Micropropagation of Agave angustifolia "Espadín" Used in the Production of Mezcal.

This chapter presents the methodological approach for the in vitro propagation of Agave angustifolia "espadin," the base material to produce mezcal. The protocol used in each stage of the crop is addressed in detail, considering the changes in the culture medium and the characteristics of the plant material at each stage. The importance of integrated management between the multiplication and growth phase, as part of the in vitro selection strategy, is mentioned.

Pulsed Electric Field-Assisted Extraction of Inulin from Ecuadorian Cabuya (Agave americana).

Inulin is a carbohydrate that belongs to fructans; due to its health benefits, it is widely used in the food and pharmaceutical industries. In this research, cabuya (Agave americana) was employed to obtain inulin by pulsed electric field-assisted extraction (PEFAE) and FTIR analysis confirmed its presence. The influence of PEFAE operating parameters, namely, electric field strength (1, 3 and 5 kV/cm), pulse duration (0.1, 0.2 and 0.5 ms), number of pulses (10,000, 20,000 and 40,000) and work cycle (20, 50 and 80%) on the permeabilization index and energy expenditure were tested. Also, once the operating conditions for PEFAE were set, the temperature for conventional extraction (CE) and PEFAE were defined by comparing extraction kinetics. The cabuya meristem slices were exposed to PEFAE to obtain extracts that were quantified, purified and concentrated. The inulin was isolated by fractional precipitation with ethanol to be characterized. The highest permeabilization index and the lowest energy consumption were reached at 5 kV/cm, 0.5 ms, 10,000 pulses and 20%. The same extraction yield and approximately the same amount of inulin were obtained by PEFAE at 60 °C compared to CE at 80 °C. Despite, the lower amount of inulin obtained by PEFAE in comparison to CE, its quality was better because it is mainly constituted of inulin of high average polymerization degree with more than 38 fructose units. In addition, TGA analyses showed that inulin obtained by PEFAE has a lower thermal degradation rate than the obtained by CE and to the standard.

Effects of dietary supplementation of cobiotic based on Agave fructans on growth performance, blood parameters, oxidative damage and immune status of broiler.

This study evaluated the effect of cobiotic (CO) composed of organic fructans powder of Agave tequilana and turmeric powder of Curcuma longa L. as an alternative of antibiotic growth promoters (AGPs) on growth performance, blood parameters, intestinal pH, oxidative stress, and cytokines serum levels of broiler chickens. A total of 135 one-day-old Ross 308 broilers distributed to five experimental groups, which included starter or finisher standard diets without AGPs (CON), CON + 0.25 COLI-ZIN g/kg feed (AGP), CON + 0.1 g Agave fructans/kg feed (AF), CON + 0.5 g turmeric powder/kg feed (TP) and CON + 0.1 g AF + 0.5 g TP /kg feed (CO), for 49 days. AF followed by TP, decreased feed intake, obtaining the best FCR. AGP increased the heterophil-lymphocyte ratio compared to other groups. CO significantly decreased the pH of the cecal content. AF increased IL-10 levels, while TP decreased it. AF decreased the IL-1ß levels. The present study showed that including a cobiotic based on AF and TP or components separately in a broilers diet improved growth performance, modified intestinal and cecum pH, and stimulated the immune system, which suggests CO as a safe alternative to AGP.

Health-Related Composition and Bioactivity of an Agave Sap/Prickly Pear Juice Beverage.

In this study, a beverage made from a combination of Agave sap (AS) and prickly pear juice (PPJ) was analyzed for its nutrients and bioactive and potentially health-promoting compounds. The beverage was evaluated for its ability to act as an antioxidant, regulate glycemic properties, and undergo gut bacterial fermentation in vitro. The major mono- and oligosaccharides present in the beverage were galacturonic acid (217.74 ± 13.46 mg/100 mL), rhamnose (227.00 ± 1.58 mg/100 mL), and fructose (158.16 ± 8.86 mg/mL). The main phenolic compounds identified were protocatechuic acid (440.31 ± 3.06 mg/100 mL) and catechin (359.72 ± 7.56 mg/100 mL). It was observed that the beverage had a low glycemic index (<40) and could inhibit digestive carbohydrases. The combination of ingredients also helped to reduce gas production during AS fermentation from 56.77 cm3 to 15.67 cm3. The major SCFAs produced during fermentation were butyrate, acetate, and propionate, with valerate being produced only during the late fermentation of the AS. This beverage is rich in bioactive compounds, such as polyphenols and dietary fiber, which will bring health benefits when consumed.

