A mixed mushroom control material to facilitate inter-laboratory harmonization of mushroom composition analyses

A wide range of nutrients and health-promoting non-nutrient components in mushrooms are a subject of international research, but specific reference materials to facilitate comparison of results among laboratories are lacking. Commercially available food matrix reference materials do not contain components unique to mushrooms (e.g., ergosterol, vitamin D₂, chitin, beta-glucans, agaritine, ergothioneine). A mixed mushroom control material (CM) (homogeneous mixture of 15 types of mushrooms) was prepared and characterized for selected components, including proximates (moisture, protein, ash), total folate, folate vitamers, ergosterol, ergosterol metabolites, vitamin D₂ (ergocalciferol), amino acids, total dietary fiber, agaritine, elements (sodium, potassium, phosphorous, magnesium, calcium, iron, copper, manganese, zinc), riboflavin, niacin, thiamin, vitamin B6, pantothenic acid. Subsamples of the CM are available to qualified laboratories from the Food Analysis Laboratory Control Center at Virginia Tech (Blacksburg, VA, USA), to be assayed concurrently with mushroom samples for which food composition data will be published along with results for the CM. Implementation of this CM should facilitate comparison of published data on mushroom composition and health benefit among species, and biodiversity within species by serving as common control sample that allows the separation of analytical variability from true differences in sample composition determined at different laboratories.     

Esterified sterols and their contribution to the total sterols in edible mushrooms

Mushrooms are a valued source of sterols, which can be present in free form and esterified to fatty acids. In addition to the principal ergosterol, several minor sterols occur. Ergosterol and ergosteryl ester were determined directly by gas chromatography coupled to mass spectrometry (GC/MS) in the trimethylsilylated lipid extract of each six samples of button mushrooms, oyster mushrooms, king trumpet mushrooms and shiitake. Free ergosterol was highest in button mushrooms 415–544 mg/100 g dry matter (d.m.) and lowest in oyster and king trumpet mushrooms (<350 mg/100 g d.m.). On the other hand, concentrations of ergosteryl esters were significantly lower (5–26 mg/100 g d.m.) in all mushroom samples. The distribution of minor sterols (most importantly ergosta-7,22-dienol, ergosta-5,7-dienol, ergosta-7-enol) between free and esterified form was determined after group separation with solid phase extraction. Higher proportions of the minor sterols were found esterified to fatty acids and ergosta-7-enol was found predominantly (70%) in this form in oyster mushrooms and shiitake. The minor sterols ergosta-8,24(24¹)-dienol, ergosta-8-enol and 7-dehydro-poriferasterol could be tentatively identified in the samples based on GC/MS data. In addition, four sterols were found only in the steryl ester fraction of the mushroom samples.     

Gas chromatography with mass spectrometry detection and characterization of 27 sterols in two truffle (Tuber) species

Fungal sterols (mycosterols) were investigated in seven samples of both Tuber aestivum and Tuber borchii truffles from Italy, Spain and Romania by means of gas chromatography with mass spectrometry (GC/MS). Sterol contents varied from 130 to 590 mg/100 g dry weight (T. borchii) and 110−420 mg/100 g dry weight (T. aestivum). The sterol pattern of both truffle species was dominated by ergosterol (60–85 %) and brassicasterol (4–33 %). In addition, 25 minor sterols were detected with 27 (n = 3), 28 (n = 17), 29 (n = 3), 30 (n = 2) and 31 (n = 2) carbon atoms (Limit of detection: 2 μg/100 g dry weight). Fourteen minor sterols were described for the first time in Tuber species. Ten minor compounds were detected in all samples in varying abundances, while the others were (i) exclusively detected in all samples of one species (T. borchii: fecosterol, ergosta-8-enol; T. aestivum: 24-methyleneergosta-5,22,24-trienol) or (ii) varied strongly in abundance (≥90 %). Variations in main and selected minor sterols could be partly related to different harvest times. In addition, differences in fungisterol and ergosta-5,7-dienol between the both species were attributed to different pathways in fungal sterol biosynthesis (oneway ANOVA p ≤ 0.01). Our study indicates that sterol pattern analysis could be used to differentiate different Tuber species.     

