Current study investigated the ability of three strains of treated (autoclaved or acid) saccharomyces cerevisiae to bind high dosage (18 μg/ml) of aflatoxin B1 in simulated ruminant model and determined the fermentability of these treated yeast strains by gas production technique. Aflatoxin B1 used in current study naturally produced with Aspergillus parasiticus. Three-step in vitro procedure were used for simulating ruminant model and unbound aflatoxin B1 quantified by ELISA method. Data analysis was carried out in a 3 - 2 factorial arrangement of a completely randomized design. Treated strains used in current study showed aflatoxin B1 binding capacity. Highest binding property related to strain A (NRRL Y-567) with 56.3% of bounded aflatoxin B1 (P ≤ 0.01). Strain B (NRRL Y-53) with 28.4% and strain C (NCYC 694) with 31.7% of bounded aflatoxin B1 showed lower binding property compared to strain A (P ≤ 0.01). Acid treatment with 47.4% of bounded aflatoxin B1 showed nearly two times more binding property compared to heat treatment with 26.9% of bounded aflatoxin B1 (P ≤ 0.01). There were no interaction between yeast strains and treatments, significantly. Gas production test results showed that strain A significantly produced lower gas (ml) compared to other strains on 2 h of incubation period (P ≤ 0.01). On this time, strain C showed numerically highest gas production and there were no significant differences between strains B and C. On 4, 6, 8 and 12 h of incubation time, lowest gas production related to strain A, highest gas production related to strain C (P ≤ 0.05) and strain B shows median gas production result. Gas production results during 24 h to 96 h of incubation period showed no significant differences between yeast strains. Kind of treatments had great effect on gas production of yeast strains, as lowest gas production (ml) in all times of incubation period related to heat treatment, significantly (P ≤ 0.05). No significant interactions were observed between time and treatments over 96 hour of incubation period. Current results suggest that acid treated of yeast strain A can be candidate for adding to the ruminant ration as a mycotoxin binder.
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Key words: Aflatoxin B1, Saccharomyces cerevisiae, Gas production test
Aspergillus flavus and A. parasiticus are the main fungal strains that grow under high humid and temperature condition (26, 28). These fungi produce aflatoxin B1 (AFB1) as a most toxic and carcinogenic metabolite of aflatoxins (10, 22). Aflatoxin B1 is quickly absorbed from digestive system of ruminant by passive mechanism (17) and appears as the aflatoxin M1 in blood and milk (14, 16, 17). Presence of aflatoxin M1 as a carcinogenic metabolite of aflatoxin B1 in edible product of animal (especially milk) is associated with human health (17).
Inclusion of organic mycotoxin binder based on yeast cell wall extract to animal rations is a routine method for reducing aflatoxins absorption from gastrointestinal tract. This practical method minimizes the risk of mycotoxins with any adverse effects on nutrient bioavailability of feed or adverse effects on environment (5, 17, 28). Saccharomyces cerevisiae cell wall consist of main chain of β-1,3 glucans branched with β-1,6 chains that linked to glycosylated mannoproteins (22). Many binding sites supplied with these glucans can trap mycotoxins easily (22).
Ruminal fermentability of these organic compounds is a key factor for assessing the usefulness of these mycotoxin binders. In vitro gas production test is an appropriate technique for estimating feeds fermentation in rumen. In this method, an organic compound is incubated with buffered rumen liqour and gas production measurement used as an indirect measure of fermentation kinetics (19). These kinetics present useful information about each organic matter for feed formulation (24) and degradation (11, 19).
Regarding that effective mycotoxin adsorbent should be tightly trapping the mycotoxin in contaminated feed without disassociating in the gastrointestinal tract of the animal (5),the objectives of current study were to screen three strains of treated yeasts for aflatoxin B1 binding ability in simulated ruminant model and assessing their in vitro rumen fermentability using gas production technique.
