Impact of Nitrogen Supplementation and Reduced Particle Size on Alcoholic Fermentation and Aroma in Nitrogen-Poor Apple and Pear Mashes

Abstract

The aim of this study was to enhance the nitrogen supply through three different mash treatments and to investigate their effects on fermentation dynamics, yeast biomass accumulation, and the concentration of aroma-active volatiles in nitrogen-poor apple and pear mashes. In terms of nitrogen supplementation, the addition of diammonium phosphate (DAP) and amino acids (AS) accelerated fermentation and reduced the fermentation duration by 4–6 days in three out of four investigated fruit varieties. One pear variety showed sluggish fermentation, which was slightly improved by reducing the particle size (<3 mm) and significantly improved by nitrogen addition. Notably, AS supplementation resulted in a significant reduction in residual sugar concentrations and led to the highest yeast biomass accumulation across all four fruit mashes. Nitrogen supplementation significantly altered the composition of aroma-active volatiles, notably by increasing higher alcohols such as propyl alcohol, 2-methylpropanol, isoamyl alcohol, and 2-methylbutanol. The addition of AS was more effective in increasing higher alcohols, such as isoamyl alcohol and phenethyl alcohol, while decreasing the off-flavor acetaldehyde.

1. Introduction

The nutrient supply in fruits harvested from meadow orchards is inconsistent and varies with each harvest year, as these meadows are not fertilized [1]. In the production of fruit spirits, yeast assimilable nitrogen (YAN) in particular is crucial for ensuring optimal fermentation and maintaining product quality [2]. Insufficient YAN is a common cause of poor cell growth [3] and fermentation problems such as sluggish or stuck fermentation [4]. To ensure a complete fermentation, the addition of YAN, such as diammonium phosphate (DAP) or amino acids (AS), is a common practice. The supplementation results in an increased fermentation rate and improved alcohol yield in must [5,6]. Also, in mead [7] and tequila production, additional YAN has the potential to increase biomass accumulation and to enhance the fermentation rate.

The type of nitrogen used, whether inorganic or organic, therefore makes a significant difference [8]. In tequila production, adding inorganic YAN (ammonium sulfate) led to a higher sugar consumption rate compared to organic YAN (glutamic acid) or a mixture of both. Conversely, the highest yeast biomass was achieved with a combination of inorganic and organic YAN [9]. In synthetic media, adding organic YAN (a mixture of amino acids) resulted in higher biomass accumulation and sugar consumption compared to media containing mixed YAN sources or inorganic YAN [4].

Therefore, it is not possible to generalize whether organic or inorganic YAN is best, as yeasts like Saccharomyces cerevisiae can utilize a wide range of nitrogen compounds. However, due to a mechanism known as nitrogen catabolite repression, YAN sources can be categorized as preferred and less preferred nitrogen sources [10,11]. Preferred nitrogen sources such as the amino acids glutamine and asparagine, as well as ammonium, are absorbed more efficiently, better supporting cell growth than less preferred nitrogen sources like proline [12]. Therefore, to create optimal fermentation conditions, both the quantity and composition of YAN are crucial.

In addition to its role in amino acid and protein synthesis, YAN is involved in the biosynthesis of higher alcohols, thiols, and esters during fermentation [13,14,15]. Consequently, the concentration of aroma-active volatiles as well as the sensory profile of alcoholic beverages are influenced by YAN availability. Numerous studies have demonstrated a positive correlation between YAN concentration and the synthesis of volatile compounds in must [14,15,16,17]. In particular, the production of higher alcohols is stimulated by a high YAN supply [18]. In synthetic media, the addition of single amino acids correlates positively with the synthesis of aroma compounds derived from these amino acids, which allows the aroma of the product to be controlled. However, as the complexity of the substrate increases, it becomes more challenging to establish clear correlations between YAN addition and sensory properties [14]. Numerous studies have shown that the addition of DAP in wine increases the formation of esters and volatile fatty acids [15,19]. In mead, DAP addition has been shown to enhance the production of volatile fatty acids and phenols, contributing to the fruity character of the distillate [7]. The effect of YAN supplementation is clearly influenced by the substrate and the specific nitrogen sources present. To date, little is known about the impact of organic and inorganic YAN addition on the concentration of volatiles in nitrogen-poor apple and pear mashes.

For this study, we selected two different apple and pear varieties with poor YAN supply and compared fermentation dynamics, yeast biomass accumulation, and the concentration of common aroma compounds in two different YAN mash treatments (DAP and AS) against a control mash. Previous research on fruits from meadow orchards indicated fermentation issues such as sluggish fermentation and high residual sugar, even with DAP addition. Therefore, we included a third mash treatment involving the reduction of the particle size (<3 mm) through intensive fruit crushing. A smaller particle size increases the surface area, which can improve the extraction of fermentable sugars, vitamins, and minerals [20,21], potentially enhancing fermentation efficiency without the need for chemical yeast additives. Fermentation parameters were assessed by measuring sugar consumption dynamics over a 14-day fermentation period, as well as the concentrations of residual sugar, ethanol, and the percentage of sugar consumed at the end of fermentation. Furthermore, the contents of ammonia nitrogen (inorganic YAN), amino nitrogen (organic YAN), and total YAN were determined.

