to Functional Biopolymers: Characterization of Chitin and Chitosan Produced from Prepupae of Black Soldier Fly Reared with Different Food Waste-Based Diets.

The use of food waste as a rearing substrate to grow insects is an ecofriendly and sustainable alternative to food waste disposal. In the present research, Hermetia illucens prepupae were reared with a standard diet, different food waste-based diets based on vegetables, fruits, and meat, and a mixed one, where the previous three components were present equally. The demineralization and deproteination of the prepupae allowed for the obtainment of chitin that was then deacetylated to produce chitosan. Also, the bleaching of chitosan was attempted for further purification. The yield of the different reactions was investigated, and the infrared spectra of the obtained materials were analyzed to obtain information on the quantity and acetylation degree trend of the chitin and chitosan as a function of the diet. The possibility to slightly modulate the yield and acetylation degree of both biopolymers thanks to the specific diet was enlightened. Interestingly, the standard diet resulted in the highest fraction of chitin having the highest acetylation degree, and in the highest fraction of chitosan having the lowest acetylation degree.

Macrophage polarization in vitro and in vivo modified by contact with fragmented chitosan hydrogel.

We have previously shown that implantation of a fragmented chitosan hydrogel suspension (chitosan-FPHS) into a traumatic spinal cord lesion in adult rats led to significant axon regrowth and functional recovery, which was associated to a modulation of inflammation. Using an in vitro culture system, we show here that polarization of bone marrow-derived macrophages is indeed modified by direct contact with chitosan-FPHS. Reducing the degree of acetylation (DA) and raising the concentration of chitosan (Cp, from 1.5% to 3%), favors macrophage polarization toward anti-inflammatory subtypes. These latter also migrate and adhere efficiently on low, but not high DA chitosan-FPHS, both in vitro and in vivo, while inflammatory macrophages rarely invade a chitosan-FPHS implant in vivo, no matter the DA. Our in vitro model setup should prove a valuable tool for screening diverse biomaterial formulations and combinations thereof for their inflammatory potential prior to implantation in vivo.

Chitosan/gelatin films with jatobá resin: Control of properties by vegetal resin inclusion and degree of acetylation modification.

Blending chitosan and gelatin, two biodegradable and non-toxic polymers, is a recurrent choice in food coating or biomaterials development. The incorporation of vegetal extracts into chitosan/gelatin films can improve or introduce some properties to these materials. Jatobá resin is a product of Hymenaea genus trees with antimicrobial and anti-inflammatory activities, interesting properties for films applied in several areas. The chitosan degree of acetylation (DA) influences the inter and intramolecular interactions of this polymer and, therefore, also implicates in changes of its properties. This research aims to study the influence of jatobá resin inclusion and chitosan DA modification on chitosan/gelatin films properties. Both jatobá resin and chitosan DA affected physicochemical, antimicrobial and barrier properties of the films, allowing the control of these properties by changes in these parameters. Jatobá resin incorporation and the decrease in chitosan DA significantly improved antimicrobial activity and water vapor permeability of films with the reduction of water solubility and swelling.

Bioinspired pH-sensitive riboflavin controlled-release alkaline hydrogels based on blue crab chitosan: Study of the effect of polymer characteristics.

Recently, the application of natural biocompatible polymeric hydrogels for the conception of drug delivery matrices has attracted widespread interest. Thus, in the present study, riboflavin pH-sensitive drug delivery hydrogels were developed based on blue crab chitosan (Cs), via direct dissolution in alkali/urea aqueous solution at low temperatures. First, the effect of Cs characteristics in terms of acetylation degree (AD) and molecular weight (Mw) on the structural, mechanical, thermal, swelling and in vitro biodegradation of Cs-based hydrogels were studied. Data from overall analysis revealed that Cs with low AD and high Mw exhibited improved mechanical properties, as evidenced by the compressive and rheological behaviors tests, thermal resistance, swelling behavior and in vitro degradation kinetics. However, hydrogels pore sizes were reduced with the AD decrease and Mw increase. Additionally, hydrogels in PBS (pH 5.5) underwent quicker degradation, compared to those immersed in PBS (pH 7.4). In the drug delivery model, the kinetics of Riboflavin release, through the Cs-based hydrogels were monitored. The Riboflavin release exhibited typical deliverance patterns, with significantly higher released amounts in more acidic systems. Therefore, drug encapsulation within the conceived pH-sensitive Cs-based hydrogels could provide suitable and promoting microenvironment for drugs delivery.

Study of chitosan with different degrees of acetylation as cardboard paper coating.