Development of a sustainability-oriented university laboratory: Insight into adsorption kinetics models for the removal of pollutants from aqueous solution.

The aim of the present research is to show the development of a sustainability-oriented lab that teaches adsorption concepts in a virtual environment based on the premise "learning-through-play". Kinetic results in the virtual environment are contrasted to those obtained experimentally when diverse adsorbents prepared from Agave Bagasse (Raw Fibers, Hydrothermal Fibers, and Activated Fibers) were synthesized. Comparison between virtual and real-life experiments involving removal of methylene blue in solution showed that a pseudo-first-order model could describe adsorption kinetics satisfactorily. The study is complemented with a characterization of the adsorbents through SEM, nitrogen adsorption isotherms, FTIR and Raman. In addition, the environmental impact of the synthesis of adsorbents was evaluated through well-known methodologies (GAPI, NEMI, and Eco-Scale), which agree that raw fibers are the most eco-friendly material. This research provides an exciting opportunity to advance our knowledge on developing new technologies for teaching in engineering and to compliment real-life practices that consider environmental impacts with virtual experiments.

PGPB Consortium Formulation to Increase Fermentable Sugar in Agave tequilana Weber var. Blue: A Study in the Field.

Agave tequilana Weber var. Blue is used as the primary raw material in tequila production due to its fructans (inulin) content. This study evaluates the formulation of a plant-growth-promoting bacteria (PGPB) consortium (Pseudomonas sp. and Shimwellia sp.) to increase sugars in A. tequilana under field conditions. A total of three doses were tested: low (5 L ha-1), medium (10 L ha-1), and high (15 L ha-1), with a cellular density of 1 × 108 CFU mL-1 and one control treatment (without application). Total reducing sugars (TRS), inulin, sucrose, glucose, fructose, and plant growth were measured in agave plants aged 4-5 years at 0 (T0), 3 (T3), 6 (T6), and 12 (T12) months. Yield was recorded at T12. The TRS increased by 3%, and inulin by 5.3% in the high-dose treatment compared to the control at T12. Additionally, a low content of sucrose, glucose, and fructose (approximately 1%) was detected. At T12, the weight of agave heads increased by 31.2% in the medium dose and 22.3% in the high dose compared to the control. The high dose provided a higher inulin content. The A. tequilana plants were five years old and exhibited growth comparable to the standards for 6-7-year-old plants. This study demonstrates a sustainable strategy for tequila production, optimizing the use of natural resources and enhancing industry performance through increased sugar content and yield.

Effect of Agavins and Agave Syrup Use in the Formulation of a Synbiotic Gelatin Gummy with Microcapsules of Saccharomyces Boulardii.

Agavins are reserve carbohydrates found in agave plants; they present texture-modifying properties and prebiotic capacity by increasing the viability of the intestinal microbiota. Through its hydrolysis, agave syrup (AS) can be obtained and can be used as a sweetener in food matrices. The objective of this work was to evaluate the effect of the variation in the content of agavins and AS on the physical, structural, and viability properties of Saccharomyces boulardii encapsulates incorporated into gelatin gummies. An RSM was used to obtain an optimized formulation of gelatin gummies. The properties of the gel in the gummy were characterized by a texture profile analysis and Aw. The humidity and sugar content were determined. A sucrose gummy was used as a control for the variable ranges. Alginate microcapsules containing S. boulardii were added to the optimized gummy formulation to obtain a synbiotic gummy. The viability of S. boulardii and changes in the structure of the alginate gel of the microcapsules in the synbiotic gummy were evaluated for 24 days by image digital analysis (IDA). The agavins and agave syrup significantly affected the texture properties (<1 N) and the Aw (>0.85). The IDA showed a change in the gel network and an increase in viability by confocal microscopy from day 18. The number of pores in the gel increased, but their size decreased with an increase in the number of S. boulardii cells. Agavins and cells alter the structure of capsules in gummies without affecting their viability.

Optimizing cellulose fraction for enhanced utility: Comparative pre-treatment of Agave tequilana Weber var. blue bagasse fiber for sustainable applications.

