Large-scale production of microalgae and their bioactive compounds has steadily increased in response to global demand for natural compounds. Spirulina, in particular, has been used due to its high nutritional value, especially its high protein content. Promising biological functions have been associated with Spirulina extracts, mainly related to its high value added blue pigment, phycocyanin. Phycocyanin is used in several industries such as food, cosmetics, and pharmaceuticals, which increases its market value. Due to the worldwide interest and the need to replace synthetic compounds with natural ones, efforts have been made to optimize large-scale production processes and maintain phycocyanin stability, which is a highly unstable protein. The aim of this review is to update the scientific knowledge on phycocyanin applications and to describe the reported production, extraction, and purification methods, including the main physical and chemical parameters that may affect the purity, recovery, and stability of phycocyanin. By implementing different techniques such as complete cell disruption, extraction at temperatures below 45 °C and a pH of 5.5–6.0, purification through ammonium sulfate, and filtration and chromatography, both the purity and stability of phycocyanin have been significantly improved. Moreover, the use of saccharides, crosslinkers, or natural polymers as preservatives has contributed to the increased market value of phycocyanin.

Microbial production of hyaluronic acid (HA) is an area of research that has been gaining attention in recent years due to the increasing demand for this biopolymer for several industrial applications. Hyaluronic acid is a linear, non-sulfated glycosaminoglycan that is widely distributed in nature and is mainly composed of repeating units of N-acetylglucosamine and glucuronic acid. It has a wide and unique range of properties such as viscoelasticity, lubrication, and hydration, which makes it an attractive material for several industrial applications such as cosmetics, pharmaceuticals, and medical devices. This review presents and discusses the available fermentation strategies to produce hyaluronic acid.

A trypsin-like serine protease was purified by gel filtration and anion-exchange chromatography from the excretory-secretory products of parasitic phase Steinernema carpocapsae. The purified protease exhibited a molecular mass of about 29 kDa by SDS–PAGE and displayed a pI of 6·3. This protease exhibited high activity with trypsin-specific substrate N-Ben-Phe-Val-Arg-p-nitroanilide and was highly sensitive to aprotinin and benzamidine. The purified trypsin protease digested the chromogenic substrate N-Ben-Phe-Val-Arg-p-nitroanilide with K_m, V_max and k_cat values of 594·2 μm, 0·496 μm/min and 22·8/s, respectively. The optimal pH and temperature for protease activity were 9 and 30°C, respectively. Internal amino acid sequencing yielded 150 amino acids and these were homologous to other trypsin sequences. In vitro investigation was carried out to monitor prophenoloxidase suppression in Galleria mellonella by the purified protease; about 38·9–52·6% suppression of prophenoloxidase was observed. The purified protease affected insect haemocyte spreading, causing cells to become spherical or round. Protease-treated actin filaments were highly disorganized in haemocytes. In vitro, G. mellonella haemocytes recognized infective juveniles of Heterorhabditis bacteriophora; however, S. carpocapsae and Steinernema glaseri were not recognized. We provide experimental evidence that the purified trypsin has the potential to alter host haemocytes, actin filaments and to inhibit host haemolymph melanization.

Steinernema carpocapsae is a nematode pathogenic in a wide variety of insect species. The great pathogenicity of this nematode has been ascribed to its ability to overcome the host immune response; however, little is known about the mechanisms involved in this process. The analysis of an expressed sequence tags (EST) library in the nematode during the infective phase was performed and a highly abundant contig homologous to serine protease inhibitors was identified. In this work, we show that this contig is part of a 641-bp cDNA that encodes a BPTI-Kunitz family inhibitor (Sc-KU-4), which is up-regulated in the parasite during invasion and installation. Recombinant Sc-KU-4 protein was produced in Escherichia coli and shown to inhibit chymotrypsin and elastase activities in a dose-dependent manner by a competitive mechanism with K_i values of 1.8 nM and 2.6 nM, respectively. Sc-KU-4 also inhibited trypsin and thrombin activities to a lesser extent. Studies of the mode of action of Sc-KU-4 and its effects on insect defenses suggest that although Sc-KU-4 did not inhibit the activation of hemocytes or the formation of clotting fibers, it did inhibit hemocyte aggregation and the entrapment of foreign particles by fibers. Moreover, Sc-KU-4 avoided encapsulation and the deposition of clotting materials, which usually occurs in response to foreign particles. We show by protein-protein interaction that Sc-KU-4 targets recognition proteins of insect immune system such as masquerade-like and serine protease-like homologs. The interaction of Sc-KU-4 with these proteins explains the ability of the nematode to overcome host reactions and its large pathogenic spectrum, once these immune proteins are well conserved in insects. The discovery of this inhibitor targeting insect recognition proteins opens new avenues for the development of S. carpocapsae as a biological control agent and provides a new tool to study host-pathogen interactions.