Effect of copper stress on growth characteristics and fermentation properties of Saccharomyces cerevisiae and the pathway of copper adsorption during wine fermentation

Food Chemistry 192 (2016) 43–52

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Effect of copper stress on growth characteristics and fermentation properties of Saccharomyces cerevisiae and the pathway of copper adsorption during wine fermentation Xiangyu Sun a,1, Lingling Liu a,1, Yu Zhao a, Tingting Ma b, Fang Zhao a, Weidong Huang a, Jicheng Zhan a,⇑ a b

College of Food Science and Nutritional Engineering, Beijing Key Laboratory of Viticulture and Enology, China Agricultural University, Beijing 100083, PR China College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an 710062, PR China

a r t i c l e

i n f o

Article history: Received 24 March 2015 Received in revised form 28 June 2015 Accepted 29 June 2015 Available online 30 June 2015 Keywords: Copper Saccharomyces cerevisiae Cell growth Fermentation performance Adsorption pathway

a b s t r a c t The effect of copper stress on the fermentation performance of Saccharomyces cerevisiae and its copper adsorption pathway during alcoholic fermentation were investigated in this study. At the limits imposed by the regulations of the European Union and South African (620 mg/l), copper had no effect on the cell growth of S. cerevisiae, but its fermentation performance was inhibited to a certain extent. Therefore, the regulated limit should be further reduced (612.8 mg/l). Under 9.6–19.2 mg/l copper stress, S. cerevisiae could absorb copper; the copper removal ratio and the unit strain adsorption were 60–81% and 2.72–9.65 mg/g, respectively. S. cerevisiae has a non-biological adsorption of copper, but compared with biological (living yeast) adsorption, the non-biological adsorption was very low. The copper adsorption way of S. cerevisiae was primarily via biological (living yeast) adsorption, which was a two-step process. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Copper is one of the heavy metals present in wines and is unavoidable in winemaking. There are many sources for copper throughout the wine production process. First, cupric pesticides, such as the Bordeaux mixture, have been used in unlimited high doses in vineyards for a long time. Copper rarely degrades or moves in arable soil layers, which causes the copper enrichment of vineyard soils. In some vineyards, the copper content in the soil has extended far beyond the European Union (EU) regulation limit of 140 mg/kg, in some cases reaching 1500 mg/kg (Ash, Vacek, Jaksik, Tejnecky, & Drabek, 2012; García-Esparza, Capri, Pirzadeh, & Trevisan, 2006; Mirlean, Roisenberg, & Chies, 2007). Excessive amounts of copper are transported to the grapes from the roots through the transpiration system, and can affect the quality of the grapes, grape must and wines (Mirlean et al., 2007). In addition, bronze wine brewing equipment and the copper sulphate or copper citrate added to wines to reduce the odour caused by hydrogen sulphide, mercaptan, etc. will also introduce copper into wines (OIV, 2013). The EU and South African (S.A.) have stipulated that the copper content in grape must not exceed 20 mg/l and 1 mg/l ⇑ Corresponding author. 1

E-mail address: [email protected] (J. Zhan). These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.foodchem.2015.06.107 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

in wines (Ferreira, Toit, Toit, Toit, & Toit, 2006; García-Esparza et al., 2006). However, the copper content exceeding this limit occurs occasionally in grape must and wines. García-Esparza et al. (2006) found that approximately 13% of grapes and 18% of wines exceeded the maximum copper residue limit in Italy. This has also occurred in Piedmont, Australian and in China (Marengo & Aceto, 2003; Sauvage, Frank, Stearne, & Millikan, 2002; Sun et al., 2015a). Because the Bordeaux mixture pesticide is difficult to replace in a short period of time, the copper contents of vineyard soils and grapes will continue to increase. At low concentrations, copper is a necessary trace element that plays an important positive role for nearly all organisms (Azenha, Vasconcelos, Moradas-Ferreira, Toit, & Toit, 2000; Ferreira et al., 2006). However, in excess of the beneficial range, it can have inhibitory effects on cells, even toxicity (Robinson & Winge, 2010). In wine making, a high copper content also affects the wine fermentation process and quality. Studies have shown that when the concentration of copper is greater than 20 mg/l, the growth of Saccharomyces cerevisiae (S. cerevisiae) is inhibited, which delays fermentation and reduces alcohol production. Moreover, a series of reactions that influence the quality of wines such as copper browning and wine oxidative browning can occur (Azenha et al., 2000; Ferreira et al., 2006; Robinson & Winge, 2010). Excessive amounts of copper in grapes will result in a high copper surplus in wines. If wines contain a high copper content, it could be

