Academic literature on the topic 'Bradford assay'

Glomalin is a soil protein resembling heat shock protein (HSP) 60 and exerting high affinity to metals, causing retention of water in the environment and improving mechanical stability of soil. Currently, glomalin is determined in the soil or other samples by combination of autoclaving extraction and total protein determination typically by the Bradford method. In this paper, a piezoelectric biosensor was prepared to determine glomalin in a label-free measurement. The biosensor contained antibodies immobilized on quartz crystal microbalance (QCM), and the recognition layer was stabilized by iron oxide nanoparticles. The assay was tested on real soil samples and compared with the standard Bradford assay. Limit of detection of the assay was equal to 2.4 µg/g for a soil extract with a volume of 50 µl. The assay takes approximately half of an hour and was fully correlated to the Bradford assay. The biosensor had significant advantages than the other methods: it worked in a label-free mode and was fully applicable for practical samples.

An inhibitive enzyme assay using α-chymotrypsin was developed. This serine protease was assayed using Bradford-protease casein assay system. This assay is sensitive to several metals such as mercury Hg2+, Zn2+, and Cr6+. The IC50 (concentration of toxicant giving 50% inhibition) of Hg2+, Zn2+, and Cr6+ is 1.34 mg/l, 2.49 mg/l and 2.19 mg/l respectively. The principle of the protein assay using casein as a substrate relies upon the inability of the Bradford dye to stain polypeptide with less than a molecular weight of 2 kDa. In the presence of heavy metals that can inhibit this enzyme, casein is not being degraded and it is stained by the Bradford dye reagent giving a blue colour. On the other hand, in the absence of inhibitors, casein is hydrolyzed to polypeptides with molecular weight of 2 kDa and below which can’t be stained by Bradford dye-binding reagent. Therefore, solution remains brown in colour. The synergistic effect of combined heavy metals was studied and the results obtained shown that there were elevation of percentage of inhibition several folds. The combination of Hg2+ (0.3 mg/l) with Zn2+ (0.8 mg/l), Hg2+ (0.3 mg/l) with Cr6+ (1.8 mg/l), Zn2+ (0.8 mg/l) with Cr6+ (1.8 mg/l) increased 15.6, 73.0, 78.8 % respectively. Biomonitoring of heavy metals using an inhibitive α-chymotripsin assay was carried out. There sites for biomonitoring were Prai Industrial Areas, Bukit Tengah Industrial Area, Juru Industrial Area, Melaka River, Kuyoh River and Endau Rompin National Park. Many of the samples gave positive inhibitory effect on this enzyme. Those samples were analysed by inductively coupled plasma-optical emission spectrometry (ICP-OES) for confirmation of the presence of heavy metals that inhibited this enzyme.

Honey has been used to heal wounds since ancient times. There are many references in ancient literature that cite honey for its medicinal uses. It is used as an alternative agent to cure infections of wounds, burns, ulcers etc. Researchers have shown some of the antimicrobial properties of honey when used as an ointment. When applied to an affected area, it helps to promote the growth of healthy tissue. One of the factors on which the quality of the honey depends, is its geographical origin. Based on the location, honey types can vary as much as 100-fold from each other in color, aroma, viscosity, and antimicrobial properties. The important components in honey that play an essential part in healing wounds and contributing to the antimicrobial properties are enzymes. Their presence allows honey to kill various types of pathogenic bacteria, viruses, fungi etc. A higher antimicrobial effect is seen in monofloral honey (when a single plant species is the source of nectar), which is often more potent than other types of honey in terms of antibacterial activity. Resistance of pathogens to these antimicrobial actions has never been shown, which makes honey a more promising source of antimicrobial research. Presently, infections of burns and wounds are very challenging to treat, especially when they are caused by antibiotic-resistant bacteria. The purpose of this study was to examine the antimicrobial properties of honey from Utah and other locales, and to identify promising antimicrobial activities that could be useful in treating infections caused by resistant bacteria.

