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- Chitosan mouthwash: toxicity and in vivo validationPublication . Costa, E. M.; Silva, S.; Costa, M. R.; Pereira, M.; Campos, D. A.; Odila, J.; Madureira, A. R.; Cardelle-Cobas, A.; Tavaria, F. K.; Rodrigues, A. S.; Pintado, M. M.A previous study showed that a chitosan mouthwash would be a valid alternative to current mouthwashesas it demonstrated, in vitro, significantly higher antibiofilm activity than two commercial mouthwashes.As such, the aim of this work was to verify the safety of the developed product and to validate, in vivo, thebiological activity ascertained in vitro. Chitosan mouthwash safety was evaluated through Ames, MTT andV79 chromosomal aberration assay while antimicrobial activity was evaluated through in vivo assays. Theresults showed that the chitosan mouthwash was safe, presenting lower cytotoxicity than a commercialmouthwash, and that it effectively reduced viable counts of Streptococcus spp. and Enterococcus spp. by ca.5.5 log of CFU. Furthermore, in direct comparison with a commercial mouthwash the chitosan mouthwashpossessed significantly higher antimicrobial activity. The conjunction of these results proves that thechitosan mouthwash is a safe, effective, natural alternative to the existent chemical mouthwashes.
- Edible films as carrier for lactic acid bacteriaPublication . Pereira, Joana Odila; Soares, José; Sousa, Sérgio; Madureira, Ana Raquel; Gomes, Ana M. P.; Pintado, M. E.The use of edible coatings and films formulated with bioactive compounds in food products in order to convey new functionalities or extend shelf-life opens new possibilities as a carrier for functional lactic acid bacteria. In this work the main objective was to study the stability of probiotic microorganisms, viz. Bifidobacterium animalis Bb-12® and Lactobacillus casei-01, in edible film formulations based on whey protein isolate (WPI). The results demonstrated a loss of bacterial cell viability of ca. 3 log cycles (reaching 106 CFU/g film) until 60 d at both 23 and 4 C, noting that the most marked decrease was at 23 C for both strains. Bifidobacterium animalis Bb-12® remained viable for a longer period of time and with less decrease in its cell numbers (108 CFU/g film). Physical properties, namely color, water activity, thickness, young’s modulus, tensile strength, elongation at break and the molecular structure of WPI films were maintained stable throughout the storage period at both temperatures tested. Edible films incorporated with probiotics can be good carriers for these to be ingested together with food products.
- Antihypertensive effect of spent brewer yeast peptidePublication . Amorim, M.; Marques, C.; Pereira, J. O.; Guardão, L.; Martins, M. J.; Osório, H.; Moura, D.; Calhau, C.; Pinheiro, H.; Pintado, M.Numerous studies have investigated dietary approaches to prevent chronic lifestyle-related diseases, including hypertension. Spent brewer's yeast is the second largest byproduct originated by the brewing industry and it deserves considerable attention because of its high nutritional value, ca. 40% of its dry mass is rich in protein which can be hydrolyzed into biologically active peptides. To upgrade this byproduct, the aim of this study was initially in vitro assessment of biological properties, e.g. ACE inhibition and antioxidant activity, and then, the in vivo effect in short-term oral antihypertensive effect of hydrolyzed yeast fractions on a well characterized model to study hypertension - Spontaneously Hypertensive Rats (SHR). Here, it was demonstrated that the fraction with molecular weight below 3 kDa containing tri and tetra- peptides with hydrophobic amino acid residues - SPQW, PWW and RYW, causes the most noticeable decrease in systolic, diastolic and mean blood pressure of SHR and shows highest antioxidant effect. These properties highlight the potential use of yeast extract as nutraceutical or functional food ingredient for the management and treatment of hypertension with antioxidant effect.
