Abstract
This study aimed to evaluate the efficiency of various mash acidification techniques in alcoholic fermentation for the production of apple spirit. Lachancea thermotolerans and Lactiplantibacillus plantarum strains were selected to conduct pre-fermentative acidification of the mash alongside a conventional chemical acidification approach. The results confirm that L. thermotolerans and L. plantarum possess acidifying potential and can serve as effective biotools for the protection of apple mash during fermentation. Through their outstanding lactic acid production (1.05–1.26 g L−1), they were able to reduce the pH of the mash by 0.29–0.40 pH units. Moreover, the sequential fermentation of these strains with Saccharomyces cerevisiae is a powerful strategy to modulate the aroma profile of apple spirits. Spirits were characterised by an enhanced ester profile with particularly increased ethyl lactate concentrations.
1 Introduction
The production of fruit spirits, particularly alcoholic fermentation, raises various concerns, including spoilage and undesirable flavour changes associated with the metabolic activity of unwanted microorganisms (Jeon et al., 2015). The growth of microorganisms, including several spoilage species, is generally favoured at high pH levels (Payan et al., 2023). Distilleries employ various techniques to prevent anomalous fermentation and microbial spoilage, including pH adjustment, temperature control, anaerobic environment setup, and microbial population management (Bovo et al., 2012). The most common practice of acidity management worldwide involves the addition of organic (malic, tartaric, citric, or lactic acid) and inorganic (sulphuric or ortho-phosphoric acid) acids at the beginning of alcoholic fermentation (Da Porto, 2002; Bovo et al., 2012; Blumenthal et al., 2021). Physical methods, like electrodialysis and ion exchange resins, are also used in winemaking (Lucio et al., 2016). Nowadays, with science and industry embracing innovation, attention is directed towards specific microorganisms capable of providing natural acidification through the production of organic acids.
An early study by Ribéreau-Gayon et al. (1975) revealed intriguing characteristics of Lachancea thermotolerans, including its high production of L-lactic acid, low production of volatile acidity, moderate alcohol production, and no off-flavour generation. Literature confirms the ability of L. thermotolerans to act as an acidifying agent during wine and beer production (Kapsopoulou et al., 2007; Postigo et al., 2023). The acidifying potential of L. thermotolerans strains ranges from 1 to 9 g L−1 in terms of lactic acid and from 1 to 6 g L−1 in terms of total acidity (Benito, 2018). The factors influencing these variations are the selected strain, fermentation conditions, and inoculation method (Vicente et al., 2022).
Moreover, lactic acid bacteria strains, namely Lactiplantibacillus plantarum, exhibit similar acidifying capacity in wine fermentation (Onetto and Bordeu, 2015). Lb. plantarum can generate lactic acid from malic acid degradation and sugar metabolism without the risk of acetic acid synthesis (Pardo and Ferrer, 2018). Lucio et al. (2016) claimed that lactic acid produced by Lactobacillus strains may be a precursor of aromatic compounds such as ethyl lactate, thus enhancing the aromatic profile of the beverage. Lactic acid and its ethyl ester enhance the aroma profile when present in moderate quantities, but at higher levels, they impart a pronounced fatty and cheesy aroma (Spaho et al., 2023).
The acidifying potential of these strains in fruit spirit production has not been exploited yet. Therefore, this study aims to assess the efficiency of various biological and chemical acidification techniques during apple mash fermentation and determine their impact on the aroma quality of the resulting spirit.
2 Materials and methods
2.1 Raw material, microorganisms, and chemicals
Apples (Malus domestica ‘Gala’) were purchased from a local producer in Hungary in September 2023. The yeast strains Saccharomyces cerevisiae (Uvaferm 228) and Lachancea thermotolerans (LaktiaTM), and the bacterial strain Lactiplantibacillus plantarum (WildBrew Sour PitchTM), produced by Lallemand Oenology (Montréal, QC, Canada), were acquired from Kokoferm Ltd. (Gyöngyös, Hungary). Lb. plantarum (Smartbrev Harvest LB-1) was provided by Chr. Hansen A/S (Hoersholm, Denmark). All analytical grade chemicals and standards were supplied by Sigma-Aldrich (Steinheim, Germany).
