Research Article Received: 4 October 2013
Revised: 15 March 2014
Accepted article published: 3 April 2014
Published online in Wiley Online Library: 12 May 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6683
Ochratoxin A released back into the medium by Saccharomyces cerevisiae as a function of the strain, washing medium and fermentative conditions Antonio Bevilacqua,* Leonardo Petruzzi, Maria Rosaria Corbo, Antonietta Baiano, Carmela Garofalo and Milena Sinigaglia Abstract BACKGROUND: This study was aimed at investigating the removal of ochratoxin A (OTA) by two wild strains of Saccharomyces cerevisiae (W20 and W30) in a semi-synthetic medium under two temperatures (25, 30 ∘ C) and sugar levels (200, 250 g L−1 ), as well as the stability of OTA–yeast complex by evaluating the amount of bound toxin released back after some washing treatments with phosphate-buffered saline (PBS) or model wine (MW). In addition, the main products of fermentation were studied. RESULTS: Both W20 and W30 strains reduced OTA with removal percentages of 5.41–49.58%, and this process was affected by temperature and sugar concentration. Concerning the stability of the OTA–yeast complex, the amount of bound toxin decreased by 20–99% after five passes of washing, with a strong strain dependence and an effect of temperature and sugar concentration only for the W30 isolate. In addition, the two strains showed interesting technological properties in terms of fermentation products in a semi-synthetic medium (high ethanol yield, volatile acidity as acetic acid < 1.2 g L−1 ; glycerol production exceeding 5.2 g L−1 ). CONCLUSIONS: Apart from the removal of OTA, release of the toxin is a variable process and relies upon the strain effect; a significance of the other factors of the design (sugar concentration, temperature) was found only for a single isolate. Thus evaluation of the stability of the complex yeasts/OTA should be an additional trait to select promising functional yeasts. © 2014 Society of Chemical Industry Keywords: wine; alcoholic fermentation; Saccharomyces cerevisiae; ochratoxin A removal; ochratoxin A release
INTRODUCTION
J Sci Food Agric 2014; 94: 3291–3295
∗
Correspondence to: Antonio Bevilacqua, Department of the Science of Agriculture, Food and Environment, University of Foggia, Via Napoli 25, 71122, Foggia, Italy. E-mail: [email protected] Department of the Science of Agriculture, Food and Environment, University of Foggia, Via Napoli 25, 71122, Foggia, Italy
www.soci.org
© 2014 Society of Chemical Industry
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Ochratoxin A (OTA) is one of the most common naturally occurring mycotoxins; it is receiving increasing attention owing to its toxic effects and high incidence in a wide range of food products.1 Traditionally, black Aspergillus species (section Nigri) were considered the main source of contamination on grapes.2 A variety of physical and chemical approaches to counteract the OTA problem were proposed;3 however, some limitations including the loss of nutritional and organoleptic qualities,4 the high cost5 and some practical difficulties6 were reported. The most recent approach is the use of microorganisms able to bind OTA.3 Yeasts have been used in wine as adsorbing tools for undesirable molecules such as fatty acids and sulfur products.7 In recent years several studies have been published about the existence of cell-binding phenomena associated with OTA removal both by viable8,9 or by heat- and acid-killed yeasts.10 The sites of the adsorption of OTA could be localized on the yeast wall,8 with polysaccharides (glucan, mannan), proteins and lipids playing a significant role.9 When evaluating a microorganism as a potential decontaminating agent, assessment of the stability of the toxin–cell complex could be a key trait, as toxin release would have negative health implications.11 A drawback in the literature is that no data
are available on the stability of OTA–yeast complex under winemaking conditions, although this aspect is very important due to the fact that OTA binding to yeast cell wall could be the result of weak hydrogen bonding, ionic or hydrophobic interactions.9 Therefore, this research was aimed at: (i) investigating the ability of two wild strains of Saccharomyces cerevisiae to remove OTA from a semi-synthetic medium, added with two sugar levels (200, 250 g L−1 ) and incubated at 25 or 30 ∘ C; (ii) evaluating the stability of the OTA–yeast complex by determining the amount of bound toxin released back after repeated washing steps with phosphate-buffered saline or model wine buffer; (iii) determining the effects of the washing medium and fermentative conditions on the release of OTA. In addition, the technological performance of the yeasts (sugar consumption, ethanol, glycerol and volatile acidity production) was assessed.
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MATERIALS AND METHODS Yeasts Two wild strains of Saccharomyces cerevisiae (W20 and W30), from the Culture Collection of the Laboratory of Predictive Microbiology (Dep. SAFE, University of Foggia) were used throughout this research; the strains were isolated from a typical grapevine cultivar (Uva di Troia) of the Apulian region (southern Italy), identified by polymerase chain reaction–restriction fragment length polymorphism and internal transcribed spacer sequencing and attributed to two different biotypes.12 The sequences are freely available with the following accession numbers: KF441720 (S. cerevisiae W20) and KF441721 (S. cerevisiae W30). Before each assay, the strains were grown in YPD broth (yeast extract, 10 g L−1 ; bacteriological peptone, 20 g L−1 ; glucose 20 g L−1 ; yeast extract and peptone were purchased from Oxoid (Milan, Italy), and the glucose was from C Erba (Milan, Italy), incubated at 30 ∘ C for 24 h. Fermentation trials Small-scale fermentation experiments were carried out in duplicate by using the method proposed by Lopes et al.,13 modified as follows. A semi-synthetic medium with the following composition was used: 100 g L−1 glucose, 100 g L−1 fructose (Sigma-Aldrich, Milan, Italy), 10 g L−1 yeast extract, 1 g L−1 ammonium sulfate (JT Baker, Milan, Italy), 1 g L−1 potassium phosphate (C Erba), 1 g L−1 magnesium sulfate (JT Baker). A second test was run using 250 g L−1 of sugars (glucose:fructose, 1:1). The fermentation was carried out in 150 mL flasks containing 100 mL of the medium. After the sterilization, the pH of the medium was decreased to 3.5 through a solution of citric acid (10 g L−1 ) (Sigma-Aldrich); then, the medium was inoculated with yeasts to 6 log cfu mL−1 , added with OTA (2 μg L−1 ) (Sigma-Aldrich) and the surface was covered with a thin layer of sterilized paraffin oil (10 mL per flask) (C Erba) in order to avoid air contact. A control sample was prepared with media containing OTA but without yeast. All the samples were incubated at 25 and 30 ∘ C without shaking. The fermentation process was monitored daily by weight loss as a result of CO2 escaping from the system, until the weight was constant. After the fermentation, the paraffin oil was carefully removed and the flasks were shaken to thoroughly mix the contents and get all the cells and the products in suspension; thereafter, the samples were analyzed to assess: (i) OTA content and the main fermentation products, after centrifugation at 2100 × g for 10 min at 10 ∘ C; (ii) the viable cell count.
