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Abstract: An early diagnosis has the potential to greatly decrease cancer mortality. For that purpose, specific cancer biomarkers have been molecularly targeted by aptamer sequences to enable an accurate and rapid detection. Aptamer-based biosensors for cancer diagnostics are a promising alternative to those using antibodies, due to their high affinity and specificity to the target molecules and advantageous production. Synthetic nucleic acid aptamers are generated by in vitro Systematic Evolution of Ligands by Exponential enrichment (SELEX) methodologies that have been improved over the years to enhance the efficacy and shorten the selection process. Aptamers have been successfully applied in electrochemical, optical, photoelectrochemical and piezoelectrical-based detection strategies. These aptasensors comprise a sensitive, accurate and inexpensive option for cancer detection being used as point-of-care devices. This review highlights the recent advances in cancer biomarkers, achievements and optimizations made in aptamer selection, as well as the different aptasensors developed for the detection of several cancer biomarkers.

Enrichment and diagnosis tools for pathogens currently available are time consuming, thus the development of fast and highly sensitive alternatives is desirable. In this study, a novel approach was described that enables selective capture of bacteria expressing hydrolyzed collagen-binding adhesins with hydrolyzed collagen-coated magnetic nanoparticles (MNPs). This platform could be useful to shorten the time needed to confirm the presence of a bacterial infection. MNPs were synthesized by a simple two-step approach through a green co-precipitation method using water as solvent. These MNPs were specifically designed to interact with pathogenic bacteria by establishing a hydrolyzed collagen-adhesin linker. The bacterial capture efficacy of hydrolyzed collagen MNPs (H-Coll@MNPs) for bacteria expressing collagen binding adhesins was 1.3 times higher than that of arginine MNPs (Arg@MNPs), herein used as control. More importantly, after optimization of the MNP concentration and contact time, the H-Coll@MNPs were able to capture 95% of bacteria present in the samples. More importantly, the bacteria can be enriched within 30 min and the time for bacterial identification is effectively shortened in comparison to the "gold standard" in clinical diagnosis. These results suggest that H-Coll@MNPs can be used for the selective isolation of specific bacteria from mixed populations present, for example, in biological samples.

10 pags., 2 figs., 1 tab., Zymomonas mobilis ZM4 is an attractive host for the development of microbial cell factories to synthesize high-value compounds, including prebiotics. In this study, a straightforward process to produce fructooligosaccharides (FOS) from sucrose was established. To control the relative FOS composition, recombinant Z. mobilis strains secreting a native levansucrase (encoded by sacB) or a mutated β-fructofuranosidase (Ffase-Leu196) from Schwanniomyces occidentalis were constructed. Both strains were able to produce a FOS mixture with high concentration of 6-kestose. The best results were obtained with Z. mobilis ZM4 pB1-sacB that was able to produce 73.4 ± 1.6 g L-1 of FOS, with a productivity of 1.53 ± 0.03 g L-1 h-1 and a yield of 0.31 ± 0.03 gFOS gsucrose-1. This is the first report on the FOS production using a mutant Z. mobilis ZM4 strain in a one-step process. KEY POINTS: • Zymomonas mobilis was engineered to produce FOS in a one-step fermentation process. • Mutant strains produced FOS mixtures with high concentration of 6-kestose. • A new route to produce tailor-made FOS mixtures was presented., The study received financial support from Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the project FoSynBio—Synthetic biology approaches to design and construct microbial cell factories for the production of fructooligosaccharides (POCI-01–0145-FEDER-029549).

For the first time, an aptamer that specifically binds to cathepsin B (CatB)-overexpressing prostate cancer cells was identified by combining SELEX and computational tools.