3. Materials and methods
3.1. Saccharomyces cerevisiae strains and culture conditions
Three strain of saccharomyces cerevisiae (strain A: NRRL Y-567; strain B: NRRL Y-53, strain C: NCYC 694)were obtained from Persian Type Culture Collection (PTCC) as lyophilized powder. Strains grown inYPD broth tube (24 h at 30 °C) were used as inoculums. 250 mlErlenmeyer flasks containing 100 ml of YPD [1% (w/v) yeast extract, 2% (w/v) bacteriological peptone and 2% (w/v) glucose] medium were inoculated at 3% (v/v) with inoculums and incubated at 30 °C and 200 rpm till reaching 2-107 cells ml−1 for cell treatments(22, 29).
3.2. Treatment of cells
Always on Time
Marked to Standard
Harvested cells were centrifuged (5000 - g, 10 min, 5 °C) and washed twice with phosphate buffered saline (PBS, 4 ml, pH 6.0, 0.01 M) (9, 22). The flasks of each strain were divided into two groups. The culture of each flask (10 7 CFU/ml) of first group was autoclaved (120 °C for 20 min), while the second group was acid treated (incubated in 4 ml of 2 M HCl for 1 h) with mild shaking. Treated yeasts were then washed twice (4 ml of PBS), all yeast samples were centrifuged, and the supernatant was removed prior to aflatoxin B1 binding assays (9).
3.3. Preparation of Aflatoxin B1 working solution
Aflatoxinwas produced via fermentation of rice as described by Shotwellet al. (1966). Briefly, Inoculum was prepared by inoculating tubes ofCzapek agar with spores of Aspergillus parasiticus PTCC 5286 were obtained from Persian Type Culture Collection related to IROST.Inoculated slants were incubated for 21 days at 28 °C. During this period, heavy crop of green conidia become visible that removed by adding 3.0 ml of 0.005% Triton X-100 per slant. After scraping the spores and making a uniform suspension, 0.5 ml of this suspension was used to inoculate each 50 g of white rice containing in Erlenmeyer flasks. Twenty five milliliters of tap water was added to each flask; the rice was autoclaved (120 °C for 20 min), cooled and shaken to prevent kernel adhesion. Incubated flasks placed on an incubator shaker (200 rpm and 28°C). Two milliliters of sterile water was added to each flask at 24 and 45 h. Incubation was continued for 6 d, and for preventing mycelium formation during this period, rice grains were kept separated (23).
At the end of fermentation process, aflatoxins were extracted by soaking rice in chloroform during overnight (at room temperature) for three times. Each extract was filtered through cheesecloth, pooled, concentrated by rotary evaporator to ~150 ml, and dried by anhydrous sodium sulfate (50 g). Sodium sulfate was removed by filtration, and the clarified filtrated chloroform was concentrated by rotary evaporator (23). Finally, for preparing aflatoxin working solution, chloroform evaporated in vacuo completely and then suspended in methanol. Differential analysis for aflatoxin B1, B2, G1, G2 was done by HPLC method in a professional mycotoxins detection lab. That result showed that from the total aflatoxins content in final solution, 84.64 % was aflatoxin B1 and 15.36% was aflatoxin G1. Aflatoxin B2 and aflatoxin G2 were not detected.
3.4. Aflatoxin B1 binding assay
A solution of 18 μg/ml aflatoxin B1 was prepared in PBS (pH 6.5) and the methanol was evaporated by heating in a water bath (80°C, 15 min). Yeast cell pellets were suspended in 1.5 ml of the working solution of aflatoxin B1 (18 μg/ml) and incubated in simulated ruminant gastrointestinal situation based on Calsamiglia and Stern, (1995); and Gargallo et al. (2006) methods butsolutions were used at higher concentration/lower volume to maintain the toxin level in different step of this procedure equal to starting level.