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OTA release after washing The method described by Serrano-Niño et al. was used,14 modified as follows: yeast pellet from the fermentation trial was suspended in 10 mL phosphate-buffered saline (PBS buffer, pH 7.4; Sigma-Aldrich), mixed for 15 s and incubated at 37 ∘ C for 5 min; thereafter, yeast cells were centrifuged (2100 × g, 10 min, 10 ∘ C) and the supernatant collected to assess the amount of OTA released back into the washing medium. This protocol was repeated up to five times. In addition, to simulate the wine conditions, a yeast pellet from the fermentation trial was washed with a model wine buffer with the following composition: ethanol (10 g L−1 ), tartaric acid (4 g L−1 ), malic acid (3 g L−1 ), acetic acid (0.1 g L−1 ), potassium sulfate (0.1 g L−1 ) and magnesium sulfate (0.025 g L−1 );15 the pH of the model wine was 3.0. As reported above, the protocol was repeated up to five times.
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A Bevilacqua et al.
Analytical determinations and cell viability OTA content was determined by quantitative enzyme-linked immunosorbent assay (ELISA), using the kit by Ridascreen® (Ochratoxin A 30/15 ELISA kit, Art. No. R1311; R-Biopharm, Darmstadt, Germany). The absorbance at 450 nm was assessed by using an ELISA 96-well plate reader (Model No.680; Bio-Rad Laboratories, Hercules, CA, USA). Residual sugars, ethanol, glycerol and volatile acidity were determined by Fourier transform infrared (FTIR) spectroscopy by employing a WineScan FT120 instrument (software version 2.2.1; FOSS Analytical, Hillerød, Denmark) according to the supplier’s instructions. Yeast count was assessed on YPD agar plates, incubated at 30 ∘ C for 2–5 days. Statistical analysis The experiments were performed in duplicate over two different batches. The results of OTA removal were modeled as removal percentage referred to the initial content, as follows: removal (%) =
OTA0 − OTAs × 100 OTA0
where OTAs and OTA0 are, respectively, the content of OTA in the different samples after the fermentation and the initial concentration of the toxin. Moreover, the data referred to the washing treatments were modeled as OTA release referred to the toxin removed by yeasts throughout the fermentation: release (%) =
OTAreleased × 100 OTA0 − OTAs
where OTAreleased is the amount of toxin released after washing. The results of the chemical determinations (ethanol, glycerol and volatile acidity) were analyzed by one-way analysis of variance (ANOVA), using Tukey’s test as the post hoc comparison test (P < 0.05). The percentage of OTA removal before washing and the amount of toxin released back after the five washing treatments were analyzed by the one-way and two way ANOVA and Tukey’s test; the content of sugar and the temperature were used as independent factors for the two-way ANOVA. Statistical analyses were performed using Statistica for Windows (software version 10.0.1011.0; Statsoft, Tulsa, OK, USA).
RESULTS AND DISCUSSION Yeasts were studied for their ability to remove OTA in a semi-synthetic medium to avoid the interference of must components, as it is well known that the toxin could interact with some components of grape, such as phenolic compounds, being adsorbed.9 Two different biotypes of S. cerevisiae (W20 and W30) were used to assess the intra-specific variability acting on OTA removal and release. Figure 1 shows the effect of temperature and sugar concentration on OTA removal after the fermentation; both strains were able to reduce the content of the toxin, with removal percentages of 5.41–49.58%. As expected, the process was significantly affected by sugar concentration and temperature;16 namely, maximum OTA removal was found at 30 ∘ C and with 250 g L−1 of sugar. However, temperature and sugar concentration acted as single factors, as the interactive effect was not significant. Concerning
© 2014 Society of Chemical Industry
J Sci Food Agric 2014; 94: 3291–3295
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11.78 ± 2.24bcd 9.34 ± 1.40c 16.66 ± 2.91 cd 11.37 ± 0.94bcd 10.20 ± 0.00abc 7.36 ± 1.23bc 12.57 ± 0.00bc 8.08 ± 1.85bcd 8.63 ± 2.23ab 3.43 ± 1.38ab 7.51 ± 1.42ab 6.77 ± 1.84abcd Steps: 1, OTA released after first washing; 2, OTA released after second washing; etc. Letters indicate significant differences for each row (one-way ANOVA and Tukey’s test, P < 0.05).