The world economy is currently moving towards more sustainable approaches. Lignocellulosic biomass has been widely used as a substitute for fossil sources since it is considered a low-cost bio-renewable resource due to its abundance and continuous production. Compost habitats presenting high content of lignocellulosic biomass are considered a promising source of robust lignocellulose-degrading enzymes. Recently, several novel biocatalysts from different environments have been identified using metagenomic techniques. A key point of the metagenomics studies is the extraction and purification of nucleic acids. Nevertheless, the isolation of high molecular weight DNA from soil-like samples, such as compost, with the required quality for metagenomic approaches remains technically challenging, mainly due to the complex composition of the samples and the presence of contaminants like humic substances. In this work, a rapid and cost-effective protocol for metagenomic DNA extraction from compost samples composed of lignocellulosic residues and containing high content of humic substances was developed. The metagenomic DNA was considered as representative of the global environment and presented high quality (>\thinspace99\\% of humic acids effectively removed) and sufficient quantity (10.5--13.8 \textmug g1 of compost) for downstream applications, namely functional metagenomic studies. The protocol takes about 4 h of bench work, and it can be performed using standard molecular biology equipment and reagents available in the laboratory., 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 the Project LIG-NOZYMES—Metagenomics approach to unravel the potential of lignocellulosic residues towards the discovery of novel enzymes (POCI-01–0145-FEDER-029773)., info:eu-repo/semantics/publishedVersion

Microbial exopolysaccharides (EPS) are promising alternatives to synthetic polymers in a variety of applications. Their high production costs, however, limit their use despite their outstanding properties. The use of low-cost substrates such as agro-industrial wastes in their production, can help to boost their market competitiveness. In this work, an alternative low-cost culture medium (CSLM) was developed for EPS production by

" Available online 22 May 2022", This study explores the structural characterization, antioxidant and prebiotic activities of hydrolysates containing xylooligosaccharides (XOS) produced by different strategies: direct fermentation of beechwood xylan (FermBX) and enzymatic treatment of beechwood (EnzBX) and rice husk (EnzRH) xylans. EnzBX and EnzRH showed XOS with a backbone of (1 4)-linked-xylopyranosyl residues and branches of arabinose, galactose, and uronic acids. FermBX presented the highest content of total phenolic compounds (14 mg GAE/g) and flavonoids (0.6 mg QE/g), which may contribute to its antioxidant capacity 39.1 mol TE/g (DPPH), 45.7 mol TE/g (ABTS), and 79.9 mol Fe II/g (FRAP). The fermentation of hydrolysates decreased the abundance of microorganisms associated with intestinal diseases from Eubacteriales, Desulfovibrionales and Methanobacteriales orders, while stimulating the growth of organisms belonging to Bacteroides, Megamonas and Limosilactobacillus genera. The production of short-chain fatty acids, ammonia, and CO2 suggested the prebiotic potential. In conclusion, hydrolysates without previous purification and obtained from non-chemical approaches demonstrated promising biological activities for further food applications., This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) – Finance Code 001; by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) – Grant numbers 423285/2018-1 and 304857/2018-1; by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit; by the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte; by the COMPETE 2020 (POCI-01-0145- FEDER-006684); by the FoSynBio (POCI-01-0145-FEDER-029549); and LAQV/REQUIMTE (UIDB/50006/2020, UIDP/50006/2020) through national founds and, where applicable, co-financed by the FEDER, within the PT2020 Partnership Agreement. GG acknowledges the Universidade Federal do Rio Grande for the financial support from Postgraduate Student Mobility Program (PROPESP/FURG). BC, CA, and SPS acknowledge their grants (SFRH/BD/132324/2017, 2020.00293 CEECIND, and SFRH/BD/136471/2018) from FCT. LB also acknowledge FCT for the junior research contract (CEECIND/03280/2020). EC thanks the research contract (CDL-CTTRI-88-ARH/2018 – REF. 049-88-ARH/2018) funded by national funds (OE), through FCT, in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19., info:eu-repo/semantics/publishedVersion

