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Whereas industrial laboratory systems for Fourier-Transform Infrared (FTIR) spectroscopic characterization have been available for decades, dedicated FTIR solutions for in-line process control or for industrial at-line analysis of protein quality are still scarce. Thus, the present study aimed to compare two industrially viable sampling techniques, namely attenuated total reflectance (ATR) and dry film FTIR spectroscopy, for qualitative and quantitative analysis of liquid protein solutions. For this purpose, two sample sets were acquired: set 1 consisted of 95 protein hydrolysate samples produced in the laboratory from salmon processing by-products, and set 2 consisted of 133 protein hydrolysate samples obtained from an industrial processing plant of poultry by-products. Average molecular weights (AMW) and Brix measurements of protein hydrolysates were used as reference values for obtaining regression models. AMW was predicted with higher accuracy and lower estimation errors using dry film FTIR compared to ATR measurements for both sample types. The difference in predictive performance was higher in poultry hydrolysates because the protein and tissue complexities are higher than in salmon hydrolysates, and information from the amide I region in the FTIR spectra is needed to provide good calibration results. ATR, on the other hand, was more reliable for the prediction of Brix values of protein hydrolysates. The study also showed that FTIR spectroscopy can be used to predict protein quality features of industrially produced protein hydrolysates with a sensitivity of high industrial relevance. Therefore, developing industrially viable portable instruments for at-line process analysis in the food, feed, and biotech industries is a natural next development stage.

The aim of this study was to investigate the role of compositional variations in side-streams from the chicken and turkey industry on product characteristics after enzymatic protein hydrolysis (EPH). Variation was obtained using four raw materials, namely chicken and turkey carcasses, and mechanically deboned chicken and turkey residues. Alcalase 2.4 L, Corolase 2TS and Flavourzyme, three commercial proteases with different specificities, were used to investigate impact of protease choice on product characteristics. Besides protein yield, effects on product characteristics such as nutrient and amino acid composition, size distribution of peptides, degree of hydrolysis (DH%), rheological properties, and angiotensin-I-converting enzyme (ACE) inhibition, were investigated. Results show that choice of protease and industrially relevant variations in poultry raw material composition both have major effect on product composition and the other quality parameters studied. Protein yield, proximate composition, and bioactivity were more affected by protease than raw material variation. Raw material variation had a bigger impact on quality parameters such as peptide size distribution and amino acid composition, exemplified by the sum of essential amino acids. This means that raw material variation should be handled and accounted for to realize the true potential that exists in tailor-making EPH products with specified properties for targeted applications.

A study of the effects of single and combined protease hydrolysis on myofibrillar versus collagenous proteins of poultry by-products has been conducted. The aim was to contribute with knowledge for increased value creation of all constituents of these complex by-products. A rational approach was implemented for selecting proteases exhibiting the most different activity towards the major protein-rich constituents of mechanically deboned chicken residue (MDCR). An initial activity screening of 18 proteases on chicken meat, turkey tendons and MDCR was conducted. Based on weight yield, size exclusion chromatography (SEC) and SDS-PAGE, stem Bromelain and Endocut-02 were selected. Studies on hydrolysis of four different poultry by-products at 40 °C, evaluated by protein yield, SEC, and SDS-PAGE, indicate that the proteases’ selectivity difference can be utilized in tailor-making hydrolysates, enriched in either meat- and collagen-derived peptides or gelatin. Three modes of stem Bromelain and Endocut-02 combinations during hydrolysis of MDCR were performed and compared with single protease hydrolysis. All modes of the protease combinations resulted in a similar approximately 15% increase in product yield, with products exhibiting similar SEC and SDS-PAGE profiles. This shows that irrespective of the modes of combination, the use of more than one enzyme in hydrolysis of collagen-rich material can provide means to increase the total protein yield and ultimately contribute to increased value creation of poultry by-products.

Enzymatic protein hydrolysis (EPH) is an invaluable process to increase the value of food processing by-products. In the current work the aim was to study the role of standard thermal inactivation in collagen solubilization during EPH of poultry by-products. Hundred and eighty hydrolysates were produced using two proteases (stem Bromelain and Endocut-02) and two collagen-rich poultry by-products (turkey tendons and carcasses). Thermal inactivation was performed with and without the sediment to study the effect of heat on collagen solubilization. A large difference in molecular weight distribution profiles was observed when comparing hydrolysate time series of the two proteases. In addition, it was shown that 15 min heat treatment, conventionally used for inactivating proteases, is essential in solubilizing collagen fragments, which significantly contributes to increasing the protein yield of the entire process. The study thus demonstrated the possibility of producing tailored products of different quality by exploiting standard heat inactivation in EPH. Financial support from the Norwegian Fund for Research Fees on Agricultural Products through the project “Precision Food Production” (no. 314111), and from the Norwegian Research Council through the project “Notably” (no. 280709) is greatly acknowledged. Internal financing from Nofima through the project “Peptek” is also greatly acknowledged.