5.49 ± 2.22a 2.45 ± 0.00ab 5.50 ± 1.42a 3.53 ± 0.91ab 13.36 ± 0.00d 6.38 ± 2.79abc 15.36 ± 1.46d 15.36 ± 0.95 cd
7.64 ± 7.25a 2.62 ± 2.45a 4.99 ± 2.02ab 13.39 ± 2.33a 27.94 ± 7.21b 25.46 ± 5.04b 19.40 ± 2.06c 22.58 ± 1.20a 22.85 ± 0.00b 21.91 ± 5.02b 17.95 ± 4.11c 23.43 ± 2.40a
5 1
2
3
4
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the differences between the two target strains, S. cerevisiae W30 showed higher removal percentages. After fermentation, the cells were collected and washed with PBS or MW (model wine), to assess the stability of the yeast–OTA complex. Table 1 shows the percentages of OTA released back after washing; as shown by one-way ANOVA, the amount of toxin released increased with the number of treatments, although the relationship was not linear for most of the experiments. The release of OTA into the medium confirmed that binding could be at least partially reversible, as suggested by the decrease of the removal ability reported for some strains of S. cerevisiae in synthetic media.16,17 Concerning other toxins, the binding of aflatoxins (AFs) to bacterial cells was reported to be reversible and the stability of the AF–cell complex relied upon the strain, the conditions throughout the formation of the complex and washing.18 For aflatoxin B1 (AFB1 ) Haskard et al. reported a variable amount of release by 12 strains of lactic acid bacteria (LAB),19 while Hernandez-Mendoza et al. reported that Lactobacillus casei 7R1 did not release any detectable AFB1 after three passes of washing with PBS.20 Concerning the kind of washing medium, the differences between PBS and MW were not significant and this result partially disagreed with the findings of Haskard et al., who reported that LAB released back 6–11% of AFB1 in water and 83–99% in methanol, acetonitrile, chloroform or benzene.19 The total amount of toxin released after the five passes of washing was used as the dependent variable for a two-way ANOVA; sugar concentration and temperature were used as independent factors (Fig. 2). The factors played different roles as a function
PBS treatment
Figure 1. Effects of sugar concentration and temperature on the removal of OTA by S. cerevisiae W20 (A) and W30 (B): ‘decomposition hypothesis’ for two-way ANOVA. Vertical bars denote 95% confidence intervals.
Table 1. Percentage of OTA release back after washing with PBS or MW. Mean values ± standard deviation
1
200 250 Sugar concentration (g L−1)
17.77 ± 4.18ab 16.60 ± 2.49ab 16.49 ± 2.05c 15.87 ± 1.17a
0
12.72 ± 4.32ab 14.84 ± 4.97ab 15.05 ± 4.09bc 15.88 ± 3.52a
10
7.64 ± 3.15a 4.36 ± 4.91a 2.15 ± 2.01a 13.39 ± 2.33a
2
25° C 30° C
20
W20 200 g/25 ∘ C 200 g/30 ∘ C 250 g/25 ∘ C 250 g/30 ∘ C W30 200 g/25 ∘ C 200 g/30 ∘ C 250 g/25 ∘ C 250 g/30 ∘ C
30
7.64 ± 3.15ab 13.08 ± 2.48ab 13.60 ± 2.04bc 15.04 ± 2.34a
40
11.78 ± 2.24 cd 6.37 ± 0.00abc 12.69 ± 0.00d 12.69 ± 0.94bcd
50
Temperature (T ), P < 0.01 Sugar concentration (S ), P < 0.01 T x S, P > 0.05
8.63 ± 2.23bcd 5.39 ± 1.39abc 10.05 ± 1.46 cd 10.05 ± 0.93abcd
B 60
7.05 ± 0.00abc 3.43 ± 1.38ab 9.53 ± 1.43ab 8.08 ± 1.85abc
Sugar concentration (g L−1)
3
250
MW treatment
200
2.36 ± 2.21a 1.48 ± 1.38a 7.51 ± 1.42ab 4.18 ± 1.83a
10
17.77 ± 4.18b 20.13 ± 2.50b 16.49 ± 2.05c 20.89 ± 1.19a
25° C 30° C
20
27.94 ± 0.21b 23.68 ± 2.51b 20.86 ± 4.13c 23.43 ± 2.40a
30
0
OTA removal by W30 (%)
14.95 ± 2.24d 9.34 ± 1.40c 17.69 ± 1.46 cd 14.02 ± 0.94d
5
50
Temperature (T ), P < 0.01 Sugar concentration (S ), P < 0.01 T x S, P > 0.05
4
OTA removal by W20 (%)
A 60
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33.03 ± 0.00b 27.24 ± 2.53b 19.40 ± 2.06c 22.58 ± 2.40a
Efficiency of OTA removal
www.soci.org Strain (St), P < 0.01 Sugar concentration (S ), P > 0.05 St x S, P < 0.05
OTA release in PBS (%)
A 160 140
A Bevilacqua et al.
Table 2. Technological performance of yeasts, expressed as amounts of ethanol, volatile acidity (as acetic acid) and glycerol
120 100
Strain
80 W20 W30
60
Temperature (∘ C)
W20
40 20
W30
0 200 250 Sugar concentration (g L−1)
OTA release in MW (%)
B 160 120 100 80
W20 W30
60 40 20 0 25
30 Temperature (°C)
Figure 2. Effects of sugar concentration and temperature on the release of OTA in PBS and MW by S. cerevisiae W20 and W30: ‘decomposition hypothesis’ for two-way ANOVA for the effects of strain versus sugar concentration in PBS (A) and strain versus temperature in MW (B). The vertical bars denote 95% confidence intervals.
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of the strain and the washing medium; namely, strain W20 was not affected by temperature and sugar concentration either in PBS or in MW. On the other hand, strain W30 was affected by sugar concentration in PBS and by temperature in MW, as the release of OTA was minimal with 200 g L−1 sugar (PBS) and at 30 ∘ C (MW). The controversial results found for the effects of the fermentative conditions on the release of OTA (different trend for the two strains; different roles for temperature and sugar concentration in the two washing media) could be due to the fact that OTA removal,16,17 as well as of other toxins,19,20 is strictly strain-dependent; thus we could suppose that strain dependence plays a significant role also in the release of the toxin. Moreover, the effects of sugar concentration and temperature for strain W30 in PBS and MW could be due to a significant effect of the fermentative/growth conditions on the electrical charge of yeasts, responsible for the stability of the complex cell/toxin. Parietal adsorption is different from yeast to yeast, according to structural characteristics and chemical composition of the outermost layer of cell wall. This layer contains mannoproteins, which represent 25–50% of the entire cell wall. The mannoproteins are connected with an inner matrix of amorphous 𝛽-1,3-glucan and are partly released in wine. As for other adsorption processes, the physical structure of mannoproteins – total charge, charge distribution, and accessible surface area – is the most important feature determining adsorption differences.21
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200 200 250 250 200 200 250 250
EtOH (g L−1 ) 10.64a 10.41ab 10.17bc 10.01 cd 10.45a 10.20bc 10.08c 9.77d
Volatile acidity (g L−1 )
Glycerol (g L−1 )
0.45a 0.39a 0.80bc 0.69c 0.43a 0.39a 0.89b 0.76c
6.16a 5.80b 7.29d 7.01de 6.59bc 6.30c 7.77f 7.44e
Letters indicate significant differences for each column (one-way ANOVA and Tukey’s test, P < 0.05).
