1. Introduction
In recent years, the craft beer production sector has seen widespread growth, with growing consumer interest in new beers with distinctive organoleptic characteristics [1,2,3,4].
Among the adopted strategies, in addition to the use of unconventional ingredients (e.g., alternative grains and exotic hops) and innovative brewing techniques (e.g., spontaneous fermentation and barrel aging) [5,6,7], the role of non-Saccharomyces yeasts is a trending topic for improving the sensory characteristics of beers [8]. For decades, the use of starters in the brewing industry has almost exclusively been limited to pure yeast cultures belonging to the genus Saccharomyces. This well-established technique ensures better control of the fermentation process and, as a result, helps to elevate and standardize the quality of the beers. In recent years, in the brewing sector, the need to diversify final products as a business strategy has continually grown to conquer new markets and meet the needs of increasingly demanding and quality-conscious consumers. On the basis of these considerations, especially in the craft beer industry, non-Saccharomyces yeasts represent a great source of biodiversity and open up new possibilities, compared to the traditional Saccharomyces genus, to obtain distinctive products with peculiar compositional and organoleptic characteristics [9,10,11,12,13,14,15,16]. Multiple studies in recent years have highlighted the potential of wild Saccharomyces and non-Saccharomyces yeasts, isolated from spontaneously fermented beers, as well as from nonbrewing environments (e.g., wines, vineyards, sourdoughs, and honey by-products), for the production of beers with appreciable and distinctive compositional and sensory characteristics compared to those obtained with conventional brewer’s yeasts [17,18,19,20,21,22,23]. Although the use of non-Saccharomyces yeasts in beer production is relatively recent, the genus Metschnikowia is one of the most studied because of its multiple positive contributions in winemaking processes and, recently, in beer production [24,25,26]. In particular, Metschnikowia pulcherrima is recommended in winemaking to obtain wines with reduced ethanol contents, as well as for bio-control and its contribution to the aromatic development of wines through its enzymatic activities (e.g., β-D-glucosidase and cysteine β-lyase) and the production of a wide range of metabolites (e.g., esters and higher alcohols) resulting from alcoholic fermentation [26,27,28,29].
On the basis of their low tolerance to alcohol and their enzymatic activities, recent studies have verified that selected M. pulcherrima strains are effective for producing beers with a low alcohol content, if used in pure culture, or can contribute to positively modifying the organoleptic properties of beers if used in co-culture with S. cerevisiae [30,31,32,33].
Today, at the industrial level, the physical methods applied for beer dealcoholization are vacuum evaporation, vacuum distillation, dialysis, and reverse osmosis [34]. However, removing alcohol using the above processes can negatively impact the flavor profile of beers. Therefore, the use of non-Saccharomyces yeasts such as M. pulcherrima can be a valid and viable biotechnological solution for obtaining beers with low alcohol contents but possessing pleasant sensory profiles [25,30]. In addition, the use of M. pulcherrima in co-culture with S. cerevisiae in brewing is an effective strategy for developing new products that meet the growing consumer demand for innovative goods, especially in the craft beer sector [31]. However, while in the wine sector there is a limited availability of commercial strains belonging to this species, in the beer sector this availability does not yet exist. In light of this situation, we aimed to contribute to the selection of M. pulcherrima strains proposed as novel starter cultures in beer production.
Therefore, the present study aims to evaluate three M. pulcherrima strains, previously isolated from vineyards located in Albania [35], specifically targeted for the brewing industry. After a preliminary screening, M. pulcherrima 62 was selected as a starter for craft beer production. Specifically, the suitability of this yeast for use in sequential inoculation with S. cerevisiae for the production of an American IPA-style beer was evaluated.
2. Materials and Methods
2.1. Yeast Strains and Growth Condition
For this study, M. pulcherrima 62, 82, and 86 strains (GenBank accession numbers: PP922572.1, PP922568.1, and PP922571.1), which belonged to the culture collection of the Agri-Food Research Centre of the Faculty of Biotechnology and Food of Agriculture University of Tirana, were used. These strains were previously isolated from autochthonous Albanian red grapes [35]. For the preliminary characterization and brewing trials, the commercial S. cerevisiae S-04 (Fermentis, Lesaffre, France) strain was used as a reference. For the beer fermentation, the yeasts were cultured aerobically at 28 °C in YEPD broth (Merck Millipore, Darmstadt, Germany), and after 48 h, the broth cultures were centrifuged at 8000 rpm for 10 min at 4 °C. Finally, the cell pellet was washed twice with saline solution (0.9% w/v NaCl) and used as a starter. The cell density of the inoculum was assessed using a Thoma Counting Chamber (Thermo Fisher Scientific, Waltham, MA, USA).
2.2. Preselection Trials
2.2.1. Carbon Assimilation Profiles
Carbon source assimilation profiles were evaluated using the API 20 C AUX system (Biomèrieux, Montalieu-Vercieu, France) based on 19 carbohydrate assimilation tests plus a negative control, read by assessing cupules for turbidity. The kit was used in accordance with the guidelines given by the manufacturer. The yeast strains before use were cultured in YEPD broth at 28 °C for 48 h. The strips were read after 48 and 72 h of incubation at 30 °C.