2.2 Fruit mashing, fermentation, and distillation
For mash preparation, 110 kg of apples were manually sorted, washed, and crushed in a blender. The resulting apple mash was divided into 27 5 L Erlenmeyer flasks, each containing 4 kg of mash. To enhance the liquefaction of the mash, the enzyme LallzymeTM HC (Lallemand Oenology, Montréal, QC, Canada) was added at a dose of 3 g/100 kg.
Chemical and biological approaches were used to acidify the mash. pH adjustment to 3.0 was achieved in the chemical approach using phosphoric and lactic acid solutions (25% v/v) at different ratios: 100:0, 90:10, 80:20, 70:30, and 60:40, respectively. The control flask remained untreated, while the others were subjected to the pre-prepared acid solutions. Subsequently, the mash was supplemented with 40 g/100 kg of UvavitalTM (Lallemand Oenology, Montréal, QC, Canada) yeast nutrients. Fermentations were initiated by inoculating the yeast S. cerevisiae (Uvaferm 228, 40 g/100 kg) into the mash. The biological approach involved inoculating the mash with L. thermotolerans (Laktia, 25 g/100 kg), Lb. plantarum (Sour Pitch, 35 g/100 kg), and Lb. plantarum (LB-1, 35 g/100 kg). The pre-fermentation took place at room temperature. S. cerevisiae (Uvaferm 228) was added 24 h later to the mash. The flasks were closed with airlocks, and the fermentation runs were conducted in triplicate at a temperature of 16 ± 1 °C for 15 days.
The distillation of fermented mashes was performed using a batch distillation apparatus consisting of a glass column filled with copper Raschig rings and a 3-L round-bottomed flask heated with a controlled heating mantle. The dephlegmator, placed above the column, had its outlet temperature regulated by controlling the water flow from the upper part of the spiral cooler. The distillate fractions (head, heart, and tail) were separated organoleptically, and the heart fractions were subjected to further analysis (Table 1).
Characteristics of heart fractions obtained after distillation
| Distillate (Heart fraction) | Volume (mL) | Alcohol content (% v/v) |
| Control | 35.0 ± 0.78 | 85.3 ± 3.40 |
| 100P:0L | 36.0 ± 0.25 | 84.6 ± 7.22 |
| 90P:10L | 34.0 ± 0.98 | 87.3 ± 1.89 |
| 80P:20L | 37.0 ± 0.33 | 82.2 ± 6.54 |
| 70P:30L | 42.0 ± 0.24 | 85.0 ± 2.31 |
| 60P:40L | 35.0 ± 0.99 | 87.2 ± 9.76 |
| LB-1 | 34.0 ± 0.12 | 89.0 ± 5.14 |
| Laktia | 35.0 ± 0.75 | 83.5 ± 8.60 |
| Sour Pitch | 35.0 ± 0.14 | 87.1 ± 4.95 |
2.3 Chemical analysis
During fermentation, the total soluble solids (PAL-1 Refractometer, Atago, Tokyo, Japan), pH (FE20-Kit FiveEasy™ Benchtop pH Meter, Mettler Toledo, Greifensee, Switzerland), and reducing sugar content (Schoorl and Regenbogen, 1917) were monitored daily. The titratable acidity was measured by potentiometric titration with 0.2N NaOH. The volatile acidity in the fermented mashes was assessed by steam distillation and titration with 0.1N NaOH. The ethanol content of the distillates was quantified by a DMA 35N density meter (Anton Paar, Graz, Austria).
2.4 Analysis of sugars and organic acids
To assess the sugar and organic acid concentrations in the mash during fermentation, the Surveyor Plus HPLC System (Thermo Fisher Scientific, Waltham, MA, USA) equipped with Refractive Index (RI) and Photodiode Array (PDA) detectors was used. The analyses were carried out following the method described by Fejzullahu et al. (2021).