44

X. Sun et al. / Food Chemistry 192 (2016) 43–52

harmful to the health of consumers, particularly in combination with other heavy metals such as iron, manganese, zinc, nickel, lead and scandium (Naughton & Petróczi, 2008). However, if copper has any effect on S. cerevisiae and wine production or not, when the concentration in grapes is less than 20 mg/l, remains unclear. The current method to remove excessive copper in wines is to add an adsorbent material, such as glue, and then remove it by filtration. For example, wine makers add potassium ferrocyanide to wines, which combines with copper and iron ions forming insoluble compounds that precipitate and can be removed by filtering. However, when the iron ions in wines are not sufficient, highly toxic cyanide may remain in wine, which is detrimental to consumer health (Benítez, Castro, & Barroso, 2002). The OIV also allows the addition of gum arabic, bentonite, polyvinylimidazole and polyvinylpyrrolidone copolymers, chitin and chitosan to wines to reduce excessive copper, but these additives affect wine sensory qualities to different degrees (Benítez et al., 2002). In recent years, it has been found that yeast has the ability to adsorb heavy metals. In particular, S. cerevisiae has shown good adsorption for several heavy metal ions (Brady & Duncan, 1994; Chen, Wen, & Wang, 2014; Suh et al., 1998; Vasudevan, Padmavathy, & Dhingra, 2002, 2003). Using S. cerevisiae to complete alcoholic fermentation and remove redundant copper ions simultaneously could not only ensure the safety and quality of wines but also help to retain their original colours and flavours. Additionally, the use of this organism conforms to the requirements of organic wine production, and, is thus, an effective method and functions in environmental protection. Yeast adsorption of heavy metals can be accomplished through non-biological (dead yeast) adsorption and biological (living yeast) adsorption. Living yeast adsorption can be further divided into extracellular and intracellular adsorption (Chen et al., 2014). There have been a number of reports on yeast biological adsorption of heavy metals, mainly focused on the factors which influence the heavy metal adsorption properties (Brady & Duncan, 1994; Vasudevan et al., 2002), dynamic models of adsorption (Vasudevan et al., 2003), adsorption mechanisms (Suh et al., 1998), the pretreatment and immobilisation of the biological adsorbent, the cell morphology and subcellular structure changes (Chen et al., 2014; Sun et al., 2015b), et al. Most of these reports pertained to industrial wastewater treatment systems. Very few reports exist on wine fermentation systems, and the few papers have focused only on the adsorption rate and adsorption capacity (Ferreira et al., 2006; Vasudevan et al., 2003). At present, in wine fermentation systems, yeast adsorption of heavy metals has been mainly through dead yeast adsorption or living yeast adsorption. To our knowledge, this is the first report of extracellular or intracellular living yeast adsorption of copper. For this study, we chose one industrial S. cerevisiae strain (AWRI R2) and one laboratory S. cerevisiae strain (BH8) which showed both good copper resistance and copper adsorption capacity in our preliminary experiment (Sun et al., 2015b). We studied the growth activity and fermentation performance of these strains under a copper concentration of less than 20 mg/l copper to gain a better understanding of the effects of low concentrations of copper on S. cerevisiae and wine production. Furthermore, the dead yeast adsorption and the living yeast adsorption were compared. Additionally, we used a laser scanning confocal microscope (LSCM) and Phen Green™ SK cell staining techniques to observe the fluorescence staining of living cells to differentiate the extracellular and intracellular position of the copper ions. With these results, we can better understand the copper adsorption pathway of S. cerevisiae and provide a theoretical basis for further regulation of the performance of yeast in copper adsorption during alcohol fermentation, thereby providing a potential improvement to the wine industry.

2. Materials and methods 2.1. Test strains Two S. cerevisiae strains were used. The laboratory strain, BH8 (B), was obtained from ‘‘BeiHong’’ grape, stored at the laboratory (China Agricultural University, Beijing), and identified as S. cerevisiae by the Institute of Microbiology, Chinese Academy of Sciences (Du et al., 2012; Li, Du, Xiao, & Huang, 2011). The second was an industrial strain, AWRI R2 (A; Maurivin Co., Australia), commonly used by Chinese winemakers for its good fermentation performance. Strain B showed characteristics of copper resistance and a good copper adsorption capacity in our preliminary experiment (Sun et al., 2015b). The yeasts, maintained on slants, were pre-cultured aerobically to mid-log phase (OD600 was approximately 1.4) in shaking flasks containing 60 ml YPD medium (1% yeast extract, 2% peptone, and 2% glucose) at 28 °C, 120 r/m (Du et al., 2012). 2.2. Medium and reagents A model synthetic medium (MSM), simulating components of standard grape juice, was used to study the fermentation characteristics of wine yeast (Marullo, Bely, Masneuf-Pomarede, Aigle, & Dubourdieu, 2004). Standards of D-trehalose, D-glucose, D-fructose, sulphuric acid, and ethanol (chromatographically pure) were obtained from Sigma–Aldrich (St. Louis, MI, USA). Phen Green™ SK (Diacetate), Phen Green™ SK (Dipotassium Salt), and Ò HEPES buffer (pH 7.2–7.5) were obtained from Molecular Probes (Life Technologies Co., USA).

2.3. Fermentation experiments CuSO4
5H2O was added to the MSM in a graded Cu2+ series of 0 mM (control), 0.15 mM (9.6 mg/l), 0.20 mM (12.8 mg/l), 0.25 mM (16.0 mg/l) and 0.30 mM (19.2 mg/l). Four millilitres of yeast, pre-cultured in YPD medium to mid-log phase (OD600 was approximately 1.4), was inoculated in 500 ml flasks containing 400 ml MSM to obtain a density of 5  105 cells/ml (Li et al., 2011). Flasks were sealed with glass capillary stoppers and filled with concentrated H2SO4 to prevent weight loss due to water evaporation. Cultures were constantly shaken at 120 r/m at 28 °C in a thermostatic shaker (Du et al., 2012). Fermentation experiments were separated into two groups: one group for weighing, and another group for sampling. The experiments were conducted in triplicate and samples were taken regularly and stored in 40 °C before testing. 2.4. Determination of cell growth and fermenting property Cell growth was quantified by measuring the OD600 of the fermenting MSM with a UV1800 spectrophotometer (Shimadzu, Japan) (Liu et al., 2015; Sun et al., 2015b). MSM, free of Cu2+, was used as the blank control. The fermenting property was measured using the carbon dioxide (CO2) weightlessness method (Brandolini et al., 2002). Mass loss caused by CO2 evolution was monitored by weighing the fermentation flasks every 24 h. Fermentation was considered to have stopped when the mass loss was less than 0.02 g for 3 days. 2.5. Determination of the alcohol produced by the fermentation and the reducing sugar residual after metabolism The remaining reducing sugars and ethanol content in samples during alcoholic fermentation were determined using A Waters