Despite recent preclinical progress with oncolytic bacteria in cancer therapy, dose-limiting toxicity has been a long-standing challenge for clinical application. Genetic and chemical modifications for enhancing the bacterial tumor-targeting ability have been unable to establish a balance between increasing its specificity and effectiveness while decreasing side effects. Herein, we report a simple, highly efficient method for rapidly self-assembling a clinically used lipid on bacterium and for reducing its minimum effective dose and toxicity to normal organs. The resultant bacteria present the ability to reverse-charge between neutral and acidic solutions, thus enabling weak interactions with the negatively charged normal cells, hence increasing their biocompatibility with blood cells and with the immune system. Additionally, the lipid-coated bacteria exhibit a longer blood circulation lifetime and low tissue trapping compared with the wild-type strains. Thereby, the engineered bacteria show enhanced tumor specificity and effectiveness even at low doses. Multiple visualization techniques are used for vividly demonstrating the time course of bacterial circulation in the blood and normal organs after intravenous administration. We believe that these methods for biointerfacial lipid self-assembly and evaluation of bacterial systemic circulation possess vast potential in exquisitely fabricating engineered bacteria for cancer therapy in the future., This research was supported by the National Natural Science Foundation of China (grant no. 81971726), the Natural Science Foundation of Jiangsu Province (grant no. BK20191351), the China Postdoctoral Science Foundation (grant no. 2020M670091ZX), and the Nanjing Healthcare Science and Technology Development Special Funded Project (YKK20191)., info:eu-repo/semantics/publishedVersion

The milk-derived bovine lactoferrin (bLf) is an iron-binding glycoprotein with remarkable selective anticancer activity towards highly metastatic cancer cells displaying the proton pump V-ATPase at the plasma membrane. As studies aiming to dissect the bLf mechanisms of action are critical to improve its efficacy and boost its targeted clinical use, herein we sought to further uncover the molecular basis of bLf anticancer activity. We showed that bLf co-localizes with V-ATPase and cholesterol-rich lipid rafts at the plasma membrane of highly metastatic cancer cells. Our data also revealed that bLf perturbs cellular trafficking, induces intracellular accumulation of cholesterol and lipid rafts disruption, downregulates PI3K, and AKT or p-AKT and inhibits glycolysis of cancer cells harbouring V-ATPase at the plasma membrane lipid rafts. Altogether, our results can lay the foundation for future bLf-based targeted anticancer strategies as they unravel a novel cascade of molecular events that explains and further reinforces bLf selectivity for cancer cells displaying plasmalemmal V-ATPase.

Lactoferrin (Lf) is a versatile natural milk-derived protein that exhibits multiple interesting biological activities. Since it is safe for human administration and currently manufactured using low cost and well-established large-scale processes, the Lf scientific community has been devoted at dissecting its mechanisms of action towards its more rational and efficient use for various applications. Emerging literature has identified proton pumping ATPases as molecular targets of Lf in different cellular models linked to distinct activities of this natural protein. Information on this subject has not been systematically analysed before, hence herein we review the current state of art on the effect of Lf on proton pumping ATPases. Though structurally different, we propose that Lf holds a proton pump inhibitor (PPI)-like activity based on the functional resemblance with the classical inhibitors of the stomach H+/K+-ATPase. The downstream events and outcomes of the PPI-like activity of Lf, as well as its impact for the development of improved Lf applications are also discussed., FCT -Fundação para a Ciência e a Tecnologia (UIDB/04050/2020), info:eu-repo/semantics/publishedVersion