The biodegradability of chitosan is significant for packaging systems. Another relevant property of chitosan is its degree of acetylation (DA), which affects other properties, such as crystallinity and hydrophobicity. The DA can be modulated by chitin deacetylation or even chitosan reacetylation. The novelty of this paper is the application of reacetylated chitosan as a coating for cardboard paper surfaces to improve the barrier and mechanical properties of the paper. Chitosan with 2% DA was reacetylated to yield chitosan with 48% DA. Both samples were applied as cardboard paper coating, and the coated materials were characterized. The paper-film system of chitosan with 2% DA had better water barrier and mechanical resistance. Heterogeneous deacetylation of chitin reduced the solubility of chitosan because molecular groups were distributed in blocks, increasing the hydrophobicity of the polymer.

Chitosan Acetylation Degree Influences the Physical Properties of Polysaccharide Nanoparticles: Implication for the Innate Immune Cells Response.

The aim of the present contribution is twofold as it reports (i) on the role played by chitosan acetylation degree for the stability of nanoparticles (NPs) formed with hyaluronan and (ii) on the effect of the interaction of such NPs with immune cells. Chitosans with similar viscosity-average molecular weight, [Formula: see text], (i.e., 200 000) and different fractions of acetylated units ( FA) together with low-molecular-weight hyaluronan were chosen for developing a select library of formulations via electrostatic complex coacervation. The resulting NPs were analyzed in terms of size, polydispersity, surface charge, and stability in physiological-mimicked media by dynamic light scattering. Only medium acetylated chitosan ( FA = 0.16) guaranteed the stability of NPs. To explore the effect of NPs interaction with immune cells, the release of proinflammatory cytokines and the reactive oxygen species production by human macrophages and neutrophils, respectively, were evaluated. Strikingly, a structure-function relationship emerged, showing that NPs made of chitosans with FA = 0.02, 0.25, 0.46, and 0.63 manifested a proinflammatory activity, linked to the instability of the system. Conversely, NPs made of chitosan with FA = 0.16 neither modified the functional response of macrophages nor that of neutrophils. Of note, such NPs were found to possess additional properties potentially advantageous in applications such as delivery of therapeutics to target inflamed sites: (i) they are devoid of cytotoxic effects, (ii) they avoid engulfment during the early stage of interaction with macrophages, and (iii) they are muco-adhesive, thereby providing for site-specificity and long-residence effects.

ß-Chitin samples with similar microfibril arrangement change mechanical properties varying the degree of acetylation.

Chitin is widespread in nature and is increasingly used in synthetic process for the production of new biomaterials. Chitin degree of acetylation, crystalline structure and microfibril arrangement differentiate chemical, physical and mechanical properties. Nevertheless, no information are available on the relationship between the mechanical properties and the degree of acetylation (DA) in chitin samples in which the microfibril arrangement does not change. Here, samples of ß-chitin with decreasing DA, up to chitosan, were prepared using the squid pen of Loligo vulgaris. These samples were characterized by CP-MAS NMR spectroscopy, scanning electron microscopy, thermal analyses, synchrotron X-ray fiber diffraction and tensile tests. The results showed a similar microfibril arrangement decreasing the DA, except for the chitosan sample. The mechanical properties showed an increase of the maximum strain and a reduction of the maximum stress and Young's modulus, decreasing the DA. These changes, not linear with the DA, were related to structural changes at molecular structure level. The knowledge deriving from this study is of interest both for the understanding of the mechanical properties of chitinous biological samples, but also for the design and synthesis of new biomacromolecular materials.

The role played by the molecular weight and acetylation degree in modulating the stiffness and elasticity of chitosan gels.

A broad library of chitosans was produced varying the molecular weight and the fraction of acetylated units, FA. The produced chitosans were used for the formation of wall-to-wall cylindrical gels through a controlled external gelation using tripolyphosphate (TPP) as cross-linker. The resulting gels were analyzed by rheometry. Viscosity average degree of polymerization (DPv¯) > 152 was shown to be required for the formation of stable gels. Both gel stiffness and gel rupture strength were proportional to the molecular weight regardless of the applied deformation. Increasing acetylation produced a marked reduction of the shear modulus, but, in parallel, switched the networks from rigid and brittle to weak and elastic. Intriguingly, gels made of chitosan with FA = 0.37 displayed notable elasticity, i.e. up to 90% of applied strain falls into linear regime. These findings suggest that the frequency of glucosamine (D unit) and N-acetyl-glucosamine (A unit) contribute to a subtle structure-property relationship in chitosan-TPP gels.

Cytotoxicity of chitosans with different acetylation degrees and molecular weights on bladder carcinoma cells.

