An Alternative Approach to Evaluate the Quality of Protein-Based Raw Materials for Dry Pet Food

The majority of dry pet food currently on the market is produced using fresh meats (FMs) and especially meat meals (MMs) as the main protein source. The transport and storage conditions of the raw materials, together with thermal and mechanical treatments in the case of MMs, may result in undesirable alterations of food products and their protein content. This study was conducted to analyze the protein component of three different kinds of raw materials used for dry pet food production, i.e., chicken, pork, and salmon. The quantitative analysis of the protein component was determined using the traditional Kjeldahl method and near-infrared (NIR) spectroscopy, and an alternative method, i.e., the Bradford assay, while the qualitative analysis was performed through SDS-PAGE, followed by Coomassie Blue staining. The amino acid (AA) profile was also evaluated by quadrupole time-of-flight liquid chromatography/mass spectrometry (Q-TOF LC/MS). In addition, the digestibility was tested through in vitro gastric and small intestine digestion simulation. Statistical analysis was performed by the Student’s t-test, and data are reported as mean ± SEM, n = 10 (p < 0.05). The results showed that the MMs are lower in quality compared to FMs, both in terms of protein bioavailability and digestibility, having a lower soluble protein (SP) content (chicken MM = 8.6 g SP/100 g dry sample; pork MM = 6.2 g SP/100 g dry sample; salmon MM = 7.9 g SP/100 g dry sample) compared to FMs (chicken FM = 14.6 g SP/100 g dry sample; pork FM = 15.1 g SP/100 g dry sample; salmon FM = 13.7 g SP/100 g dry sample). FMs appear, therefore, to be higher-quality ingredients for pet food production. Moreover, the Bradford assay proved to be a quick and simple method to better estimate protein bioavailability in the raw materials used for dry pet food production, thanks to its correlation with the in vitro digestibility

Biopolymer Nanoparticles for Nose-to-Brain Drug Delivery: A New Promising Approach for the Treatment of Neurological Diseases

Diseases affecting the central nervous system (CNS) are among the most disabling and the most difficult to cure due to the presence of the blood–brain barrier (BBB) which represents an impediment from a therapeutic and diagnostic point of view as it limits the entry of most drugs. The use of biocompatible polymer nanoparticles (NPs) as vehicles for targeted drug delivery to the brain arouses increasing interest. However, the route of administration of these vectors remains critical as the drug must be delivered without being degraded to achieve a therapeutic effect. An innovative approach for the administration of drugs to the brain using polymeric carriers is represented by the nose-to-brain (NtB) route which involves the administration of the therapeutic molecule through the neuro-olfactory epithelium of the nasal mucosa. Nasal administration is a non-invasive approach that allows the rapid transport of the drug directly to the brain and minimizes its systemic exposure. To date, many studies involve the use of polymer NPs for the NtB transport of drugs to the brain for the treatment of a whole series of disabling neurological diseases for which, as of today, there is no cure. In this review, various types of biodegradable polymer NPs for drug delivery to the brain through the NtB route are discussed and particular attention is devoted to the treatment of neurological diseases such as Glioblastoma and neurodegenerative diseases

An Alternative Approach to Evaluate the Quality of Protein-Based Raw Materials for Dry Pet Food

The majority of dry pet food currently on the market is produced using fresh meats (FMs) and especially meat meals (MMs) as the main protein source. The transport and storage conditions of the raw materials, together with thermal and mechanical treatments in the case of MMs, may result in undesirable alterations of food products and their protein content. This study was conducted to analyze the protein component of three different kinds of raw materials used for dry pet food production, i.e., chicken, pork, and salmon. The quantitative analysis of the protein component was determined using the traditional Kjeldahl method and near-infrared (NIR) spectroscopy, and an alternative method, i.e., the Bradford assay, while the qualitative analysis was performed through SDS-PAGE, followed by Coomassie Blue staining. The amino acid (AA) profile was also evaluated by quadrupole time-of-flight liquid chromatography/mass spectrometry (Q-TOF LC/MS). In addition, the digestibility was tested through in vitro gastric and small intestine digestion simulation. Statistical analysis was performed by the Student’s t-test, and data are reported as mean ± SEM, n = 10 (p < 0.05). The results showed that the MMs are lower in quality compared to FMs, both in terms of protein bioavailability and digestibility, having a lower soluble protein (SP) content (chicken MM = 8.6 g SP/100 g dry sample; pork MM = 6.2 g SP/100 g dry sample; salmon MM = 7.9 g SP/100 g dry sample) compared to FMs (chicken FM = 14.6 g SP/100 g dry sample; pork FM = 15.1 g SP/100 g dry sample; salmon FM = 13.7 g SP/100 g dry sample). FMs appear, therefore, to be higher-quality ingredients for pet food production. Moreover, the Bradford assay proved to be a quick and simple method to better estimate protein bioavailability in the raw materials used for dry pet food production, thanks to its correlation with the in vitro digestibility

