Introduction

The seed skins are a by-product generated by roasting or blanching hazelnuts. The hazelnut seed skins (HSS) are material which does not have direct second utilization1. Hazelnuts (Corylus avellana L.) produce approximately 1 million tons of shelled seeds2. HSS are a by-product of the confectionery industry. They are generated by roasting or blanching hazelnuts. Even though it constitutes about 1% of the weight of the whole nut, it is produced in large quantities and poses an economic and environmental disposal problem1. However, HSS have low moisture content. It makes it effortless to transport and store, which is usually a major problem in the processing of food by-products3. It is worth noting that the polyphenols in hazelnuts are mostly concentrated in seed skins and the largest part are flavonoids and phenolic acids4,5. Scientific studies have shown the beneficial effects of polyphenols from nuts in maintaining well-being and preventing chronic diseases through their antioxidant, anti-inflammatory, cardioprotective, and hypolipidemic properties6. Moreover, polyphenolic compounds show bacteriostatic properties against a wide spectrum of pathogenic and saprophytic bacteria7,8. Within food products, polyphenolic compounds exert their activity by scavenging free radicals, significantly contributing to the prevention of lipid oxidation, thereby extending a product's shelf life and preserving sensory quality9.

Currently, more and more application works are being created. Mainly, concern the addition of HSS to yoghurts, sweet snacks, pasta or meat products to increase their health-promoting properties and stabilisation of the transformation of nutrients4,10,11,12. Moreover, in the latest scientific reports, HSS are used to feed animals to improve meat quality or as an element of compost when growing plants13,14. However, understanding of the properties of HSS at the molecular level is needed. Up to now, there are several works describing properties of HSS other than the polyphenol content. Importantly, hazelnuts are among the food products that most often cause allergies. There is data that technological processes can reduce the allergenicity of hazelnuts15. Thus, the content of allergens contained in HSS should be assessed. Furthermore, the sensory properties of HSS and the main chemical compounds responsible for them have not been specified so far. Analysing the sensory properties of the food ingredients is extremely important because it can modulate the holistic sensory profiles of the food product. However, HSS have a mainly unfavourable impact on the sensory properties of food products1. It is necessary to characterize this material to use it in food without introducing negative sensory characteristics. Therefore, developing an efficient and sustainable extraction process is necessary. It will allow for easy application of this material into food products. Further, the chemical composition of HSS, including the high polyphenols content, can be a bacteriostatic and preservative factor that protects food against pathogens and oxidation. Therefore, there is a need for research on these properties of HSS.

The application of HSS can contribute to the by-products management from the food industry and increase the nutritional value of food products and safety. Nonetheless, it is required to evaluate the mentioned properties and analyse the advantages and risks of HSS1. Moreover, food industry residue management can support environment protection and introduce sustainability practices by reusing generated by-products in the food system16,17.

The hypothesis of the work was that HSS had properties that would improve food quality and safety if added to it. The objective of this study was a comprehensive analysis of the potential reuse of HSS in food processing, in particular to determine the possibility of HSS extraction, polyphenolic compounds content analysis, assessment of antioxidant capacity, estimation of bacteriostatic properties, analysis of allergen content and evaluation of sensory properties using instrumental methods.

Results and discussion

Extraction efficiency

The selected extraction method was used due to its environmental non-invasiveness and ease of use. No extractants other than water were used, which means there is no additional chemical contamination or generation of chemical waste. This approach is important in recent green sustainable chemistry18. The analysis of the dry matter shows that extraction efficiency was higher in the E5 sample than in the E10 (p < 0.05) (Table 1; description of the abbreviations E5 and E10 can be found in the materials and methods section). The acquired results demonstrate that the adaptation extraction method to the HSS allowed for a lower extraction yield than in the original paper where the extraction method was first described19. Regarding this phenomenon, extraction efficiency depends on the soluble fraction content that can migrate to the solvent20. The HSS is composed of insoluble fibre (46.5–67.1 g per 100 g), lipids (11.0–21.2 g per 100 g), and approximately 4.3–8.6 g water per 100 g1,21. Therefore, the chemical composition of HSS limited the achievement of higher extraction yields due to the high content of non-water-soluble molecules such as insoluble fibre and lipids. However, recovery from HSS was quite effective considering the amount of insoluble compounds in this by-product. Significantly higher values ​​were obtained in the case of the E5 sample and the conditions used in this sample can be applied for efficient extraction of HSS. Also, it has been shown that a smaller amount of extracting material allows for a higher level of extraction. This phenomenon is caused by better penetration of the solvent into the extracted material and, consequently, greater extraction efficiency. As stated in a comprehensive review of extraction methods by Zhang et al.20, the higher the solvent-to-solid ratio, the higher the extraction yield. For this reason, the extraction efficiency was higher for sample E5 than E10.