Briefly, for simulating rumen situation, samples were incubated for 16 h in PBS that have similar pH (6.5) to rumen of high producing dairy cows that consume high concentrate ration. Then by adding 1 N HCl solution containing pepsin (10g/l) to each sample, pH adjusted to 1.9 and were incubated for 1 h to mimicking abomasum situations. After this incubation phase, pH was neutralized with 10N NaOH and then a buffer-pancreatine solution (5 M phosphate solution, pH 7.8, containing 30 g/L) were added and incubation were continued for 24 h to mimicking small intestine situation. All incubated were carried out with constant rotation at 39 °C. All assays were performed in triplicate and a yeast control (yeast suspended in PBS) and an aflatoxin B1 control (18 μg/ml of aflatoxin B1 in PBS) were also incubated for all six treatments (4, 8, 22).
3.5. Quantification of unbound aflatoxin B1 by ELISA
Yeast strains were pelleted (5000 - g, 10 min, 5°C) and the supernatant fluid containing unbound aflatoxin B1 were collected and analyzed by microtitre plate enzyme linked immunosorbent assay (ELISA) method.
Sample preparation was performed by diluting supernatant fluid (12,000 times) containing unbound aflatoxin B1 by methanol (33%). For standing the OD of samples among the range of standards OD in ELISA kit, this dilution was done. 50 µl of each diluted sample were used per well in this assay (25).
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According to test kit manual, 200 µl of distilled water and 50 µl of methanol (33%) placed into the blank and zero standard wells respectively. Then, 50 µl of aflatoxin standard solutions and 50 µl prepared test samples were added into separate wells of micro-titer plate in duplicate. Then, 50 µl of the enzyme conjugate and 100 µl of anti- aflatoxin B1 antibody were added to each well except the blank wells and mixed gently and incubated for 20 minutes at room temperature (20 - 25 °C) in the dark. The liquid was then removed completely from the wells (tap the microwell holder upside down against absorbent laboratory paper to ensure complete liquid removal) and then each well was washed with 300 µl washing buffer (PBS-Tween Buffer, pH 7.2). The washing procedure was repeated for a total of five times. After the washing step, 200 µl of chromogen (tetramethyl-benzidine) was added to each well and incubated for 20 minutes at room temperature in the dark. Finally, 50 µl of the stop solution (1 M H2SO4) were added to each well and the absorbance was measured at 450 nm in ELISA plate reader (Bio-Tek Inst.) (2, 25).
The unknown values for aflatoxins concentration in samples are determined from a calibration curve by following below steps; at first, subtract the mean absorbance value for blanks from the absorbance value of all other wells. Second, divide the mean absorbance value of standards and samples (B) by the mean absorbance value of the zero standards (Bo) and multiple by 100 (for converting to percentage). Then, B/Bo (%) values calculated for each standard in a semi-logarithmic system of coordinates against the total aflatoxin standard concentration draw the standard curve and take the B/Bo (%) values for each sample and interpolate the corresponding concentration from the calibration curve. As, samples have been diluted prior to assay, the read concentrations (ng/ml) from the standard curve must be multiplied by the dilution factor to obtain the effective aflatoxin B1 concentration in original samples.
The percentage of aflatoxin B1 bound by the treated yeast suspension was calculated using the following formula (2, 6):
3.6. Gas production test
Regarding that yeast cell wall enters to the rumen as a first part of gastrointestinal tract and stay in this part for a long time (nearly to 16-24 h in high producing dairy cows) and exposed to the anaerobic condition and diverse microorganisms that produced variety of enzymes, we hypothesized that treated yeasts can be affected by lysis and fermentation. So, gas production test were done as an indirect index for fermentation process in rumen during 96 hour of incubation. In vitro gas production was done according to the procedure described by Theodorouet al. (1994). All laboratory handling of rumen fluids, buffer and mineral solution were carried out under a continuous flow of CO2 in pre-warmed flask and bottles.