In recent decades, natural fibers have emerged as an alternative to synthetic fibers due to their renewable nature, lower environmental impact, and comparable strength properties. Agave bagasse, a byproduct of agave juice extraction in Mexico, stands out for its potential in various industrial applications, notably biocomposite production. Bagasse is rich in cellulose, along with hemicellulose and lignin. Cellulose is the most suitable to be converted into valuable products, and it is versatile, renewable, and biodegradable. An effective pre-treatment is crucial to enrich its fraction. This study aims to determine the optimal pre-treatment conditions for the agave bagasse. Three different pre-treatments were tested, acid (H2SO4), enzymatic (Cellic® HTec2 enzymatic preparation), and sequence of acid-enzymatic (sulfuric acid and Cellic® HTec2), to determine which pre-treatment got the optimal cellulose fraction increase. The acid pre-treatment was conducted over three time ranges (5, 10, and 15 min) at different acid concentrations (1%, 1.5%, and 2%). Enzymatic reactions were conducted over 24 h, testing three different enzyme concentrations (1.5%, 3%, 4.5%). The sequential pre-treatment utilized the optimal conditions derived from the acid experiments (1.5% H2SO4 for 10 min), followed by enzymatic reactions carried out over three different durations (6, 12, and 24 h). The findings revealed that a 1.5% acid concentration applied for 10 min was the most efficient pre-treatment method. This pre-treatment resulted in a 1.9-fold increase in the cellulose fraction while reducing hemicellulose content by 30%. The hemicellulose reduction was confirmed through Fourier Transform IR spectroscopy (FTIR) analysis, complemented by scanning electron microscopy (SEM) observations highlighting physical alterations in the fiber structure. Furthermore, thermogravimetric analysis (TGA) demonstrated improved thermal stability, suggesting potential use in biocomposites. Future research should evaluate the environmental impact of optimized pre-treatment methods for agave bagasse.

Characterization of the complete chloroplast genome sequence of Agave durangensis (Asparagales: Asparagaceae: Agavoideae).

Agave durangensis commonly known as agave cenizo, is an endemic Agave species in Mexico used for mescal production, yet its taxonomic delimitation is still controversial. This study aimed to enhance taxonomic clarity by characterizing its chloroplast genome. Chloroplast DNA was isolated from 2-year-old A. durangensis leaves. The complete chloroplast genome size was 156,441 bp, comprising a large single-copy region (LSC), a pair of inverted repeat regions (IR), and a small single-copy region (SSC). Annotation revealed 87 protein-coding genes, 38 tRNAs, and 8 rRNAs, with notable gene inversions. Phylogenetic analysis suggests, A. durangensis forms a separate lineage within the Agave genus.

Agave angustifolia Haw. Leaves as a Potential Source of Bioactive Compounds: Extraction Optimization and Extract Characterization.

The leaves of Agave angustifolia Haw. are the main agro-waste generated by the mezcal industry and are becoming an important source of bioactive compounds, such as phenolic compounds, that could be used in the food and pharmaceutical industries. Therefore, the extraction and identification of these phytochemicals would revalorize these leaf by-products. Herein, maceration and supercritical carbon dioxide (scCO2) extractions were optimized to maximize the phenolic and flavonoid contents and the antioxidant capacity of vegetal extracts of A. angustifolia Haw. In the maceration process, the optimal extraction condition was a water-ethanol mixture (63:37% v/v), which yielded a total phenolic and flavonoid content of 27.92 ± 0.90 mg EAG/g DL and 12.85 ± 0.53 µg QE/g DL, respectively, and an antioxidant capacity of 32.67 ± 0.91 (ABTS assay), 17.30 ± 0.36 (DPPH assay), and 13.92 ± 0.78 (FRAP assay) µM TE/g DL. Using supercritical extraction, the optimal conditions for polyphenol recovery were 60 °C, 320 bar, and 10% v/v. It was also observed that lower proportions of cosolvent decreased the polyphenol extraction more than pressure and temperature. In both optimized extracts, a total of 29 glycosylated flavonoid derivatives were identified using LC-ESI-QTof/MS. In addition, another eight novel compounds were identified in the supercritical extracts, showing the efficiency of the cosolvent for recovering new flavonoid derivatives.

