Utilizing Nordic Forest Plant and Fungi Extracts: Bioactivity assessment with bacterial whole-cell biosensors

Luonnonmateriaalien sisältämien biologisesti aktiivisten komponenttien seulontaan tarvitaan uusia tehokkaita menetelmiä. Tässä tutkimuksessa metsästä saatavien uutteiden bioaktiivisuuden määrittämiseen käytettiin kokosolubiosensoreita. Tätä vaihtoehtoista seulontamenetelmää on tutkittu tarkoituksessa vain vähän, mutta herkkyytensä vuoksi se on varteenotettava. Tutkimuksessa aktiivisuushavaintoja täydentämään käytettiin myös perinteisempiä menetelmiä kuten antioksidanttikuoppalevytestejä. Lisäksi aineenvaihduntatuotteet määritettiin bioaktiivisiksi havaituista endofyyttisieniuutteista nestekromatografia-massaspektrometriaa (LC-MS) käyttäen. Tutkimuksen osana kehitettiin kaksi biosensorimenetelmää Escherichia coli kokosolubiosensoreille. Ensimmäinen sovellus parantaa aiemmin kuvattua antioksidanttisuuden mittaamisen menetelmää sen seulonnan tehopotentiaalia lisäämällä. Toinen esittelee menetelmän UV-säteilysuojan mittaamiseksi määrittäen sekä biologista että absorbanssipohjaista suojauskykyä. Menetelmällä voidaan saada samalla tietoa myös näytteen genotoksisuudesta. Kehitettyjen menetelmien toimivuus vahvistettiin malliyhdisteitä käyttäen, ja ne todettiin toimiviksi myös luonnonaineuutteilla joillakin rajoituksilla. Bakteeribiosensoreita voidaan käyttää etenkin konsentraatioltaan pienien näytteiden ja fraktioiden aktiivisuus- tai inaktiivisuusseulonnoissa, jolloin minimoidaan rajoitukset, kuten näytematriisin signaalin peittovaikutus ja induktioviive. Endofyyttisienet elävät kasvien sisällä aiheuttamatta oireilua isäntäkasvissa, ja ne voivat lisätä esimerkiksi isäntien kasvua ja stressinsietokykyä. Endofyyttisienet sisältävät suuren määrän toistaiseksi hyödyntämättömiä bioaktiivisia yhdisteitä, joita voidaan käyttää erilaisiin tarkoituksiin. Tutkimuksessa männyn taimien juurista eristettiin Acephala applanata, Philocephala fortinii ja Humicolopsis cephalosporioides/ Coniochaeta mutabilis -endofyyttisienilajit, ja niistä löydettiin suuri määrä yhdisteitä LC-MS: lla (yht. 318 kpl). Yhdisteistä tunnistettiin eri varmuusasteilla 220 kpl. Sienilajien uutteiden ja fraktioiden antioksidantti- ja antibakteerisuuspotentiaali seulottiin käyttäen E. coli -biosensorimenetelmiä sekä täydentäviä menetelmiä ja havaittiin useita aktiivisuuksia. Johtopäätöksenä endofyyttisienet sisältävät valtavan määrän mahdollisesti toiminnallisia aineenvaihduntatuotteita, ja kokosolubiosensorimenetelmät soveltuvat erilaisten luontopohjaisten näytteiden tutkimiseen antaen samanaikaisesti tietoa sekä näytteen bioaktiivisuudesta että biosaatavuudesta, mikäli myös niiden heikkoudet huomioidaan mittausasettelussa.Efficient methods are needed in the search for novel biologically active components from natural sources. Bacterial whole-cell biosensors offer a less-examined alternative with high sensitivity. In this thesis, forest-derived extracts were screened for their bioactivity with bacterial whole-cell biosensors and complementary measures, such as antioxidant microplate tests. The main metabolites were also identified from endophytic fungi extracts using liquid chromatography-mass spectrometry (LC-MS). Two novel applications of biosensors are described for Escherichia coli whole-cell biosensors. The first application provides an improved antioxidant method via increased high-throughput screening potential and the other describes a method for measuring the UV protection capacity for both the biological and absorbance-based shield. There is also an opportunity to obtain simultaneous information of the genotoxicity of the sample. The methodologies were validated using reference compounds and were found to be usable in the field of forest-derived extracts with certain limitations. The bacterial biosensors were used in an activity/inactivity type of screening. By using small concentrations of forest-derived extracts or fractionated samples, the limitations, such as possible sample matrix masking effect and induction delay phenomenon, can be reduced. Endophytic fungi live asymptomatically inside a plant host and are often beneficial for the host plant growth and stress resistance. They comprise a large untapped source of potential bioactive compounds to be utilized for different purposes. Many metabolites (318) were discovered using an LC-MS methodology from Acephala applanata, Phialocephala fortinii and Humicolopsis cephalosporioides/ Coniochaeta mutabilis isolated from the roots of Scots pine seedlings. Out of the metabolites, 220 were identified with varying degree of certainty. The antioxidant and antimicrobial potential of the extracts and fractions were screened with the biosensors and complementary methods and various bioactivities were discovered. In conclusion, endophytic fungi are a rich source of potential functional metabolites and whole-cell biosensor methodologies are usable for nature-based samples and provide valuable insights into both the bioactivity and bioavailability of the material, if their limitations are also considered