Strain (St), P < 0.01 Temperature (T ), P > 0.05 St x T, P < 0.05
140
30 25 30 25 30 25 30 25
Sugar (g L−1 )
The differences amongst wine yeasts in OTA adsorption during winemaking may depend on the different mannosyl phosphate content, but also on dissimilar fermentation and cell sedimentation dynamics, cell dimension and flocculence.21 Yeast strain is one of the factors acting on the amount of mannoproteins released during winemaking. In addition, environmental factors such as temperature, carbon source or the level of the initial colloid content of the medium have been shown to influence the amount of cell wall polysaccharides secreted into the fermenting medium.22 A difference in the composition of the cell wall could be the factor leading to a different trend between the two strains in response to temperature and sugar concentrations. For example, a high chitin content decreased the flexibility of cell wall with a consequent lower capacity to form complexes with zearalenone in S. cerevisiae;23 however, further investigations are required to confirm this hypothesis. As a final step in the research, the fermentative performance of the two strains was assessed (Table 2). Both strains showed: (i) high ethanol yield associated with an efficient conversion of sugars; (ii) volatile acidity as acetic acid < 1.2 g L−1 (threshold according to European legislation);24 (iii) glycerol production exceeding 5.2 g L−1 (breakpoint for a significant effect on the sweetness, body and fullness of wines),25 with higher production for the strain W30 (P < 0.05); (iv) residual sugars at levels below 2 g L−1 (data not shown); (v) cell count of 5–6 log cfu mL−1 at the end of fermentation (data not shown). The results of the fermentative performance indicated that the two strains, apart from their ability to remove OTA, showed some interesting traits in terms of ethanol, glycerol and volatile acidity production, thus suggesting that potentially they did not negatively affect the quality of the products. In conclusion, this research is the first report on the release of OTA by yeasts after its biological removal throughout fermentation; release of the toxin was strictly strain dependent, although the results showed a significant effect of temperature and sugar concentration for a single strain. The stability of the yeast–OTA complex and release of the toxin after fermentation should be an additional trait for the selection of suitable functional starter cultures of S. cerevisiae, along with technological performance; moreover, a future perspective could be a focus on the role of the cell wall charge and fermentative conditions on the complex phenomena of OTA removal and release to minimize the amount of the toxin released back into the medium by the starter.
© 2014 Society of Chemical Industry
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Efficiency of OTA removal
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REFERENCES 1 Pozo-Bayón MÁ, Monagas M, Bartolomé B and Moreno-Arribas MV, Wine features related to safety and consumer health: an integrated perspective. Crit Rev Food Sci Nutr 52:31–54 (2012). 2 Esti M, Benucci I, Liburdi K and Acciaro G, Monitoring of ochratoxin A fate during alcoholic fermentation of wine-must. Food Control 27:53–56 (2012). 3 Quintela S, Villarán MC, de Armentia IL and Elejalde E, Ochratoxin A removal in wine: a review. Food Control 30:439–445 (2013). 4 Fernandes A, Ratola N, Cerdeira A, Alves A and Venâncio A, Changes in ochratoxin A concentration during winemaking. Am J Enol Viticult 58:92–96 (2007). 5 Piotrowska M, Nowak A and Czyzowska A, Removal of ochratoxin A by wine Saccharomyces cerevisiae strains. Eur Food Res Technol 236:441–447 (2013). 6 Patharajan S, Reddy KRN, Karthikeyan V, Spadaro D, Lore A, Gullino ML et al., Potential of yeast antagonists on in vitro biodegradation of ochratoxin A. Food Control 22:290–296 (2011). 7 Pradelles R, Alexandre H, Ortiz-Julien A and Chassagne D, Effects of yeast cell-wall characteristics on 4-ethylphenol sorption capacity in model wine. J Agric Food Chem 56:11854–11861 (2008). 8 Caridi A, Galvano F, Tafuri A and Ritieni A, Ochratoxin A removal during winemaking. Enzyme Microb Tech 40:122–126 (2006). 9 Cecchini F, Morassut M, Moruno EM and Di Stefano R, Influence of yeast strain on ochratoxin A content during fermentation of white and red must. Food Microbiol 23:411–417 (2006). 10 Bejaoui H, Mathieu F, Taillandier P and Lebrihi A, Ochratoxin A removal in synthetic and natural grape juices by selected oenological Saccharomyces strains. J Appl Microbiol 97:1038–1044 (2004). 11 Peltonen K, El-Nezami H, Haskard C, Ahokas J and Salminen S, Aflatoxin B1 binding by dairy strains of lactic acid bacteria and bifidobacteria. J Dairy Sci 84: 2152–2156 (2001). 12 Petruzzi L, Bevilacqua A, Corbo MR, Garofalo C, Baiano A and Sinigaglia M, Selection of autochthonous Saccharomyces cerevisiae strains as wine starters using a polyphasic approach and ochratoxin A removal. J Food Prot (in press). 13 Lopes CA, Rodríguez ME, Sangorrín M, Querol A and Caballero AC, Patagonian wines: the selection of an indigenous yeast starter. J Ind Microbiol Biotechnol 34:539–546 (2007). 14 Serrano-Niño JC, Cavazos-Garduño A, Hernandez-Mendoza A, Applegate B, Ferruzzi MG, San Martin-González MF et al., Assessment of
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probiotic strains ability to reduce the bioaccessibility of aflatoxin M1 in artificially contaminated milk using an in vitro digestive model. Food Control 31:202–207 (2013). Martínez-Rodriguez AJ, Carrascosa AV and Polo MC, Release of nitrogen compounds to the extracellular medium by three strains of Saccharomyces cerevisiae during induced autolysis in a model wine system. Int J Food Microbiol 68:155–160 (2001). Petruzzi L, Bevilacqua A, Baiano A, Beneduce L, Corbo MR and Sinigaglia M, Study of Saccharomyces cerevisiae W13 as a functional starter for the removal of ochratoxin A. Food Control 35:373–377 (2014). Petruzzi L, Sinigaglia M, Corbo MR, Beneduce L and Bevilacqua A, Ochratoxin A removal by Saccharomyces cerevisiae strains: effects of wine-related physicochemical factors. J Sci Food Agric 93:2110–2115 (2013). Elsanhoty RM, Ramadan MF, El-Gohery SS, Abol-Ela MF and Azeke MA, Ability of selected microorganisms for