2.5 Analysis of volatile compounds
Chromatographic analyses of selected volatile compounds were carried out in triplicate using a GC-FID system (Perichrom PR2100, Alpha MOS, Toulouse, France), according to the method outlined by Rodríguez Madrera and Suárez Valles (2007). The compounds were separated on a CP-WAX 57CB capillary column (Agilent Technologies, Santa Clara, CA, USA) with dimensions of 50 m × 0.32 mm × 0.2 µm. Helium served as the carrier gas at a flow rate of 3 mL min−1. External standards were used to identify and quantify the components in the sample. The concentration of volatile compounds was expressed in mg L−1 100% v/v ethanol.
2.6 Statistical analysis
Data are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) followed by Tukey's HSD post hoc test was employed to determine the difference between means using SPSS software (Version 20.0, SPSS Inc., Chicago, IL, USA). Statistical differences were considered significant at P < 0.05.
3 Results and discussion
3.1 Acidification kinetics
The pH of the fresh apple mash was adjusted from 3.69 to 3.0 in the chemically acidified samples (Fig. 1). During fermentation, a slight pH variation ranging between 2.94 and 3.10 was observed in these samples, indicating sustained inhibition of harmful microorganisms. The bioregulators effectively reduced the pH of the fermentation medium. Laktia stood out for lowering the pH by 0.4 units to 3.29. Morata et al. (2019) highlighted a specific L. thermotolerans strain for its ability to lower the pH by 0.5 units. Among Lb. plantarum strains, LB-1 distinguished itself for reducing the mash pH to 3.31. Likewise, Lucio et al. (2016) reported that Lb. plantarum strains can lower the pH by approximately 0.35 units. Samples inoculated with Laktia, LB-1, and Sour Pitch yielded the lowest alcohol content, likely due to carbohydrate diversion for bacterial growth and lactic acid production (Narendranath et al., 1997). No significant differences were observed in volatile acid production among the samples (Table 2).
pH changes during the fermentation process
Citation: Acta Alimentaria 53, 3; 10.1556/066.2024.00039
Physico-chemical parameters of fresh and fermented apple mashes
| Refraction (%w/w) | Reducing sugars (g L−1) | Total sugars (g L−1) | Titratable acidity (g L−1) | Volatile acidity (g L−1) | Ethanol (%v/v) | Sugars' consumption (%) | |
| Fresh apple mash | 11.90 ± 0.25 | 89.80 ± 2.97 | 110.38 ± 5.27 | 3.17 ± 0.26 | n.a. | n.a. | n.a. |
| Fermented apple mash | |||||||
| Control | 4.30 ± 0.10 a | 7.20 ± 0.21 a | 7.65 ± 0.25 ab | 4.33 ± 0.21 d | 0.52 ± 0.06 a | 6.10 ± 0.10 ab | 92.53 ± 1.49 a |
| 100P:0L | 4.10 ± 0.17 a | 6.96 ± 0.12 ab | 7.42 ± 0.14 abc | 5.77 ± 0.26 b | 0.61 ± 0.01 a | 6.30 ± 0.10 a | 92.73 ± 1.35 a |
| 90P:10L | 4.30 ± 0.20 a | 7.16 ± 0.16 a | 7.74 ± 0.19 a | 6.15 ± 0.27 ab | 0.46 ± 0.04 a | 6.20 ± 0.17 a | 92.45 ± 1.26 a |
| 80P:20L | 4.10 ± 0.17 a | 6.58 ± 0.27 bcd | 7.12 ± 0.26 bc | 5.96 ± 0.13 ab | 0.55 ± 0.09 a | 5.90 ± 0.30 abc | 92.99 ± 1.27 a |
| 70P:30L | 4.00 ± 0.10 a | 6.10 ± 0.33 d | 6.19 ± 0.23 e | 6.63 ± 0.36 a | 0.46 ± 0.03 a | 6.20 ± 0.26 a | 93.80 ± 1.48 a |
| 60P:40L | 4.20 ± 0.10 a | 6.84 ± 0.14 abc | 7.31 ± 0.14 abc | 6.25 ± 0.27 ab | 0.46 ± 0.06 a | 5.60 ± 0.10 bc | 92.82 ± 1.22 a |
| LB-1 | 4.12 ± 0.10 a | 6.35 ± 0.11 cd | 6.87 ± 0.12 cd | 5.00 ± 0.07 cd | 0.57 ± 0.06 a | 5.40 ± 0.17 c | 93.21 ± 1.34 a |
| Laktia | 4.00 ± 0.20 a | 6.30 ± 0.19 cd | 6.30 ± 0.19 e | 5.58 ± 0.27 bc | 0.52 ± 0.08 a | 5.40 ± 0.18 c | 93.71 ± 1.40 a |
| Sour Pitch | 4.10 ± 0.20 a | 6.40 ± 0.18 bcd | 6.42 ± 0.16 de | 4.81 ± 0.19 d | 0.49 ± 0.06 a | 5.40 ± 0.10 c | 93.60 ± 1.41 a |
Data are expressed as mean ± standard deviation; n.a.: not analysed. Values with different letters in the same column are significantly different according to Tukey's HSD test (P < 0.05).