Valorizing Assorted Logging Residues: Response Surface Methodology in the Extraction Optimization of a Green Norway Spruce Needle-Rich Fraction To Obtain Valuable Bioactive Compounds

During stemwood harvesting, substantial volumes of logging residues are produced as a side stream. Nevertheless, industrially feasible processing methods supporting their use for other than energy generation purposes are scarce. Thus, the present study focuses on biorefinery processing, employing response surface methodology to optimize the pressurized extraction of industrially assorted needle-rich spruce logging residues with four solvents. Eighteen experimental points, including eight center point replicates, were used to optimize the extraction temperature (40–135 °C) and time (10–70 min). The extraction optimization for water, water with Na2CO3 + NaHSO3 addition, and aqueous ethanol was performed using yield, total dissolved solids (TDS), antioxidant activity (FRAP, ORAC), antibacterial properties (E. coli, S. aureus), total phenolic content (TPC), condensed tannin content, and degree of polymerization. For limonene, evaluated responses were yield, TDS, antioxidant activity (CUPRAC, DPPH), and TPC. Desirability surfaces were created using the responses showing a coefficient of determination (R2) > 0.7, statistical significance (p ≤ 0.05), precision > 4, and statistically insignificant lack-of-fit (p > 0.1). The optimal extraction conditions were 125 °C and 68 min for aqueous ethanol, 120 °C and 10 min for water, 111 °C and 49 min for water with Na2CO3 + NaHSO3 addition, and 134 °C and 41 min for limonene. The outcomes contribute insights to industrial logging residue utilization for value-added purposes

Metabolic Profiling of Water-Soluble Compounds from the Extracts of Dark Septate Endophytic Fungi (DSE) Isolated from Scots Pine (Pinus sylvestris L.) Seedlings Using UPLC–Orbitrap–MS

Endophytes are microorganisms living inside plant hosts and are known to be beneficial for the host plant vitality. In this study, we isolated three endophytic fungus species from the roots of Scots pine seedlings growing on Finnish drained peatland setting. The isolated fungi belonged to dark septate endophytes (DSE). The metabolic profiles of the hot water extracts of the fungi were investigated using Ultrahigh Performance Liquid Chromatography with Diode Array Detection and Electron Spray Ionization source Mass Spectrometry with Orbitrap analyzer (UPLC–DAD–ESI–MS–Orbitrap). Out of 318 metabolites, we were able to identify 220, of which a majority was amino acids and peptides. Additionally, opine amino acids, amino acid quinones, Amadori compounds, cholines, nucleobases, nucleosides, nucleotides, siderophores, sugars, sugar alcohols and disaccharides were found, as well as other previously reported metabolites from plants or endophytes. Some differences of the metabolic profiles, regarding the amount and identity of the found metabolites, were observed even though the fungi were isolated from the same host. Many of the discovered metabolites have been described possessing biological activities and properties, which may make a favorable contribution to the host plant nutrient availability or abiotic and biotic stress tolerance