Enzymatic protein hydrolysis can be used industrially for refining peptides from poultry by-products. The aim is to introduce these peptides for human consumption, but today the majority is used for feed or pet food. One main barrier for entering the human market is unknown and unstable peptide product qualities. Reliable analytical methods for characterising the peptide fraction is therefore vital for optimizing and controlling the product quality. In this work, we investigate the potential of using a large database of dry-film Fourier-transform Infrared (FTIR) fingerprints to gain new insight in quality variations of peptide products. The database cover >1300 samples from laboratory experiments performed over several years. We found that the FTIR fingerprints contain patterns that are stable across experiments and time, and that variations in the fingerprint can be related to raw material composition and processing factors. We also show that the database can be used for evaluating industrial samples. By comparing the FTIR fingerprints of industrial samples with those in the database, we could see systematic changes in product quality over time and make hypotheses about the underlying causes for change. These results lay the foundation for using dry-film FTIR spectroscopy as an industrial tool for product and process optimisation and control.

In this work, a new magnetic ligand fishing probe for discovery of DPP-IV inhibitory ligands was developed and it was tested as a proof of concept on the fruit extract of Vaccinium vitis-idaea (lingonberry). The ligands were shown to have appreciable dipeptidyl peptidase IV (DPP-IV) inhibitory activity (IC50: 31.8 μg mL-1).) Inhibition of DPP-IV is a well-known therapeutic approach for management of type 2 diabetes (T2D). DPP-IV was successfully immobilized onto magnetic beads and was shown to retain its catalytic activity and selectivity over a model mixture. A total of four ligands were successfully fished out and identified as cyanidin-3-galactoside (2), cyanidin-3-arabinoside (3), proanthocynidin A (4), and 10-carboxyl-pyranopeonidin 3-O-(6″-O-p-coumaroyl)-glucoside (5) using HPLC/HRMS.

The frequency of foodborne outbreaks epidemiologically associated with Listeria monocytogenes in fresh produce has increased in recent years. Although L. monocytogenes may be transferred from the environment to vegetables during farming, contamination of food products most commonly occurs in food processing facilities, where L. monocytogenes has the ability to establish and persist on processing equipment. The current study was undertaken to collect data on the occurrence of L. monocytogenes and the identity of the endogenous microbiota in a fresh produce processing facility, for which information has remained scarce. L. monocytogenes was not detected in the facility. Experiments simulating conditions in the processing environment were performed, including examination of bacterial growth in nutrients based on vegetables (salad juice) compared to in other types of nutrients (fish, meat). Results showed that the endogenous microbiota (dominated by Pseudomonas) grew well in iceberg lettuce and rocket salad juice at low temperatures, while growth inhibition of L. monocytogenes was observed, particularly in rocket salad juice. The anti-listerial activity in rocket salad juice was retained in a polar chromatographic fraction containing several metabolites. Characterization of this active fraction, using LC-MS/MS, led to identification of 19 compounds including nucleosides and amino acids. Further work is necessary to determine the molecular mechanism responsible for the inhibitory activity of rocket salad constituents. The study nevertheless suggests that the available nutrients, as well as a low temperature (3 °C) and the in-house bacterial flora, may influence the prevalence of L. monocytogenes in fresh produce processing facilities.

While the harmonized INFOGEST model provides a physiologically relevant platform for simulated digestion, it needs to be combined with adequate analytical methods to enable quantification and comparison of protein digestibility in different food matrices. We have shown that size exclusion chromatography (SEC) can be used to estimate the proportion of small peptides potentially available for uptake. Combined with determination of total dissolved protein, the % of small peptides per total protein was calculated as a physiologically relevant estimate of protein digestibility (DSEC). Values for DSEC differed for casein (87.6%), chicken mince (72.6%), heated pea protein concentrate (67.8%), bread (63%), beef entrecote (57.7%) and pea protein concentrate (57.8%). In contrast to existing methods (TCA soluble protein, free NH2-groups), the proposed SEC based method gives separate insight into the two fundamental processes during protein digestion (solubilization and break-down), while maintaining the ability to rank digestibility of very different food proteins.

Immobilization of enzymes onto magnetic particles can potentially allow for enzyme reuse, which can significantly reduce the cost associated with e.g. enzymatic protein hydrolysis (EPH) of fish and meat by-products. Here, we report glutaraldehyde-mediated immobilization of a food-grade protease (Subtilisin A) onto magnetic silica particles using three different amine ligands; a short, brush-like linker (aminopropyl trimethoxysilane), a long, flexible linker (Jeffamine), and a gel-like coating (chitosan). The three coupling strategies were evaluated and compared with respect to the amount of immobilized protease, enzyme activity and catalytic performance in the hydrolysis of chicken meat and turkey tendons, respectively. The particle systems showed high reusability (≤85% activity remaining after six consecutive cycles) and storage stability (≤93% activity remaining after 25 months storage). Particle-immobilized enzyme systems were able to catalyze degradation and extraction of protein from chicken meat and turkey tendons.

