Plant Bioactive Compounds in Foods and Food Packages.

There has been growing interest in the use of numerous plant bioactive compounds (PBCs) in food and nutrition technology due to their properties that promote human health by reducing the risk of various serious diseases [...].

Applications of Plant Bioactive Compounds as Replacers of Synthetic Additives in the Food Industry.

According to the Codex Alimentarius, a food additive is any substance that is incorporated into a food solely for technological or organoleptic purposes during the production of that food. Food additives can be of synthetic or natural origin. Several scientific evidence (in vitro studies and epidemiological studies like the controversial Southampton study published in 2007) have pointed out that several synthetic additives may lead to health issues for consumers. In that sense, the actual consumer searches for "Clean Label" foods with ingredient lists clean of coded additives, which are rejected by the actual consumer, highlighting the need to distinguish synthetic and natural codded additives from the ingredient lists. However, this natural approach must focus on an integrated vision of the replacement of chemical substances from the food ingredients, food contact materials (packaging), and their application on the final product. Hence, natural plant alternatives are hereby presented, analyzing their potential success in replacing common synthetic emulsifiers, colorants, flavorings, inhibitors of quality-degrading enzymes, antimicrobials, and antioxidants. In addition, the need for a complete absence of chemical additive migration to the food is approached through the use of plant-origin bioactive compounds (e.g., plant essential oils) incorporated in active packaging.

Enzymatic oxidation of oleuropein and 3-hydroxytyrosol by laccase, peroxidase, and tyrosinase.

The oxidation of oleuropein and 3-hydroxytyrosol by oxidases laccase, tyrosinase, and peroxidase has been studied. The use of a spectrophotometric method and another spectrophotometric chronometric method has made it possible to determine the kinetic parameters Vmax and KM for each enzyme. The highest binding affinity was shown by laccase. The antioxidant capacities of these two molecules have been characterized, finding a very similar primary antioxidant capacity between them. Docking studies revealed the optimal binding position, which was the same for the two molecules and was a catalytically active position. PRACTICAL APPLICATIONS: One of the biggest environmental problems in the food industry comes from olive oil mill wastewater with a quantity of approximately 30 million tons per year worldwide. In addition, olive pomace, the solid residue obtained from the olive oil production, is rich in hydroxytyrosol and oleuropein and the action of enzymatic oxidases can give rise to products in their reactions that can lead to polymerization. This polymerization can have beneficial effects because it can increase the antioxidant capacity with potential application on new functional foods or as feed ingredients. Tyrosinase, peroxidase, and laccase are the enzymes degrading these important polyphenols. The application of a spectrophotometric method for laccase and a chronometric method, for tyrosinase and peroxidase, allowed us to obtain the kinetic information of their reactions on hydroxytyrosol and oleuropein. The kinetic information obtained could advance in the understanding of the mechanism of these important industrial enzymes.

Synergistic Antimicrobial Activities of Combinations of Vanillin and Essential Oils of Cinnamon Bark, Cinnamon Leaves, and Cloves.

Plant bioactive compounds have antimicrobial and antioxidant activities that allow them to be used as a substitute for synthetic chemical additives in both food and food packaging. To improve its sensory and bactericidal effects, its use in the form of effective combinations has emerged as an interesting possibility in the food industry. In this study, the antimicrobial activities of essential oils (EOs) of cinnamon bark, cinnamon leaves, and clove and the pure compounds vanillin, eugenol, and cinnamaldehyde were investigated individually and in combination against Listeria monocytogenes and Escherichia coli O157:H7. The possible interactions of combinations of pure compounds and EOs were performed by the two-dimensional checkerboard assay and isobologram methods. Vanillin exhibited the lowest antimicrobial activity (MIC of 3002 ppm against L. monocytogenes and 2795 ppm against E. coli O157:H7), while clove and cinnamon bark EOs exhibited the highest antimicrobial activity (402-404 against L. monocytogenes and 778-721 against E. coli O157:H7). For L. monocytogenes, pure compound eugenol, the main component of cinnamon leaves and clove, showed lower antimicrobial activity than EOs, which was attributed to the influence of the minor components of the EOs. The same was observed with cinnamaldehyde, the main component of cinnamon bark EO. The combinations of vanillin/clove EO and vanillin/cinnamon bark EO showed the most synergistic antimicrobial effect. The combination of the EOs of cinnamon bark/clove and cinnamon bark/cinnamon leaves showed additive effect against L. monocytogenes but indifferent effect against E. coli O157:H7. For L. monocytogenes, the best inhibitory effects were achieved by cinnamon bark EO (85 ppm)/vanillin (910 ppm) and clove EO (121 ppm)/vanillin (691 ppm) combinations. For E. coli, the inhibitory effects of clove EO (104 ppm)/vanillin (1006 ppm) and cinnamon leaves EO (118 ppm)/vanillin (979 ppm) combinations were noteworthy. Some of the tested combinations increased the antimicrobial effect and would allow the effective doses to be reduced, thereby offering possible new applications for food and active food packaging.