removing aflatoxins in vitro and fate of aflatoxins in contaminated wheat during baladi bread baking. Food Control 33:287–292 (2013). Haskard CA, El-Nezami HS, Kankaanpää PE, Salminen S and Ahokas JT, Surface binding of aflatoxin B1 by lactic acid bacteria. Appl Environ Microbiol 67:3086–3091 (2001). Hernandez-Mendoza A, Garcia HS and Steele JL, Screening of Lactobacillus casei strains for their ability to bind aflatoxin B1 . Food Chem Toxicol 47:1064–1068 (2009). Caridi A, New perspectives in safety and quality enhancement of wine through selection of yeasts based on the parietal adsorption activity. Int J Food Microbiol 120:167–172 (2007). Giovani G, Canuti V and Rosi I, Effect of yeast strain and fermentation conditions on the release of cell wall polysaccharides. Int J Food Microbiol 137:303–307 (2010). Yiannikouris A, François J, Poughon L, Dussap CG, Bertin G, Jeminet G et al., Adsorption of Zearalenone by 𝛽-D-glucans in the Saccharomyces cerevisiae cell wall. J Food Prot 67:1195–200 (2004). Vilela A, Schuller D, Mendes-Faia A and Côrte-Real M, Reduction of volatile acidity of acidic wines by immobilized Saccharomyces cerevisiae cells. Appl Microbiol Biotechnol 97:4991–5000 (2013). Noble AC and Bursick GF, The contribution of glycerol to perceived viscosity and sweetness in white wine. Am J Enol Viticult 39:110–112 (1984).
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J Sci Food Agric 2014; 94: 3291–3295
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/jsfa
Revised: 15 March 2014
Accepted article published: 3 April 2014
Published online in Wiley Online Library: 12 May 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6683
Ochratoxin A released back into the medium by Saccharomyces cerevisiae as a function of the strain, washing medium and fermentative conditions Antonio Bevilacqua,* Leonardo Petruzzi, Maria Rosaria Corbo, Antonietta Baiano, Carmela Garofalo and Milena Sinigaglia Abstract BACKGROUND: This study was aimed at investigating the removal of ochratoxin A (OTA) by two wild strains of Saccharomyces cerevisiae (W20 and W30) in a semi-synthetic medium under two temperatures (25, 30 ∘ C) and sugar levels (200, 250 g L−1 ), as well as the stability of OTA–yeast complex by evaluating the amount of bound toxin released back after some washing treatments with phosphate-buffered saline (PBS) or model wine (MW). In addition, the main products of fermentation were studied. RESULTS: Both W20 and W30 strains reduced OTA with removal percentages of 5.41–49.58%, and this process was affected by temperature and sugar concentration. Concerning the stability of the OTA–yeast complex, the amount of bound toxin decreased by 20–99% after five passes of washing, with a strong strain dependence and an effect of temperature and sugar concentration only for the W30 isolate. In addition, the two strains showed interesting technological properties in terms of fermentation products in a semi-synthetic medium (high ethanol yield, volatile acidity as acetic acid < 1.2 g L−1 ; glycerol production exceeding 5.2 g L−1 ). CONCLUSIONS: Apart from the removal of OTA, release of the toxin is a variable process and relies upon the strain effect; a significance of the other factors of the design (sugar concentration, temperature) was found only for a single isolate. Thus evaluation of the stability of the complex yeasts/OTA should be an additional trait to select promising functional yeasts. © 2014 Society of Chemical Industry Keywords: wine; alcoholic fermentation; Saccharomyces cerevisiae; ochratoxin A removal; ochratoxin A release
INTRODUCTION
J Sci Food Agric 2014; 94: 3291–3295
∗
Correspondence to: Antonio Bevilacqua, Department of the Science of Agriculture, Food and Environment, University of Foggia, Via Napoli 25, 71122, Foggia, Italy. E-mail: [email protected] Department of the Science of Agriculture, Food and Environment, University of Foggia, Via Napoli 25, 71122, Foggia, Italy
www.soci.org
© 2014 Society of Chemical Industry
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Ochratoxin A (OTA) is one of the most common naturally occurring mycotoxins; it is receiving increasing attention owing to its toxic effects and high incidence in a wide range of food products.1 Traditionally, black Aspergillus species (section Nigri) were considered the main source of contamination on grapes.2 A variety of physical and chemical approaches to counteract the OTA problem were proposed;3 however, some limitations including the loss of nutritional and organoleptic qualities,4 the high cost5 and some practical difficulties6 were reported. The most recent approach is the use of microorganisms able to bind OTA.3 Yeasts have been used in wine as adsorbing tools for undesirable molecules such as fatty acids and sulfur products.7 In recent years several studies have been published about the existence of cell-binding phenomena associated with OTA removal both by viable8,9 or by heat- and acid-killed yeasts.10 The sites of the adsorption of OTA could be localized on the yeast wall,8 with polysaccharides (glucan, mannan), proteins and lipids playing a significant role.9 When evaluating a microorganism as a potential decontaminating agent, assessment of the stability of the toxin–cell complex could be a key trait, as toxin release would have negative health implications.11 A drawback in the literature is that no data
are available on the stability of OTA–yeast complex under winemaking conditions, although this aspect is very important due to the fact that OTA binding to yeast cell wall could be the result of weak hydrogen bonding, ionic or hydrophobic interactions.9 Therefore, this research was aimed at: (i) investigating the ability of two wild strains of Saccharomyces cerevisiae to remove OTA from a semi-synthetic medium, added with two sugar levels (200, 250 g L−1 ) and incubated at 25 or 30 ∘ C; (ii) evaluating the stability of the OTA–yeast complex by determining the amount of bound toxin released back after repeated washing steps with phosphate-buffered saline or model wine buffer; (iii) determining the effects of the washing medium and fermentative conditions on the release of OTA. In addition, the technological performance of the yeasts (sugar consumption, ethanol, glycerol and volatile acidity production) was assessed.
