Friday, February 23, 2024

Assessment of mycotoxin sequestration efficacy in Saccharomyces cerevisiae by-products cultured in wheat bran and whey protein medium – Scientific Reports

Yeast cell cultivation

The culture medium composition plays a pivotal role in yeast growth and expression. Commercial yeast growth mediums typically consist of glucose as a carbon source, peptone, and yeast extract as nitrogen sources. Noteworthy, these cultivation mediums are expensive on the industrial scale12. In contrast, lignocellulosic compounds offer an attractive alternative as they are natural, cost-effective, and renewable carbon sources13. These compounds have three principal units in their structures: cellulose, hemicellulose, and lignin with cellulose being the primary component interconnected to hemicellulose units by lignin, these compounds are not readily accessible to microorganisms. Consequently, pre-purification stages are required to make monosaccharide of the cellulose and hemicellulose accessible to the microorganisms in the medium14,16. This approach can be Facilitate their bioavailability to the microorganisms and making it a cost-effective approach. The initial investment in the pre-treatment processes is mitigated by the low cost of the raw materials and the high nutrient yield, paving the way for widespread application on a large scale. In this study, wheat bran as a lignocellulosic source was purified twice through acid hydrolysis and after filtration that supernatant utilized as the carbon source in two culture mediums, Wb/Ye and Wb/Wp. Remarkably, all three formulated mediums, YPG, Wb/Ye, and Wb/Wp, demonstrated an increase in biomass. The comparison of growth process in three culture medias over 72 h of incubation was monitored by measuring absorbance at OD 600 nm at intervals (0, 24, 48, and 72 h), as depicted in Fig. 1. The yeast biomass growth in all three mediums exhibited an exponential pattern. Interestingly, the Wb/Wp culture medium, with whey protein as the nitrogen source, showed the highest absorbance after 72 h of incubation, indicating a higher yeast cell growth rate in this medium. Overall, the results suggest that the wheat bran pretreatment process is highly effective and makes the glucose in the cellulose and hemicellulose structures available to the growing cells.

Figure 1

Absorbance analysis of three distinct formulated culture media at 600 nm wavelength post 72-h incubation period.

Characterization of disrupted yeast cells

Microscope photos

Figure 2 shows microscopic visualizations that confirm the efficiency of the physical method (Ph) in homogenizing yeast cells, which results in thorough disintegration of the cell wall. This ensures that the internal cell components and organelles are effectively released. Figure 2a,b show clusters of YCW polymers. These clusters highlight the dominant presence of hydrophobic β-glucans, as shown by the studies of Bertsch et al.17 and further confirmed by Bzducha-Wróbel et al.18. It is therefore obvious that these methods are very suitable for producing fragmented yeast cells and at the same time facilitating the purification of cytosolic elements. In contrast, Fig. 2c shows cells that remain largely intact. This indicates that only a portion of the cells were digested during the hydrolysis process. The lower efficiency of this method and its implications are discussed in more detail in the following sections.

Figure 2
figure 2

Comparative analysis of yeast cell disruption through various approaches: (a) Physical lysis (Ph); (b) combined hydrolysis-physical (H-Ph); (c) hydrolysis (H).

Yeast cell disruption analysis

The effectiveness of the different disruption methods in releasing intracellular components of yeast cells was evaluated by two different analyzes. In the first analysis, culturing yeast suspensions on agar plates showed significant differences in cell viability after disruption. In the samples where physical lysis was performed, there were almost no surviving cells 48 h after incubation (Fig. 3a,b). In contrast, suspensions obtained by hydrolytic disruption still had living cells after 48 h incubation (Fig. 3c). During analysis, absorbance was measured at OD 260 and 280 nm to monitor the extent of cell disruption and the resulting release of cell contents such as nucleic acids, proteins, and polysaccharides (Fig. 4). The data showed that after 48 h lysis period, samples processed by the Ph method using glass beads had higher absorbance than samples processed just by the H method. At this stage of analysis, the yield of solid residues excreted after disruption was considered an important marker for the release of intracellular space. Synthesis of the observations revealed that the hybrid hydrolytic-physical method is a superior strategy for cell disruption because it allows optimal recovery of intracellular contents compared to the other individual methods. This seems to agree with the findings of Běehalová and Beran19 and Takalloo et al.20, who indicated that enzymatic hydrolysis mainly attacks the cell wall and facilitates the conversion of proteins into smaller peptides that can leave the cell. Therefore, the combination of pH lysis method and H lysis method may increase the efficiency of the process and allow a higher yield in the extraction of intracellular constituents.

