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Chitosan (CH) was N-alkylated via Schiff base formation and further reduced via sodium borohydride. The reaction was carried out at room temperature, in a homogeneous aqueous medium, using as a source of alkyl group an essential oil (Eucalyptus staigeriana) containing an unsaturated aldehyde (3,7-dimethylocta-2,6-dienal). Derivatives were characterized by Infrared Spectroscopy, proton and carbon Nuclear Magnetic Resonance, XRD, particle size distribution and zeta potential. Chitosan hydrophobization evidence was given by FTIR as new bands at 2929 cm−1 due to methyl groups, along with the presence of strong band at 1580 cm−1 owing to N substitution. Moreover, carbon and proton NMR corroborated the insertion of methyl groups in chitosan backbone. The degree of substitution was found to be in the range 0.69–1.44. X-ray diffractograms revealed that the insertion of alkyl substituents in chitosan backbone led to a less crystalline material. Data from antibacterial activity revealed that chitosan and derivatives were effective against Gram-positive bacteria, whereby derivatives exhibited greater inhibitory effect than CH. Derivatives are likely candidates for use as carriers for active principles of interest of food, pharmacy and medicine.

There is a critical unmet need to improve microbial safety of fresh fruits and vegetables. Current sanitation approaches cannot achieve >2 log inactivation of bacteria on fresh produce. Thus, there is a need to develop antimicrobial strategies that can consistently achieve >2 logs of bacterial inactivation on the surface of diverse fresh produce. Furthermore it is highly desired that these antimicrobial strategies have reduced environmental impact and are clean label solutions for food products. In this study, we evaluated the combination of curcumin and UV-A light radiation for the inactivation of inoculated E. coli O157:H7 and L. innocua bacterial cells on the surface of spinach, lettuce and tomatoes. Curcumin was deposited on the surface of fresh produce by either aerosolization or conventional spray-atomization methods before exposing the contaminated produce to UV-A light for 10 min (total light fluence of 20.4 kJ m−2). Results showed that the proposed combination of aerosolized or sprayed curcumin and UV-A light radiation can reduce the initial Escherichia coli O157:H7 and Listeria innocua load from 6 log CFU cm−2 to approximately 3 log CFU cm−2 on spinach, lettuce and tomato surfaces. Furthermore, there was no significant difference in bacterial reduction between the different types of inoculated fresh produce surfaces (P > .05). Interestingly, subsequent curcumin deposition and UV-A light exposure cycles were not able to further reduce the bacterial load below the observed threshold of approximately 3 log CFU cm−2. Lastly, the combination of aerosolized curcumin and UV-A light radiation did not affect the color or the texture of the treated fresh produce samples. The findings described in this study illustrate the potential of applying aerosolized or sprayed curcumin under UV-A light illumination to improve microbial safety of fresh produce products.

Sanitation of fresh produce is critical for reducing microbial load and preventing the risk of foodborne illness. This study evaluated an alternative approach for the inactivation of bacteria in wash water and fresh produce using curcumin in combination with UV-A or visible light. This study characterized the inactivation of Escherichia coli O157:H7 and Listeria innocua under different experimental conditions, such as curcumin concentration, time of light exposure, incubation temperature, pH and chemical oxygen demand content (COD). The combination of UV-A light and curcumin was also evaluated to prevent cross-contamination of fresh produce from contaminated wash water during simulated spinach and tomato washing. It was observed that UV-A light in combination with low concentrations of curcumin (1–10 mg L−1) was able to inactivate more than 5 log CFU mL−1 of E. coli O157:H7 and L. innocua in solution regardless of the incubation temperature. In addition, reduced pH increased the antimicrobial activity of curcumin in combination with light against both E. coli O157:H7 and L. innocua. This enhancement in antimicrobial activity reduced the time required for the inactivation of 5 log CFU mL−1 of E. coli O157:H7 from 10 min to 2 min of treatment. Combination of curcumin with UV-A light was more effective than its combination with visible light for achieving the same level of reduction in bacterial plate count. Although the presence of COD in simulated wash water decreased the antimicrobial photo-activity of curcumin, more than 5 log CFU mL−1 of bacteria could still be inactivated in the presence of high COD (1000 mg L−1). Furthermore, the combination of UV-A light and curcumin was able to significantly inhibit bacterial cross-contamination from contaminated wash water to produce surfaces. In summary, the results demonstrate significant potential of curcumin in combination with light for the inactivation of bacteria in food systems including sanitation of wash water and fresh produce.