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MATERIALS AND METHODS Yeasts Two wild strains of Saccharomyces cerevisiae (W20 and W30), from the Culture Collection of the Laboratory of Predictive Microbiology (Dep. SAFE, University of Foggia) were used throughout this research; the strains were isolated from a typical grapevine cultivar (Uva di Troia) of the Apulian region (southern Italy), identified by polymerase chain reaction–restriction fragment length polymorphism and internal transcribed spacer sequencing and attributed to two different biotypes.12 The sequences are freely available with the following accession numbers: KF441720 (S. cerevisiae W20) and KF441721 (S. cerevisiae W30). Before each assay, the strains were grown in YPD broth (yeast extract, 10 g L−1 ; bacteriological peptone, 20 g L−1 ; glucose 20 g L−1 ; yeast extract and peptone were purchased from Oxoid (Milan, Italy), and the glucose was from C Erba (Milan, Italy), incubated at 30 ∘ C for 24 h. Fermentation trials Small-scale fermentation experiments were carried out in duplicate by using the method proposed by Lopes et al.,13 modified as follows. A semi-synthetic medium with the following composition was used: 100 g L−1 glucose, 100 g L−1 fructose (Sigma-Aldrich, Milan, Italy), 10 g L−1 yeast extract, 1 g L−1 ammonium sulfate (JT Baker, Milan, Italy), 1 g L−1 potassium phosphate (C Erba), 1 g L−1 magnesium sulfate (JT Baker). A second test was run using 250 g L−1 of sugars (glucose:fructose, 1:1). The fermentation was carried out in 150 mL flasks containing 100 mL of the medium. After the sterilization, the pH of the medium was decreased to 3.5 through a solution of citric acid (10 g L−1 ) (Sigma-Aldrich); then, the medium was inoculated with yeasts to 6 log cfu mL−1 , added with OTA (2 μg L−1 ) (Sigma-Aldrich) and the surface was covered with a thin layer of sterilized paraffin oil (10 mL per flask) (C Erba) in order to avoid air contact. A control sample was prepared with media containing OTA but without yeast. All the samples were incubated at 25 and 30 ∘ C without shaking. The fermentation process was monitored daily by weight loss as a result of CO2 escaping from the system, until the weight was constant. After the fermentation, the paraffin oil was carefully removed and the flasks were shaken to thoroughly mix the contents and get all the cells and the products in suspension; thereafter, the samples were analyzed to assess: (i) OTA content and the main fermentation products, after centrifugation at 2100 × g for 10 min at 10 ∘ C; (ii) the viable cell count.
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OTA release after washing The method described by Serrano-Niño et al. was used,14 modified as follows: yeast pellet from the fermentation trial was suspended in 10 mL phosphate-buffered saline (PBS buffer, pH 7.4; Sigma-Aldrich), mixed for 15 s and incubated at 37 ∘ C for 5 min; thereafter, yeast cells were centrifuged (2100 × g, 10 min, 10 ∘ C) and the supernatant collected to assess the amount of OTA released back into the washing medium. This protocol was repeated up to five times. In addition, to simulate the wine conditions, a yeast pellet from the fermentation trial was washed with a model wine buffer with the following composition: ethanol (10 g L−1 ), tartaric acid (4 g L−1 ), malic acid (3 g L−1 ), acetic acid (0.1 g L−1 ), potassium sulfate (0.1 g L−1 ) and magnesium sulfate (0.025 g L−1 );15 the pH of the model wine was 3.0. As reported above, the protocol was repeated up to five times.
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A Bevilacqua et al.
Analytical determinations and cell viability OTA content was determined by quantitative enzyme-linked immunosorbent assay (ELISA), using the kit by Ridascreen® (Ochratoxin A 30/15 ELISA kit, Art. No. R1311; R-Biopharm, Darmstadt, Germany). The absorbance at 450 nm was assessed by using an ELISA 96-well plate reader (Model No.680; Bio-Rad Laboratories, Hercules, CA, USA). Residual sugars, ethanol, glycerol and volatile acidity were determined by Fourier transform infrared (FTIR) spectroscopy by employing a WineScan FT120 instrument (software version 2.2.1; FOSS Analytical, Hillerød, Denmark) according to the supplier’s instructions. Yeast count was assessed on YPD agar plates, incubated at 30 ∘ C for 2–5 days. Statistical analysis The experiments were performed in duplicate over two different batches. The results of OTA removal were modeled as removal percentage referred to the initial content, as follows: removal (%) =
OTA0 − OTAs × 100 OTA0
where OTAs and OTA0 are, respectively, the content of OTA in the different samples after the fermentation and the initial concentration of the toxin. Moreover, the data referred to the washing treatments were modeled as OTA release referred to the toxin removed by yeasts throughout the fermentation: release (%) =
OTAreleased × 100 OTA0 − OTAs
where OTAreleased is the amount of toxin released after washing. The results of the chemical determinations (ethanol, glycerol and volatile acidity) were analyzed by one-way analysis of variance (ANOVA), using Tukey’s test as the post hoc comparison test (P < 0.05). The percentage of OTA removal before washing and the amount of toxin released back after the five washing treatments were analyzed by the one-way and two way ANOVA and Tukey’s test; the content of sugar and the temperature were used as independent factors for the two-way ANOVA. Statistical analyses were performed using Statistica for Windows (software version 10.0.1011.0; Statsoft, Tulsa, OK, USA).