- Impact of whey protein coating incorporated with Bifidobacterium and Lactobacillus on sliced ham propertiesPublication . Pereira, Joana Odila; Soares, José; Monteiro, Maria João P.; Gomes, Ana; Pintado, ManuelaEdible coatings/films with functional ingredients may be a solution to consumers' demands for high-quality food products and an extended shelf-life. The aim of this work was to evaluate the antimicrobial efficiency of edible coatings incorporated with probiotics on sliced ham preservation. Coatings was developed based on whey protein isolates with incorporation of Bifidobacterium animalis Bb-12® or Lactobacillus casei-01. The physicochemical analyses showed that coating decreased water and weight loss on the ham. Furthermore, color analysis showed that coated sliced ham, exhibited no color change, comparatively to uncoated slices. The edible coatings incorporating the probiotic strains inhibited detectable growth of Staphylococcus spp., Pseudomonas spp., Enterobacteriaceae and yeasts/molds, at least, for 45 days of storage at 4 °C. The sensory evaluation demonstrated that there was a preference for the sliced coated ham. Probiotic bacteria viable cell numbers were maintained at ca. 108 CFU/g throughout storage time, enabling the slice of ham to act as a suitable carrier for the beneficial bacteria.
- Development of a cashew nut coated with bioactive whey peptide extract with antihipertensivePublication . Pereira, Joana Odila; Amorim, Manuela; Silva, L. B.; Ormenese, R.C.S.C.; Pacheco, M.T.B.; Pintado, ManuelaThere is a growing concern in the consumer’s habits seeking healthy foods, which has opened new markets for functional foods. This global demand led to the development of functional foods, searching new ingredients to incorporate into conventional foods. Whey proteins are studied not only from the nutritional and functional properties, but also as a source of peptides that may exert biological functions, particularly antihypertensive activity. These bioactive peptides are released by enzymatic hydrolysis and according the final composition they may exert biological activities. Cashew nut was used as matrix for incorporation functional ingredients due to its nutritional properties conveyed by the high content of folic acid and essential fatty acids. The aim of this study was to develop a cashew nut coated with peptide fraction obtained from whey and to evaluate the antihypertensive activity and consumer acceptance of the new functional cashew nuts. The fraction with MW < 3000 Da was obtained by hydrolysis of whey with Cynara cardunculus followed by nanofiltration to obtain low MW fraction, exhibited very high ACE-inhibitory activity, IC50 12.8 μg /mL protein. The incorporation of peptides (2 %) in cashew nut led to a reduction to values of ACEinhibitory activity, 532.2 μg/mL. However, this is close the values reported by other products in the market claiming antihypertensive activity. In the formulation there is space to increase concentration and biological activity. Nevertheless, it is important to highlight that the antihypertensive activity was not lost even when high temperatures during coating and processing of the product were used. The sensory analysis of the functional cashew nut showed high acceptability by the consumers. Our results suggest that the application of these bioactive peptide extracts with antihypertensive activity in the development of a new snacks with reduce salt content is promising in the improvement of new value-added food products.
- Characterization of edible films based on alginate or whey protein incorporated with Bifidobacterium animalis subsp. lactis BB-12 and prebioticsPublication . Pereira, Joana Odila; Soares, José; Costa, Eduardo; Silva, Sara; Gomes, Ana; Pintado, ManuelaRecently, edible films were shown to be an effective strategy for the delivery of functional ingredients, such as probiotics and prebiotics. With that in mind, two soluble fibres (inulin and fructooligosaccharides) were selected as prebiotic elements, in whey protein isolate (WPI) and alginate (ALG) matrices plasticized with glycerol and used for the incorporation of Bifidobacterium animalis subsp. lactis BB-12. The results obtained showed that the viability of the B. animalis subsp. lactis BB-12 probiotic strain was maintained within the minimum threshold (10(6) CFU/g) necessary to act as a probiotic throughout 60 days of storage at 23 degrees C. The incorporation of prebiotic compounds improved B. animalis subsp. lactis BB-12 viability, with inulin showing the best performance, as it maintained the viability at 7.34 log CFU/g. The compositional characteristics (biopolymer type and prebiotics addition) of the film forming solutions had no significant impact upon the viability of the probiotic strain. The incorporation of probiotics and prebiotics did not modify the infrared spectra, revealing that the molecular structure of the films was not modified. The moisture content and water solubility decreased positively in WPI- and ALG-based films with the addition of prebiotics compounds. Overall, the results obtained in this work support the use of WPI films containing inulin as a good strategy to immobilize B. animalis subsp. lactis BB-12, with potential applications in the development of functional foods.
- 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.