Table 1 Characteristics of antioxidant properties, the content of individual phenolic compounds and allergens in hazelnut seed skins (HSS) and their extracts (E5, E10) and extraction efficiency, (n = 3 for HPLC and extraction efficiency, n = 8 for colorimetric methods).
Full size table

Antioxidant properties, the polyphenols content and composition

The antioxidant properties and the content of the polyphenols in the HSS and their extracts are presented in Table 1. The antioxidant properties of E5 and E10 extract were similar to the HSS. The statistical difference between the used methods of the antioxidant activity (ABTS, DPPH) and total polyphenols content (TPC) by Folin–Ciocalteu (F–C) method of the HSS, E5 and E10 was not significant (p > 0.05). However, the differences between HSS and their extracts in the sum of the polyphenols were observed in the HPLC method (p < 0.05). The total content of the polyphenols was lower in the E5 and E10. It shows that the water high-temperature extraction had a detrimental effect on the polyphenols in extract samples22,23.

The antioxidant activity of extracts and HSS is directly related to the content of polyphenols in them—the higher antioxidant capacity and polyphenol content had HSS in comparison with E5 and E10 (Table 1). Generally, the antioxidant activity of the analysed samples was similar to those already described5,24. However, the authors of the cited works emphasize that the laboratory conditions and the origin of the samples have a considerable impact on antioxidant activity. For this reason, the differences between the obtained results of the analyses in the same type of materials were noticed.

Polyphenol analysis by HPLC showed that gallic acid was present in the highest concentration in all tested samples (Table 1). The results are consistent with those reported in the articles, where gallic acid and catechin are the predominant phenolic compounds found in HSS11,25. The content of the rest of the phenolic compounds was various. Some polyphenols were lost during aqueous extraction of HSS to obtain the E5 and E10, i.e. caffeic acid, p-coumaric acid, ferulic acid, glycoside-3-O-kaempferol, quercetin, and kaempferol. It was due to high-temperature treatment during E5 and E10 preparation (90 ℃, 10 min, water extraction). Literature data reported that polyphenols are sensitive compounds for temperature processing which can cause their degradation or change chemically and biologically active form25. Nevertheless, based on the data obtained and the literature, the water extraction method was effective and easy to employ18,19.

It can be stated that the content of polyphenols in hazelnut seed coats varies depending on the origin of the raw material, the method of roasting the nuts and the temperature of this process24. The literature presents various levels of the content of polyphenolic compounds in HSS and their extracts. For example in the paper by Del Rio et al. and Taş and Gökmen5,26, the researchers using the F–C method obtained results ranging from 41 mg (gallic acid equivalent) GAE g−1 to 203 mg GAE g−1. The results presented in this study coincide with those described in the literature. Similar results to ours, regarding the polyphenols content and composition in the HSS are presented by Pelvan et al.25. Also, in the study by Król et al.27 based on the HPLC analysis were detected similar content and composition of polyphenols. Even though the obtained research results are consistent with other studies, it should be emphasized that the available data contain a significantly wide range of outcomes regarding the content of polyphenols and their composition. The selection of the polyphenol extraction method for analysis is important22. The authors indicated the direct influence of the origin of hazelnuts and their seed coats on the content of polyphenols. In these studies, the influence of the hazel variety on the content of these compounds was also analysed in the hazelnut's skins5,26,27. Important is that the research material in this study was industrial HSS, which makes the obtained results reflect the content of bioactive compounds found in this material to industrial conditions.