RESULTS AND DISCUSSION Yeasts were studied for their ability to remove OTA in a semi-synthetic medium to avoid the interference of must components, as it is well known that the toxin could interact with some components of grape, such as phenolic compounds, being adsorbed.9 Two different biotypes of S. cerevisiae (W20 and W30) were used to assess the intra-specific variability acting on OTA removal and release. Figure 1 shows the effect of temperature and sugar concentration on OTA removal after the fermentation; both strains were able to reduce the content of the toxin, with removal percentages of 5.41–49.58%. As expected, the process was significantly affected by sugar concentration and temperature;16 namely, maximum OTA removal was found at 30 ∘ C and with 250 g L−1 of sugar. However, temperature and sugar concentration acted as single factors, as the interactive effect was not significant. Concerning
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11.78 ± 2.24bcd 9.34 ± 1.40c 16.66 ± 2.91 cd 11.37 ± 0.94bcd 10.20 ± 0.00abc 7.36 ± 1.23bc 12.57 ± 0.00bc 8.08 ± 1.85bcd 8.63 ± 2.23ab 3.43 ± 1.38ab 7.51 ± 1.42ab 6.77 ± 1.84abcd Steps: 1, OTA released after first washing; 2, OTA released after second washing; etc. Letters indicate significant differences for each row (one-way ANOVA and Tukey’s test, P < 0.05).
5.49 ± 2.22a 2.45 ± 0.00ab 5.50 ± 1.42a 3.53 ± 0.91ab 13.36 ± 0.00d 6.38 ± 2.79abc 15.36 ± 1.46d 15.36 ± 0.95 cd
7.64 ± 7.25a 2.62 ± 2.45a 4.99 ± 2.02ab 13.39 ± 2.33a 27.94 ± 7.21b 25.46 ± 5.04b 19.40 ± 2.06c 22.58 ± 1.20a 22.85 ± 0.00b 21.91 ± 5.02b 17.95 ± 4.11c 23.43 ± 2.40a
5 1
2
3
4
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the differences between the two target strains, S. cerevisiae W30 showed higher removal percentages. After fermentation, the cells were collected and washed with PBS or MW (model wine), to assess the stability of the yeast–OTA complex. Table 1 shows the percentages of OTA released back after washing; as shown by one-way ANOVA, the amount of toxin released increased with the number of treatments, although the relationship was not linear for most of the experiments. The release of OTA into the medium confirmed that binding could be at least partially reversible, as suggested by the decrease of the removal ability reported for some strains of S. cerevisiae in synthetic media.16,17 Concerning other toxins, the binding of aflatoxins (AFs) to bacterial cells was reported to be reversible and the stability of the AF–cell complex relied upon the strain, the conditions throughout the formation of the complex and washing.18 For aflatoxin B1 (AFB1 ) Haskard et al. reported a variable amount of release by 12 strains of lactic acid bacteria (LAB),19 while Hernandez-Mendoza et al. reported that Lactobacillus casei 7R1 did not release any detectable AFB1 after three passes of washing with PBS.20 Concerning the kind of washing medium, the differences between PBS and MW were not significant and this result partially disagreed with the findings of Haskard et al., who reported that LAB released back 6–11% of AFB1 in water and 83–99% in methanol, acetonitrile, chloroform or benzene.19 The total amount of toxin released after the five passes of washing was used as the dependent variable for a two-way ANOVA; sugar concentration and temperature were used as independent factors (Fig. 2). The factors played different roles as a function
PBS treatment
Figure 1. Effects of sugar concentration and temperature on the removal of OTA by S. cerevisiae W20 (A) and W30 (B): ‘decomposition hypothesis’ for two-way ANOVA. Vertical bars denote 95% confidence intervals.
Table 1. Percentage of OTA release back after washing with PBS or MW. Mean values ± standard deviation
1
200 250 Sugar concentration (g L−1)
17.77 ± 4.18ab 16.60 ± 2.49ab 16.49 ± 2.05c 15.87 ± 1.17a
0
12.72 ± 4.32ab 14.84 ± 4.97ab 15.05 ± 4.09bc 15.88 ± 3.52a
10
7.64 ± 3.15a 4.36 ± 4.91a 2.15 ± 2.01a 13.39 ± 2.33a
2
25° C 30° C
20
W20 200 g/25 ∘ C 200 g/30 ∘ C 250 g/25 ∘ C 250 g/30 ∘ C W30 200 g/25 ∘ C 200 g/30 ∘ C 250 g/25 ∘ C 250 g/30 ∘ C
30
7.64 ± 3.15ab 13.08 ± 2.48ab 13.60 ± 2.04bc 15.04 ± 2.34a
40
11.78 ± 2.24 cd 6.37 ± 0.00abc 12.69 ± 0.00d 12.69 ± 0.94bcd
50
Temperature (T ), P < 0.01 Sugar concentration (S ), P < 0.01 T x S, P > 0.05
8.63 ± 2.23bcd 5.39 ± 1.39abc 10.05 ± 1.46 cd 10.05 ± 0.93abcd
B 60
7.05 ± 0.00abc 3.43 ± 1.38ab 9.53 ± 1.43ab 8.08 ± 1.85abc
Sugar concentration (g L−1)
3
250
MW treatment
200
2.36 ± 2.21a 1.48 ± 1.38a 7.51 ± 1.42ab 4.18 ± 1.83a
10
17.77 ± 4.18b 20.13 ± 2.50b 16.49 ± 2.05c 20.89 ± 1.19a
25° C 30° C
20
27.94 ± 0.21b 23.68 ± 2.51b 20.86 ± 4.13c 23.43 ± 2.40a
30
0
OTA removal by W30 (%)
14.95 ± 2.24d 9.34 ± 1.40c 17.69 ± 1.46 cd 14.02 ± 0.94d
5
50
Temperature (T ), P < 0.01 Sugar concentration (S ), P < 0.01 T x S, P > 0.05
4
OTA removal by W20 (%)
A 60
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33.03 ± 0.00b 27.24 ± 2.53b 19.40 ± 2.06c 22.58 ± 2.40a
Efficiency of OTA removal
www.soci.org Strain (St), P < 0.01 Sugar concentration (S ), P > 0.05 St x S, P < 0.05
OTA release in PBS (%)
A 160 140
A Bevilacqua et al.