This study reports the feasibility of using Raman spectroscopy for quantification of soluble solids and individual sugars in apples. Six different commercial apple varieties were measured by Raman spectroscopy at three different steps: 1) Intact apples with skin, 2) apples without skin and 3) juices obtained from apples. Results indicated that it is possible to measure Raman signals to a depth of 8 mm into the apple with a wide area Raman probe. Multivariate calibration models were established to evaluate how well Raman spectra can be used to estimate the quality parameters SSC (%), total sugars, glucose, fructose and sucrose. Estimation accuracy for SSC was comparable with what is achievable with near-infrared spectroscopy: Root mean square error of cross-validation (RMSECV) = 0.66, 0.46 and 0.72 % and coefficients of determination (R2) = 0.70, 0.85 and 0.63 for intact apples, apples without skin and juices, respectively. Sucrose and glucose were well estimated with RMSECV of 2.8, 1.9, 2.1 mg/mL for glucose and 5.8, 3.9 and 3.7 mg/mL for sucrose, for the three sample cases, respectively. Coefficient of determination was higher than 0.82 for all models. Regression coefficients for all calibration models highlighted identifiable Raman bands that could be related to the target sugars.

In this study we explore the potential of using Fourier-transform infrared (FTIR) spectra of trifluoroacetate-protein and peptide complexes for monitoring proteolytic reactions. The idea of treating dry-films of protein hydrolysates with trifluoroacetic acid (TFA) prior to FTIR analysis is based on the unique properties of TFA. By adding a large excess of TFA to protein hydrolysate samples, the possible protonation sites of the proteins and peptides will be saturated. In addition, TFA has a low boiling point when protonated as well as complex-forming abilities. When forming TFA-treated dry-films of protein hydrolysates, the excess TFA will evaporate and the deprotonated acid (CF3COO−) will interact as a counter ion with the positive charges on the sample materials. In the study, spectral changes in TFA-treated dry-films of protein hydrolysates from a pure protein and poultry by-products, were compared to the FTIR fingerprints of untreated dry-films. The results show that time-dependent information related to proteolytic reactions and, consequently, on the characteristics of the protein hydrolysates can be obtained. With additional developments, FTIR on dry-films treated with TFA may be regarded as a potential future tool for the analysis of all types of proteolytic reactions in the laboratory as well as in industry.

Enzymatic protein hydrolysis of food processing by-products is a well-recognized strategy for producing peptide-rich formulations of added value. The biochemical complexity of this process poses a significant challenge in the use of classical analytical methods as process monitoring tools in both laboratory and industrial setups. In the present study, we are reporting an FTIR-based multivariate approach for monitoring the change in the molecular size distribution of proteins during enzymatic hydrolysis of chicken fillets and processing by-products. For 129 protein hydrolysates, weight-average molecular weight derived from size-exclusion chromatographic analysis was established as a pragmatic measure of the extent of hydrolysis. FTIR spectra acquired from dry films of the hydrolysates were used to build multivariate calibration models using partial least squares (PLS) regression. The best predictions were obtained when the data from two different raw materials, i.e., chicken fillets and mechanical chicken deboning residues, were modeled individually. A good prediction model was also achieved for the combined data from the two raw materials using canonical PLS (CPLS) regression or variable selection. The results from the current study underline the potential of FTIR spectroscopy as a rapid analytical tool for monitoring enzymatic protein hydrolysis of complex raw materials.

Type 2 diabetes (T2D) constituted 90% of the global 387 million diabetes cases in 2014. The enzyme protein-tyrosine phosphatase 1B (PTP1B) has been recognized as a therapeutic target for treatment of T2D and its adverse complications. With the aim of accelerating the investigation of complex natural sources, such as crude plant extracts, for potential PTP1B inhibitors, we have developed a bio-analytical platform combining high-resolution PTP1B inhibition profiling and high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy, i.e., HR-bioassay/HPLC-HRMS-SPE-NMR. Human recombinant PTP1B enzyme was used for the microplate-based PTP1B inhibition assay, which was optimized for pH and substrate concentration to be compatible with rate measurements within the 10 min incubation time. Subsequently, analytical-scale HPLC-based microfractionation followed by colorimetric microplate-based PTP1B bioassaying enabled construction of a high-resolution inhibition profile corresponding to the HPLC profile. The high-resolution PTP1B inhibition profiling was validated using an artificial mixture of known PTP1B inhibitors and non-inhibiting compounds as negative controls. Finally, a proof-of-concept study with a real sample was performed using crude ethyl acetate extract of the phytochemically hitherto unexplored plant Eremophila lucida. This led to the identification of the first viscidane type diterpene, i.e., 5-hydroxyviscida-3,14-dien-20-oic acid (9) as PTP1B inhibitor with an IC50 value of 42.0 ± 5.9 μM. In addition, a series of flavonoids, i.e., luteolin (1), dinatin (3a), tricin (3b), 3,6-dimethoxyapigenin (4), jaceidin (5), and cirsimaritin (6) as well as a cembrene diterpene, (3Z, 7E, 11Z)-15-hydroxycembra-3,7,11-trien-19-oic acid (8), were also identified for the first time from E. lucida.