Production of activated carbon from agave residues and its synergistic application in a hybrid adsorption-AOPs system for effective removal of sulfamethazine from aqueous solutions.

Tequila production in Mexico generates large quantities of agave bagasse (AB), a waste that could be used more efficiently. AB has a high cellulose, hemicellulose, and lignin content, which allows its use as a precursor for synthesizing carbonaceous materials. In the present work, the synthesis of activated carbon impregnated with Fe2+ (AG-Fe-II) and Fe3+ (AG-Fe-III) was carried out and evaluated in a hybrid adsorption-AOP (advanced oxidation process) methodology for sulfamethazine removal (SMT). The materials were characterized before and after the process to determine their morphological, textural, and physicochemical properties. Subsequently, the effect of the main operational variables (pH, initial SMT concentration, mass, and activator dosage) on the hybrid adsorption-degradation process was studied. The Fenton-like reaction was selected as the AOP for the degradation step, and potassium persulfate (K2S2O8) was used as an activating agent. The main iron crystallographic phases in AG-Fe-II were FeS, with a uniform distribution of iron particles over the material's surface. The main crystallographic phase for AG-Fe-III was Fe3O4. The hybrid process achieved 61% and 78% removal efficiency using AG-Fe-II and AG-Fe-III samples, respectively. The pH and initial SMT concentration were the most critical factors for removing SMT from an aqueous phase. Finally, the material was successfully tested in repeated adsorption-degradation cycles.

Lignocellulosic Membranes Grafted with N-Vinylcaprolactam Using Radiation Chemistry: Load and Release Capacity of Vancomycin.

Radiation chemistry presents a unique avenue for developing innovative polymeric materials with desirable properties, eliminating the need for chemical initiators, which can be potentially detrimental, especially in sensitive sectors like medicine. In this investigation, we employed a radiation-induced graft polymerization process with N-vinylcaprolactam (NVCL) to modify lignocellulosic membranes derived from Agave salmiana, commonly known as maguey. The membranes underwent thorough characterization employing diverse techniques, including contact angle measurement, degree of swelling, scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier-transform infrared-attenuated total reflectance spectroscopy (FTIR-ATR), nuclear magnetic resonance (CP-MAS 13C-NMR), X-ray photoelectron spectroscopy (XPS), and uniaxial tensile mechanical tests. The membranes' ability to load and release an antimicrobial glycopeptide drug was assessed, revealing significant enhancements in both drug loading and sustained release. The grafting of PNVCL contributed to prolonged sustained release by decreasing the drug release rate at temperatures above the LCST. The release profiles were analyzed using the Higuchi, Peppas-Sahlin, and Korsmeyer-Peppas models, suggesting a Fickian transport mechanism as indicated by the Korsmeyer-Peppas model.

Plant Regeneration of Agave cupreata by Somatic Embryogenesis in a Temporary Immersion System with Silver Nanoparticles.

Somatic embryogenesis in Agave genus has been induced; however, it is desirable to increase the rate of growth to get a more efficient propagation system. In this chapter, we present in detailed a protocol for somatic embryogenesis in Agave cupreata and the use of silver nanoparticles in a temporary immersion system. This is an efficient method that can be used commercially to improve the production and germination of somatic embryos.

Draft genomes of four Kluyveromyces marxianus isolates retrieved from the elaboration process of henequen (Agave fourcroydes) mezcal.

We report the draft genomes of four Kluyveromyces marxianus isolates obtained from the elaboration process of henequen (Agave fourcroydes) mezcal, a Mexican alcoholic beverage. The average nucleotide identity analysis revealed that isolates derived from agave plants are distinct from those from other environments, including agave fermentations.

Optimization of cost-effective enzymatic saccharification using low-cost protic ionic liquid as pretreatment agent in Agave bagasse.

