Pro-Health and Anti-Cancer Activity of Fungal Fractions Isolated from Milk-Supplemented Cultures of Lentinus (Pleurotus) Sajor-caju

The present study aimed to demonstrate Lentinus (formerly Pleurotus) sajor-caju (PSC) as a good source of pro-health substances. It has also shown that supplementation of its culture medium with cow milk may further improve its beneficial properties. Intracellular fractions from fungi grown on a medium supplemented with cow milk were analyzed using various biochemical methods for determination of the nutrient composition. Furthermore, anti-cancer properties of selected extracts were investigated on colorectal cancer cell lines (HT-29, LS 180, and SW948) in vitro. Biochemical analysis showed enrichment in health-enhancing compounds, such as proteins or polysaccharides (about 3.5- and 4.5-fold increase in concentration of proteins and carbohydratesin extracts of mycelia cultured on whole milk (PSC2-I), respectively), with a decrease in the level of free radicals (10-fold decrease in extract grown on milk and medium mixture (1:1) (PSC3-II)), which was related to increased catalase and superoxide dismutase activity (7.5-fold increase in catalase activity and 5-fold in SOD activity in PSC3-II compared to the control). Moreover, the viability of the cancer cells was diminished (to 60.0 ± 6.8% and 40.0 ± 8.6% of the control, on HT-29 and SW948 cells, respectively), along with pro-apoptotic (to 18.8 ± 11.8 and 14.7 ± 8.0% towards LS 180 and SW948 cells, respectively) and NO-secreting effects (about 2-fold increase) of the extracts. This study suggests that PSC has multiple nutritional and anti-cancer properties and can be used as a source of healthy biomolecules in modern medicine or functional foods.


Introduction
Fungal material has been used by humankind for centuries, especially due to its gastronomic and pro-health properties. Edible mushrooms are also widely used in daily diet, mainly due to their unique organoleptic properties (characteristic taste and smell), and in ethnomedicine. A significant influence of fungi and their metabolites is particularly evident in natural medicine, especially in Asia. Mushrooms are considered as a very rich source of bioactive compounds. Furthermore, they are easy and cheap to culture and harvest. They can grow in competitive conditions, e.g., in low light, and have many Biotechnology, Maria Curie-Skłodowska University, Lublin, Poland. Fungal cultures were cultivated on Lindeberg-Czapek medium [15]. After four weeks, mycelium was collected and transferred into four medium variant flasks: 1-Lindeberg-Holm medium; 2-commercial whole milk (lactose 5.3%, casein 3.2%, fat 5.5%, total proteins 4.2%); 3-whole milk and Lindeberg-Holm medium 1:1 v/v; 4-whole milk and Lindeberg-Holm medium in 1:2 v/v. Then, the cultures were harvested after 2 (variant I) or 4 weeks (variant II) of growth. The mycelium was separated from the culture medium and washed with distilled water. Afterwards, 1 g/mL of mycelium was homogenized in a Potter Homogenizer (Sartorius, Goettingen, Germany) (1200 rpm for 5 min), centrifuged, and freeze-dried. The lyophilized samples were weighed and dissolved in sterile RPMI 1640 medium (Sigma Aldrich, St. Louis, MO, USA) to obtain a 100 mg/mL stock solution diluted to the concentrations used in the subsequent analyses.

Evaluation of Biochemical Properties 2.3.1. Determination of Amino Acids, Proteins, Carbohydrates, and Phenolic Compounds
The ninhydrin method was employed to assess the concentration of free amino acids. The amount of free amino acids was evaluated spectrophotometrically with glycine as standard substrate [20]. Protein concentrations were determined using the Bradford reagent and bovine serum albumin as a standard [21,22]. Assessment of total sugar content was performed with the phenol-sulfuric acid assay using D-glucose as a standard [23,24]. The complete range of phenolic compounds was determined with diazosulfanilamide using the DASA test, where absorbance was measured at 500 nm, and vanillic acid was used as a standard [24,25].

