The role of biospectroscopy and chemometrics as enabling technologies for upcycling of raw materials from the food industry.

It is important to utilize the entire animal in meat and fish production to ensure sustainability. Rest raw materials, such as bones, heads, trimmings, and skin, contain essential nutrients that can be transformed into high-value products. Enzymatic protein hydrolysis (EPH) is a bioprocess that can upcycle these materials to create valuable proteins and fats. This paper focuses on the role of spectroscopy and chemometrics in characterizing the quality of the resulting protein product and understanding how raw material quality and processing affect it. The article presents recent developments in chemical characterisation and process modelling, with a focus on rest raw materials from poultry and salmon production. Even if some of the technology is relatively mature and implemented in many laboratories and industries, there are still open challenges and research questions. The main challenges are related to the transition of technology and insights from laboratory to industrial scale, and the link between peptide composition and critical product quality attributes.

FTIR-based prediction of collagen content in hydrolyzed protein samples.

Fourier transform infrared spectroscopy (FTIR) is a powerful analytical tool that has been used for protein and peptide characterization for decades. In the present study, the objective was to investigate if FTIR can be used to predict collagen content in hydrolyzed protein samples. All samples were obtained from enzymatic protein hydrolysis (EPH) of poultry by-products providing a span in collagen content from 0.3% to 37.9% (dry weight), and the FTIR analysis was performed using dry film FTIR. Since nonlinear effects were revealed by calibration using standard partial least squares (PLS) regression, Hierarchical Cluster-based PLS (HC-PLS) calibration models were constructed. The HC-PLS model provided a low prediction error when validated using an independent test set (RMSE = 3.3% collagen), while validation using real industrial samples also showed satisfying results (RMSE = 3.2%). The results corresponded well with previously published FTIR-based studies of collagen, and characteristic spectral features for collagen were well identified in the regression models. Covariance between collagen content and other EPH related processing parameters could also be ruled out in the regression models. To the authors' knowledge, this is the first time that collagen content has been systematically studied in solutions of hydrolysed proteins using FTIR. This is also one of few examples where FTIR is successfully used to quantify protein composition. The dry-film FTIR approach presented in the study is expected to be an important tool in the growing industrial segment that is based on sustainable utilization of collagen-rich biomass.

Dietary intake of micronized avian eggshell membrane in aged mice reduces circulating inflammatory markers, increases microbiota diversity, and attenuates skeletal muscle aging.

Introduction: Avian eggshell membrane (ESM) is a complex extracellular matrix comprising collagens, glycoproteins, proteoglycans, and hyaluronic acid. We have previously demonstrated that ESM possesses anti-inflammatory properties in vitro and regulates wound healing processes in vivo. The present study aimed to investigate if oral intake of micronized ESM could attenuate skeletal muscle aging associated with beneficial alterations in gut microbiota profile and reduced inflammation. Methods: Elderly male C57BL/6 mice were fed an AIN93G diet supplemented with 0, 0.1, 1, or 8% ESM. Young mice were used as reference. The digestibility of ESM was investigated using the static in vitro digestion model INFOGEST for older people and adults, and the gut microbiota profile was analyzed in mice. In addition, we performed a small-scale pre-clinical human study with healthy home-dwelling elderly (>70 years) who received capsules with a placebo or 500 mg ESM every day for 4 weeks and studied the effect on circulating inflammatory markers. Results and discussion: Intake of ESM in elderly mice impacted and attenuated several well-known hallmarks of aging, such as a reduction in the number of skeletal muscle fibers, the appearance of centronucleated fibers, a decrease in type IIa/IIx fiber type proportion, reduced gene expression of satellite cell markers Sdc3 and Pax7 and increased gene expression of the muscle atrophy marker Fbxo32. Similarly, a transition toward the phenotypic characteristics of young mice was observed for several proteins involved in cellular processes and metabolism. The digestibility of ESM was poor, especially for the elderly condition. Furthermore, our experiments showed that mice fed with 8% ESM had increased gut microbiota diversity and altered microbiota composition compared with the other groups. ESM in the diet also lowered the expression of the inflammation marker TNFA in mice and in vitro in THP-1 macrophages. In the human study, intake of ESM capsules significantly reduced the inflammatory marker CRP. Altogether, our results suggest that ESM, a natural extracellular biomaterial, may be attractive as a nutraceutical candidate with a possible effect on skeletal muscle aging possibly through its immunomodulating effect or gut microbiota.

