Fabrício Luiz Tulini

Purification and characterization of antimicrobial peptides from fish isolate Carnobacterium maltaromaticum C2: Carnobacteriocin X and carnolysins A1 and A2.

Carnobacterium maltaromaticum C2, isolated from Brazilian smoked fish (Surubim, Pseudoplatystoma sp.), was found to exert antimicrobial activity against Listeria monocytogenes, an important foodborne pathogen. In this study, the bacteriocins produced by C. maltaromaticum C2 were purified via an extraction with XAD-16 resin, a C18 solid phase extraction, followed by reversed-phase fast protein liquid chromatography. The purified active fractions were characterized using tandem mass spectrometry, permitting the identification of multiple bacteriocins. Carnobacteriocins BM1, B1, and a variant of carnobacteriocin B2 were all found, providing much of the antilisterial activity. Additionally, we herein report the first isolation of the previously predicted antimicrobial peptide carnobacteriocin X. Moreover, C. maltaromaticum C2 produces a novel two-component lantibiotic, termed carnolysin, homologous to enterococcal cytolysin. This lantibiotic is antimicrobially inactive when tested against the non-bacteriocinogenic strain C. maltaromaticum A9b-, likely requiring an additional proteolytic cleavage to reach maturity.

Development of solid lipid microparticles loaded with a proanthocyanidin-rich cinnamon extract (Cinnamomum zeylanicum): Potential for increasing antioxidant content in functional foods for diabetic population

Cinnamon proanthocyanidins play an important role on the attenuation of complications associated to diabetes, but the daily ingestion of these compounds is not always satisfactory due to several aspects such as low stability and unpleasant taste. Thus, in the present study, a proanthocyanidin-rich cinnamon extract (Cinnamomum zeylanicum) was incorporated into solid lipid microparticles (SLMs) by spray chilling technique using vegetable fat as carrier. The microparticles were characterized with regard to their physical and chemical properties, morphology, proanthocyanidin stability and sensory properties. SLMs were spherical with a unimodal size distribution between 60 and 130μm, and proanthocyanidins were highly stable in SLM stored for up to 90days at 5, 25 and 37°C. Moreover, SLMs were able to mask the bitter taste and astringent sensation of proanthocyanidins and other polyphenols from cinnamon extract. Taken together, these results show the potential of SLM loaded with cinnamon proanthocyanidins for improving functional properties in new foods.

Screening for antimicrobial and proteolytic activities of lactic acid bacteria isolated from cow, buffalo and goat milk and cheeses marketed in the southeast region of Brazil

Lactic acid bacteria (LAB) can be isolated from different sources such as milk and cheese, and the lipolytic, proteolytic and glycolytic enzymes of LAB are important in cheese preservation and in flavour production. Moreover, LAB produce several antimicrobial compounds which make these bacteria interesting for food biopreservation. These characteristics stimulate the search of new strains with technological potential. From 156 milk and cheese samples from cow, buffalo and goat, 815 isolates were obtained on selective agars for LAB. Pure cultures were evaluated for antimicrobial activities by agar antagonism tests and for proteolytic activity on milk proteins by cultivation on agar plates. The most proteolytic isolates were also tested by cultivation in skim milk followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fermented milk. Among the 815 tested isolates, three of them identified as Streptococcus uberis (strains FT86, FT126 and FT190) were bacteriocin producers, whereas four other ones identified as Weissella confusa FT424, W. hellenica FT476, Leuconostoc citreum FT671 and Lactobacillus plantarum FT723 showed high antifungal activity in preliminary assays. Complementary analyses showed that the most antifungal strain was L. plantarum FT723 that inhibited Penicillium expansum in modified MRS agar (De Man, Rogosa, Sharpe, without acetate) and fermented milk model, however no inhibition was observed against Yarrowia lipolytica. The proteolytic capacities of three highly proteolytic isolates identified as Enterococcus faecalis (strains FT132 and FT522) and Lactobacillus paracasei FT700 were confirmed by SDS-PAGE, as visualized by the digestion of caseins and whey proteins (β-lactoglobulin and α-lactalbumin). These results suggest potential applications of these isolates or their activities (proteolytic activity or production of antimicrobials) in dairy foods production.