2.2.2. Cryotolerance
The cryotolerance was evaluated as reported by Iorizzo et al. [11]. Each strain pre-grown overnight in YEPD at 25 °C was inoculated at an initial concentration of 1 × 102 CFU/mL into Erlenmeyer flasks (100 mL capacity) containing 80 mL of YEPD, maintained under stirring using a digital orbital shaker (Heathrow Scientific, Vernon Hills, IL, USA) at 4 °C. The growth was determined visually after 24 h of incubation.
2.2.3. Biogenic Amines Production
Biogenic amines production was carried out as described by Barbosa et al. [36] with some modification. For this purpose, a culture media containing 3% (w/v) yeast extract, 1% (w/v) glucose, 2% (w/v) amino acid precursor (histidine, tyrosine, ornithine, phenylalanine, and histidine), and 0.015 g/L bromocresol purple was used. The pH was adjusted to 5.2. Medium without an amino acid precursor was used as a negative control. The decarboxylation of the amino acids to the corresponding biogenic amines results in an increase in pH, detected by the culture medium color change. All reagents were purchased from Merck KGaA (Darmstadt, Germany).
2.2.4. Hydrogen Sulfide Production
Hydrogen sulfide (H2S) production by M. pulcherrima strains was evaluated according to Comitini et al. [37] using BIGGY agar (Bismuth Sulphite Glucose Glycine Yeast; Thermo Fisher Scientific, Waltham, MA, USA) as the medium. After 3 days, H2S-negative strains showed white colonies, while H2S-producing colonies were characterized by a brown or dark brown color. For the results, the different color intensities of the colonies indicated the different levels of H2S production, and the following color scale was considered: 1 (white color, no H2S production), 2 (light brown, low H2S production), 3 (brown color, moderate H2S production), 4 (dark brown, high H2S production), 5 (black color, very high H2S production).
2.2.5. Pulcherrimin Production
Pulcherrimin production was evaluated according to the method described by Mažeika et al. [38] with some modifications and using a culture medium with the following composition: 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, and 2% (w/v) agar. After sterilization, the medium was supplemented with a sterile 0.05% FeCl3 solution (w/v) and poured into Petri dishes. Finally, 10 µL of yeast cultures (106 cells/mL) was spotted onto the medium, and the plates were incubated for 3 days at 28 °C. After incubation, colonies surrounded by reddish halos were recorded as positive results. All reagents used in the experiment were purchased from Merck KGaA (Darmstadt, Germany).
2.3. Enzymatic Activities
2.3.1. API ZYM Assay
Screening of enzymatic activities was evaluated using the API ZYM system (Biomèrieux, Montalieu-Vercieu, France) according to the manufacturer’s instructions. The yeast strains were cultured in YEPD broth at 28 °C. After 48 h, 60 µL of the yeast cell suspensions was transferred into the wells of the API ZYM strips and incubated at 37 °C for 4 h. The color changes observed in the wells indicated positive enzymatic reactions and were used for evaluation of the results on the basis of the API ZYM color chart.
2.3.2. Proteolytic Activity
The proteolytic activity was detected qualitatively by using the skim milk agar hydrolysis method as previously described by Gut et al. [39] with some modifications. Briefly, 10 µL of yeast suspensions in YEPD broth (106 cells/mL) was spotted onto SDA (Sabouraud Dextrose Agar) containing 10% (w/v) skim milk (Merck KGaA, Darmstadt, Germany), with a final medium pH of 7.3. Plates were incubated at 28 °C for 5 days. Proteolytic activity was indicated by the presence of a clear zone around the colony.
2.3.3. β-Glucosidase Activity
A qualitative assay of β-glucosidase activity was performed as reported by Testa et al. [40]. For this purpose, 10 µL of yeast suspensions in YEPD broth (106 cells/mL) was spotted onto a culture medium with the following composition: 2 g/L glucose, 1 g/L peptone, 1 g/L yeast extract, 0.3 g/L esculin, 0.01 g/L ferric-ammonium citrate, and 15 g/L agar (Merck KGaA, Darmstadt, Germany). Plates were incubated at 28 °C for 3 days. The β-glucosidase activity was indicated by the appearance of a black zone around the colonies, signifying the hydrolysis of esculin.
2.3.4. β-Lyase Activity
Qualitative screening of β-lyase activity was conducted as described by Belda et al. [41] using a medium containing 0.1% (w/v) S-methyl-l-cysteine, 0.01% (w/v) pyridoxal-5′-phosphate, 1.2% (w/v) Yeast Carbon Base, and 2% (w/v) agar with a final medium pH of 3.5. Then, 10 µL of yeast suspensions in YEPD broth (106 cells/mL) was spread onto the plate surface and incubated at 25 °C for 72 h. The growth of yeast cultures after 72 h of incubation indicated the presence of β-lyase activity [42]. All reagents were purchased from Merck KGaA (Darmstadt, Germany).