Effect of Citric Acid Cross Linking on the Mechanical, Rheological and Barrier Properties of Chitosan.

In this study, acetic acid (AA-2% w/v), a combination of acetic acid and citric acid (AA-1% w/v + CA-1% w/w), and three different concentrations of citric acid (CA-2, 4 and 6% w/w) were used to create chitosan solution. The FTIR analysis showed the presence of residual CA in all the CA-containing samples where no trace of AA was observed. The tensile strengths of the CA-containing samples were lower than the AA samples. Whereas the values for the elongation at break of the CA samples were higher than the AA samples, which kept increasing with an increasing CA content due to the plasticizing effect from residual citric acid. The elongation at break values for 4 and 6% CA-containing samples were 98% higher than the AA samples. The samples prepared with CA showed shorter LVE regions that reduced with an increasing CA concentration compared to the AA samples. Different acid concentrations did not have a large effect on the gelation time. However, CA-containing samples showed higher viscosities as compared to the AA-containing solution, which increased with an increasing CA content. The water vapour transmission rates of the CA-containing samples were lower than the others. All the chitosan solutions suppressed the growth of the two test strains, and none of the variants reached an abs 600 nm at 0.2.

Post-enzymatic hydrolysis heat treatment as an essential unit operation for collagen solubilization from 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.

Improved estimation of in vitro protein digestibility of different foods using size exclusion chromatography.

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.

Characterization of Collagen Structure in Normal, Wooden Breast and Spaghetti Meat Chicken Fillets by FTIR Microspectroscopy and Histology.

Recently, two chicken breast fillet abnormalities, termed Wooden Breast (WB) and Spaghetti Meat (SM), have become a challenge for the chicken meat industry. The two abnormalities share some overlapping morphological features, including myofiber necrosis, intramuscular fat deposition, and collagen fibrosis, but display very different textural properties. WB has a hard, rigid surface, while the SM has a soft and stringy surface. Connective tissue is affected in both WB and SM, and accordingly, this study's objective was to investigate the major component of connective tissue, collagen. The collagen structure was compared with normal (NO) fillets using histological methods and Fourier transform infrared (FTIR) microspectroscopy and imaging. The histology analysis demonstrated an increase in the amount of connective tissue in the chicken abnormalities, particularly in the perimysium. The WB displayed a mixture of thin and thick collagen fibers, whereas the collagen fibers in SM were thinner, fewer, and shorter. For both, the collagen fibers were oriented in multiple directions. The FTIR data showed that WB contained more ß-sheets than the NO and the SM fillets, whereas SM fillets expressed the lowest mature collagen fibers. This insight into the molecular changes can help to explain the underlying causes of the abnormalities.

Magnetic ligand fishing using immobilized DPP-IV for identification of antidiabetic ligands in lingonberry extract.

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.

Microdochium majus and other fungal pathogens associated with reduced gluten quality in wheat grain.

