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- The progress of essential oils as potential therapeutic agents: a reviewPublication . Baptista-Silva, Sara; Borges, Sandra; Ramos, Oscar. L.; Pintado, Manuela; Sarmento, BrunoEssential oils are products obtained from plants, by steam distillation, mechanical processes of citrus fruit epicarp, or dry distillation after separation of the aqueous phase by physical processes. They are usually composed of secondary metabolites of aromatic plants with oxygenated structures such as alcohols, ketones, aldehydes, and esters, presenting therapeutic properties such as antibacterial, antifungal and antioxidant activities. Essential oils are used in the pharmaceutical, food, and fragrance industries. The increasing use of plants by the pharmaceutical industry makes the study of essential oils crucial to design new bioactive delivery systems. This paper presents aliterature review that summarizes the best advanced data regarding the use of essential oils and their volatile constituents for biomedical applications with focuses on innovative pharmaceutical formulations. Nonetheless, it seems clear that more clinical evaluations are required until essential oils can be considered as possible applications in pharmacy or as adjuvants to current medications.
- Cytotoxicity and genotoxicity of chitooligosaccharides upon lymphocytesPublication . Fernandes, João C.; Borges, Margarida; Nascimento, Henrique; Bronze-da-Rocha, Elsa; Ramos, Oscar S.; Pintado, Manuela E.; Malcata, F. Xavier; Santos-Silva, AliceTwo COS mixtures and a low molecular weight chitosan (LMWC) were tested for potential cytotoxicity and genotoxicity upon human lymphocytes. Genotoxicity was evaluated in vitro by cytokinesis-blocked micronucleus and alkaline comet assays, while cytotoxicity was assessed by flow cytometry analysis. Our results suggest that COS do not exhibit any genotoxicity upon human lymphocytes, independently of MW or concentration. However, above 0.07 mg/mL COS induced strong cytotoxic effects. According to the concentration used, such cytotoxicity will induce cell death, essentially by necrosis (>0.10 mg/mL) and/or apoptosis (<0.10 mg/mL). The level of necrosis/apoptosis induced by high COS concentrations, suggests a promising use as apoptosis inducers in specific cancer situations.
- Features and performance of edible films, obtained from whey protein isolate formulated with antimicrobial compoundsPublication . Ramos, Óscar L.; Silva, Sara I.; Soares, José C.; Fernandes, João C.; Poças, M. Fátima; Pintado, Manuela E.; Malcata, F. XavierThe goal of this research effort was to assess the efficacy of edible films produced fromwhey protein isolate (WPI) and glycerol, including incorporation of lactic acid (LA) and propionic acid (PRO), chitooligosaccharideswith nominal MW of 3 kDa (COS) and natamycin (NA) as antimicrobial agents. Their features were evaluated in vitro via agar diffusion and viable cell counting, against spoilage microflora often found contaminating cheese surfaces. The effect of incorporating the aforementioned compounds upon thickness, moisture content (MC), solubility (S), density (ρs),water activity (aw) and water vapor permeability (WVP), as well as upon tensile and optical properties of those films were also evaluated. Films formulated with LA, PRO or COS exhibited antimicrobial activity against all microorganisms tested, yet the viable cell count assay was more sensitive and reproducible. COS was the most active against Gram-negative bacteria, whereas LA was the most active against Gram-positive ones. NA was not active against bacteria, but displayed the strongest effect against yeasts. Incorporation of said antimicrobial compounds did not significantly (pN0.05) affect film thickness, yet it significantly (pb0.05) reduced tensile strength (TS). Incorporation of LA and NA in particular did not significantly (pb0.05) affect MC, S, ρs, WVP, elongation at break (EB) and Young's modulus (YM) values; however, a statistically significant increase (pb0.05) of MC, S andWVP, together with a statistically significant decrease (pb0.05) of ρs were attained upon incorporation of PRO or COS. Moreover, PRO produced the highest variation (pb0.05) in EB, TS and YM,whereas COS produced the highest change (pb0.05) in optical properties.