The purpose of this research was to evaluate the cytotoxicity of chitosans with different degrees of acetylation (DA) and molecular weights (MW), as well as the effect of their positive ionic charges controlled by pH on bladder carcinoma cells (RT112 and RT112cp) using the tetrazolium salt colorimetric (MTT) assay. Our data showed that all chitosan samples were cytotoxic on RT112 and RT112cp cells with a higher cytotoxicity obtained at lower pH. Further, it was found that the toxicity increased with increasing DA. However, no significant difference in cytotoxicity between chitosans with different molecular weights was observed. Annexin V-FITC staining test was then used to study and quantify the induction of apoptosis. Data shows that chitosans induce apoptosis of RT112 and RT112cp cells with the same dependence with DA.

Pectin content and composition from different food waste streams.

In the present paper, 26 food waste streams were selected according to their exploitation potential and investigated in terms of pectin content. The isolated pectin, subdivided into calcium bound and alkaline extractable pectin, was fully characterized in terms of uronic acid and other sugar composition, methylation and acetylation degree. It was shown that many waste streams can be a valuable source of pectin, but also that pectin structures present a huge structural diversity, resulting in a broad range of pectin structures. These can have different physicochemical and biological properties, which are useful in a wide range of applications. Even if the data could not cover all the possible batch by batch and country variabilities, to date this represents the most complete pectin characterization from food waste streams ever reported in the literature with a homogeneous methodology.

Use of a fractional factorial design to study the effects of experimental factors on the chitin deacetylation.

Chitosan is obtained by deacetylation of chitin. Chitosan versatility is directly related to the polymer's characteristics depending on the deacetylation process. The aim of this research was to study the parameters influencing deacetylation and to elucidate their effect on acetylation degree (DA) and molecular weight (MW). The effect on chitosan DA was investigated using a fractional factorial design 2(7-3) with seven factors and two variation levels. The tested factors were: X1=number of successive baths, X2=reaction time, X3=temperature, X4=alkali reagent, X5=sodium borohydride, X6=the atmospheric conditions and X7=alkali concentration. A mathematical model was investigated corresponding to the following relation y=7.469-1.344X1-1.094X2-3.094X3+1.906X4+0.656X5+0.906X6-1.031X7+0.469X1X2-0.781X3X4+0.906X1X3X4 with R2=0.99. This model allows fixing experimental conditions for each desired DA. To study the effect on chitosan MW, only atmospheric conditions and use of sodium borohydride as an oxygen scavenger were investigated. The use of sodium borohydride and nitrogen atmosphere was found to have a protective effect against chitosan degradation during deacetylation.

Simple and validated quantitative ¹H NMR method for the determination of methylation, acetylation, and feruloylation degree of pectin.

The knowledge of pectin esterification degree is of primary importance to predict gelling and other properties of pectin from different sources. This paper reports the development of a simple and rapid (1)H NMR-based method for the simultaneous quantitative determination of methylation, acetylation, and feruloylation degree of pectin isolated from various food sources. Pectin esters are hydrolyzed in NaOH/D2O, and the obtained methanol, acetic acid, and ferulic acid are directly measured by (1)H NMR. High accuracy, repeatability, and reproducibility of the method were obtained, and the analysis time is reduced as compared to conventional chromatography- or titration-based methods.

Influence of acetylation degree and molecular weight of homogeneous chitosans on antibacterial and antifungal activities.

The results given in the literature are conflicting when considering the relationship between antimicrobial activity and chitosan characteristics. To be able to clarify, we prepared fifteen homogeneous chitosans with different acetylation degrees (DA) and molecular weights (MW) by reacetylation of a fully deacetylated chitin under homogeneous conditions. They were tested at different pH values for their antimicrobial activities against four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella typhi), four Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis and Micrococcus luteus) and three fungi (Aspergillus niger, Fusarium oxysporum and Alternaria solani). Chitosans markedly inhibited growth of most bacteria and fungi tested, although the inhibitory effect depends on the type of microorganism and on the chitosan characteristics (DA and MW) with minimum inhibitory concentrations in the range of 0.001 to 0.1 w%. Considering chitosan efficiency on bacteria, our series of data clearly show that the lower DA and the lower pH give the larger efficiency. Antibacterial activity was further enhanced for Gram-negative bacteria with decreasing MW, whereas, opposite effect was observed with the Gram-positive. Concerning the antifungal activity, the influence of chitosan characteristics was dependent on the particular type of fungus. Fungal growth decreased with increasing MW for F. oxysporum and decreasing DA for A. solani, but no MW or DA dependences were observed with A. niger.