The bread-making quality of wheat depends on the viscoelastic properties of the dough in which gluten proteins play an important role. The quality of gluten proteins is influenced by the genetics of the different wheat varieties and environmental factors. Occasionally, a near complete loss of gluten strength, measured as the maximum resistance towards stretching (Rmax), is observed in grain lots of Norwegian wheat. It is hypothesized that the loss of gluten quality is caused by degradation of gluten proteins by fungal proteases. To identify fungi associated with loss of gluten strength, samples from a selection of wheat grain lots with weak gluten (n = 10, Rmax < 0.3 N) and strong gluten (n = 10, Rmax ≥ 0.6 N) was analyzed for the abundance of fungal operational taxonomic units (OTUs) using DNA metabarcoding of the nuclear ribosomal Internal Transcribed Spacer (ITS) region ITS1. The DNA quantities for a selection of fungal pathogens of wheat, and the total amount of fungal DNA, were analyzed by quantitative PCR (qPCR). The mean level of total fungal DNA was higher in grain samples with weak gluten compared to grain samples with strong gluten. Heightened quantities of DNA from fungi within the Fusarium Head Blight (FHB) complex, i.e. Fusarium avenaceum, Fusarium graminearum, Microdochium majus, and Microdochium nivale, were observed in grain samples with weak gluten compared to those with strong gluten. Microdochium majus was the dominant fungus in the samples with weak gluten. Stepwise regression modeling based on different wheat quality parameters, qPCR data, and the 35 most common OTUs revealed a significant negative association between gluten strength and three OTUs, of which the OTU identified as M. majus was the most abundant. The same analysis also revealed a significant negative relationship between gluten strength and F. avenaceum detected by qPCR, although the DNA levels of this fungus were low compared to those of M. majus. In vitro growth rate studies of a selection of FHB species showed that all the tested isolates were able to grow with gluten as a sole nitrogen source. In addition, proteins secreted by these fungi in liquid cultures were able to hydrolyze gluten substrate proteins in zymograms, confirming their capacity to secrete gluten-degrading proteases. The identification of fungi with potential to influence gluten quality can enable the development of strategies to minimize future problems with gluten strength in food-grade wheat.

Fourier-transform infrared spectroscopy for monitoring proteolytic reactions using dry-films treated with trifluoroacetic acid.

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.

Average molecular weight, degree of hydrolysis and dry-film FTIR fingerprint of milk protein hydrolysates: Intercorrelation and application in process monitoring.

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.

FTIR-based hierarchical modeling for prediction of average molecular weights of protein hydrolysates.

In the presented study, Fourier-transform infrared (FTIR) spectroscopy is used to predict the average molecular weight of protein hydrolysates produced from protein-rich by-products from food industry using commercial enzymes. Enzymatic protein hydrolysis is a well-established method for production of protein-rich formulations, recognized for its potential to valorize food-processing by-products. The monitoring of such processes is still a significant challenge as the existing classical analytical methods are not easily applicable to industrial setups. In this study, we are reporting a generic FTIR-based approach for monitoring the average molecular weights of proteins during enzymatic hydrolysis of by-products from the food industry. A total of 885 hydrolysate samples from enzymatic protein hydrolysis reactions of poultry and fish by-products using different enzymes were studied. FTIR spectra acquired from dry-films of the hydrolysates were used to build partial least squares regression (PLSR) models. The most accurate predictions were obtained using a hierarchical PLSR approach involving supervised classification of the FTIR spectra according to raw material quality and enzyme used in the hydrolysis process, and subsequent local regression models tuned to specific enzyme-raw material combinations. The results clearly underline the potential of using FTIR for monitoring protein sizes during enzymatic protein hydrolysis in industrial settings, while also paving the way for measurements of protein sizes in other applications.

The use of Fourier-transform infrared spectroscopy to characterize connective tissue components in skeletal muscle of Atlantic cod (Gadus morhua L.).

In the present study, Fourier-transform infrared spectroscopy (FTIR) is investigated as a method to measure connective tissue components that are important for the quality of Atlantic cod filets (Gadus morhua L.). The Atlantic cod used in this study originated from a feeding trial, which found that fish fed a high starch diet contained relative more collagen type I, while fish fed a low starch (LS) diet contained relative more glycosaminoglycans (GAGs) in the connective tissue. FTIR spectra of pure commercial collagen type I and GAGs were acquired to identify spectral markers and compare them with FTIR spectra and images from connective tissue. Using principal component analysis, high and LS diets were separated due to collagen type I in the spectral region 1800 to 800 cm-1 . The spatial distribution of collagen type I and GAGs were further investigated by FTIR imaging in combination with immunohistochemistry. Pixel-wise correlation images were calculated between preprocessed connective tissue images and preprocessed pure components spectra of collagen type I and GAGs, respectively. For collagen, the FTIR images reveal a collagen distribution that closely resembles the collagen distribution as imaged by immunohistochemistry. For GAGs, the concentration is very low. Still, the FTIR images detect the most GAGs rich regions.