Fourier-transform infrared (FTIR) spectroscopy was applied to predict the degree of hydrolysis (DH%) and weight-average molecular weight (Mw) in milk protein hydrolysates. Both DH% and Mw are important quality parameters of protein hydrolysates. Measuring these parameters and following their development during proteolytic reactions is therefore essential for process control and optimization in industry. In the present study the intercorrelation and the complimentary nature of these parameters were investigated and a partial least squares regression (PLSR) model was developed for the prediction of DH% from molecular weight distributions. Finally, we developed PLSR models based on dry-film FTIR spectroscopy for the prediction of both DH% and Mw. Here spectral changes in the amide region were found to be important for the two calibration models, underlining the advantage of dry-film FTIR measurement. This shows that dry-film infrared spectroscopy is a promising tool for dual prediction of DH% and Mw.

The success of a given biotechnological process lies in the ability to produce a specific product with a desired quality and optimal yield. It is therefore vital to apply appropriate analytical strategies for measuring raw material composition, monitoring the process, and evaluating product qualities. Such analytical strategies are critical elements in the recovery of protein from vegetable, marine, and animal by-products, largely due to the inevitable composition variations of by-products. There have been several analytical developments to measure important process and quality parameters, such as degree of protein hydrolysis, peptide size distribution, and protein yield. This chapter covers the classical analytical methodologies used to measure these parameters, as well as emerging approaches of using rapid spectroscopic techniques as process control and optimization tools. Moreover, key factors and process considerations in relation to the use of proteases for recovery of peptides are discussed.

Plants are well-recognized sources of inhibitors for -glucosidase - a key target enzyme for management of type 2 diabetes. Recently, two advanced bioactivity-profiling techniques, i.e., ligand fishing and high-resolution inhibition profiling, have shown great promises for accelerating identification of -glucosidase inhibitors from complex plant extracts. Non-specific affinities and non-specific inhibitions are major sources of false positive hits from ligand fishing and highresolution inhibition profiling, respectively. In an attempt to minimize such false positive hits, we describe a new screening approach based on ligand fishing and high-resolution inhibition profiling for detection of high-affinity ligands and assessment of inhibitory activity, respectively. The complementary nature of ligand fishing and high-resolution inhibition profiling was explored to Manuscript for Talanta 3 identify-glucosidase inhibitory ligands from a complex mixture, and proof-of-concept was demonstrated with crude ethyl acetate extract of Ginkgo biloba. In addition to magnetic beads with a 3-carbon aliphatic linker, -glucosidase was immobilized on magnetic beads with a 21-carbon aliphatic linker; and the two different types of magnetic beads were compared for their hydrolytic activity and fishing efficiency.

A bioanalytical platform combining magnetic ligand fishing for α-glucosidase inhibition profiling and HPLC-HRMS-SPE-NMR for structural identification of α-glucosidase inhibitory ligands, both directly from crude plant extracts, is presented. Magnetic beads with N-terminus-coupled α-glucosidase were synthesized and characterized for their inherent catalytic activity. Ligand fishing with the immobilized enzyme was optimized using an artificial test mixture consisting of caffeine, ferulic acid, and luteolin before proof-of-concept with the crude extract of Eugenia catharinae. The combination of ligand fishing and HPLC-HRMS-SPE-NMR identified myricetin 3-O-α-l-rhamnopyranoside, myricetin, quercetin, and kaempferol as α-glucosidase inhibitory ligands in E. catharinae. Furthermore, HPLC-HRMS-SPE-NMR analysis led to identification of six new alkylresorcinol glycosides, i.e., 5-(2-oxopentyl)resorcinol 4-O-β-d-glucopyranoside, 5-propylresorcinol 4-O-β-d-glucopyranoside, 5-pentylresorcinol 4-O-[α-d-apiofuranosyl-(1→6)]-β-d-glucopyranoside, 5-pentylresorcinol 4-O-β-d-glucopyranoside, 4-hydroxy-3-O-methyl-5-pentylresorcinol 1-O-β-d-glucopyranoside, and 3-O-methyl-5-pentylresorcinol 1-O-[β-d-glucopyranosyl-(1→6)]-β-d-glucopyranoside.

In our ongoing efforts of finding natural fungicides to fight food and feed spoilage during production and storage, the antifungal potential of Ghanaian Uvaria chamae P. Beauv. was investigated, with emphasis on plant metabolites targeting the fungal plasma membrane (PM) H + -ATPase. Ethyl acetate extract of U. chamae was subjected to high-resolution fungal PM H + -ATPase inhibition screening followed by structural elucidation by high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy (HPLC–HRMS–SPE–NMR). This led to identification of a series of uncommon o -hydroxybenzylated flavanones and chalcones, i.e., chamanetin ( 8 ), isochamanetin ( 9 ), isouvaretin ( 10 ), uvaretin ( 11 ), dichamanetin ( 12 ), and diuvaretin ( 15 ). Preparative-scale isolation of the active metabolites allowed determination of IC 50 values for inhibition of the PM H + -ATPase, and growth inhibition of Saccharomyces cerevisiae and Candida albicans . These revealed a strong correlation between o -hydroxybenzyl substituents and PM H + -ATPase activity, with dichamanetin being the most potent compound, but showing moderate activity in the fungal growth inhibition assays.

