Biobased Composites of Poly(Lactic Acid) Melt Compounded with Bacterial and Vegetal Nanocelluloses Incorporated through Different Strategies.

In the current contribution, bacterial nanocellulose obtained from a by-product of Kombucha tea production and vegetal nanocellulose isolated from milled rice husks were employed as fillers of PLA-based composites prepared by intensive mixing followed by compression molding. Given the challenges associated with the incorporation of nanocelluloses-initially obtained as aqueous suspensions-into melt compounding processes, and also with achieving a proper dispersion of the hydrophilic nanofillers within PLA, three different nanofibrils incorporation strategies were studied: i.e., direct mixing of dried milled nanocelluloses and PLA; masterbatching by solvent casting of native nanocelluloses followed by melt compounding; and masterbatching by solvent casting of acetylated nanocelluloses followed by melt compounding. Composites with varying filler content (from 0.5 wt.% to 7 wt.%) were characterized in terms of morphology, optical properties, and mechanical performance. Results revealed the relative suitability of each strategy employed to promote nanocelluloses dispersion within the PLA matrix. PLA/nanocellulose masterbatches prepared by solvent casting proved to be particularly useful for feeding the nanocelluloses into the processing equipment in a dry state with limited hornification. Acetylation also contributed to a better dispersion of both nanocelluloses within the PLA matrix, although no clear positive impact on the mechanical properties of the films was observed. Finally, filler loading played an important role in the films' properties by increasing their stiffness while reducing their translucency.

Antioxidant Edible Films Based on Pear Juice and Pregelatinized Cassava Starch: Effect of the Carbohydrate Profile at Different Degrees of Pear Ripeness.

Edible films based on fruit and vegetable purees combined with different food-grade biopolymeric binding agents (e.g., pectin, gelatin, starch, sodium alginate) are recognized as interesting packaging materials that benefit from the physical, mechanical, and barrier properties of biopolymers as well as the sensory and nutritional properties of purees. In the current contribution, edible antioxidant films based on pear juice and pregelatinized cassava starch were developed. In particular, the suitability of using pregelatinized cassava starch for the non-thermal production of these novel edible films was evaluated. In addition, the effects on the films' properties derived from the use of pear juice instead of the complete puree, from the content of juice used, and from the carbohydrate composition associated with the ripening of pears were all studied. The produced films were characterized in terms of their total polyphenol content, water sensitivity, and water barrier, optical, mechanical and antioxidant properties. Results showed that the use of pear juice leads to films with enhanced transparency compared with puree-based films, and that juice concentration and carbohydrate composition associated with the degree of fruit ripeness strongly govern the films' properties. Furthermore, the addition of pregelatinized cassava starch at room temperature discloses a significant and favorable impact on the cohesiveness, lightness, water resistance, and adhesiveness of the pear-juice-based films, which is mainly attributed to the effective interactions established between the starch macromolecules and the juice components.

Patents involving nanocellulose: Analysis of their evolution since 2010.

During the last two decades cellulosic nanomaterials have been the subject of much research around the world. Moreover, in the last few years, increasing industrial interest on the field enabled the setting-up of the first facilities producing commercial quantities of nanocelluloses; whereas a number of inventions involving cellulose nano-objects are claimed every year. In this context, the current article describes the recent evolution (from 2010 till 2017) of published patents which explicitly include in their title, abstract and/or claims references to cellulose nano-objects such as cellulose nanocrystals, cellulose nanofibrils and bacterial nanocellulose. Results evidence the astonishing increase in nanocellulose patents since 2010, and specially within the last three years surveyed (i.e. 2015-2017), when published documents accounted for ca. 70 % of the total number of patents published since 2010. Besides patent timelines, data is analysed in terms of patent owners, countries of application, and citing number.

UV Protective, Antioxidant, Antibacterial and Compostable Polylactic Acid Composites Containing Pristine and Chemically Modified Lignin Nanoparticles.

Polylactic acid (PLA) films containing 1 wt % and 3 wt % of lignin nanoparticles (pristine (LNP), chemically modified with citric acid (caLNP) and acetylated (aLNP)) were prepared by extrusion and characterized in terms of their overall performance as food packaging materials. Morphological, mechanical, thermal, UV-Vis barrier, antioxidant and antibacterial properties were assayed; appropriate migration values in food simulants and disintegration in simulated composting conditions were also verified. The results obtained indicated that all lignin nanoparticles succeeded in conferring UV-blocking, antioxidant and antibacterial properties to the PLA films, especially at the higher filler loadings assayed. Chemical modification of the fillers partially reduced the UV protection and the antioxidant properties of the resulting composites, but it induced better nanoparticles dispersion, reduced aggregates size, enhanced ductility and improved aesthetic quality of the films through reduction of the characteristic dark color of lignin. Migration tests and disintegration assays of the nanocomposites in simulated composting conditions indicated that, irrespectively of their formulation, the multifunctional nanocomposite films prepared behaved similarly to neat PLA.