Figure 3
figure 3

Comparative growth of yeast cells on YPG media after 24-h incubation. Plate (a) shows growth after physical lysis (Ph), plate (b) after combined hydrolysis-physical lysis (H-Ph) and plate (c) after hydrolysis (H).

Figure 4
figure 4

Monitoring cell damage and cytoplasmic component leakage in the supernatant: (a) absorbance measured at 260 nm; (b) absorbance measured at 280 nm.

Effect of analyzed disruption methods on the content of total nitrogen and saccharides in the cell wall preparation

Protein release

The release of proteins is a critical aspect of yeast cell disruption procedures21. From the Kjeldahl analysis data (see Table 3); the protein content of the cell wall sample obtained by Ph disruption was about 37.49%. In contrast, the H disruption yielded a significantly higher protein content of about 41.65% in the cell wall. Interestingly, both values are higher than the protein content of the yeast cell wall samples obtained by H-Ph disruption, which was about 28.54%. In Ph disruption methods, cell fission leads to the best fragmentation of cells, which helps to effectively release proteins and other intracellular compounds. However, there is a possibility of significant contamination by proteins derived from other cytoplasmic components such as mitochondria and ribosomes22.

Table 3 Results of the Kjeldahl analysis of cell walls obtained using different disruption methods.

However, in the duration of the disruption method using enzyme (H), the cell wall is only porous, and the hydrolytic activity of the enzyme is gradually steered toward the proteins on the cell surface20. Also, this method of disruption is a time-consumer and costly process.

The lowest protein content observed in the cell wall with the H-Ph method compared to the Ph and H methods alone could be due to the initial enzymatic pretreatment phase. This phase could mitigate the introduction of unwanted protein impurities. As noted by Iten and Matile23, this enzymatic phase facilitates the partial hydrolysis of extracellular proteins on the cell surface, thereby possibly protein removing gradually occurs from cytoplasm under the influence of the hydrolytic activity of the enzyme24. Hence, it can be claimed that partial physical lysis after hydrolytic pretreatment can lead to the completion of cell disruption process, and fragmentation of the cell, as well as the exit of the cell contents and their entry into the extract with the lowest protein contamination of various other intracellular organelles.

Saccharide content

Figure 5 shows that the cell wall preparations obtained by the H-Ph disruption method contain an average 56% total saccharides. This figure is consistent with the observed decrease in protein content in the YCW compared to the whole cell. In contrast, the Ph disruption method yielded an average total saccharide content of about 49%. It is worth noting that the use of glass beads with a size between 0.5 and 1 mm in the physical disruption method could lead to similar results in terms of protein and total saccharide content18. The H disruption method gave an average total saccharide content of about 41%. During the autolytic or hydrolytic process, the polysaccharides of YCW are gradually degraded. This process alleviates the osmotic pressure in the cell cytoplasm and keeps the cellular polysaccharides largely intact, albeit at the expense of a partial loss of structural polymers in the cell wall. Therefore, the extraction of β-glucans from these cell wall fractions proves to be suboptimal25. Considering the higher total saccharide content and lower protein concentration in the cell wall samples obtained by the H-Ph disruption method (compared to the other methods), this cell wall fraction was preferred for β-glucan extraction. This approach appears to ensure comprehensive YCW fragmentation while minimizing protein contamination by other cell organelles.