The risk of high mercury accumulation in edible mushrooms cultivated on contaminated substrates

Mercury (Hg) exposures represent a significant worldwide health issue. At the same time its content in cultivated mushrooms is not effectively regulated. The present study investigated how substrate contamination with Hg (0.1–0.5 mM) affects its accumulation in stipes and caps of Agaricus bisporus E58, Pleurotus ostreatus H195 and Hericium erinaceus HE01, mushroom growth and composition of macronutrients. The greatest Hg accumulation was demonstrated for caps. Generally, Hg uptake increased in a concentration-dependent manner and exceeded 44 mg kg⁻¹ (P. ostreatus), 116 mg kg⁻¹ (A. bisporus) and 53 mg kg⁻¹ (H. ercinaceus) in caps after 0.5 mM was added to the substrate. Importantly, an increase in Hg accumulation was also significant and potentially hazardous for human health at the lowest assayed concentration. A. bisporus and P. ostreatus revealed high resistance to Hg and declined its biomass only at 0.4 and 0.5 mM concentration. The presence of Hg did not alter the macronutrient composition (total carbohydrates, proteins and fats). These results highlight the significant role of proper substrate selection in mushroom cultivation to avoid exposing consumers to harmful Hg levels and further health consequences.     

Single-run analysis of vitamin D photoproducts in oyster mushroom (Pleurotus ostreatus) after UV-B treatment

A single-run high performance liquid chromatography (HPLC) with diode array detector (DAD) based method was developed for the separation, identification and comprehensive quantification of degradation products of ergosterol formed in oyster mushroom (Pleurotus ostreatus) after UV-B exposure. After 60 min, 10 substances involved in the photoprocess were separated, identified by their characteristic DAD spectrum and distinguished by their molecular weight, in cases where spectra were identical: vitamin D₂, previtamin D₂, tachysterol₂, lumisterol₂ and ergosterol, and, in minor quantity, their structural analogues of the 22,23-dihydroergocalciferol (vitamin D₄) series. Sample preparation protocol affected the total yields and the ratios of previtamin and vitamin D₂/D₄. Hot alkaline hydrolysis resulted in the best digestion of the mushroom matrix and accordingly gave the highest vitamin D yield (D₂: 141.32 μg g⁻¹ dry matter, DM; D₄: 22.72 μg g⁻¹ DM). Limit of detection for vitamin D_2/4 was 0.02 μg g⁻¹ dry matter (DM) and was estimated for previtamin D_2/4 (0.06 μg g⁻¹ DM), tachysterol_2/4 (0.02 μg g⁻¹ DM) and lumisterol_2/4 (0.06 μg g⁻¹ DM). Recovery of spiked vitamin D₂ was 97 ± 0.7%. The study provides an analytical tool to assess the process of vitamin D generation after UV-B treatment for the production of oyster mushrooms with a balanced nutritional profile of vitamin D compounds.     

Determination of total phenol content, antioxidant activity and acetylcholinesterase inhibition in selected mushrooms from Turkey

In the present study, we investigated the acetylcholinesterase (AChE) inhibitory activity of the ethanolic extracts of a number of mushroom species growing in Turkey, including mainly Polyporus species (Polyporus gilvus, Polyporus sulphureus, Polyporus annosus, Polyporus radiatus, Polyporus pinicola, Polyporus volvatus, Polyporus fomentarius, Polyporus stevenii, Polyporus badius), as well as Cantharellus cibarius, Lactarius deliciosus, and Trametes versicolor. The analyses were carried out using the spectrophotometric method of Ellman in ELISA microplate reader at 500 μg/ml. Since Alzheimer's disease (AD) is associated with oxidative degeneration of cells, several methods of antioxidant activity were applied to the mushroom extracts such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, ferrous ion-chelating effect, ferric-reducing antioxidant power (FRAP), and beta-carotene bleaching tests. Total phenol contents of the extracts were also determined by Folin-Ciocalteu reagent. In anti-AChE assay, the most active one was P. sulphureus having 31.44% of inhibition. P. voluatus showed the best scavenging effect against DPPH, while all of the extracts exerted low capacity in chelating ferrous ions and reducing ferric ion.     