The high number of foodborne outbreaks in ready-to-eat (RTE) food such as cooked sausages illustrate the importance of controlling microbial safety during post-processing stages of food products. Edible antimicrobial coatings can be applied to food after processing and sanitation with the goal of preventing microbial cross-contamination, decreasing the risk of foodborne illness and increasing the product shelf-life. In this study, edible hydrogel coatings that can present strong antimicrobial activity when combined with UV-A light were prepared. The hydrogels coatings were prepared using either turmeric residue and gelatin hydrogels (TGH) or cassava starch and gelatin hydrogels with added purified curcumin (CGH). The coatings were characterized regarding their thickness, encapsulated curcumin concentration and water swelling, in addition to their light-activated antimicrobial activity against different initial loads of Listeria innocua at different incubation temperature. Additionally, the coatings were applied to the surface of cooked sausages and evaluated for their ability to prevent bacterial cross-contamination. It was observed that UV-A light-exposed hydrogels coatings could inactivate more than 5 log CFU/mL of L. innocua after light treatments as short as 5 min. In addition, the light-activated antimicrobial activity of the hydrogel coatings were not affected by the incubation temperature. Hydrogel-coated sausages exposed to UV-A light experienced a reduction from 4 log CFU/mL of incubated bacteria to levels below the detection limit of 1 log CFU/mL after 5 and 15 min of light exposure, for CGH and TGH, respectively. Further mechanistic studies suggested that L. innocua inactivation was due to the photo-irradiation of low levels of curcumin released from the coatings to solution. Lastly, it was shown that the combination of curcumin-loaded hydrogels coatings and UV-A light have great potential as antimicrobial coatings to prevent cross-contamination of L. innocua in refrigerated sausages.

Possible mechanisms behind the enhanced antimicrobial activity of gallic acid (GA) and its ester propyl gallate (PG) in the presence of UV-A light against Escherichia coli O157:H7 were investigated. GA by itself is a mild antimicrobial and has a pro-oxidant ability. We found that the presence of UV-A light increases the uptake of GA by the bacteria. Once GA is internalized, the interaction between GA and UV-A induces intracellular ROS formation, leading to oxidative damage. Concurrently, GA + UV-A also inhibits the activity of superoxide dismutase (SOD), magnifying the imbalance of redox status of E. coli O157:H7. In addition to ROS induced damage, UV-A light and GA also cause injury to the cell membrane of E. coli O157:H7. UV-A exposed PG caused oxidative damage to the cell and significantly higher damage to the cell membrane than GA + UV-A treatment, explaining its higher effectiveness than GA + UV-A treatment. The findings presented here may be useful in developing new antimicrobial sanitation technologies for food and pharmaceutical industries.

The objective of this study was to investigate synergistic antibacterial activity based on a combination of UV-A light and three classes of food grade compounds: benzoic acid derivatives, cinnamic acid derivatives, and gallates. By using Escherichia coli O157:H7 as the model strain, it was observed that three cinnamic acid derivatives (ferulic acid, coumaric acid, and caffeic acid) and one benzoic acid derivative (2,5-dihydroxybenzoic acid) presented strong synergistic antibacterial activity with UV-A light radiation, where 1 mM levels of these compounds plus with 15 min of UV-A light (total light dose of 6.1 cm−2) led to more than 7-log CFU mL−1 of bacterial inactivation. In contrast, synergistic antibacterial activity between UV-A light and most benzoic acid derivatives (benzoic acid, gallic acid, vanillic acid, and 2,5-dimethoxybenzoic acid) were only observed after higher concentrations of these compounds were applied (10 mM). Lastly, from the three gallates tested (methyl gallate, ethyl gallate, and propyl gallate), only propyl gallate showed strong antibacterial synergism with UV-A light, where 10 mM of propyl gallate plus 15 min of UV-A light led to approximately 6.5-log of bacterial reduction. Presence of antioxidant compounds mitigated the light-mediated antibacterial activity of gallic acid, 2,5-dihydroxybenzoic acid, and propyl gallate. Similarly, the light-mediated antibacterial activity of these compounds was significantly (P