Metabolic Profiling of Water-Soluble Compounds from the Extracts of Dark Septate Endophytic Fungi (DSE) Isolated from Scots Pine (Pinus sylvestris L.) Seedlings Using UPLC–Orbitrap–MS

Endophytes are microorganisms living inside plant hosts and are known to be beneficial for the host plant vitality. In this study, we isolated three endophytic fungus species from the roots of Scots pine seedlings growing on Finnish drained peatland setting. The isolated fungi belonged to dark septate endophytes (DSE). The metabolic profiles of the hot water extracts of the fungi were investigated using Ultrahigh Performance Liquid Chromatography with Diode Array Detection and Electron Spray Ionization source Mass Spectrometry with Orbitrap analyzer (UPLC–DAD–ESI–MS–Orbitrap). Out of 318 metabolites, we were able to identify 220, of which a majority was amino acids and peptides. Additionally, opine amino acids, amino acid quinones, Amadori compounds, cholines, nucleobases, nucleosides, nucleotides, siderophores, sugars, sugar alcohols and disaccharides were found, as well as other previously reported metabolites from plants or endophytes. Some differences of the metabolic profiles, regarding the amount and identity of the found metabolites, were observed even though the fungi were isolated from the same host. Many of the discovered metabolites have been described possessing biological activities and properties, which may make a favorable contribution to the host plant nutrient availability or abiotic and biotic stress tolerance

Correction: Tienaho, J., et al. Metabolic Profiling of Water-Soluble Compounds from the Extracts of Dark Septate Endophytic Fungi (DSE) Isolated from Scots Pine (Pinus sylvestris L.) Seedlings Using UPLC–Orbitrap–MS. Molecules 2019, 24, 2330

The authors wish to make the following corrections to this paper published in Molecules [...

Correction: Tienaho, J., et al. Metabolic Profiling of Water-Soluble Compounds from the Extracts of Dark Septate Endophytic Fungi (DSE) Isolated from Scots Pine (Pinus sylvestris L.) Seedlings Using UPLC–Orbitrap–MS. Molecules 2019, 24, 2330

The authors wish to make the following corrections to this paper published in Molecules [...

Field-Grown and In Vitro Propagated Round-Leaved Sundew (Drosera rotundifolia L.) Show Differences in Metabolic Profiles and Biological Activities

Drosera rotundifolia L. is a carnivorous plant used in traditional medicine for its therapeutic properties. Because of its small size, its collection in nature is laborious and different cultivation methods have been studied to ensure availability. However, only a few studies exist where the lab-grown sundew tissue and field-grown sundew would have been compared in their functionality or metabolic profiles. In this study, the antioxidant and antiviral activities of lab-grown and field-grown sundew extracts and their metabolic profiles are examined. The effect of drying methods on the chromatographic profile of the extracts is also shown. Antioxidant activity was significantly higher (5-6 times) in field-grown sundew but antiviral activity against enterovirus strains coxsackievirus A9 and B3 was similar in higher extract concentrations (cell viability ca. 90%). Metabolic profiles showed that the majority of the identified compounds were the same but field-grown sundew contained higher numbers and amounts of secondary metabolites. Freeze-drying, herbal dryer, and oven or room temperature drying of the extract significantly decreased the metabolite content from -72% up to -100%. Freezing was the best option to preserve the metabolic composition of the sundew extract. In conclusion, when accurately handled, the lab-grown sundew possesses promising antiviral properties, but the secondary metabolite content needs to be higher for it to be considered as a good alternative for the field-grown sundew.</p