Determination of Free Radicals
The level of free radicals was evaluated by the NBT assay according to the Malarczyk and Wilkołazka method. Mycelial samples were mixed with 50 µL of 4% nitro blue tetrazolium, 250 µL 1N NaOH, and 1,5 mL H 2 O and incubated at room temperature for 30 min. Afterwards, the samples were measured spectrophotometrically at 560 nm [26,27].

Electrophoretic Assessment of Catalase and Superoxide Dismutase Activity
Homogenized mycelia were used for electrophoretic analysis. Electrophoretic separation was carried out on 12.5% polyacrylamide gel, with 25 µL of samples loaded per well. The electrophoresis was held at 4 • C, 140 V. Densitometric semi-quantitative analyses of electropherograms were performed with ImageJ software ver. 1.52a (NIH, Bethesda, MD, USA). For analysis of catalase activity, gels were rinsed 3 times in distilled water and Biomolecules 2021, 11, 1089 4 of 15 incubated in 0.003% H 2 O 2 for 10 min. The gels were rinsed and placed in a mixture of equal volumes of 2% K 2 Fe(CN) 6 and 2% FeCl 3 and incubated for 10 min. Superoxide dismutase activity was measured using the NBT method. The gels were incubated for 30 min with NBT (nitrotetrazolium blue), rinsed, and placed in a mixture of methionine, riboflavin, and K 2 HPO 4 [28,29]

NR Uptake Assay
In the NR method, after 24-h incubation of the cells in 96-well plate (100 µL of 1 × 10 5 cells/mL) with the studied extracts, the medium was removed and 100 µL of 40 mg/mL NR were added to each well. After 3-h incubation, the cells were fixed with 200 µL of 0.5% formalin in 1% CaCl 2 . Subsequently, 100 µL of solvent (1% acetic acid in 50% ethanol) were added for 20 min. The plates were measured at 540 nm.

May-Grünwald-Giemsa Staining
Cells at a density of 1 × 10 5 cells/mL were cultured in Petri dishes (35 mm). After 24-h incubation with the studied extracts, the medium was discarded, and the MGG staining protocol was initiated. The cells were stained and fixed with the May-Grünwald dye for 3 min and another 3 min in the dye diluted with an equal volume of water. Thereafter, the dye was removed and Giemsa stain, previously diluted (1:19 in water), was added for 20 min. The dishes were rinsed three times with distilled water and dried. The observation was performed under a light microscope Olympus BX51 (Olympus, Tokio, Japan) with 100× magnification and analyzed. Changes in the number and morphology of the cells were verified.

Nitric Oxide Secretion Measurement
The indirect measurement of NO was conducted using the spectrophotometric method based on the Griess method.
Cells at a density of 1 × 10 5 cells/mL were cultured in 24-well plates. After 24-h incubation with the studied extracts (with or without 2-h pre-incubation with E. coli LPS (10 µg/mL) (Sigma Aldrich, St. Louis, MO, USA) the medium was collected and frozen at −80 • C. For the analysis, the samples were thawed, and 100 µL of samples and standards (1, 5, and 10 µM of NaNO 2 (Sigma Aldrich, St. Louis, MO, USA) were added to the wells of the 96-well plate. Afterwards, 100 µL of Griess reagent (1% sulfanilamide/0.1% N-(1-naphthyl) ethylenediamine dihydrochloride (POCH, Gliwice, Poland) in 3% H 3 PO 4 (POCH, Gliwice, Poland) were added to each well. After 10-min incubation at room temperature, the plates were read using a Microplate Reader, and absorbance was measured at 570 nm. Cells at a density of 1 × 10 5 cells/mL were cultured in Petri dishes (35 mm). After 24-h incubation with the extracts at a concentration of 200 µg/mL, 5 µL of a Hoechst 33342 and PI mixture were added to the medium in each dish. The samples were incubated for 5 min, washed with PBS, and observed under a fluorescence microscope (Olympus BX51, Tokio, Japan ) at magnification 100×. More than 1000 cells in randomly selected areas were counted, and the ratios of apoptotic and necrotic cells were assessed.