Our objective was to compare the effects of additional organic and inorganic YAN with a mash treatment without chemical yeast additives on fermentation and cell growth. Given that the quality of fruit distillates is primarily influenced by their aroma, we aimed to determine the impact of all mash treatments on the concentration of aroma-active volatiles. Therefore, we analyzed the concentrations of common aldehydes, higher alcohols, esters, and acids in the mashes at the end of the fermentation period. Understanding how different mash treatments influence the fermentation process and the concentration of aroma compounds can enhance the quality and sensory characteristics of the resulting fruit spirits. Consequently, this research provides valuable insights that can improve fermentation efficiency and product quality in the production of fruit spirits from apples and pears, despite variations in quality and nutrient supply.

2. Materials and Methods

2.1. Raw Material

For this study, 130–180 kg of the two apple varieties Gewürzluiken (rootstock M9) and Berlepsch (rootstock Sämling) and the pear varieties Prevorster Bratbirne (rootstock Sämling) (Prevorster) and Champagner Bratbirne (rootstock Old Home Farmingdale 87) (Ch. Bratbirne) were used. The fruits were harvested from non-fertilized meadow orchards located in Schlat (73114, Göppingen, Germany) and were provided by Manufaktur Jörg Geiger GmbH (73114, Göppingen, Germany).

2.2. Mash Preparation

The fruits were first washed thoroughly and left to air-dry. They were then crushed to a particle size of less than 15 mm using a food processor (Helmut Rink, Amtzell, Germany). To achieve a finer mash consistency for the mash with a reduced particle size, one-quarter of each fruit batch was further processed using a Thermomix (Vorwerk Deutschland Stiftung & Co. KG, Wuppertal, Germany). The Thermomix was operated in pulse mode for three cycles of 6 s each, reducing the particle size to below 3 mm. The resulting fruit mash was distributed into four 25.0 L plastic barrels, and 100.0 mg/L of pectin lyase (product No. 5015, Schliessmann, Schwäbisch Hall, Germany) was added to promote liquefaction. The pH of the mash was adjusted to 3.2 by adding a mixture of lactic acid and malic acid in a 1:1 mass ratio, each acid present at a concentration of 8% by mass (product No. 5850, Schliessmann, Schwäbisch Hall, Germany). For fermentation, 150.0 mg/L of dry yeast Saccharomyces cerevisiae AROMA plus (AN-CHOR-Weinhefe NT 116, Schliessmann, Schwäbisch Hall, Germany) was pre-dissolved in tap water (1:10) at 35.0 °C and subsequently added to the mashes. For the inorganic nitrogen treatment (DAP mash), 566.0 mg/L of diammonium phosphate (DAP) (≥98% purity, Carl Roth GmbH+Co KG, Karlsruhe, Germany) was added to provide 120.0 mg/L of yeast assimilable nitrogen (YAN). For the organic nitrogen treatment (AS mash), a mixture of L-glutamic acid (≥99% purity, Carl Roth GmbH+Co KG, Karlsruhe, Germany), L-asparagine (≥99% purity, Carl Roth GmbH+Co KG, Karlsruhe, Germany), L-lysine (≥98.5% purity, Carl Roth GmbH+Co KG, Karlsruhe, Germany), and L-serine (≥99% purity, Carl Roth GmbH+Co KG, Karlsruhe, Germany), in a 1:1:1:1 ratio was used to deliver 120.0 mg/L of organic YAN. Following mash preparation, three portions of approximately 3.0 kg were transferred into glass flasks for laboratory fermentation, while the remaining mash was kept in 25.0 L plastic barrels. Both the flasks and barrels were sealed with fermentation locks, and fermentation was carried out at a controlled temperature of 18.0 ± 0.5 °C for 14 days.

2.3. Fermentation Analysis

To monitor the fermentation process, we measured the sugar consumption dynamics, the concentration of residual sugar and ethanol, and the percentage of sugar consumed on day 14 of the fermentation period. Therefore, the concentrations of glucose, fructose, and ethanol were analyzed over the 14-day period every 24 h from day 0 to day 4 and for every 48 h from day 4 to day 14. On day 14, ethanol content was measured to ensure it reached ≤ 8.0% v/v, which is typical for apple and pear mashes and necessary for accurate aroma compound analysis. After each sampling, the glass flasks were CO₂-gassed to prevent oxygen input. The mash samples were centrifuged for 10.0 min × 3600 rpm and filtered with a syringe filter (pore size 0.2 µm) to eliminate particles and yeast. To analyze the concentrations of fructose, glucose, and ethanol, an HPLC system (1260 Infinity II LC System, Agilent Technologies Inc., Santa Clara, CA, USA) was used equipped with a refractive index detector (RID) and a column of the brand Rezex (Phenomex, Torrance, CA, USA) with an ROA-Organic Acid H+ (8%) phase with a particle size of 8.0 μm at 80.0 °C. Sulfuric acid (0.005 N) was used as a mobile phase at a flow rate of 0.6 mL/min.