2.4. Craft Beer Brewing Process
After the preselection tests, M. pulcherrima 62 was chosen as the starter for American IPA-style beer production using a Grainfather G series brewing system (Bevie Handcraft NZ Limited, Nelson, New Zealand) at the Department of Agricultural, Environmental and Food Sciences (University of Molise; Campobasso, Italy). For the American IPA-style beer production, pale ale and cara crystal malts (Château Pale ale, Castle Malting, Lambermont, Belgium) were used. Amarillo and Cascade hops (Barth-Hass, Nürnberg, Germany) were added during the boiling and dry hopping phases. The flowchart shown in Figure 1 illustrates the main steps of the brewing process.
The main analytical characteristics of the wort, meeting the requirements established by the Beer Judge Certification Program (BJCP) [43] for American IPA, are reported in Table 1. Fermentation tests were carried out at 20 ± 1 °C using thermoregulated stainless steel tanks (30 L capacity) containing 20 L of wort. In detail, Test A was initially inoculated with M. pulcherrima 62 and after 48 h with S. cerevisiae S0-4 (sequential inoculation), while Test B was inoculated only with S. cerevisiae S0-4. The starter cultures were inoculated at an initial concentration of approximately 106 CFU/mL, and the fermentations were conducted at 20 ± 1 °C. The fermentations were performed in triplicate. At the end of the primary fermentation, 3.5 g/L of sucrose was added as a primer for the secondary fermentation, which took place in 330 mL dark brown glass bottles. After 40 days of maturation at 20 °C, the beers were subjected to chemical analysis.
2.5. Monitoring of Fermentation Kinetics
During the primary fermentation, density values (g/cm3) were monitored. To determine yeast cell viability (CFU/mL), WL agar containing 100 mg/L chloramphenicol (Merck KGaA, Darmstadt, Germany) was used as the culture medium. The plates were incubated at 28 °C under aerobic conditions. After 72 h, colonies were evaluated based on color and topography to distinguish S. cerevisiae from M. pulcherrima [29].
2.6. Beer Chemical Analysis
A pH meter (Crison basic 20, Barcelona, Spain) was used to measure the pH. The density (g/cm3), alcohol content (% v/v), FAN, and IBU were determined according to the analytical procedures described by the European Brewery Convention (EBC) [44]. Glycerol (mg/L), acetic acid (mg/L), acetaldehyde (mg/L), L-malic acid (g/L), and L-lactic acid (g/L) were determined using enzymatic kits (Steroglass, Perugia, Italy) according to the manufacturer’s instructions. All measurements were conducted in triplicate.
2.7. Analysis of Volatile Compounds by GC-FID
The volatile compounds were determined by gas chromatography with flame ionization detection (GC-FID) using a GC2010 Plus apparatus with an FID-2010 detector equipped with a headspace autosampler (HS-20) (Shimadzu Corporation, Kyoto, Japan) and a CP-WAX 57 CB column (50 m × 0.32 mm × 0.2 μm) (Agilent Technologies, Santa Clara, CA, USA). Quantification of volatile compounds was performed using external standards based on a standard curve with five calibration points (the coefficient of determination R2 was greater than or equal to 0.999). The limit of detection was 0.1 mg/L. The analysis method was conducted according to Paszkot et al. [45]. All measurements were conducted in triplicate.
2.8. Statistical Analysis
The results were expressed as mean value ± standard deviation (SD). Statistical analyses were performed using the t-test and one-way analysis of variance ANOVA (IBM SPSS Statistics 21), along with Tukey post hoc tests, at a significance level of p < 0.05.
3. Results and Discussion
3.1. Technological and Biochemical Properties
The results of the API 20 C AUX test are shown in Figure 2 as a heat map. The assimilation profiles of the 19 tested sugars tested were similar; however, the inability of M. pulcherrima 82 to ferment D-Xylose and of M. pulcherrima 86 to ferment D-Xylose and D-Melezitose were detected. All M. pulcherrima strains tested were able to assimilate glucose, maltose, and saccharose, which represent the main sugars present in beer wort. These assimilative capacities make them suitable for potential use as starters in beer production.
The results on the H2S and pulcherrimin production, cryotolerance, and β-glucosidase, β-lyase, and protease activities of the three M. pulcherrima strains are reported in Table 2.
M. pulcherrima 62 was able to grow at 4 °C, while M. pulcherrima 86 and 82 showed very weak growth capacity at this temperature. Low-temperature fermentation by Saccharomyces spp. is believed to lead to the production of wines and beers with improved taste and aroma due to the increased production of volatile aromatic compounds [46,47].
Therefore, the cryotolerance of M. pulcherrima 62 could be beneficial for the production of low-temperature fermented products, such as lager beers. Regarding the qualitative tests we conducted to evaluate the production of biogenic amines, our results showed that the three M. pulcherrima strains do not produce these nitrogenous compounds. In alcoholic beverages, biogenic amines are primarily formed by the decarboxylation or transamination of precursor amino acids, which are directly influenced by the activity of amino acid decarboxylase in yeasts and lactic acid bacteria (LAB) [48,49,50].
Biogenic amines in food may pose a potential public health risk due to their physiological and toxicological effects. In food, their concentrations typically increase during processing and storage because of exposure to microorganisms that catalyze their formation [51]. In recent years, there has been an increase in the number of cases of food poisoning related to biogenic amines in foods and beverages [52]. Therefore, it is extremely important to monitor the level of biogenic amines in food and alcoholic beverages, such as beer. The European legislation does not specify a threshold for biogenic amines, but the European Food Safety Authority (EFSA) has developed a scientific opinion on the risk associated with the formation of these compounds in fermented products [53].