Figure 5
figure 5

Analysis of sugar content in yeast cell walls derived from various disruption techniques. The lowercase letters (ac) indicate significant differences between the treatments. Values are means ± standard error.

FTIR spectroscopy of the optimal cell wall and extracted β-glucan

FTIR analysis was carried out to explore the architecture of the optimal YCW after cell lysis (H-Ph disruption method), and the β-glucan derived from it. Additionally, this analysis allowed for the comparison of structural alterations to the β-glucan post-extraction from the cell wall. The recorded FTIR spectra of two samples are shown in Fig. 6, where “a” represents YCW from the optimal sample after cell lysis and “b” represents extracted β-glucan from the optimal YCW sample. The structural changes due to the conversion of the cell wall to β-glucan can be clearly seen in the graph of “b”. The bands in the 750–950 cm−1 and 950–1200 cm−1 ranges (C–C, C–O, and C–O–C stretching bands) are characteristic of polysaccharides, which can be seen in the graph of samples “a” and “b”, respectively. These regions represent the anomeric and sugar regions, respectively20,26. The bands in the range 1450–1650 cm−1 are associated with the stretching vibrations of C=O, C–N and N–H. According to Novák et al.27, these bands indicate amide bonds and aromatic rings. The 1650 cm−1 band in sample “b” (after extraction of β-glucan from the wall) is smaller than that of sample “a”, which is due to a decrease in protein content and an increase in the purity of saccharides after extraction of β-glucans. In addition, the bands in the 2850–2921 cm−1 ranges are indicators of aliphatic C–H groups in pyranoid rings (C–H stretching bands) seen individually in both samples. These observations are consistent with those of Hromádková et al.28. Also, the visible bands in the region of 3300 cm−1 in sample “a” and 3750 cm−1 in sample “b” are indicative of OH stretching vibrations4.

Figure 6
figure 6

Infrared (IR) spectra of two analytical samples: (a) post cell disruption yeast cell wall preparation; (b) β-glucan extracted from yeast cell wall.

Antibacterial assay

The antibacterial activity of the optimized cell wall, β-glucan, and their combination against the growth of E. coli and S. aureus is shown in Fig. 7. The combination of extracted β-glucan and YCW had the most pronounced effect on the bacteria studied. This can be attributed to the low molecular weight of the extracted β-glucan, which can easily penetrate the bacteria and disrupt their metabolism. The minimum concentration of the combination of β-glucan and YCW that inhibited the growth of E. coli and S. aureus was measured to be 250 µg/mL and 500 µg/mL, respectively (Table 4). All results showed that the studied yeast derivatives exhibited stronger antibacterial activity against Gram-negative bacteria than against Gram-positive ones. Our results agree with previous studies29. The hardness and resistance caused by the thick peptidoglycan of the Gram-positive bacteria may be the reason26,30.

Figure 7
figure 7

Evaluation of the antibacterial potency of the yeast derivatives studied: remarkable diameter of the inhibition zones formed by the mixture of β-glucan and cell wall. The lowercase letters (a, b, c) indicate significant differences between the treatments for S. aureus, while the uppercase letters (A, B, C) indicate significant differences between the treatments for E. coli. Values are means ± standard error.

Table 4 MIC and MBC values of yeast derivatives investigated against S. aureus and E. coli.