HexA-Enzyme Coated Polymer Nanoparticles for the Development of a Drug-Delivery System in the Treatment of Sandhoff Lysosomal Storage Disease

Lysosomal storage disorders (LSDs) are a set of metabolic diseases caused by mutations in genes that are in charge of the production of lysosomal enzymes, resulting in the buildup of non-degraded substrates and the consequent systemic damage that mainly involves the Central Nervous System (CNS). One of the most widely used and studied treatments is Enzyme Replacement Therapy, which is based on the administration of the recombinant deficient enzyme. This strategy has often proved fallacious due to the enzyme instability in body fluids and its inability to reach adequate levels in the CNS. In this work, we developed a system based on nanotechnology that allows a stable enzyme to be obtained by its covalent immobilization on nanoparticles (NPs) of polylactic acid, subsequently administered to a cellular model of LSDs, i.e., Sandhoff disease, caused by the absence or deficiency of the β-d-N-acetyl-hexosaminidase A (HexA) enzyme. The HexA enzymes, loaded onto the polymeric NPs through an immobilization procedure that has already been investigated and validated, were found to be stable over time, maintain optimal kinetic parameters, be able to permeate the plasma membrane, hydrolyze HexA’s natural substrate, and restore enzyme activity close to the levels of healthy cells. These results thus lay the foundation for testing the HexA-NPs in animal models of the disease and thus obtaining an efficient drug-delivery system

Biogenic Amine Content Analysis of Three Chicken-Based Dry Pet Food Formulations

The pet food market is constantly expanding, and more and more attention is paid to the feeding of pets. Dry foods stand out and are often preferred due to their long shelf life, ease of administration, and low cost. In this context, dry foods are formulated from fresh meats, meat meals, or a mix of the two. These raw materials are often meat not fit for human consumption; they might be subject to contamination and proliferation of microorganisms which, by degrading the organic component, can lead to the formation of undesirable by-products such as biogenic amines. These nitrogenous compounds obtained by decarboxylation of amino acids can therefore be found in high-protein foods, and their ingestion in large quantities can cause intoxication and be harmful. This study aims at analyzing the possible presence of biogenic amines in three different formulations of chicken-based kibbles for pets: one obtained from fresh meat, one from meat meal, and one from a mix of the two. This study is also focused on the presence of free amino acids as they represent the key substrate for decarboxylating enzymes. Mass spectrometry (Q-TOF LC/MS) was used to analyze the presence of biogenic amines and free amino acids. The results show that fresh-meat-based products have a lower content of biogenic amines, and at the same time a higher quantity of free amino acids; on the contrary, meat-meal- and mix-based products have a greater quantity of biogenic amines and a lower concentration of free amino acids, suggesting that there has been a higher microbial proliferation as proved by the total aerobic mesophilic bacteria counts. It is therefore clear that fresh-meat-based kibbles are to be preferred when they are used for preparing dry pet food due to the lowest concentration of biogenic amines

The Hard Choice about Dry Pet Food: Comparison of Protein and Lipid Nutritional Qualities and Digestibility of Three Different Chicken-Based Formulations

Dry pet food, made of fresh meats and especially meat meals, represents one of the main types of complete food available on the market by virtue of its practicality and long shelf life. The kibble production process includes mixed thermal and mechanical treatments that help to improve the palatability and durability of the final product but may have undesirable effects on nutrient bioavailability and digestibility. An analysis of the protein and lipid content of different dry pet food formulations, together with an in vitro digestibility analysis, can reveal which formulation can provide a more nourishing diet for pets. In this study, a quantitative and qualitative analysis was performed on three different formulations of chicken-based dry pet food, consisting of fresh meats, meat meals, or a mix of these two. The soluble protein concentration was determined by the Bradford assay, while the crude protein content was assessed through the Kjeldahl method. Quadrupole time-of-flight liquid chromatography/mass spectrometry (Q-TOF LC/MS) was used to analyze the amino acid (AA) and lipid compositions. Finally, a gastric and small intestinal digestion simulation was used to determine the in vitro digestibility. The results show that dry pet food consisting only of chicken fresh meats has the highest content of soluble protein; it also contains more Essential AAs, Branched-Chain AAs, and Taurine, as well as a greater quantity of monounsaturated and polyunsaturated fatty acids. In addition, its in vitro digestibility was the highest, exceeding 90% of its dry weight, in agreement with the soluble protein content. These findings thus make the fresh-meat-based formulation a preferable choice as dry pet food