During fermentation, yeasts are responsible for the production of several sulfur compounds, including H2S. This compound can have an undesirable impact by directly affecting the flavor profile or masking other flavor compounds found in beer [20]. H2S is a highly volatile compound with a very low flavor threshold level (11–80 μg/L), reminiscent of rotten eggs [54]. Our results from semi-quantitative tests highlighted the low production of H2S by the M. pulcherrima strains.
Pulcherrimin-producing yeast species like M. pulcherrima are considered effective antimicrobial agents against various microorganisms, with great potential for biocontrol applications [55]. The M. pulcherrima strains tested in this study were found to produce pulcherrimin. Pulcherrimin has inhibitory activity against several yeast species, but S. cerevisiae does not seems to be affected by this antimicrobial activity [56,57,58]. This appears to be confirmed in the beer production trials discussed below, which showed that M. pulcherrima 62 did not cause any interference with alcoholic fermentation by S. cerevisiae S0-4. The use of non-Saccharomyces yeasts, possessing specific enzymatic activities, is still an innovative concept in beer production, and opens up possibilities to produce beers with distinctive sensory characteristics compared to the use of single cultures of Saccharomyces species [32]. The results of the API ZYM test are presented in Figure 2 as a heat map. All the M. pulcherrima strains exhibited the following enzymatic activities: phosphohydrolase, α-glucosidase, β-glucosidase, acid phosphatase, valine, and leucine arylamidase.
Leucine arylamidase is involved in the production of leucine, which is needed for cyclodileucine (cyclo(Leu-Leu)) formation—the precursor of pulcherriminic acid [59].
M. pulcherrima 62 and M. pulcherrima 82 possess esterase lipase (C8) and esterase (C4) activities. Esterases are the enzymes involved in the release of phenolic compounds from plant cell walls [60]. Moreover, previous studies suggest a crucial link between esterase activity and aroma production during fermentation [16,61].
Some non-Saccharomyces yeasts, like M. pulcherrima, possess the β-glucosidase enzyme, which can hydrolyze glycoconjugate precursors and promote the release of active aromatic compounds [62]. In our study, it was found that all three M. pulcherrima strains exhibited β-glucosidase activity. During the alcoholic fermentation of beer wort, the yeasts produced volatile compounds, such as higher alcohol and esters, which directly contribute to the organoleptic characteristics of the final product [11]. In addition to these compounds, β-glucosidase activity results in the release of monoterpene alcohols like linalool, α-terpineol, β-citronellol, geraniol, and nerol [62]. Terpenes can have diverse flavor impacts (citrus and floral), and higher levels of these compounds are associated with greater overall hop aroma intensity. In wort, these compounds are often present in glycosidically bound forms and aromatically inactive [63]. Additionally, our study detected β-lyase activity produced by the tested M. pulcherrima strains. This enzymatic activity results in the release of volatile flavor-active thiols from their conjugated (glutathionylated or cysteinylated) and therefore aroma-inactive forms present in hops [64]. Volatile thiols are active at very low flavor thresholds and impart tropical, citrus, and other fruity aromas to beers [65,66]. Our results showed that M. pulcherrima 62 and M. pulcherrima 86 possess protease activity. Yeast extracellular proteases have the potential to stabilize beer by facilitating filtration and clarification. In addition, protein degradation results in amino acids that serve as a source of nitrogen for yeast growth and are precursors for the biosynthesis of higher alcohols, which significantly influence the aroma and flavor of beers [67]. Based on the screening results, M. pulcherrima 62 was chosen as the starter for the production of an American IPA-style beer.
3.2. Main Chemical Parameters of Beers
The values of the main physical–chemical parameters of the beers in this study are shown in Table 3. The ethanol values (% v/v) were 5.2% in the beer obtained in Test A and 5.0% in the beer obtained in Test B, showing no significant differences. Therefore, the sequential inoculation did not affect the ethanol level in the beer. In fact, S. cerevisiae S0-4, both in co-culture and as a single starter, successfully completed alcoholic fermentation after 10 days. These data confirm findings from other studies that the antimicrobial activity of M. pulcherrima does not interfere with the completion of alcoholic fermentation by S. cerevisiae [56,68].