Combined magnetic ligand fishing and high-resolution inhibition profiling for identification of α-glucosidase inhibitory ligands: A new screening approach based on complementary inhibition and affinity profiles.

Plants are well-recognized sources of inhibitors for α-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 α-glucosidase inhibitors from complex plant extracts. Non-specific affinities and non-specific inhibitions are major sources of false positive hits from ligand fishing and high-resolution 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 identify α-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, α-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.

Quantification of 1,3-ß-D-glucan from yeast added as a functional ingredient to bread.

Due to their immunomodulatory effect, 1,3-ß-G from yeast are used as functional ingredients, but reliable methods for their detection in foods are lacking. We have adapted a method based on fluorescence detection with aniline blue to quantify the amount of five commercial yeast ß-glucan preparations added to crisp or yeast-leavened bread. This assay detected yeast ß-glucan preparations added to different breads with an average recovery of 90, 96, 99 and 105%, while one of the preparations was overestimated, with an average recovery of 157%. The presence of cereal 1,3-1,4-ß-D-glucans did not interfere with assay performance. The addition of 1,3-ß-G at 0.2 and 0.5 g/100g is low compared to the recommended dose of 1,3-ß-G per serving demonstrating assay sensitivity. However, more research is needed to fully understand the effect of 1,3-ß-G conformation/structure on aniline blue interaction as well as the effect of baking on structure and dissolution properties of yeast ß-glucans.

Fourier-transform infrared spectroscopy for characterization of protein chain reductions in enzymatic reactions.

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.

Effect of sodium bicarbonate and varying concentrations of sodium chloride in brine on the liquid retention of fish (Pollachius virens L.) muscle.

BACKGROUND: Negative health effects associated with excessive sodium (Na) intake have increased the demand for tasty low-Na products (<2% NaCl) rather than traditional heavily salted fish products (∼20% NaCl). This study investigates the causes of improved yield and liquid retention of fish muscle brined with a combination of salt (NaCl) and sodium bicarbonate (NaHCO3 ). RESULTS: Water characteristics and microstructure of saithe (Pollachius virens L.) muscle brined in solutions of NaCl and NaHCO3 or NaCl alone were compared using low-field nuclear magnetic resonance (LF-NMR) T2 relaxometry, microscopy, salt content, liquid retention and colorimetric measurements. Saithe muscle was brined for 92 h in 0, 30, 60, 120 or 240 g kg(-1) NaCl or the respective solutions with added 7.5 g kg(-1) NaHCO3 . NaHCO3 inclusion improved the yield in solutions ranging from 0 to 120 g kg(-1) NaCl, with the most pronounced effect being observed at 30 g kg(-1) NaCl. The changes in yield were reflected in water mobility, with significantly shorter T2 relaxation times in all corresponding brine concentrations. Salt-dependent microstructural changes were revealed by light microscopy, where NaHCO3 supplementation resulted in greater intracellular space at 30 and 60 g kg(-1) NaCl. CONCLUSION: Sodium bicarbonate addition to low-salt solutions can improve yield and flesh quality of fish muscle owing to altered water mobility and wider space between the muscle cells.

Influence of temperature during grain filling on gluten viscoelastic properties and gluten protein composition.