Packaging of Fresh Sliced Mushrooms with Essential Oils Vapours: A New Technology for Maintaining Quality and Extending Shelf Life.

The use of vapour of essential oils (EOs) through an innovative pilot-plant packaging device was studied to preserve the quality of sliced mushrooms during storage. A mix of EOs (eugenol, bergamot EO, and grapefruit EO) was vaporized (100 and 125 µL L-1) and applied during packaging of sliced mushrooms under modified atmosphere packaging (MAP); then, the product quality was studied during cold storage up to 12 days. The highest colour changes of EOs125 samples, which were observed in the mushroom stipe, were not observed with the EOs100 atmosphere. Thus, the high polyphenoloxidase activity observed in untreated samples after 5-7 days was highly controlled with the vapour EOs atmospheres. Furthermore, the visual appearance scores of EOs100 samples were still over the limit of usability, while untreated samples were already below this threshold after 5 days of storage. A strong bacteriostatic effect was achieved with vapour EOs, reducing the Pseudomonas spp. (the main microbial genus in cultivated mushrooms) growth by ≈1.7 log CFU g-1, regardless of the EOs dose, after 12 days. The activity of phenyl ammonia lyase was also reduced up to ≈0.4 enzymatic units with the EOs100 treatment. Conclusively, packaging of sliced mushrooms under an atmosphere enriched with 100 µL L-1 EOs vapour highly controlled the quality loss of sliced mushrooms owing to their enzymatic inhibition and high bacteriostatic effect.

Antioxidant and Antimicrobial Effect of Plant Essential Oils and Sambucus nigra Extract in Salmon Burgers.

The antioxidant capacity of oregano (OEO) and clove (CLEO) essential oils and black elderberry (Sambucus nigra) flower extract (SNE) were compared with butylhydroxytoluene (BHT) regarding its protection against lipid peroxidation and microbial counts in salmon burgers stored at 4 °C for 14 days and after cooking. The content of total phenols was 5.74% in OEO, 2.64% in CLEO and 2.67 % in the SNE. The total phenolic content and the antioxidant capacity were significantly higher (p < 0.05) for SNE and OEO. Both essential oils showed a similar IC50 and inhibition percentage of lipid peroxidation to BHT. The combination of OEO and SNE reduced 29% of thiobarbituric acid reactive substances (TBARS), while BHT reduced 31% of TBARS generated during refrigeration storage in salmon burgers in relation to the control sample without antioxidants. Additionally, the microbial counts after 14 days of refrigeration were the lowest in burgers when the combination of OEO and SNE was used. This study concludes that OEO and SNE can be used as inhibitors of lipid oxidation in salmon products and as natural candidates to replace commonly used synthetic antioxidants and antimicrobials in these food products.

The Application of Essential Oil Vapors at the End of Vacuum Cooling of Fresh Culinary Herbs Promotes Aromatic Recovery.

Aroma is an important quality parameter of fresh culinary herbs that may be highly affected after postharvest treatments. The innovative technology of vapor essential oil (EO) application under vacuum conditions may recover aroma lost during the postharvest processing of plant products like aromatic herbs. Hence, this study assessed the aroma recovery effect of vapor EOs applied during vacuum cooling on curly parsley and dill. The volatile organic compounds (VOCs) profiles of these aromatic herbs were studied by static headspace solid-phase microextraction (SPME), and the VOCs sorption kinetics onto the SPME stir-bar coating were modeled by the Baranyi model. At the pilot plant scale, the total VOCs contents of parsley and dill (whose extractability was increased by 10-20% after a single vacuum process) were enhanced by 4.5- and 2-fold, respectively, when vapor EOs were applied. In particular, 1,3,8-p-menthatriene and carvone (parsley) increased by 18.7- and 7.3-fold, respectively, while dill ether (the characteristic VOC of dill) augmented by 2.4-fold after vapor EOs were applied under vacuum conditions. The aroma recovery of culinary herbs was successfully validated at an industrial level in an installation developed by our group to apply vapor EOs within a vacuum cooling system, reaching total VOC recoveries of 4.9- and 2.3-fold in parsley and dill, respectively.