The reduction of microbial load in food and water systems is critical for their safety and shelf life. Conventionally, physical processes such as heat or light are used for the rapid inactivation of microbes, while natural compounds such as lactic acid may be used as preservatives after the initial physical process. This study demonstrates the enhanced and rapid inactivation of bacteria based on a synergistic combination of sublethal levels of stresses induced by UV-A light and two food-grade organic acids. A reduction of 4.7 ± 0.5 log CFU/ml in Escherichia coli O157:H7 was observed using a synergistic combination of UV-A light, gallic acid (GA), and lactic acid (LA), while the individual treatments and the combination of individual organic acids with UV-A light resulted in a reduction of less than 1 log CFU/ml. Enhanced inactivation of bacteria on the surfaces of lettuce and spinach leaves was also observed based on the synergistic combination. Mechanistic investigations suggested that the treatment with a synergistic combination of GA plus LA plus UV-A (GA+LA+UV-A) resulted in significant increases in membrane permeability and intracellular thiol oxidation and affected the metabolic machinery of E. coli . In addition, the antimicrobial activity of the synergistic combination of GA+LA+UV-A was effective only against metabolically active E. coli O157:H7. In summary, this study illustrates the potential of simultaneously using a combination of sublethal concentrations of natural antimicrobials and a low level of physical stress in the form of UV-A light to inactivate bacteria in water and food systems. IMPORTANCE There is a critical unmet need to improve the microbial safety of the food supply, while retaining optimal nutritional and sensory properties of food. Furthermore, there is a need to develop novel technologies that can reduce the impact of food processing operations on energy and water resources. Conventionally, physical processes such as heat and light are used for inactivating microbes in food products, but these processes often significantly reduce the sensory and nutritional properties of food and are highly energy intensive. This study demonstrates that the combination of two natural food-grade antimicrobial agents with a sublethal level of physical stress in the form of UV-A light can greatly increase microbial load inactivation. In addition, this report elucidates the potential mechanisms for this synergistic interaction among physical and chemical stresses. Overall, these results provide a novel approach to develop antimicrobial solutions for food and water systems.

Sustainable control of gastrointestinal nematodes (GIN) in small ruminants has been based on the use of alternative methods, including targeted selective treatment, such as FAMACHA. Another GIN control alternative is the use of herbal medicines, although in many cases their use is based on empirical knowledge. Biopolymer nanoformulations has been investigated to maximize the essential oil effects against sheep gastrointestinal nematodes. The aim of the present study was to combine a Eucalyptus staigeriana essential oil nanoemulsion (EsNano) with FAMACHA as an alternative control for sheep haemonchosis. The study was performed over six months at a commercial sheep farm located in a semiarid region of Northeast Brazil. Initially, a fecal egg count reduction test (FECRT) in sheep with levamisole, ivermectin and oxfendazole in sheep was performed used to determine the most effective anthelmintic to use as the positive control. Levamisole has been selected because it showed efficacy superior to 95%. EsNano was obtained and then its physicochemical properties were characterized. The average (±SE) size of the particles in the nanoemulsion was 276.8 (±12.3) nm with bimodal distribution and polydispersity. Nine visits were performed, from April to September 2013, with an interval of 17 days. One hundred sixty-two male and female sheep were divided into three groups (n=54 each) and were treated when FAMACHA score was 3, 4, or 5: G-EsNano 250mg kg-1 EsNano; G-Lev 7.5mg kg-1 levamisole (positive control), and G-Neg was not treated (negative control). Feces from sheep were collected to quantify the number of eggs per gram of feces (epg) and to identify nematode genera. Sheep weight gain was monitored. The epg data for each group and the average sheep weight gains were analyzed by variance analysis and compared with the Tukey’s test (P0.05). The epg variation was similar in the G-EsNano and G-Lev groups on visits (P>0.05), except the second and fifth evaluation in the epg groups were significantly different (P0,05). A variação opg foi semelhante para os grupos G-EsNano e G-Lev nas vistas (P>0,05), excetuando a segunda e quinta avaliação em que os opg dos grupos foram significativamente diferentes (P>0,05). O nematoide Haemonchus spp. foi o mais prevalente. Não houve aumento significativo de peso em qualquer um dos grupos tratados (P>0,05). Assim, a combinação de fitoterapia e FAMACHA pode ser uma alternativa para minimizar o uso anti-helmínticos sintéticos para controlar populações resistentes NGI em pequenos ruminantes.