Yarrowia lipolytica: a model yeast for citric acid production.

Every year more than 2 million tons of citric acid (CA) are produced around the world for industrial uses. Although initially extracted from citrus, the low profitability of the process and the increasing demand soon stimulated the search for more efficient methods to produce CA. Currently, most world CA demand (99%) is satisfied by fermentations with microorganisms, especially filamentous fungi and yeasts. CA production with yeasts has certain advantages over molds (e.g. higher productivity and easier cultivation), which in the last two decades have triggered a clear increase in publications and patents devoted to the use of yeasts in this field. Yarrowia lipolytica has become a model yeast that proved to be successful in different production systems. Considering the current interest evidenced in the literature, the most significant information on CA production using Y. lipolytica is summarized. The relevance on CA yields of key factors such as strains, media formulation, environmental conditions and production regimes is thoroughly discussed, with particular focus on increasing CA productivity. Besides, the possibility of tuning the mentioned variables to reduce concomitant isocitric acid production-the biggest disadvantage of using yeasts-is analyzed. Available methods for CA purification/quantification are also discussed.

Simple citric acid-catalyzed surface esterification of cellulose nanocrystals.

A simple straightforward route for the surface esterification of cellulose nanocrystals (CNC) is herein proposed. CNC obtained from microcrystalline cellulose were acetylated using as catalyst citric acid, a α-hydroxy acid present in citrus fruits and industrially produced by certain molds in sucrose or glucose-containing medium. No additional solvent was added to the system; instead, the acylant (acetic anhydride) was used in sufficient excess to allow CNC dispersion and proper suspension agitation. By tuning the catalyst load, CNC with two different degree of substitution (i.e. DS=0.18 and 0.34) were obtained. Acetylated cellulose nanocrystals were characterized in terms of chemical structure, crystallinity, morphology, thermal decomposition and dispersion in a non-polar solvent. Results illustrated for the first time the suitability of the protocol proposed for the simple surface acetylation of cellulose nanocrystals.

Acetylation of bacterial cellulose catalyzed by citric acid: Use of reaction conditions for tailoring the esterification extent.

Bacterial cellulose (BC) nanoribbons were partially acetylated by a simple direct solvent-free route catalyzed by citric acid. The assay of reaction conditions within chosen intervals (i.e. esterification time (0.5-7h), catalyst content (0.08-1.01mmol/mmol AGU), and temperature (90-140°C)), illustrated the flexibility of the methodology proposed, with reaction variables which can be conveniently manipulated to acetylate BC to the required degree of substitution (DS) within the 0.20-0.73 interval. Within this DS interval, characterization results indicated a surface-only process in which acetylated bacterial cellulose with tunable DS, preserved fibrous structure and increased hydrophobicity could be easily obtained. The feasibility of reusing the catalyst/excess acylant in view of potential scale-up was also illustrated.

Organocatalytic acetylation of starch: effect of reaction conditions on DS and characterisation of esterified granules.

Starch acetates with varying degree of substitution (DS) were prepared by a novel solvent-free organocatalytic methodology. The acetylation protocol involved a non-toxic biobased α-hydroxycarboxylic acid as catalyst, and proceeded with high efficiency in absence of solvents. The effect of reaction conditions including reaction temperature (90-140 °C), catalyst load (0-2.3 g/g starch), acetic anhydride/starch weight ratio (6.5-13.5 g/g), and starch moisture content (0.6-14.8%) on the DS of the esters was evaluated. The analysis performed showed that the increase of temperature and catalyst concentration resulted in higher DS values, and evidenced a beneficial contribution of native starch moisture content on the substitution level achieved. Variation of reaction conditions allowed starch esters to be obtained with DS in the 0.03-2.93 range. Starch esters were characterised in terms of morphology, chemical structure, thermal properties, and distribution in polar/non polar liquid systems.

Surface esterification of cellulose nanofibers by a simple organocatalytic methodology.

Bacterial cellulose nanofibers were esterified with two short carboxylic acids by means of a simple and novel organic acid-catalyzed route. The methodology proposed relayed on the use of a non-toxic biobased α-hydroxycarboxylic acid as catalyst, and proceeded under moderate reaction conditions in solventless medium. By varying the esterification interval, acetylated and propionized bacterial cellulose nanofibers with degree of substitution (DS) in the 0.02-0.45 range could be obtained. Esterified bacterial cellulose samples were characterized by means of Solid-State CP/MAS (13)C Nuclear Magnetic Resonance spectroscopy (CP/MAS (13)C NMR), Fourier Transform Infrared spectroscopy (FTIR), X-ray Diffraction (XRD), Thermogravimetric Analysis (TGA) and chosen hydrophobicity test assays. TGA results showed that the esterified nanofibers had increased thermal stability, whereas XRD data evidenced that the organocatalytic esterification protocol did not alter their crystallinity. The analysis of the ensuing modified nanofibers by NMR, FTIR, XRD and TGA demonstrated that esterification occurred essentially at the surface of bacterial cellulose microfibrils, something highly desirable for changing their surface hydrophilicity while not affecting their ultrastructure.