DAPI/SR101 Staining
To visualize morphological changes in the nuclei and protein localization, DAPI/SR101 staining was performed. The blue-fluorescent DAPI stained cell nuclei, and the redfluorescent SR101stained cell proteins.
After-24 h incubation with the studied extracts (200 µg/mL), the cells were fixed and incubated with a DAPI/SR101 (Sigma Aldrich, St. Louis, MO, USA) mixture. The cells were observed and photographed at magnifications 100× (for normal cells) and 200× (for cancer cells) at 460-500 nm (DAPI) and 600 nm (SR101) using a fluorescence microscope Olympus BX51. The intensity of the red fluorescence of proteins was evaluated with ImageJ software ver. 1.52a (NIH, Bethesda, MD, USA). Six random areas were measured from every image; results from 3 repeats were presented as mean ± SD.

Statistical Analysis
All analyses were performed in at least 3 replications, and the data were analyzed using GraphPad Prism ver. 7.01 (GraphPad Software, San Diego, CA, US). The results were presented as mean ± SD. Statistical significance was evaluated with the one-way Anova test and post-hoc Dunnett's test.

Screening Selection of Samples
During the screening analysis, three extracts of L. sajor-caju were selected for further analysis: PSC2-I, PSC3-II, and PSC4-II. These were extracts cultured on whole milk only for 2 weeks (PSC2-I), cultured on whole milk and Lindeberg-Holm medium 1:1 v/v for 4 weeks (PSC3-II), and cultured on whole milk and Lindeberg-Holm medium 1:2 v/v for 4 weeks (PSC4-II). The screening was performed on CCD 841 CoTr and HT-29 cells using the NR method. The extracts were selected due to their stronger effect towards cancer cells with a concurrently milder effect on normal cells. The biochemical properties of extracts from the fungus grown on milk or milk-supplemented medium were compared to the control, i.e., fungal mycelium cultured on the standard mineral medium (Lindeberg-Holm) for 2 weeks (control for PSC2-I) and 4 weeks (control for PSC3-II and PSC4-II), respectively. The controls are presented as green bars in the figures in the biochemical section. The control in the cell culture section for the anticancer activity investigations were cells cultured in the medium with FBS only (with no extracts added).

Determination of Proteins, Amino Acids, Carbohydrates, Phenolic Compounds, and Free Radicals
The determination of proteins showed that all selected extracts had a higher protein concentration than the control ( Figure 1A). The level of free amino acids (aa) determined in the extracts was lower than in the control, with the lowest aa concentration in PSC4-II ( Figure 1B). The analysis of carbohydrates showed that the PSC2-I and PSC3-II extracts had a significantly higher level of these substances than the control. In turn, the concentration of carbohydrates in the PSC4-II extract was lower than in the control ( Figure 1C). The analysis of the level of phenolic compounds revealed their significantly higher concentrations in all selected extracts, compared to the control ( Figure 1D).
The analysis of the concentration of free radicals revealed their significantly lower levels in all extracts in comparison with the control. The lowest level was determined in PSC2-I, compared to the other extracts. In turn, the highest free radical level was determined in PSC3-II; however, their concentration was lower than in the control ( Figure 1E). The concentrations of the compounds are presented in the supplementary material (Table  S1).

Electrophoretic Analysis of Catalase and SOD Activity
The electrophoretic analysis of catalase activity revealed that all studied extracts increased the enzyme activity several-fold, compared to the controls. The strongest effect was exhibited by PSC3-II ( Figure 2A). The analysis of the concentration of free radicals revealed their significantly lower levels in all extracts in comparison with the control. The lowest level was determined in PSC2-I, compared to the other extracts. In turn, the highest free radical level was determined in PSC3-II; however, their concentration was lower than in the control ( Figure 1E). The concentrations of the compounds are presented in the (Supplementary Materials (Table S1)).

