The interactive effect of fungicide residues and yeast assimilable nitrogen on fermentation kinetics and hydrogen sulfide production during cider fermentation

Apple juice

Commercially produced pasteurized apple juice was used to ensure the consistency of juice across samples and to best represent large‐scale cidermaking practices. The juice used was White House Fresh Pressed Natural Apple Juice (National Fruit Product Co., Winchester, VA, USA). Multiple bottles of the juice were combined into one homogenous lot then stored in 1‐L aliquots at −20 °C until use, and then thawed to 22 °C prior to yeast inoculation. The juice was measured to have 12.9 °Brix by refractometer, pH 3.7 and titratable acidity 3.4 g L⁻¹ malic acid equivalent using standard methods,27 and 53 mg L⁻¹ YAN. YAN was quantified using commercially available Megazyme (Wicklow, Ireland) kits for primary amino nitrogen (K‐PANOPA) and ammonium ion concentration (Ammonia‐Rapid).

Fungicide additions

S⁰ was obtained in the form of Microthiol Disperss fungicide (80% sulfur; Nufarm Australia Ltd, Victoria, Australia) and added at concentrations of 5 and 20 mg L⁻¹ S⁰. Fludioxonil was obtained in the form of Scholar SC fungicide (20.4% fludioxonil; Syngenta Crop Protection LLC, Greensboro, NC, USA) and added at concentrations of 0.2 and 0.4 mg L⁻¹ fludioxonil. Fenbuconazole was obtained in the form of Indar 2 F fungicide (23.5% fenbuconazole; Dow Agrosciences LLC, Indianapolis, IN, USA) and added at concentrations of 2.5 and 5.0 mg L⁻¹ fenbuconazole. Commercially available juice with no fungicides added was used as a control. The final fungicide concentrations in juice were not confirmed through analysis of juice after fungicide addition, which is a limitation of the present study. Furthermore, the original juice (commercially available apple juice) was not screened for fungicide residue. The results of the present study should therefore be interpreted from the perspective that the reported concentrations of fungicides added represent an additional amount to any fungicide residue possibly present in the original juice. The USDA stated limits for fungicide residues in apple juice are 0.4 mg L⁻¹ for fludioxonil and 5.0 mg L⁻¹ for fenbuconazole, which were set as the maximum added concentrations studied in the present study. Interactive effects of YAN and fenbuconazole residues were examined by adding YAN to the apple juice at concentrations of 25 mg N L⁻¹ in the form of Fermaid K (Scott Laboratories, Inc., Petaluma, CA, USA) for the control, and further supplementing base apple juice with 100 and 200 mg N L⁻¹ using diammonium phosphate to 153 and 253 mg N L⁻¹ for the medium and high YAN concentration treatments (Scott Laboratories, Inc.). Each treatment was then supplemented with fenbuconazole at concentrations of 0 (control), 0.2 and 0.4 mg L⁻¹ each.

Fermentations

Experimental fermentations were carried out in triplicate with two yeast strains for each treatment. Prise de Mousse Saccharomyces bayanus EC1118 (Lallemand, Montreal, Canada) was selected to represent a low‐H₂S producing strain and Montrachet S. cerevisiae UCD522 (Lallemand, Montreal, Canada) was selected to represent a high‐H₂S producing strain. Juice was inoculated with 0.05 g of the active dry yeast rehydrated in 35 °C water for 20 min. Yeast nutrient was added in the form of Fermaid K (Scott Laboratories, Inc.) to all fermentations at concentrations of 25 mg L⁻¹. This added an additional 25 mg L⁻¹ nitrogen to the juice, bringing total juice YAN to 78 mg L⁻¹ for the control because the endogenous 53 mg N L⁻¹ in the apple juice was not sufficient to complete fermentation. Fermentations were carried out in accordance with the method described by Ugliano and Henschke28 for rapid determination of H₂S formation during alcoholic fermentation. These were conducted in 250‐mL Erlenmeyer flasks fitted with a one‐hole rubber stopper to which a H₂S detector tube was affixed (see below). Fermentations were not aerated but were stirred twice per day at 800 r.p.m. for 5 min to prevent yeast settling. Fermentations were carried out at 18 °C in triplicate. The fermentation rate was monitored by measuring the mass of the fermentation vessel as a proxy for CO₂ evolution. Finished cider was analyzed to determine pH, titratable acidity, residual YAN and residual sugar. Residual sugar was analyzed using a d‐fructose/d‐glucose (K‐FRUGLU) enzymatic kit (Megazyme).