Discrimination of major and minor streptococci incriminated in bovine mastitis by MALDI-TOF MS fingerprinting and 16S rRNA gene sequencing.

The current work investigated the discriminatory potential of MALDI-TOF MS fingerprinting towards most-relevant major (Streptococcus agalactiae, S. dysgalactiae, S. uberis) and minor (S. canis, S. parauberis, S. salivarius, S. equinus and S. gallolyticus) streptococci involved in bovine mastitis (BM), in comparison to 16S rRNA gene sequencing (GS)-based identification. The MALDI-TOF MS-generated spectral fingerprints were recruited for eliciting a detailed proteomic map that demonstrated clear variability for inter- and intra-species-specific biomarkers. Besides, a phyloproteomic dendrogram was evolved and comparatively analyzed against the phylogenetic one obtained from 16S rRNA GS in order to assess the differentiation of streptococci of bovine origin based on variability of protein fingerprints versus the variation of 16S rRNA gene homology. Results showed that the discrimination of BM-implicated streptococci can be obtained by both approaches; however MALDI-TOF MS was superior, achieving more variability at both intra- and sub-species levels. MALDI-TOF MS spectral analytics revealed that Streptococcus spp. exhibited three genus-specific biomarkers (peaks with m/z values at 2112, 4452 and 5955) and all streptococci exhibited spectral variability at both species and subspecies levels. Remarkably, MALDI-TOF MS fingerprinting was found to be at least as robust as 16S rRNA GS-based identification, allowing much cheaper and faster analysis, and additionally exhibiting high reliability for characterization of BM-implicated streptococci, thus proving to be a powerful tool that can be used independently within dairy diagnostics.

Kinetic characterization of the oxidation of catecolamines and related compounds by laccase.

The pathways of melanization and sclerotization of the cuticle in insects are carried out by the action of laccases on dopamine and related compounds. In this work, the laccase action of Trametes versicolor (TvL) on catecholamines and related compounds has been kinetically characterized. Among them, dopamine, l-dopa, l-epinephrine, l-norepinephrine, dl-isoprenaline, l-isoprenaline, dl-α-methyldopa, l-α-methyldopa and l-dopa methylester. A chronometric method has been used, which is based on measuring the lag period necessary to consume a small amount of ascorbic acid, added to the reaction medium. The use of TvL has allowed docking studies of these molecules to be carried out at the active site of this enzyme. The hydrogen bridge interaction between the hydroxyl oxygen at C-4 with His-458, and with the acid group of Asp-206, would make it possible to transfer the electron to the T1 Cu-(II) copper centre of the enzyme. Furthermore, Phe-265 would facilitate the adaptation of the substrate to the enzyme through Π-Π interactions. To kinetically characterize these compounds, we need to take into consideration that, excluding l-dopa, l-α-methyldopa and dl-α-methyldopa, all compounds are in hydrochloride form. Because of this, first we need to kinetically characterize the inhibition by chloride and, after that, calculate the kinetic parameters KM and VmaxS. From the kinetic data obtained, it appears that the best substrate is dopamine. The presence of an isopropyl group bound to nitrogen (isoprenaline) makes it especially difficult to catalyse. The formation of the ester (l-dopa methyl ester) practically does not affect catalysis. The addition of a methyl group (α-methyl dopa) increases the rate but decreases the affinity for catalysis. l-Epinephrine and l-norepinephrine have an affinity similar to isoprenaline, but faster catalysis, probably due to the greater nucleophilic power of their phenolic hydroxyl.

Development of a method to measure laccase activity on methoxyphenolic food ingredients and isomers.

We studied the laccase-catalysed oxygenation of methoxyphenolic food ingredients, such as 2-methoxyphenol (guaiacol) and 2,6-dimethoxyphenol (syringol), isomers such as 3- and 4-methoxyphenol, and 2,3-, 3,4- and 3,5-dimethoxyphenol. These methoxyphenolic substrates generate unstable free radicals, which leads to the erroneous determination of steady state rates. The addition of small quantities of ascorbic acid as coupling reagent generates a lag period because it reduces free radicals to methoxyphenols. Measurement of the length of the lag period provides the reliable determination of true steady state rates. We describe the application of this chronometric method to the kinetic characterization of the oxidation of the above methoxyphenolic substrates by Trametes versicolor laccase.

Combined use of thermo-ultrasound and cinnamon leaf essential oil to inactivate Saccharomyces cerevisiae in culture broth and natural orange juice.