Analysis of a preferential action of α-amylase from B. licheniformis towards amorphous regions of waxy maize starch.

Waxy maize starch was subjected to α-amylase (Bacillus licheniformis) hydrolysis in buffered medium to determine the evolution of reaction in quantitative terms and also in terms of the morphology and crystallinity of the partially hydrolyzed starch granules. Gathered data allowed studying the pattern of action of this α-amylase over waxy maize starch granules, with particular focus on a preferential hydrolysis of the amorphous regions of starch. Results showed that waxy maize starch hydrolysis followed a two-stage kinetic profile with an initial stage characterized by high reaction rate, followed by a slower second stage. The change of hydrolysis rate occurred at approximately 6h of reaction, a time for which X-ray diffraction data quantitatively analyzed by three different techniques showed a maximum of crystallinity in partially hydrolyzed granules. Scanning electron microscopy images illustrated the action of α-amylases which implied the exoerosion of the granules surface, the entry of α-amylases into the granules through radial channels, their endoerosion towards the granule exterior, and their fragmentation. Fragmentation of waxy maize starch granules revealed internal layered structures of starch which were interpreted as hydrolyzed/non-hydrolyzed growth rings. Under the conditions chosen, kinetic, electron microscopy and X-ray data all gave evidence of a preferential action of α-amylase from Bacillus licheniformis towards the less ordered regions of waxy maize starch. Results showed that, provided the proper hydrolysis time is chosen, starch granules with increased crystallinity can be obtained by a pure enzymatic treatment.

Cross-linked enzyme aggregates (CLEAs) of selected lipases: a procedure for the proper calculation of their recovered activity.

In the last few years, synthesis of carrier-free immobilized biocatalysts by cross-linking of enzyme aggregates has appeared as a promising technique. Cross-linked enzyme aggregates (CLEAs) present several interesting advantages over carrier-bound immobilized enzymes, such as highly concentrated enzymatic activity, high stability of the produced superstructure, important production costs savings by the absence of a support, and the fact that no previous purification of the enzyme is needed. However, the published literature evidences that a) much specific non-systematic exploratory work is being done and, b) recovered activity calculations in CLEAs still need to be optimized. In this context, this contribution presents results of an optimized procedure for the calculation of the activity retained by CLEAs, based on the comparison of their specific activity relative to their free enzyme counterparts. The protocol implies determination of precipitable protein content in commercial enzyme preparations through precipitation with ammonium sulphate and a protein co-feeder. The identification of linear ranges of activity versus concentration/amount of protein in the test reaction is also required for proper specific activity determinations. By use of mass balances that involve the protein initially added to the synthesis medium, and the protein remaining in the supernatant and washing solutions (these last derived from activity measurements), the precipitable protein present in CLEAs is obtained, and their specific activity can be calculated. In the current contribution the described protocol was applied to CLEAs of Thermomyces lanuginosa lipase, which showed a recovered specific activity of 11.1% relative to native lipase. The approach described is simple and can easily be extended to other CLEAs and also to carrier-bound immobilized enzymes for accurate determination of their retained activity.

Lipase-catalyzed acidolysis of tripalmitin with capric acid in organic solvent medium: Analysis of the effect of experimental conditions through factorial design and analysis of multiple responses.

The acidolysis of tripalmitin with capric acid catalyzed by an immobilized form of a 1,3-positionally selective lipase (Rhizomucor miehei) showed to be effective for the synthesis of structured lipids of the MLL and MLM type. The effects that reaction parameters such as substrate molar ratio (N), biocatalyst load (E), and reaction temperature (T) have on selected responses variables (i.e. total conversion of tripalmitin, selectivity and yield of the desired structured lipid, hydrolysis yield, and acyl migration importance), were evaluated by use of an experimental factorial design of three factors and three levels with two central points and with a confidence level of 95%. The range of each parameter was selected as follows: N=3-9, E=5-15wt%, T=50-70°C. The statistical analysis of results was addressed by use of both simple linear models and more complicated quadratic models using specific commercial software. The results obtained showed that a proper selection of reaction conditions is needed in order to maximize not only the yield of the desired structured lipid, but also to minimize the generation of hydrolysis and acyl migration by-products.