Introduction

The human digestive system is a complex microbial ecology comprised of trillions of fungi, archaea, bacteria, and viruses [1]. Starting at birth, a unique microbial population dominates one that plays a crucial function in host physiology throughout the lifespan. The host, the microbiota, the released metabolites, and the active metabolic pathways are all playing more important roles as progress has been achieved. The role of quorum sensing (QS), a sort of cell–cell interaction, in the gut microbiota and its influence on human metabolism and nutrition are still insufficiently understood [2]. Despite significant progress in the field of biology, more research has to be done to reveal the real role of bacteria in the gastrointestinal milieu, including their reaction to communicating with the host cells and creating a variety of bioactive substances. The optimal function of the intestinal microbiota is affected by a number of variables, including the host's microbiota, the host's food, and the host's health. Research also shows that dead or nonliving microbial cells, their biostructural components, and their metabolites may activate particular signaling pathways in host cells and perform certain biological or physiological actions [3]. Bioactive metabolites (postbiotics) made by intestinal microbiota are an intriguing strategy for offering therapeutic benefits that play an important role in creating eubiosis circumstances because they do things like prevent the growth of pathogens, keep the intestinal mucosa integrity and functioning properly, alter the composition of the intestinal microbiota, and moderate immune responses and certain key inflammatory pathways. Postbiotics, by virtue of their peculiar structure, interfere with the processes by which the immune system and the neurological system exert their controlling influence on host cells. Examples of this issue include improving the efficiency of the innate immune system, decreasing the inflammatory responses caused by the presence and activity of pathogenic germs with inflammation-inducing activity and carcinogenic agents (especially those derived from food processing), and bolstering the effectiveness of the intestinal barrier [4, 5].

The term “postbiotic” is used to describe the consumption of deactivated microbial cells, cellular bioactive elements/metabolites that are produced primarily through the fermentation process by potential probiotic microorganism cultures or in response to ecological context and, that when ingested in sufficient quantities, provide the host multiple biological health benefits [3, 6]. Furthermore, postbiotic compounds of specific chemical compositions may modify physiological mechanisms, regulatory pathways, and/or behavioral reactions associated with the host's commensal microbiota functioning. Exopolysaccharides, glycoproteins, peptides, proteins, peptidoglycans, linoleic acid, lactic acid, and short-chain fatty acids are only some of the many postbiotic metabolites that have been shown to have significant antioxidant, antiinflammation, and antibacterial effects [6,7,8].

Saccharomyces cerevisiae is a functional probiotic yeast that is often used as a fermenting dough starter and has been recognized for its probiotic and functional properties. Fungemia can occur in both humans and animals when exposed to certain strains of this yeast (under special conditions such as individuals with a suppressed immune system, dysbiosis status in the gut microbiome, etc.), which exhibited opportunistic pathogenic behavior. Several studies have lately suggested using the postbiotic form to take advantage of probiotics and health-promoting advantages in a safe and controllable manner [9].

Based on the aforementioned considerations, the present study characterized and investigated some of the main functional biologic dimensions of postbiotics derived from Saccharomyces cerevisiae (PSC) in generating significant antioxidant, antibacterial, α-glucosidase inhibition, cholesterol-lowering, cytotoxic, and apoptosis responses under in vitro circumstances.

Materials and Methods

Materials

Yeast peptone dextrose (YPD), yeast malt broth (YMB), agar–agar, absolute ethanol, and chloroform were purchased from Merck Millipore, (Darmstadt, Germany). ABTS (2,2′-azino-bis [3-ethylbenzothiazoline-6-sulfonic acid] diammonium salt), gallic acid, and quercetin were procured from Sigma-Aldrich Co. (St Louis, MO, USA). The culture media Mueller Hinton agar (MHA), Mueller Hinton broth (MHB), Sabouraud dextrose agar (SDA), mannitol salt agar (MSA), eosin methylene blue (EMB), plate count agar (PCA), brain heart infusion (BHI), Luria Bertani (LB), and cefixime tellurite sorbitol MacConkey (CT-SMAC) were purchased from Merck Co. (Darmstadt, Germany).

Preparation and Maintenance of Probiotic Yeast Strain

Iran’s industrial center of bacteria and fungi collection (Iran’s scientific, research, and industrial organization) supplied Saccharomyces cerevisiae (PTCC 5269) and was cultivated in YPD (20 g/L dextrose, 4 g/L yeast extract, 3 g/L bacterial peptone, 2 g/L triammonium citrate, 1 g/L polysorbate 80, 1 g/L KH2PO4, and 0.8 g/L MgSO4) for 24–48 h at 30 °C while being stirred at 200 rpm. Saccharomyces cerevisiae suspensions were standardized using an ultraviolet–visible spectrophotometer (UV3600, Japan) at 600-nm wavelength region if needed [10].

Preparation of Yeast Cell-Free Supernatant Solution

The first stage in the generation of cell-free supernatant (CFS) postbiotics is the aerobic culture of S. cerevisiae in YMB for 48 h at a temperature of 37 ± 1 °C. This procedure was carried out in 2000-mL Erlenmeyer flasks in order to get sufficient and appropriate quantities of CFSs because different microbial strain growth circumstances and extraction techniques significantly affect the postbiotic efficiency. Briefly, CFS was extracted by 24–48 h of incubation at a temperature of 37 °C, followed by 10 min of centrifugation at 4 °C at 4500 rpm. The yeast medium supernatant was collected, the pH was modified to 7.2, and it was purified through a 0.22-μm Millipore filter (Sigma-Aldrich, MilliporeSigma Co., Germany) before being used to treat the samples [10]. The filtrate was collected for freeze-drying. After, the harvested CFS were frozen at − 80 °C for 24 h. The CFS was lyophilized (Lyophilization Systems, Inc, USA) from − 40 to − 30 °C, 0.2 mbar. The entire freeze-drying process was performed in 24 h, and the freeze-dried powders were stored at − 20 °C. They were then rehydrated with sterile deionized water prior to use.