Background. Osteosarcoma is a highly malignant bone tumor, most frequently occurring in the rapid bone growth phase. Effective treatment of this disease is hindered by the lack of specific probes for early diagnosis and the fast cancer widespread. Methods. To find such probes, the cell-Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) methodology was implemented against the human osteosarcoma MG-63 cell line towards the selection of new specific aptamers. After 10 rounds of selection, the aptamer DNA pool was Sanger sequenced and the sequences were subjected to a bioinformatic analysis that included sequence alignment, phylogenetic relationship, and secondary structure prediction. Results. A DNA aptamer (OS-7.9), with a dissociation constant (Kd) value in the nanomolar range (12.8\thinspace\textpm\thinspace0.9 nM), revealed high affinity against the target cells at the physiological temperature. Furthermore, the selected aptamer also recognized lung carcinoma and colon colorectal adenocarcinoma cell lines, which are reported as common metastasis sites of osteosarcoma. Conclusions. These results suggest that OS-7.9 could recognize a common protein expressed in these cancer cells, possibly becoming a potential molecular probe for early diagnosis and targeted therapies for metastatic disease. Moreover, to the best of our knowledge, this was the first attempt to generate a DNA aptamer (OS-7.9 aptamer) against the MG-63-cell line by cell-SELEX., This work was supported by the Hacettepe University Scientifc Research Projects Coordination Unit (project no. FHD 2018–17245) and by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020unit. Diana A. Sousa (DAS) acknowledge FCT for the grant PD/BD/139083/2018. Débora Ferreira (DF) is recipient of a fellowship supported by a doctoral advanced training (call NORTE 69–2015-15) funded by the European Social Fund under the scope of Norte2020—Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Cellobiose 2-epimerases are interesting enzymes that can catalyze the production of lactose-based prebiotics, namely, epilactose and lactulose, through a single substrate reaction with noteworthy yields. In this work, the GRAS yeast Saccharomyces cerevisiae was for the first time used as a host for the heterologous production of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus. The enzyme showed an optimal temperature and an optimal pH of 80 °C and 7.5, respectively. Under these conditions, the time-course production of epilactose from lactose (50 g/L) was evaluated. The maximum yield and productivity obtained were 27\% and 9 g L1 h1, respectively. This epilactose yield represents the highest value obtained from the several approaches reported using cellobiose 2-epimerase from C. saccharolyticus. In conclusion, it was shown that the enzyme produced by S. cerevisiae is suitable for the production of epilactose-enriched prebiotic mixtures with potential applications in the food industry., FCT -Fundação para a Ciência e a Tecnologia(Lisboa2020), info:eu-repo/semantics/publishedVersion

Lactoferrin (Lf), a bioactive milk protein, exhibits strong anticancer and antifungal activities. The search for Lf targets and mechanisms of action is of utmost importance to enhance its effective applications. A common feature among Lf-treated cancer and fungal cells is the inhibition of a proton pump called V-ATPase. Lf-driven V-ATPase inhibition leads to cytosolic acidification, ultimately causing cell death of cancer and fungal cells. Given that a detailed elucidation of how Lf and V-ATPase interact is still missing, herein we aimed to fill this gap by employing a five-stage computational approach. Molecular dynamics simulations of both proteins were performed to obtain a robust sampling of their conformational landscape, followed by clustering, which allowed retrieving representative structures, to then perform protein-protein docking. Subsequently, molecular dynamics simulations of the docked complexes and free binding energy calculations were carried out to evaluate the dynamic binding process and build a final ranking based on the binding affinities. Detailed atomist analysis of the top ranked complexes clearly indicates that Lf binds to the V1 cytosolic domain of V-ATPase. Particularly, our data suggest that Lf binds to the interfaces between A/B subunits, where the ATP hydrolysis occurs, thus inhibiting this process. The free energy decomposition analysis further identified key binding residues that will certainly aid in the rational design of follow-up experimental studies, hence bridging computational and experimental biochemistry., Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding UIDP/04378/2020, UIDB/04378/2020, UIDB/04469/2020 and UIDB/04050/2020; and by the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Cátia Santos-Pereira acknowledges the PhD fellowship PD/BD/128032/2016 funded by FCT under the scope of the doctoral program in Applied and Environmental Microbiology (DP_AEM). Juliana F. Rocha is the recipient of the SFRH/BD/136746/2018 fellowship supported by FCT. Henrique S. Fernandes acknowledges FCT for his Ph.D. grant SFRH/BD/115396/2016. Some of the calculations were produced with the support of INCD funded by FCT and FEDER under the project 01/SAICT/2016 number 022153., info:eu-repo/semantics/publishedVersion