Survey of ergosterol, zearalenone and trichothecene contamination in maize from Nigeria

The contents of ergosterol, zearalenone (ZEA) and a spectrum of A- and B-types of 13 trichothecene toxins in maize samples destined for human consumption from farmers’ stores and markets in Nigeria in 2005 and 2006 were determined. Ergosterol was analysed by HPLC with UV-detection, ZEA by HPLC with fluorescence and UV-detection, while trichothecenes were determined by gas chromatography/mass spectrometry. Ergosterol was detected in 105 of the 106 samples at concentrations ranging from 0.1 to 24.2 μg g^?1. ZEA was found in 4 out of 69 samples at 2–13 μg kg^?1. Out of the 32 samples analysed for trichothecenes, only one sample was contaminated with 15-monoacetoxyscirpenol (15-MAS) at 4 μg kg^?1, and two samples were contaminated with T-2 tetraol at levels of 73 and 280 μg kg^?1. Nivalenol (NIV), scirpentriol (SCIRP), deoxynivalenol (DON), fusarenon-X (FUS-X), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON), T-2 triol, neosolaniol (NEO), 4,15-diacetoxyscirpenol (4,15-DAS), HT-2 and T-2 toxins (HT-2, T-2) were not detected in any of the samples. Thus in spite of the limited number of samples investigated, it seems to be obvious that zearalenone and trichothecene toxins do not appear to be major contaminants of Nigerian maize. The significantly higher ergosterol levels in maize that had been stored for 9 months compared with those of maize that had been stored for 4 months or less, point to the inadequacy of currently used storage methods in preventing deterioration. This is the first report of ergosterol levels in Nigerian maize.     

Vitamin D2 content and antioxidant properties of fruit body and mycelia of edible mushrooms by UV-B irradiation

Our objective was to study the effect of ultraviolet-B (UV-B) light irradiation on the vitamin D₂ content of several edible fruit bodies and mycelia and their antioxidant properties. Eleven species of fresh mushroom fruiting bodies, including species from each of the six genera, Agaricus, Agrocybe, Auricularia, Hypsizigus, Lentinula and Pholiota, and five species from Pleurotus genus, were irradiated with UV-B light for 2 h. For three species of mushroom fruiting bodies with excellent vitamin D₂ yield, their mycelia were obtained by liquid culture, and subjected to the same time as the UV-B irradiation. Vitamin D₂ content of irradiated fruit bodies significantly increased from 0–3.93 to 15.06–208.65 μg/g, of which the amount in golden oyster mushroom increased by a maximum of 204.7 μg/g. Vitamin D₂ content in irradiated mycelia of golden oyster, oyster and pink oyster mushrooms increased from 0.28–5.93 to 66.03–81.71 μg/g, respectively. The three irradiated mycelium polysaccharide contents decreased in a range from 1.3% to 24.6%. Overall, EC₅₀ values of non-irradiated and irradiated fruiting bodies and mycelia were 0.92–4.94, 0.20–6.90 and 0.02–0.84 mg/mL for reducing power, scavenging ability and chelating ability, respectively. Although UV-B irradiation influenced the content of ergothioneine, flavonoids and total phenols, these irradiated samples still contained a sufficient amount of these antioxidant components.     

Investigation of differentiation of metal contents of Agaricus bisporus,Lentinula edodes and Pleurotus ostreatus sold commercially in Poland between 2009 and 2017

Agaricus bisporus, Pleurotus ostreatus and Lentinula edodes are the most commercially important mushroom species cultivated for human consumption. This study compared the content of major elements (ME) and trace elements (TE) in fruiting bodies of these three mushrooms available on the Polish market between 2009 and 2017. It demonstrates that the studied mushrooms, particularly A. bisporus, can be a valuable source of macro- and micro-nutrients such as K, P, Cu, Fe and Se (means: 28,500; 6150; 14.3; 49.3 and 2.59 mg kg dry weoght, respectively) and contain a beneficial Na/K ratio (1:76). The mushrooms were not found to contain increased levels of toxic metals with the ominous exception of As, levels of which were found to be elevated.     

