In the last two decades, the application and processing of proteins in food, biological, pharmaceutical and chemistry industries have been increased due to their applications such as food supplement (whey protein) [1, 2], as hormones, vaccines, antibiotics, antibodies and enzymes in biopharmaceutical [3], among others. Obtaining high quality proteins to delivery to the final consumers is a major concern of the industries and, for this, processing them is necessary. Some of used unitary operations are purification, concentration, mixing, pumping, heating, and delivery. These procedure steps can induce protein denaturation and as consequence the loss of biological activity. [4, 5]. The commonly technique used to guarantee good quality of products is to add additives, preservatives, buffer, etc. However, these additional components can induce side effects.
Chromatography and membrane separations are commonly used for the purification of proteins during the processing [6]. The use of each is based upon the process and economic constrains. Using chromatography techniques, proteins can be purified considering their size, shape, total charge, hydrophobic groups at the surface and the binding capacity with the stationary phase. They are widely used due to their high degree of purity and recovery of protein [7]. However, chromatography is expensive, difficult to scale up or work in batch operations [6].
Membrane processes, on the other hand, are a greater alternative. Their advantages include low energy consumption, high selectivity, continuous possibility of separation, mild conditions of operation and the possibility to avoid any additional chemicals (additives). Another important feature is the possibility to adjust and vary the membrane properties in order to be the best fit for the process. [8]. However, membrane processes are susceptible to fouling, that is, the deposition of undesired particle on the membrane pores or surface, decreasing the permeate flux and limiting the mass transfer. Commonly, membrane fouling presents simultaneously inorganic/organic, particulate, and microbial fouling. [9]. Various studies address the problem and propose different solutions such as: use a hydrophilic membrane [10], cross flow filtration [11], low-temperature plasma treatment [12] or increase feed solution velocity to limit polarization layer [13]. Beside the purification step, in order to produce a large quantity of purified proteins, the solution must be concentrated.
Ultrafiltration (UF) is a common choice for membrane process as it can be used concomitantly for protein concentration, buffer exchange, desalting, protein purification and viruses and bacteria clearance [14]. It is a pressure driven process for molecules with a molecular weight from 500 g.mol− 1 to 500 kg.mol− 1 and diameter of 1-100 nm. [15]. Considering these values, it can retain solutes such as nanoparticles, colloids, and large molecules. Besides that, depending on the feeding solution, it is possible to choose different membrane materials varying from organic to inorganic ones. Inorganic membranes, for example made of ceramic material, are more stable than organic ones at high temperature and pH conditions and that is the reason of their higher price. [16].
In UF method, protein retention is very high but the efficiency of ultrafiltration can be affected by the characteristics of the membrane (material, pore size, surface charge, hydrophilicity), the protein solution (concentration, species, pH) and by the operating conditions (pressure, temperature, fluid velocity, etc.). [17]
Nevertheless, during the manufacturing process, the proteins are also subjected to changes in temperature, pH, pressures, to different organic solvents, to shearing, shaking etc. For example, during purification by membrane processes, the protein can be exposed to shear rates between 1000 and 10 000 s− 1. [18]. High shear can deform the three-dimensional structure of the protein and affect its inherent biological activity. Charm and Wong reported that by increasing the shear (shear rate 1155s− 1), the catalase enzyme suffers a decrease in activity up to 45%. [19]. Bowen and Gan explain the loss of activity by the membrane-enzyme interaction resulting from the shear induced deformation of the enzyme structure during microfiltration. [20]. During filling or packaging, proteins can also be subjected to agitation [21, 22] or shear stress (shear rates up to 20 000 s− 1 for 20 gauges by 10 cm needle, 0.5 ml.s− 1 rate) [18].
Lysozyme is a globular protein composed of 129 amino acids sequence and its molecular weight is approximately 14.6 kDa. It comes from hen egg white and is high natural abundant. It presents antibacterial action destroying the bacterial cell wall by polysaccharide hydrolysis, it’s also used as active ingredient in antivirus and antitumoral pharmaceutical drugs. [23–25].
Considering the possible influence of protein conformation into its biological activity, the aim of this work is to evaluate the behavior and antibacterial activity of lysozyme when ultrafiltration is used as a method for concentration. Different membranes and applied pressures were tested and antibacterial tests against Micrococcus Lysodeikticus were performed to understand the effect of processing conditions into protein applications.