2.4. YAN Analysis

Samples for analyzing amino nitrogen (organic YAN) and ammonia nitrogen (inorganic YAN) were collected immediately after crushing the fruits, before adding any yeast additives. For ammonia nitrogen analysis, samples were centrifuged at 3600 rpm for 10 min, filtered through a 0.2 µm syringe filter, and frozen at −20 °C. The concentration of ammonia nitrogen was analyzed with a cuvette test kit (HACH LANGE GmbH, Berlin, Germany) using ammonia (HACH LANGE GmbH, Berlin) for standard curve. Ammonium ions react at pH 12.6 with hypochlorite ions and salicylic ions in the in the presence of nitroprusside sodium (Na₂[Fe(CN)₅NO]) as a catalyzer to form indophenol blue. The samples were mixed with the reagents, incubated for 10 min, and analyzed photometrically at 694.0 nm. Samples for amino nitrogen analysis were frozen without any pretreatment at −20 °C. The concentration of amino acids was analyzed by the Core Facility at the University of Hohenheim according to European Commission Regulation (EC) No 152/2009 III F [22]. Amino nitrogen was calculated based on the concentration and molecular weight of the amino acids. The concentrations of amino nitrogen and ammonia nitrogen were combined to determine total YAN.

2.5. Biomass Accumulation Analysis

For yeast biomass analysis, 5.0 g of mash was mixed with 5.0 mL of 50% v/v glycerol and frozen at −20.0 °C. The defrosted samples were passed through a 1.0 mm sieve to remove big particles and a syringe filter with 30.0 µm pore size (Carl Roth GmbH+Co KG, Karlsruhe, Germany) was used to remove smaller particles. Samples were washed and centrifuged four times (at 3600 rpm, 3000 rpm, 3000 rpm, and 2000 rpm) for purification. The resulting pellet was resuspended in 5.0 mL of distilled water, and the OD₆₀₀ was measured photometrically.

2.6. Aroma-Active Volatiles Analysis

The samples for measuring aroma-active volatiles were taken from the laboratory setup at the end of the fermentation period on day 14 and frozen at −20.0 °C. Prior to analysis, the defrosted samples were heated in a drying oven for 40 min at 80.0 °C to prevent further fermentation during the measurement. The analysis was performed using a headspace gas chromatograph (GC-2010 Plus, Shimadzu Scientific Instruments, Kyoto, Japan) equipped with a flame ionization detector (FID) and an Rtx-volatiles column (Restek Corp., Bellefonte, PA, USA). For calculating the concentration of all volatiles, the standard curves were generated at an ethanol concentration of 8.0% v/v, corresponding to the ethanol concentration of the mash samples. A six-point calibration curve was constructed within the concentration range 1.0–500.0 mg/L of the following volatiles: acetaldehyde, hexanal, propyl alcohol, 2-methylpropanol, n-butyl alcohol, isoamyl alcohol, 2-methylbutanol, hexyl alcohol, phenethyl alcohol, ethyl acetate, hexyl acetate, ethyl 2-methylbutyrate, ethyl butyrate, and 2-methylbutric acid. All standard substances were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.7. Statistical Analysis

Statistical analyses were performed using SPSS Statistics 27 software (IBM, Armonk, NY, USA). A two-way analysis of variance (ANOVA) was applied to the collected data, followed by post hoc pairwise comparisons using Tukey’s HSD test. For null-hypothesis testing, results were deemed statistically significant at p < 0.05, very significant at p < 0.01, and highly significant at p < 0.001.

3. Results and Discussion

3.1. Fermentation Parameters

Sugar consumption in mash is one of the primary parameters used to describe the fermentation process. Under normal fermentation conditions, there is a steady decrease in sugar concentration, indicating active fermentation. Figure 1 illustrates the sugar consumption dynamics of the apple mashes Gewürzluiken and Berlepsch. The mashes of both apple varieties exhibited faster fermentation with YAN addition compared to those without supplementation. For the apple variety Gewürzluiken, the fermentation duration was reduced by 4 days with YAN supplementation, in apple variety Berlepsch by 4–6 days. In the Berlepsch apple mash, a difference between the control mash and the particles mash was observed, due to a higher initial sugar concentration in the particles mash.

However, by the end of the fermentation period (day 14), no differences in sugar consumption, residual sugar concentration, or ethanol content were detected between both approaches (

Table 1. Fermentation parameters at the end of the fermentation period, day 14, n = 3, mean ± SD. Different letters in the same row of one parameter indicate statistically significant differences among the mash treatments obtained from ANOVA followed by post hoc pairwise comparison using Tukey-HSD (p < 0.05).

		Residual Sugar [g/L]	Sugar Consumed [%]	Ethanol [% v/v]
Gewürzluiken	control	5.41 ± 0.05 a	96.08 ± 0.06 a	7.19 ± 0.07 a,b
	DAP	6.20 ± 0.06 b	95.51 ± 0.07 b	7.61 ± 0.12 a
	AS	4.65 ± 0.01 c	96.62 ± 0.10 c	7.08 ± 0.01 b
	particles	5.43 ± 0.06 a	96.24 ± 0.07 a,c	7.52 ± 0.06 a
Berlepsch	control	7.96 ± 0.01 a,b	93.50 ± 0.01 a	6.96 ± 0.01 a
	DAP	7.62 ± 0.04 a,c	93.78 ± 0.03 a	6.51 ± 0.02 b
	AS	7.29 ± 0.04 c	94.06 ± 0.02 a	6.41 ± 0.05 b
	particles	8.35 ± 0.16 b	93.78 ± 0.23 a	6.64 ± 0.11 a,b
Prevorster	control	30.66 ± 0.05 a	79.51 ± 0.86 a	6.61 ± 0.01 a
	DAP	9.58 ± 0.03 b	93.59 ± 0.30 b,c	7.89 ± 0.03 b
	AS	7.85 ± 0.37 c	94.77 ± 0.02 c	8.07 ± 0.50 b
	particles	12.24 ± 0.36 d	91.64 ± 0.29 b	7.45 ± 0.20 a,b
Ch. Bratbirne	control	4.63 ± 0.16 a	96.26 ± 0.14 a	6.47 ± 0.19
	DAP	5.19 ± 0.03 a,b	95.81 ± 0.02 a,b	6.50 ± 0.04
	AS	5.88 ± 0.01 b	95.26 ± 0.00 b	6.61 ± 0.01
	particles	5.75 ± 0.35 b	95.38 ± 0.28 a,b	6.84 ± 0.43

