Maria Manuela Pintado

Bovine whey proteins : Overview on their main biological properties

Abstract Whey, a liquid by-product, is widely accepted to contain many valuable constituents. These include especially proteins that possess important nutritional and biological properties – particularly with regard to promotion of health, as well as prevention of diseases and health conditions. Antimicrobial and antiviral actions, immune system stimulation, anticarcinogenic activity and other metabolic features have indeed been associated with such whey proteins, as α-lactalbumin, β-lactoglobulin, lactoferrin, lactoperoxidase, and bovine serum albumin. The most important advances reported to date pertaining to biological properties of whey proteins are reviewed in this communication.

Optimization of the production of solid Witepsol nanoparticles loaded with rosmarinic acid.

During the last decade there has been a growing interest in the formulation of new food and nutraceutical products containing compounds with antioxidant activity. Unfortunately, due to their structure, certain compounds such as polyphenols, in particular rosmarinic acid (RA) are not stable and may interact easily with matrices in which they are incorporated. To overcome such limitations, the formulation of loaded polyphenols nanoparticles can offer an efficient solution to protect such compounds. Based on this rationale, the aim of this study was to prepare solid lipid nanoparticles (SLNs) loaded with RA using a hot melt ultrasonication method, where Witepsol H15 was used as lipid and Polysorbate 80 (Tween 80) as surfactant, following a 3(2) fractional factorial design, resulting in the use of 3 different percentages of surfactant (viz. 1, 2 and 3%, v/v) and lipid (0.5, 1.0 and 1.5%, w/v). The stability of the nanoparticles systems were tested during 28 d in aqueous solution stored at refrigeration temperature (ca. 5 °C), tracking the mean particle size of different formulations by photon correlation spectroscopy. To confirm RA entrapment, thermal analyses of the nanoparticles by DSC and FTIR were performed. The association efficiencies percentages (AE%) were determined using HPLC to quantitatively assess the RA in supernatants. Results showed that Witepsol H15 produced nanoparticles with initial mean diameters between 270 and 1000 nm, yet over time, a slight increase occurred, but without occurrence of aggregation. The AE% showed a high percentage of encapsulation (ca. 99%), which reveals low polyphenol releases from SLNs throughout storage time. In general, results showed a successful production of SLNs with properties that can be used to food applications.

Characterization of solid lipid nanoparticles produced with carnauba wax for rosmarinic acid oral delivery

In the last decade, research studies have increased on the development of delivery systems for polyphenols, for protection, improvement of stability and increase of their bioavailability. Rosmarinic acid is a polyphenol with described bioactivities, such as antioxidant, anti-mutagenic, anti-bacterial and anti-viral capabilities. Thus, the aim of this research work was to produce stable solid lipid nanoparticles (SLN) using carnauba wax as lipidic matrix, for delivery of rosmarinic acid, to be further incorporated into food matrices. Hence, different concentrations of wax (0.5, 1 and 1.5%, w/v) and percentages of surfactant (1, 2 and 3%, v/v) were tested. Physical properties, surface morphology and association efficiencies were studied at time of production and after 28 day at refrigerated storage. Thermal properties and the nature of the chemical interactions between the lipids waxes and rosmarinic acid were also evaluated. The particles showed range size between 35–927 nm and zeta potentials of ca. −38 to 40, showing high stability, with no risk of aggregation due to electric repulsion of SLN. High association efficiencies % (ca. 99%) were obtained. FTIR analyses proved the association of rosmarinic acid and lipidic matrix. The low lipid and high surfactant concentrations leads to small SLN. The surfactant, polysorbate 80 decreases the interfacial tension in the SLN surfaces, preventing aggregation, leading to the development of small particles. These properties were maintained throughout the 28 day of refrigerated storage, and no rosmarinic acid was released by the particles during refrigeration, indicating good compatibility between rosmarinic acid and the waxy core of SLN. The optimum range values to obtain the desirable features for incorporation in a functional food suggest formulations containing 1.0 and 1.5% (w/v) of lipid and 2% (v/v) of surfactant.