This study demonstrated an innovative processing approach based on synergistic antimicrobial activity of two phenolic acids with mild thermal and non-thermal processing technologies. The two selected model phenolic acids were gallic acid (GA; 10 mM) and ferulic acid (FA; 1 mM). The processing technologies evaluated for processing of a model clarified apple juice were UV-A light, mild heat (55 °C) and moderate pressure (250 MPa), with processing times ranging from 1 to 30 min. The results demonstrated that combinations of selected phenolic acids and a mild physical processing were able to lower E. coli O157:H7 and Listeria innocua counts from 6-log CFU mL−1 to below the detection limit of 1-log CFU mL−1. Bacterial inactivation was significantly enhanced by the combination of UV-A light processing and FA, where 10 min of treatment enhanced bacterial inactivation by 5-log as compared to light processing alone, which presented no bacterial inactivation. In contrast, the combination of GA and mild-temperature thermal processing (55 °C) or mild-levels of high-pressure processing (250 MPa), enhanced bacterial inactivation by 4-log as compared to the physical treatments alone, which presented only 1-log of inactivation. The influence of these synergistic combinations on bacterial membrane damage was assessed by selective plating technique under osmotic pressure. Furthermore, the total intracellular thiol content was also measured to assess for thiol oxidation. Overall, the results demonstrated enhanced bacterial inactivation based on synergistic interactions of selected phenolic acids with both mild-thermal and non-thermal technologies in a model food system and illustrate potential to create diversity of novel antimicrobial strategies for food processing. Industrial relevance This study showed that the presence of naturally-based compounds can significantly reduce intensity levels of physical processing required to inactivate model gram-positive and gram-negative bacteria in apple juice. In this study two phenolic acids, ferulic acid and gallic acids were selected as these compounds are naturally present in many fruits and other food products including grains. Furthermore, the levels of these natural phenolic acids used in this study are comparable to the levels naturally present in some of the food materials.

The anthelmintic activity of Eucalyptus staigeriana essential oil has previously been inferred through both in vitro and in vivo tests. Thus, the encapsulation process generally improves oil stability, promotes controlled release in target organs, reduces dosage, and increases efficacy. The aims of this study were to analyze and encapsulate E. staigeriana essential oil and to verify its anthelmintic activity in sheep. The encapsulation process was accomplished through emulsion using a 4 % chitosan solution as the matrix. Anthelmintic activity was established through controlled testing using 18 sheep that were separated into three groups: group 1 was treated with a single dose of 365 mg/kg of E. staigeriana encapsulated oil, group 2 was treated with 200 μg/kg of ivermectin, and group 3 was treated with a 4 % chitosan solution as a negative control. The sheep were euthanized and necropsied 13 days posttreatment to evaluate worm burden. Limonene was the major oil component (72.91 %). The final product was a hydrogel with 36.5 % (m/m) E. staigeriana essential oil per gram. Its efficacy on gastrointestinal nematodes was 60.79 %. The highest efficacy was against abomasal nematodes, with 83.75 % efficacy. Further studies are necessary to explore the possibility of increasing the hydrogel efficacy; nevertheless, we can state that E. staigeriana encapsulated oil had anthelmintic activity and can be used in gastrointestinal nematode control.