This work studied the optimization of enzymatic saccharification of Agave tequilana bagasse (ATB) pretreated with the low-cost protic ionic liquid (PIL) ethanolamine acetate ([EOA][OAc]) using the highly available and cost-effective mixture of the enzymatic cocktails Celluclast 1.5L-Viscozyme L. Response surface methodology (RSM) was employed to maximize the sugars concentration and yield. The RSM optimization conditions of the enzymatic saccharification of pretreated ATB that achieved the maximum reducing sugars (RS) concentration were: 11.50 % w/v solids loading, 4.26 pH with 0.76 and 1.86 mg protein/mL buffer of Viscozyme L and Celluclast 1.5L, respectively. Similarly, the conditions that maximize the sugar yield (SY) were solids loading of 5.62 % w/v, and 4.51 pH as well as 1.07 and 2.03 mg protein/mL buffer of Viscozyme L and Celluclast 1.5L, respectively. Saccharification performance of the first-generation and low-cost enzyme mixture Celluclast 1.5L-Viscozyme L was compared with that reached by a second-generation and higher-cost CTec2, where Celluclast 1.5L-Viscozyme L achieved 60.86 ± 2.66 % y 79.25 ± 3.34 % of the sugars released by CTec2 at the same hydrolysis time (12 h) for the sugar concentration and yield models, respectively. These results are encouraging since they positively contribute to cost reduction and availability issues, which are key parameters to consider when thinking about scaling-up the process.

Yeast diversity in open agave fermentations across Mexico.

Yeasts are a diverse group of fungal microorganisms that are widely used to produce fermented foods and beverages. In Mexico, open fermentations are used to obtain spirits from agave plants. Despite the prevalence of this traditional practice throughout the country, yeasts have only been isolated and studied from a limited number of distilleries. To systematically describe the diversity of yeast species from open agave fermentations, here we generate the YMX-1.0 culture collection by isolating 4524 strains from 68 sites with diverse climatic, geographical, and biological contexts. We used MALDI-TOF mass spectrometry for taxonomic classification and validated a subset of the strains by ITS and D1/D2 sequencing, which also revealed two potential novel species of Saccharomycetales. Overall, the composition of yeast communities was weakly associated with local variables and types of climate, yet a core set of six species was consistently isolated from most producing regions. To explore the intraspecific variation of the yeasts from agave fermentations, we sequenced the genomes of four isolates of the nonconventional yeast Kazachstania humilis. The genomes of these four strains were substantially distinct from a European isolate of the same species, suggesting that they may belong to different populations. Our work contributes to the understanding and conservation of an open fermentation system of great cultural and economic importance, providing a valuable resource to study the biology and genetic diversity of microorganisms living at the interface of natural and human-associated environments.

Antimicrobial activities of Agave fructans against multi-resistant and biofilm-producing Staphylococcus aureus isolated from bovine mastitis.

Bovine mastitis is an emerging disease that causes large economic losses. Staphylococcus aureus its main etiological agent, is multi-resistant to antimicrobials and produces biofilm. The objective of this study was to investigate the effect of Agave fructans (AF), a type of prebiotic, on multi-resistant and biofilm-forming isolates of S. aureus. Ten isolates of S. aureus from bovine subclinical mastitis previously characterized as highly resistant to antimicrobials and biofilm formers were used in this study. The growth kinetics of S. aureus in the presence of AF was evaluated by the Baranyi and Roberts microbial growth model using the DMFit program. The antibacterial activity of AF against S. aureus was studied by the well-diffusion method and the effect on biofilm formation by the crystal violet method. All assays were performed in triplicate for each isolate and an ANOVA with Tukey's post hoc was performed considering p < 0.05 as significant. The AF showed a decrease in maximum growth rate (µmax) and OD max levels (Ymax) in all isolates with all concentrations. Also, zones of inhibition were observed due to the effect of all AF concentrations in all isolates in a dose-dependent manner. Interestingly, S. aureus biofilm formation was inhibited by all AF concentrations assessed in this study. More investigations are required to elucidate the mechanisms of action of AF on S. aureus as well as in vivo studies to evaluate its therapeutic efficacy for bovine mastitis.

How to awaken a sleeping giant: antagonistic expression of Flowering locus T homologs and elements of the age-related pathway are associated with the flowering transition in Agave tequilana.