Type 2 diabetes (T2D) is an endocrine metabolic disease with a worldwide prevalence of more than 8%, and an expected increase close to 50% in the next 15-20years. T2D is associated with severe and life-threatening complications like retinopathy, neuropathy, nephropathy, and cardiovascular diseases, and therefore improved drug leads or functional foods containing α-glucosidase inhibitors are needed for management of blood glucose. In this study, leaves of Myrcia palustris were investigated by high-resolution α-glucosidase inhibition profiling combined with HPLC-HRMS-SPE-NMR. This led to identification of casuarinin, myricetin 3-O-β-d-(6″-galloyl)galactopyranoside, kaempferol 3-O-β-d-galactopyranoside, myricetin, and quercetin as α-glucosidase inhibitors. In addition, four acetylated ellagic acid rhamnosides, i.e., 4-O-(2″,4″-O-diacetyl-α-l-rhamnopyranosyl)ellagic acid, 4-O-(2″,3″-O-diacetyl-α-l-rhamnopyranosyl)ellagic acid, 4-O-(3″,4″-O-diacetyl-α-l-rhamnopyranosyl)ellagic acid, and 4-O-(2″,3″,4″-O-triacetyl-α-l-rhamnopyranosyl)ellagic acid were identified.

Type 2 diabetes is a metabolic disorder affecting millions of people worldwide, and new drug leads or functional foods containing selective α-glucosidase inhibitors are needed. Crude extract of 24 plants were assessed for α-glucosidase inhibitory activity. Methanol extracts of Cinnamomum zeylanicum bark, Rheum rhabarbarum peel, and Rheum palmatum root and ethyl acetate extracts of C. zeylanicum bark, Allium ascalonicum peel, and R. palmatum root showed IC50 values below 20 μg/mL. Subsequently, high-resolution α-glucosidase profiling was used in combination with high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy for identification of metabolites responsible for the α-glucosidase inhibitory activity. Quercetin (1) and its dimer (2), trimer (3), and tetramer (4) were identified as main α-glucosidase inhibitors in A. ascalonicum peel, whereas (E)-piceatannol 3'-O-β-D-glucopyranoside (5), (E)-rhapontigenin 3'-O-β-D-glucopyranoside (6), (E)-piceatannol (8), and emodin (12) were identified as main α-glucosidase inhibitors in R. palmatum root.

There is an increasing focus on the recovery of proteinaceous components from marine biomass. In the future it is expected that more food and feed will come globally from the oceans. A lot of marine biomass is today under-utilized or used for low value products. In other cases, the biomass is from under-utilized marine resources such as seaweed, low trophic species or mesopelagic species. The reason why they are not being used can be due to difficult catching or harvesting methods or due to insufficient availability of processes and infrastructure. Much of this biomass is suitable for human consumption; however, the right processing technologies are not put into place in order to make these products available. In this chapter, the focus will be on marine biomass that is currently of low value or currently wasted, and possible solutions for use of these will be evaluated. An overview of possible products from a variety of marine biomasses is presented, the desired properties of the different products towards specific markets are described and the possibilities of these products becoming a commodity that can be produced in large volumes is evaluated based on technological and market possibilities.

The potential of dry-film Fourier-transform infrared (FTIR) measurements as a monitoring tool for enzymatic hydrolysis of protein-based substrates is explored in this study. As a proof-of-concept, the enzymatic digestion of bovine serum albumin using Alcalase was monitored. To evaluate the analytical approach on complex substrates with industrial relevance, salmon- and chicken-based substrates were digested for 80 minutes using Alcalase and a total of 12 FTIR spectra were acquired during the course of the hydrolysis. The observed changes in the IR spectral features as a function of hydrolysis time were found to be in agreement with the breakdown of the amide backbone and formation of amino and carboxylate terminals. Some of the most consistent markers for hydrolysis time were the bands at 1516 cm−1 (–NH3+) and ∼1400 cm−1 (–COO−). Moreover, principal component analysis (PCA) of the FTIR spectra was used to demonstrate the systematic relationship of the hydrolysis time with key variables (wavelengths) in the protein backbone region (800–1800 cm−1). Scores in the first principal component versus the hydrolysis time have been shown to provide an overview of the process dynamics related to protein structural changes. The herein presented results suggest that dry-film FTIR measurements have potential as a rapid tool for monitoring industrial protein hydrolysis processes.

The original version of this article unfortunately contained a mistake. The spelling of the fourth author was incorrect. The correct name is Themistoklis Altintzoglou.

The metabolite production of the five members of the fungal species complex Heterobasidion annosum s.l., i.e. H. annosum s.s., H. abietinum , H. parviporum , H. occidentale and H. irregulare , was analyzed by LC–HRMS. The five members are described to have differences in host preferences: H. annosum s.s. and H. irregulare are pine infecting species, and H. parviporum , H. occidentale and H. abietinum are non-pine infecting. Principal component analysis (PCA) of the LC–HRMS data showed that samples from the five species could be separated into five groups and in accordance with the differences in host preferences. Twenty-three compounds, important to the observed PCA grouping, were isolated and identified. The main contributor to the separation of the pine infecting species from the non-pine infecting species in PC1 was the benzohydrofuran fomannoxin, which was only detected in the pine infecting species H. annosum s.s. and H. irregulare . These two species were further separated in PC3, and one major contributor here was the sesquiterpene deoxyfomannosin A. The three non-pine infecting species were separated in PC2, by epoxydrimenol that was detected in only two of the species and further in PC4, where a few fomannoxin related compounds were important for the grouping. During the work, three unknown compounds were isolated and described: 3-hydroxy-2-(1,3-dihydroxypropan-2-yl)-2,3-dihydrobenzofuran-5-carbaldehyde, 3-hydroxy-2-(1,2,3-trihydroxypropan-2-yl)-2,3-dihydrobenzofuran-5-carbaldehyde and 3-hydroxy-2,3-dihydrobenzofuran-5-carboxylic acid.