Assessment of the Total Phenolic Content

An approach developed by Abeysekera and colleagues (2013) was used to evaluate the total phenolic content of the PSC using the Folin-Ciocalteu reagent [11]. Sodium carbonate solution (70 μL) and Folin–Ciocalteu reagent (110 μL) have been utilized to charge the PSC (20 μL). The mixture's absorbance was measured at 765 nm after being kept for 30 min at 25 °C. The standard curve was generated using gallic acid (0.06–1 mg/mL), and the total phenol content of PSC was calculated as mg gallic acid equivalent (GAE) per gram PSC.

Assessment of the Total Flavonoid Content

The aluminium chloride technique was used to determine PSC's total flavonoid content. After being incubated at 25 °C for 10 min, the combination of PSC (100 μL) and aluminium chloride mixture (100 μL; 2% w/v in methanol) was measured for absorbance at 367 nm. The total flavonoid concentration was reported as milligram quercetin equivalent (QE) per gram PSC, and quercetin (7.81–125 mg/mL) was used to generate the calibration curve [11].

Antioxidant Activity Assessment of the Prepared Postbiotics

The technique of Nantitanon and colleagues [12] was modified to test the antioxidant impact of PSC against ABTS free radicals. The 7 mM ABTS solution was oxidized with 2.45 mM K2S2O8 to produce the ABTS radical monocation. After being kept at room temperature and kept in the dark for 12 h, the mixture was diluted with ethanol until it had an absorbance of 0.7 ± 0.2 at 750 nm. The ABTS solution (1.8 mL) was then mixed with the PSC and control (0.2 mL), and a spectrophotometer was utilized to determine the absorbance of the mixture at 750 nm. The following formula was used to determine PSC's antioxidant activity: 

$$Antioxidant\;activity\;(\%)=\left(1-\frac{Absorbance\;of\;sample}{Absorbance\;of\;control}\right)\times100$$
(1)

Antibacterial Activity Assessment of the Prepared Postbiotics

Under In Vitro Condition

The disc diffusion agar (DDA), minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and well diffusion agar (WDA) were employed to assess the postbiotics' antibacterial efficacy against Salmonella typhi ATCC 6539, Escherichia coli ATCC 25922, Streptococcus mutans ATCC 25175, and Listeria monocytogenes ATCC19112 using the techniques outlined by Sabahi and colleagues [13]. In the DDA test, the antibiotics tetracycline, gentamicin, and chloramphenicol were used to compare their antibacterial effects.

Under Food Models

The minimal effective concentration (MEC) of postbiotics was calculated using the same manner as previously stated by Hartmann and colleagues (2011) [14]. To get the final population to an adequate level (∼ 3.2 and 4.1 (E. coli) and 3.5 and 4.6 (L. monocytogenes) log10 CFU/mL or gram of milk and meat, respectively), E. coli ATCC 25922 and L. monocytogenes ATCC19112 were incorporated into 10 mL of pasteurized milk in a bottle and 100 g of ground meat in sterile wrappers, respectively. Both the milk and the meat samples, which had PSC added at doses ranging from 10 to 60 mg/mL, were fully homogenous. Six days were spent storing all samples at 4 °C. The examined pathogens were cultured and counted using cefixime tellurite sorbitol MacConkey agar and PALCAM Listeria selective agar. MEC was defined as the PSC concentration that decreases the initial microbial population to below the detection limit of 10 bacteria for milk and 100 bacteria for meat over the course of 3 days of preservation at 4 °C. Instead of a food matrix, comparable experiments were conducted using BHI broth and LB broth.

Determination of Growth-Promoting Properties of the Prepared Postbiotics

The effects of PSC on the proliferation of probiotic strains were studied to ascertain their growth-promoting qualities [15]. Lactobacillus casei L431, Lactobacillus brevis TD4, Lactobacillus acidophilus ATCC 4356, and Bifidobacterium bifidum ATCC 35914 were employed for this experiment. For 24 h, MRS broth medium was used to cultivate Lactobacilli and Bifidobacteria at 37 °C in anaerobic circumstances. We prepared sugar-free MRS (pH 5.7) with 0.5% PSC as the carbon source to determine whether it would stimulate growth. Positive controls consisted of an MRS medium comprising 0.5% glucose, whereas negative controls consisted of a sugar-free MRS medium. The cell density change was measured using a spectrophotometer (Cary-4000, Agilent, Technologies, USA) at 600 nm after 48 h of inoculation of probiotic strains (106 CFU/mL) from the previous culture into PSC-containing MRS media.

Determination of α-Glucosidase Inhibitor Activities of the Prepared Postbiotics

The activity of PSC to inhibit α-glucosidase was measured according to the protocol laid forth by Kazeem and colleagues [16]. This was accomplished by incubating 100 μL of α-glucosidase (1.0 U/mL, from Saccharomyces cerevisiae, Sigma, USA) with 50 μL of PSC (0.5, 1.0, and 2.0 mg/mL) at 37 °C for 10 min. The process was initiated by adding 50 μL of 3.0 mM 4-nitrophenly α-D-glucopyranoside (pNPG) diluted in 20 mM phosphate buffer, incubating for 20 min at 37 °C, and then adding 2 mL of 0.1 M Na2CO3. The wavelength of the yellow paranitrophenol emitted by pNPG was determined to be 405 nm (Multiskan Go, Thermo Fisher Scientific, Waltham, MA, USA). The percentage of α-glucosidase inhibition was determined using the following formula: 

$$\alpha-glu\cos idase\;inhibitor\;activity\;\left(\%\right)\;=\;\left[\left(A_{Control}-A_{Sample}\right)/A_{Control}\right]\times100$$
(2)

where AControl is the solution without the specimen and ASample is the solution with the various PSC concentration samples.