3.2 Metabolism of sugars and organic acids in apple mash
HPLC measurements revealed no major differences in residual sugar concentrations among the samples at the end of the fermentation process (Fig. 2). In each variant, negligible saccharose (<0.50 g L−1), glucose (<1.13 g L−1), and fructose (<3.63 g L−1) contents were detected, confirming the completion of the process by the yeast.
Kinetics of fructose (A), glucose (B), and saccharose (C) consumption during fermentation
Citation: Acta Alimentaria 53, 3; 10.1556/066.2024.00039
Variations in total acidity and pH during fermentation can be attributed to unique patterns of organic acid production in each sample (Fig. 3). The highest amount of succinic acid was produced in the sample 100P:0L (0.73 g L−1), while the lowest levels were noted in the samples inoculated with bioregulators, in particular Sour Pitch (0.66 g L−1). Lactobacillus can produce succinic acid by utilising citric acid generated during the tricarboxylic acid cycle (Li et al., 2021). Malic acid was consumed throughout the fermentation, with the fastest consumption rate observed in samples inoculated with bioregulators. The amount of lactic acid formed corresponds to the amount of malic acid consumed. The highest lactic acid concentrations were measured in the sample Laktia (1.26 g L−1). Hranilovic et al. (2021) reported lactic acid levels ranging from 1.0 to 8.1 g L−1, depending on the L. thermotolerans strain used in sequential fermentation with S. cerevisiae. Lb. plantarum strains, LB-1 and Sour Pitch, also showed high lactic acid producing potential, 1.22 and 1.05 g L−1, respectively. In winemaking, the combined fermentation of S. cerevisiae and Lb. plantarum yielded 2.66 g L−1 of lactic acid and 0.40 g L−1 of acetic acid (Onetto and Bordeu, 2015). In our study, lower acetic acid levels were observed in the samples LB-1 (0.34 g L−1) and Sour Pitch (0.32 g L−1). Even lower amounts of acetic acid were noted in the sample Laktia (0.25 g L−1), indicating that these strains effectively mitigate the increase of acetic acid.
The concentration of malic (A), lactic (B), succinic (C), and acetic (D) acids in mash during fermentation
Citation: Acta Alimentaria 53, 3; 10.1556/066.2024.00039
3.3 Aroma profile of the obtained distillates
The use of different mash acidification techniques led to significant variations in the volatile profiles of the resulting distillates (Table 3). Acetaldehyde is the main carbonyl compound in fruit spirits. Higher acetaldehyde concentrations were noted in the acidified samples compared to the Control. Likewise, Su et al. (2023) reported increased acetaldehyde levels in apple spirits acidified with malic, lactic, and citric acids.