).

When comparing mashes with YAN addition, those with AS had lower residual sugar and higher sugar consumption than those with DAP for both apple varieties. How-ever, this result was significant only in apple Gewürzluiken, indicating that AS were more effective in enhancing sugar utilization in this variety.

A clearer dependence on YAN supplementation was observed in the pear variety Prevorster (Figure 2). Mashes without either AS or DAP addition showed sluggish and incomplete fermentation by day 14, with high residual sugar concentration and low sugar consumption (Table 1). With YAN addition, the fermentation duration was shortened and completed between day 8 and 10. As already seen in apple mashes, the mash with AS addition exhibited significantly lower residual sugar and slightly higher sugar consumption than the DAP mash. Although fermentation of the particles mash remained active on day 14, the results for residual sugar content, consumed sugar, and ethanol content were significantly different compared to the control mash. Thus, a reduced particle size proved beneficial for fermentation dynamics in cases of sluggish fermentation. One explanation for this result is that more intensive crushing of the fruits increases the surface area, allowing yeast better contact with fermentable sugars and nutrients [21]. While this effect may be minor and not noticeable under optimal fermentation conditions, it can significantly improve fermentation kinetics in cases of sluggish fermentation. Smaller particle sizes facilitate a more rapid release of nutrients and fermentable sugars [21,23], which is particularly beneficial when the initial mash conditions are suboptimal. In contrast, the pear variety Ch. Bratbirne demonstrated optimal fermentation kinetics with and without YAN addition. However, all four fruits exhibited higher fermentation dynamics with the addition of YAN, particularly at the beginning of the fermentation. In both apple varieties and the pear variety Prevorster, the addition of AS resulted in lower residual sugar compared to mashes with DAP, suggesting that AS hold more potential for optimal sugar utilization.

Xu et al. [24] also concluded that amino acids are somewhat more effective in improving fermentation kinetics than DAP in apple cider. They found that adding 60 mg/L and 240 mg/L of an amino acid mixture resulted in a lower residual sugar concentration and a faster fermentation rate compared to the same amount of DAP. Also, in must, the addition of amino acids reduced the fermentation duration more effectively than ammonium chloride [25]. However, in tequila, supplementation with ammonium sulfate had a greater effect on the sugar consumption rate than either glutamic acid or a mixture of glutamic acid and ammonium sulfate [9]. Determining the most suitable YAN source depends on the yeast strain and the medium, making it difficult to generalize whether amino acids or DAP is more effective. The apple and pear varieties included in this study exhibited a higher sugar utilization rate with the use of AS.

3.2. YAN Concentration

Yeast assimilable nitrogen (YAN) consists primarily of the nitrogen sources ammonia nitrogen and amino nitrogen [26]. The results for ammonia nitrogen, amino nitrogen, and total YAN are shown in

Table 2. Concentration of ammonia nitrogen, amino nitrogen, and YAN in all apple and pear mashes at the beginning of the fermentation period, day 0, n = 3, mean ± SD. Different letters in the column of one parameter and one variety indicate statistically significant differences among the mash treatments obtained from ANOVA followed by post hoc pairwise comparison using Tukey-HSD (p < 0.05).

		Ammonia Nitrogen [mg/L]	Amino Nitrogen [mg/L]	YAN [mg/L]
Gewürzluiken	control	1.87 ± 0.02 a	4.43 ± 0.31 a	6.31 ± 0.29 a
	DAP	124.89 ± 0.16 b	4.43 ± 0.31 a	129.32 ± 0.18 b
	AS	1.87 ± 0.02 a	88.00 ± 6.87 b	89.88 ± 6.86 c
	particles	1.13 ± 0.01 a	6.58 ± 0.54 a	7.71 ± 0.53 a
Berlepsch	control	1.81 ± 0.01 a	8.01 ± 0.81 a	9.82 ± 0.81 a
	DAP	113.52 ± 0.77 b	8.01 ± 0.81 a	121.54 ± 1.08 b
	AS	1.81 ± 0.01 a	101.70 ± 5.33 b	103.51 ± 5.34 c
	particles	2.53 ± 0.03 a	10.10 ± 0.91 a	12.63 ± 0.89 a
Prevorster	control	1.14 ± 0.02 a	11.34 ± 1.09 a	12.48 ± 1.09 a
	DAP	64.86 ± 0.42 b	11.34 ± 1.09 a	76.21 ± 1.11 b
	AS	1.14 ± 0.02 a	78.84 ± 0.86 b	79.98 ± 0.88 b
	particles	0.89 ± 0.01 a	9.89 ± 2.33 a	10.78 ± 2.34 a
Ch. Bratbirne	control	1.40 ± 0.15 a	15.06 ± 1.70 a	16.47 ± 1.81 a
	DAP	95.61 ± 0.22 b	15.06 ± 1.70 a	110.67 ± 1.42 b
	AS	1.40 ± 0.15 a	109.91 ± 6.71 b	111.31 ± 6.56 b
	particles	1.25 ± 0.04 a	11.07 ± 2.46 a	12.32 ± 2.48 a