Determination of Cholesterol Removal Capabilities of the Prepared Postbiotics

The cholesterol-removing capacity of the synthesized PSC was evaluated according to the protocol laid forth by Soh and colleagues [17]. Different concentrations of 0.1% PSC (0.5, 1.0, and 2.0 mg/mL) and 30 μg cholesterol were combined in a 1-mL reaction mixture, which was then incubated at 25 °C for 20 min before 50 μL of hexadecyl trimethyl ammonium bromide was added. At 500 nm, the optical density of the supernatant was determined after centrifuging the mixture at 12,500 × g. The following equation was used to determine cholesterol-removing potential: 

$$Cholesterol-removing\;capability\;(\%)\;=\;\left[\left(A_{Control}-A_{Sample}\right)/A_{Control}\right]\times100$$
(3)

where AControl is the cholesterol solution without PSC and ASample is the cholesterol solution containing 0.1% PSC.

Determination of Anticancer Activity of the Prepared Postbiotics

Cell Culture

A 5% CO2 incubator at 37 °C was used to cultivate SW480 colon cancer cell lines that had been obtained from the Pasteur Institute, National Cell Bank of Iran, Tehran, Iran. The medium included 10% (v/v) fetal bovine serum, 100 mg/mL of streptomycin, and 10 U/mL of penicillin. Cells were detached using trypsin at 80–90% confluency, and while seeding in the appropriate culture plates, the culture medium was changed at regular intervals over a period of 2–3 days. After 3–4 passages, we used the cells for experimental analyses [18].

Cytotoxicity Assay

To determine PSC's cytotoxic potential, our research used the cytotoxicity test described in [19, 20]. In brief, 96-well microtiter plates were seeded with the examined cell lines (180 μL) at a density of 7 × 103 cells per milliliter, and the plates were incubated at 37 °C for 24 h. Then, 24-, 48-, and 72-h incubations at 37 °C were performed with PSC at doses of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 μg/mL, along with a 7 μL/well addition of 5-Fluorouracil (5-FU) (50 mg/mL) as a positive control. Following the designated time in culture, 20 μL of MTT solution (5 mg/mL) was added to each well, and the plates were incubated for another 4 h at the same temperature as the growth condition. After discarding the growth medium, we added 150 μL each of dimethyl sulfoxide (Sigma, St. Louis, MO, USA) and Sorenson buffer to the produced blue formazan crystals to dissolve them. The ELISA reader (ELx 800; Biotek, Winooski, VT, USA) was used to measure the absorbance at 570 nm. It is worth noting that every experiment was repeated three times.

Cell Cycle Analysis

Research into the anticancer properties of biomolecules produced from gut microbes sometimes includes cell cycle assay as a supplemental and confirmatory test. We used flow cytometry to examine the effects of 48 h of treatment with postbiotic metabolites on SW480 cells, looking for evidence of cell cycle arrest responses in addition to cytotoxicity [21]. It is important to note that the IC50 values obtained from the cytotoxicity test serve as the basis for selecting the time or/and concentration variables in this experiment. A six-well cell culture plate was seeded with 3 × 106 SW480 cells per mL, and the plate was placed in a 5% CO2 incubator at 37 °C for 5 h. After 48 h in a growth medium, the cells were treated with 24 μL of postbiotic metabolites, harvested from the wells, centrifuged at 3000 rpm for 10 min, and given two washes. Following a final fixation step in cold ethanol (70%), the cells were incubated for 30 min at 37 °C in 1 mL of PI master mix solution comprising 950 μL of phosphate-buffered saline (PBS) (pH 7.4), 10 μL of RNase, and 40 μL of the PI solution. Following this, flow cytometry (Becton Dickinson, San Jose, CA) was used to analyze the cells’ phases of division.

Annexin V/PI Binding Assay

Annexin V-FITC/propidium iodide (PI) cell apoptosis kit (eBioscience, BD Biosciences, San Diego, CA, USA) was used to quantify apoptosis in SW480 cell lines as per the manufacturer's instructions. In a nutshell, cells were seeded at a density of 0.5 × 106 cells per well on six-well culture plates and then treated to PSC (23.58 μg/mL) for 24 and 48 h. The cells were then removed, washed with PBS, incubated at room temperature in the dark for 30 min with annexin V-FITC binding solution, and then incubated for 5 min with PI [22]. FACSCalibur (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) was used to measure annexin V and PI expression.

RNA Extraction and qRT-PCR

The SW480 cells were grown in RPMI medium with 10% fetal bovine serum. After adding the cell suspension (~ 2 × 105 SW480 cells) to the 6-well plates, we put them in an incubator at 37 °C, 5% CO2, and humidified air for 48 h. PSC and 5-FU were used to treat the cells independently at the IC50 concentration. Total RNA was isolated from the cells using TRIzol Reagent (Invitrogen, USA) after 8, 24, and 48 h of incubation at 37 °C under the aforementioned conditions, and cDNA was generated as described below. After incubating at 65 °C for 5 min, 1 μg of mRNA, 1 μL of random hexamer primers, 1 μL of dNTP, and DEPC water up to 12.5 μL were combined. The reaction volume was adjusted to 20 μL with DEPC water before the addition of 1 μL of MMLV reverse transcriptase, 0.5 μL of RNase inhibitor, and 4 μL of reaction buffer. Both the 10-min and 60-min portions of the reverse transcriptions were performed at 25 °C. At 72 °C for 5 min; the process was stopped. Thermo Scientific was used for the procurement of all reverse transcription reagents.

SYBR Master Mix and a Bio-Rad IQ5 real-time PCR detection equipment (Bio-Rad, Hercules, CA, USA) were used to quantify the expression levels of the target mRNAs. First, the qRT-PCR was run at 94 °C for 10 min. Then, 45 cycles of annealing at various temperatures (Table 1) for 20 s and extension at 72 °C for 15 s were performed. Specificity was determined once PCR was completed by analyzing melting curves. Amplification of cDNA at 10 different concentrations was performed using quantitative real-time PCR to create the standard curve. Using the PCR cycle number (CT), we determined the expression level and then normalized the mRNA expression level using the GAPDH gene (as an endogenous control gene). Standard 2−ΔΔCT calculations demonstrated the ability to quantitatively express relationships. There were three separate runs of each response [23].