 У оквиру ове докторске дисертације испитан је хемијски састав и биолошке активности ЕtOH, H₂Oи CHCl₃  екстраката четири врсте гљива рода Ganoderma  (Basidiomycota):  G. applanatum,  G. lucidum,G. pfeifferi,  G. resinaceum  са територије Војводине.Хемијски састав анализираних врста је одређенприменом: ААЅ методе (састав макро-  имикроелемената у сувим остацима гљива) и LC-MS/MS технике (квантитативни састав фенолних једињења и флавоноида) при чему је детектовано 12 једињења. Спектрофотометријским методама је одређен садржај протеина, шећера, укупних фенола и флавоноида, код којих је највећи садржај протеина утврђен за ЕtOH екстракте  G. applanatum  и  G. pfeifferi. Испитивања биолошких активности екстраката обухватила су: одређивање   in vitro   и  in vivo антиоксидантне, антимикробне, антиинфламаторне, антипролиферативне и антијабето гене   aктивности.      Антиоксидантна активност (способност неутрализације слободних радикала и редукциони потенцијал) је одређена спектрофотометријским методама, при којој су најбољу активност остварили Н₂О екстракти  G. applanatum. Антимикробнa активност  анализираних екстраката одређена је испитивањем антибактеријског, антифунгалног и антивиралног потенцијала где се издвојила G. pfeifferi врста. Антиинфламаторни потенцијал EtOH и  CHCl₃ екстраката одређен је  ex vivo  методом мерењем способности инхибиције продукције медијатора инфламације  (продукти  метаболизма арахидонске киселине) при којој су бољу активност испољили CHCl₃ екстракти.Ефекат EtOH и H₂O екстраката врста рода Ganoderma   на раст MCF ћелијске линије испитан је MTT тестом, а посебно су се издвојили  EtOH  екстракти врста после 72h.Остварена антидијабетогена активност EtOH и Н₂О екстраката врста   G. pfeifferi   и  G. resinaceum  код алоксан-индукованог  D. mell itus-a  на  експерименталним  животињама  праћена je регенерацијом  ß- ћелија  Лангерхансових острваца панкреаса. Као потенцијални нефро-  и  хепатопротективни агенси се издвајају екстракти  G. resinaceum.Сумарно, укупни биопотенцијал анализираних врста рода  Ganoderma  на основу спроведених анализа хемијске   kарактеризације и биолошке активности упућује  на  могућност њихове потенцијалне примене као нутрацеутика и додатака исхрани, у будућности уз неопходност додатних микохемијских истраживања ових врста, посебно терпеноида и полисахарида, као и других биолошких активности као што је неуропротективна.
 U okviru ove doktorske disertacije ispitan je hemijski sastav i biološke aktivnosti EtOH, H₂Oi CHCl₃  ekstrakata četiri vrste gljiva roda Ganoderma  (Basidiomycota):  G. applanatum,  G. lucidum,G. pfeifferi,  G. resinaceum  sa teritorije Vojvodine.Hemijski sastav analiziranih vrsta je određenprimenom: AAЅ metode (sastav makro-  imikroelemenata u suvim ostacima gljiva) i LC-MS/MS tehnike (kvantitativni sastav fenolnih jedinjenja i flavonoida) pri čemu je detektovano 12 jedinjenja. Spektrofotometrijskim metodama je određen sadržaj proteina, šećera, ukupnih fenola i flavonoida, kod kojih je najveći sadržaj proteina utvrđen za EtOH ekstrakte  G. applanatum  i  G. pfeifferi. Ispitivanja bioloških aktivnosti ekstrakata obuhvatila su: određivanje   in vitro   i  in vivo antioksidantne, antimikrobne, antiinflamatorne, antiproliferativne i antijabeto gene   aktivnosti.      Antioksidantna aktivnost (sposobnost neutralizacije slobodnih radikala i redukcioni potencijal) je određena spektrofotometrijskim metodama, pri kojoj su najbolju aktivnost ostvarili N₂O ekstrakti  G. applanatum. Antimikrobna aktivnost  analiziranih ekstrakata određena je ispitivanjem antibakterijskog, antifungalnog i antiviralnog potencijala gde se izdvojila G. pfeifferi vrsta. Antiinflamatorni potencijal EtOH i  CHCl₃ ekstrakata određen je  ex vivo  metodom merenjem sposobnosti inhibicije produkcije medijatora inflamacije  (produkti  metabolizma arahidonske kiseline) pri kojoj su bolju aktivnost ispoljili CHCl₃ ekstrakti.Efekat EtOH i H₂O ekstrakata vrsta roda Ganoderma   na rast MCF ćelijske linije ispitan je MTT testom, a posebno su se izdvojili  EtOH  ekstrakti vrsta posle 72h.Ostvarena antidijabetogena aktivnost EtOH i N₂O ekstrakata vrsta   G. pfeifferi   i  G. resinaceum  kod aloksan-indukovanog  D. mell itus-a  na  eksperimentalnim  životinjama  praćena je regeneracijom  ß- ćelija  Langerhansovih ostrvaca pankreasa. Kao potencijalni nefro-  i  hepatoprotektivni agensi se izdvajaju ekstrakti  G. resinaceum.Sumarno, ukupni biopotencijal analiziranih vrsta roda  Ganoderma  na osnovu sprovedenih analiza hemijske   karakterizacije i biološke aktivnosti upućuje  na  mogućnost njihove potencijalne primene kao nutraceutika i dodataka ishrani, u budućnosti uz neophodnost dodatnih mikohemijskih istraživanja ovih vrsta, posebno terpenoida i polisaharida, kao i drugih bioloških aktivnosti kao što je neuroprotektivna.
Whitin this doctoral thesis the chemical composition and biological activity of EtOH, H ₂ O and CHCl₃ extracts of four fungal species which belong to genus Ganoderma  (phylum Basidiomycota) :  G. applanatum,  G. lucidum,  G. pfeifferi,  G. resinaceum  were determinated. The samples were collected from different localities in Vojvodina. Chemical characterization included: AAS methods (compositon of macro- and  microelements in d.w. of fungi) and LC-MS/MS technique (quantitative analysis of phenolic compounds and flavonoids) wherein the 12 selected phenolic compounds were detected. The total proteins, sugars, phenolics and flavonoids content were    determined using spectrophotometric methods. The highest protein content was determined in EtOH extracts of  G. applanatum   and G. pfeifferi  species. In order to assess the biological potential, the in vitro  and in vivo antioxidant, antimicrobial, anti-inflammatory, antiproliferative and antidiabetic activities of the extracts were investigated.    The antioxidant activity (the ability of neutralizing free radicals and reduction potential) estimated byspectrophotometric methods. The highest   antioxidant potential was noticed in H₂O extracts of  G. applanatum. Evaluation of antimicrobial activity included the estimation of antibacterial, antifungal and antiviral activity, whereby the  species  G. pfeifferi  showed the highest potential The anti-inflammatory activity of EtOH and  CHCl₃  extracts was determined by  ex vivo  method measuring the ability of production inhibition of inflammation mediators  (products of arachidonic acid metabolism), where the CHCl₃  extracts were exhibited better activity.   The effect of EtOH and H₂O extracts of  Ganoderma species on the growth of the cell line MCF-7, has been examined using MTT assay (stand out ethanolic extracts of analyzed species after 72h incubation period).   Achieved antidiabetic activity of EtOH and H₂O extracts of  G. pfeifferi   and G. resinaceum  at alloxan-i nduced D. mellitus in experimental animals was followed by regeneration of  cells of Langerhans pancreatic islets. Extracts of  G.   resinaceum  were allocated as a potential nephro- and  hepatoprotective agents.In summary, the overall biological potential of the analyzed species of the genus  Ganoderma  based on results for chemical and biological characterization indicate that they could be used  as a nutraceuticals and food supplements in the future, with further the necessity of additional mycochemical investigation (especially terpenoids and polysaccharides) and other biological activity such as neuroprotective.

