Hyperimmunised bovine milk and whey: influence of pH and enzymatic treatments on the antigen-binding capacity of immunoglobulin G.

BACKGROUND: Hyperimmunised bovine milk and whey (whole and defatted) were submitted at 37°C to different pH values (between 1 and 10) and enzymes (pepsin, trypsin and chymotrypsin) at their optimum pH and the IgG immunoactivity against Campylobacter jejuni was measured by means of ELISA assays. RESULTS: The kinetic antigen-binding capacity (ABC) losses follow a hyperbolic-type equation. The ABC of IgG is strongly reduced at low pH (1 and 2) and the effect is lower at alkaline pH (8 and 10). The presence of pepsin (at their optimum pH of 2) almost completely reduced the IgG ABC after 2 h of treatment. The ABCs are higher in whole products (milk and whey). The influence of trypsin and quimotrypsin on the ABCs is moderate (ABC losses lower that 25%). CONCLUSIONS: The ABC of IgG obtained from hyperimmunised bovine defatted milk and whey is largely reduced in conditions similar to those found in the human digestive tract. Only whole milk can maintain around 40% of their initial ABC. IgG encapsulation or other methods to protect the immunoglobulin activities could be an alternative to use these type of products in final foods formulae.

Caseinomacropeptide behaviour in a whey protein fractionation process based on α-lactalbumin precipitation.

This work studied the behaviour of caseinomacropeptide (CMP) in a whey protein fractionation process based on the selective precipitation of α-lactalbumin (α-la) in an acid medium. Three different acids (hydrochloric, citric and lactic) and different operating conditions (protein concentration, temperature and pH) were considered to perform the precipitation step. Under the optimised precipitation conditions obtained for α-la (pH 4, 55°C, initial α-la concentration around 12 g/l) CMP presents quite similar behaviour to that observed for ß-lactoglobulin (ß-lg), namely remaining in the supernatant fraction. However, at a lower pH value (3.5) the amount of precipitated CMP increases up to 72% when citric acid is added. This behaviour could be due to the fact that CMP is close to its isoelectric point, which allows a supernatant fraction enriched in ß-lg that is almost free from the rest of proteins in sweet whey.

Utilization of blood by-products: An in silico and experimental combined study for BSA usage.

In order to exploit industrial discards, protein enzymatic hydrolysis is a currently popular methodology for obtaining bioactive peptides. However, once released, most promising peptides have to be selected from the mixture. In this work, the suitability of pepsin (EC 3.4.23.1) to hydrolyse serum albumin in order to obtain bioactive peptides was assessed. Then, a suitable process to obtain best separation of bioactive peptides was evaluated, using polyethersulfone membranes at different pH values. Serum albumin was easily hydrolysed by pepsin, reaching a DH value of the 65.64 ± 1.57% of the maximum possible. A 23.25% of the identified peptides possessed high bioactivity scores (greater than 0.5), and one of them had reported bioactivity (LLL). Charge mechanisms always predominated over the sieve effect, and best transmission was accomplished at pH values close to the peptides isoelectric points. Basic and neutral peptides with the highest scores were always the most transmitted. Membrane material had greater influence than NMWCO in determining peptide transmission. In order to obtain purified fractions rich in peptides with high bioactivity scores from serum albumin, polyethersulfone membranes (applicable to industrial scale) of 5 kDa MWCO should be used at basic pH values after pepsin digestion.

Influence of heat pre-treatment on BSA tryptic hydrolysis and peptide release.

In contrast with other food proteins, such as ß-lactoglobulin or caseins, intensely studied for bioactive peptide production, relatively little attention has been paid to serum albumin, the main blood protein, even though blood disposal is a severe problem for meat processors. In this study, serum albumin was hydrolysed with trypsin after several heat treatments and using different enzyme concentrations. The degree of hydrolysis reached and the peptide sequences released over time were evaluated. Large differences in enzyme-to-substrate ratios (1:50, 1:100 and 1:200) led to similar degree of hydrolysis values (31.92±1.43%, 31.08±3.09% and 26.21±0.71%), and did not alter the number of peptides released. However, thermal treatment enhanced significantly (p<0.05) both the degree of hydrolysis (up to 50.41±1.90%) and the number and amount of the majority of peptides obtained, all with potential bioactivity (28 peptides in the native hydrolysate, 39 in the thermally treated).

VY6, a ß-lactoglobulin-derived peptide, altered metabolic lipid pathways in the zebra fish liver.

Today enormous research efforts are being focused on alleviating the massive, adverse effects of obesity. Short peptides are key targets for research as they can be generated from natural proteins, like milk. Here we conducted trypsinogen digestion of beta-lactoglobulin (ß-lg), the major mammalian milk protein, to release the hexamer VY6. It was assayed in vivo for its activities on lipid metabolism using zebra fish as a vertebrate model. Zebra fish juveniles were injected with two different doses of the peptide: 100 and 800 µg per g fish and left for 5 days before sacrificing. Lipid measurements showed significant reduction in liver triglycerides and free cholesterol, as well as increased liver HDL cholesterol. Dose-dependent increases of the mRNA levels of the genes coding for the enzymes acyl coenzyme A oxidase 1 (acox1) and lipoprotein lipase (lpl) were also found. The complete results suggest significant anti-obesity activity of the ß-lg-derived VY6 peptide. Its use as a nutraceutical has been discussed.

Enzymatic cleaning of inorganic ultrafiltration membranes fouled by whey proteins.

The aim of this work was to study the cleaning of inorganic membranes fouled with whey protein solutions using the enzymatic formulation Alcalase (Novo Nordisk A/S). Hydraulic and chemical methods were considered to characterize the cleanliness of the membranes. Cleaning efficiency was observed to be a function of the operating conditions. The operating conditions tested were the following: recycling versus non-recycling of permeate, pH of the cleaning solution, addition of alkali to regulate the pH, enzymatic agent concentration, and cleaning time. The best conditions to perform the cleaning were related to the best conditions to hydrolyze whey proteins in a discontinuous reactor using the same enzyme preparations. Very high cleaning efficiencies (>90%) were achieved in short operating times (20 min). However, residual matter was observed on the membrane surface.

ATR-FTIR spectroscopy for the determination of Na4EDTA in detergent aqueous solutions.

Fourier transform infrared spectroscopy in the attenuated total reflectance mode (ATR-FTIR) combined with partial last square (PLS) algorithms was used to design calibration and prediction models for a wide range of tetrasodium ethylenediaminetetraacetate (Na4EDTA) concentrations (0.1 to 28% w/w) in aqueous solutions. The spectra obtained using air and water as a background medium were tested for the best fit. The PLS models designed afforded a sufficient level of precision and accuracy to allow even very small amounts of Na4EDTA to be determined. A root mean square error of nearly 0.37 for the validation set was obtained. Over a concentration range below 5% w/w, the values estimated from a combination of ATR-FTIR spectroscopy and a PLS algorithm model were similar to those obtained from an HPLC analysis of NaFeEDTA complexes and subsequent detection by UV absorbance. However, the lowest detection limit for Na4EDTA concentrations afforded by this spectroscopic/chemometric method was 0.3% w/w. The PLS model was successfully used as a rapid and simple method to quantify Na4EDTA in aqueous solutions of industrial detergents as an alternative to HPLC-UV analysis which involves time-consuming dilution and complexation processes.