The survival of Sacharomyces cerevisiae in Trypticase Soy Broth and natural orange juice processed by combined use of thermo-ultrasound and cinnamon leaf essential oil has been evaluated and modelled. Minimal inhibitory concentration of cinnamon leaf essential oil against S. cerevisiae was determined using absorbance measurements based on the microtiter plate assay. The resistance of S. cerevisiae cells to the combined action of thermal treatment with ultrasound was analyzed in Trypticase Soy Broth with different concentrations of cinnamon leaf essential oil at 30, 40 and 50 °C. The best conditions of inactivation in TSB to study the inactivation of S. cerevisiae in natural orange juice. Experimental data were fitted by using the "shoulder + log-linear" and "Weibull" models (GInaFiT). The combined use of thermo-ultrasound and cinnamon leaf essential oil enhanced the inactivation of S. cerevisiae in TSB and natural orange juice.

Control of Native Spoilage Yeast on Dealcoholized Red Wine by Preservatives Alone and in Binary Mixtures.

In order to preserve a commercial dealcoholized red wine (DRW), a study with 4 preservatives and binary mixtures of them were performed against 2 native spoilage yeasts: Rhodotorula mucilaginosa and Saccharomyces cerevisiae. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for potassium sorbate, sodium benzoate, sodium metabisulfite and dimethyl dicarbonate (DMDC) were evaluated in DRW stored at 25 °C. MICs of potassium sorbate and sodium metabisulfite were 250 and 60 mg/kg, respectively for both target strains. However for sodium benzoate, differences between yeasts were found; R. mucilaginosa was inhibited at 125 mg/kg, while S. cerevisiae at 250 mg/kg. Regarding MFC, differences between strains were only found for sodium metabisulfite obtaining a MFC of 500 mg/kg for R. mucilaginosa and a MFC of 250 mg/kg for S. cerevisiae. Potassium sorbate and sodium benzoate showed the MFC at 1000 mg/kg and DMDC at 200 mg/kg. Regarding the effect of binary mixtures the Fractional Fungicidal Concentration Index (FFCi ) methodology showed that binary mixtures of 100 mg/kg DMDC/200 mg/kg potassium sorbate (FFCi = 0.7) and 50 mg/kg DMDC / 400 mg/kg sodium benzoate (FFCi = 0.65) have both synergistic effect against the 2 target strains. These binary mixtures can control the growth of spoilage yeasts in DRW without metabisulfite addition. The results of this work may be important in preserving the health of DRW consumers by eliminating the use of metabisulfite and reducing the risk of growth of R. mucilagosa, recently recognized as an emerging pathogen.

Dealcoholized wines by spinning cone column distillation: phenolic compounds and antioxidant activity measured by the 1,1-diphenyl-2-picrylhydrazyl method.

Spinning cone column (SCC) distillation has been shown to be a commercially suitable technique for dealcoholized wine (DW) manufacturing, but there are not enough studies about its influence on the DW quality. So, the effect of this technique on the antioxidant activity (% of remaining 1,1-diphenyl-2-picrylhydrazyl radical) and the phenolic compound composition of red, rose, and white DW, obtained at pilot plant scale, has been analyzed. Nineteen raw wines (RWs) from different grape varieties and five different Spanish viticultural regions have been studied before and after dealcoholization. The total phenolic content, flavonols, tartaric esters, and anthocyanins, was determined by spectrophotometry, while the content of phenolic compounds such as stilbenes (trans- and cis-resveratrol), flavonols (rutin, quercetin, and myricetin), flavan-3-ols [(+)-catechin and (-)-epicatechin], anthocyanins (malvidin 3-glucoside), and non-flavonoids (gallic, caffeic, and p-coumaric acids) was determined by high-performance liquid chromatography (HPLC). The resveratrol contents in red wines were between 1.81 and 34.01 mg/L in RWs and between 2.12 and 39.57 mg/L in DWs, Merlot being the grape producing the RWs and DWs with higher resveratrol content. In general, the percent of remaining DPPH(*) was similar or slightly higher (until 5 units of % of remaining DPPH(*)) in DWs versus RWs. This small difference may be due to removal of SO2 (that is an antioxidant) from RWs during distillation. DWs and RWs show similar contents of the studied phenolic compounds, with a tendency, in some cases, to exhibit increases after dealcoholization, caused by the concentration effect via removal of the ethanol. From this work, we can deduce that SCC distillation is a dealcoholization technique minimally destructive with the wine phenolic compounds.