Volatile aroma compounds identified in the apple distillates
| Compounds (mg L−1 100% v/v ethanol) | Control | 100P:0L | 90P:10L | 80P:20L | 70P:30L | 60P:40L | LB-1 | Laktia | Sour Pitch |
| Acetaldehyde | 11.50 ± 1.12 c | 13.13 ± 0.86 bc | 12.32 ± 1.04 bc | 14.76 ± 0.88 b | 14.29 ± 1.25 b | 13.25 ± 0.46 bc | 18.13 ± 0.84 a | 18.94 ± 1.09 a | 18.48 ± 0.84 a |
| Methanol | 3642.80 ± 126.63 b | 3332.52 ± 96.83 bc | 3082.38 ± 130.49 c | 3355.55 ± 80.11 bc | 2570.55 ± 85.54 de | 2347.11 ± 120.03 e | 4423.12 ± 102.61 a | 3548.30 ± 88.88 b | 2703.23 ± 151.93 d |
| 1-Propanol | 1742.32 ± 96.23 ab | 1472.27 ± 123.16 bc | 972.80 ± 85.25 de | 1554.65 ± 116.17 b | 1185.47 ± 67.17 cd | 837.98 ± 113.28 e | 1168.39 ± 105.50 cd | 1873.22 ± 124.77 a | 1129.82 ± 123.30 de |
| 1-Butanol | 159.04 ± 19.84 b | 166.93 ± 17.37 ab | 74.60 ± 7.95 d | 165.67 ± 21.61 ab | 149.57 ± 13.70 bc | 77.39 ± 8.40 d | 75.02 ± 5.50 d | 210.82 ± 30.54 a | 104.19 ± 13.38 cd |
| 1-Hexanol | 68.95 ± 3.42 cd | 82.94 ± 4.99 bc | 21.57 ± 1.03 ef | 90.00 ± 2.32 b | 63.21 ± 3.91 d | 19.24 ± 1.19 ef | 14.66 ± 0.93 f | 127.61 ± 13.26 a | 33.86 ± 3.68 e |
| 2-Methyl-1-propanol | 1162.65 ± 104.47 a | 957.65 ± 88.65 abc | 677.95 ± 149.82 cde | 973.05 ± 78.18 ab | 633.57 ± 71.87 de | 542.04 ± 88.13 e | 677.46 ± 100.68 cde | 1068.34 ± 128.22 ab | 839.30 ± 64.92 bcd |
| 3-Methyl-1-butanol | 1882.60 ± 94.42 b | 1898.97 ± 115.58 b | 830.98 ± 119.51 e | 1998.22 ± 87.68 b | 1513.90 ± 111.21 c | 772.97 ± 65.55 e | 675.05 ± 51.47 e | 2519.55 ± 131.66 a | 1129.96 ± 109.67 d |
| 2-Methyl-1-butanol | 477.71 ± 21.19 ab | 440.77 ± 23.97 b | 207.04 ± 12.83 d | 425.56 ± 25.48 b | 295.11 ± 30.14 c | 188.98 ± 29.08 d | 190.49 ± 25.60 d | 534.85 ± 48.65 a | 302.04 ± 20.82 c |
| Phenethyl alcohol | 0.24 ± 0.01 a | n.d. | n.d. | n.d. | 0.04 ± 0.00 d | n.d. | 0.19 ± 0.01 b | n.d. | 0.10 ± 0.00 c |
| Ethyl acetate | 213.77 ± 41.37 b | 200.19 ± 15.42 b | 168.46 ± 25.85 b | 313.42 ± 36.16 a | 83.71 ± 17.55 cd | 151.28 ± 28.49 bc | 59.76 ± 14.62 d | 327.68 ± 15.26 a | 180.80 ± 16.02 b |
| Ethyl propionate | 0.18 ± 0.01 b | 0.16 ± 0.01 b | 0.20 ± 0.01 b | 0.19 ± 0.02 b | 0.32 ± 0.01 a | 0.08 ± 0.00 c | 0. 17 ± 0.01 b | 0.09 ± 0.01 c | 0.19 ± 0.02 b |
| Ethyl butyrate | 0.57 ± 0.02 c | 0.25 ± 0.01 ef | 0.20 ± 0.01 f | 0.30 ± 0.02 e | 0.18 ± 0.00 f | 0.46 ± 0.01 d | 0.85 ± 0.05 b | 0.01 ± 0.00 g | 1.06 ± 0.09 a |