X. Sun et al. / Food Chemistry 192 (2016) 43–52

Alliance 2695 HPLC system with Waters 2414 RID Detector (Waters Corp., Milford, Massachusetts, USA) and an Aminex HPX-87H ion exchange column (300  7.8 mm, BIO-RAD, Berkeley, California, USA) protected with a Bio-Rad guard cartridge (30  4.6 mm, BIO-RAD, Berkeley, California, USA) (the column was eluted with 5 mM sulphuric acid (H2SO4) at 55 °C, 0.5 ml/min, sample quantity 10 ll) (Du et al., 2012). 2.6. Copper content determination 2.6.1. Sample pretreatment Fermentation broth (10 ml) was filtered with a 0.45 lm water membrane and the filtrate was collected into a 10 ml centrifuge tube. The wet digesting method was used. Briefly, 4 ml fermentation broth was placed into a 50 ml conical flask and was baked in a dust-free oven at 105 °C until sticky. Following baking, 5 ml HNO3–HClO4 (4:1, excellent level of purity) was mixed into the conical flask, which was then covered with a lid and heated at 80 °C for 2 h on an electric hot plate, then 120 °C for 2 h, then 190 °C until the mixture did not emit white smoke and the solution was colourless or light yellow. After this, the mixture was cooled to the ambient temperature and diluted to 25 ml with ultrapure water (diluted multiples 6.25). All glass wares were soaked overnight with 20% HNO3, washed with ultrapure water and dried prior to use (Katona, Abranko, & Stefanka, 2012). After fermentation, the yeast mycelium were collected using a centrifuge, washed twice with deionized water, and weighed after drying to a constant weight at 105 °C. A suitable amount of dried yeast mycelium was then diluted with ultrapure water to 25 ml after wet digestion. The wet digesting method was same to the method used for the fermentation broth. 2.6.2. ICP-OES determination The copper content was detected using the inductively coupled plasma optical emission spectrometer (ICP-OES, Optima 7000DV, Perkin Elmer) method (Yilmaz, Arslan, Hazer, & Yilmaz, 2014). Diluted copper standard stock solutions at different concentrations (0.00, 5.00, 10.00, 20.00 mg/l series copper standard solution with 5% HNO3) were used to draw the standard curve. The instrument drew the standard curve automatically, and the correlation coefficient of the standard curve was greater than 0.9999. 2.7. Determination of the copper removal ratio (g) and copper adsorption efficiency (A) of S. cerevisiae The copper removal ratio g (%) and the adsorption efficiency A (mg/g) of yeast were calculated according to the equations g = (C0 C1)  100%/C0 and A = (C0 C1)  V/M, where C0 and C1 are the initial and final Cu concentrations of the MSM ferment, respectively, V represents the volume of the sample, and M represents the dry weight of yeast separated by centrifuge from the sample (Zu, Zhao, & Hu, 2006). 2.8. Determination of copper adsorption by dead yeast Strains A and B were cultured in 400 ml MSM medium at 28 °C, shaking at 120 r/m to the OD600 maximum (OD600 was approximately 2.1). After this, the yeast mycelium were collected by centrifuge, re-suspended in a sterile phosphate buffer, and sterilized at a high temperature for a short time to death. This solution was then diluted with different concentration of Cu2+ (as fermentation experiments) and sterile phosphate buffer to 400 ml, and shaken at 28 °C and 120 r/m. Samples were collected regularly and stored in 40 °C before testing.

45

2.9. Determination of extracellular and intracellular copper positioning The determination of copper positioning was performed on strain B. Strain B was cultured in MSM with different concentrations of Cu2+, and 4 ml samples were collected at 24 h and 48 h. For extracellular (cell surface) copper positioning dyeing, 100 ll fermentation broth was placed on a microscope slide, and 100 ll Phen Green™ SK (Dipotassium Salt) (30 lM) was added and the slide held for 30 min. For intracellular copper positioning dyeing, 100 ll fermentation broth was placed on a microscope slide, and 100 ll Phen Green™ SK (Diacetate) (7 lM) was added. After 30 min, the excess dye solutions were washed away and the slides were incubated for 30 min with HBSS buffer solution. The microscope slide was placed under an LSCM (Leica TCS SP5, Leica, Germany), the lamp-house wavelength was adjusted to 488 nm, and the fluorescence intensity was measured at 505 nm (Shingles, Wimmers, & McCarty, 2004; Zu et al., 2006). 2.10. Statistical analysis Experimental results are presented as means ± SD of three parallel measurements. Statistical analyses were performed using the program PASW Statistics 18. 3. Results 3.1. Influence of copper stress on cell growth The results of cell growth and survival rate of strains A and B in MSM with different copper concentrations are presented in Fig. 1(A, B). Under all copper concentration, strains A and B grew well. After the strains were pre-cultured in YPD medium to mid-log phase and inoculated on the MSM with copper stress, the two strains continued logarithmic growth, without readapting to the stress environments, and entered the stable phase on the 4th day (OD600 was approximately 2.0). With different copper concentrations, the cell growth conditions were slightly different, and the growth of the yeast was slower as the initial copper concentration increased. However, there was no significant difference in the ultimate OD600 values, indicating that when the concentration is less than 20 mg/l, copper has a negligible impact on the cell growth of S. cerevisiae. With different strains, the growth speed, and the amount of yeast strain B was higher than strain A; thus, strain B had better copper resistance. In the 19.2 mg/l copper treatment, the fermentation period of strain A lasted 77 day and there was no delay in entering the stable phase (the OD600 of the 4th day was approximately 1.8). This indicated that under the 19.2 mg/l copper concentration, the alcoholic fermentation property was restrained, but the cell growth of the strain was not restrained. 3.2. Influence of copper stress on fermenting property The fermenting property of strains B and A is presented in Fig. 1(C, D). In all treatments, the fermenting property of S. cerevisiae was lower than in the control group. In general, the fermenting property of the yeast was weaker with the initial copper concentration increased. In the control group, the fermentation of strains B and A concluded on the 18th day, and the production of CO2 was approximately 36 g; therefore, strains B and A both had an adequate and consistent fermenting property. Compared with the results of cell growth (Fig. 1(A, B)), even the when the concentration was less than 20 mg/l, copper impacted the fermenting property of S. cerevisiae.