- Antimicrobial effects of chitosans and chitooligosaccharides, upon Staphylococcus aureus and Escherichia coli, in food model systemsPublication . Fernandes, João C.; Tavaria, Freni K.; Soares, José C.; Ramos, Óscar S.; Monteiro, M. João; Pintado, Manuela E.; Malcata, F. XavierThe objective of this study was to elucidate the controversial relationship between the molecular weight (MW) of chitosans and their antibacterial activity (upon different inoculum levels, at several concentrations). The influence of food components on the activity was also ascertained, as well as acceptance by a sensory panel. All the compounds tested exhibited antibacterial activity against Staphylococcus aureus and Escherichia coli. This activity was shown to be closely dependent on the inoculum level, MW and concentration used. Within 4 h at 10³ cells/mL, all five compounds, at every concentration (0.5%, 0.25% and 0.1%, w/v), proved to be bactericidal; for higher inocula, 0.1% (w/v) was only bacteriostatic; at 107 or 105 cells/mL, and independently of the inoculum level, 0.25% (w/v) of any chitooligosaccharide (COS) mixture was sufficient to reduce the E. coli initial population by at least 3 log cycles; COS never exhibited bactericidal action over S. aureus, unlike high and medium MW chitosans—which, at 0.5% (w/v), presented a bactericidal effect even against 107 cells/mL. When incorporated in liquid food matrices, medium and high MW chitosans maintained their activity, for both matrices and bacteria, although a slower activity was noticeable in milk; however, COS lost their activity upon both bacteria in milk after 4–8 h. Furthermore, addition of chitosans to apple juice led to several unpleasant off-flavors, such as astringency and after taste—which increased in magnitude with MW.
- Active whey protein edible films and coatings incorporating lactic acid bacteria for fungi control in cheesePublication . Guimarães, Ana; Ramos, Oscar. L.; Cerqueira, Miguel; Venâncio, Armando; Abrunhosa, Luís
- Nanostructures of whey proteins for encapsulation of food ingredientsPublication . Ramos, Oscar L.; Pereira, Ricardo N.; Simões, Lívia S.; Madalena, Daniel A.; Rodrigues, Rui M.; Teixeira, José A.; Vicente, António A.The most current and high-level research is being taken on the use of nanoscience and nanotechnology due to its varied application in numerous fields of science. Food nanotechnology, and in particular, the development and application of bio-based nanostructures are an emerging area having a high potential to engender new products and processes in the food industry. This chapter intends to discuss whey protein-based nanostructured systems (i.e., whey protein isolate, whey protein concentrate, β-lactoglobulin, and α-lactalbumin) for encapsulation of food ingredients. These protein nanostructures have unique properties, such as a high nutritional value, GRAS nature, gelling capability, and can be easily prepared and controlled. They have also the ability to conjugate a large variety of food ingredients (e.g., antioxidants, vitamins, minerals, flavors, and odors) via amino groups or ionic and hydrophobic interactions. This behavior will prevent the degradation of sensitive bioactives, while permitting a site-specific action and controlled delivery rate due to the swelling behavior of the gel in reaction to external and physical stimuli such as temperature, enzymes, pH, or ionic strength), thus contributing to an improved bioavailability of such ingredients. The potential of whey protein nanostructures for encapsulation and controlled delivery of food ingredients will be addressed in a critical manner in this chapter. Moreover, various techniques used for their nanoencapsulation and evaluation of their stability during storage will also be discussed. The behavior and bioavailability of whey nanostructures and their associated/encapsulated food ingredients will be discussed using insights from in vitro and in vivo gastrointestinal systems together with potential cytotoxicity, cellular uptake, and allergenicity via in vitro cell lines. Finally, examples of such nanostructures applied in food matrices will be described, as well as the main challenges for their commercial use.
- Effect of composition of commercial whey protein preparations upon gelation at various pH valuesPublication . Ramos, Óscar S.; Pereira, Joana O.; Silva, Sara I.; Amorim, Maria M.; Fernandes, João C.; Lopes-da-Silva, José A.; Pintado, Manuela E.; Malcata, F. XavierThe major goal of this research effort was to comprehensively characterize various whey protein products available in the market — including one whey protein isolate (WPI) and three whey protein concentrates (two forms of WPC 80, and WPC 50), with regard to the effects of specific components (e.g. lecithin and minerals) and concentration of β-lactoglobulin (β-Lg) and α-lactalbumin upon thermal and gelation properties at various pH values (using micro differential scanning calorimetry, μDSC, and oscillatory rheometry). At pH values far from the isoelectric point of whey proteins, denaturation and aggregation appeared as one single endothermic peak in the corresponding μDSC heating thermograms, for WPI and both WPC 80; however, they appeared as separate transitions at pH 5. Acidic conditions increased the temperature of occurrence of the dominant endothermic transition associated to β-Lg, thus increasing the thermal stability of WPI, WPC 80A and WPC 80B. Gelation took place at the lowest temperature when pH was set at 5. WPI, WPC 80A and WPC 80B exhibited the highest G′ values at pH 5 — whereas WPI led to stronger gels than WPC, irrespective of pH. In the case of WPC 50, gelation did not occur at all.