Allergens content

Hazelnuts are one of the most common food allergens. Therefore, an allergen analysis was conducted and the results are shown in Table 1. The analysis of profilins and Bet v 1 showed that they were inactivated as a result of thermal processes which were subjected to HSS and tested extracts. The content of allergens was below the detection level of the applied research method. This means that profilines and Bet v 1 have been inactivated already at the stage of hazelnut processing in the industry. Other groups of allergens in HSS and its extracts need to be investigated in the future. Several identified and described proteins are responsible for this allergenicity (Cor a 1, Cor a 2, Cor a 8, Cor a 9, Cor a 10, Cor a 11, Cor a 12, Cor a 14, Cor a 15 and Cor a TLP)28,29. Cor a 1 belongs to the Bet v 1 allergen group, while Cor a 2 belongs to profilines. Both of these groups of proteins show a decrease in allergenicity under the influence of high temperatures (above 120 ℃)28. Based on the performed analysis, it can be concluded that roasting as a high-temperature treatment is sufficient to inactivate these two groups of hazelnut allergens. Similar results were obtained by López et al.15 who, by autoclaving hazelnuts, reduced their allergenicity. Studies have already shown that roasting and other food processing can directly affect the allergenicity of nuts. It should be emphasized that neither roasting nor any other treatment makes the nuts safe for allergy sufferers and loss of allergenicity was achieved only in a few cases30. In clinical trials, it has been proven that the roasting process reduces the allergenicity of nuts and some of the test subjects did not experience an allergic reaction. Despite this, the effect in these studies was not obtained on all study members or the effect was not clinically significant31,32. However, all the cited research results focus on the allergenicity of hazelnuts and not HSS. It is undeniably necessary to test HSS for the occurrence of allergens from other Cor groups, as such data have not been published yet. Referring again to López et al.15, it was noted that the autoclaving process significantly inactivated allergens. It can be concluded that HSS are the parts of the hazelnut most exposed to heat during roasting, and at the same time, the inactivation of allergenic compounds in this by-product may be the greatest.

Bacteriostatic activity

The bacteriostatic activity of the extracts is presented in Table 2. The minimum inhibitory concentration (MIC) level was achieved for all tested pathogenic and saprophytic bacterial strains. Lactiplantibacillus plantarum 299v and Lacticaseibacillus rhamnosus ATCC 53,103 did not have sensitivity to the tested concentrations range so the MIC is under 10 mg g−1 and it has not been achieved. The E5 and E10 had a similar effect on the tested bacteria. Usually, the E5 and E10 extracts inhibited the growth of the same strain of tested bacteria. There were a few exceptions where E5 had better bacterial inhibitory activity. It was in the case of the Bacillus spizizenii ATCC 6633 and Escherichia coli ATCC 11775. The E10 had only a better inhibitory effect on Staphylococcus aureus ATCC 25923. Many studies present the mechanisms of polyphenolic compounds that cause bacteriostatic or bactericidal effects7,33. The phenolic acids identified in our samples interfere with the tightness and permeability of the bacterial cell membrane. The disruption of the cell membrane is explained as local hyperacidification, which causes an increase in the permeability of the cell membrane and then denaturation of intracellular proteins and, as a result, inactivation of the bacterial cell7,33. The mechanism of antimicrobial action of flavonoids such as catechins, kaempferol and quercetin is focused on the changes in the cell membrane, and cell wall but also the inhibition of DNA synthesis and increased oxidation stress in the bacterial cell. The effect of flavonoids, on cell membranes, is different than in the case of phenolic acids. They probably damage phospholipid bilayers, inhibition of the respiratory chain or ATP synthesis34. Studies indicate that the action of flavonoids on the cell wall causes disturbances in its integrity and, as a result, lyses the cell wall and releases the cytoplasmic contents of the bacteria35. On the other hand, it was found that quercetin was able to inhibit the function of DNA gyrase, thus leading to the disruption of DNA synthesis36. In another study, it was observed that catechins may increase the content of reactive oxygen species in the cell and disrupt the antioxidant economy of E. coli cells37. This property can be linked to the reaction between catechins and oxygen present in the culture, which results in the formation of hydrogen peroxide and then highly reactive hydroxyl radicals. The activity of these radicals leads to irreversible changes in bacterial cells, through the oxidation of proteins, disturbances in DNA synthesis38. It has been found that phenolic compounds have a synergistic effect when used as mixes gallic acid is shown to have significantly greater antimicrobial activity when combined with hydroxytyrosol and other polyphenols than as a single compound39. In our previous work, we showed that HSS powders have bacteriostatic potential. HSS without extraction showed lower bacteriostatic activity compared to the results obtained in this study11. In other studies, extracts from the seed coats of peanuts were analysed. Klompong and Benjakul40 obtained high microbiological activity against S. aureus, E. coli and B. cereus. Microscopic images, cell damage and the release of intracellular substances into the external environment were observed, which indicates an analogy to the mechanisms described above. An important result is the lack of effect of the extracts used on the tested probiotic bacteria. Other studies tested the effect of HSS on the growth of L. plantarum P17630 and Lactobacillus crispatu P17631. Montella et al.41 found that HSS and their extracts are rich in soluble and insoluble dietary fibre, which stimulates the effect on the growth of probiotic bacteria. It is assumed that the extracts used by our research team contained soluble fibre. The high fibre content in this case could significantly influence the high metabolic activity of probiotic bacteria and stimulate their growth. Moreover, probiotic bacteria can metabolize polyphenolic compounds as prebiotics and have a highly adapted enzymatic apparatus for their catabolism42. It has been shown that polyphenolic compounds addition to in vitro large intestine models or their dietary consumption cause an increasing number of probiotic bacteria and a simultaneous reduction in the development of proteobacteria8.