. Without YAN supplementation, both apple and pear varieties had a very low YAN availability compared to fruits in other studies, which reported YAN concentrations of 59.0 to 159.0 mg/L in apple mash [27,28]. Januszek et al. [29] classified apples from conventional fruit cultivation as low in nitrogen when their YAN levels ranged between 13.7 and 61.7 mg/L. Based on this definition, all fruits included in this study should be categorized as low in YAN. In mashes with additional YAN, the target concentration of >120 mg/L YAN was reached in only one case, while deviations of up to 55 mg/L YAN between the calculated and measured values were observed in the others. Fruit varieties with the highest demand for YAN, whether organic or inorganic, such as apple Gewürzluiken and pear Prevorster, showed particularly low concentrations. This discrepancy may result from chemical interactions with mash components and rapid nitrogen uptake by yeast shortly after YAN addition, leading to substance loss during sampling and preparation. Combined with matrix effects, these factors likely contributed to the reduced YAN concentrations in the mash.

The reduction in particle size had a minimal effect on the total YAN amount in both apple varieties. In the apple variety Gewürzluiken, the concentration of amino nitrogen increased, whereas in the variety Berlepsch, both ammonia and amino nitrogen concentrations increased slightly. However, in both pear varieties, the reduction in particle size led to a lower content of ammonia nitrogen, amino nitrogen, and total YAN. Salari et al. [23] reported that particle size primarily influenced the concentration of fermentable sugars in apples, while the concentration of proteins and vitamins was either unaffected or decreased with reduced particle size. Further studies examining the relationship between particle size and the content of vitamins or minerals did not find a clear correlation [30,31], which makes addition of YAN necessary.

Among all investigated fruit varieties, the control mash of the apple variety Gewürzluiken had the lowest YAN level, resulting in a fermentation process that took 4 days longer than that of the apple variety Berlepsch, which had a higher YAN concentration. In the control mash of pear Prevorster, the YAN content was 4.0 mg/L lower than in pear Ch. Bratbirne, resulting in a sluggish fermentation that was not completed by day 14. Notably, the concentration of amino nitrogen was low in the pear variety Prevorster. Despite all four fruit varieties having less YAN than the 140 mg/L limit known from viticulture [26], fermentation proceeded well in both apple varieties and the pear variety Ch. Bratbirne without any YAN addition. Apples usually contain less than the required 140 mg/L of YAN and exhibit a normal fermentation process [28]. Apples had a lower total YAN content compared to both pear varieties, but the concentration of amino acids was higher. The composition of YAN, specifically the ratio between organic and inorganic YAN [32] and the composition of amino acids [12,14], plays a crucial role in cell growth and sugar consumption. In apple cider, a higher ratio of amino acids relative to ammonium ions is associated with a higher fermentation efficiency [28]. Arias-Gil et al. [33] demonstrated that the fermentation with only inorganic YAN took 6 days longer compared to fermentation with organic and inorganic YAN, but further increase in inorganic YAN did not change the fermentation duration. A favorable composition of YAN and amino acids, such as in the selected apples, can result in effective fermentation even at lower YAN concentrations.

3.3. Biomass Accumulation

As normal cell growth of Saccharomyces cerevisiae is influenced by nutrient availability and is essential for optimal fermentation [4,34], the biomass of all mash samples was measured during the entire fermentation period. Among both apple varieties (Figure 3), the highest biomass was observed in the mashes supplemented with AS. In the apple variety Gewürzluiken, the biomass in the AS and DAP mashes was significantly higher than in the particles and control mashes. These results correspond with the sugar consumption dynamics (Figure 1), indicating that mashes with higher biomass, such as those with AS and DAP, ferment faster than those with lower biomass, like the particles and control mashes. In the apple variety Berlepsch, the biomass differed between all four mash treatments. The control mash and the particles mash exhibited the lowest biomass. While DAP addition slightly improved biomass accumulation, the most significant increase was seen with AS addition. However, the higher biomass in the AS mashes compared to the DAP mash did not lead to differences in the fermentation dynamics between the AS and DAP mashes (Figure 1).

