Incorporation of copaiba and oregano essential oils on the shelf life of fresh ground beef patties under display: Evaluation of their impact on quality parameters and sensory attributes.

The preservative effect of the addition of different essential oils (copaiba and oregano) on meat quality parameters and sensorial acceptability was analyzed for fresh ground beef patties over 21 days of display. Five treatments were assessed: control (CON) without antioxidants; addition of the synthetic additive butylated hydroxytoluene (BHT); addition 0.05% of copaiba essential oil (CEO); 0.05% of oregano essential oil (OEO); or blend of 0.025% copaiba and 0.025% oregano essential oils (BEO). The lowest cooking losses and greatest tenderness (P <0.05) were reached with the blend (BEO). The inclusion of oregano essential oil presented a more intense chroma (P <0.05), with the best color retained during display. Oregano essential oil (OEO) and the blend (BEO) showed the highest antioxidant activity, reducing the lipid oxidation of beef patties during display (P < 0.05). Consumers preferred the odor of beef patties with essential oils (OEO and BEO) to the CON; however, the flavor from OEO had the lowest acceptability and the worst scores for overall acceptability (P < 0.05). Patties with the blend addition (BEO) were the best scored on overall acceptability assessments. In conclusion, the oregano and copaiba essential oils blend had a good preservative effect on fresh beef patties during display and increased sensory acceptability of the product, thus being a possible alternative for replacing synthetic compounds in processed foods.

Determination of the Nutritional Value of Diet Containing Bacillus subtilis Hydrolyzed Feather Meal in Adult Dogs.

Feathers are naturally made up of non-digestible proteins. Under thermal processing, total tract digestibility can be partially improved. Furthermore, Bacillus subtilis (Bs) has shown a hydrolytic effect In vitro. Then, a Bs FTC01 was selected to hydrolyze enough feathers to produce a meal, and then test the quality and inclusion in the dog's diet to measure the apparent total tract digestibility coefficient (ATTDC) in vivo and the microorganism's ability to survive in the gastrointestinal tract. A basal diet was added with 9.09% hydrolyzed Bs feather meal (HFMBs) or 9.09% thermally hydrolyzed feather meal (HFMT). Nine adult dogs were randomized into two 10-day blocks and fed different diets. Microbial counts were performed on feather meal, diets and feces. The Bs was less effective in digesting the feathers, which reduced the ATTDC of dry matter, crude protein, energy and increased the production of fecal DM, but the fecal score was maintained (p > 0.05). The digestible energy of HFMT and HFMBs was 18,590 J/kg and 9196 J/kg, respectively. Bacillus subtilis showed limitation to digest feather in large scale, but the resistance of Bs to digestion was observed since it grown on feces culture.

Microbial bioconversion of feathers into antioxidant peptides and pigments and their liposome encapsulation.

OBJECTIVES: The co-encapsulation of bioactive peptides obtained from degradation of chicken feathers and flexirubin-type pigment produced by Chryseobacterium sp. kr6 into phosphatidylcholine liposomes was investigated. RESULTS: Control empty liposomes showed mean diameter of 168.5 nm, varying to 185.4, 102.0 and 98.5 nm after the encapsulation of peptides, pigment and their co-encapsulation, respectively. Control liposomes presented zeta potential of - 20.9 mV, while the formulations containing the bioactive compounds showed values of - 30 mV or higher in magnitude. Infrared analysis revealed typical spectra for phosphatidylcholine, suggesting that no new chemical bonds were formed after encapsulation. ABTS radical scavenging assay showed that the antioxidant activity of the compounds was maintained after encapsulation. CONCLUSIONS: Feather waste can be a valuable substrate for simultaneous production of antioxidant peptides and pigment by Chryseobacterium sp. kr6, and their encapsulation into liposomes may be a suitable alternative for delivery of these natural antioxidants.

Buffalo cheese whey hydrolyzed with Alcalase as an antibrowning agent in minimally processed apple.