KEY MESSAGE: Antagonistic expression of Flowering locus T proteins and the ageing pathway via miRNAs and sugar metabolism regulate the initiation of flowering in A. tequilana. Flowering in commercial plantations of Agave tequilana signals that plants are ready to harvest for tequila production. However, time of flowering is often unpredictable and a detailed understanding of the process would be beneficial in the field, for breeding and for the development of future research. This report describes the functional analysis of A. tequilana FLOWERING LOCUS T (FT) genes by heterologous expression in A. thaliana and in situ hybridization in agave plants. The gene structures of the Agave tequilana FT family are also described and putative regulatory promoter elements were identified. Most Agave species have monocarpic, perennial life cycles that can last over 25 years during which plants do not respond to the normal environmental signals which induce flowering, suggesting that the ageing pathway as described in Arabidopsis may play an important role in determining flowering time in these species. Elements of this pathway were analyzed and in silico data is presented that supports the regulation of SQUAMOSA PROMOTER BINDING LIKE proteins (SPL), APETALA2 (AP2) proteins and members of Plant Glycoside Hydrolase Family 32 (PGHF32) by interactions with miRNAs 156, 172 and 164 during the initiation of flowering in A. tequilana.

Pre-contact Agave domesticates - living legacy plants in Arizona's landscape.

BACKGROUND AND SCOPE: Agaves played a central role as multi-use plants providing food, fibre and beverage to pre-contact and historical Mesoamerican cultures. However, their importance to Indigenous Peoples in the Southwest USA and northern Mexico, where they occur because of adaptations such as CAM photosynthesis, is less well known. Archaeological research indicates the Hohokam and other pre-contact Southwestern agrarian people increased agricultural potential in this region by engineering riverine terraces and bajadas for agave dry farming. Agricultural features such as terraces and rock piles were especially characteristic of post-1000 CE with the increase of dense, aggregated populations. We present an overview of six pre-contact agave domesticates (PCADs) the Hohokam and other cultures cultivated, and their ecological and cultural attributes. These PCADs are Agave murpheyi, A. delamateri, A. phillipsiana, A. sanpedroensis, A. verdensis and A. yavapaiensis. CONCLUSION: Pre-contact agriculturists cultivated at least six once cryptic domesticated agave species in the modern Arizona landscape associated with pre-contact agricultural features, such as rock structures. Because of the longevity and primarily asexual reproduction of these agaves, relict clones have persisted to the present day, providing an opportunity to study pre-contact nutrition, trade, migration and agricultural practices. Taxonomic data imply that pre-contact farmers selected desirable attributes, initiating domestication processes that resulted in discrete lineages. These agaves are morphologically and genetically distinct from Southwest US and northern Mexico wild agaves and Mesoamerican wild and domesticated species. Additionally, the remnant clones present a rare opportunity to examine domesticates virtually unchanged since they were last cultivated prehistorically. These discoveries underline the need to view landscapes and some plant species from a cultural, rather than 'natural', perspective and discern potential cryptic species veiled by traditional taxonomic treatments. Protecting and understanding the distribution, and ecological and cultural roles of these plants require interdisciplinary collaboration between botanists, archaeologists, federal agencies and Indigenous Peoples.

Thermoplastic Starch Biocomposite Films Reinforced with Nanocellulose from Agave tequilana Weber var. Azul Bagasse.

In this work, cellulose nanocrystals (CNCs), bleached cellulose nanofibers (bCNFs), and unbleached cellulose nanofibers (ubCNFs) isolated by acid hydrolysis from Agave tequilana Weber var. Azul bagasse, an agro-waste from the tequila industry, were used as reinforcements in a thermoplastic starch matrix to obtain environmentally friendly materials that can substitute contaminant polymers. A robust characterization of starting materials and biocomposites was carried out. Biocomposite mechanical, thermal, and antibacterial properties were evaluated, as well as color, crystallinity, morphology, rugosity, lateral texture, electrical conductivity, chemical identity, solubility, and water vapor permeability. Pulp fibers and nanocelluloses were analyzed via SEM, TEM, and AFM. The water vapor permeability (WVP) decreased by up to 20.69% with the presence of CNCs. The solubility decreases with the presence of CNFs and CNCs. The addition of CNCs and CNFs increased the tensile strength and Young's modulus and decreased the elongation at break. Biocomposites prepared with ubCNF showed the best tensile mechanical properties due to a better adhesion with the matrix. Images of bCNF-based biocomposites demonstrated that bCNFs are good reinforcing agents as the fibers were dispersed within the starch film and embedded within the matrix. Roughness increased with CNF content and decreased with CNC content. Films with CNCs did not show bacterial growth for Staphylococcus aureus and Escherichia coli. This study offers a new theoretical basis since it demonstrates that different proportions of bleached or unbleached nanofibers and nanocrystals can improve the properties of starch films.