Compared to both apple varieties the analyzed biomass in pears, presented in Figure 4, was lower. In the pear variety Ch. Bratbirne, the DAP and AS mashes exhibited higher biomass compared to the control and particles mashes, resulting in a higher fermentation rate in both mashes during the initial fermentation period (day 2–6). From day 8 onwards, the control mash, with the lowest biomass, and the AS mash, with the highest biomass and the most significant improvement, stood out. Also, in the pear variety Prevorster, the control mash exhibited the lowest biomass, likely causing sluggish fermentation (Figure 4). Surprisingly, adding DAP did not significantly impact biomass compared to the control and particles mashes, despite significant differences in sugar dynamics among the three treatments. In the AS mash, the highest biomass was observed, which significantly differed from the other three mashes. However, despite the differences in biomass between the AS and DAP mashes, the sugar consumption dynamics were similar. A similar result was observed in the apple variety Berlepsch, where significant biomass differences between the AS and DAP mashes did not lead to differences in sugar consumption. This missing positive correlation between cell biomass and fermentation rate has been reported already [4,35,36]. Factors such as nitrogen deficiency [37], inhibitors, or high concentrations of sugar and ethanol [38] can disrupt the relationship between biomass and fermentation rate. In addition to these factors, supplementary YAN can impact fermentation in two ways, either by increasing the sugar utilization rate in yeast cells or by increasing the cell population [6,39]. In the DAP mashes of pear variety Prevorster and apple variety Berlepsch, the sugar utilization rate was more influenced than cell growth. Conversely, in the AS mashes, additional YAN primarily affected cell growth. Studies have shown that in wine must, organic YAN consistently leads to higher biomass accumulation compared to inorganic YAN at the same concentration, and that the enhancement of the fermentation rate also depends on the nature of YAN [4]. Under nitrogen-deficient conditions, maintaining a higher cell biomass is more crucial for achieving an optimal fermentation rate than increasing metabolic activity [3]. This suggests that adding AS would be the better approach to optimize fermentation in the case of a YAN deficiency.

3.4. Aroma-Active Volatiles

The sensory profile of fruit distillates is shaped by a complex interplay between various compounds derived from the raw materials and those synthesized during fermentation. Volatiles that significantly contribute to the aroma profile of distillates include aldehydes, higher alcohols, esters, and acids. In both apple varieties studied, the addition of YAN altered the concentration of volatiles similarly, notably by increasing higher alcohols (

Table 3. Concentration of the most common aroma compounds in apple mash Gewürzluiken and Berlepsch on day 14 of the fermentation period, n = 3, mean ± SD. Different letters in the same row of one parameter indicate statistically significant differences among the mash treatments obtained from ANOVA followed by post hoc pairwise comparison using Tukey-HSD (p < 0.05).

	Control	DAP	AS	Particles
Gewürzluiken
aldehydes
acetaldehyde (mg/L)	18.07 ± 0.21 a,b	11.70 ± 2.18 a	8.94 ± 1.10 a	27.79 ± 4.66 b
hexanal (mg/L)	1.06 ± 0.01 a	0.98 ± 0.00 b	1.06 ± 0.01 a	1.09 ± 0.01 a
alcohols
propyl alcohol (mg/L)	20.48 ± 0.65 a	36.30 ± 1.45 b	43.56 ± 0.94 c	26.88 ± 0.54 d
2-methylpropanol (mg/L)	31.91 ± 0.55 a	47.79 ± 1.26 b	41.45 ± 0.58 c	28.04 ± 0.05 a
n-butyl alcohol (mg/L)	11.71 ± 0.30	12.08 ± 0.45	11.89 ± 0.41	11.31 ± 0.16
isoamyl alcohol (mg/L)	94.68 ± 1.96 a	115.00 ± 3.34 b	144.18 ± 2.30 c	98.54 ± 0.89 a
2-methylbutanol (mg/L)	41.49 ± 0.76 a	47.98 ± 1.20 b	47.39 ± 0.78 b	40.93 ± 0.19 a
hexyl alcohol (mg/L)	6.31 ± 0.14 a,b	6.11 ± 0.14 a,b	5.70 ± 0.03 a	6.34 ± 0.08 b
phenethyl alcohol (mg/L)	17.68 ± 0.05 a	16.40 ± 0.15 b	19.46 ± 0.01 c	14.92 ± 0.39 d
esters
ethyl acetate (mg/L)	113.99 ± 1.05 a	44.07 ± 1.34 b,c	37.73 ± 0.58 b	50.53 ± 1.85 c
ethyl 2-methylbutyrate (mg/L)	0.84 ± 0.01 a,b	0.86 ± 0.02 a,b	0.88 ± 0.01 a	0.80 ± 0.01 b
ethyl butyrate (mg/L)	0.66 ± 0.01 a	0.72 ± 0.03 a	0.67 ± 0.02 a	0.48 ± 0.01 b
Berlepsch
aldehydes
acetaldehyde (mg/L)	31.96 ± 2.57 a	22.82 ± 0.27 a,b	16.29 ± 0.50 b	28.60 ± 1.86 a
hexanal	1.01 ± 0.01	1.02 ± 0.03	1.05 ± 0.01	1.06 ± 0.01
alcohols
propyl alcohol (mg/L)	21.67 ± 0.12 a	39.23 ± 0.24 b	40.24 ± 0.39 b	25.70 ± 0.53 c
2-methylpropanol (mg/L)	36.35 ± 0.11 a	50.12 ± 0.37 b	50.66 ± 0.26 b	34.99 ± 0.61 a
n-butyl alcohol (mg/L)	16.29 ± 0.09	16.30 ± 0.14	16.86 ± 0.09	16.35 ± 0.31
isoamyl alcohol (mg/L)	102.22 ± 0.50 a	115.21 ± 0.79 b	148.52 ± 0.43 c	109.37 ± 1.99 b
2-methylbutanol (mg/L)	42.83 ± 0.19 a	46.01 ± 0.47 b	49.81 ± 0.08 c	43.97 ± 0.71 a,b
hexyl alcohol (mg/L)	9.14 ± 0.07 a	9.40 ± 0.17 a,b	9.97 ± 0.06 b,c	10.41 ± 0.21 c
phenethyl alcohol (mg/L)	15.48 ± 0.15 a	17.65 ± 0.35 a	24.13 ± 0.89 b	15.55 ± 0.18 a
esters
ethyl acetate (mg/L)	116.84 ± 0.13 a	59.61 ± 3.61 b	93.71 ± 0.28 c	110.05 ± 4.13 a
ethyl 2-methylbutyrate (mg/L)	0.77 ± 0.01	0.77 ± 0.01	0.82 ± 0.02	0.75 ± 0.02
ethyl butyrate (mg/L)	0.82 ± 0.03 a	0.62 ± 0.06 a,b	0.84 ± 0.04 a	0.59 ± 0.02 b