Moreover, M. pulcherrima appears to have a significant oxygen-consuming capacity [69], which could promote fermentation while suppressing aerobic respiration by S. cerevisiae. Recent studies on wine have shown that aerobic conditions and sequential inoculation times play a crucial role in achieving alcohol reduction using M. pulcherrima in co-culture with S. cerevisiae [28,70]. In a study by Postigo et al. [71] examining the impact of non-Saccharomyces wine yeast strains on beer characteristics and sensory profiles in sequential fermentation, M. pulcherrima CLI 457 was first inoculated, followed by S. cerevisiae S-04 after five days. This produced a beer with an alcohol content of 5.67 ± 0.17, compared to 5.93 ± 0.58 in beer made with S. cerevisiae S-04 as a single starter. In our experiment, fermentation was carried out under anaerobic conditions, and sequential inoculation with S. cerevisiae S-04 took place 48 h after the initial inoculation with M. pulcherrima 62. As a result, our conditions did not lead to significant differences in alcohol content between Test A and Test B. However, in our study, the glycerol content was significantly higher in Test A (1026.02 ± 28.21) than in Test B (892.66 ± 9.60). Complex interactions and substantial differences have been observed in the metabolism of S. cerevisiae when in single culture versus co-culture with non-Saccharomyces yeasts [72,73]. As reported in previous studies, there appears to be an increase in glycerol production when M. pulcherrima is used in mixed culture with S. cerevisiae under controlled oxygenation during the first 48 h of fermentation, followed by anaerobic conditions [73,74]. These studies suggest that M. pulcherrima may exhaust its oxygen supply during the 48 h prior to the inoculation of S. cerevisiae. Reduced oxygen availability could explain the modulation of glyceropyruvic fermentation and the shift in S. cerevisiae metabolism towards pyruvate dehydrogenase (PDH) bypass, leading to increased glycerol production [75]. Glycerol influences beer taste due to its sweetness, improves foam stability, enhances aroma volatility, and increases the retention of the worty off-flavor [76]. Its content in beer is usually higher than that of many other flavor compounds (e.g., higher alcohols, esters, and organic acids), and the addition of glycerol to beer above and below the threshold level (10 g/L) has been found to modify the flavor of the product [77]. No significant difference in lactic acid content was detected in beers, while lower amounts of L-malic acid (160.33 ± 2.51 mg/L) were found in beer from in Test A compared to the beer produced using S. cerevisiae S-04 as a single starter (Test B), which contained 202.33 ± 7.50 mg/L of L-malic acid. In a previous study, it was shown that some M. pulcherrima strains can metabolize malic acid in wine [78], which could explain the significant differences in these two organic acids in the beers analyzed. Finally, our analyses found that the quantitative values of acetaldehyde and acetic acid were not significantly different in the beers from Tests A and B, confirming that M. pulcherrima produces low amounts of these two compounds [37,79,80]. Acetic acid, the main component of the volatile acids in beer, has a flavor threshold range of 71 to 200 mg/L [81]. Therefore, the quantities measured in our fermentation trials (Test A: 60.66 ± 3.51 mg/L; Test B: 58.83 ± 1.25 mg/L) are lower than these values. Acetic acid is a by-product of alcoholic fermentation, produced via the pyruvate dehydrogenase (PDH) bypass. As pointed out above, under anaerobic conditions, M. pulcherrima influences the expression of genes involved in the PDH bypass in S. cerevisiae during mixed fermentation, which should lead to increased production of glycerol and acetic acid [75,82]. However, as highlighted in previous studies [28,83], it can be hypothesized that M. pulcherrima may consume part of the acetate produced by S. cerevisiae in sequential fermentation to synthesize acetate esters during mixed fermentation. Acetaldehyde (25 mg/L threshold) is the carbonyl compound present in beer in the highest concentration at the conclusion of primary fermentation, produced by the decarboxylation of pyruvate, and is an intermediate in ethanol formation during glycolysis [84,85]. In our study, this compound was present in all beers and, in low concentrations such as those detected (Test A: 7.66 ± 0.80; Test B: 7.70 ± 0.19), it can positively contribute to the sensory character of the beers, imparting notes of green apple, pumpkin pulp/seed, and unripe avocado [14,86,87].
3.3. Fermentation Kinetics
The pH and density trends monitored during alcoholic fermentation are illustrated in Figure 3. The corresponding numeric data are reported in Table S1 (Supplementary Materials).
The pH, starting from an initial value of approximately 5.50, gradually decreased to final values of 4.35 in Test A and 4.31 in Test B, with no significant differences between the two. The reduction in pH during fermentation is due to the yeast’s consumption of compounds with buffering capacity (e.g., amino acids) and the production of organic acids.
In Test A, initially inoculated with M. pulcherrima 62, the density decreased after 48 h of fermentation, from 1.047 to 1.042 g/cm3. Following the sequential inoculation with S. cerevisiae S-04, density values rapidly decreased, reaching 1.007 g/cm3 at the end of alcoholic fermentation (10 days). In Test B, the fermentative vigor of S. cerevisiae S-04 caused a greater decrease in density after 24 h (1.022 g/cm3). After 10 days of fermentation, the density values were not significantly different in the beers obtained from Tests A and B. Our results demonstrated that the use of M. pulcherrima 62 as an initial starter did not negatively affect the fermentation activity of S. cerevisiae S-04. Regarding the yeast viable cell count (Table 4), the use of WL agar medium allowed us to evaluate the populations of viable yeasts present during the alcoholic fermentation, and in particular confirmed the aptitude of this culture medium to differentiate S. cerevisiae from M. pulcherrima based on colony color and colony topographic characteristics (Figure 4). Validation of the taxonomic identification of yeast species was performed using a 26S rDNA D1/D2 domain sequence analysis, employing NL1 (5′-GCA TAT CAATAA GCG GAG GAA AAG-3′) and NL4 (3′-GGT CCG TGT TTC AAG ACGG-5′) primers [29].
