Screening of fungal sources of -galactosidase with potential for the synthesis of prebiotics

Book of Abstracts of CEB Annual Meeting 2017[Excerpt] β-Galactosidases (EC 3.2.1.23), also known as lactases, are a family of enzymes able to catalyse two different types of reactions, namely hydrolysis and transgalactosylation. The hydrolytic activity is commonly applied in the food industries to reduce the lactose content of dairy products, preventing lactose crystallization problems and increasing sweetness, flavour and solubility. On the other hand, transgalactosylation reactions have been explored in the synthesis of lactose-based prebiotics, such as galacto-oligosaccharides (GOS), lactosucrose [1] and lactulose [2], with potential application in the pharmaceutical and food industry. These prebiotics are enzymatically produced through the hydrolysis of lactose and further transfer of a galactosyl residue to a suitable acceptor, i.e. fructose for the disaccharide lactulose; sucrose for the trisaccharide lactosucrose; and lactose for GOS. The sources of βgalactosidase are extensively distributed in nature, namely in microorganisms, plants and animal organs. [...]info:eu-repo/semantics/publishedVersio

Enzymatic synthesis of monosaccharide fatty acid esters

The non-ionic and biodegradable surfactant family known as sugar fatty acid esters (SFAEs) includes a wide range of products broadly used in several industrial applications. They are considered as important compounds in the food due to their remarkable technological properties. SFAEs are composed of lipophilic fatty acid moieties and hydrophilic sugar head groups which result in particular physicochemical characteristics that are the basis for their broad range of applications. A great variety of coupling possibilities between hydrophilic sugar head groups and hydrophobic alkyl chains can be explored, leading to the production of different SFAEs with promising industrial features. SFAEs can be obtained by chemical esterification. However, the alternative synthesis by enzymatic route using lipases as biocatalysts has gained increased attention in the last decades. When enzymes are used as catalysts, reactions are carried out in mild conditions, avoiding product degradation. Moreover, enzymes are biodegradable and present higher specificity, thus minimizing the formation of undesirable side-products. In this work, the enzymatic synthesis of SFAEs in t-butanol was followed and qualitatively evaluated by thin layer chromatography. The esterification between different mono-, di- or trisaccharides and butyric acid was performed by commercial immobilized Lipase B from Candida antarctica, at 60°C. The results showed that SFAEs were successfully biosynthesized under these conditions using monosaccharides.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020. BB acknowledges her PhD grant from FCT (2020.05612.BD).info:eu-repo/semantics/publishedVersio

β-glucan from brewers spent yeast as a techno-functional food ingredient

Brewer’s spent yeast (BSY) is a by-product generated during beer production. After heat inactivation, large quantities of BSY are discarded or sold as a low-cost animal feed supplement. Fortunately, BSY can be a good source of valuable compounds such as β-glucan, which has several biological and techno-functional properties for application as a food ingredient. Practical application of β-glucan from BSY requires disruption cell wall and purification steps that significantly influences the yield, cost, biological, physic-chemical, and technological characteristics of this compound. This mini-review presents the use of BSY as a source of β-glucan, the available methods to extract it, and its biological and techno-functional properties.The authors acknowledge the CNPq, CAPES, FAPEG, IF Goiano and UFRJ by support. SS acknowledges the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and by LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020.info:eu-repo/semantics/publishedVersio

Exploitation of olive oil mil wastewater for esterase production

[Excerpt] Development of bio-sustainable and renewable resource technologies is extremely important on environmental contexts. Waste can contain many valuable substances and, through a suitable process or technology, this material can be converted into value-added products or raw materials that can be used in secondary processes. To reduce the environmental problems caused by olive oil mill wastewater (OOMW), which is the liquid effluent produced during the extraction process of olive oil, this waste can be applied to bioproducts production. (...