Electrophoretic Analysis of Catalase and SOD Activity
The electrophoretic analysis of catalase activity revealed that all studied extracts increased the enzyme activity several-fold, compared to the controls. The strongest effect was exhibited by PSC3-II (Figure 2A).
Similarly, SOD activity was also several-fold increased by all the extracts, with the highest effect exerted by PSC4-II ( Figure 2B). Similarly, SOD activity was also several-fold increased by all the extracts, with the highest effect exerted by PSC4-II ( Figure 2B).

Cytotoxicity Assessment
The NR method indicated that only PSC2-I showed significant cytotoxic activity. It inhibited the viability of HT-29 in a dose-dependent manner. The IC50 value was approximately 323.3 µg/mL. The extract also inhibited proliferation of CCD 841 CoTr and SW948 cells. LS 180, however, seemed to be invulnerable ( Figure 3A, upper panel). The results obtained with the MTT method appear slightly different. They showed that the effect of the extracts was associated with disruption of cell metabolism rather than disruption of membrane integrity. The extract reduced the viability of all examined cell lines, excluding HT-29. However, the viability reduction in the CCD 841 CoTr cells was no higher than 10% ( Figure 3A, bottom panel). The images produced by May-Grünwald-Giemsa staining confirm the NR and MTT assays; there were visible morphological changes in the cells, as well as dead cells, compared to the control ( Figure 3B). The PSC3-II and PSC4-II extracts did not exert a significant cytotoxic effect. They induced proliferation or inhibited viability to a small extent (excluding the effect of PSC4-II on CCD 841 CoTr measured with the MTT method ( Figure 3A, upper panel). The results were confirmed by staining the cells with the May-Grünwald-Giemsa method. There were slightly greater numbers of morphologically changed and dead cells compared to the control, but the changes were not distinct ( Figure 3B).

Apoptosis and Necrosis Evaluation
All studied extracts exhibited the desired activity. They increased the apoptosis rate in all cancer lines with no significant rise in apoptotic CCD 841 CoTr cells. Furthermore, the higher the stage of cancer was, the higher the pro-apoptotic effect was. The most pronounced effect was exerted by PSC4-II on HT-29 and LS 180 and by PSC3-II on SW948 ( Figure 3C). The cell necrosis rate was insignificant, i.e., lower than 1% in most cases (with the highest necrotic rate of 1.1% in the SW948 cells incubated with PSC2-I).

Evaluation of Morphological Changes in Nuclei and the Concentration and Localization of Proteins
No significant changes in the CCD 841 CoTr cells after the incubation with the extracts were observed.

Cytotoxicity Assessment
The NR method indicated that only PSC2-I showed significant cytotoxic activity. It inhibited the viability of HT-29 in a dose-dependent manner. The IC 50 value was approximately 323.3 µg/mL. The extract also inhibited proliferation of CCD 841 CoTr and SW948 cells. LS 180, however, seemed to be invulnerable ( Figure 3A, upper panel). The results obtained with the MTT method appear slightly different. They showed that the effect of the extracts was associated with disruption of cell metabolism rather than disruption of membrane integrity. The extract reduced the viability of all examined cell lines, excluding HT-29. However, the viability reduction in the CCD 841 CoTr cells was no higher than 10% ( Figure 3A, bottom panel). The images produced by May-Grünwald-Giemsa staining confirm the NR and MTT assays; there were visible morphological changes in the cells, as well as dead cells, compared to the control ( Figure 3B). The PSC3-II and PSC4-II extracts did not exert a significant cytotoxic effect. They induced proliferation or inhibited viability to a small extent (excluding the effect of PSC4-II on CCD 841 CoTr measured with the MTT method ( Figure 3A, upper panel). The results were confirmed by staining the cells with the May-Grünwald-Giemsa method. There were slightly greater numbers of morphologically changed and dead cells compared to the control, but the changes were not distinct ( Figure 3B).