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.seppur.2018.07.062., Esterases catalyze the cleavage and formation of ester bonds of a broad range of substrates presenting a widespread spectrum of industrial applications. This work aimed to partially purify and characterize an esterase from Aureobasidium pullulans LABIOTEC 01 produced in a culture medium containing olive mill wastewater. Esterase purification was evaluated using different strategies, namely the enzyme recovery by PEG-salt aqueous two-phase systems (ATPSs); and the enzyme precipitation with ammonium sulfate, acetone, and ethanol. The best purification factor (18±2) was obtained when the ATPS composed of 20% (w/w) polyethylene glycol (PEG) 6000 and 5.8% (w/w) potassium phosphate buffer (PPB) pH 8.0 was combined with acetone precipitation. The partially purified enzyme presented an optimum pH of 5.0, although it remained active in the pH range of 4.5 to 7.5 ( 50% relative activity). The optimum temperature was found to be 60°C. Furthermore, the addition of salts such as FeCl3, CuSO4 and MnCl2 promoted an increase in the enzymatic activity (above 100%). The enzyme was found to be stable and showed high activity when exposed to polar solvents such as dimethyl sulfoxide, dimethylformamide, and methanol. The use of an integrated strategy of purification combining simple purification methods such as ATPS and precipitation was herein reported for the first time for esterase. This strategy proved to be an interesting approach to partially purify the esterase produced under submerged fermentation by A. pullulans. Furthermore, the enzyme showed potential to be applied in industrial biocatalytic processes using high temperature and different salts or solvents. Also, the production of esterase using olive mill wastewater as substrate demonstrated to be a suitable alternative to reduce and valorize agro-industrial residues., ACL acknowledges his post-doctoral grant and research funds from CAPES and CNPq (Brazilian funding agencies). SCS and LRR acknowledge their grants SFRH/BPD/88584/2012 and SFRH/BSAB/142873/ 2018, respectively, from the Portuguese Foundation for Science and Technology (FCT). The study also received financial support from FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), the Project MultiBiorefinery - Multi‐purpose strategies for broadband agro‐forest and fisheries by‐products valorisation: a step forward for a truly integrated biorefinery (POCI-01-0145-FEDER-016403) and the Project Lignozymes - Metagenomics approach to unravel the potential of lignocellulosic residues towards the discovery of novel enzymes (POCI-010145-FEDER-029773). The authors also acknowledge financial support from the BioTecNorte operation (NORTE-01-0145-FEDER-000004) fundedbytheEuropeanRegionalDevelopment Fundunderthescopeof Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Most biosurfactants are obtained using costly culture media and purification processes, which limits their wider industrial use. Sustainability of their production processes can be achieved, in part, by using cheap substrates found among agricultural and food wastes or byproducts. In the present study, crude glycerol, a raw material obtained from several industrial processes, was evaluated as a potential low-cost carbon source to reduce the costs of surfactin production by Bacillus subtilis #309. The culture medium containing soap-derived waste glycerol led to the best surfactin production, reaching about 2.8 g/L. To the best of our knowledge, this is the first report describing surfactin production by B. subtilis using stearin and soap wastes as carbon sources. A complete chemical characterization of surfactin analogs produced from the different waste glycerol samples was performed by liquid chromatographymass spectrometry (LC-MS) and Fourier transform infrared spectroscopy (FTIR). Furthermore, the surfactin produced in the study exhibited good stability in a wide range of pH, salinity and temperatures, suggesting its potential for several applications in biotechnology., National Science Centre, Poland, project 2020/37/B/NZ9/ 01519. The Article Processing Charge (APC) was financed under the Leading Research Groups support project from the subsidy increased for the period 2020–2025 in the amount of 2% of the subsidy referred to in Art. 387 (3) of the Law of 20 July 2018 on Higher Education and Science, obtained in 2019. This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte, info:eu-repo/semantics/publishedVersion