| Ethyl lactate | 0.24 ± 0.01 e | 0.16 ± 0.01 f | 0.39 ± 0.01 d | 0.54 ± 0.01 bc | 0.58 ± 0.03 abc | 0.40 ± 0.01 d | 0.65 ± 0.02 a | 0.61 ± 0.06 ab | 0.52 ± 0.01 c |
| Ethyl benzoate | 0.83 ± 0.02 a | 0.32 ± 0.01 b | 0.04 ± 0.00 c | 0.02 ± 0.00 cd | 0.03 ± 0.01 cd | 0.02 ± 0.01 cd | 0.02 ± 0.00 cd | 0.02 ± 0.01 cd | 0.01 ± 0.00 d |
| Ethyl octanoate | 2.66 ± 0.10 e | 4.86 ± 0.13 c | 3.55 ± 0.23 d | 6.01 ± 0.36 b | 1.40 ± 0.09 g | 1.23 ± 0.02 g | 1.93 ± 0.07 f | 5.32 ± 0.16 c | 6.85 ± 0.13 a |
| Ethyl decanoate | 0.95 ± 0.08 c | 1.43 ± 0.05 b | 0.18 ± 0.01 e | 0.49 ± 0.01 d | 3.84 ± 0.18 a | 0.30 ± 0.01 de | 1.03 ± 0.08 c | 0.98 ± 0.04 c | 0.94 ± 0.08 c |
| Ethyl myristate | 0.15 ± 0.01 c | n.d. | 0.20 ± 0.00 b | n.d. | 0.15 ± 0.01 c | n.d. | 0.37 ± 0.00 a | 0.03 ± 0.00 d | n.d. |
| Ethyl hexanoate | 1.75 ± 0.22 a | 1.13 ± 0.19 c | 1.29 ± 0.04 bc | 1.59 ± 0.18 ab | 0.56 ± 0.17 d | 0.50 ± 0.07 d | 1.39 ± 0.02 abc | 0.57 ± 0.04 d | 0.63 ± 0.02 d |
| Ethyl phenylacetate | 0.01 ± 0.01 f | 0.10 ± 0.01 d | 0.19 ± 0.02 b | 0.03 ± 0.00 f | 0.18 ± 0.01 b | 0.06 ± 0.00 e | 0.13 ± 0.01 c | 0.25 ± 0.01 a | 0.05 ± 0.00 e |
| Diethyl succinate | 0.01 ± 0.00 e | 0.14 ± 0.00 c | 0.02 ± 0.00 e | 0.04 ± 0.01 d | 0.03 ± 0.00 de | 0.02 ± 0.00 de | 0.32 ± 0.02 a | 0.26 ± 0.00 b | 0.03 ± 0.00 de |
| Isoamyl acetate | 3.16 ± 0.04 c | 2.83 ± 0.14 c | 4.71 ± 0.23 b | 5.52 ± 0.20 a | 1.47 ± 0.07 e | 2.87 ± 0.19 c | 2.06 ± 0.12 d | 1.06 ± 0.12 e | 2.17 ± 0.24 d |
| Propyl acetate | 0.10 ± 0.01 a | 0.05 ± 0.01 bc | 0.04 ± 0.01 cd | 0.05 ± 0.00 bcd | 0.04 ± 0.01 bcd | 0.03 ± 0.00 d | 0.03 ± 0.01 d | 0.06 ± 0.01 b | 0.05 ± 0.00 bcd |
| Isobutyl acetate | n.d. | 0.17 ± 0.01 d | 0.05 ± 0.01 e | 0.32 ± 0.02 c | 0.68 ± 0.02 b | 0.05 ± 0.01 e | 0.09 ± 0.00 e | 0.27 ± 0.02 c | 1.09 ± 0.07 a |
| Butyl acetate | 0.07 ± 0.00 d | 0.13 ± 0.03 c | 0.18 ± 0.00 b | 0.27 ± 0.01 a | 0.03 ± 0.00 e | 0.16 ± 0.00 bc | 0.02 ± 0.01 e | 0.03 ± 0.00 e | 0.02 ± 0.01 e |
| 2-Phenethyl acetate | 0.14 ± 0.00 b | 0.21 ± 0.01 a | n.d. | 0.04 ± 0.00 c | 0.21 ± 0.02 a | n.d. | n.d. | 0.02 ± 0.00 c | n.d. |
Values with different letters in the same row are significantly different according to Tukey's HSD test (P < 0.05); n.d.: not detected.