Apoptosis and Necrosis Evaluation
All studied extracts exhibited the desired activity. They increased the apoptosis rate in all cancer lines with no significant rise in apoptotic CCD 841 CoTr cells. Furthermore, the higher the stage of cancer was, the higher the pro-apoptotic effect was. The most pronounced effect was exerted by PSC4-II on HT-29 and LS 180 and by PSC3-II on SW948 ( Figure 3C). The cell necrosis rate was insignificant, i.e., lower than 1% in most cases (with the highest necrotic rate of 1.1% in the SW948 cells incubated with PSC2-I).
phological changes, e.g., enlarged cells probably with cytoplasm leakage (potentially cells in the state of necrosis) and deformed or enlarged nuclei. There were also fewer cells undergoing division. The red color was less intense, which may indicate cytoplasm leakage (with proteins), probably caused by disruption of cell membrane integrity or a decrease in the protein concentration. The strongest anti-cancer effect was exhibited by PSC2-I and PSC3-II on the SW948 cells and by all extracts on the LS 180 cells. The HT-29 cells, however, seemed unaffected ( Figure 4A).  Figure S2); * p < 0,05, ** p < 0,01, *** p < 0,005, one-way ANOVA, Dunnett's test.

Evaluation of Morphological Changes in Nuclei and the Concentration and Localization of Proteins
No significant changes in the CCD 841 CoTr cells after the incubation with the extracts were observed.
The effect of the PSC2-I, PSC3-II, and PSC4-II extracts on cancer cells included morphological changes, e.g., enlarged cells probably with cytoplasm leakage (potentially cells in the state of necrosis) and deformed or enlarged nuclei. There were also fewer cells undergoing division. The red color was less intense, which may indicate cytoplasm leakage (with proteins), probably caused by disruption of cell membrane integrity or a decrease in the protein concentration. The strongest anti-cancer effect was exhibited by PSC2-I and PSC3-II on the SW948 cells and by all extracts on the LS 180 cells. The HT-29 cells, however, seemed unaffected ( Figure 4A).

Discussion
A significant increase in lifestyle diseases has been observed over the past decades. Modern medicine is not always efficient enough and alternatives are being sought. Therefore, the interest in agents used in folk medicine for centuries is growing today. Many of such substances, especially those derived from mushrooms, exhibit pro-health and anti-  Tables S2-S4); * p < 0,05, ** p < 0,01, *** p < 0,005, one-way ANOVA, Dunnett's test.

NO Secretion Measurement
The cytotoxic effect of the studied extracts was reflected in a rise in the level of the secreted NO. It has been demonstrated that agents inducing NO leakage have the highest cytotoxicity [30,31]. The results are presented in Figure 4B  On the other hand, a drastic decrease in the NO level may also be regarded as anti-cancer activity. Such activity was exhibited by 100 µg/mL PSC3-II towards LS 180 and SW948.

