Enzymatic processing of animal by-products: production of antioxidant hydrolysates with Bacillus sp. CL18 crude protease.

Protein hydrolysates might display diverse bioactivities with potential relevance to human and animal health and food technology. Enzymatic hydrolysis of agro-industrial by-products is increasingly focused. In this study, a crude protease from Bacillus sp. CL18 was applied to obtain antioxidant protein hydrolysates from porcine, bovine, poultry, and fish by-products. The crude enzyme hydrolyzed all the twelve investigated by-products, as detected by increased soluble protein contents after 4 h of proteolysis. Hydrolysates exhibited higher radical-scavenging, Fe2+-chelating and reducing power capacities than non-hydrolyzed by-products. Hydrolysis times (0-8 h) and enzyme-to-substrate (E/S) ratios (384, 860, and 1,400 U/g) were assessed to produce antioxidant bovine lung hydrolysates. The highest E/S ratio accelerated both hydrolysis and increases in antioxidant activities; however, it did not result in bioactivities higher than hydrolysates obtained with the intermediate E/S ratio. Optimal antioxidant activities could be reached after 6 h of hydrolysis using 860 U/g. Animal by-products are interesting sources of bioactive protein hydrolysates, which could be produced with a non-commercial bacterial protease. This might represent a promising strategy for the valorization of animal by-products generated in large amounts by the agri-food sector.

Characterization and application of a crude bacterial protease to produce antioxidant hydrolysates from whey protein.

Bacillus sp. CL14 crude protease was partially characterized and applied to obtain antioxidant whey protein isolate (WPI) hydrolysates. Optimal activity occurred at pH 9.0 and 60 °C. Ca2+, Mg2+, and Mn2+ (5 mM) enhanced activity (12-26%), whereas Co2+, Cu2+, Fe2+, and Zn2+ inhibited it (50-94%). At 1% (v/v), Tween 20 and Triton X-100 enhanced activities (21-27%), ß-mercaptoethanol decreased it (15%), and dimethyl sulfoxide (DMSO) had no effect. Sodium dodecyl sulfate (SDS; 0.1%, w/v) increased activity by 36%. Complete inhibition by phenylmethylsulfonyl fluoride (PMSF), and 85% inhibition by ethylenediaminotetraacetic acid, indicates its serine protease character and the importance of cations for activity/stability. With 5 mM Ca2+, protease was optimally active at 65 °C and completely stable after 20 min at 40-55 °C. Crude protease preferentially hydrolyzed WPI and soy protein, followed by casein. WPI hydrolysis was then performed (55 °C, pH 9.0, 5 mM Ca2+) for 0-180 min. Contents of trichloroacetic acid (TCA)-soluble proteins in WPI hydrolysates (HWPI) increased from 29% (0 min) to 50-52% (60-180 min), accompanied by enhanced radical scavenging activity (14%, 0 min; ∼34%, 60-180 min) and Fe2+-chelating ability (56%, 0 min; ∼74%, 45-180 min). CL14 protease might represent an alternative biocatalyst to obtain antioxidant hydrolysates from WPI and, potentially, from other food proteins.

Beyond plucking: Feathers bioprocessing into valuable protein hydrolysates.

The livestock production and subsequent processing of meat results in huge quantities of solid waste such as viscera, bones, skin and keratin-rich materials, including feathers, hair, wool, claws and hooves. In particular, the continuous growth of poultry industry generates massive amounts of feathers as major waste material. The conversion of such by-products into materials with increased value has been studied. Hydrothermal, chemical or biological approaches have been investigated to achive effective conversion of highly recalcitrant proteins that are abundant in animal waste, but increasing interest is devoted to the development of biotechnological methods. The processing of feathers and other by-products into protein hydrolysates may have industrial and commercial significance. Therefore, this review comprehensively addresses the postulated applications of hydrolysates obtained from keratinous biomasses. Examples on the utilization of feather hydrolysates as organic soil fertilizers, feed ingredients, cosmetic formulations and biofuel production are described in the literature. Microbial feather hydrolysis can generate bioactive peptides as well. The use of protein-rich waste from meat industry to produce hydrolysates with biological activities constitutes a point of utmost interest for development of functional ingredients with elevated value.