Improved adsorption-desorption extraction applied to the partial characterization of the antilisterial bacteriocin produced by Carnobacterium maltaromaticum C2

Bacteriocins are ribosomally produced peptides useful for food biopreservation. An improved adsorption-desorption process is proposed for the partial purification of the bacteriocin produced by the fish isolate Carnobacterium maltaromaticum C2. Analyzis of extract by SDS-PAGE indicated this method may offer an alternative to improve the yield of purification of bacteriocins.

Evaluation of the release profile, stability and antioxidant activity of a proanthocyanidin-rich cinnamon (Cinnamomum zeylanicum) extract co-encapsulated with α-tocopherol by spray chilling

Cinnamon has many health improving compounds such as proanthocyanidins, which also have potential for the prevention of damages caused by diabetes. Similarly, α-tocopherol is a natural antioxidant with important role on protection of fatty acids in membranes and lipoproteins. However, the addition of antioxidants in food may result in interaction with food matrix, low stability and unpleasant taste. In the present study, a proanthocyanidin-rich cinnamon extract (PRCE) (Cinnamomum zeylanicum) was co-encapsulated with α-tocopherol into solid lipid microparticles (SLMs) by spray chilling. The microparticles were characterized with regard to the physical and chemical properties, morphology, proanthocyanidin stability and release profile. SLMs were spherical with an average diameter of ca. 80μm. Proanthocyanidins were highly stable in SLMs stored for up to 90days at 5, 25 and 37°C. Moreover, SLMs gradually released proanthocyanidins in simulated gastrointestinal fluids by a diffusional process, following a Korsmeyer-Peppas kinetic. Analyses of the antioxidant compounds indicated that PRCE components exhibited a higher scavenging capacity against reactive oxygen species (ROS) and reactive nitrogen species (RNS). Thus, the SLMs produced in the present study have potential for application in the development of new functional foods and nutraceuticals, also providing an alternative for the controlled release of proanthocyanidins and α-tocopherol into the intestine.

Proteolytic activity of Enterococcus faecalis VB63F for reduction of allergenicity of bovine milk proteins

With the aim of screening proteolytic strains of lactic acid bacteria to evaluate their potential for the reduction of allergenicity of the major bovine milk proteins, we isolated a new proteolytic strain of Enterococcus faecalis (Ent. faecalis VB63F) from raw bovine milk. The proteases produced by this strain had strong activity against caseins (αS1-, αS2-, and β-casein), in both skim milk and sodium caseinate. However, only partial hydrolysis of whey proteins was observed. Proteolysis of Na-caseinate and whey proteins, observed after sodium dodecyl sulfate-PAGE, was confirmed by analysis of peptide profiles by reversed-phase HPLC. Inhibition of proteolysis with EDTA indicated that the proteases produced by Ent. faecalis VB63F belonged to the group of metalloproteases. The optimal conditions for their activity were 42°C and pH 6.5. The majority of assessed virulence genes were absent in Ent. faecalis VB63F. The obtained results suggest that Ent. faecalis VB63F could be efficient in reducing the immunoreactivity of bovine milk proteins.

Microcapsules loaded with the probiotic Lactobacillus paracasei BGP-1 produced by co-extrusion technology using alginate/shellac as wall material: Characterization and evaluation of drying processes

Microcapsules containing Lactobacillus paracasei BGP-1 were produced by co-extrusion technology using alginate and alginate-shellac blend as wall materials. Sunflower oil and coconut fat were used as vehicles to incorporate BGP-1 into the microcapsules. The microcapsules were evaluated with regard the particle size, morphology, water activity and survival of probiotics after 60days of storage at room temperature. Fluidized bed and lyophilization were used to dry the microcapsules and the effect of these processes on probiotic viability was also evaluated. Next, dried microcapsules were exposed to simulated gastrointestinal fluids to verify the survival of BGP-1. Microcapsules dried by fluidized bed had spherical shape and robust structures, whereas lyophilized microcapsules had porous and fragile structures. Dried microcapsules presented a medium size of 0.71-0.86mm and aw ranging from 0.14 to 0.36, depending on the drying process. When comparing the effects of drying processes on BGP-1 viability, the fluidized bed was less aggressive than lyophilization. The alginate-shellac blend combined with coconut fat as core effectively protected the encapsulated probiotic under simulated gastrointestinal conditions. Thus, the production of microcapsules by co-extrusion followed by drying using the fluidized bed is a promising strategy for protection of probiotic cells.