Higher alcohols, the predominant aroma compounds in distillates, can impart various notes, from fruity and floral to strong and pungent, depending on their type and concentration (Stanzer et al., 2023). Laktia exhibited the highest contents of amyl alcohols (2519.55 mg L−1 and 534.85 mg L−1), 1-propanol (1873.22 mg L−1), 1-butanol (210.82 mg L−1), 1-hexanol (127.61 mg L−1), and 2-methyl-1-propanol (1068.34 mg L−1). Lower amounts of these alcohols were detected in apple brandies fermented with S. cerevisiae (Januszek et al., 2020). The presence of two Lb. plantarum strains, particularly LB-1, in the mash during alcoholic fermentation led to reduced production of all higher alcohols compared to the Control. Chen et al. (2023) noted higher alcohol content increases in ciders sequentially fermented with Lb. plantarum compared to the S. cerevisiae control. Phenethyl alcohol (rose-like odour) was found in small amounts in the samples LB-1 (0.19 mg L−1), Sour Pitch (0.10 mg L−1), and 70P:30L (0.04 mg L−1).
Esters are formed during fermentation and are crucial compounds for the aroma quality of alcoholic beverages (Stanzer et al., 2023). Ethyl acetate was the most prevalent ester. In concentrations up to 100 mg L−1, ethyl acetate contributes desirable nuances, while higher levels can result in a solvent-like aroma (Sumby et al., 2010). Accordingly, the acidification of the mash with LB-1 (59.76 mg L−1) and 70P:30L (83.71 mg L−1) can modulate the production of ethyl acetate, ensuring that positive characteristics prevail in the final product.
The metabolism of S. cerevisiae was greatly influenced by the phosphoric and lactic acid ratios used for mash acidification. Specifically, the 80P:20L ratio positively impacted the production of isoamyl acetate (5.52 mg L−1), ethyl hexanoate (1.59 mg L−1), and butyl acetate (0.27 mg L−1). While the 70P:30L ratio resulted in elevated levels of ethyl decanoate (3.84 mg L−1), ethyl propionate (0.32 mg L−1), and 2-phenethyl acetate (0.21 mg L−1). Bioregulators positively influenced the production of ethyl lactate. A phenomenon favoured by the presence of the precursor, lactic acid, in the mash (Lucio et al., 2016). LB-1 was characterised by the highest levels of ethyl lactate at 0.65 mg L−1, followed by Laktia (0.61 mg L−1) and Sour Pitch (0.52 mg L−1). The ability of L. thermotolerans to produce significant quantities of ethyl lactate and yield higher concentrations of total esters compared to S. cerevisiae controls has been reported in the literature (Fejzullahu et al., 2024). Our study aligns with these findings, as we observed higher ethyl octanoate, ethyl decanoate, ethyl phenylacetate, diethyl succinate, and isobutyl acetate levels in the sample Laktia compared to the Control. Distinct ester profiles were noted in samples LB-1 and Sour Pitch. The Sour-Pitch strain prompted higher amounts of ethyl acetate, ethyl propionate, ethyl butyrate, ethyl octanoate, isoamyl acetate, propyl acetate, and isobutyl acetate compared to LB-1.
4 Conclusions
The employed mash acidification technique had a significant effect on the fermentation performance of S. cerevisiae and the resulting aroma bouquet. This study confirms that L. thermotolerans and Lb. plantarum strains can naturally acidify the apple mash, causing a rapid pH drop primarily through the production of lactic acid. Moreover, when sequentially inoculated with S. cerevisiae, these strains may contribute to the development of a unique aroma profile in the resulting distillates. L. thermotolerans promoted the formation of higher alcohols and esters. Lb. plantarum strains reduced the levels of higher alcohols and ethyl acetate and contributed to higher amounts of esters. The positive attributes offered by these strains are of technological interest in spirit production.
Acknowledgement
We acknowledge the support provided by the Stipendium Hungaricum Program and the Doctoral School of Food Science at the Hungarian University of Agriculture and Life Sciences.
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