Buffalo whey was hydrolyzed with Alcalase for different times ti (i = 0, 0.5, 1, 2, 3, 4 or 6 h) and the browning inhibition of minimally processed apples was investigated. The hydrolysis process was followed by determination of the degree of hydrolysis. In order to understand possible modes of action on the enzymatic browning, whey was submitted to the analysis of antioxidant activity (ABTS·+ radical sequestration, Fe2+ chelating activity and reducing power), reactivity with quinones and inhibitory activity on polyphenol oxidases (PPO) extracted from Red Delicious apples. Buffalo whey showed significant increase in degree of hydrolysis, antioxidant activity, reactivity with quinones and PPO-inhibitory activity as a function of the hydrolysis time. Maximum PPO-inhibitory activity was observed from 4 h hydrolysis (t4h hydrolysate), reaching about 50% inhibition. Then, slices of minimally processed apples were immersed in a buffered solution of the t4h hydrolysate, packed and subjected to instrumental color evaluation during storage for up to 6 days. As for the ability to inhibit the browning of the minimally processed apples, the hydrolyzate kept the L∗ parameter of the apples during 6 days of storage, not statistically differing from the metabisulfite. In addition to the luminosity, the hydrolyzed whey was able to maintain the browning index of the apples at lower values during this storage time compared to the non-hydrolyzed whey. These results evidence possible applications of buffalo whey hydrolyzed with Alcalase as a natural substitute for additives conventionally used in the control of enzymatic browning in foods.

Mineral fortification modifies physical and microstructural characteristics of milk gels coagulated by a bacterial enzymatic pool.

An enzymatic pool from the Amazonian bacterium Bacillus sp. P7 was used as milk coagulant. Discovery of novel coagulants is of great interest in dairy industry for the development of new textures in cheese. Color, mechanical and microstructural characterization of milk gels induced by the bacterial enzymatic pool was carried out. Effect of mineral fortification on these characteristics was studied. Whiter gels with smaller pore diameters were obtained in the presence of Ca2+ or Mg2+. These characteristics seemed to be influenced by the effect of ionic strength on casein structure which was also evidenced by digital texture features analysis. On the other hand, specific affinity of the assayed cations for milk proteins showed to be important in the development of the mechanical texture of the gels. Firmness and fracture force of milk gels obtained in the presence of Zn2+ or Ca2+ were higher than in the presence of Mg2+ and Na2+.

Effects of Achyrocline satureioides Inflorescence Extracts against Pathogenic Intestinal Bacteria: Chemical Characterization, In Vitro Tests, and In Vivo Evaluation.

Three Achyrocline satureioides (AS) inflorescences extracts were characterized: (i) a freeze-dried extract prepared from the aqueous extractive solution and (ii) a freeze-dried and (iii) a spray-dried extract prepared from hydroethanol extractive solution (80% ethanol). The chemical profile, antioxidant potential, and antimicrobial activity against intestinal pathogenic bacteria of AS extracts were evaluated. In vitro antioxidant activity was determined by the total reactive antioxidant potential (TRAP) assay. In vivo analysis and characterization of intestinal microbiota were performed in male Wistar rats (saline versus treated animals with AS dried extracts) by high-throughput sequencing analysis: metabarcoding. Antimicrobial activity was tested in vitro by the disc diffusion tests. Moisture content of the extracts ranged from 10 to 15% and 5.7 to 17 mg kg-1 of fluorine. AS exhibited antioxidant activity, especially in its freeze-dried form which also exhibited a wide spectrum of antimicrobial activity against intestinal pathogenic bacteria greater than those observed by the antibiotic, amoxicillin, when tested against Bacillus cereus and Staphylococcus aureus. Antioxidant and antimicrobial activities of AS extracts seemed to be positively correlated with the present amount of flavonoids. These findings suggest a potential use of AS as a coadjuvant agent for treating bacterial-induced intestinal diseases with high rates of antibiotic resistance.

Inhibition of mycotoxin-producing fungi by Bacillus strains isolated from fish intestines.

Bacillus strains isolated from the aquatic environment of the Brazilian Amazon region were tested for their activity against mycotoxigenic fungi. All tested bacteria showed antifungal activity, inhibiting at least 7 indicator fungi. Four Bacillus strains showing promising antifungal results were subsequently evaluated for their activity in reducing mycelial growth rate, sporulation, spore germination percentage, and mycotoxin production. Bacillus sp. P1 and Bacillus sp. P11 had a remarkable antifungal effect on toxigenic fungi. Washed bacterial cell suspension of strains P1 and P11 (107CFU/ml) reduced by >70% the fungal colony diameters, including a complete inhibition of ochratoxin A (OTA) producing Aspergillus spp. Significant reduction of growth rate, sporulation and spore germination were also observed. The bacteria influenced the production of mycotoxins, causing a reduction around 99 and 97% in AFB1 and OTA concentration, respectively. Chromatographic analysis revealed the presence of lipopeptides (iturin A and surfactin isomers) in butanol extracts of cell-free supernatants and cell pellets of strains P1 and P11. Furthermore, antifungal activity of these extracts was confirmed against A. flavus A12 and A. carbonarius ITAL293, producers of AFB1 and OTA, respectively. These bacterial strains could be promising biocontrol agents against toxigenic fungi.

Milk protein suspensions enriched with three essential minerals: Physicochemical characterization and aggregation induced by a novel enzymatic pool.