46

X. Sun et al. / Food Chemistry 192 (2016) 43–52

Fig. 1. Changes of cell growth (OD600) (A, B) and fermentation efficiency (mass loss caused by CO2) (C, D) during fermentation under different copper concentrations.

For strain B, under all copper concentrations, only the 19.2 mg/l treatment significantly delayed (day 45) the fermentation and reduced the CO2 release (14% less than the control). The trends of fermentation of the other three concentrations were similar, and the fermenting property was inversely proportional to the copper concentration. The fermentation of the 9.6 mg/l and 12.8 mg/l groups concluded on the 18th day, and the 16.0 mg/l group extended to the 26th day. In addition, the diffusion of CO2 of the 9.6 mg/l, 12.8 mg/l and 16.0 mg/l groups showed no significant difference with the control group. With copper treatment, the fermenting property of strain A was significantly weaker than strain B. The 9.6 mg/l treatment extended the fermentation period. When the initial concentration of copper increased to 12.8 mg/l, the diffusion of CO2 (33.87 g) was noticeably lower than in the control group (35.90 g). Additionally, with the 16.0 mg/l treatment, the fermentation showed a significant inhibition at the early stage (1–25 days), and the fermentation period lasted 58 days. With the 19.2 mg/l treatment, the yeast showed an adjustment period at the beginning of fermentation. The diffusion of CO2 was only 16.75 g, and the fermentation period lasted to 77 days; however, the final diffusion of CO2 was consistent with the 16.0 mg/l treatment. In general, 9.6–12.8 mg/l copper concentrations showed indistinctive inhibition on the fermenting property of strains B and A. The 16.0 mg/l copper concentration had no effect on the fermenting property of the copper resistant strain B, but significantly inhibited the fermenting property of strain A. Both copper resistant strain B and industrial strain A’s fermenting properties were significantly inhibited under 19.2 mg/l.

3.3. Influence of copper stress on the reducing sugar utilisation and alcohol production The HPLC chromatography of the standard of the fermentation products is shown in Appendix. The calibration curves are as follows, D-trehalose: y = 980574x 1589.4 (R2 = 0.9998); D-glucose: y = 133468x 2648.1 (R2 = 0.9999); D-fructose: y = 15346 3x 2515.5 (R2 = 0.9999); ethanol, y = 393515x + 4262.4 (R2 = 0. 9994). The correlation coefficients of standard curve were greater than 0.9999 and showed a strong linear relationship. The effect of copper stress on the reducing sugar utilisation and alcohol production is shown in Fig. 2(A, B). Along with the fermentation, the reducing sugar content declined. The speed of the decline and the total reduction of reducing sugar were inversely proportional to the copper concentrations, but was in synchrony with the trend of CO2 release (Fig. 1(C, D)). In the control group, the reducing sugar content was stable at the lowest level on the 10th day. Similar to the result of CO2 release (Fig. 1(C, D)), strain B showed a higher utilisation of reducing sugar than strain A. In the 19.2 mg/l treatment group, the reducing sugar was not completely used (residual sugar was 29.69 g/l) by strain B; however, under the 16.0 mg/l treatment, the residual sugar of strain A was high (39.00 g/l). The effect of copper stress on alcohol production is shown in Fig. 2(C, D). In agreement with the results of CO2 release (Fig. 1(C, D)) and reducing sugar utilisation (Fig. 2(A, B)), under copper stress, both the speed and the amount of alcohol production of S. cerevisiae were inhibited. Furthermore, the restraining level was consistent with the copper concentration, which was different from the results of the cell growth experiment (Fig. 1(A, B)).

X. Sun et al. / Food Chemistry 192 (2016) 43–52

47

Fig. 2. Changes of ethanol concentrations (A, B) and reducing sugar concentrations (C, D) during fermentation under different copper concentrations.

Under the 9.6 mg/l treatment, the alcohol production of strains A and B were the same as the control group (10.90% or so), indicating that 9.6 mg/l copper had an unnoticeable influence on alcohol production. In the 19.2 mg/l treatment, the alcohol generation was the slowest and lowest; the alcohol yield on the 21st day was only 8.74% for strain B and 4.34% for strain A, and 9.63% for strain B and 9.19% for strain A when the fermentation stopped. Under different copper concentrations, the trend of the alcohol generation curve of strain B was similar; all groups increased rapidly for the first 10 days, and then slowly increased. In addition, the alcohol content of 0–16.00 mg/l was approximate, which indicated that when the copper concentration was less than 16 mg/l, it only delayed the fermentation, but did not reduce the amount of ethanol production of strain B. Only the 19.2 mg/l treatment significantly reduced the amount of ethanol production of strain B. In contrast, under different copper concentrations, the trend of the alcohol generation curve of strain A differed significantly. The 12.8 mg/l copper treatment not only delayed the fermentation but also reduced the amount of ethanol production. For the 12.8 mg/l and 16.0 mg/l treatments, the rapid growth in the early stage of fermentation was very short (0–3 days), the alcohol generation was very slow, and the fermentation period was greatly extended. Under the 16 mg/l treatment, the trend of the alcohol generation curve still basically conformed to the logarithmic growth curve. However, for the 19.2 mg/l treatment, the growth of the alcohol content was briefly stalled at the fermentation metaphase, and the rate of increase was severely lower after this stagnation. After the long-term slow growth in late fermentation, the alcohol content increased to the level of the 16.0 mg/l group. All these data indicated that a copper content of 19.2 mg/l severely inhibited the alcohol fermentation of strain A. In general, the performance of reducing sugar utilisation and alcohol production of the copper resistant strain B were much better than the industrial strain A. For strain A, the 16.0 mg/l copper