Discussion
A significant increase in lifestyle diseases has been observed over the past decades. Modern medicine is not always efficient enough and alternatives are being sought. Therefore, the interest in agents used in folk medicine for centuries is growing today. Many of such substances, especially those derived from mushrooms, exhibit pro-health and anti-cancer properties [32][33][34].
The present study demonstrated the biochemical composition and anti-cancer effects of extracts from the arboreal medicinal fungus Lentinus sajor-caju cultivated on milksupplemented medium on human colon cancer cells HT-29, LS 180, and SW948, and on human normal epithelial cells CCD 841 CoTr as a control.
So far, this species has not been studied widely in this regard or has not been studied at all. In turn, the effect of supplementation of culture media with cow milk on anti-cancer activity has been evaluated. Cow milk is an extremely widespread product in the diet of modern society. However, milk consumption raises considerable controversy and seems to be a source of health problems related to allergic reactions to its ingredients in a certain group of consumers. The research presented in our work shows new possibilities of using its potential. Previous research conducted by our team for the first time showed the role of fungal extracts of Cerrena unicolor and Pycnoporus sanguineus as health-related factors used to improve the pro-health properties of cow milk and its effect on extract activity [35].
The fungus was grown on normal medium (1), medium supplemented with milk (2 and 3), and milk only (4) for two (I) or four weeks (II). After this time, the extracts from all variants were prepared and biochemical analysis was performed, followed by evaluation of the activity of the extracts towards cancer and normal cells. PSC2-I exhibited the best anti-cancer properties, inducing a cytotoxic effect towards cancer cells, increasing the apoptosis ratio, and causing morphological and physiological changes in the cells.
The PSC2-I extract had different biochemical properties than the other extracts and the control. It showed the highest concentration of proteins, free amino acids, carbohydrates, and phenolic compounds and the lowest level of free radicals compared to the other extracts. It also exhibited the strongest anti-cancer properties. The present results may indicate a correlation between these properties. Probably, PSC2-I produced antitumor proteins, thus the cytotoxic effect on the cancer cell lines was observed. On the other hand, the extract promoted proliferation of the normal cells, which was demonstrated by the NR and MTT methods.
Fungal proteins are known for their anti-cancer properties. Lin et al. (2010) studied antitumor and immunomodulatory effects of protein isolated from Ganoderma microsporum towards lung cancer A549 cells. It appeared that the protein, called GMI (Ganoderma microsporum im-munomodulatory), inhibited epidermal growth factor-mediated invasion of cancer cells [36]. PSC2-I and PSC4-II also exhibited elevated levels of phenolic compounds, i.e., substances with confirmed anti-cancer properties, in contrast to PSC3-II. Many phenolic and polyphenolic compounds, such as flavonoids, tannins, lignans, and stilbens, occur in fungal cells. Due to their structure and chemical properties, phenolic compounds have high antioxidant potential [37][38][39]. The antioxidant properties of phenols constitute one of the mechanisms that protect cells against neoplastic changes. They may also exhibit cytotoxic properties, e.g., hericenons A and B from Hericium erinaceus on HeLa cells [40]. Gallic acid, i.e., an organic phenolic acid derived from fungi, has been demonstrated to exhibit antitumor properties against prostate cancer and human glioblastoma cells [41][42][43]. It was also found that cinnamic acid isolated from Clitocybe alexandri inhibited cell growth in non-small cell lung cancer (NCI-H460), colon cancer (HCT15), colon adenocarcinoma (CaCo-2), and cervical cancer (HeLa) [44][45][46]. Furthermore, polyphenolic compounds are linked to scavenging reactive oxygen forms. We demonstrated that the extracts exhibited potential to reduce the level of free radicals, especially PSC2-I and PSC4-II. Such an effect may be related to the higher level of phenolic compounds and the higher CAT and SOD activity. Similar effects of Lentinula edodes and Lentinus sajor-caju on HEp-2 and HeLa (cervical cancer) and L. sajor-caju on HeLa and HepG2 (hepatocellular cancer) were demonstrated by Finimundy et al. [47,48]. In another study conducted by Seedevi et al., free radical scavenging activity was exhibited by polysaccharide isolated from L. sajor-caju [49].
In the present study, we demonstrated an anti-cancer effect of PSC extracts on colon cancer cells HT-29, LS 180, and SW948. A decrease in viability, induction of apoptosis, and morphological changes were observed. Cytotoxic and pro-apoptotic effects of L. sajorcaju extracts were demonstrated by Finimundy et al.: the extract decreased the viability of HCT-116 cells, induced apoptosis, and affected the cell cycle, leading to its arrest in the G2/M phase [50]. Chaturvedi et al. reported similar results revealing cytotoxic and pro-apoptotic effects of PSC water extract on HCT-116 cells [51]. The fungus was studied on other models, as well, and was shown to affect human cervical cancer HEp-2 and HeLa cells [47], breast cancer MCF-7 and non-small cell lung cancer NCI-H460 cells [48], and human gastric cancer (AGS) and hepatocellular carcinoma cells (HepG2) [49]. Many more mushrooms and derived compounds have been demonstrated to possess anti-cancer activity. For instance, sesquiterpene Hirsutanol A from Chandrostereum sp. had an effect on SW620 cells [52], diterpene cyathin Q from Cyathus africanus on HCT116 cells [53], Lactarius flavidulus and Ganoderma capense on hepatoma HepG2 and leukemic L1210 cells [54,55], and Pholiota adiposa and Hericum erinaceum on hepatoma HepG2 and breast cancer MCF7 cells [56,57].
Another activity responsible for the PSC anti-cancer effect may be related to an increased NO concentration. Nitric oxide may act as a pro-carcinogenic agent inducing mutagenesis, proliferation, angiogenesis, and metastasis; on the other hand, the anti-cancer effect of NO achieved mainly through apoptosis induction is known, as well. It is thought that low concentrations of nitric oxide are pro-cancerous, while higher concentrations favor the anti-cancer effect [30,31]. Therefore, a number of compounds capable of NO releasing or inducing its production are being developed as anti-cancer agents [31,58]. We demonstrated that extracts PSC2-II and PSC4-II had the strongest activities associated with a decrease in cell viability and an increase in NO secretion. Similar results were obtained by Chaturvedi et al. (2020), who demonstrated a rise in the level of intracellular ROS after treatment of HCT-116 cells with PSC extract [51].
A very important aspect of the present study was to evaluate the effect of various culture conditions: time of growth and composition of the medium-the presence (and amount) or absence of cow milk on biochemical and anti-cancer properties. We have previously demonstrated such a correlation in the case of Cerrena unicolor and Pycnoporus sanguineus [35]. A similar correlation was observed in the case of Lentinus sajor-caju. The shorter the incubation with milk was (variant I, 2 weeks of culturing on milk), the milder the effect on both normal and cancer cells was. In turn, the longer the growth was (variant II, 4 weeks of culturing of milk), the stronger the effect on both models was exerted. It appears that the best properties are acquired by the mushroom during shorter culturing on milk only (2 weeks, variant I) or longer growth on milk mixed with medium (4 weeks, variants II). Probably, prolonged fermentation of milk by PSC contributes to accumulation of cytotoxic agents with limited specificity (cytotoxic effect towards both normal and cancer cells). Alternatively, at a specific time point, the fungus assimilates pro-health agents, which are fermented or transformed beyond that point. This question is to be answered in future studies.
In the present study, we demonstrated the biochemical composition and anti-cancer properties of Lentinus sajor-caju extracts, and the effect of culturing the fungus on full cow milk. The results seem to suggest that the mushroom has anti-cancer activity, which is enhanced by cultivation on milk. However, the exact mechanisms responsible for such activity are to be explained in the future.