Briefly, rumens fluids were collected from two fistulated Holstein cows fed a diet to meet maintenance requirement (18). The ration was fed twice daily at 0800 and at 1500 h. Rumen fluids were collected before the morning feeding into a pre-warmed flask, and after transfer to lab, strained through 4 layers of cheesecloth then added to the buffered mineral solution (1:2 v/v). In vitro gas tests were done using a manual pressure transducer technique. Approximately, 200 mg of each dried sample (yeast strain were cultured, harvested, treated as described above and dried in 50 °C for 3 days) was weighed into a serum bottle (totally five samples) before the injection of 30 ml rumen fluid-buffer mixture into each bottle followed by incubation at 39°C. During of experimental period, bottles were gently rolled to facilitate mixing and to maximize contact of the inoculums with the samples. Gas pressure measured by a digital pressure gauge (model SEDPGB0015PG5 sensor unit, SenSym, Milpitas, Calif.) having a 0.01 lb/in2 sensitivity. Three bottles with only buffered rumen fluid are incubated and considered as the blanks. Measurements of the pressure and gas production were done at 2, 4, 6, 8, 12, 24, 36, 48, 72 and 96 h after the incubation. Total gas volumes were corrected for the blanks incubation which contained only the buffered rumen fluid without any sample (1, 24).
2.7. Statistical analysis
All experiments and analyses for binding assay and gas production were performed in triplicate and quintuplicate, respectively. Data analysis was carried out by GLM procedure of SAS statistical package (v. 9.1) in a 3 - 2 factorial arrangement of a completely randomized design and Duncan's mean comparison tests were done to identify significant differences between means. P values ≤ 0.05 were considered to be significant (21).
3. Results and discussion
The focus of this study was the comparison between three strains of treated saccharomyces cerevisiae for naturally produced aflatoxin B1 binding ability after passing in vitro simulated ruminant gastrointestinal tract and gas production testing for determining the fermentability of these treated strains in rumen situation. So, to avoid repetition of previous experiments and reducing the laboratory costs, some previous findings used as principles of current study. First; previous studies shows that maximum aflatoxin B1 binding occurs by treated yeasts harvested in exponential phase of growth. This variation of aflatoxin binding related to change of cell wall component during yeast growth cycle(22). Second;concluded that among different treatments on yeast cells, autoclaving and acid treatment shown highest pure aflatoxin B1 binding results (3, 22). Third, different study concluded that concentration of microorganism cells/ml has important role in mycotoxins binding and recommended that minimum 107 yeast cells/ml was required for aflatoxin B1 binding significantly (29).
Three strains of saccharomyces cerevisiaetested in current study shows significant (P ≤ 0.01) aflatoxin B1 binding property but the binding levels showed specificity to strain (Table 1). Strain A by more than 56% aflatoxin B1 binding showed higher binding level than two other strains but there were no significant differences between strains B and C. Strain specificity for aflatoxin B1 binding showed in current study reflect the cell wall component diversity in different strain of saccharomyces cerevisiae growing in similar culture situation. (12). Yeast cell wall forming about 30% of weight of yeasts cell (DM basis). Two main component of yeast cell wall are mannoprotein (outer surface of the cell wall) and beta 1,3 glucan (inner surface of the cell wall). Beta 1,3 glucan branched with beta 1,6 glucan that communicate inner and outer surface of cell wall (12, 15).Developed beta 1,6 glucan around the main chain of beta 1,3 glucan, increase the binding site of aflatoxin B1 (28). Chitin (beta 1,4 N-acetylglucosamine) chains is another component of yeast cell wall attached to beta 1,3 and 1,6 glucans (15). There were close relation between beta glucans and mycotoxins sequestering capacity of yeast cell wall mainly related to nature of mycotoxins (streochemical, electrical charge, solubility and size). Previously, high affinity of beta glucans binding sites for aflatoxin B1 was demonstrated. Aflatoxin B1 sequestering is not only related to beta glucans content, but chitin content have an important role too, as lower chitin content forms more flexible conformation of cell wall that increase mycotoxins sequestering efficiency (28). So, better binding results for strain A could be due to higher amount of beta glucans and/or lower amount of chitin in yeast cell wall structure. These cause higher amount of flexible glucans that appears more binding sites for trapping of aflatoxin B1 in the single helix of beta 1,3 glucan chain that followed by covering the aflatoxin B1 by beta 1,6 glucan chain and maintain toxin molecule inside the helix (28).