concentration was unacceptable to finish wine fermentation in a timely manner. For strain B, 16.0 mg/l was acceptable, but 19.2 mg/l was too high to finish wine fermentation in a timely manner. 3.4. Copper adsorption by S. cerevisiae in MSM during the fermentation process The results of the copper adsorption of S. cerevisiae in MSM during the fermentation process are shown in Fig. 3. In all treatment groups, the copper content dropped rapidly at the early stage of fermentation and increased slowly as fermentation proceeded (Fig. 3(A)). At 3–5 days, the rate of decline in the copper content was positively correlated with the initial copper concentration and also with the final copper content after fermentation. After 5 days, the copper content of strain B groups increased rapidly; the rebound speeds of the 9.6–16.0 mg/l treatments were nearly identical. In contrast, in the 19.2 mg/l group, because the fermentation period was greatly extended, the copper content showed a fluctuating trend in late fermentation. Compared with the lowest copper content during fermentation, the extent of the rebound in final copper content after fermentation was smaller. The rate of decline in copper content for strain B was faster than strain A, and the rebound speed and its extent in late fermentation was smaller than strain A. Therefore, strain A had faster and more stable copper removal ability. Fig. 3(B, C) shows the copper removal ratio (g) of S. cerevisiae in MSM after the fermentation process. For strain B, the copper removal ratio of the different copper concentration groups showed no significant differences; the g value were all near 60%. For strain A, the highest copper removal ratio was in the 12.8 mg/l treatment group (81%), while the copper removal ratio of the 9.6 mg/l group agreed with 16.0 mg/l group (76%), and the copper removal ratio of the 19.2 mg/l group was the lowest (72%), though still higher than

48

X. Sun et al. / Food Chemistry 192 (2016) 43–52

Fig. 3. Changes of the copper content (A), the copper removal ratio (g) (B, C) and the adsorption efficiency of copper (D) under different copper concentrations.

strain B. In general, strain A’s copper removal rate was higher than that of strain B. Fig. 3(D) shows the copper adsorption efficiency (A) of S. cerevisiae in MSM after the fermentation process. As shown in Fig. 3(D), the copper adsorption efficiency was not in strict accordance with the results of the copper removal ratio. Under different copper concentrations, the copper adsorption efficiency of strain B was different; the higher the initial copper concentration, the higher the adsorbed copper per unit. For strain A, except in the 16.0 mg/l treatment group, the adsorbed copper per unit strain of all other groups was lower than for strain B. The copper removal ratio (g) was defined as the change in copper content in the fermented liquid at the beginning and at the end of fermentation, and the adsorption efficiency reflected the unit strain’s adsorption of copper. The total amount and the living rate of strains were different in each treatment group, leading to inconsistent results between g and A. The speed of the decline in copper

content (Fig. 3(A)) and the removal ratio of copper (Fig. 3(B, C)) of strain A were higher than for strain B, which indicated that the copper removal ability of strain A was better. In contrast, the adsorbed copper per unit strain (Fig. 3(D)) of strain A was lower than strain B. One likely cause for this was that the cell specific surface area and the survival rate of strain A were lower than strain B at the same weight. This phenomenon was in agreement with the result that cell growth of strain A was weaker than strain B (Fig. 1(A, B)). All these results indicate that the copper removal and adsorption mechanism of S. cerevisiae is complex and related to many factors such as S. cerevisiae strains, the living yeast quantity, the cell specific surface area and the survival rate. 3.5. Copper adsorption by dead yeast Fig. 4(A, B) shows the changes in copper content of when dead yeast strains B and A were used in sterile phosphate buffer with

X. Sun et al. / Food Chemistry 192 (2016) 43–52

different concentrations of copper during shaking. Fig. 4(C) shows the changes of the removal ratio of copper. In the experiment, the samples were carried through until the end of fermentation (same to Fig. 1(C, D)), but the results showed no significant changes from 3 to 4 days, so only the results of the prophase are shown in Fig. 4. Statistical analysis showed a significant difference before adding dead yeast mycelium (0 day) and after adsorption (4 day for strain B, 3 day for strain A) with copper content (P < 0.01), which indicates that dead yeast mycelium were able to adsorb the copper in the sterile phosphate buffer, and demonstrated that S. cerevisiae do have a non-biological copper adsorption for capacity. Compared with Fig. 3, it was obvious that copper adsorption by dead yeast was very low. The removal ratio of strain B was 1.05– 19.93%, and strain A was 17.07–23.36%. The speed and removal ratio of strain A was faster and larger than strain B. In the different copper concentrations groups, the removal ratio of strain A was basically the same (approximately 20%), which was higher than for strain B. In the 16.0 mg/l and 19.2 mg/l groups, the removal ratio of strain B was significantly lower. A reason for this might be that the adsorption sites were saturated. In general, the adsorption property by the dead yeast of strain A was more stable, and the non-biological adsorption capacity for copper of strain A was stronger than strain B. 3.6. Extracellular and intracellular copper positioning As strains B and A are both S. cerevisiae, we chose only to use only strain B to analyse the adsorption pathway. The results of the experiment testing extracellular and intracellular copper positioning after 24 h and 48 h are shown in Fig. 5. The concentration of copper was consistent with the degree of green fluorescence quenching (inconsistent with the fluorescence intensity) (Shingles et al., 2004). As in Fig. 5, in all copper concentration treatment groups, the fluorescence intensity of extracellular adsorption was higher than intracellular accumulation, suggesting that the copper concentration in intracellular was higher than in extracellular. This indicates that under copper stress, S. cerevisiae relied more on intracellular accumulation. The higher the initial concentration of copper stress, the lower the intracellular green fluorescence. This behaviour showed that for groups under 9.6–19.2 mg/l copper stress, the higher the initial copper concentration, the adsorbed copper per unit strain was higher, which was inconsistent with the results of the copper adsorption efficiency (Fig. 3(D)) assay. At 24 h, with the initial copper concentration increasing, the fluorescence intensity of the extracellular test decreased; the fluorescence intensity of intracellular decreased too, but not as much. At 24 h, with an increase in the initial copper concentration, the intracellular accumulation and extracellular adsorption groups both increased; however, the extracellular adsorption group increased more. After 48 h, the fluorescence intensity of the extracellular group was larger than that of the intracellular group, where the green fluorescence was almost all quenched. This result showed that in 24–48 h, copper moved from the cell surface into intracellular spaces, and the copper on the cell surface was reduced. This indicated that under copper stress, the biological adsorption pathway of S. cerevisiae might be to adsorb copper on the cell surface first, and then transport it to intracellular spaces. The extracellular adsorption occurred relatively quickly (the time was short), and achieved a balance faster; in contrast, the intracellular accumulation was an ongoing slow process. This conformed to the theory of microbial accumulation of heavy metals occurs in two steps, firstly through rapid extracellular adsorption, then slower intracellular accumulation (Brady & Duncan, 1994; Norris & Kelly, 1977).