).

When DAP or AS were added, the concentrations of propyl alcohol, 2-methylpropanol, isoamyl alcohol, and 2-methylbutanol significantly increased compared to the control mashes. Moderate concentrations of higher alcohols like 2-methylbutanol and n-butyl alcohol enhance a fruity profile [40,41], while isoamyl alcohol adds a banana-like note [42]. Furthermore, the off-flavors acetaldehyde and ethyl acetate, known for their solvent-like character [43,44], were reduced through YAN supplementation.

Differences between organic and inorganic YAN sources were also observed. Mashes supplemented with AS exhibited significantly higher concentrations of isoamyl alcohol and phenethyl alcohol, which imparts a rosy, honey-like aroma [45], and slightly lower concentrations of acetaldehyde compared to those supplemented with DAP. Conversely, mashes with a reduced particle size showed the lowest concentrations of esters and higher alcohols, and the highest concentration of acetaldehyde, indicating that this method was the least effective for enhancing desirable flavor compounds in apples.

While YAN supplementation influenced the same higher alcohols in both apple varieties studied, its effects on aroma-active volatiles in pears varied between both varieties (

Table 4. Concentration of the most common aroma compounds in pear mash Prevorster and Ch. Bratbirne on day 14 of the fermentation period, n = 3, mean ± SD. Different letters in the same row of one parameter indicate statistically significant differences among the mash treatments obtained from ANOVA followed by post hoc pairwise comparison using Tukey-HSD (p < 0.05).

	Control	DAP	AS	Particles
Prevorster
aldehydes
acetaldehyde (mg/L)	46.15 ± 2.30 a	36.79 ± 1.49 a,b	25.16 ± 1.92 b	29.56 ± 4.46 b
alcohols
propyl alcohol (mg/L)	18.20 ± 1.01 a	20.14 ± 0.62 a,b	21.22 ± 0.59 a,b	22.69 ± 0.77 b
2-methylpropanol (mg/L)	19.58 ± 0.79 a	37.84 ± 0.78 b	40.03 ± 0.73 b	23.85 ± 0.71 a
n-butyl alcohol (mg/L)	1.94 ± 0.07 a	1.30 ± 0.01 b	1.61 ± 0.05 c	1.47 ± 0.04 b,c
isoamyl alcohol (mg/L)	63.94 ± 3.11 a	97.60 ± 1.77 b	113.21 ± 2.32 c	87.11 ± 2.77 b
2-methylbutanol (mg/L)	27.64 ± 1.22 a,b	26.92 ± 0.43 a	31.74 ± 0.56 a,b	32.42 ± 0.92 b
hexyl alcohol (mg/L)	4.20 ± 0.19 a	3.14 ± 0.03 b	3.27 ± 0.06 b,c	3.82 ± 0.07 a,c
phenethyl alcohol (mg/L)	9.38 ± 0.23 a	11.58 ± 0.47 a,b	13.78 ± 0.40 b	9.28 ± 0.61 a
esters
ethyl acetate (mg/L)	424.73 ± 13.49 a	191.55 ± 1.74 b	355.27 ± 0.11 c	199.28 ± 9.28 b
hexyl acetate (mg/L)	0.53 ± 0.00 a	0.45 ± 0.01 b	0.48 ± 0.01 b	0.46 ± 0.00 b
ethyl 2-methylbutyrate (mg/L)	1.15 ± 0.02 a	0.85 ± 0.01 b	0.96 ± 0.00 c	0.85 ± 0.01 b
acids
2-methylbutric acid (mg/L)	1.19 ± 0.05 a	0.97 ± 0.01 b	1.07 ± 0.03 a,b	1.02 ± 0.00 b
Ch. Bratbirne
aldehydes
acetaldehyde (mg/L)	13.70 ± 1.45	20.39 ± 3.77	13.44 ± 0.58	13.87 ± 0.31
alcohols
propyl alcohol (mg/L)	26.44 ± 1.22 a	48.88 ± 0.62 b	56.35 ± 0.25 c	29.63 ± 0.13 a
2-methylpropanol (mg/L)	31.30 ± 1.17 a	35.39 ± 0.35 b	34.24 ± 0.49 a,b	34.13 ± 0.07 a,b
n-butyl alcohol (mg/L)	7.71 ± 0.31 a	7.60 ± 0.15 a	6.89 ± 0.09 a,b	6.45 ± 0.04 b
isoamyl alcohol (mg/L)	56.89 ± 2.38 a	52.82 ± 0.42 a,b	49.52 ± 0.81 b	52.43 ± 0.14 a,b
2-methylbutanol (mg/L)	25.87 ± 0.96 a	20.72 ± 0.11 b	18.79 ± 0.32 b	24.48 ± 0.05 a
hexyl alcohol (mg/L)	4.58 ± 0.16 a	4.15 ± 0.02 a,b	4.03 ± 0.06 b	4.10 ± 0.11 a,b
phenethyl alcohol (mg/L)	11.94 ± 0.64 a	7.69 ± 0.49 b,c	7.16 ± 0.84 c	11.21 ± 0.37 a,b
esters
ethyl acetate (mg/L)	524.54 ± 1.82 a	300.20 ± 24.55 b	286.87 ± 4.02 b	493.58 ± 11.15 a
hexyl acetate (mg/L)	0.56 ± 0.01	0.53 ± 0.05	0.57 ± 0.00	0.53 ± 0.00
ethyl 2-methylbutyrate (mg/L)	1.11 ± 0.01 a	1.01 ± 0.01 b	0.90 ± 0.01 c	0.61 ± 0.00 d
acids
2-methylbutric acid (mg/L)	0.96 ± 0.00	0.94 ± 0.03	0.93 ± 0.01	0.94 ± 0.01