A polymeric floating system composed of Alginate (ALG) and Cashew gum (CG), loaded with an essential oil (Lippia sidoides-Ls) was prepared by ionotropic gelation, characterized regarding its physical-chemistry properties and evaluated on its potential as a controlled release system. The influence of process parameters on the buoyancy, loading, swelling and in vitro and in vivo release kinetics, was investigated. Results showed that beads produced with carbonate and Ls at high level contents exhibit good floatability (up to 5 days) and loading capacity (15.2-23.8%). In vitro release data showed a Fickian diffusion profile and in vivo experiments showed that ALG-CG floating system presented a superior and prolonged larvicide effect, in comparison with non-floating ones, presenting larvae mortality values of 85% and 33%, respectively, after 48 h. These results indicate that ALG-CG floating beads loaded with Ls presented enhanced oil entrapment efficiency, excellent floating ability, and suitable larvicide release pattern.

Alginate/cashew gum nanoparticles were prepared via spray-drying, aiming at the development of a biopolymer blend for encapsulation of an essential oil. Nanoparticles were characterized regarding to their hydrodynamic volume, surface charge, Lippia sidoides essential oil content and release profile, in addition to being analyzed by infrared spectroscopy (FT-IR), thermal analysis (TGA/DSC) and X-ray diffractometry. Nanoparticles in solution were found to have averaged sizes in the range 223–399 nm, and zeta potential values ranging from −30 to −36 mV. Encapsulated oil levels varied from 1.9 to 4.4% with an encapsulation efficiency of up to 55%. The in vitro release profile showed that between 45 and 95% of oil was released within 30–50 h. Kinetic studies revealed that release pattern follow a Korsmeyer–Peppas mechanism.

Nanogels based on chitosan and cashew gum were prepared and loaded with Lippia sidoides oil. Several parameters such as cashew gum concentration and relative oil content in the matrix had their influence on nanogel properties investigated. Nanogels were characterized regarding their morphologies, particle size distributions, zeta potential, Fourier transform infrared spectroscopy and essential oil contents. The release profile was investigated by UV/vis spectroscopy and its efficacy was determined through bioassays. Results showed that samples designed using relative ratios matrix:oil 10:2, gum:chitosan 1:1 and 5% gum concentration showed high loading (11.8%) and encapsulation efficiency (70%). Nanogels were found to exhibit average sizes in the range 335–558nm. In vitro release profiles showed that nanoparticles presented slower and sustained release. Bioassays showed that larval mortality was related mainly to oil loading, with samples presenting more effective larvicide efficacies than the pure L. sidoides oil.