Table 1 Sequence and annealing specifics of the primers utilized for qRT-PCR amplification
Full size table

Statistical Analysis

All data were analyzed using GraphPad Prism (GraphPad Software; San Diego, California, USA). The Levene test was carried out to ensure the equality of variances. Distinctions between the groups were evaluated using unpaired, two-tailed t-tests or one-way analysis of variance (ANOVA) for normally distributed data. Results are shown as mean ± SD (n = 3). The threshold for significance was set at P < 0.05.

Results

Total Phenols and Flavonoids of the Prepared Postbiotics

The PSC has a high concentration of phenolic (102.46 ± 0.25 mg GAE/g) and flavonoid (19.87 ± 75.32 mg QE/g) components. Indeed, the content and quality of the final obtained PSC solution are affected by growth and extraction circumstances, along with primary stock preparation procedures. Taking into account all of the effective elements, the examined PSC included a large quantity of flavonoid and phenolic substances.

Antioxidant Activity of the Prepared Postbiotics

Since polyphenols contain natural redox capabilities that may neutralize free radicals, these bioactive substances have the ability to strengthen the immune system while also having preventative benefits on a number of degenerative illnesses, including diabetes and cardiovascular disorders [24]. The immediate oxidation of ABTS using K2S2O8 to produce a stable form of ABTS●+ with blue color is the basis for the ABTS assessment. Next, at 750 nm, the radical solution’s degree of decolorization (reduction) is assessed. The PSC had a remarkably strong antioxidant capacity (87.34 ± 0.56%).

Antibacterial Activity of the Prepared Postbiotics

Under In Vitro Condition

The antimicrobial property of the PSC was examined in this study using antimicrobial assays, such as DDA, WDA, MIC, and MBC, against some spoilage and pathogenic bacterial species. The type of yeast varieties affected the PSC’s antibacterial activity, and the microbial byproducts frequently affected Gram-positive bacteria more than Gram-negative bacteria (Tables 2 and 3). For Gram-positive bacteria, the average inhibition zone was 19.61 mm and 23.24 mm in the DDA and WDA tests, respectively, whereas the mean inhibition zones for Gram-negative bacteria were 10.90 mm and 15.16 mm, in the DDA and WDA tests, respectively. Similar findings were obtained from the MIC and MBC assays, and less PSC was required to kill or limit the development of the Gram-positive bacteria than the Gram-negative bacteria (Table 3).

Table 2 The mean inhibition zone diameter (mm) of postbiotics of Saccharomyces cerevisiae PTCC 5269 (PSC), on some pathogenic microorganisms by disc diffusion agar (DDA) method
Full size table
Table 3 The well diffusion agar (WDA), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of the postbiotics of Saccharomyces cerevisiae PTCC 5269 (PSC) on some pathogenic microorganisms
Full size table

Under Food Models

The minimum effective concentration of an antimicrobial substance is the concentration required to prevent the development of a pathogen in a food product. Table 4 shows the estimated MECs of PSC against L. monocytogenes and E. coli in a variety of challenge matrices. It was found that the MECs of PSC were significantly higher in ground beef and milk in the L. monocytogenes inoculation model, respectively, but that the MECs varied widely among the various food models. Similar to in vitro conditions, PSC showed weak antibacterial activity (50 mg/mL in food models) when exposed to an E. coli inoculated model. This highlighted the significance of structural differences between Gram-positive and Gram-negative bacteria on the antimicrobial properties of PSC.

Table 4 Minimal effective concentration (MEC) (mg/mL) of the postbiotics of Saccharomyces cerevisiae PTCC 5269 (PSC) on E. coli and L. monocytogenes in whole milk, ground meat, and culture media
Full size table

Growth-Promoting Properties of the Prepared Postbiotics

After 48 h of incubation, PSC was more effective than glucose at supporting B. bifidum ATCC 35914 growth. These findings suggested that PSC was used by B. bifidum ATCC 35914 for energy metabolism (Fig. 1). After 5 h, L. casei L431 began utilizing the synthesized PSC, and after 30 h, the cell density reached that of the positive control as well as the growth behavior of L. casei L431 to the presence of PSC in the media has been demonstrated in a diauxic growth curve form. However, cell density increased only slightly in the second logarithmic stage of the growth curve (Fig. 1). In regard to L. brevis TD4, it was detected that the PSC can be utilized as a carbon and energy source; however, its growth-promoting effect has significant difference (P < 0.05) from the glucose source in the positive control at the end of the incubation (Fig. 1). By the end of the incubation period, cell density had reached the same level as the positive control when PSC was added to the growth medium of L. acidophilus ATCC 4356. Therefore, it can be stated that in the case of L. acidophilus ATCC 4356, the growth and proliferation processes have demonstrated a strong positive response to the existence of PSC in the milieu, and in a way, it shows the importance of the capability of various strains to use PSC carbon sources, which can possess a significant impact like a glucose source (Fig. 1).

Fig. 1
figure 1

The growth curve of treated probiotic strains with the MRS medium containing postbiotics derived from Saccharomyces cerevisiae (PSC), MRS medium containing 0.5% glucose (positive control), and sugar-free MRS medium (negative control). All the experiments were performed, at least, in triplicate. All the data are presented as mean ± SD (n = 3)

Full size image

α-Glucosidase Inhibition Activities of the Prepared Postbiotics

In this research, PSC was synthesized and utilized to suppress α-glucosidase activity. There was a dose-dependent increase in enzyme inhibition in all replicates within the treatment group (P < 0.05) (Fig. 2A).

Fig. 2
figure 2

The α-glucosidase inhibitory (A) and cholesterol-lowering (B) activities of postbiotics derived from Saccharomyces cerevisiae (PSC) at different concentrations (0.5, 1, and 2 mg/mL). All the data are presented as mean ± SD (n = 3). *P ≤ 0.05 and **P ≤ 0.01 indicate significant and highly significant differences between the concentrations at each PSC at A and PSC at B

Full size image

Cholesterol Removal Capabilities of the Prepared Postbiotics

Cholesterol was reduced by the tested PSC, although the degree to which it reduced cholesterol varied with PSC concentration and the milieu pH value (Fig. 2B). The maximum cholesterol-lowering activity (67.58%) was seen with 2 mg/mL PSC at pH 4.0, whereas the lowest activity (30.27%) was observed with 0.5 mg/mL PSC at pH 7.0 (P < 0.05).