Feeds by passing through ruminant gastrointestinal tract exposed to different specific situation (pH, enzymes, retention time, initial toxin concentration, etc.) in each part that can affect aflatoxin B1 binding results. Most of in vitro studies assessed strains aflatoxin B1 binding properties only in one or more pH separately without paying attention to other variables and this make difficult generalizing their results for in vivo application. Given that change of pH through gastrointestinal tract could alter the geometry of beta glucans and reduce possibility of forming mycotoxins-beta glucan complexat high and low pH (8 and 3, respectively) whereas highest aflatoxin B1 adsorption shows in close to neutral pH condition (pH=6) (28), we got on to evaluate aflatoxin B1 binding of treated yeast strains after a continuous incubation period.In this method, simulated rumen (pH=6.5) starts first part of continuous incubation period for 16 h (4, 8). Referring to previous reports, this pH can make maximum aflatoxin B1 adsorption but entering to simulated abomasum and intestine parts (acidic and alkaline pH)can showaflatoxin B1 desorptive effects (28). Although expected that time remaining in second part of simulated gastrointestinal tract, had lowest recovery effect on bonded aflatoxin B1 based on previous finding that time of exposing to hydrochloric acid had little effect on metal recovery (7). So, for predicting that how amount of aflatoxin B1 can be bound to yeasts cell wall after passing gastrointestinal tract, remained aflatoxin B1 analyzed after this continuous incubation.
Yeasts in dead or alive form, rapidly adsorb aflatoxin B1 molecule (22) but increasing the incubation time for more contacting between molecules (rumen situation) can reduce recovery levels of aflatoxin B1 post ruminally by changing in pH, likely(7) that can be due to the penetration of aflatoxin B1 to the inner layer of cell wall (7). In another study shows that ochratoxin binding by heat and acid treated s. cerevisiae reach to maximum percentage after five minutes and no toxin recovery observed after 72 h (17). Also, initial dosage of aflatoxin B1 can be effective on its recovery when expose to gastric acid (HCl) in a multilayer adsorption process. High dosage of aflatoxin B1 likely make easier desorption from yeast cell wall or change the binding sites affinities (7). As previously shown by Cr (III), it's likely that in low dosage of initial concentration of toxin, binding sites with higher affinity prefer to bind aflatoxin B1 whereas initial concentration of aflatoxin B1 is increased, lower affinity binding sites is participated to adsorption of toxin. This part of aflatoxin B1 that bound to lower affinity sites of glucans can be release easier in post ruminal situation by decreased pH (7). High initial dosage of aflatoxin B1 in current study may be caused to release some aflatoxin B1 in simulated post ruminal parts of gastrointestinal tract. So, it's expected that in field and real farm situations that cows consume lower level of aflatoxin B1 normally, these strains show higher aflatoxin B1 binding properties, because 16 to 24 h remaining in rumen that have pH value near to neutral pH situation can cause high and strong binding in inner layer of yeast cell wall and subsequently can reduced recovery of aflatoxin B1 post ruminally, so the efficiency of these strains (specially strain A) in in vivo situation can be increased.
El-Nezami et al, (2002)(6) shows that using mixture of toxins (zearalenone and α-zearalenone, 1:1 ratio) decreased the binding percentageof lactobacilli that can confirmsthe possibility that treated yeasts have many binding sites for different mycotoxins and the binding sites for many of mycotoxins are the same. So, presence of aflatoxin G1 in addition to aflatoxin B1 is another reason for decreased aflatoxin B1 binding in current study compared to studies that used pure aflatoxin B1.