Adsorbing capacity of studied mycotoxin’s binders

The binding efficiency of mycotoxins to YCW adsorbents is significantly influenced by their physical attributes. The YCW is an intricate lattice primarily formed by polysaccharides. It is coated on the outside with richly glycosylated mannoproteins that play a key role in cell recognition and enhance the mycotoxin adsorption mechanism. Additionally, the thickness and composition of the YCW are vital determinants, with increased thickness and higher content correlating with enhanced detoxification capabilities against mycotoxins. Utilization of yeasts and their derivatives has been recognized as a preventive strategy against the detrimental effects of toxins on both human and animal health31,32. Within the scope of this discourse, the adsorption mechanism of the YCW is underscored by the critical interaction between β-(1,3)-d-glucans and mycotoxins. The principal interactions facilitating mycotoxin adsorption are Van der Waals forces, which manifest between the aromatic cycles of the mycotoxins and the β-d-glucopyranose rings within the YCW. This is supplemented by hydrogen bonds forming between the hydroxyl, ketone, and lactone functional groups of the mycotoxins and the hydroxyl constituents of the glucose molecules found in the β-D-glucans of the YCW33. Moreover, the detoxification potential is modulated by the physical parameters of the YCW, where an increment in wall thickness and biochemical constituency is directly proportional to an increase in mycotoxin detoxification efficacy34. As Table 5 shows, when AFB1 was absorbed by two developed binders, the mixture of β-glucan and YCW was assigned a higher absorption capacity (P < 0.05). Apparently, the hydroxyl, ketone, phenyl, and lactone groups in β-glucans are involved in the formation of hydrogen and van der Waals bonds between AFB1 and glucan, and the increased groups in a mixture of β-glucan and YCW could explain the observed results11,35. The YCW used as a binder for AFB1 showed the lowest binding rate and did not exceed 50%. Despite this observation, a significant difference was observed between the absorption properties of YCW and the combination of β-glucan and YCW in AFB1 absorption (P < 0.05). In agreement with our results on the binding of AFB1 to the YCW and its components, Hojati et al.35. reported that YCW products have an average efficiency in AFB1 absorption in vitro. According to the results of OTA absorption by the tested binders, it was also shown that the mixture of β-glucan with the cell wall had a higher absorption capacity for this toxin (P < 0.05). Bornet and Tessedre36 showed that chitin and β-glucan mixtures, as well as wall hydrolysis, have a relatively high ability (64–74%) to remove OTA from OTA-contaminated wine. In addition, Piotrowska and Masek4 showed that YCW components, especially glucan extracted from them, are responsible for OTA adsorption at nearly neutral pH.

Table 5 The mycotoxins adsorption ability of the yeast-derived products.

As the observed results on ZEN adsorption show, the absorption of this mycotoxin by two types of binders was higher than that of other mycotoxins. Among the components of the YCW, β-glucans are the main molecules responsible for the absorption of ZEN, and the absorption capacity of YCW strongly depends on its β-glucan content5. Generally, the properties of adsorbed toxins such as polarity, solubility and charge distribution play an important role in the binding process and are manifested in various binding mechanisms such as non-covalent interactions, hydrogen bonding, etc37,38. ZEN is a macrocyclic molecule and much more hydrophobic than aflatoxin. The presence of diphenolic groups made it a weak acid. Consequently, the adsorbent must also have polarity in order for the bond between ZEN and the binder to be properly established2. A structural change in the composition of the β-glucan when these two molecules come into contact with each other enhances the binding process5.

Of the two types of our binders, the highest absorption rate for ZEN is associated with the mixture of β-glucan and YCW, and in general this mixture can be chosen as the optimal binder among the other binders used. The stronger binding of these binders can be justified by the report of Yiannikouris et al.5. According to this report, the first bound molecules of ZEN open the helix structure of β-glucans and thus bind more molecules of the toxin. In general, various functional groups in β-glucans, including C=O, O–H, C–O–C, C–C, etc., have high absorption capacity for various mycotoxins. On the other hand, the combination of pure β-glucan with YCW, which also contains a few polysaccharides such as mannan-oligosaccharides, β-glucans, and chitin, increases the absorption of mycotoxins. This is because the combination of these two binders brings a greater diversity of functional groups that cause the formation of different bonds with mycotoxins. Therefore, the combination of the YCW with β-glucan is likely to increase the amount of glucan accessible to the toxins and consequently cause higher binding.

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