Table 2 Bacteriostatic effect on different species of the bacteria of the tested hazelnut seed skins extracts (E5, E10) based on the minimum inhibitory concentration (MIC); n = 3.
Full size table

Instrumental sensory analysis

Instrumental sensory methods allow the detection of small differences between the tested samples and permit the analysis of products that are not usually consumed, such as by-products. The volatile molecules in the tested HSS extracts obtained using e-nose are presented in Fig. 1a and the supplementary materials in Table S1. Both extracts (E5 and E10) contained the highest proportion of 2-hexanol (61.1–62.8%), acetaldehyde (7.6–7.6%), 2.3—pentanedione (5.7–6.1%) and myristicin (3.8–6.8%). These compounds were mainly responsible for the fatty, fruity, woody, spicy, aldehydic and nutty aromas. An important feature of the E5 extract is that it contained aromatic compounds that did not occur in the E10 extract. It was but-(E)-2-enal in the E5 sample with a floral and green sensory characteristic. The analysis of the taste by e-tongue (Fig. 1b) shows that bitter, sour, umami and universal taste are the highest intensity in the tested samples. The taste profiles of the tested extracts were consistent and similar to each other. The statistical difference between the e-tongue sensor response occurred only in the saltiness sensor (CTS) (p < 0.05). The PCA analysis defined the impact of individual sensory descriptors contributed by the identified compounds and the obtained intensity of taste on the sensory profile. The amount of HSS in the extraction process affected the sensory profile of the obtained extract. The E10 extract was more bitter, salty, sour, baked, caramelized, roasted, nutty, almond, earthy and nutty. The E5 extract was more floral, fatty and fresh. These properties were directly associated with the content of the but-(E)-2-enal with floral and green sensory descriptors and it was identified only in sample E5. The E5 extract had also a higher amount of the n-nonanal, (E)—cinnamaldehyde and 2-acetyl-naphtalene which had a citrus, floral, candy and sweet aroma. The PCA analysis has shown the dependency between these selective aromas (Fig. 1c). The vectors which are longer and situated closer themselves have a bigger correlation to each other. Cases representing test samples, marked with green or blue points and ellipses, are significantly distant from each other in the plane of the coordinate system and correlated with other sensory descriptors represented by black lines (Fig. 1c). This result indicates sensory differences in the tested samples. The samples mainly contained the same aroma compounds and had similar taste profiles with the comparable intensity of the analysed tastes. Nonetheless, thanks to instrumental sensory methods and statistical analysis like PCA, it is possible to obtain and present the sensitive difference between tested samples if they are similar43,44.