49

4. Discussion Under copper stress, strain B had better cell growth and a better fermenting property than strain A. This might be related to the growing environment of the strains; the laboratory strain B was subjected to a rigorous and adverse environment (separated from Chinese wine grape ‘‘BeiHong’’, which was grown in a cool climate and under summer rains, and the Bordeaux mixture pesticide was largely used) and showed resistance to many different stresses (Du et al., 2012; Li et al., 2011). The industrial strain A was from Australia, where the climate was semi-arid and fungal diseases rarely occur, as a result the Bordeaux mixture pesticide was used less. Under 9.6–19.2 mg/l copper stress, copper has little impact on the cell growth of strains B and A, which was different from the results of high copper stress (32–96 mg/l) (Du, Li, Li, Zhan, & Huang, 2010; Sun et al., 2015b). Therefore, within the limitation of the regulations of the EU and S.A. (620 mg/l), copper has no effect on the cell growth of S. cerevisiae. In contrast, though the cell growth was good, the fermentation performance of strains B and A were inhibited to some extent. All results of the fermenting property (Fig. 1(C, D)), the alcohol production (Fig. 2(A, B)), and reducing sugar utilisation (Fig. 2(C, D)) showed that the fermentation performance of strains B and A were reduced with increasing copper concentration. Overall, strain B performed better than strain A, which agreed with our results for high copper stress (32–96 mg/l) (Du et al., 2010; Sun et al., 2015b). For strain B, 19.2 mg/l copper stress obviously inhibited alcoholic fermentation; while for strain A, under 12.8 mg/l the alcoholic production was reduced. In general, copper stress under the limitation of the regulations of the EU and S.A. (620 mg/l) might not guarantee safety in wine production; therefore, the limitation should be further reduced (612.8 mg/l). Donmez and Aksu (1999) and Volesky and Holan (1995) reported that S. cerevisiae could remove copper effectively (the unit strain’s adsorption on copper was 2–40 mg/g). In the current experiment, under 9.6–19.2 mg/l copper stress, S. cerevisiae could also remove copper; the copper removal ratio and the unit strain’s adsorption were 60–81% and 2.72–9.65 mg/g, respectively. In terms of the copper removal ratio, strain A was better; for the unit strain’s adsorption, strain B was better. In Brandolini et al. (2002), the cell growth and fermentation performance of a copper resistant yeast strain behaved better than the normal yeast strain, and could absorb more copper ions. In contrast, in this experiment, strain B behaved better in terms of cell growth and fermentation performance under copper stress, but for the copper removal ratio, strain A was better. Sun et al. (2015b) also reported similar results. This difference might be related to particular features of the tested strains. With different initial copper concentrations, the copper adsorption of yeast strains was different. In Brady and Duncan (1994), the adsorption capacity of S. cerevisiae was related to the ratio of copper concentration and biomass. In general, the initial concentration of copper increased, and then the adsorption quantity increased. With the increase of adsorption site saturation, the adsorption gradually stopped (Brady & Duncan, 1994). In this experiment, under 9.6–19.2 mg/l copper stress, with the increase in the initial concentration of copper, the copper toxicity increased and the biomass of the yeast decreased. As a result, the copper removal ratio was reduced, but the unit strain’s adsorption increased. This might be because under 9.6–19.2 mg/l copper stress, the adsorption sites of S. cerevisiae were still unsaturated. In the test of copper adsorption by dead yeast in the sterile phosphate buffer, the copper concentration obviously decreased, and then slowly increased (Fig. 4(A, B)). Different from living yeast

50

X. Sun et al. / Food Chemistry 192 (2016) 43–52

Fig. 4. Changes of copper concentration of buffer with dead cells (A, B), and the removal ratio of copper for dead cells (C) under different copper concentrations.