Anticancer Activity of the Prepared Postbiotics

Cytotoxic Assay

The effect of different PSC doses on SW480 cell viability at 24 and 48 h is shown in Fig. 3. The IC50 of PSC was calculated to be 34.27 μg/mL after 24-h treatments and 23.58 μg/mL after 48-h treatments. There was a concentration-dependent, statistically significant (P < 0.05), and a significant decrease in colon cancer cell viability as a result of PSC's cytotoxic action. PSC significantly (P < 0.05) reduced cell viability of SW480 cells after 48 h compared to other circumstances (24-h treatment of PSC and 24–48-h exposure to 5-FU). This means that PSC is cytotoxic to human adenocarcinoma colon cells.

Fig. 3
figure 3

MTT assay of different concentrations of postbiotics derived from Saccharomyces cerevisiae (PSC) and 5-FU (positive control) on SW480 cancer cell lines after 24- and 48-h treatment. All the experiments were performed, at least, in triplicate. All the data are presented as mean ± SD (n = 3)

Full size image

Cell Cycle Analysis

After 48 h of treatment with PSC, it was found that the majority of SW480 cells had entered the sub-G1 (G0/G1) (73.45%) and G2M (15.23%) phase of the cell cycle, indicating that exposure to postbiotic metabolites inhibited the division and growth of cancer cells in the early stages of their cell cycle (Fig. 4).

Fig. 4
figure 4

Effect of postbiotics derived from Saccharomyces cerevisiae (PSC) and treatment on the cell cycle of SW480 cells by flow cytometry. Values are mean ± SD, *P < 0.05 v/s untreated cells

Full size image

Annexin V/PI Binding Assay

The strongest barrier against the proliferation of cancer is apoptosis, the programmed death of cells that regulate homeostasis. The regulation of apoptosis, which might represent among the most significant techniques for avoiding colorectal cancer, may be triggered, according to various studies, by probiotic microorganisms and their generated biological metabolites. We labeled cells with annexin V-FITC/PI to show that PSC induced substantial apoptotic responses in SW480 cells (Fig. 5A). After being exposed to 23.58 μg/mL of PSC for 24 and 48 h, we found that the apoptosis rate was significantly greater in the treatment groups than in the control group (P < 0.001, Fig. 5B).

Fig. 5
figure 5

Apoptotic effects of postbiotics derived from Saccharomyces cerevisiae (PSC), on SW480 cell line. SW480 cells were treated with the concentration of 23.58 μg/mL of PSC for 24 and 48 h. Apoptotic cells were stained with annexin V-FITC/propidium iodide (PI) for flow cytometry assay. A The raw flow cytometry figures. B The apoptosis rates. Data are expressed as mean ± SD (n = 3). a,bMeans for each value without a common letter differ significantly (P < 0.05). c,dMeans for each value without a common letter differ significantly (P < 0.001)

Full size image

qRT-PCR Results

Cells were treated to an IC50 dosage of PSC and 5-FU for varying amounts of time before RNA was extracted, and qRT-PCR was done to determine the effects of the treatments on Akt/NF-κB-induced apoptosis. IkB mRNA expression peaked after 8 h and declined steadily during the next 48 h. At 48 h, there was a significant (P < 0.01) reduction in IkB mRNA expression in both treatment groups (Fig. 6). After 48 h of treatment, PTEN expression was observed to be statistically (P < 0.01) greater in the PSC- and 5-FU-treated groups compared to the untreated group. In addition, PSC was more effective than 5-FU at inducing PTEN mRNA expression (Fig. 6). At 8 h, the mRNA expressions of RelA were significantly (P < 0.01) reduced by both treatments, followed by a time-dependent upregulation (Fig. 6). The mRNA expression of Bcl-XL was higher in the PSC- and 5-FU treated group at 8 h compared to the untreated group due to cell resistance [27]. Both treatments resulted in a significant (P < 0.01) decrease of Bcl-XL at 48 h compared to 8 h, as was to be expected (Fig. 6).

Fig. 6
figure 6

The expression levels of IkB, PTEN, RelA, and Bcl-XL genes in the postbiotics derived from Saccharomyces cerevisiae (PSC)- and 5-FU-treated SW480 cells during 8, 24, and 48 h. The data are expressed as fold changes. Target genes were normalized to GAPDH as housekeeping control gene. All the experiments were performed, at least, in triplicate. All the data are presented as mean ± SD (n = 3). *P ≤ 0.05 and **P ≤ 0.01 indicate significant and highly significant versus the control group, respectively

Full size image

Discussion

Reducing mortality from all causes is a top target for the Sustainable Development Goals in 2023. In this context, a unique therapeutic method is the use of microbial bioactive substances with features like high compatibility and tight contact with the host immune system. Postbiotics with distinct structures and functions are critical mediators between the gut microbiota and human cellular processes/metabolic pathways. Healthcare systems (especially in developing nations) will benefit greatly from a better understanding of the nature of parent microbial cells and elements affecting their metabolic pathways, as this will allow for the development of postbiotics with unique efficiencies [25]. In this context, the most usual yeast probiotic strain (S. cerevisiae) utilized in the fermentation process has been selected as parent microbial cells for generating postbiotic metabolites in this study, and in the following, their potential biological activities have been assessed under in vitro and food circumstances.