Addition of probiotic bacteria in a semi-hard goat cheese (coalho): Survival to simulated gastrointestinal conditions and inhibitory effect against pathogenic bacteria

In this study, the survival of the probiotics Lactobacillus acidophilus (LA-5), Lactobacillus casei subsp. paracasei (L. casei 01) and Bifidobacterium lactis (BB12) incorporated in a Brazilian semi-hard goat cheese (coalho) when exposed to in vitro simulated conditions of digestion was assessed. The inhibitory effects of these probiotic bacteria were also evaluated against Listeria monocytogenes and Staphylococcus aureus in the goat coalho cheese during refrigerated storage. At the end of the in vitro digestion, all of the probiotic tested strains presented decreased (p<0.05) viable cell counts (5.5-6.0logcfu/g) with respect to those determined before exposure to the mouth conditions (7-8logcfu/g). L. casei subsp. paracasei presented inhibition rate of 7.87% and 23.63% against S. aureus on the 14th and 21st day of storage at 10°C, respectively; against L. monocytogenes these values were 12.96 and 32.99%. Positive inhibition rates of B. lactis toward S. aureus were found on the 1st, 14th and 21st days of storage (16.32%, 10.12% and 3.67%, respectively); and against L. monocytogenes only on the 1st day of storage (3.28%). From these results, goat coalho cheese could be an interesting carrier of probiotic strains of L. acidophilus, L. casei subsp. paracasei and B. lactis. Moreover, L. casei subsp. paracasei, could be used as protective culture for delaying the growth of S. aureus and L. monocytogenes in goat coalho cheese.

Encapsulation of probiotic strains in plain or cysteine‐supplemented alginate improves viability at storage below freezing temperatures

Four probiotic bacteria (Lactobacillus paracasei L26, L. casei‐01, L. acidophilus Ki, and Bifidobacterium animalis BB‐12®) were encapsulated in plain alginate or alginate supplemented with L‐cysteine·HCl, and resulting microcapsules were stored at different temperatures, namely 21, 4, −20, or −80°C for a period of up to 6 months. The results showed that the encapsulation in calcium alginate microcapsules was only effective in promoting protection at freezing temperatures, independently of the sensitivity of the strain. Storage of calcium alginate microcapsules at −80°C indicated a protective effect upon viability of all four probiotic strains and the presence of L‐cysteine·HCl in the alginate matrix improved protection upon cell viability of B. animalis BB‐12®. An increase in storage temperature of encapsulated bacteria caused an increase in rate of loss in their viability that was strain dependent. This study suggests that microencapsulation of probiotic cells in calcium alginate can be suitable for sustaining the viability of probiotics in food products that require storage below freezing temperatures, even in the absence of cryoprotectors, contributing to an increased shelf life.

Bioactivity of probiotic whey cheese: Characterization of the content of peptides and organic acids

BACKGROUND Probiotic whey cheeses have been produced for several years. It is recognized that several bacterium-mediated metabolic activities contribute differently to the final sensory and nutritional profiles of dairy products. Hence the metabolic activity of probiotic strains in a whey cheese and their contribution to the bioactivity of such matrices were investigated here, including in particular Bifidobacterium animalis, Lactobacillus acidophilus and Lactobacillus casei. RESULTS Both L. casei and B. animalis produce lactic and acetic acids, whereas L. acidophilus produce mainly lactic acid; these metabolites may be considered bioprotection factors. Water-soluble extracts (WSE) obtained from these cheese matrices were subjected to ultrafiltration through a 3 kDa cut-off membrane, and the eluted peptides were resolved by high-performance liquid chromatography. Different qualitative and quantitative profiles were obtained, depending on the strain. WSE were further assayed for their ability to inhibit angiotensin-converting enzyme; the <3 kDa fraction exhibited higher activities in the case of L. casei and B. animalis than the control and L. acidophilus. CONCLUSION Whey cheeses with higher nutritional value were those inoculated with L. casei.

Insights into the protective role of solid lipid nanoparticles on rosmarinic acid bioactivity during exposure to simulated gastrointestinal conditions.

The evaluation of the digestion effects on bioactive solid lipid nanoparticles (SLN) was performed. For this purpose, witepsol and carnauba SLN loaded with rosmarinic acid (RA) were exposed to the simulated gastrointestinal tract (GIT) conditions prevailing in stomach and small intestine. The simulation of intestinal epithelium was made with a dialysis bag and intestinal cell culture lines. Changes on SLN physical properties, RA release and absorption profiles were followed at each step. Combination of digestion pH and enzymes showed a significant effect upon SLN physical properties. Zeta potential values increased at stomach conditions and decreased at small intestine simulation. Also, at intestine, SLN increased their sizes and released 40-60% of RA, maintaining its initial antioxidant activity values. Sustained release of 40% of RA from SLN was also observed in dialysis tube. At CaCo-2 cell line, both types of SLN showed similar absorbed RA % (ca. 30%). Nevertheless, in CaCo-2/HT29x mix cell lines, for carnauba SLN a lower adsorption RA % was observed than for witepsol SLN. Solid lipid nanoparticles protected RA bioactivity (in terms of antioxidant activity) until reaching the intestine. A controlled release of RA from SLN was achieved and a significant absorption was observed at intestinal cells. Overall, SLN produced with witepsol showed a higher stability than carnauba SLN.