Triple-negative breast cancer is the most aggressive subtype of invasive breast cancer with a poor prognosis and no approved targeted therapy. Hence, the identification of new and specific ligands is essential to develop novel targeted therapies. In this study, we aimed to identify new aptamers that bind to highly metastatic breast cancer MDA-MB-231 cells using the cell-SELEX technology aided by high throughput sequencing. After 8 cycles of selection, the aptamer pool was sequenced and the 25 most frequent sequences were aligned for homology within their variable core region, plotted according to their free energy and the key nucleotides possibly involved in the target binding site were analyzed. Two aptamer candidates, Apt1 and Apt2, binding specifically to the target cells with Kd values of 44.3 ± 13.3 nM and 17.7 ± 2.7 nM, respectively, were further validated. The binding analysis clearly showed their specificity to MDA-MB-231 cells and suggested the targeting of cell surface receptors. Additionally, Apt2 revealed no toxicity in vitro and showed potential translational application due to its affinity to breast cancer tissue sections. Overall, the results suggest that Apt2 is a promising candidate to be used in triple-negative breast cancer treatment and/or diagnosis. © 2021, The Author(s)., Tis 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 BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. Débora Ferreira (DF) is the recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020—Programa Operacional Regional do Norte. Joaquim Barbosa (JB) and Diana A. Sousa (DAS) acknowledge FCT for the Grants SFRH/BD/51109/2010 and PD/BD/139083/2018, respectively., info:eu-repo/semantics/publishedVersion

Innovation of pastry products towards higher nutritional and commercial value remains a challenge to the growing field of healthy food. In this study, the prebiotic supplementation and sugar reduction were explored in a widely consumed pastry product with a low level of innovation. The prebiotic potential of commercial agave inulin and galacto-oligosaccharides (GOS) was evaluated and compared by an in vitro model using human fecal inocula. Rice muffins containing 100% of sugar or 75% of sugar supplemented with 0.8% GOS were produced and compared with commercial rice muffins regarding their physical and textural properties. GOS fermentation led to the highest production of lactate and short-chain fatty acids, besides the most significant reduction of the final pH value and of the ammonia and methane production. Inulin presented a higher selectivity towards Lactobacillaceae (51 ± 1% of all), while GOS are more efficient to stimulate Bifidobacteriaceae growth (65 ± 7% of all)., 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 BioTecNorte operation (NORTE-01-0145-FEDER000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte; COMPETE 2020 (POCI-01-0145-FEDER-006684), FoSynBio (POCI-01-0145-FEDER-029549) and NewFood (NORTE-01-0246-FEDER000043). CA and BBC acknowledge their grants (UMINHO/BPD/4/2019 and SFRH/BD/132324/2017) from FCT. RM acknowledges the financial support to the Xunta de Galicia and FEDER by the project (EDD431B 2019/01)., info:eu-repo/semantics/publishedVersion

Consumers are conscientiously changing their eating preferences toward healthier options, such as functional foods enriched with pre- and probiotics. Prebiotics are attractive bioactive compounds with multidimensional beneficial action on both human and animal health, namely on the gastrointestinal tract, cardiometabolism, bones or mental health. Conventionally, prebiotics are non-digestible carbohydrates which generally present favorable organoleptic properties, temperature and acidic stability, and are considered interesting food ingredients. However, according to the current definition of prebiotics, application categories other than food are accepted, as well as non-carbohydrate substrates and bioactivity at extra-intestinal sites. Regulatory issues are considered a major concern for prebiotics since a clear understanding and application of these compounds among the consumers, regulators, scientists, suppliers or manufacturers, health-care providers and standards or recommendation-setting organizations are of utmost importance. Prebiotics can be divided in several categories according to their development and regulatory status. Inulin, galactooligosaccharides, fructooligosaccharides and lactulose are generally classified as well established prebiotics. Xylooligosaccharides, isomaltooligosaccharides, chitooligosaccharides and lactosucrose are classified as "emerging" prebiotics, while raffinose, neoagaro-oligosaccharides and epilactose are "under development." Other substances, such as human milk oligosaccharides, polyphenols, polyunsaturated fatty acids, proteins, protein hydrolysates and peptides are considered "new candidates." This chapter will encompass actual information about the non-established prebiotics, mainly their physicochemical properties, market, legislation, biological activity and possible applications. Generally, there is a lack of clear demonstrations about the effective health benefits associated with all the non-established prebiotics. Overcoming this limitation will undoubtedly increase the demand for these compounds and their market size will follow the consumer's trend.