with biological adsorption, there was no issue of biological absorption stopping for the dead yeast. One possible reason was that the morphology of dead yeast cells could not be maintained for a long time; after cell decomposition, the cell wall structure was damaged and the adsorption sites that could bind copper was reduced, resulting in released copper (Kratochvil & Volesky, 1998). Fig. 4 demonstrates that S. cerevisiae do have a non-biological adsorption pathway for copper, but compared with the biological (living yeast) adsorption (Fig. 3(A)), the copper adsorption was very low. Hence, the copper adsorption ways of S. cerevisiae were mainly by biological (living yeast) adsorption. This could explain why the copper content dropped rapidly at the early stage of fermentation and increased slowly as the fermentation progressed (Fig. 3(A)). For the biological (living yeast) adsorption mechanism of S. cerevisiae to copper in a waste water environment, Brady and Duncan (1994) reported that the process was divided into two steps; the first step was rapid, usually 10 min, while the second step was quite slow, especially when the concentration ratio of copper to yeast was less than 100 nmol/mg. When this situation arose, the second step did not occur. Donmez and Aksu (1999) also reported that the mechanism of living yeast adsorbing copper was through two independent processes, the first a quick adsorption process. In this first process, copper adsorbed to the cell surface and did not require metabolism to provide energy. The second process was related to metabolism and was much slower, with copper accumulating in the cell. In this experiment, the non-biological (dead yeast) adsorption and biological (living yeast) adsorption both achieved a maximum within 24 h (Figs. 3(A) and 4(A, B)), performed as a rapid adsorption, but were saturated in a low concentration. All of these trends conform to the characteristics of the first stage of biological accumulation process (Norris & Kelly, 1977). The

copper accumulated in intracellular spaces and increased with time (Fig. 5). After 24 h of this fermentation and onwards, the removal speed was declining, the removal process existed until the late fermentation (Fig. 3(A)). This phenomenon conformed to the characteristics of the second stage of the biological accumulation process, intracellular accumulation (Norris & Kelly, 1977). Different from the present experiment’s results, Sun used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) combined with an electronic differential system (EDS) to analyse the elemental profiles of extracellular and intracellular surfaces of S. cerevisiae cells and did not find intracellular copper (Sun et al., 2015b). This might be due to the fact that before direct electron microscope observation was possible, a metal had to be sprayed onto the sample, used to mask to copper element, and the detection limit of EDS was higher. It was not possible to prove that no copper had accumulated in the intracellular space. On the other hand, their investigation showed that LSCM is more suitable for metal adsorption research than the EDS analysis. With regarding to the pathway of S. cerevisiae transported copper into intracellular, some research showed that this might be associated with an ion exchange mechanism. During the process of yeast absorbing copper, 70% of the potassium content of the yeast cell was quickly released, and then 60% magnesium ion quickly released (Brady & Duncan, 1994). A SEM-EDS observation found that when the copper stress reached 96 mg/l, the yeast had nitrogen on the surface, and the nitrogen peak signal increased with time, indicated that the copper on the cell surface might be complexed with the nitrogen groups (Sun et al., 2015b). Fernandes, Peixoto, and Sá-Correia (1998) reported that the H+-ATPase activity of yeast plasma membrane increased under copper stress. Because the H+-ATPase of yeast plasma membrane could produce the transmembrane proton electromotive force by

X. Sun et al. / Food Chemistry 192 (2016) 43–52

51

Fig. 5. Copper distribution of yeast strain B after fermentation for 24 h and 48 h. A: 0.00 mg/l (control); B: 9.6 mg/l; C: 12.8 mg/l; D: 16.0 mg/l; E: 19.2 mg/l.

pumping H+, and activate the other ion channels of yeast to promote the ions into the cell (Morsomme & Boutry, 2000). As a result, as copper could promote the H+-ATPase activity, and provide a means for copper exchange into intracellular through ion exchange mechanism. In summary, within the limitation of the regulations of the EU and S.A. (620 mg/l), copper had no effect on the cell growth of S. cerevisiae, but its fermentation performance was inhibited to some extent. Therefore, the limitation should be further reduced (612.8 mg/l). Under 9.6–19.2 mg/l copper stress, S. cerevisiae could remove copper to a certain extent; and the copper removal ratio and the unit strain adsorption were 60–81% and 2.72–9.65 mg/g, respectively. S. cerevisiae can perform a non-biological adsorption

of copper, but compared with biological (living yeast) adsorption, the non-biological adsorption was very low. Thus, the copper adsorption way of S. cerevisiae was mainly via biological (living yeast) adsorption. The biological (living yeast) copper adsorption pathway of S. cerevisiae is a two-step process. The pathway of S. cerevisiae intracellular copper transport might be associated with an ion exchange mechanism, but this requires further in-depth study. Acknowledgement This study was supported by the National Nature Science Foundation Project (31471835).

52

X. Sun et al. / Food Chemistry 192 (2016) 43–52

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2015. 06.107.