As mentioned above, yeast cell wall structure considered as a key factor in strain diversity and main goal of treatment for improving aflatoxin B1 binding (12). Enhancing of ochratoxin (3), aflatoxin B1 (22) andzearalenone (6) removal, previously reported by heat and acid treated of S. cerevisiae compared with viable cells. However, two types of treatments shown inconsistent results among literature. Our result shows that kind of treatment had significant (P ≤ 0.01) effect on binding capacity of yeast strains as highest binding level were related to acid treatment of yeasts strains (Table 2). Acid treatment caused that aflatoxin B1 binding capacity of yeast strains approximately two times increased. Data analysis showed that there were no interactions between yeast strains and treatments (Data not shown). Acid and heat treatments affected polysaccharides and peptidoglycans as two main components of yeast cell wall structure. Glycosidic linkage in polysaccharide and amide linkage in proteins and peptides affected by acid and respectively caused releasing of some glucose(that further fragmented to aldehydes); peptides and amino acids (3, 6).Acid concentration and cell wall structure of strains likely affected the hydrolysis of glycosidic linkages (22).Protein (peptide) denaturation or browning reaction occurs between polysaccharides and peptides by heat treatment (3, 6) by reacting of amino group with an aldehyde (20). These changes in cell wall structure by decreasing thickness of peptidoglycans and creating a more porous network with increased pore size, increased surfaces for aflatoxin B1 binding by appearing more adsorption sites that are not available in intact cells (3, 6).
Current gas production results showed that strain A significantly (P ≤ 0.01) produced lower gas (ml) compared to other strains on time 2h. On this time, strain C showed numerically highest gas production while there were no differences between strains B and C, significantly (Table 3). On times 4, 6, 8 and 12h, lowest gas production related to strain A, highest gas production related to strain C,significantly and strain B showed median gas production (P ≤ 0.05). Our results showed that from 24h of incubation till end of incubation period, there were no significant differences between yeast strains for gas production measurements although strain B showed lower gas production numerically in these times.These results confirm structural differences in three strains used in current study. As shown, during first 24 h of incubation, lowest gas production belongs to the strain that had highest aflatoxin B1 binding percentage. This interaction showed that strain A had highest amount of glucans and chitin plays a minor role in binding results because chitin constitute lowest part of yeast cell wall that could be the minor effect on gas production. Strain specificity for gas production removed after 24 hour of incubation (Figure 1) that had minimal effects in binding process, because feeds retention time in rumen is close to this time.
Our results showed that treatments (autoclave or acid) had great effect on gas production of yeast strains. Based on these results, lowest gas production (ml) in all time of incubation period related to heat treatment (Table 4). These results are consistent with strain specificity of gas production results, because in spite of significant and drastic differences between two types of treatments until 24h of incubation period,at and after this time the differences reduced, while significant differences still remained. Acid treatment approximately produced 2 times more gas on 2, 4, 6 and 8h of incubation period (Figure 2). Our results showed no interactions between time of incubation and treatments for all time of incubation period.As mentioned above, autoclave and acid treatments create a more porous network and increased the pore size of yeast cell wall network, by two different ways. In heat process, maillard reaction occurs without releasingmonomers and by inducing additional potent linkage between peptides and saccharides can be reduced nitrogen digestibility of yeast cell walls in one hand(same as reduced protein digestibility of forage when exposed to excessive heat) and reduced fermentability of glucans in another hand (27). Degree of polymerization of yeast cell wall component plays an important role in bacterial utilization as degree of polymerization decreased, count of bacteria increased that shows more utilization of cell wall component by some bacteria and increased their fermentability (13).Acid hydrolysation process, produce the products with lower degree of polymerization (20) by releasing monomers, so despite higher binding property, higher fermentability and gas production also be expected.
We concluded thatin vitroaflatoxin B1 binding results cannot be the sole criterion for selection of a strain for using as a mycotoxin binder (single compound or in combination with other compound). In addition, kind of treatment on yeast cells and their rumen fermentability are important parameters for choosing an applied mycotoxin binder in ruminant nutrition based on yeast cell wall. Strain A by two times more aflatoxin B1 binding capacity and lower gas production compared to other strains, were the certain strain selection. Despite that heat treatment caused lower gas production, acid treatment selected as an appropriate treatment for strain A, because the amount of cumulative gas production on 24 h (close to real retention time of feed in rumen) of incubation period are not drastically differed (19.3 vs. 22.8; P ≤ 0.04)for heat and acid treatments and regarding to binding results, acidtreatment can induce more aflatoxin B1 binding results in farm situation.