H₂S detector tubes

The H₂S detection and quantification method was as described by Ugliano and Henschke.28 Detector tubes were obtained from Komyo Kitagawa (Tokyo, Japan). Tubes were inserted into a one‐hole rubber stopper to obtain a gas‐tight seal. CO₂ produced during fermentation carried H₂S through the detector tube. H₂S reacts with the lead acetate (Tube 120SB, 120SD) or silver nitrate (Tube 120SF) contained in the tube, creating a discolored band. The different tubes have different capacities for total H₂S quantification, and were selected based on total H₂S production in a given treatment as determined by preliminary experimentation. The length of the discolored band is proportional to the amount of purged H₂S. Readings were taken twice daily to determine the H₂S production rate. If, at any reading point, the tube appeared to be near saturation, the tube was replaced with a new tube. This method may have allowed a small amount of H₂S gas to escape, although it was employed consistently across treatments in the present study.

Determination of fermentation rate, duration and H₂S production rate

Maximum fermentation rate was determined by taking the slope of the fermentation curve during the exponential phase of yeast growth corresponding to the highest constant rate of CO₂ production, as reported previously.29 Steeper slopes correspond to faster fermentation rate. Fermentations were determined to be complete when the CO₂ production rate decreased to less than 0.2 g day⁻¹. Fermentation duration is expressed as total hours starting from inoculation. Total H₂S production was determined by calculating the sum of H₂S production over the time course of fermentation. To determine the relative rate of H₂S production over the time course of fermentation, the fermentation duration was divided into four quartiles of equal time. The percentage of H₂S produced in each quartile out of the total H₂S produced for a given fermentation was compared to determine whether there was a significant difference in the H₂S production over the time course of fermentation across treatments.

Statistical analysis

Values were compared using a one‐way analysis of variance (ANOVA), with P < 0.05 being considered statistically significant, followed by parametric mean testing using Tukey's honestly significant difference (HSD) using Prism, version 6 (GraphPad, La Jolla, CA, USA). Analyses comparing the interaction between yeast strain/YAN concentration and fungicide residues were analyzed using a two‐way ANOVA, with P < 0.05 being considered statistically significant, and post‐hoc testing by Tukey's HSD.

Fermentation kinetics

Total H₂S production

Relative rate of H₂S production

The time course of H₂S production during fermentation may vary because H₂S can be effectively stripped from the fermenting media by CO₂ gas evolved during fermentation,35 especially during the period of maximum fermentation rate when CO₂ evolution is also at its maximum rate. It is possible that a cider in which less total H₂S had been produced but was subsequently produced later in the time course of fermentation could retain more of the unwanted H₂S character in the final product. This could impart a greater sensory impact on the finished product. The addition of S⁰, especially at high concentrations, tended to alter the rate of H₂S production (Fig. 5A, D). The percentage of the total production of H₂S for a given treatment was greater in the third and fourth quartiles at all concentrations of S⁰ in the strain UCD522 (P < 0.05) compared to the control. At concentrations of 20 mg L⁻¹, H₂S production was greater in the third quartile in the strain EC1118 (P < 0.01). The greatest proportion of the total H₂S was produced in the second quartile for the majority of fermentations. This correlates with the logarithmic phase of fermentation, which also corresponds to the highest rate of SRS activity in the yeast, leading to the observable spike in H₂S production during the highest rate of fermentation.36 When S⁰ is present in the fermenting juice, however, H₂S is produced throughout the duration of the fermentation because SRS activity is not the primary cause of H₂S production.37 The addition of fludioxonil did not affect the rate of H₂S production (Fig. 5B, E). H₂S production decreased in the second quartile when fenbuconazole was added at 0.2 mg L⁻¹ in both yeast strains (P < 0.05) (Figs 4C, F). However, higher concentrations of fenbuconazole did not significantly impact total H₂S production. It is possible that yeast cell density is correlated with a change in the relative rate of H₂S production and that fenbuconazole may decrease total cell biomass achieved during fermentation. The mechanism by which fenbuconazole affects H₂S production may be determined in future studies by monitoring cell density throughout the time course of fermentation and corresponding H₂S production.

Interactive effect of fenbuconazole and YAN

The fermentation kinetics results of the present study clearly illustrate that residual fenbuconazole can lead to sluggish and stuck cider fermentations. Insufficient YAN is also widely known to result in stuck and sluggish fermentations.9 The generally accepted minimum recommended concentration of YAN to successfully complete fermentation is 140 mg L⁻¹ for wine.38 However, this recommendation is not universally accepted. The optimal concentration of pre‐fermentation YAN required for a given fermentation is difficult to determine as a result of interactions with numerous factors including but not limited to yeast strain,1, 2 micronutrient concentrations,6 biotin concentration,6 pantothenic acid concentration6 and temperature.6 The apple juice used in the present study contained an initial YAN concentration of 53 mg L⁻¹ and was supplemented to 78 mg L⁻¹ in all of the treatments. This relatively low YAN concentration is typical of apple juice.39 However, this concentration may still be insufficient to prevent sluggish or stuck fermentations and may have influenced the initial findings of the present study with respect to the impact of fenbuconazole on fermentation rate and duration.