- Emergent food proteins – towards sustainability, health and innovationPublication . Fasolin, L. H.; Pereira, R. N.; Pinheiro, A. C.; Martins, J. T.; Andrade, C. C. P.; Ramos, O. L.; Vicente, A. A.There is an increasing demand for alternative and sustainable protein sources, such as vegetables, insects and microorganisms, that can meet the nutritional and sensory pleasantness needs of consumers. This emergent interest for novel protein sources, allied with “green” and cost-effective processing technologies, such as high hydrostatic pressure, ohmic heating and pulsed electric fields, can be used as strategies to improve the consumption of proteins from sustainable sources without compromising food security. In addition to their nutritional value, these novel proteins present several technological-functional properties that can be used to create various protein systems in different scales (i.e., macro, micro and nano scale), which can be tailored for a specific application in innovative food products. However, in order for these novel protein sources to be broadly used in future food products, their fate in the human gastrointestinal tract (e.g., digestion and bioavailability) must be assessed, as well as their safety for consumers must be clearly demonstrated. In particular, these proteins may become novel allergens triggering adverse reactions and, therefore, a comprehensive allergenicity risk assessment is needed. This review presents an overview of the most promising alternative protein sources, their application in the production of innovative food systems, as well as their potential effects on human health. In addition, new insights on sustainable processing strategies are given.
- Effect of whey protein purity and glycerol content upon physical properties of edible films manufactured therefromPublication . Ramos, Óscar L.; Reinas, Isabel; Silva, Sara I.; Fernandes, João C.; Cerqueira, Miguel A.; Pereira, Ricardo N.; Vicente, António A.; Poças, M. Fátima; Pintado, Manuela E.; Malcata, F. XavierThis manuscript describes the detailed characterization of edible films made from two different protein products e whey protein isolate (WPI) and whey protein concentrate (WPC), added with three levels of glycerol (Gly) e i.e. 40, 50 and 60%(w/w). The molecular structure, as well as barrier, tensile, thermal, surface and optical properties of said films were determined, in attempts to provide a better understanding of the effects of proteinaceous purity and Gly content of the feedstock. WPI films exhibited statistically lower (p < 0.05) moisture content (MC), film solubility (S), water activity, water vapor permeability (WVP), oxygen and carbon dioxide permeabilities (O2P and CO2P, respectively) and color change values, as well as statistically higher (p < 0.05) density, surface hydrophobicity, mechanical resistance, elasticity, extensibility and transparency values than their WPC counterparts, for the same content of Gly. These results are consistent with data from thermal and FTIR analyses. Furthermore, a significant increase (p < 0.05) was observed in MC, S, WVP, O2P, CO2P, weight loss and extensibility of both protein films when the Gly content increased; whereas a significant decrease (p < 0.05) was observed in thermal features, as well as in mechanical resistance and elasticity e thus leading to weaker films. Therefore, fundamental elucidation was provided on the features of WPI and WPC germane to food packaging e along with suggestions to improve the most critical ones, i.e. extensibility and WVP.
- Evaluation of antimicrobial edible coatings from a whey protein isolate base to improve the shelf life of cheesePublication . Ramos, Ó. L.; Pereira, J. O.; Silva, S. I.; Fernandes, J. C.; Franco, M. I.; Lopes-da-Silva, J. A.; Pintado, M. E.; Malcata, F. X.The objective of this work was to evaluate the effectiveness of antimicrobial edible coatings to wrap cheeses, throughout 60 d of storage, as an alternative to commercial nonedible coatings. Coatings were prepared using whey protein isolate, glycerol, guar gum, sunflower oil, and Tween 20 as a base matrix, together with several combinations of antimicrobial compounds—natamycin and lactic acid, natamycin and chitooligosaccharides (COS), and natamycin, lactic acid, and COS. Application of coating on cheese decreased water loss (~10%, wt/wt), hardness, and color change; however, salt and fat contents were not significantly affected. Moreover, the antimicrobial edible coatings did not permit growth of pathogenic or contaminant microorganisms, while allowing regular growth of lactic acid bacteria throughout storage. Commercial nonedible coatings inhibited only yeasts and molds. The antimicrobial edible coating containing natamycin and lactic acid was the best in sensory terms. Because these antimicrobial coatings are manufactured from food-grade materials, they can be consumed as an integral part of cheese, which represents a competitive advantage over nonedible coatings.