Immobilizing Enzymes on a Commercial Polymer: Performance Analysis of a GOx-Laccase Based Enzymatic Biofuel Cell Assembly

Enzymatic Biofuel Cell (EBC) represents a promising green source since it is capable of harvesting electricity from renewable and abundantly available biofuels using enzymes as catalysts. Nevertheless, nowadays long-term stability and low power output are currently the main concerns. To this end, several research studies focus on using complex tridimensional and highly expensive nanostructures as electrode support for enzymes. This increases cell performance whilst drastically reducing the economic feasibility needed for industrial viability. Thus, this paper analyzes a novel flow-based EBC consisting of covalent immobilized GOx (bioanode) and Laccase (biocathode) on a commercial flat conductive polymer. A suitable immobilization technique based on covalent ligands is carried out to enhance EBC durability. The experimental characterization demonstrates that the cell generates power over three weeks, reaching 590 mV and 2.41 &micro;W cm&minus;2 as maximum open circuit voltage and power density, respectively. The most significant contributions of this configuration are definitely ease of implementation, low cost, high scalability, and reproducibility. Therefore, such a design can be considered a step forward in the viable EBC industrialization process for a wide range of applications

Optimizing Covalent Immobilization of Glucose Oxidase and Laccase on PV15 Fluoropolymer-Based Bioelectrodes

Enzymatic biofuel cells (EBCs) represent a promising technology for biosensors, biodevices, and sustainable green energy applications, thanks to enzymes&rsquo; high specificity and catalytic efficiency. Nevertheless, drawbacks such as limited output power and short lifetime have to be solved. Nowadays, research is addressed to the use of 3D electrode structures, but the high cost and the industrialization difficulties of such electrodes represent a key issue. The purpose of the paper is thus to describe the use of a low-cost commercial conductive polymer (Sigracell&reg; PV15) as support for the covalent immobilization of glucose oxidase and laccase, for bioanode and biocathode fabrication, respectively. Efficient immobilization protocols were determined for the immobilized enzymes in terms of employed linkers and enzyme concentrations, resulting in significant enzymatic activities for units of area. The analysis focuses specifically on the optimization of the challenging immobilization of laccase and assessing its stability over time. In particular, an optimum activity of 23 mU/cm2 was found by immobilizing 0.18 mg/cm2 of laccase, allowing better performances, as for voltage output and electrochemical stability, and a direct electron transfer mechanism to be revealed for the fabricated biocathode. This study thus poses the basis for the viable development of low-cost functional EBC devices for biomedical applications

Amphiphilicity-Controlled Localization of Red Emitting Bicationic Fluorophores in Tumor Cells Acting as Bio-Probes and Anticancer Drugs

Small organic molecules arouse lively interest for their plethora of possible biological applications, such as anticancer therapy, for their ability to interact with nucleic acids, or bioimaging, thanks to their fluorescence emission. Here, a panchromatic series of styryl-azinium bicationic dyes, which have already proved to exhibit high water-solubility and significant red fluorescence in water, were investigated through spectrofluorimetric titrations to assess the extent of their association constants with DNA and RNA. Femtosecond-resolved transient absorption spectroscopy was also employed to characterize the changes in the photophysical properties of these fluorophores upon interaction with their biological targets. Finally, in vitro experiments conducted on tumor cell lines revealed that some of the bicationic fluorophores had a peculiar localization within cell nuclei exerting important antiproliferative effects, others were instead found to localize in the cytoplasm without leading to cell death, being useful to mark specific organelles in light of live cell bioimaging. Interestingly, this molecule-dependent behavior matched the different amphiphilicity featured by these bioactive compounds, which are thus expected to be caught in a tug-of-war between lipophilicity, ensured by the presence of aromatic rings and needed to pass cell membranes, and hydrophilicity, granted by charged groups and necessary for stability in aqueous media