Structural changes of casein micelles and their aggregation induced by a novel enzymatic pool isolated from Bacillus spp. in the presence of calcium, magnesium or zinc were investigated. The effect of cations on milk protein structure was studied using fluorescence and dynamic light scattering. In the presence of cations, milk protein structure rearrangements and larger casein micelle size were observed. The interaction of milk proteins with zinc appears to be of a different nature than that with calcium or magnesium. Under the experimental conditions assayed, the affinity of each cation for some groups present in milk proteins seems to play an important role, besides electrostatic interaction. On the other hand, the lowest aggregation times were achieved at the highest calcium and zinc concentrations (15 mM and 0.25 mM, respectively). The study found that the faster the aggregation of casein micelles, the less compact the gel matrix obtained. Cation concentrations affected milk protein aggregation kinetics and the structure of the aggregates formed.

Soy protein hydrolysis with microbial protease to improve antioxidant and functional properties.

Soybean proteins are widely used as nutritional and functional food ingredients. This investigation evaluated through a 2(3) central composite design the effect of three variables (pH, temperature and enzyme/substrate (E/S) ratio) on the production of soy protein isolate (SPI) hydrolysates with a microbial protease. Soluble peptides, antioxidant activity, and foaming and emulsifying capabilities of the hydrolysates were analyzed. All variables, as well as their interactions, were significant for the soluble peptides content of SPI hydrolysates. Optimal conditions for obtaining soluble peptides were around 30-35 °C, pH 6.5-9.5, and E/S ratios of 1,650-6,300 U g(-1). SPI hydrolysates produced at 30-45 °C, pH 8.0-9.5, and E/S ratios of 4,000-8,000 U g(-1) showed higher capacity to scavenge the 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical. Models for soluble peptides and ABTS activity of hydrolysates were obtained. In the range studied, the variables had not significant influence on the ability of hydrolysates to scavenge the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. SPI hydrolysates also presented reducing power and ability to chelate iron. Hydrolysis temperature was significant for the Fe(2+)-chelating ability of hydrolysates. Temperature of hydrolysis was significant for the foaming capacity of hydrolysates, with higher values observed at 45 °C and 8,000 U g(-1). For emulsifying capacity, only E/S ratio presented a significant effect. Temperature and E/S ratio appeared to be more significant variables influencing the properties of the SPI hydrolysates. The results of this study indicate that specific hydrolysis conditions should be selected to obtain SPI hydrolysates with preferred characteristics.

Hydrolysates of sheep cheese whey as a source of bioactive peptides with antioxidant and angiotensin-converting enzyme inhibitory activities.

Enzymatic proteolysis may be employed to release bioactive peptides, which have been investigated for potential benefits from both technological and human health perspectives. In this study, sheep cheese whey (SCW) was hydrolyzed with a protease preparation from Bacillus sp. P7, and the hydrolysates were evaluated for antioxidant and angiotensin I-converting enzyme (ACE)-inhibitory activities. Soluble protein and free amino acids increased during hydrolysis of SCW for up to 4h. Antioxidant activity of hydrolysates, evaluated by the 2,2'azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid radical scavenging method, increased 3.2-fold from 0 h (15.9%) to 6h of hydrolysis (51.3%). Maximum Fe(2+) chelation was reached in 3h hydrolysates, and the reducing power peaked at 1h of hydrolysis, representing 6.2 and 2.1-fold increase, respectively, when compared to that of non-hydrolyzed SCW. ACE inhibition by SCW (12%) was improved through hydrolysis, reaching maximal values (55% inhibition) in 4h, although 42% inhibition was already observed after 1h hydrolysis. The peptide LAFNPTQLEGQCHV, derived from ß-lactoglobulin, was identified from 4-h hydrolysates. Such a biotechnological approach might be an interesting strategy for SCW processing, potentially contributing to the management and valorization of this abundant dairy byproduct.

Infection in a rat model reactivates attenuated virulence after long-term axenic culture of Acanthamoeba spp.

Prolonged culturing of many microorganisms leads to the loss of virulence and a reduction of their infective capacity. However, little is known about the changes in the pathogenic strains of Acanthamoeba after long culture periods. Our study evaluated the effect of prolonged culturing on the invasiveness of different isolates of Acanthamoeba in an in vivo rat model. ATCC strains of Acanthamoeba, isolates from the environment and clinical cases were evaluated. The in vivo model was effective in establishing the infection and differentiating the pathogenicity of the isolates and re-isolates. The amoebae cultured in the laboratory for long periods were less virulent than those that were recently isolated, confirming the importance of passing Acanthamoeba strains in animal models.