Crude extracts of 33 plant species were assessed for fungal plasma membrane (PM) H(+)-ATPase inhibition. This led to identification of 18 extracts showing more than 95% inhibition at a concentration of 7.5 mg/mL and/or a concentration-dependent activity profile. These extracts were selected for semi-high-resolution fungal PM H(+)-ATPase inhibition screening, and, on the basis of these results, Haplocoelum foliolosum (Hiern) Bullock and Sauvagesia erecta L. were selected for investigation by high-resolution fungal PM H(+)-ATPase inhibition screening. Structural analysis performed by high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLC-HRMS-SPE-NMR) led to identification of chebulagic acid (1) and tellimagrandin II (2) from H. foliolosum. Preparative-scale isolation of the two metabolites allowed determination of IC50 values for PM H(+)-ATPase, and growth inhibition of Saccharomyces cerevisiae and Candida albicans. Chebulagic acid and tellimagrandin II are both potent inhibitors of the PM H(+)-ATPase with inhibitory effect on the growth of S. cerevisiae.

This work describes the coupling of a microplate-based antioxidant assay with a hyphenated system consisting of high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy, i.e., HPLC-SPE-NMR/high-resolution antioxidant assay, for the analysis of complex food extracts. The applicability of the microplate-based antioxidant assay for high-resolution screening of common food phenolics as well as parameters related to their trapping efficiency, elution behavior, and recovery on/from SPE cartridges are described. It was found that the microplate-based high-resolution antioxidant assay is an attractive and easy implementable alternative to direct on-line screening methods. Furthermore, it was shown that Resin SH and Resin GP SPE material are superior to RP C18HD for trapping of phenolic compounds. Proof-of-concept study was performed with caper bud extract, revealing the most important antioxidants to be quercetin, kaempferol, rutin, kaempferol-3-O-β-rutinoside and N(1),N(5),N(10)-triphenylpropenoyl spermidine amides. Targeted isolation of the latter, and comprehensive NMR experiments showed them to be N(1),N(10)-di-(E)-caffeoyl-N(5)-p-(E)-coumaroyl spermidine, N(1)-(E)-caffeoyl-N(5),N(10)-di-p-(E)-coumaroyl spermidine, N(10)-(E)-caffeoyl-N(1),N(5)-di-p-(E)-coumaroyl spermidine, and N(1),N(5),N(10)-tri-p-(E)-coumaroyl spermidine amides.

The high-resolution radical scavenging profile of an extract of the endophytic fungus Penicillium namyslowskii was used to target analysis by high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy, i.e., HPLC–HRMS–SPE–NMR, for identification of anti-oxidative secondary metabolites. This revealed the two chromatographic peaks with the highest relative response in the radical scavenging profile to be griseophenone C and peniprequinolone. The HPLC–HRMS–SPE–NMR analysis was performed in the tube-transfer mode using a cryogenically cooled NMR probe designed for 1.7 mm NMR tubes. To further explore the potential of the above HPLC–HRMS–SPE–NMR platform for analysis of endophytic extracts, six peaks displaying no radical scavenging activity were also analyzed. This allowed unambiguous identification of six metabolites, i.e., dechlorogriseofulvin, dechlorodehydrogriseofulvin, griseofulvin, dehydrogriseofulvin, mevastatin acid, and mevastatin. The high mass sensitivity of the 1.7 mm cryogenically cooled NMR probe allowed for the first time acquisition of direct detected 13C NMR spectra of fungal metabolites, i.e., dechlorogriseofulvin and griseofulvin, directly from crude extract via HPLC–HRMS–SPE–NMR. Dechlorodehydrogriseofulvin was reported for the first time from nature.

Large volumes of protein-rich residual raw materials, such as heads, bones, carcasses, blood, skin, viscera, hooves and feathers, are created as a result of processing of animals from fisheries, aquaculture, livestock and poultry sectors. These residuals contain proteins and other essential nutrients with potentially bioactive properties, eligible for recycling and upgrading for higher-value products, e.g. for human, pet food and feed purposes. Here, we aim to cover all the important aspects of achieving optimal utilization of proteins in such residual raw materials, identifying those eligible for human consumption as co-products and for feed applications as by-products. Strict legislation regulates the utilization of various animal-based co- and by-products, representing a major hurdle if not addressed properly. Thorough understanding and optimization of all parts of the production chain, including conservation and processing, are important prerequisites for successful upgrading and industrial implementation of such products. This review includes industrially applied technologies such as freezing/cooling, acid preservation, salting, rendering and protein hydrolysis. In this regard, it is important to achieve stable production and quality through all the steps in the manufacturing chain, preferably supported by at- or online quality control points in the actual processing step. If aiming for the human market, knowledge of consumer trends and awareness are important for production and successful introduction of new products and ingredients.