A method for the simultaneous extraction and quantitation of lipophilic antioxidants in Triticum sp. by HPLC-DAD/FLD-MSn

A method for the simultaneous determination of carotenoids, tocopherols, tocotrienols, alk(en)ylresorcinols, and steryl ferulates from whole grain wheat flour was developed and validated using HPLC-DAD/FLD-MSⁿ. After comparing various solvents and extraction techniques, samples were extracted with acetone without previous saponification. Extraction recoveries ranged from 93.2 to 101.6% for carotenoids, tocopherols, and steryl ferulates, while 83.4–99.4% were determined for alkylresorcinols. The inter-day CV for all analytes was below 5%. HPLC separation was compared on porous particle C₁₈- and C₃₀- as well as a core–shell particle C₁₈-column, achieving optimum separation on the latter within 40 min after gradient optimization. The limit of detection (LOD) ranged from 0.03 to 20.41 ng on column for lutein and tricosylresorcinol, respectively. Further validation parameters are reported in detail. HPLC–MSⁿ supported the identification of minor compounds, when standards were unavailable. This method is proved to be suitable for high-throughput screening of a large number of lipophilic compounds in wheat.     

Study of mercury content in wild edible mushrooms and its contribution to the Provisional Tolerable Weekly Intake in Spain

The study determined total mercury (Hg) content of 10 wild edible mushroom species collected in southern Spain. Results indicated that the highest Hg level corresponded to Boletus aereus with 10.28 ± 2.92 mg/kg DW (dry weight), while the lowest Hg level was found in Terfezia arenaria with 0.09 ± 0.08 mg/kg DW. Regarding the anatomic parts of the mushrooms, caps showed significantly higher Hg concentrations than stems in B. aereus, Amanita caesarea and Macrolepiota procera. The percentage of contribution to the Provisional Tolerable Weekly Intake (PTWI) for Hg was also calculated. Based on consumption data in Spain (0.011 kg/person/week), %PTWI was calculated ranging between 0.06 and 3.5% for mean Hg levels. When a high level of consumption was assumed (0.100 kg/person/week), %PTWI ranged between 0.58 and 31%. Results suggest that wild edible mushrooms in southern Spain could contribute with high Hg levels to the Spanish diet. However, the lack of consumption data for wild edible mushrooms made it difficult to give more definitive conclusions; hence further studies including specific consumption data will be needed.     

Determination of free quinic acid in food matrices by Hydrophilic Interaction Liquid Chromatography with UV detection

A new method using Hydrophilic Interaction Liquid Chromatography and UV detection was developed for the determination of free quinic acid in fourteen commercial beverages (fruit juices, red wine, coffee, vinegar, and edible mushroom extract). Samples were directly injected into the HPLC system after dilution and centrifugation. All separations were carried out by gradient elution on a ZIC^®-cHILIC column, using an eluent consisting of acetonitrile and aqueous phosphate buffer at pH 6. Quinic acid eluted in 12 min as a symmetric peak well separated from all other peaks in the chromatograms. The total turnaround time per sample was 60 min. The method was validated using cranberry juice as model beverage matrix. Linearity was demonstrated on spiked juice between 200 and 600 μg/mL (r² = 0.999), and precision and accuracy were satisfactory (RSD ≤ 10% and recovery 100 ± 1%, respectively).The method was proven to be suitable for its purpose. Quinic acid levels ranged from undetectable (<15 μg/mL in red grape juice, elderberry juice, and in red wine vinegar) to 740 ± 30 μg/mL (in mushroom extract). The results were discussed in comparison with the previous reports on similar matrices using different HPLC separation modes.     

Ergosterol as an indicator of mould growth on building materials

Ergosterol was used as a specific indicator of fungal biomass to determine and assess mould growth on damp building material. The samples were saponified, cleaned up on a silica gel column and the sterols silylated and analysed by gas chromatography and mass spectrometry in MS/MS mode. Ninety-one samples of building materials from damp rooms were analysed including plaster, plaster/paint, paint, wood-chip wallpaper and paper wall covering. The concentrations of ergosterol ranged from 0.1 to 130 microg/g dry mass and depended on carbon content of the material. The highest concentrations were determined for wallpaper, the lowest for plaster and intermediate ones for paint. Based on ergosterol content and inspection of the room during sampling a rough classification of mould infestation is presented. The applicability of the ergosterol method was further tested in two case studies on the spatial distribution of fungi on damp walls and irregular distributions were found. With few exceptions the concentration of ergosterol in building materials was found to be a suitable indicator to estimate fungal biomass.     