In Test A, the population of M. pulcherrima averaged 6.93 log CFU/mL after 2 days of fermentation then decreased and was not detected 5 days after inoculation. In both tests, the density of viable S. cerevisiae cells increased after inoculation to values greater than 7 log CFU/mL from day 4 until the end of alcoholic fermentation. The fermentation kinetics reported in Table 4 confirm, as found in previous studies, that M. pulcherrima, while possessing multiple antimicrobial activities, does not negatively affect the viability and metabolic activity of S. cerevisiae [28,30,56,88].
3.4. Volatile Compounds
The concentrations of volatile compounds identified in beers by GC-FID are shown in Table 5. The flavor and aroma of the beers originate from the raw materials (malt, hops, and yeast) as well as the metabolic reactions that occur during fermentation, which generate ethanol and other co-products, primarily volatile compounds such as higher alcohols, esters, acids, and aldehydes [84,89].
The production of higher alcohols by yeast occurs through the Ehrlich pathway either from amino acids transported across the cell membrane or through de novo biosynthesis of amino acids. After the initial transamination, α-keto acid intermediates are excreted into the growth medium, and yeasts convert them into alcohols or acids via the Ehrlich pathway. In our tests, the identified alcohols, regardless of the initial starter culture, included isobutanol, isoamyl alcohol, 2-phenylethanol, and 1-hexanol. Our results confirmed that these compounds are the most abundant higher alcohols found in beer [90,91]. However, the amounts of these compounds were significantly different in the beers obtained in Tests A and B (Table 5). For isoamyl alcohol, produced using M. pulcherrima 62 as the initial starter culture (Test A), the amount (101.83 ± 1.55 mg/L) was well above its flavor threshold in beer (70 mg/L) [14]. This alcohol also possesses some banana flavor characteristics and has been identified above its threshold in banana, orange, mango, pineapple, and passion fruit [90]. As for isobutanol, the highest concentration was obtained in the sequential fermentation with M. pulcherrima 62 (42.26 ± 0.75 mg/L), which has been previously reported as a high isobutanol producer [32,71]. Higher alcohols not only impact flavor but also provide the alcohol moiety required for the synthesis of esters, which represent the largest and possibly most important group of flavor-active compounds in beer [84].
Flavor-active esters represent an important group of compounds that impart fruity and flowery aromas to beer [92]. These volatile compounds are primarily formed during the active phase of the primary fermentation through the enzymatic condensation between activate fatty acids (acyl-CoA or acetyl-CoA) and higher alcohols [90]. Among the esters, we detected ethyl acetate and isoamyl acetate in the highest concentrations. Specifically, in the beers obtained in Tests A and B, the following concentrations were quantified: ethyl acetate at 1.31 ± 0.18 mg/L (Test A) and 0.68 ± 0.04 mg/L (Test B) and isoamyl acetate at 2.50 ± 0.14 mg/L (Test A) and 1.88 ± 0.04 mg/L (Test B). The threshold concentration of ethyl acetate in beer is 30 mg/L, but for lager-type beers the recommended concentration is <5 mg/L [93]. Therefore, the concentrations detected in the beers we produced were well below these threshold levels of perception. M. pulcherrima has been described as a good producer of isoamyl acetate in beer [71]. This ester is formed by the condensation of acetyl CoA and isoamyl alcohol during fermentation, and its intensive ‘fruity’ aroma (banana, apple, and pear) is perceived at concentrations >1.2 mg/L [84,94]. In our study, other esters were present at concentrations below their threshold values. However, the presence of different esters can have a synergistic effect on individual flavors, meaning that esters as a whole can also affect the flavor of beer well below their individual threshold values [14,95]. In our study, among the volatile compounds, butyric, hexanoic, octanoic, and decanoic fatty acids were detected. However, the quantities present in the beers produced were all below the sensory perception thresholds [96,97]. Fatty acids have a beneficial effect on yeast growth during fermentation but can negatively affect the organoleptic properties of beer and the stability of beer foam [98,99]. These compounds and their oxidized forms cause unpleasant, off-flavor beer, such as rancid, cheesy, soapy, and fatty notes [100,101]. Finally, no significant difference in the content of diacetyl and acetoin was found between the beers obtained in Test A and those obtained in Test B. This indicates that the use of M. pulcherrima 62 as a starter does not result in an increase in the production of these substances. Diacetyl (butanedione or butane-2,3-dione) is a vicinal diketone generated as a by-product of amino acid metabolism in yeast during wort fermentation and is secreted into beer, imparting aroma characteristics described as butter, butterscotch, or buttermilk when detected above its flavor threshold > 0.1 mg/L [85]. During the maturation process, the yeast reabsorbs diacetyl and converts it to acetoin and subsequently to 2,3-butanediol. Both acetoin and 2,3-butanediol can escape the cell, but neither contribute significantly in terms of flavor.