Figure 1
figure 1

(a-c) The odour profile of the analysed extracts (E5, E10) based on volatile compounds and their sensory descriptors in e-nose analysis, the percentage share in the total volatile compounds depending on the chromatographic column (MTX-5, MXT-1701) used for the separation, n = 3 (a); the taste profile of the hazelnut seed skins extract obtained by e-tongue, the abbreviations mean the sensors that are responsible for detecting the taste assigned to them; the value is expressed in units of the scale of indefinite taste intensity, n = 8 (b); E5 means extract where 5 g of HSS was extracted in 100 g of water, and E10 means extract where 10 g of HSS was extracted in 100 g of water, full explanations on the preparation of extracts are in section "Extraction method and extraction efficiency".; principal component analysis of the instrumental sensory evaluation by e-nose and e-tongue of extracts, projection of variables (sensory descriptors of detected volatile compounds by e-nose and taste descriptors detected by e-tongue) and cases (tested samples) onto the plane of the principal components (c); error bars represent the confidence interval (p < 0.05), these results were confirmed by t-test or Tukey's HSD post hoc test after ANOVA analysis (p < 0.05).

Full size image

So far, a similar sensory analysis has not been published. Nevertheless, the aromatic and taste profile of HSS extracts is other than that of hazelnuts. However, both contain similar features such as nut, fatty or sweet aromas. Noteworthy, HSS is removed during roasting, therefore a large part of the published studies concern the impact of the roasting process on the aromatic parameters of nuts1,45. For this reason, the results are difficult to compare with each other. On the other hand, many studies suggest that HSS and their extracts have a high potential for food applications. HSS was added to various food matrices such as yoghurts, chicken burgers, and date bars1,10,11. The authors of these works consistently conclude that this material can be a functional food additive, and in appropriate dosage, it did not change the sensory characteristics of food. In addition, there are suggestions in the literature that a deeper understanding of the properties of HSS and their extracts from the molecular sensory characteristics will contribute to their facile application to food in the future1.

Conclusion and future perspectives

A comprehensive study of HSS and their extracts yielded new knowledge about these residues. It has been demonstrated that HSS contain a high concentration of polyphenolic and antioxidant compounds. Additionally, HSS exhibit broad bacteriostatic and sensory properties that do not discriminate against their use in further processing in the food industry. Another significant finding is the reduced level of allergenicity, which has been proven.

This holistic analytical approach paves the way for further scientific research under HSS. Consequently, the allergenic activity of HSS and their extracts must be assessed across all allergen groups. Alternative methods of extracting bioactive compounds may provide further insight into the properties of this material. It will be essential to apply the bacteriostatic properties of HSS in various food matrices. The sensory properties of HSS should be evaluated by classical quantified descriptive analysis to establish the addition of the HSS on an acceptable level in food. Future investigation of toxic substances formed during high-temperature roasting is essential. Studies on HSS stability, sensory properties, and safety in different food matrices will be crucial.

The research indicates that HSS extracts have significant potential for use in food products. The study also provides a diverse and broad knowledge base that can be utilised by other researchers or industries seeking various substances with a natural bacteriostatic effect, creating sensory properties, high concentrations of bioactive substances and reduced content of allergens.

Materials and methods

The scheme of the experiment is presented in Fig. 2.

Figure 2
figure 2

The scheme of the experiment.

Full size image

Hazelnut seed skins

The 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 efficiency

The 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 capacity

The 100 mg of HSS or HSS extracts (freeze-dried) were mixed with 5 mL of 80% methanol (Chempur, Piekary Śląskie, 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 Śląskie, 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 Śląskie, 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 determination

Polyphenols 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, Poznań, Poland) and 55 mL 100 mL−1 acetonitrile (phase B) (Sigma-Aldrich, Poznań, Poland. Both phases were acidified by ortho-phosphoric acid (Sigma-Aldrich, Poznań, 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, Poznań, 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, Poznań, 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, Poznań, 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, Poznań, Poland). The inactive powdered radicals were mixed with pure < 99.5% methanol (Chempur, Piekary Śląskie, 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 content

The determination was performed according to Słowianek 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, Poznań, Poland), antibodies against the rabbit immunoglobulin conjugate with alkaline phosphatase (Sigma-Aldrich, Poznań, 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, Poznań, Poland) as the substrate for the alkaline phosphatase, and 3 M NaOH (Sigma-Aldrich, Poznań, Poland) as the stop reagent was used. As a washing solution, the PBS with 0.1 mL 100 mL−1 Tween 20 (Sigma-Aldrich, Poznań, 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 inhibition

Determination 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; Poznań, 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 nose

Volatile 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 tongue

The 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 analysis

Statistical 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.