Fungal production of polyunsaturated fatty acids (PUFAs) is a highly potential approach in biotechnology. Currently the main focus is directed towards screening of hundreds strains in order to select of few potential ones. Thus, a reliable method for screening a high number of strains within a short period of time is needed. Here, we present a novel method for screening of PUFA-producing fungi by high-throughput microcultivation and FTIR spectroscopy. In the study selected Mucor fungi were grown in media with different carbon sources and fatty acid profiles were predicted on the basis of the obtained spectral data. FTIR spectra were calibrated against fatty acid analysis by GC-FD. The calibration models were cross-validated and correlation coefficients (R2) from 0.71 to 0.78 with RMSECV (root mean squared error) from 2.86% to 6.96% (percentage of total fat) were obtained. The FTIR results show a strong correlation to the results obtained by GC analysis, where high total contents of unsaturated fatty acids (both PUFA and MUFA) were achieved for Mucor plumbeus VI02019 cultivated in canola, olive and sunflower oil and Mucor hiemalis VI01993 cultivated in canola and olive oil.

Mucoromycota fungi are important producers of low- and high-value lipids. Mortierella alpina is used for arachidonic acid production at industrial scale. In addition, oleaginous Mucoromycota fungi are promising candidates for biodiesel production. A critical step in the development of such biotechnological applications is the selection of suitable strains for lipid production. The aim of the present study was to use the Duetz-microtiter plate system combined with Fourier transform infrared (FTIR) spectroscopy for high-throughput screening of the potential of 100 Mucoromycota strains to produce low- and high-value lipids. With this reproducible, high-throughput method, we found several promising strains for high-value omega-6 polyunsaturated fatty acid (PUFA) and biodiesel production purposes. Gamma-linolenic acid content was the highest in Mucor fragilis UBOCC-A-109196 (24.5% of total fatty acids), and Cunninghamella echinulata VKM F-470 (24.0%). For the first time, we observed concomitant gamma-linolenic acid and alpha-linolenic acid (up to 13.0%) production in psychrophilic Mucor flavus strains. Arachidonic acid was present the highest amount in M. alpina ATCC 32222 (41.1% of total fatty acids). Low cultivation temperature (15 °C) activated the temperature sensitive ∆17 desaturase enzyme in Mortierella spp., resulting in eicosapentaenoic acid production with up to 11.0% of total fatty acids in M. humilis VKM F-1494. Cunninghamella blakesleeana CCM-705, Umbelopsis vinacea CCM F-539 and UBOCC-A-101347 showed very good growth (23–26 g/L) and lipid production (7.0–8.3 g/L) with high palmitic and oleic acid, and low PUFA content, which makes them attractive candidates for biodiesel production. Absidia glauca CCM 451 had the highest total lipid content (47.2% of biomass) of all tested strains. We also demonstrated the potential of FTIR spectroscopy for high-throughput screening of total lipid content of oleaginous fungi. The use of Duetz-microtiter plate system combined with FTIR spectroscopy and multivariate analysis, is a feasible approach for high-throughput screening of lipid production in Mucoromycota fungi. Several promising strains have been identified by this method for the production of high-value PUFA and biodiesel.

Oleaginous fungi can accumulate lipids by utilizing a wide range of waste substrates. They are an important source for the industrial production of omega-6 polyunsaturated fatty acids (gamma-linolenic and arachidonic acid) and have been suggested as an alternative route for biodiesel production. Initial research steps for various applications include the screening of fungi in order to find efficient fungal producers with desired fatty acid composition. Traditional cultivation methods (shake flask) and lipid analysis (extraction-gas chromatography) are not applicable for large-scale screening due to their low throughput and time-consuming analysis. Here we present a microcultivation system combined with high-throughput Fourier transform infrared (FTIR) spectroscopy for efficient screening of oleaginous fungi. The microcultivation system enables highly reproducible fungal fermentations throughout 12 days of cultivation. Reproducibility was validated by FTIR and HPLC data. Analysis of FTIR spectral ester carbonyl peaks of fungal biomass offered a reliable high-throughput at-line method to monitor lipid accumulation. Partial least square regression between gas chromatography fatty acid data and corresponding FTIR spectral data was used to set up calibration models for the prediction of saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, unsaturation index, total lipid content and main individual fatty acids. High coefficients of determination (R2 = 0.86–0.96) and satisfactory residual predictive deviation of cross-validation (RPDCV = 2.6–5.1) values demonstrated the goodness of these models. We have demonstrated in this study, that the presented microcultivation system combined with rapid, high-throughput FTIR spectroscopy is a suitable screening platform for oleaginous fungi. Sample preparation for FTIR measurements can be automated to further increase throughput of the system.

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.

In the present study, the possibility of employing spatially offset Raman spectroscopy (SORS) in the qualitative and quantitative characterization of quality parameters of salmon through the skin has been explored. A laboratory-based SORS setup comprising an 830 nm laser was employed, and intact samples and model samples made of salmon tissue constituents were used to investigate the penetration of Raman signals through the dark and light part of salmon skin. Intact salmon samples with both dark and light skin were measured at different spatial offsets. When using spatial offsets in the range of 5–6 mm, the results clearly show that information regarding fatty acid composition and carotenoid content could be obtained from both dark and light parts of the skin. Similar information could not be obtained using conventional backscattering Raman spectroscopy. Model samples of ground salmon spiked with either solutions of carotenoids or a range of vegetable oils were also measured, and at a spatial offset of 5 mm, a clear relationship between Raman carotenoid band intensities and carotenoid concentrations in the model samples was revealed. In addition, high correlations for the estimation of iodine values (i.e., fatty acid unsaturation) could be obtained for SORS measurements through light and dark parts of the salmon skin. A crude estimate suggested that information from around 5 mm beneath the surface area of the salmon skin could be obtained. The choice of a laser line in the near-infrared region is a major prerequisite for successful through-skin analysis of salmon. This feasibility study could pave the way for future Raman analysis of intact salmon.

In the present study a novel approach for Fourier transform infrared (FT-IR) characterization of the fatty acid composition of milk based on dried film measurements has been presented and compared to a standard FT-IR approach based on liquid milk measurements. Two hundred and sixty-two (262) milk samples were obtained from a feeding experiment, and the samples were measured with FT-IR as dried films as well as liquid samples. Calibrations against the most abundant fatty acids, CLA (i.e., 18:2 cis-9, trans-11), 18:3 cis-9, cis-12, cis-15, and summed fatty acid parameters were obtained for both approaches. The estimation errors obtained in the dried film calibrations were overall lower than the corresponding liquid sample calibrations. Similar and good calibrations (i.e., R² ranges from 0.82 to 0.94 (liquid samples) and from 0.88 to 0.97 (dried films)) for short-chain fatty acids (6:0–14:0), 18:1 cis-9, SAT, MUFA, and iodine value were obtained by both approaches. However, the dried film approach was the only approach for which feasible calibrations (i.e., R² ranges from 0.78 to 0.93) were obtained for the major saturated fatty acids 16:0 and 18:0, the minor fatty acid features 4:0, CLA (i.e., 18:2 cis-9, trans-11), PUFA, and the summed 18:1 trans isomers. For the dried film approach, logical spectral features were found to dominate the respective fatty acid calibration models. The preconcentration step of the dried film approach could be expected to account for a major part of the prediction improvements going from predictions in liquid milk to predictions in dried films. The dried film approach has a significant potential for use in high-throughput applications in industrial environments and might also serve as a valuable supplement for determination of genetic and breeding factors within research communities.