The scheme of the experiment is presented in Fig. 2.Figure 2The scheme of the experiment.Hazelnut seed skinsThe research material was HSS (Ferrero; Belsk Duży, Poland), a by-product material remaining after the roasting of hazelnuts and water extracts of this material were used for the research. The HSS before extraction and all analyses were ground to obtain a powder by laboratory grinder (Chemland; Stargard; Poland). Material was stored at − 20 °C.Extraction method and extraction efficiencyThe extraction method was adapted according to Ferysiuk et al.19. Two types of samples were researched, with different amounts of the HSS substrate used for extraction. The 5 g HSS per 100 g of water (E5) and 10 g HSS per 100 g of water (E10) were weighed separately into the 100 mL tubes. The tubes were filled with 90 ℃ water and sonicated in the ultrasound bath (IS-6; 35 kHz; Inter Sonic; Olsztyn, Poland) for 10 min at 90 ℃. Next, the extracts were centrifugated (Eppendorf Centrifuge 5804 R; Hamburg, Germany) at 6000 rpm and 4 ℃ for 15 min. The extracts were filed into a separate beaker and froze at – 80 ℃. Next, samples were freeze-dried using Labconco freeze-drier (− 45 ℃, 0.11 mBar) for 6 days. Freeze-dried the extracts were stored tightly closed at − 20 ℃.Extraction level efficiency was obtained by the weight method. Liquid extracts were used in this evaluation. Empty glass weighing dishes were weighed, filled extracts (E5 and E10) and weighed again. Samples were dried at 100 °C, at atmospheric pressure, for 24 h in a laboratory dryer. After this time samples were cooled and put to the exicator to stabilise weight for 24 h. Samples were weighted on the laboratory scale (AS 60/220.R2; Radwag; Radom; Poland). Weighting was performed accurately to within 0.00001 g. Extraction level efficiency was calculated like g dry mater extracted from 100 g of the HSS. Experiments were performed in 3 independent biological replicates.Total polyphenol content, individual polyphenols and antioxidant capacityThe 100 mg of HSS or HSS extracts (freeze-dried) were mixed with 5 mL of 80% methanol (Chempur, Piekary ÅšlÄ…skie, Poland). The samples were extracted in an ultrasonic bath (IS-6; 35 kHz; Inter Sonic; Olsztyn, Poland) at 30 °C for 10 min. The samples were centrifuged using an Eppendorf Centrifuge 5804 R (Eppendorf; Hamburg, Germany) for 10 min at 10,000 rpm and 0 °C. The extract was stored at − 20 °C in the dark until use.Total polyphenol content (TPC)The Attard46 method was used to determine TPC. The extract of the sample was diluted in ultrapure water (HPL 20UV; Hydrolab; Straszyn, Poland). 20 µL of the prepared sample dilution was poured into the 96-well plate (NEST Biotechnology; Wuxi, China), and 100 µL of F–C (Folin-Ciocalteu’s phenol reagent; Chempur, Piekary ÅšlÄ…skie, Poland) was added. The plate was left for 5 min at room temperature (20 °C) in the dark. 80 μL of the (7.5 g 100 g−1) sodium carbonate (Chempur, Piekary ÅšlÄ…skie, Poland) solution in H2O was poured into the wells and mixed at 150 rpm for 5 min. The samples were left for 2 h in the dark. The absorption was measured at wavelength λ = 750 nm using the SpectraMax iD3 reader (Molecular Devices, San Jose, CA, USA). Before the measurement, the plate was shaken for 1 min by the auto-shaker of the SpectraMax iD3 reader. Experiments were performed in 8 independent biological replicates.HPLC polyphenol determinationPolyphenols according to Król et al.27. Shimadzu equipment was used for analysis (USA Manufacturing Inc, USA, two LC-20AD pumps, a CBM-20A controller, a CTD-20AC oven, a SIL-20AC autosampler, and a UV/Vis SPD-20AV detector). The samples were filtered before being analysed by 0.22 μm. 20 μL of the sample was injected into the HPLC Synergi Fusion-RP 80i Phenomenex column (250 mm × 4.60 mm; Torrance, CA, USA). Polyphenols