The interactive effect of YAN and fenbuconazole on fermentations kinetics was investigated in fermentations conducted using yeast strain EC1118. One yeast strain and fungicide combination was selected to carry forward, with the experimental treatment now comprising an added residual fungicide concentration, YAN concentration, and the interaction of those two factors. The fermentation rate increased and fermentation duration decreased with an increasing YAN concentration (P < 0.05) (Fig. 6). This is in agreement with prior findings indicating that increasing YAN is correlated with increasing fermentation rate and decreasing fermentation duration.8 RS decreases with increasing YAN when fenbuconazole was present in concentrations of 0.4 mg L⁻¹ (Fig. 7D), although RS was not significantly affected by increasing YAN for the control (no fenbuconazole) or at 0.2 mg L⁻¹ fenbuconazole. This corroborates previous research indicating that increasing YAN minimizes sluggish or stuck fermentations,8, 9, 10 although the findings of the present study emphasize the influence of interactive effect of YAN with other parameters.

There was an interactive effect between the addition of fenbuconazole and YAN on H₂S production (P < 0.0001) (Fig. 7E). Increasing the YAN from 153 to 253 mg L⁻¹ decreased H₂S production in all fermentations (P < 0.001) (Fig. 8). There was no detectable H₂S when the YAN concentration was 253 mg L⁻¹. H₂S production decreases with an increasing YAN concentration at 0.4 mg L⁻¹ fenbuconazole (Fig. 8), with both factors significantly correlating (P = 0.0012) (Fig. 7E). This corroborates previous findings indicating that insufficient YAN supplementation can lead to increased H₂S production.2 The results of the present study show that a minimum YAN concentration of 140 mg L⁻¹ does not always prevent the occurrence of stuck or sluggish fermentations. Furthermore, a minimum YAN concentration of 140 mg L⁻¹ does not always prevent the formation of H₂S during fermentation, even with the relatively low starting sugar concentration of apple juice (compared to grape juice, comprising the media in which most YAN investigations have been conducted).

The addition of both 0.2 and 0.4 mg L⁻¹ fenbuconazole lowered the fermentation rate in juice at both 78 and 153 mg L⁻¹ YAN (P < 0.05) (Table 3). However, there was no difference in fermentation rate for any concentration of fenbuconazole at 253 mg L⁻¹ YAN. At YAN concentrations of 153 mg L⁻¹, fermentations with added fenbuconazole at both concentrations demonstrated a slower fermentation rate than the control with no fenbuconazole (P < 0.01). However, fermentation duration was not different among fenbuconazole concentrations when the YAN concentration was 253 mg L⁻¹. The interaction of YAN and fenbuconazole concentration was not significant in terms of fermentation rate or duration (Fig. 7A, B). With all other variables held constant, increasing YAN concentrations correlate with an increase in yeast biomass.40 It is possible that residual fenbuconazole inhibited yeast growth and promoted yeast mortality. One possible explanation for the results of the present study is that increasing the YAN concentration may overcome this effect by allowing yeast biomass to increase to the point where the impact of residual fenbuconazole on yeast cell populations becomes negligible. Interestingly, fenbuconazole still adversely impacted fermentation kinetics when the YAN concentration was 153 mg L⁻¹, which is higher the minimum concentration 140 mg L⁻¹ typically considered sufficient to complete fermentations in wine and cider. Therefore, the minimum YAN concentration required for fermentation is greater than the standard recommendation when fenbuconazole is present in the juice.

The RS concentration increased with increasing concentrations of fenbuconazole at YAN concentrations of 78 mg L⁻¹ and 153 mg L⁻¹ (Table 3). However, with a concentration of 153 mg L⁻¹ YAN, the RS concentration was ten‐ to 20‐fold lower compared to juices with the same fenbuconazole concentration fermented at 78 mg L⁻¹ YAN. When the YAN concentration was 153 mg L⁻¹, all treatments fermented completely. Finally, there was no difference in RS concentration at any concentration of fenbuconazole when the YAN concentration was 253 mg L⁻¹ (Table 3). There was an interaction effect of YAN and fenbuconazole on RS (P = 0.0006) (Fig. 7D).The effect of fenbuconazole on RS at a low YAN concentration becomes negligible at high YAN concentrations (Fig. 7D). This further corroborates the finding that increasing YAN lessens the impact of fenbuconazole on yeast growth and metabolism. There was no difference in CO₂ when fenbuconazole was added to juice with153 or 253 mg L⁻¹ YAN (Table 3). Even though CO₂ production did not decrease with an increasing YAN from 153 to 253 mg L⁻¹, there was a significant correlation between an increasing YAN and a decreasing CO₂ when fenbuconazole was added at either concentration (P < 0.01) (Fig. 7C). This supports the hypothesis of the present study suggesting that increasing the YAN concentration contributes to an increased fermentation rate, even when fenbuconazole is present. The addition of fenbuconazole did not affect H₂S production for any of the YAN treatments.