The fish and shellfish industry processes 851,984 tonnes of fish per year worldwide. However, only 43% of that is consumed, and valuable proteins are processed as waste. Protein hydrolysates are widely used in food technology for their nutritional and functional properties. The goal of this project is to extract protein from whelk by-products derived from the shellfish processing industry and create protein hydrolysates that have marketable value. The by-products were divided into two types: raw (R) and cooked byproduct (C). The proteins were extracted using the pH shift method and quantified using the Bradford assay. It was possible to extract a maximum of 455 mg/g at a neutral pH, for which R had the highest protein yield. Proteins were also qualified using reverse phase high-performance liquid chromatography (RP-HPLC) that showed that R has more hydrophilic proteins while the C extracted protein showed more peaks in the hydrophobic phase. The Fourier-transform infrared spectroscopy (FTIR) indicated the presence of glutamine, tyrosine, and serine in the extracted proteins. Extracted proteins were then hydrolyzed using Alcalase and α-Chymotrypsin. It was possible to obtain higher degrees of hydrolysis (DH) using Alcalase. The hydrolysates were tested for antioxidant activity using the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical antioxidant assay. Alcalase hydrolysates showed to have overall lower IC50 for stabilization of the DPPH radical than α-Chymotrypsin, the lowest one being 13.92±1.57 µg/mL for the Alcalase hydrolyzed neutral proteins. The IC50 results obtained are significantly lower than the ones described in other studies using the same enzymes or other marine species. This can indicate that more heterogenous mixtures of by-product can originate extracted proteins that when hydrolyzed lead to higher radical scavenging activity, thus making shellfish industry by-product a sustainable and valuable source of antioxidant peptides. Keywords: Shellfish; Bioactive peptides; Protein extraction; Protein hydrolysates, Waste management, Nutraceuticals