Table 5Volatile compounds (mg/L) detected by GC-FID in the beer produced using M. pulcherrima 62 and S. cerevisiae S0-4 in sequential inoculation (Test A) and in the beer produced using S. cerevisiae S0-4 as a single starter (Test B).
| Class of Organic Compounds | Volatile Compounds * | Test A | Test B | Threshold Values * | Reference |
|---|---|---|---|---|---|
| Higher alcohols | Isobutanol | 42.26 ± 0.75 a | 11.44 ± 0.55 b | 200 | [102] |
| Isoamyl alcohol | 101.83 ± 1.55 a | 53.02 ± 2.01 b | 70 | [102] | |
| 1-Hexanol | nd | 1.23 ± 0.10 a | 4 | [102] | |
| β-phenylethanol | 21.10 ± 1.86 b | 34.77 ±1.71 a | 125 | [102] | |
| Esters | Ethyl acetate | 1.31 ± 0.18 a | 0.68 ± 0.04 b | 30 | [102] |
| Ethyl isovalerate | 0.24 ± 0.06 a | 0.17 ± 0.03 a | 1.3 | [102] | |
| Ethyl butirate | 0.24 ± 0.05 a | 0.34 ± 0.03 a | 0.4 | [102] | |
| Ethyl lactate | nd | 0.56 ± 0.06 a | 250 | [102] | |
| Isoamyl acetate | 2.50 ± 0.14 a | 1.88 ± 0.04 b | 1.2 | [102] | |
| Ethyl hexanoate | 0.03 ± 0.01 a | 0.05 ± 0.01 a | 8 | [102] | |
| Ethyl octanoate | 0.13 ± 0.02 b | 0.24 ± 0.01 a | 0.9 | [102] | |
| Diethyl succinate | 0.34 ± 0.04 a | 0.22 ± 0.02 b | 1.2 | [103] | |
| Fatty acids | Butyric acid | 1.80 ± 0.08 a | 0.11 ± 0.02 b | 1.9–3 | [104] |
| Hexanoic acid | 2.30 ± 0.12 a | 0.65 ± 0.05 b | 6.8 | [105] | |
| Octanoic acid | 1.13 ± 0.10 b | 1.94 ± 0.05 a | 4.3 | [105] | |
| Decanoic acid | 0.47 ± 0.08 a | 0.30 ± 0.04 a | 1.3 | [105] | |
| Aldehydes/ketones | Diacetyl | 0.11 ± 0.04 a | 0.12 ± 0.03 a | 0.15 | [106] |
| Acetoin | 0.84 ± 0.05 a | 0.64 ± 0.08 b | 50 | [102] |
* Expressed as mg/L. All values are presented as mean ± standard deviation (n = 3). Different superscript letters in each row indicate significant differences (p < 0.05).
4. Conclusions
The use of non-Saccharomyces yeasts is an increasingly pursued biotechnological strategy in brewing processes to achieve greater diversification of beers and improve their sensory characteristics. In the present study, we presented the results obtained from the production of an American IPA-style beer using M. pulcherrima 62 in sequential inoculation with S. cerevisiae S-04.
The higher concentrations of glycerol, isoamyl alcohol, and isoamyl acetate in beer produced by the sequential inoculation of M. pulcherrima 62 with S. cerevisiae S-04 compared to beer made using S. cerevisiae S0-4 as a single starter are remarkable. These findings support the potential of M. pulcherrima 62 as a promising starter in beer production, but further validation is needed through investigations on the volatile compounds in beer, employing more advanced techniques such as gas chromatography–mass spectrometry (GC-MS) along with sensory evaluation of the final product. In the future, it may also be interesting to assess the influence of M. pulcherrima 62 on the quality of other beer styles.
Conceptualization, M.I., B.T. and J.K.; methodology, B.T. and J.K.; software, F.L. (Francesco Letizia); validation, M.I., K.S., F.C. and R.K.; formal analysis, B.T. and J.K.; data curation, M.I., B.T. and J.K.; writing—original draft preparation, J.K.; writing—review and editing, M.I., B.T., F.L. (Francesco Letizia) and J.K.; visualization, O.K., N.X., M.R. and F.L. (Fatbardha Lamçe); supervision, M.I., R.K. and K.S. All authors have read and agreed to the published version of the manuscript.
Not applicable.
Not applicable.
The original contributions presented in the study are included in the article and
The authors declare no conflicts of interest.
Footnotes
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![View Image - Figure 2. Heatmap visualization of the test results for carbohydrate assimilation and enzymatic activities of M. pulcherrima strains. Red square: [Image omitted. Please see PDF.] negative; blue square: [Image omitted. Please see PDF.] positive.](https://media.proquest.com/media/hms/THUM/4/gTNMa?_a=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%3D%3D&_s=mYPUy30YM5YlOwtRGiapaWLcKzE%3D "View Image - Figure 2. Heatmap visualization of the test results for carbohydrate assimilation and enzymatic activities of M. pulcherrima strains. Red square: [Image omitted. Please see PDF.] negative; blue square: [Image omitted. Please see PDF.] positive.")
Enlarge this image.Figure 2. Heatmap visualization of the test results for carbohydrate assimilation and enzymatic activities of M. pulcherrima strains. Red square: [Image omitted. Please see PDF.] negative; blue square: [Image omitted. Please see PDF.] positive.