BACKGROUND: The aim of this study was to investigate the effects of low to moderate temperatures on gluten functionality and gluten protein composition. Four spring wheat cultivars were grown in climate chambers with three temperature regimes (day/night temperatures of 13/10, 18/15 and 23/20 °C) during grain filling. RESULTS: The temperature strongly influenced grain weight and protein content. Gluten quality measured by maximum resistance to extension (Rmax ) was highest in three cultivars grown at 13 °C. Rmax was positively correlated with the proportion of sodium dodecyl sulfate-unextractable polymeric proteins (%UPP). The proportions of ω-gliadins and D-type low-molecular-weight glutenin subunits (LMW-GS) increased and the proportions of α- and γ-gliadins and B-type LMW-GS decreased with higher temperature, while the proportion of high-molecular-weight glutenin subunits (HMW-GS) was constant between temperatures. The cultivar Berserk had strong and constant Rmax between the different temperatures. CONCLUSION: Constant low temperature, even as low as 13 °C, had no negative effects on gluten quality. The observed variation in Rmax related to temperature could be explained more by %UPP than by changes in the proportions of HMW-GS or other gluten proteins. The four cultivars responded differently to temperature, as gluten from Berserk was stronger and more stable over a wide range of temperatures.

High-throughput biochemical fingerprinting of Saccharomyces cerevisiae by Fourier transform infrared spectroscopy.

Single-channel optical density measurements of population growth are the dominant large scale phenotyping methodology for bridging the gene-function gap in yeast. However, a substantial amount of the genetic variation induced by single allele, single gene or double gene knock-out technologies fail to manifest in detectable growth phenotypes under conditions readily testable in the laboratory. Thus, new high-throughput phenotyping technologies capable of providing information about molecular level consequences of genetic variation are sorely needed. Here we report a protocol for high-throughput Fourier transform infrared spectroscopy (FTIR) measuring biochemical fingerprints of yeast strains. It includes high-throughput cultivation for FTIR spectroscopy, FTIR measurements and spectral pre-treatment to increase measurement accuracy. We demonstrate its capacity to distinguish not only yeast genera, species and populations, but also strains that differ only by a single gene, its excellent signal-to-noise ratio and its relative robustness to measurement bias. Finally, we illustrated its applicability by determining the FTIR signatures of all viable Saccharomyces cerevisiae single gene knock-outs corresponding to lipid biosynthesis genes. Many of the examined knock-out strains showed distinct, highly reproducible FTIR phenotypes despite having no detectable growth phenotype. These phenotypes were confirmed by conventional lipid analysis and could be linked to specific changes in lipid composition. We conclude that the introduced protocol is robust to noise and bias, possible to apply on a very large scale, and capable of generating biologically meaningful biochemical fingerprints that are strain specific, even when strains lack detectable growth phenotypes. Thus, it has a substantial potential for application in the molecular functionalization of the yeast genome.

A high-throughput microcultivation protocol for FTIR spectroscopic characterization and identification of fungi.

Characterization and identification of fungi in food industry is an important issue both for routine analysis and trouble-shooting incidences. Present microbial techniques for fungal characterization suffer from a low throughput and are time consuming. In this study we present a protocol for high-throughput microcultivation and spectral characterization of fungi by Fourier transform infrared spectroscopy. For the study 11 species of in total five different fungal genera (Alternaria, Aspergillus, Mucor, Paecilomyces, and Phoma) were analyzed by FTIR spectroscopy. All the strains were isolated from trouble-shooting incidents in the production of low and high acid beverages. The cultivation was performed in malt extract broth (liquid medium) in a Bioscreen C system, allowing high-throughput cultivation of 200 samples at the same time. Mycelium was subsequently investigated by high-throughput Fourier transform infrared spectroscopy. Four spectral regions, fatty acids + lipid (3200-2800 cm(-1), 1300-1000 cm(-1)), protein-lipid (1800-1200 cm(-1)), carbohydrates (1200-700 cm(-1)) and "finger print" (900-700 cm(-1)) were evaluated for reproducibility and discrimination ability. The results show that all spectral regions evaluated can be used as spectroscopic biomarkers for differentiation of fungi by FTIR. The influence of different growth times on the ability of species discrimination by FTIR spectroscopy was investigated, and an optimal separation of all five genera was observed after five days of growth. This work presents a novel concept for high-throughput cultivation of fungi for FTIR spectroscopy that enables characterization or identification of hundreds of strains per day.