were separated under gradient conditions with a flow rate of 1 mL min−1. As liquid phases were employed an aqueous solution of 10 mL 100 mL−1 acetonitrile (phase A) (Sigma-Aldrich, PoznaÅ„, Poland) and 55 mL 100 mL−1 acetonitrile (phase B) (Sigma-Aldrich, PoznaÅ„, Poland. Both phases were acidified by ortho-phosphoric acid (Sigma-Aldrich, PoznaÅ„, Poland) to pH 3.0. The analysis lasted 38 min. The phases changed as follows: 1.00–22.99 min 95% phase A and 5% phase B, 23.00–27.99 min 50% phase A and 50% phase B, 28.00–35.99 min 80% phase A and 20% phase B, and 36.00–38.00 min 95% phase A and 5% phase B. The wavelengths used for detection were 250 nm for flavonols and 370 nm for phenolic acids. Identifying individual phenolics was established on Sigma–Aldrich and Fluka external standards with a 99.00–99.99% purity. Experiments were performed in 3 independent replicates.ABTS· + (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)ABTS· + (Sigma-Aldrich, PoznaÅ„, Poland) was prepared 24 h before the determination. The powder ABTS· + radicals (7 mM L−1) were mixed with K2S2O8 salt (2.45 mM L−1) (Sigma-Aldrich, PoznaÅ„, Poland) in deionized water. The solution was stored at room temperature in the dark. Before the determination, the ABTS· + solution was diluted with PBS (phosphate-buffered saline; Sigma-Aldrich, PoznaÅ„, Poland), and the absorbance of the radicals was adjusted to 0.7 ± 0.02 at 734 nm. The samples were diluted in demineralised water. 50 μL of each of the test sample solutions and 150 μL of the ABTS· + radical solution were poured into a well of a 96-well plate (NEST Biotechnology; Wuxi, China). The reaction was performed for 6 min and then immediately measured at 734 nm with a SpectraMax iD3 reader (Molecular Devices, San Jose, CA, USA). The determination was carried out with limited access to light. Experiments were performed in 8 independent replicates.DPPH (1,1-Diphenyl-2-picrylhydrazyl)The antioxidant activity was determined using the radicals DPPH (Sigma-Aldrich, PoznaÅ„, Poland). The inactive powdered radicals were mixed with pure < 99.5% methanol (Chempur, Piekary ÅšlÄ…skie, Poland). The solution was diluted to the absorbance of 1.1 ± 0.05 using methanol at the wavelength λ = 517 nm. The DPPH solution was stored in the dark. The samples were diluted in demineralised water. The tested sample contained 150 µL of DPPH solution and 2 µL of the sample extract. The absorbance was measured after 30 min from the initiation of the reaction. The sample was stored in the dark with constant shaking at 150 rpm throughout the incubation period. Absorbance was measured at a wavelength of λ = 517 nm using a SpectraMax iD3 reader (Molecular Devices, San Jose, CA, USA). The determination was carried out with limited access to light. Experiments were performed in 8 independent replicates.Allergens contentThe determination was performed according to SÅ‚owianek et al.47. The following reagents were used to assess the allergenic potential of the samples: mouse antibodies against Bet v1 (Dendritics, Dardilly, France), rabbit antibodies against profilin (Dendritics, Dardilly, France), the conjugate of antibodies against mouse immunoglobulins with alkaline phosphatase (Sigma-Aldrich, PoznaÅ„, Poland), antibodies against the rabbit immunoglobulin conjugate with alkaline phosphatase (Sigma-Aldrich, PoznaÅ„, Poland). 