Phaeophyceae and rhodophyceae macroalgae from the Antarctic: A source of selenium

This study reports the development of a voltammetric method applied for Se determination in Phaeophyceae (A. utricularis, C. jacquinotii, A. mirabilis, D. anceps, H. grandifolius) and Rhodophyceae (G. confluens, C. racovitzae, I. cordata) macroalgae from the Antarctic. Evaluation of the instrumental parameters showed the square wave cathodic stripping voltammetry provided greater sensitivity (deposition potential, -0.4 V; deposition time, 420 s; amplitude, 0.08 V; frequency, 60 Hz) than influence of differential pulse cathodic stripping voltammetry. The matrix effect and the influence of Cu concentration on the determinations were also assessed. After microwave-assisted digestion of the samples, the quantification limit was from 5.21 × 10⁻³ (G.confluens) to 9.85 × 10⁻³ mg kg^-1 (I. cordata). The quantification of Se was carried out over the concentration range from 0.23 (C. jacquinotii) to 1.22 mg kg^-1 (A. mirabilis). The method accuracy was by analysing the reference material of peach leaves (SRM 1547).     

Direct metabolic fingerprinting of olive oils using STELDI-MS

A new and rapid approach for analysis of olive oil has been developed using sorptive tape-like extraction in combination with laser desorption ionization mass spectrometry (STELDI-MS). This powerful combination has some great advantages, as no-separation steps, solvent-free, matrix-free, and no sample preparation. The olive oil compounds are analyzed by LDI-MS, directly from the sample spot in a thin-layer chromatography (TLC) plate. Chosen samples represent products commonly used as adulterants in extra virgin olive oil (EVOO) and the main monitored ions were lipid adulteration markers. Analytical procedures consisted of profiling the main fatty acids (m/z 255 – palmitic acid, 279 – linoleic acid, 281 – oleic acid and 283 – stearic acid), triacylglycerols (m/z 901 – LLL and 907 – OOO) and some phenolic compounds (m/z 169 – gallic acid, 193 – ferulic acid and 195 – 2(4-hydroxyphenyl) ethyl acetate) in extra virgin olive oil (EVOO), olive oil (OO), hazelnut oil (HO) and soybean oil (SO). Compound identification was confirmed by analysis of collision-induced dissociation (CID) products in positive (ion [M+Na]⁺) and negative mode (ion [M−H]⁻). This method is simple, fast and efficient in identifying compounds that can be used to recognize different levels of adulteration, oxidation and hydrolysis of vegetables oils.     

Selective extraction of Cu+ and Cu2+ ions from mushroom and lichen samples prior to analysis by micro-volume UV-Vis spectrophotometry: Application of a novel poly (SMIm)-Tris-Fe3O4 nanocomposite

In this study, a novel poly (SMIm)-Tris-Fe₃O₄ nanocomposite was prepared for selective extraction of Cu⁺ and Cu²⁺ ions by ultrasound assisted-cloud point extraction (UA-CPE). The nanocomposite was characterized by analysis of ATR-FT-IR, ¹H NMR and XRD. After optimization of the extraction conditions, the copper ions were independently detected against sample blank at 347 nm by micro-volume UV–vis spectrophotometer. Under the optimal conditions, good linear relationship was obtained in the ranges of 0.3−150 and 10−350 μg L⁻¹ for each ion at pH 7.0 and pH 5.0, respectively, with a better regression coefficient than 0.992. The method detection limits, accuracy and precision were 0.095 and 3.03 μg L⁻¹, 91.5−96.0 % and 93.0−98.5 %, and 2.5–4.5 and 3.8–7.1 % (n:5, 25 and 100 μg L⁻¹) for Cu⁺ and Cu²⁺ ions, respectively. A preconcentration of 70-fold was obtained from 35-mL of sample. After validation, the method was successfully applied to determination of total Cu levels in lichen and mushroom samples after pre-reduction at pH 7.0, and the recoveries in the range of 90−96 % were obtained by two calibration approaches after spiking with 10 μg L⁻¹. The results were also statistically compared with those obtained by FAAS analysis to ensure accuracy and precision.     