The seed oil of Euphorbia lathyris L. contains a series of macrocyclic diterpenoids known as Euphorbia factors. They are the current industrial source of ingenol mebutate, which is approved for the treatment of actinic keratosis, a precancerous skin condition. Here, we report an alcohol dehydrogenase-mediated cyclization step in the biosynthetic pathway of Euphorbia factors, illustrating the origin of the intramolecular carbon-carbon bonds present in lathyrane and ingenane diterpenoids. This unconventional cyclization describes the ring closure of the macrocyclic diterpene casbene. Through transcriptomic analysis of E. lathyris L. mature seeds and in planta functional characterization, we identified three enzymes involved in the cyclization route from casbene to jolkinol C, a lathyrane diterpene. These enzymes include two cytochromes P450 from the CYP71 clan and an alcohol dehydrogenase (ADH). CYP71D445 and CYP726A27 catalyze regio-specific 9-oxidation and 5-oxidation of casbene, respectively. When coupled with these P450-catalyzed monooxygenations, E. lathyris ADH1 catalyzes dehydrogenation of the hydroxyl groups, leading to the subsequent rearrangement and cyclization. The discovery of this nonconventional cyclization may provide the key link to complete elucidation of the biosynthetic pathways of ingenol mebutate and other bioactive macrocyclic diterpenoids.

According to the International Diabetes Federation, type 2 diabetes (T2D) has reached epidemic proportions, affecting more than 382 million people worldwide. Inhibition of protein tyrosine phosphatase-1B (PTP1B) and α-glucosidase is a recognized therapeutic approach for management of T2D and its associated complications. The lack of clinical drugs targeting PTP1B and side effects of the existing α-glucosidase drugs, emphasize the need for new drug leads for these T2D targets. In the present work, dual high-resolution PTP1B and α-glucosidase inhibition profiles of Eremophila gibbosa, E. glabra, and E. aff. drummondii "Kalgoorlie" were used for pinpointing α-glucosidase and/or PTP1B inhibitory constituents directly from the crude extracts. A subsequent targeted high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLC-HRMS-SPE-NMR) analysis and preparative-scale HPLC isolation led to identification of 21 metabolites from the three species, of which 16 were serrulatane-type diterpenoids (12 new) associated with either α-glucosidase and/or PTP1B inhibition. This is the first report of serrulatane-type diterpenoids as potential α-glucosidase and/or PTP1B inhibitors.

1. The metabolism of midazolam was investigated in vivo in locusts in order to evaluate the presence of an enzyme with functionality similar to human CYP3A4/5. 2. Hydroxylated metabolites of midazolam identical to human metabolites were detected in locusts and the apparent affinities (Km values) were in the same range as reported in humans (in locusts: 7–23 and 33–85 µM for the formation of the 1′-OH and 4-OH metabolites, respectively). 3. The formation of hydroxylated metabolites could successfully be inhibited by co-administration of ketoconazole, a known CYP3A4/5 inhibitor. 4. Besides phase I metabolites, a number of conjugated metabolites were detected using high-resolution mass spectrometry. The most abundant metabolites detected were structurally identified by 1H NMR as two N-glucosides. NMR analysis strongly suggested that the glycosylation occurred at the two nitrogens (either one in each case) of the imidazole ring. 5. Distribution of midazolam and the glucose conjugates were successfully measured using desorption electrospray mass spectrometry imaging revealing time-dependent changes in distribution over time. 6. In conclusion, it appears that an enzyme with functionality similar to human CYP3A4/5 is present in locusts. However, it appears that conjugation with glucose is the main detoxification pathway of midazolam in locusts.

In this work, development of a new microplate-based high-resolution profiling assay using recombinant human aldose reductase is presented. Used together with high-resolution radical scavenging and high-resolution α-glucosidase assays, it provided the first report of a triple aldose reductase/α-glucosidase/radical scavenging high-resolution inhibition profile - allowing proof of concept with Radix Scutellariae crude extract as a polypharmacological herbal drug. The triple bioactivity high-resolution profiles were used to pinpoint bioactive compounds, and subsequent structure elucidation was performed with hyphenated high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy. The only α-glucosidase inhibitor was baicalein, whereas main aldose reductase inhibitors in the crude extract were baicalein and skullcapflavone II, and main radical scavengers were ganhuangemin, viscidulin III, baicalin, oroxylin A 7-O-glucuronide, wogonoside, baicalein, wogonin, and skullcapflavone II.