Polyethylene glycol 8000+ citrate salts aqueous two-phase systems: Relative hydrophobicity of the equilibrium phases

The Gibbs free energy of transfer of a methylene group, G*(CH2), is reported as a measure of the relative hydrophobicity of the equilibrium phases. Furthermore, G*(CH2) is a characteristic parameter of each tie-line, and for that reason can be used for comparing different tie-lines of a given aqueous two-phase system (ATPS) or even to establish comparisons among different ATPSs. In this work, the partition coefficients of a series of four dinitrophenylated-amino acids were experimentally determined, at 23 °C, in five different tie-lines of PEG8000(sodium or potassium) citrate ATPSs. G*(CH2) values were calculated from the partition coefficients and used to evaluate the relative hydrophobicity of the equilibrium phases. PEG8000potassium citrate ATPSs presented larger relative hydrophobicity than PEG8000sodium citrate ATPSs. Furthermore, the results obtained indicated that the PEG-rich phase (top phase) has higher affinity to participate in hydrophobic hydration interactions than the salt-rich phase (bottom phase).This work was partially supported by FCT/MEC and FEDER under Programe PT2020 (Project UID/EQU/50020/2013), and co-financed by QREN, ON2 and FEDER (Projects NORTE-07-0162FEDER-000050 and NORTE-07-0124-FEDER-0000011), for which the authors are thankful. SCS alsoa cknowledges her post-doc grant (SFRH/BPD/88584/2012) from FCT

b-galactosidase from Aspergillus lacticoffeatus: production, characterization and potential application in prebiotic synthesis

The enzyme b-galactosidase, also known as lactase, is widely used in the dairy industry to produce lactose-free milk. This enzyme is able to hydrolyse lactose from milk into galactose and glucose, thus enabling the consumption of milk by lactose-intolerant people. Under suitable conditions, some b-galactosidases can also catalyze transgalactosylation reactions and produce interesting compounds with recognized prebiotic effect, namely galactooligosaccharides (GOS) or lactulose. The enzyme can be obtained from different biological sources such as microorganisms, plants and animals. Nevertheless, the most interesting b-galactosidases for technological applications are those obtained through microbial routes since higher production yields can be achieved. In this study, the fungus Aspergillus lacticoffeatus is described as a new and promising source of b-galactosidase. Preliminary chromogenic tests performed in agar plates suggested that this strain was able to produce the enzyme and additional studies carried out under submerged fermentation conditions confirmed the presence of b-galactosidase in the fermentation broth, as well as in the cell extract obtained after ultrasonic cell disruption. The enzyme production was evaluated in different fermentation media: synthetic medium composed by lactose (20 g/L), yeast extract (4g/L), peptone (4g/L) and salts; and fermentation media with some industrial byproducts as cheese whey and/or corn steep liquor. However, the higher values of enzymatic activity (444 U/L) were obtained using the synthetic medium. The enzyme presented a molecular weight around 130 kDa and optimal pH and temperature in the range 3.5-4.5 and 50-55 ºC, respectively. The effect of some metal ions (Na+, K+, Li+, Ba2+, Fe2+, Mg2+, Zn2+, Mn2+, Co2+ and Cu2+), detergents (Triton, SDS and Tween), additives (EDTA, PMSF and ascorbic acid) and sugars (glucose, fructose and galactose) on the enzymatic activity was also evaluated. Afterwards, the potential of the enzyme for the synthesis of prebiotics was studied and it was demonstrated that b-galactosidase from A. lacticoffeatus is able to catalyze the transfer reactions involved in the formation of lactulose and GOS

Simultaneous synthesis of prebiotic mixtures containing GOS and FOS

FCT (UID/BIO/04469/2013 unit); COMPETE 2020 (POCI-01-0145-FEDER- 006684); project BioTecNorte (NORTE-01-0145-FEDER-000004) and project MultiBiorefinery (POCI-01-0145-FEDER-016403) funded by the European Regional Development Fund under the scope of Norte2020. SCS acknowledges her grant (SFRH/BPD/88584/2012) from FCTinfo:eu-repo/semantics/publishedVersio

Using agro-industrial byproducts for a more sustainable production of natural pigments