- Use of whey peptide fraction in coated cashew nut as functional ingredient and salt replacerPublication . Amorim, M.; Pereira, J. O.; Silva, L. B.; Ormenese, R. C. S. C.; Pacheco, M. T. B.; Pintado, M.Consumers are increasingly concerned about healthy eating habits. The incorporation and stability of 2% antihypertensive whey peptide extract in a new coating of cashew nuts with reduced salt (less 15 and 30%) was studied. The evaluation of nutritional value, in vitro antihypertensive activity and consumer acceptance of final products was assessed. Incorporation of peptide fraction assured the production of a snack with an ACE-inhibitory activity (532.2 μgmL−1 IC50 value). The amount of lipids present in coated cashew nuts was composed mainly by essential fatty acids, mostly monounsaturated. Glutamic acid, leucine, arginine and aspartic acid were the most abundant essential aminoacids. 70% of the consumers considered both samples (15 and 30%) as “ideal taste”. The results suggest that the new coating allowed the development of a new snack with reduced salt content, opening new opportunities as carrier of other ingredients to develop more diversified and efficient functional foods.
- In vitro ACE-inhibitory peptide KGYGGVSLPEW facilitates noradrenaline release from sympathetic nerve terminals: relationship with the lack of antihypertensive effect on spontaneous hypertensive ratsPublication . Marques, Cláudia; Amorim, Maria Manuela; Pereira, Joana Odila; Guardão, Luísa; Martins, Maria João; Pintado, Manuela Estevez; Moura, Daniel; Calhau, Conceição; Pinheiro, HélderThis study aimed to validate the antihypertensive activity of the angiotensin-converting enzyme (ACE)-inhibitor whey protein hydrolysate (WPH) obtained through the action of proteolytic enzymes fromCynara Cardunculus. The antihypertensive activity of WPH fractions containing peptides with molecularweight below 3 kDa (Whey < 3 kDa) and 1 kDa (Whey < 1 kDa) along with the antihypertensive activity ofthree potent ACE-inhibitory peptide sequences (DKVGINYW, DAQSAPLRVY and KGYGGVSLPEW), previ-ously identified in WPH, were also investigated. In parallel, the influence of KGYGGVSLPEW (the mostpotent ACE-inhibitory peptide sequence) on AT1receptors (a common pharmacological target of anti-hypertensive therapies beyond ACE), was evaluated. The effect of WPH and fractions (300 mg/kg) andpeptide sequences (5 mg/kg) on systolic, diastolic and mean blood pressure was evaluated by telemetryon Spontaneously Hypertensive Rats (SHR), after single oral administration. Despite their ACE-inhibitoryeffect in vitro, neither WPH, Whey <3 kDa, Whey <1 kDa or peptide sequences exhibited antihyperten-sive activity. In addition, KGYGGVSLPEW was not only devoid of AT1receptor antagonism but, on thecontrary, had a similar effect to that of Ang II by facilitating the noradrenaline release from sympatheticnerve terminals. In vitro ACE blockade does not always correlate with antihypertensive activity and food-derived peptides cannot be classified as antihypertensive agents based exclusively on in vitro assays. Theabsence of an antihypertensive effect may also be a result of the interaction of these compounds withother components of the systems involved in the blood pressure control.
- Biodiversity and characterization of Staphylococcus species isolated from a small manufacturing dairy plant in PortugalPublication . Soares, José C.; Marques, M. Rosário; Tavaria, Freni K.; Pereira, Joana O.; Xavier Malcata, F.; Pintado, Manuela M.The level and the diversity of the staphylococcal community occurring in the environment and dairy products of a small manufacturing dairy plant were investigated. Species identification was performed using different molecular methods, viz. Multiplex-PCR, amplified ribosomal DNA restriction analysis (ARDRA), and sodA gene sequencing. The main species encountered corresponded to Staphylococcus equorum (41 isolates, 39.0%), S. saprophyticus (28 isolates, 26.7%) and S. epidermidis (15 isolates, 14.3%). Additionally, low incidence of enterotoxin genes was obtained, with only 9 strains (8.6%) being positive for one or more toxin genes. With regard to antimicrobial resistance, 57.1% of the isolates showed at least resistance against one antibiotic, and 28.6% were multi-resistant, which might accomplish resistance for up to 6 antibiotics simultaneously. These results provided evidence that the presence of Staphylococcus species in dairy environment are mostly represented by S. equorum and S. saprophyticus, and illustrate that carrying antimicrobial resistance genes has become reasonably widespread in cheese and dairy environment.