Generation and characterization of avian IgY antibodies for detecting beta-casein A1 in bovine milk.

Beta-casein is a primary milk protein that constitutes approximately 30% of the casein in bovine milk, with the two most common types in cattle being A1 and A2. The A2 protein differs from the A1 version due to a mutation in the codon at position 67, resulting in a histidine to proline substitution. However, the bioactive peptide, beta-casomorphine-7 (BCM7), which originates from partial proteolysis of the A1 variant, has been linked to several gastrointestinal disorders in humans. Production of A1 beta casein-free products is increasing demand in the milk market, worldwide. This study generated and characterized a polyclonal IgY antibody that specifically recognizes the A1 beta-casein protein present in cow's milk. A commercially available IgY anti-A1 antibody was used as a positive control, and the sensitivity and specificity of both the commercial and produced anti-A1 antibodies were evaluated. The results showed 100% sensitivity and specificity of 100% of the commercial IgY anti-A1. The in-house produced anti-A1 antibody demonstrated a sensitivity of 95.2% and a specificity of 100%, indicating its potential as a reliable and cost effective tool for detecting A1 beta-casein protein in milk samples.

In Vitro Diagnostic Assay to Detect SARS-CoV-2-Neutralizing Antibody in Patient Sera Using Engineered ACE-2 Mini-Protein.

The recent development and mass administration of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines allowed for disease control, reducing hospitalizations and mortality. Most of these vaccines target the SARS-CoV-2 Spike (S) protein antigens, culminating with the production of neutralizing antibodies (NAbs) that disrupt the attachment of the virus to ACE2 receptors on the host cells. However, several studies demonstrated that the NAbs typically rise within a few weeks after vaccination but quickly reduce months later. Thus, multiple booster administration is recommended, leading to vaccination hesitancy in many populations. Detecting serum anti-SARS-CoV-2 NAbs can instruct patients and healthcare providers on correct booster strategies. Several in vitro diagnostics kits are available; however, their high cost impairs the mass NAbs diagnostic testing. Recently, we engineered an ACE2 mimetic that interacts with the Receptor Binding Domain (RBD) of the SARS-2 S protein. Here we present the use of this engineered mini-protein (p-deface2 mut) to develop a detection assay to measure NAbs in patient sera using a competitive ELISA assay. Serum samples from twenty-one patients were tested. Nine samples (42.8%) tested positive, and twelve (57.1%) tested negative for neutralizing sera. The data correlated with the result from the standard commercial assay that uses human ACE2 protein. This confirmed that p-deface2 mut could replace human ACE2 in ELISA assays. Using bacterially expressed p-deface2 mut protein is cost-effective and may allow mass SARS-CoV-2 NAbs detection, especially in low-income countries where economical diagnostic testing is crucial. Such information will help providers decide when a booster is required, reducing risks of reinfection and preventing the administration before it is medically necessary.

Salivary ß-glucosidase as a direct factor influencing the occurrence of halitosis.

ß-Glucosidases are enzymes present in all living organisms, playing a pivotal role in diverse biological processes. These enzymes cleave ß-glycosidic bonds between carbohydrates, or between a carbohydrate and a non-carbohydrate moiety, which may result in the liberation of volatile aglycones. Released compounds execute diverse physiological roles, while the industry takes advantage of exogenously added ß-glucosidases for aroma enrichment during food and beverage production. ß-Glucosidase enzymatic activity has been reported in human saliva and given the fact that these enzymes are involved in aroma release, we investigated here the correlation between ß-glucosidase activity in human saliva and the occurrence of halitosis. Measurement of salivary enzyme activity of 48 volunteers was performed using p-nitrophenyl-ß-d-glucopyranoside as substrate. Each volunteer was clinically evaluated by a dental surgeon and clinical and laboratorial data were statistically analyzed. Gas-chromatography of saliva headspace allowed the analysis of the direct role of exogenous ß-glucosidase on aromatic /volatile profile of saliva samples. The data demonstrated a positive correlation between halitosis and enzymatic activity, suggesting that the enzyme exerts a direct role in the occurrence of bad breath. Gas-chromatography analysis demonstrated that exogenously added enzyme led to the alteration of volatile organic content, confirming a direct contribution of ß-glucosidase activity on saliva volatile compounds release. Although halitosis is a multifactorial condition, the complete understanding of all governing factors may allow the development of more effective treatment strategies. Such studies may pave the way to the use of ß-glucosidase inhibitors for halitosis clinical management.