In regard to phenolic and flavonoid contents of the prepared postbiotics, it was demonstrated that the matrix of PSC possesses significant levels of these components. It is worth noting that phenolic and flavonoid components have a significant impact on PSC's biological activities [26]. Molska and colleagues (2022) investigated the concentration of phenolic elements and their impact on radical scavenging and anti-inflammatory activities, as well as dietary fiber, in transformed buckwheat sprouts. Buckwheat seeds were modified for this purpose by introducing S. cerevisiae var. boulardii. The altered buckwheat sprouts had a greater total phenol compound content (1526 μg/g d.w.) than the seed (672 μg/g d.w.) and control group (951 μg/g d.w.). The addition of probiotic yeast to the sprouts increased their nutritional content in addition to their anti-inflammatory and antioxidant activities [27]. In addition, Wang and colleagues (2022) used three commercial lactic acid bacteria (L. plantarum 90 (Lp90), Lactobacillus helveticus 76 (Lh76), and L. acidophilus 85 (La85)) to study the impacts on the phenolic compounds, antioxidant potentials, and flavor volatiles of kiwifruit juices made from two cultivars (Actinidia chinensis cv. Hongyang and Actinidia deliciosa cv. Xuxiang). The results indicated that L. helveticus 76 significantly boosted the total flavonoids and phenolics in Hongyang and Xuxiang juices. Moreover, protocatechuic acid and catechin concentrations (two newly generated phytochemicals in fermented kiwifruit juices) were meaningfully connected with antioxidant capabilities based on DPPH, ABTS, and FRAP techniques and considerably enhanced (P < 0.05) [28]. Hence, it can be stated that probiotic yeasts/bacteria and their postbiotic metabolites possess a significant level of phenolic and flavonoid compounds that directly influence their exhibited antioxidant activities.

Byproducts of cellular metabolism include reactive oxygen species (ROS), which may play a role in cell signaling as second messengers for a number of different biological processes. Cell and tissue damage results from an imbalance between ROS formation and protective antioxidants (oxidative stress), which may be exacerbated by environmental stressors including UV-B radiation and xenobiotic substances like mycotoxins. Multiple chronic diseases, such as cardiovascular disease and cancer, have been linked to this condition [29]. Various antioxidants from exogenous origins have been investigated for their protective effects toward oxidative stress due to the crucial role it plays in the development of such clinical conditions. Antioxidants from various sources have been explored [30], but the most essential ones come from vegetables and fruits and include vitamins (E, C), carotenoids, polyphenols (e.g., flavonoids), and minerals (e.g., selenium, zinc). Some probiotics have antioxidant activity, minimizing oxidative damage [31, 32]. Studies have demonstrated that probiotics exert this effect through antioxidant capacity, metal ion sequestration, microbiota modification, and/or modulating the expression of the Nrf2 transcription factor via direct interaction with the host intestine epithelial cells; however, the exact mechanisms involved have not yet been established [33]. It is important to remember that these processes rely on microorganisms to function properly. In contrast, recent findings suggest that Lacticaseibacillus casei CRL 431's intracellular content may also mitigate the oxidative stress caused by aflatoxin B1 (AFB1) in rats [34] and acrylamide in human erythrocytes [32]. In this regard, our results exhibited that the prepared postbiotics possess significant antioxidant activity under in vitro conditions, and flavonoids, phenols, and the primary chemical components of PSC all contribute to this remarkably strong antioxidant activity [48]. Several studies have reported the PSC's antioxidant properties [5, 35,36,37]. As a result, under in vitro or/and in vivo circumstances, the PSC with their safety profile might have been employed to postpone or block the free radical reactions and related oxidative impairments.

In regard to the antibacterial activity of the PSC, L. monocytogenes and E. coli earned the maximum and lowest inhibition zones, respectively, based on the results of the DDA and WDA tests. Moreover, the WDA method's inhibitory zone was much larger than the DDA assay's. This is largely because the former approach directly contacts the PSC with the bacterial species, while the latter method does not [38,39,40]. A synergistic impact between the PSC and antibiotics may also be worth mentioning; this combination had a typically stronger antimicrobial property than the PSC alone (Table 2). These outcomes have been attributed to variations in the bacterial cell wall structure based on the results of the MIC and MBC assessments. The mucopeptide layer is found to be thicker in the first group than in the second. Moreover, the primary components of the walls of Gram-negative bacterial species include lipoproteins and lipopolysaccharides, which contribute to their greater resistance to antimicrobial substances [41]. The PSC's capacity to prevent the production of vital enzymes produced by bacteria and/or harm the bacterium's cell wall is thought to be the reason for its antibacterial action [42, 43]. In this context, 42 yeast strains were recovered from Bollo batter by Pereira and colleagues (2021). Four yeast isolates (DABRP1, DABRP2, DABRP5, and DABRP12) were obtained in this study from the first screening of the isolates with probiotic capabilities and were identified as S. cerevisiae using D1D2-LSU-rDNA sequencing. The pathogenicity of the samples was assessed using the in vitro hemolysis assessment and measurement of gelatinase and DNase activities in order to assess the safety of the samples and their postbiotic metabolites. All of the isolates were regarded as possibly safe since none of them showed hemolysis or generated DNase or gelatinase. The strongest isolate of S. cerevisiae, DABRP5, demonstrated antimicrobial properties versus the investigated pathogens (S. enterica serovar Typhi, P. aeruginosa, and E. coli) [44].

The well-known foodborne pathogen L. monocytogenes can endure a wide variety of environmental circumstances, including temperature (1–45 °C), pH (4.3–9.8), and high salt levels [45]. One of the most serious foodborne diseases is listeriosis, which is induced by the bacterium L. monocytogenes. Pregnant women, newborns, and people with impaired immune systems are particularly vulnerable to listeriosis [46]. According to a report from the Centers for Disease Control and Prevention (CDC), listeriosis has one of the highest hospitalization rates of any disease. Each year, around 1600 individuals get listeriosis, and 260 people pass away from it [47]. According to our outcomes, postbiotics of S. cerevisiae possess a significant growth inhibitory effect on L. monocytogenes that in turn enable this probiotic yeast and their derived postbiotic metabolites to develop in food biotechnology practices, especially in meat and its microbial/plant analogues, to establish microbial safety. Li and colleagues (2021) found that marinating chicken breast strips in a marinade containing S. boulardii, a leucocin C bacteriocin producer, inhibited the proliferation of L. monocytogenes in raw chicken meat. The marinade preparation was proven to sustain its antibacterial effectiveness for 38 days in this experiment. After marinating chicken breast strips contaminated with L. monocytogenes in the antibacterial marinade preparation for an overnight period, the amount of L. monocytogenes that were eliminated was determined. Leucocin C marinating decreased the cell viability of L. monocytogenes by approximately 1.6 logs from (2.2 ± 0.6) × 107 CFU/g on day 24 and by 2.2 log from (1.8 ± 0.3) × 105 CFU/g on day 38 [48]. Thus, it can be concluded that marinade preparations containing S. cerevisiae and its effective postbiotic metabolites may be thought of as a promising tool for lowering the incidence of Listeria in chicken breast strips.