Enlarge this image.Figure 3. pH and density (g/cm3) trends during alcoholic fermentation: Test A, inoculated with M. pulcherrima 62 and S. cerevisiae S-04 in sequential inoculum; Test B, inoculated with S. cerevisiae S-04 as a single starter.
Enlarge this image.Figure 4. Colony morphology of S. cerevisiae (creamy white) and M. pulcherrima (light blue) using WL agar medium.
Key parameters of the wort used for American IPA beer production.
| Density (g/cm3) | 1.047 ± 0.003 |
| *FAN (mg/L) | 208.67 ± 0.12 |
| **IBU | 54.00 ± 0.40 |
| pH | 5.50 ± 0.20 |
| °Plato | 11.70 ± 0.10 |
All values are expressed as the mean of three technical replicates ± standard deviation (n = 3). *FAN: Free Amino Nitrogen; **IBU: International Bitterness Unit.
H2S and pulcherrimin production, cryotolerance, and enzymatic activities of M. pulcherrima strains.
| M. pulcherrima Strains | H2S * | β-Glucosidase ** | β-Lyase ** | Protease ** | Pulcherrimin ** | Cryotolerance ** |
|---|---|---|---|---|---|---|
| 62 | 1 | + | + | + | + | + |
| 82 | 1 | weak | + | − | + | weak |
| 86 | 1 | weak | + | + | + | weak |
* H2S production using BIGGY agar as an indicator medium: 1 (white color, no H2S production), 2 (light brown, low H2S production), 3 (brown color, moderate H2S production), 4 (dark brown, high H2S production), 5 (black color, very high H2S production). ** Enzymatic activities: (β-glucosidase, β-lyase, and protease), pulcherrimin production, and cryotolerance (+ positive; − negative).
Main physical–chemical parameters of beer produced using M. pulcherrima 62 and S. cerevisiae S-04 in sequential inoculation (Test A) and beer produced using S. cerevisiae S-04 as a single starter (Test B).
| Physical–Chemical Parameters | Test A | Test B |
|---|---|---|
| pH | 4.35 ± 0.05 a | 4.31 ± 0.06 a |
| Alcohol (% v/v) | 5.20 ± 0.10 a | 5.00 ± 0.10 a |
| Acetic acid (mg/L) | 60.66 ± 3.51 a | 58.83 ± 1.25 a |
| L-malic acid (mg/L) | 160.33 ± 2.51 b | 202.33 ± 7.50 a |
| L-lactic acid (mg/L) | 115.66 ± 5.13 a | 115.50 ± 2.29 a |
| Glycerol (mg/L) | 1026.02 ± 28.21 a | 892.66 ± 9.60 b |
| Acetaldehyde (mg/L) | 7.66 ± 0.80 a | 7.70 ± 0.19 a |
| Density (g/cm3) | 1.007 ± 0.006 a | 1.006 ± 0.003 a |
All values are expressed as mean ± standard deviation (n = 3). Different superscript letters in each row indicate significant differences (p < 0.05).
Viability evolution (log CFU/mL) of S. cerevisiae and M. pulcherrima during alcoholic fermentation: Test A was inoculated with M. pulcherrima 62 and S. cerevisiae S-04 in sequential inoculation, while Test B was inoculated with S. cerevisiae S-04 as a single starter.
| Fermentation Time (Days) | |||||||
|---|---|---|---|---|---|---|---|
| 0 | 2 | 4 | 6 | 8 | 10 | ||
| Test A | M. pulcherrima | 6.12 ± 0.13 b | 6.93 ± 0.25 a | 6.33 ± 0.15 b | 4.02 ± 0.14 c | 0.00 ±0.00 d | 0.00 ± 0.00 d |
| S. cerevisiae | 0.00 ± 0.00 d | 6.75 ± 0.25 c | 7.54 ± 0.23 b | 8.27 ± 0.30 a | 8.85 ± 0.15 a | 7.62 ± 0.20 b | |
| Test B | S. cerevisiae | 6.83 ± 0.15 c | 6.88 ± 0.20 c | 7.86 ± 0.35 b | 8.52 ± 0.33 a | 7.88 ± 0.10 a | 7.73 ± 0.15 b |
All values are expressed as mean ± standard deviation (n = 3). Different superscript letters with each row indicate significant differences (p < 0.05).
Supplementary Materials
The following supporting information can be downloaded at
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; Testa, Bruno 4
; Letizia, Francesco 4 ; Kyçyk, Onejda 1 ; Kongoli, Renata 2 ; Ruci, Mamica 1 ; Lamçe, Fatbardha 1 ; Sulaj, Kapllan 5 ; Iorizzo, Massimo 4
1 Faculty of Biotechnology and Food, Food and Research Center, Agricultural University of Tirana, 1001 Tirana, Albania;
2 Faculty of Biotechnology and Food, Department of Agro-Food Technology, Agricultural University of Tirana, 1001 Tirana, Albania;
3 Institute of Food Sciences, National Research Council of Italy, 83100 Avellino, Italy;
4 Department of Agriculture, Environmental and Food Sciences, University of Molise, 86100 Campobasso, Italy;
5 Faculty of Biotechnology and Food, Food Science and Biotechnology Department, Agricultural University of Tirana, 1001 Tirana, Albania;