10 μl of each of the above-mentioned reagents was suspended in 10 ml of deionized water. Commercial skim milk solution (3 g 100 g−1) in deionized water and pNPP (p-Nitrophenyl Phosphate; Sigma-Aldrich, PoznaÅ„, Poland) as the substrate for the alkaline phosphatase, and 3 M NaOH (Sigma-Aldrich, PoznaÅ„, Poland) as the stop reagent was used. As a washing solution, the PBS with 0.1 mL 100 mL−1 Tween 20 (Sigma-Aldrich, PoznaÅ„, Poland) was used. The total protein extraction kit for plant tissues was used to obtain the extracts of the samples. The standard solution was prepared by serial dilutions of 0.01 mg per 1 ml Bet v1 stock solution or profilin for the standard curve. First, each well of the microplate (SPL Lifesciences, Geumgang-ro, Korea) was filled with 100 μL of the standard solution (reference curve) or the samples of the extracts diluted ten times in the carbonate buffer. The plate was then incubated at 4 °C for 12 h. Next, the wells were washed 4 times with 350 µL of the PBS solution. The plate was incubated for 2 h, and after that 400 μL of 3 g 100 g−1 skim milk in PBS solution was added to the wells. The wells were rinsed again 4 times with 350 µl PBS solution. Then, 100 μL of the antibodies against Bet v1 (1000 × diluted) or against profilin (1000 × diluted) was added and incubated for 1 h at room temperature. After that, the plate was washed 4 times with 350 μL of the washing buffer. Next, 100 μL of the anti-mouse antibody in the case of analogues Bet v 1 determination (or anti-rabbit in the case of profilin determination) conjugated to alkaline phosphatase (diluted 5000×) was added to each well and incubated for one hour. The plate was washed 4 times with 350 μL of the washing buffer. Finally, 100 μL of the substrate pNPP for the enzyme was added. After 30 min, the yellow colour was observed, and the reaction was stopped by adding 100 μL of the stopping NaOH solution. The plate was read at 405 nm by a Multiscan RC microplate reader (Labsystems, Vantaa, Finland). The results were calculated using a standard curve prepared with the Bet v 1 allergen (the range of concentration 0.5–50 ng mL−1) or profilin (range of concentration 0.5–100 ng mL−1). Bet v 1 limit detection was 0.88 ng mL−1, for profilin it was 1.2 ng mL−1. Experiments were performed in 8 independent replicates.Bacteria growth inhibitionDetermination of MIC (minimal inhibitory concentration) of the HSS extracts was carried out according to Wiegand et al.48. Each strain was activated from frozen (− 80 °C) cultures by incubation overnight at 37 °C in nutrient broth (Oxoid, Basingstoke, UK) except LAB lactic acid bacteria (LAB), Enterococcus faecalis and Clostridium strains. LAB and Enterococcus faecalis were activated with MRS broth (De Man, Rogosa, Sharpe; Biokar Diagnostic, Allonne, France). Enterococcus faecalis strains were incubated at 30 °C. LAB was incubated at 37 C. Clostridium strains were activated in the cooked meat medium (Becton, Dickinson and Company; Maryland, USA) at 37 °C. Clostridium strains were incubated in anaerobic conditions with the of use an absorbing oxygen bag (Thermo Fisher Scientific; Waltham, MA, USA). After overnight incubation, 1 mL of all strains were separately transferred to the new broth medium (9 mL) and incubated for another 24 h. MH agar (Mueller–Hinton; Bio-Rad; Hercules, CA, USA) was used for the MIC determination. Tested microorganisms are listed in Table 2. To obtain the growth of LAB and E. faecalis, 1 g of glucose (Sigma-Aldrich; PoznaÅ„, Poland) for 100 g MH medium was added. HSS extracts were mixed with sterile distillate water to 100 mg g−1 concentration. Water HSS extracts were diluted in a molten MH at 40 °C to receive tested concentration i.e., 1—10 mg g−1. After adding the extract solution, the MH agar was immediately poured onto sterile plates and cooled. Bacterial cultures were diluted in buffered peptone water (Biocorp; Warsaw, Poland). The tested strain with a density of 104 colony forming units mL−1 was spread on a growth medium. The samples were incubated for 24 h under the above conditions selected for the bacteria species. The positive control was MH agar inoculated with the bacteria without the extracts. Not inoculated plates containing a tested concentration