). In pear variety Prevorster, YAN addition increased the concentrations of the higher alcohols 2-methylpropanol and isoamyl alcohol. As observed in the apple mashes, AS mashes had higher levels of isoamyl alcohol and phenethyl acetate and lower levels of acetaldehyde than DAP mashes. However, in Ch. Bratbirne pears, YAN addition resulted in isolated effects, with higher propyl alcohol and lower ethyl acetate concentrations, while most higher alcohols and esters were most abundant in the control mash. Consequently, the positive effect of YAN, particularly AS addition, was not evident in the Ch. Bratbirne pear variety.

In general, the source of YAN had minimal impact on the types of compounds affected, as both organic and inorganic YAN influenced the concentrations of similar higher alcohols and the off-flavor acetaldehyde. Comparable findings have been reported in wine, where additional YAN, whether organic or inorganic, had a comparable impact on the levels of isoamyl alcohol and phenethyl alcohol [46], suggesting that these aroma compounds are particularly sensitive to YAN supplementation. However, AS were more effective in increasing the levels of phenethyl alcohol and isoamyl alcohol, contributing to enhanced fruity and rose-like characteristics, and decreasing the levels of the off-flavor acetaldehyde. Xu et al. [24] also found that amino acids led to higher levels of higher alcohols and acetate esters in cider compared to DAP. Research comparing the effects of organic and inorganic nitrogen sources on wine aroma has demonstrated that amino acids generally result in a more desirable aroma profile compared to ammonium alone [47].

Nevertheless, the impact of higher alcohols on the sensory profile of alcoholic beverages is a subject of ongoing debate. High concentrations of higher alcohols such as n-butyl alcohol and isoamyl alcohol can have a masking effect, diminishing desirable fruity notes and introducing solvent-like and butyric off-flavors in red wine [48]. Conversely, in concentrations below 300.0 mg/L wine, higher alcohols contribute positively to the complexity of wine flavors, adding fruity and flowery notes [49,50]. In apple [51] and plum distillates [52], higher concentrations of higher alcohols have been linked to enhanced flavor profiles. Thereby, concentrations up to 1449.0 mg/L for 2-methylpropanol and 2635.0 mg/L for 2-methylbutanol and isoamyl alcohol were associated with beneficial complexity and a fruity character [40]. Spaho [53] reported a pungent flavor at concentrations above 3500 mg/L, indicating a wide range in which higher alcohols can develop desirable fruity characteristics. Consequently, higher alcohols in the concentrations measured in this study can be considered as desirable flavor compounds. Additionally, higher alcohols can enhance the flavor of alcoholic beverages as they are precursors of acetate esters, which contribute a fruity character [41,54].

4. Conclusions

The addition of YAN was essential for accelerating sluggish fermentation in one pear variety and beneficial for improving fermentation dynamics, cell growth, and higher alcohol concentrations across all four fruit varieties. While reducing the fruit particle size offered some improvement in sluggish fermentation, it was the least effective in increasing biomass, YAN levels, or desirable volatiles.

Both DAP and AS significantly enhanced and shortened the fermentation process. AS demonstrated slightly better performance, with lower residual sugar and higher sugar consumption by the end of fermentation, and significantly greater biomass production. Both YAN sources primarily increased higher alcohols, contributing to the fruity character of distillates. However, AS resulted in notably higher concentrations of phenethyl and isoamyl alcohols while reducing the off-flavor acetaldehyde. Future research should explore whether the increased higher alcohol concentrations affect sensory perception.

In conclusion, while both DAP and AS are effective, further studies are needed to evaluate amino acids as an alternative to DAP, considering their higher cost and lack of legal approval. This study provides a new perspective on optimizing YAN supply in nitrogen-deficient fruit mashes and confirms the critical role YAN composition plays in fermentation dynamics, cell growth, and aroma development.