Conclusions
In summary, we presented the potential beneficial effects of supplementation of Lentinus sajor-caju growth medium with milk. We have demonstrated the reducing effect of mycelium extracts on the survival of selected tumor cell lines (HT-29, LS 180, and SW948). All tested extracts exerted an anti-cancer influence through reduction of cancer cell viability, a pro-apoptotic effect, disruption of cell membrane integrity and nuclei morphology, and an increase in NO secretion. There is no doubt that the above results require further and more comprehensive analyses; however, they have revealed the most desired pro-health and anti-cancer properties of the extracts. We have also shown a novel application of the nutritional potential of milk fermented by mushrooms.  Table  S1. Biochemical composition of the cow whole milk and the studied extracts of Pleurotus sajor-caju ,  Table S2. Concentration of the NO secreted by CCD 841 CoTr, HT-29, LS 180 and SW948 cells after incubation with PSC2-I extract, with and without pre-incubation with LPS, Table S3. Concentration of the NO secreted by CCD 841 CoTr, HT-29, LS 180 and SW948 cells after incubation with PSC3-II extract, with and without pre-incubation with LPS, Table S4. Concentration of the NO secreted by CCD 841 CoTr, HT-29, LS 180 and SW948 cells after incubation with PSC4-II extract, with and without pre-incubation with LPS.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.

Conflicts of Interest:
The authors declare no conflict of interest.