Pigments have a vast record of enhancing product appeal in industries like food, cosmetics, textiles, pharmaceuticals, and tanneries. A shift towards eco-consciousness has fueled the demand for biocompatible, natural pigments, prompting interest in microbial fermentation. Filamentous fungi, particularly Penicillium species, stand out as promising pigment producers. Recently, we have demonstrated that a Penicillium strain is able to produce different pigment mixtures under different fermentation conditions and culture media [1], [2]. Using agro-industrial byproducts, such as cheese whey and corn steep liquor, as substrates for microbial growth can be a sustainable approach to reduce production costs and byproduct accumulation. Both cheese whey and corn steep liquor are rich in valuable nutrients and were shown to enhance pigment production when used as medium supplements [1]. In this work, we present a comparative study involving three fermentation types (submerged, submerged with biomass immobilization, and solid-state fermentation) and employing two distinct culture media (synthetic medium composed of commercial substrates (A), and an alternative medium only composed of cheese whey and corn steep liquor (B)). Notably, we found that, under submerged fermentation either with free or immobilized biomass, the alternative medium (B) provides similar results in terms of pigment production to the reference synthetic medium (A). These results show that it is possible to obtain a value-added product exclusively using agro- industrial byproducts, which not only decreases the associated production costs but also contributes to the circular economy. Moreover, we describe a more sustainable approach to obtain natural pigments, which can also help to address environmental concerns, ethical issues, and/or consumer demands raised against synthetic pigments.info:eu-repo/semantics/publishedVersio

Construction of a novel and safe S. cerevisiae biocatalyst for lactulose production

Prebiotics are defined as substrates that are utilized by host microorganisms conferring a health benefit. These compounds have been incorporated in a wide variety of food products1. One of the most well-recognized prebiotics is lactulose. Lactulose is not found naturally so it has to be produced through different methods: chemical or enzymatic synthesis and electro-activation2. Recently, the production of lactulose through lactose isomerization catalyzed by cellobiose 2- epimerase from Caldicellulosiruptor saccharolyticus (CsCE) was reported3. This strategy is gaining attention as a preferable methodology for industrial application due to its notable yields. Saccharomyces cerevisiae, one of the most well-characterized microorganisms, is widely used for the heterologous production of several enzymes, also due to the diverse genetic manipulating tools that are currently available. Here, we propose a new and promising S. cerevisiae biocatalyst. Taking advantage of its GRAS status and using lactose as a single substrate, we believe that it can be a more economic and attractive approach for the synthesis of lactulose. The CsCE gene was cloned in the CEN.PK2-1C S. cerevisiae strain, under TEF or GAP promoters control. Both biocatalysts were used in lactulose production, reaching a concentration of 1.26 g/L (TEF promoter) and 2.15 g/L (GAP promoter). The maximum prebiotic yield was 6.20%. These promising results represent the first use of a S. cerevisiae biocatalyst for lactulose production and demonstrated its potential as a sustainable and safe approach for food application.BBC acknowledge her doctoral grant (SFRH/BD/132324/2017) from the Portuguese Foundation of Science and Technology (FCT). This study was supported by FCT under the scope of the strategic funding of UID/BIO/04469/2019 unit, the Project FoSynBio (POCI-01-0145-FEDER-029549) and BioTecNorte operation (NORTE-01-0145-FEDER000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

The use of the Collander equation as solute partition-predictive model

Efficient and economical downstream processing of biological products has been one of the main challenges of biotechnology industry. Aqueous two-phase systems (ATPSs) are long known to be a promising separation technique and a valuable alternative to the conventional approaches. Since their first application, ATPSs have been used for a wide range of applications, mainly in the separation and recovery of bioproducts. Their clear advantageous features and their potential as extraction technique has been demonstrated through the years. Regardless of their potential, only recently the use of ATPSs by the industry sector has received relevant interest. Yet, for their successful use it is important to study systems properties at molecular level and understand the mechanisms of solute partitioning. The Collander equation was proposed to describe solute partition in water-organic solvent systems, but this model has been effectively extended to correlate partition of unrelated compounds in two (or more) different ATPSs, supporting the idea that this model can be used to predict partitioning in ATPSs. So far, the use of the Collander equation to describe and predict the partition of solutes present in complex mixtures, in ATPSs, was never reported. Thus, we attempted to apply this empirical model to a real case scenario of ATPS partitioning, aiming the recovery of three natural pigments obtained by submerged fermentation of a Penicillium strain.info:eu-repo/semantics/publishedVersio