Hericium erinaceus Mycelium Ameliorates In Vivo Progression of Osteoarthritis

Osteoarthritis (OA) is an age-related disorder that affects the joints and causes functional disability. Hericium erinaceus is a large edible mushroom with several known medicinal functions. However, the therapeutic effects of H. erinaceus in OA are unknown. In this study, data from Sprague-Dawley rats with knee OA induced by anterior cruciate ligament transection (ACLT) indicated that H. erinaceus mycelium improves ACLT-induced weight-bearing asymmetry and minimizes pain. ACLT-induced increases in articular cartilage degradation and bone erosion were significantly reduced by treatment with H. erinaceus mycelium. In addition, H. erinaceus mycelium reduced the synthesis of proinflammatory cytokines interleukin-1β and tumor necrosis factor-α in OA cartilage and synovium. H. erinaceus mycelium shows promise as a functional food in the treatment of OA.     

Beta-glucans from edible and medicinal mushrooms: Characteristics,physicochemical and biological activities

One of the main active components from mushrooms was recently identified as β-glucan. Health-promoting β-glucans are an auspicious group of polysaccharides. β-Glucans from different sources such as cereals, yeast and grass have previously been documented. However, information on mushroom β-glucan is limited. This review summarizes the extraction, purification, quantification, and structural characterization of β-glucans, along with chemical and biological activities from this compound from mushrooms, and the current status of this research area with a view for future directions.     

Modeling of stable isotope and multi-element compositions of jujube (Ziziphus jujuba Mill.) for origin traceability of protected geographical indication (PGI) products in Xinjiang,China

Jujube is widely grown in Xinjiang province, China, including two high-value PGI products, Charkhlik Hui jujube and Khotan Jun jujube. Origin mislabeling and substitution of PGI jujube by inferior products seriously harms their reputation and has potential food safety risks. In this study, stable isotope (δ¹³C, δ¹⁵N, δ²H, δ¹⁸O) and elemental (Na, Mg, Al, P, K, Ca, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Sr, Mo, Cd, Sb, Ba, Pb) compositions of jujube from five different regions across Xinjiang province were analyzed by orthogonal partial least squares-discriminant analysis (OPLS-DA) to verify the geographical origin of jujube and authenticate PGI products. A total of 167 Hui jujube and 156 Jun jujube samples, together with their associated soils were collected annually from 2013 to 2018. The regional, varietal and inter-annual differences of stable isotope and elemental compositions of jujube and their correlation with soil values were analyzed using one-way ANOVA and multivariate statistics. The discriminant accuracies of OPLS-DA modeling for both Hui and Jun jujube samples collected in 2016 were higher than 90 %. Over a longer five-year (from 2013 to 2018), the discriminant accuracies decreased slightly, but were still acceptable at 85 % and 75 % for the two varieties. The most important variables for discrimination models were Na, Al, Ba, and δ¹³C for Hui jujube, δ¹⁸O and δ¹⁵N for Jun jujube, respectively. The study show that this strategy holds good promise as a tool to combat mislabeling and fraudulent conduct and has the ability to protect PGI jujube produced in Xinjiang province.     

Dried Wild-Grown Mushrooms Can Be Considered a Source of Selected Minerals

Dried mushrooms might be a source of mineral components, which are indispensable for human health. The aim of this study was to determine the contents of calcium (Ca), magnesium (Mg), iron (Fe), zinc (Zn), copper (Cu), manganese (Mn), and selenium (Se) in dried wild-grown mushrooms (Boletus edulis and Xerocomus badius) available for sale, and to evaluate these mushrooms’ contribution to the daily reference intake of the studied bioelements. The concentrations of mineral components in the mushroom samples were determined by the flame method (Ca, Mg, Fe, Zn, Cu, Mn) and the electrothermal (Se) atomic absorption spectrometry method. The mean Ca, Mg, Fe, Zn, Cu, Mn (in mg/kg), and Se concentrations (in µg/kg) in B. edulis were 82.1, 964.1, 233.4, 97.9, 25.3, 22.1, and 6501.6, respectively, whereas in X. badius: 67.5, 1060.2, 87.8, 197.2, 33.9, 19.8, and 282.4, respectively. We have shown that dried B. edulis can be considered a source of Se. In the case of the other microelements, the tested mushrooms may serve only as additional supplements. Therefore, the studied species of mushrooms cannot be regarded as potential nutritional sources of the macroelements in question. Consumers should be properly informed about this, which should be guaranteed by appropriate legal regulations.