Stability of bioactive solid lipid nanoparticles loaded with herbal extracts when exposed to simulated gastrointestinal tract conditions

ABSTRACT Solid lipid nanoparticles (SLN) can be used as vehicles for phenolic compounds rich extracts. In the present work two types of waxes — witepsol and carnauba were tested for the first time in the production of solid lipid nanoparticles (WSLN and CSLN, respectively) loaded with sage and savoury extracts. Physical characterization and association efficiencies calculation were performed. Discrimination of loaded phenolic compounds from each extract was made using HPLC assays. Antioxidant activities of SLN were characterized using two different methods — ABTS and ORAC. Finally, the phenolic compound release profile from SLN and stability when exposed to simulated gastrointestinal tract (GIT) conditions were also evaluated. Different phenolic compounds from sage and savoury extracts were entrapped in SLN. The highest antioxidant activity was obtained for the SLN loaded with savoury extract. Stomach simulated condition provokes a partial release of rosmarinic acid from SLN, whereas at small intestine simulation step, all SLN showed a release of ca. 100%. Witepsol SLN were the ones that best maintained their physical integrity during digestion, showing to be the most stable vehicles for sage and savoury extracts. These SLN show to be suitable for the production of food functional ingredients bearing antioxidant activity.

Safety profile of solid lipid nanoparticles loaded with rosmarinic acid for oral use: in vitro and animal approaches

Rosmarinic acid (RA) possesses several protective bioactivities that have attracted increasing interest by nutraceutical/pharmaceutical industries. Considering the reduced bioavailability after oral use, effective (and safe) delivery systems are crucial to protect RA from gastrointestinal degradation. This study aims to characterize the safety profile of solid lipid nanoparticles produced with Witepsol and Carnauba waxes and loaded with RA, using in vitro and in vivo approaches, focused on genotoxicity and cytotoxicity assays, redox status markers, hematological and biochemical profile, liver and kidney function, gut bacterial microbiota, and fecal fatty acids composition. Free RA and sage extract, empty nanoparticles, or nanoparticles loaded with RA or sage extract (0.15 and 1.5 mg/mL) were evaluated for cell (lymphocytes) viability, necrosis and apoptosis, and antioxidant/prooxidant effects upon DNA. Wistar rats were orally treated for 14 days with vehicle (control) and with Witepsol or Carnauba nanoparticles loaded with RA at 1 and 10 mg/kg body weight/d. Blood, urine, feces, and several tissues were collected for analysis. Free and loaded RA, at 0.15 mg/mL, presented a safe profile, while genotoxic potential was found for the higher dose (1.5 mg/mL), mainly by necrosis. Our data suggest that both types of nanoparticles are safe when loaded with moderate concentrations of RA, without in vitro genotoxicity and cytotoxicity and with an in vivo safety profile in rats orally treated, thus opening new avenues for use in nutraceutical applications.

Chitosan’s biological activity upon skin-related microorganisms and its potential textile applications

Over the years the body of work relating chitosan and its possible skin-related applications has grown, showing that chitosan is active both as a free compound and as a functional finishing of textiles. As a free molecule chitosan proved itself to be an attractive option as it is biocompatible and has a demonstrated biological activity (e.g. growth inhibition or adhesion inhibition) upon multiple skin pathogens, even upon multidrug resistant species. Furthermore, chitosan has wound healing accelerating properties, which make it a prime candidate for possible control of skin related infections. Almost inconspicuously, textiles have been one the main lines of defense of the skin against foreign threats, acting as a physical barrier to microbial colonization and infection. With the advent of textile functionalization specially designed textiles with enhanced protective characteristics, such as antimicrobial capacity, have come to the forefront. Chitosan functionalized textiles have been shown to be effective inhibitors of microbial growth with even invasive multidrug resistant species, as MRSA, being inhibited. Therefore, chitosan and chitosan functionalized textiles present themselves as both an interesting alternative to traditional antibiotics and as a possible means to enhance current treatment strategies.