Co-production of Proteases and Bioactive Protein Hydrolysates from Bioprocessing of Feather Meal

Abstract: Feather meal conversion through submerged cultivations with Bacillus strains (CL33A, CL14) yielded proteases and protein hydrolysates. After 4-day (CL33A) and 10-day (CL14) cultivations, protease activities reached 461 U/mL; hydrolysates presented antioxidant (radicals-scavenging, 57-77%; Fe2+-chelation, 14-28%; Fe3+-reduction) and antidiabetic (dipeptidyl peptidase-IV inhibition, 49-52%) potentials. The obtained bioproducts present prospective commercial/industrial applications.

Antimicrobial activity of free and liposome-encapsulated thymol and carvacrol against Salmonella and Staphylococcus aureus adhered to stainless steel.

Antimicrobial activity of thymol, carvacrol and thymol/carvacrol liposomes (TCL) was evaluated against two bacterial pools, each one consisting of four strains of Staphylococcus aureus or Salmonella enterica. TCL were prepared using thin-film hydration, showing 270.20nm average diameter (polydispersity index of 0.33) and zeta potential of +39.99mV. Minimum inhibitory concentration (MIC) of thymol, carvacrol and TCL against S. aureus pool was 0.662mg/ml, while MIC for Salmonella pool was 0.331mg/ml for thymol and carvacrol, and for TCL was 0.662mg/ml. Bacterial pools (8.0logCFU/ml), allowed in contact on stainless steel AISI 304 coupons in UHT skim milk for 15min, resulted in adhered populations of 5.6-6.1logCFU/cm2. Adhered S. aureus (±6.1logCFU/cm2) were inhibited after 1-min and 10-min treatments using thymol or carvacrol at MIC and 2.0 MIC. Reductions of 1.47-1.76logCFU/cm2 and 1.87-2.04logCFU/cm2 were obtained using 0.5 MIC of thymol and carvacrol, respectively. A 10-min contact with free (MIC and 2.0 MIC) and encapsulated (MIC) antimicrobials inhibited attached Salmonella (±6.0logCFU/cm2); however, after 1-min of contact, 2.0 MIC of thymol and carvacrol were not able to inactivate adhered Salmonella MIC of TCL inactivated S. aureus and Salmonella after 10min; however, after 1-min contact, adhered S. aureus and Salmonella populations were decreased in 1.62logCFU/cm2 and 2.01logCFU/cm2, respectively. Considering antimicrobial concentrations and contact times, thymol, carvacrol, and TCL could be employed in food-contact surfaces to prevent biofilm formation at early stages of bacterial attachment. Further investigations should be performed considering long-term antibacterial effects of TCL.

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.

Inhibition of listeria monocytogenes in minas frescal cheese by free and nanovesicle-encapsulated nisin

The effectiveness of free and nanovesicle-encapsulated nisin to control Listeria monocytogenes in Minas Frescal cheese was investigated. Commercial nisin was encapsulated into liposomes of partially purified soy lecithin. Free (0.1 mg/mL and 0.25 mg/mL) and nanovesicle-encapsulated nisin (0.25 mg/mL) were applied onto the surface of cheese samples, and L. monocytogenes was inoculated before incubation at 6-8°C for 28 days. A bactericidal effect was observed with 0.25 mg/mL free nisin; a bacteriostatic effect was observed for liposome-encapsulated nisin and 0.1 mg/mL free nisin. Free nisin was more efficient than nisin-loaded liposomes in controlling L. monocytogenes. Possible reasons for this behavior, and also the significance of nisin to soft cheeses are discussed. Nisin acted as a suitable barrier within hurdle technology, potentially extending the shelf-life and safety of fresh cheeses.