of extracts were negative control. The MIC level was established when there was no eye-visible bacterial growth. In analysing growth on agar media, single bacterial colonies were omitted in establishing the MIC. The analysis was performed in 3 replicates.Electronic noseVolatile compounds were identified using the Heracles Neo ultrafast gas chromatograph (Alpha M.O.S., Toulouse, France). The e-nose instrument has an autosampler and two capillary chromatography columns with different polarities—MXT-5 (non-polar; 10 m × 18 µm, Restek) and MXT-1701 (slightly polar; 10 m × 18 µm, Restek) and two Flame Ionization Detectors (FID). For analysis, 1 g of each HSS extract was dissolved in 100 mL of deionized water. The sample solution (1 mL) was transferred into the 20 mL headspace vials and closed with a treflon-faced silicon rubber cap. Each sample was incubated in the autosampler for 20 min at 50 °C with constant shaking speed at 500 rpm. After incubation, the headspace was collected and injected into GC. The injection volume was 1.0 cm3, the speed was 125 cm3 s−1, and the temperature was 200 °C. The temperature of the injector was 200 °C, and the detector (FID1 and FID2) was 260 °C. The content of volatile molecules is expressed as the percentage of the relative area which is the area of the chromatogram peaks. The method was calibrated using an alkane solution (C6–C16) to convert retention time in Kovats indices and identify the volatile compounds using the AroChemBase database. The AlphaSoft v 16.0 software was used to process the data. The analysis was performed in 3 replicates.Electronic tongueThe taste profiles of the water extracts of the HSS were measured using an Astree electronic tongue (Alpha MOS, Toulouse, France). The e-tongue has an autosampler and seven potentiometric chemical sensors for the detection of individual tastes (sensor set: CTS—saltiness; ANS—sweetness; PKS—universal taste; AHS—sourness; SCS—bitterness; CPS—universal taste; NMS—umami taste), a reference electrode of Ag/AgCl and data acquisition software. The potentiometric difference between each electrode and the Ag/AgCl reference electrode in the equilibrium state was recorded as a response signal. The electronic tongue sensor was pre-conditioned and calibrated with 0.01 mol L−1 hydrochloric acid solution. The diagnostic started after calibration. For diagnostic purposes, a 0.01 mol L−1 hydrochloric acid solution, monosodium glutamate and sodium chloride were used to evaluate the sensitivity and discrimination of electronic tongue sensors. The sourness, saltiness, and umami were measured using 0.1 M HCl, 0.1 M NaCl, and 0.1 M monosodium glutamate as reference materials. Samples of the HSS extracts were prepared by dissolving 1 g of each extract in 100 mL of deionized water. The solutions were transferred into the electronic tongue sample beaker. The signal of each electrode was recorded per second. The detection time was set for 120 s to ensure the sensors acquired enough signal information for the sample. Between sample analyses, the sensors were rinsed in ultrapure water for 10 s to stabilize them. Eight replicate measurements were conducted for each sample, and five of the most stable measurement points were used for data processing.Statistical analysisStatistical analysis was performed using Statistica 13.3 (StatSoft, Cracow, Poland) and Microsoft Excel 2019 (Microsoft; Redmond, WA, USA). Mean as well as standard deviation analysis was calculated. The homogeneity of variance and the normality of the distribution of results were checked. ANOVA was performed with Tukey’s post hoc test. If the number of grouping variables was too low to analyse them by the ANOVA, Student’s t-test was applied. Principal components analysis (PCA) was also performed based on the covariance matrix.