In regard to investigated food models, an isolate of Leuconostoc was shown to be effective against E. coli in ground beef in a case of CFS [49]. The organic acids produced by Leuconostoc were related by the authors to the bacterium’s antibiotic properties. It has been found that a 1.0% concentration of CFSs from L. acidophilus, B. bifidum, and L. plantarum is antibacterial against E. coli [50]. They determined that the organic acids found in CFS may be responsible for this behavior. Organic acids and potentially other undiscovered bacteriocin-like components in L. salivarius's CFS provide the necessary antibacterial activity against certain gram-positive bacteria (L. monocytogenes). For reasons including commodity complexity, solubility and adsorption of CFS to food matrix, and communications of CFS elements with food components [51], it is evident from this study that the effectiveness of CFS in culture media is significantly higher than in food matrices. Hartmann and colleagues (2011) studied the effects of CFS of several Lactobacillus spp. on the growth and survival of L. monocytogenes in different media and reported a greater MEC of the CFS towards L. monocytogenes in ground beef and milk compared to culture broth [14].

Yeasts have long played an important role in the production of a variety of fermented foods, and due to their high potential in the synthesis of postbiotic compounds with a safe profile and impressive biological activities (e.g., antimicrobial activity), they may be useful in the development of functional, nonfermented foods as well.

In regard to the growth-promoting properties of PSC, all probiotic strains evaluated in this research showed increased growth, proving the study’s hypothesis that PSC has a growth-promoting impact. The probiotic strains stimulated growth throughout the fermentation period of 48 h, but in the growth media, no fast growth was detected in the condition of the PSC compared to glucose. This was likely because the PSC's complex components took longer to hydrolyze and transfer during growth. Moreover, it is noteworthy that the growth-promoting effect of PSC is largely strain-dependent and according to this, various postbiotic preparations can be chiefly formulated for the target strain (particularly those utilized as starter culture in fermented food products).

In regard to the α-glucosidase inhibitory activity of PSC, the results demonstrated that this effect is largely concentration-dependent, and various investigated concentrations of PSC (0.5, 1.0, and 2.0 mg/mL) possess significant increments in the suppression of the enzymatic activity of α-glucosidase. Although the glucosidase inhibitory effect of the L. plantarum BR2 strain’s high molecular weight postbiotics [52] was higher (67%) than our findings, the PSC was still significantly effective.

According to the results of the cholesterol removal capabilities of PSC, it can be stated that this effect is mainly concentration- and pH-dependent. It is noteworthy that the efficiency of PSC had established a direct and inverse relationship with the concentration and pH value, respectively. The mentioned consequences can be caused by the effect of different pH values on the charge density of polysaccharide compounds and the structural and functional changes of protein and peptide elements in PSC solution. Different postbiotics have been shown to have varying degrees of success in lowering cholesterol, from 31 to 48.81% [52, 53]. Our results showed that PSC's cholesterol-lowering activities were consistent with those found in the literature, whereas the 2-mg/mL concentration of PSC at pH 4.0 showed much higher levels of cholesterol-lowering capabilities than those found in the published literature. These results imply that the PSC's ability to remove cholesterol may vary with factors such as size, charge, structure, and the presence of functional groups under the investigated circumstances.

Colon cancer is the leading cause of cancer-related mortality globally and is one of the most frequent and aggressive malignancies of the digestive system. Due to its high incidence in the West, Iran ranks fourth for females and third for males. Scientific data reveal that S. cerevisiae and their biometabolites have significant growth and proliferation-inhibiting impact on the breast, bladder, stomach, and colon cancer cells [54]. S. cerevisiae has considerable antiproliferative and antioxidant capabilities, and it also modulates immune system responses and alters the composition of the gut microbiota. We showed that S. cerevisiae postbiotic metabolites exhibited a significant cytotoxic effect against cancer cells under study, further indicating the presence of biological molecules with antitumor function. These findings are consistent with those of previous studies showing that postbiotics of B. adolescentis SPM0212 inhibited the growth of HT-29, SW-480, and Caco-2 colon cancer cell lines to a greater extent than live/heat-inactivated. The findings demonstrated that the cytotoxic effects were not only time- and dose-dependent but also selective, with the strongest responses occurring after 48 h and at a concentration of 23.58 μg/mL.

Complex protein and carbohydrate molecules compose S. cerevisiae's postbiotic metabolites. Cancer cell growth may be slowed or inhibited by taking yeast CFS, which contains beta-glucan and mannan [55]. Insoluble glucans and their derivatives are responsible for yeast cell wall extract's antiproliferative actions on cancer cells [56]. Postbiotics have been found to exhibit anti-inflammatory properties in a number of in vitro experiments employing THP-1 monocytes, Caco-2 cells, and HT-29 colonocytes in addition to their cancer-preventative effects [57]. The PSC was observed to decrease SW480 cell viability. We were prompted to finish our study by an interest in how PSC affects cell death and related gene expression in the context of these cancer cells. The strongest protection against cancer’s spread is apoptosis, a programmed cell death process that maintains homeostasis. The present evidence suggests that yeast-based probiotics may be one of the most significant treatments for reducing colorectal cancer by stimulating the regulation of apoptosis. We used annexin V-FITC/PI staining to show that PSC induces apoptosis in SW480 cells (Fig. 5A). After 24 and 48 h of exposure to 23.58 μg/mL of PSC, the apoptosis rate in the treatment groups was significantly greater than in the control group (P < 0.001, Fig. 5B).