Esferas de alginato (ALG) reticuladas com cálcio e revestidas com goma do cajueiro (GC) foram preparadas e dopadas com óleo essencial de Croton zehntneri Pax et Hoffm (Cz), visando a seu emprego como larvicida para combate ao vetor da dengue. As esferas foram caracterizadas estruturalmente por espectroscopia de infravermelho, análise térmica, microscopia eletrônica de varredura e em relação aos seus parâmetros de transporte líquido, dopagem, embebição (Q) e cinética de liberação in vitro e in vivo. Os resultados revelaram que as esferas possuem morfologia com uma alta regularidade esférica, com superfície porosa. A matriz ALG:Cz =1:1 apresentou maior dopagem e eficiência de encapsulamento e valores de Q entre 10 e 12. O perfil de liberação do óleo encapsulado apresentou-se mais prolongado nas esferas contendo goma do cajueiro, ALG-GC:Cz =1:1. A taxa de mortalidade de larvas de Stegomyia aegypti para teores de 1,81 a 4,25 mg de óleo nas esferas resultou em valores na faixa de 72 a 100%, após 24 h. A matriz polimérica mostrou-se efetiva para proteção do principio ativo até cerca de 70 dias, com perdas da ordem de 35%. A análise térmica revelou que a incorporação de Cz e GC resulta em uma matriz polimérica termicamente mais estável. Alginate beads (ALG) crosslinked with calcium and coated with cashew gum (GC) were prepared and loaded with an essential oil from Croton zehntneri Pax et Hoffm (Cz). The beads were characterized by FTIR, thermal properties, with respect to their size, shape, larvicide loading, swelling, and in vitro and in vivo release kinetics. Results showed that beads with ALG:Cz =1:1 relative ratio showed higher loading and encapsulation efficiency, although presenting a faster release kinetics. On the other hand, ALG beads coated with GC showed Q values from 10 to 12, representing a significant increase in the hydrophilic character of the matrix. Infrared spectroscopy and thermal analysis confirmed the presence of essential oil encapsulated in beads. Morphologic investigation by SEM revealed a highly spherical and porous surface. The in vitro profile showed that the Cz oil encapsulated had a prolonged release in ALG-GC beads, probably due to the crosslinking. In vivo tests showed that the mortality degree was related to the mass of the beads, where beads having oil content from 1.81 to 4.25 mg caused larvae mortalities from 72 to 100% after 24 h. The control release system was found to be effective up to 70 days. These results showed that ALG-GC beads loaded with Cz are promising candidate for the control of Stegomyia aegypti larvae.

Phytotherapy has been described as an alternative method for the control of gastrointestinal nematodes in small ruminants. Goal of the encapsulation of essential oils in biopolymer matrices is to optimize the biological effects of these oils. The aim of the present study was to evaluate the in vitro and in vivo anthelmintic activity of encapsulated Eucalyptus staigeriana essential oil (EncEs) on the eggs and larvae of Haemonchus contortus. Therefore, the egg hatching test (EHT), larval development test (LDT) and worm load evaluation were performed to evaluate Meriones unguiculatus experimentally infected with H. contortus. The chemical constituents of E. staigeriana essential oil (EsEO) and the in vitro oil release profile from the chitosan matrix at a pH of 1.2 and 7.0 were also characterized. EncEs and EsEO inhibited larval hatching by 97.19% and 99.96% at doses of 1.5 and 1.0 mg ml(-1), respectively. In the LDT, EncEs and EsEO induced a larvicidal effect greater than 95% at concentrations of 5.8 and 8 mg ml(-1), respectively. EncEs and EsEO decreased H. contortus load in M. unguiculatus by 40.51% and 46.44%, respectively. The major chemical constituents of EsEO were (+)-Limonene (72.9%), 1,8-Cineole (9.5%) and o-Cimene (4.6%). The release profile of EsEO was 30% in acid and 25% at neutral pH. The similar efficacy of EncEs and EsEO demonstrates that there was no optimization of anthelmintic action following the encapsulation process. Therefore, the use of new encapsulation matrices with controlled release in the pH of the abomasum should be investigated.

The anthelmintic resistance stimulated the search for strategies for controlling gastrointestinal nematodes, including the use of free essential oils or its nanoemulsion. This study evaluated the anthelmintic efficacy of Cymbopogon citratus essential oil (CcEO) and C. citratus essential oil nanoemulsion (CcEOn). Pysicochemical analyses were performed. The in vitro effect was determined using the egg hatch test (EHT) on Haemonchus contortus and in vivo effect was evaluated in sheep infected with gastrointestinal nematodes. The animals were treated with CcEO (500 mg/kg) or CcEOn (450 mg/kg) for the fecal egg count (FEC) and the determination of worm burden. The main component of CcEO was citral. The CcEO content in the nanoemulsion was 20% (v/v), and the mean particle size was 248 nm. In EHT, CcEO and CcEOn (1.25 mg/mL) inhibited larval hatching by 98.4 and 97.1%, respectively. Three animals treated with CcEO died whereas in the group treated with CcEOn one animal died. The FEC and total worm burden of the treated groups did not differ from the negative control (p>0.05). The CcEOn showed efficacy only on H. contortus (p