References Ash, C., Vacek, O., Jaksik, O., Tejnecky, V., & Drabek, O. (2012). Elevated soil copper content in a bohemian vineyard as a result of fungicide application. Soil and Water Research, 7, 151–158. Azenha, M., Vasconcelos, M., Moradas-Ferreira, J., Toit, M., & Toit, P. (2000). The influence of Cu concentration on ethanolic fermentation by Saccharomyces cerevisiae. Journal of Bioscience and Bioengineering, 90, 163–167. Benítez, P., Castro, R., & Barroso, C. (2002). Removal of iron, copper and manganese from white wines through ion exchange techniques: Effects on their organoleptic characteristics and susceptibility to browning. Analytica Chimica Acta, 458, 197–202. Brady, D., & Duncan, J. (1994). Bioaccumulation of metal cations by Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 41, 149–154. Brandolini, V., Tedeschi, P., Capece, A., Maietti, A., Mazzotta, D., Salzano, G., et al. (2002). Saccharomyces cerevisiae wine strains differing in copper resistance exhibit different capability to reduce copper content in wine. World Journal of Microbiology and Biotechnology, 18, 499–503. Chen, C., Wen, D., & Wang, J. (2014). Cellular surface characteristics of Saccharomyces cerevisiae before and after Ag(I) biosorption. Bioresource Technology, 156, 380–383. Donmez, G., & Aksu, Z. (1999). The effect of copper (II) ions on the growth and bioaccumulation properties of some yeasts. Process Biochemistry, 35, 135–142. Du, J., Li, H. L., Li, H., Zhan, J. C., & Huang, W. D. (2010). Influence of copper stress on Saccharomyces cerevisiae grape must. Scientia Agricultura Sinica, 43, 3259–3265. Du, G., Zhan, J. C., Li, J. Y., You, Y. L., Zhao, Y., & Huang, W. D. (2012). Effect of fermentation temperature and culture medium on glycerol and ethanol during wine fermentation. American Journal of Enology and Viticulture, 63, 132–138. Fernandes, A., Peixoto, F., & Sá-Correia, I. (1998). Activation of the H+-ATPase in the plasma membrane of cells of Saccharomyces cerevisiae grown under mild copper stress. Archives of Microbiology, 171, 6–12. Ferreira, J., Toit, M., Toit, J., Toit, M., & Toit, W. (2006). The effects of copper and high sugar concentrations on growth, fermentation efficiency and volatile acidity production of different commercial wine yeast strains. Australian Journal of Grape and Wine Research, 12, 50–56. García-Esparza, M. A., Capri, E., Pirzadeh, P., & Trevisan, M. (2006). Copper content of grape and wine from Italian farms. Food Additives & Contaminants, 23, 274–280. Katona, R., Abranko, L., & Stefanka, Z. (2012). Comparison of sample preparation techniques for multielemental analysis of wine samples by ICP-MS. Acta Alimentaria, 41, 83–91. Kratochvil, D., & Volesky, B. (1998). Advances in the biosorption of heavy metals. Trends in Biotechnology, 16, 291–300.

Li, J., Du, G., Xiao, Y., & Huang, W. (2011). Effect of proanthocyanidins on yeast metabolism, H+-ATPase activity, and wine fermentation. American Journal of Enology and Viticulture, 62, 512–518. Liu, X. Y., Jia, B., Sun, X. Y., Ai, J. Y., Wang, L. H., Wang, C., et al. (2015). Effect of initial pH on growth characteristics and fermentation properties of Saccharomyces cerevisiae. Journal of Food Science, 80, 800–808. Marengo, E., & Aceto, M. (2003). Statistical investigation of the differences in the distribution of metals in Nebbiolo-based wines. Food Chemistry, 81, 621–630. Marullo, P., Bely, M., Masneuf-Pomarede, I., Aigle, M., & Dubourdieu, D. (2004). Inheritable nature of enological quantitative traits is demonstrated by meiotic segregation of industrial wine yeast strains. FEMS Yeast Research, 7, 711–719. Mirlean, N., Roisenberg, A., & Chies, J. (2007). Metal contamination of vineyard soils in wet subtropics (southern Brazil). Environmental Pollution, 149, 10–17. Morsomme, P., & Boutry, M. (2000). The plant plasma membrane H+-ATPase: Structure, function and regulation. Biochimica et Biophysica Acta-bioenergetics, 1465, 1–16. Naughton, D., & Petróczi, A. (2008). Heavy metal ions in wines: Meta-analysis of target hazard quotients reveal health risks. Chemistry Central Journal, 3, 1–7. Norris, P., & Kelly, D. (1977). Accumulation of cadmium and cobalt by Saccharomyces cerevisiae. Journal of General and Applied Microbiology, 99, 317–324. OIV (2013). International code of oenological practices (Issue 2013/01 revised). ISBN: 979-10-91799-10-2. Robinson, N., & Winge, D. (2010). Copper metallochaperones. Annual Review of Biochemistry, 79, 537–562. Sauvage, L., Frank, D., Stearne, J., & Millikan, M. B. (2002). Trace metal studies of selected white wines: An alternative approach. Analytica Chimica Acta, 458, 223–230. Shingles, R., Wimmers, L., & McCarty, R. (2004). Copper transport across pea thylakoid membranes. Plant Physiology, 135, 145–151. Suh, J., Kim, D., Yun, J., Song, S., Kim, D., Yun, J., et al. (1998). Process of Pb2+ accumulation in Saccharomyces cerevisiae. Biotechnology Letters, 20, 153–156. Sun, X. Y., Zhao, Y., Liu, L. L., Jia, B., Zhao, F., Huang, W. D., et al. (2015b). Copper tolerance and biosorption of Saccharomyces cerevisiae during alcoholic fermentation. PLOS One. http://dx.doi.org/10.1371/journal.pone.0128611. Sun, X., Zhao, F., Ma, T., Wang, L., Wang, C., Sun, Y., et al. (2015a). Analysis of the copper content of part of China’s main wine regions. Modern Food Science and Technology, 31, 278–284. Vasudevan, P., Padmavathy, V., & Dhingra, S. (2002). Biosorption of monovalent and divalent ions on baker’s yeast. Bioresource Technology, 82, 285–289. Vasudevan, P., Padmavathy, V., & Dhingra, S. (2003). Kinetics of biosorption of cadmium on baker’s yeast. Bioresource Technology, 89, 281–287. Volesky, B., & Holan, Z. (1995). Biosorption of heavy metals. Biotechnology Progress, 11, 235–250. Yilmaz, V., Arslan, Z., Hazer, O., & Yilmaz, H. (2014). Selective solid phase extraction of copper using a new Cu(II)-imprinted polymer and determination by inductively coupled plasma optical emission spectroscopy (ICP-OES). Microchemical Journal, 114, 65–72. Zu, Y., Zhao, X., & Hu, M. (2006). Biosorption effects of copper ions on Candida utilis under negative pressure cavitation. Journal of Environmental Sciences, 18, 1254–1259.