The Akt/NF-κB, MAPK (mitogen-activated protein kinase), pro-apoptotic, and antiapoptotic signaling pathways are often modified by probiotics. In this work, we looked at the signaling pathways involved in the antiproliferative and pro-apoptotic actions of PSC and 5-FU on a colorectal cancer cell line. Figure 7 shows that NF-κB and Akt are abnormally active in cancer cells [58]. Host cell surface receptors may be used to activate the positive effects of the probiotic bacteria or its postbiotics on host signaling pathways. Decreased levels of p-Akt induce apoptotic responses [59] when postbiotic metabolites or/and 5-FU are present. Caspase activation (caspase 3 and 9), the mitochondrial apoptosis pathway (Bcl-XL and PTEN), and the nuclear factor kappa B (NF-κB) pathway (RelA and IkB) were all profoundly impacted by the lowering of p-Akt [60].

Fig. 7
figure 7

Schematic illustration of the proposed signaling pathway involved in the anticancer activity of the postbiotics derived from Saccharomyces cerevisiae (PSC) on colon cancer cells. In cancer cells, PI3K-Akt and NF-κB-AKT pathways are overactivated. In response to extracellular stimuli (e.g., chemokines, growth factors, presence of insulin), PI3K is activated by tyrosine kinase receptors or G-protein-coupled receptors and it phosphorylates PIP2 to generate PIP3 which in turn phosphorylates and activates Akt. The main function of PTEN consists in the regulation of this pathway. PTEN is a lipid phosphatase that antagonizes the action of PI3K by dephosphorylating PIP3 to generate PIP2 (thus blocking the PI3K signaling cascade). On the other hand, protein-based postbiotic metabolites can be recognized by receptors on the host cell surface and triggers its beneficial effects on the host signaling pathways. The PSC induces apoptosis through the proposed Akt-1 modulating effect on the NF-κB signaling pathway. p-Akt1 reduction can downregulate RelA mRNA through an increased expression of IkB. Reduction of p-Akt-1 and NF-κB activates apoptosis cascade

Full size image

In addition, the mRNA expression level of RelA and its inhibitor gene (IkB) was evaluated by qRT-PCR to clarify the mechanism of NF-κB-induced apoptosis, and it was discovered that after 8 h of treatment with PSC and 5-FU, the RelA level was significantly reduced as compared with the untreated control cells, as a result of lkB upregulation. It has been shown that IkB mRNA has a very short half-life (~ 30 min) [61]. After peaking at 8 h, IkB levels in this research steadily declined over the subsequent 48 h. At 8 h, the RelA level (as a functioning subunit of NF-κB) was low, which is consistent with the beginning of the apoptosis cascade given the presence of IkB and its role as an inhibitor of RelA. The paradoxical impact of NF-κB on cancer cell proliferation may be shown by the elevated level of RelA 24 and 48 h after therapy. A thorough analysis of the paradoxical impact was published in 2002 by Karin and colleagues [62]. They spoke about how decreasing NF-κB activity initiates apoptosis and how increasing NF-κB activity might encourage the body's immune system to prevent cancer. Further research is required to understand the complex role of NF-κB, which may be associated with cancer inhibition via stimulation of the immune system, as suggested by the elevated levels of NF-κB in our investigation throughout 24 and 48 h.

The expression levels of PTEN (a pro-apoptotic gene) and Bcl-XL (an antiapoptotic gene) were also measured to see whether PSC therapy may induce apoptosis through the intrinsic route (mitochondrial pathway). qRT-PCR results demonstrated an influence of PSC on the mitochondrial apoptotic pathway, with increased PTEN mRNA and decreased Bcl-XL mRNA [63]. Interestingly, the PSC-treated cells' increase in PTEN expression level was significantly greater than that seen in the 5-FU-treated cells. Previous research has documented an increased Bcl-XL mRNA level as cellular resistance to apoptosis [64]; however, this was shown to be incompatible with apoptosis in the first 8 h. In conclusion, our results suggest that postbiotic metabolites from S. cerevisiae PTCC 5269 may induce apoptosis in a colorectal cancer cell line through the Akt/NF-κB signaling pathway.

Conclusion

Biostrategies comprised of probiotics-derived bioactive substances have recently been recognized as a potential tool in preventing and complementary treatment of a broad variety of acute/chronic disorders, according to the existing data gained from preclinical and clinical investigations. Because they are derived from safe probiotics, postbiotics are structurally and functionally compatible with host cells and contribute to a number of cellular processes essential to restoring homeostasis. According to the results of this study, the postbiotic metabolites derived from Saccharomyces cerevisiae (PTCC 5269) possess multiple health-promoting effects due to their significant antioxidant capacity (87.34 ± 0.56%), antibacterial action toward both of the Gram-negative (S. typhi, E. coli) and Gram-positive (L. monocytogenes, S. mutans) bacteria under in vitro and food models (whole milk and ground meat), probiotics' growth-promoting activity, and cholesterol-lowering and α-glucosidase enzyme-inhibiting properties, as well as their capability to cause significant cytotoxicity effect (IC50 values of 23.58 μg/mL for 48 h), arrest the cell cycle in cancer cells (suppressed the initial G0/G1 phase), and induce apoptotic responses, which this effect mediated by molecular mechanisms with a positive influence on the expression levels of genes involved in the Akt/NF-κB signaling pathway–induced apoptosis process. Therefore, it can be accomplished to develop certain mixtures of postbiotics with significant efficiency by learning more about lactic acid bacteria, using developed and optimized techniques in extraction, verification, characterization, implementing metabolomic and proteomic studies. This will substantially enhance the efficacy of health systems toward a broad spectrum of acute/chronic diseases.