Using natural biomass microorganisms for drinking water denitrification.

Among the methods that are studied to eliminate nitrate from drinking water, biological denitrification is an attractive strategy. Although several studies report the use of denitrifying bacteria for nitrate removal, they usually involve the use of sewage sludge as biomass to obtain the microbiota. In the present study, denitrifying bacteria was isolated from bamboo, and variable parameters were controlled focusing on optimal bacterial performance followed by physicochemical analysis of water adequacy. In this way, bamboo was used as a source of denitrifying microorganisms, using either Immobilized Microorganisms (IM) or Suspended Microorganisms (SM) for nitrate removal. Denitrification parameters optimization was carried out by analysis of denitrification at different pH values, temperature, nitrate concentrations, carbon sources as well as different C/N ratios. In addition, operational stability and denitrification kinetics were evaluated. Microorganisms present in the biomass responsible for denitrification were identified as Proteus mirabilis. The denitrified water was submitted to physicochemical treatment such as coagulation and flocculation to adjust to the parameters of color and turbidity to drinking water standards. Denitrification using IM occurred with 73% efficiency in the absence of an external carbon source. The use of SM provided superior denitrification efficiency using ethanol (96.46%), glucose (98.58%) or glycerol (98.5%) as carbon source. The evaluation of the operational stability allowed 12 cycles of biomass reuse using the IM and 9 cycles using the SM. After physical-chemical treatment, only SM denitrified water remained within drinking water standards parameters of color and turbidity.

Kinetic and biochemical characterization of Trypanosoma evansi nucleoside triphosphate diphosphohydrolase.

Nucleoside triphosphate diphospho-hydrolases (NTPDases) catalyze the hydrolysis of several nucleosides tri and diphosphate playing major roles in eukaryotes including purinergic signaling, inflammation, hemostasis, purine salvage and host-pathogen interactions. These enzymes have been recently described in parasites where several evidences indicated their involvement in virulence and infection. Here, we have investigated the presence of NTPDase in the genome of Trypanosoma evansi. Based on the genomic sequence from Trypanosoma brucei, we have amplified an 1812 gene fragment corresponding to the T. evansi NTPDase gene. The protein was expressed in the soluble form and purified to homogeneity and enzymatic assays were performed confirming the enzyme identity. Kinetic parameters and substrate specificity were determined. The dependence of cations on enzymatic activity was investigated indicating the enzyme is stimulated by divalent cations and carbohydrates but inhibited by sodium. Bioinformatic analysis indicates the enzyme is a membrane bound protein facing the extracellular side of the cell with 98% identity to the T. brucei homologous NTPDase gene.

Substrate specificity and enzyme recycling using chitosan immobilized laccase.

The immobilization of laccase (Aspergillus sp.) on chitosan by cross-linking and its application in bioconversion of phenolic compounds in batch reactors were studied. Investigation was performed using laccase immobilized via chemical cross-linking due to the higher enzymatic operational stability of this method as compared to immobilization via physical adsorption. To assess the influence of different substrate functional groups on the enzyme's catalytic efficiency, substrate specificity was investigated using chitosan-immobilized laccase and eighteen different phenol derivatives. It was observed that 4-nitrophenol was not oxidized, while 2,5-xylenol, 2,6-xylenol, 2,3,5-trimethylphenol, syringaldazine, 2,6-dimetoxyphenol and ethylphenol showed reaction yields up 90% at 40 °C. The kinetic of process, enzyme recyclability and operational stability were studied. In batch reactors, it was not possible to reuse the enzyme when it was applied to syringaldazne bioconversion. However, when the enzyme was applied to bioconversion of 2,6-DMP, the activity was stable for eight reaction batches.