Table 2. Technological performance of yeasts, expressed as amounts of ethanol, volatile acidity (as acetic acid) and glycerol
120 100
Strain
80 W20 W30
60
Temperature (∘ C)
W20
40 20
W30
0 200 250 Sugar concentration (g L−1)
OTA release in MW (%)
B 160 120 100 80
W20 W30
60 40 20 0 25
30 Temperature (°C)
Figure 2. Effects of sugar concentration and temperature on the release of OTA in PBS and MW by S. cerevisiae W20 and W30: ‘decomposition hypothesis’ for two-way ANOVA for the effects of strain versus sugar concentration in PBS (A) and strain versus temperature in MW (B). The vertical bars denote 95% confidence intervals.
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of the strain and the washing medium; namely, strain W20 was not affected by temperature and sugar concentration either in PBS or in MW. On the other hand, strain W30 was affected by sugar concentration in PBS and by temperature in MW, as the release of OTA was minimal with 200 g L−1 sugar (PBS) and at 30 ∘ C (MW). The controversial results found for the effects of the fermentative conditions on the release of OTA (different trend for the two strains; different roles for temperature and sugar concentration in the two washing media) could be due to the fact that OTA removal,16,17 as well as of other toxins,19,20 is strictly strain-dependent; thus we could suppose that strain dependence plays a significant role also in the release of the toxin. Moreover, the effects of sugar concentration and temperature for strain W30 in PBS and MW could be due to a significant effect of the fermentative/growth conditions on the electrical charge of yeasts, responsible for the stability of the complex cell/toxin. Parietal adsorption is different from yeast to yeast, according to structural characteristics and chemical composition of the outermost layer of cell wall. This layer contains mannoproteins, which represent 25–50% of the entire cell wall. The mannoproteins are connected with an inner matrix of amorphous 𝛽-1,3-glucan and are partly released in wine. As for other adsorption processes, the physical structure of mannoproteins – total charge, charge distribution, and accessible surface area – is the most important feature determining adsorption differences.21
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200 200 250 250 200 200 250 250
EtOH (g L−1 ) 10.64a 10.41ab 10.17bc 10.01 cd 10.45a 10.20bc 10.08c 9.77d
Volatile acidity (g L−1 )
Glycerol (g L−1 )
0.45a 0.39a 0.80bc 0.69c 0.43a 0.39a 0.89b 0.76c
6.16a 5.80b 7.29d 7.01de 6.59bc 6.30c 7.77f 7.44e
Letters indicate significant differences for each column (one-way ANOVA and Tukey’s test, P < 0.05).
Strain (St), P < 0.01 Temperature (T ), P > 0.05 St x T, P < 0.05
140
30 25 30 25 30 25 30 25
Sugar (g L−1 )
The differences amongst wine yeasts in OTA adsorption during winemaking may depend on the different mannosyl phosphate content, but also on dissimilar fermentation and cell sedimentation dynamics, cell dimension and flocculence.21 Yeast strain is one of the factors acting on the amount of mannoproteins released during winemaking. In addition, environmental factors such as temperature, carbon source or the level of the initial colloid content of the medium have been shown to influence the amount of cell wall polysaccharides secreted into the fermenting medium.22 A difference in the composition of the cell wall could be the factor leading to a different trend between the two strains in response to temperature and sugar concentrations. For example, a high chitin content decreased the flexibility of cell wall with a consequent lower capacity to form complexes with zearalenone in S. cerevisiae;23 however, further investigations are required to confirm this hypothesis. As a final step in the research, the fermentative performance of the two strains was assessed (Table 2). Both strains showed: (i) high ethanol yield associated with an efficient conversion of sugars; (ii) volatile acidity as acetic acid < 1.2 g L−1 (threshold according to European legislation);24 (iii) glycerol production exceeding 5.2 g L−1 (breakpoint for a significant effect on the sweetness, body and fullness of wines),25 with higher production for the strain W30 (P < 0.05); (iv) residual sugars at levels below 2 g L−1 (data not shown); (v) cell count of 5–6 log cfu mL−1 at the end of fermentation (data not shown). The results of the fermentative performance indicated that the two strains, apart from their ability to remove OTA, showed some interesting traits in terms of ethanol, glycerol and volatile acidity production, thus suggesting that potentially they did not negatively affect the quality of the products. In conclusion, this research is the first report on the release of OTA by yeasts after its biological removal throughout fermentation; release of the toxin was strictly strain dependent, although the results showed a significant effect of temperature and sugar concentration for a single strain. The stability of the yeast–OTA complex and release of the toxin after fermentation should be an additional trait for the selection of suitable functional starter cultures of S. cerevisiae, along with technological performance; moreover, a future perspective could be a focus on the role of the cell wall charge and fermentative conditions on the complex phenomena of OTA removal and release to minimize the amount of the toxin released back into the medium by the starter.
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Efficiency of OTA removal
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