Extracts of five halophytes from southern Portugal (Arthrocnemum macrostachyum, Mesembryanthemum edule, Juncus acutus, Plantago coronopus and Halimione portulacoides), were studied for antioxidant, anti-inflammatory and in vitro antitumor properties. The most active extracts towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were the methanol extracts of M. edule (IC50 = 0.1 mg/mL) and J. acutus (IC50 = 0.4 mg/mL), and the ether extracts of J. acutus (IC50 = 0.2 mg/mL) and A. macrostachyum (IC50 = 0.3 mg/mL). The highest radical scavenging activity (RSA) against the 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical was obtained in the ether extract of J. acutus (IC50 = 0.4 mg/mL) and H. portulacoides (IC50 = 0.9 mg/mL). The maximum total phenolic content (TPC) was found in the methanol extract of M. edule (147 mg gallic acid equivalents (GAE)/g) and in the ether extract of J. acutus (94 mg GAE/g). Significant decreases in nitric oxide (NO) production were observed after incubation of macrophages with lipopolysaccharide (LPS) and the chloroform extract of H. portulacoides (IC50 = 109 mu g/mL) and the hexane extract of P. coronopus (IC50 = 98.0 mu g/mL). High in vitro cytotoxic activity and selectivity was obtained with the ether extract of J. acutus. Juncunol was identified as the active compound and for the first time was shown to display selective in vitro cytotoxicity towards various human cancer cells. SEABIOMED project [PTDC/MAR/103957/2008]; XtremeBio project - Foundation for Science and Technology (FCT); Portuguese National Budget; Danish Research Council for Independent Research, Nature and Universe [10-085264] info:eu-repo/semantics/publishedVersion

Sea aster (Aster tripolium L.) and searocket (Cakile maritima Scop.), potential ingredients in the New Nordic Diet, were analyzed by high-resolution radical scavenging and high-resolution α-glucosidase inhibition assays. Results from the two bioactivity profiles were used to guide subsequent structural analysis toward constituents with potential health-promoting effects. Structural analysis was performed by high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction and automated tube transfer nuclear magnetic resonance spectroscopy, that is, HPLC-HRMS-SPE-ttNMR. High-resolution mass spectrometry together with detailed analysis of one- and two-dimensional proton detected NMR experiments enabled unambiguous assignment of the targeted analytes. This revealed a series of caffeoyl esters (1, 2, 5), flavonoid glycosides (3, 4, 6, 11-15), flavonoids (7-9), sinapate esters (10, 16, 17), and sinapinic acid (18) associated with radical scavenging and/or α-glucosidase inhibition. In vitro assays implemented in this study showed that sea aster holds potential as a future functional food ingredient for lowering postprandial blood glucose level for diabetics, but further investigations are needed to prove the effect in vivo.

Time-based trapping of chromatographically separated compounds onto solid-phase extraction (SPE) cartridges and subsequent elution to NMR tubes was done to emulate the function of HPLC-NMR for dereplication purposes. Sufficient mass sensitivity was obtained by use of a state-of-the-art HPLC-SPE-NMR system with a cryogenically cooled probe head, designed for 1.7 mm NMR tubes. The resulting (1)H NMR spectra (600 MHz) were evaluated against a database of previously acquired and prepared spectra. The in-house-developed matching algorithm, based on partitioning of the spectra and allowing for changes in the chemical shifts, is described. Two mixtures of natural products were used to test the approach: an extract of Carthamus oxyacantha (wild safflower), containing an array of spiro compounds, and an extract of the endophytic fungus Penicillum namyslowski, containing griseofulvin and analogues. The database matching of the resulting spectra positively identified expected compounds, while the number of false positives was few and easily recognized.

Solid phase extraction (SPE) was introduced as a crucial step in the HPLC-SPE-NMR technique to enable online analyte enrichment from which proton-detected NMR experiments on submicrogram amounts from complex mixtures were possible. However, the significance of direct-detected (13)C NMR experiments is indubitable in simplifying structural elucidations. In the current study, we demonstrated direct (13)C NMR detection of triterpenoids from a Ganoderma lucidum extract in hyphenation mode. The combined advantage of a cryogenically cooled probe, miniaturization, and multiple trapping enabled the first reported application of HPLC-SPE-NMR analysis using direct-detected (13)C NMR spectra. HPLC column loading, accumulative SPE trappings, and the effect of different elution solvents were evaluated and optimized. A column loading of approximately 600 μg of a prefractionated triterpenoid mixture, six trappings, and an acquisition time of 13 h resulted in spectra with adequate signal-to-noise ratios to detect all C-13 signals.

An extract of Carthamus oxyacantha (wild safflower) was investigated using two approaches: a traditional, nontarget fractionation by VLC and HPLC, and the hyphenated technique HPLC-PDA-HRMS-SPE-NMR followed by targeted isolation of selected constituents for inclusion in a screening library of pure natural products. While the nontarget fractionation involved considerable time spent on pursuing fractions containing well-known or undesired compounds, the hyphenated analysis was considerably faster and required less solvent and other consumables. The results were used to design and execute an optimized, HPLC-HRMS-guided, targeted isolation scheme aiming exclusively at a series of identified spiro compounds. Thus, HPLC-PDA-HRMS-SPE-NMR is a dereplication technique of choice, allowing economical acquisition of comprehensive data about compounds in crude extracts, which can be used for rational, prospective decisions about further isolation efforts. A total of 15 compounds were identified in the extract. Six spiro compounds, of which four have not previously been characterized, and tracheloside (a lignin glucoside) are presented with assigned 1H and 13C chemical shifts.