Infection in a rat model reactivates attenuated virulence after long-term axenic culture of Acanthamoeba spp

Prolonged culturing of many microorganisms leads to the loss of virulence and a reduction of their infective capacity. However, little is known about the changes in the pathogenic strains of Acanthamoeba after long culture periods. Our study evaluated the effect of prolonged culturing on the invasiveness of different isolates of Acanthamoeba in an in vivo rat model. ATCC strains of Acanthamoeba, isolates from the environment and clinical cases were evaluated. The in vivo model was effective in establishing the infection and differentiating the pathogenicity of the isolates and re-isolates. The amoebae cultured in the laboratory for long periods were less virulent than those that were recently isolated, confirming the importance of passing Acanthamoeba strains in animal models.

Kinetic modeling of thermal inactivation of the Bacillus sp. protease P7.

Data from thermal stability of a keratinolytic protease produced by the Amazon isolate Bacillus sp. P7 was fitted to various mathematical models. Kinetic modeling showed that Weibull distribution was the best equation to describe the residual activity of protease P7 after heat treatment. The effects of temperature on equation parameters and on characteristics of the inactivation curves were evaluated. As expected, faster inactivation was observed at higher temperatures. The critical temperature to accelerate protease decomposition was about 70 °C. The reliable life (t(R)) of the enzyme, analogous to the D value, ranged from 1,824 to 8 min at 45-65 °C. Within these temperatures, an increase of 8.81 °C was needed to lower enzyme t(R) in one-log unit. Protease P7 is a potentially useful biocatalyst for various industrial bioprocesses, and therefore, kinetic modeling of thermal inactivation addresses an important topic aiming enzyme characterization and applications.

Production of Proteolytic Enzymes by a Keratin-Degrading Aspergillus niger.

A fungal isolate with capability to grow in keratinous substrate as only source of carbon and nitrogen was identified as Aspergillus niger using the sequencing of the ITS region of the rDNA. This strain produced a slightly acid keratinase and an acid protease during cultivation in feather meal. The peak of keratinolytic activity occurred in 48 h and the maximum proteolytic activity in 96 h. These enzymes were partly characterized as serine protease and aspartic protease, respectively. The effects of feather meal concentration and initial pH on enzyme production were evaluated using a central composite design combined with response surface methodology. The optimal conditions were determined as pH 5.0 for protease and 7.8 for keratinase and 20 g/L of feather meal, showing that both models were predictive. Production of keratinases by A. niger is a less-exploited field that might represent a novel and promising biotechnological application for this microorganism.

Evaluation of the immunogenicity and in vivo toxicity of the antimicrobial peptide P34.

Immunogenicity and toxicity of antimicrobial peptide P34 were evaluated in vivo. BALB/c mice were inoculated intraperitoneally with peptide P34 alone and associated with Freund's adjuvant. For acute toxicity testing, different concentrations of the peptide P34 (82.5, 165.0, 247.5 and 330.0mg/kg) were orally administered. To evaluate the sub-chronic toxicity the tested dose of 0.825 mg/kg/day of the peptide P34 or nisin were administered for 21 days. There were no hypersensitivity reactions or significant increase in antibody titer during the immunogenicity experiment or death of animals during the acute or sub-chronic toxicity tests. The LD(50) was higher than 332.3 ± 0.76 mg/kg. No significant changes in serum biochemical parameters were observed in the animals treated with the peptide P34 unlike nisin-treated group showed a significant increase in alanine transaminase levels in comparison to controls. The group treated with 0.825 mg/kg/day of nisin showed histological changes in the spleen, skin and liver. In the group treated with peptide P34 histological changes in the spleen were observed, with the presence of megakaryocytes. Few studies report the use of animal models to evaluate the in vivo toxicity of antimicrobial peptides and such investigation is an essential step to ensure it safe use in foods.

Production and properties of keratinolytic proteases from three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils.

The keratinolytic potential and protease properties of three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils was described. Aeromonas hydrophila K12, Chryseobacterium indologenes A22 and Serratia marcescens P3 were able to degrade feather meal, producing high amounts of soluble proteins and forming thiol groups. The proteases of strains K12, A22 and P3 had optimal pH of 8.0, 7.5 and 6.0, respectively; this last is an uncommon feature for bacterial keratinases. The optimal temperature was in the range 45-55°C. All three proteases were active towards azokeratin and were inhibited by EDTA, suggesting that they are keratinolytic metalloproteases. The proteolytic activity of K12 was stimulated by organic solvents and the detergent SDS, suggesting its potential application for detergent formulations and peptide synthesis. Strains A22, K12 and P3 have great potential for use in biotechnological processes involving hydrolysis of keratinous byproducts.