Synergistic Effect Enhances Aromatic Profile in Beer Brewing Through Mixed-Culture Fermentation of Pichia kluyveri and Saccharomyces cerevisiae var. diastaticus

Abstract

Saccharomyces cerevisiae is one of the important species of traditional fermented foods and beverages. However, incorporating non-Saccharomyces in the fermentation process is a promising strategy to improve the organoleptic profile. In this study, we assessed the potential of a wild Pichia kluyveri strain (PKL) to augment the aromatic profile in beer brewing while maintaining high fermentation attenuation through inoculation with Saccharomyces cerevisiae var. diastaticus yeast (SY) in both simultaneous (SI-PKL/SY) and sequential (SE-3-PKL/SY) approaches. The fermentation performance was analyzed by residual sugar content, volatile organic compounds, and sensory evaluation. The results indicated that both co-fermentation methods yielded residual sugar levels comparable to those of SY monoculture fermentation. The 2-phenethyl acetate, isoamyl acetate, and linalool in SE-3-PKL/SY increased 12.00, 12.37, and 1.17 folds than the SY monoculture, respectively. Furthermore, the incremental concentrations of these compounds contributed to the highest acceptability and prominent fruity notes in the SE-3-PKL/SY coculture. The current study is the first to report on the co-fermentation with Pichia kluyveri and Saccharomyces cerevisiae var. diastaticus in beer brewing. These findings highlighted the importance of Pichia kluyveri in shaping the ameliorative aroma profile of fermentation production.

1. Introduction

Aroma is one of the most important elements determining the quality and consumer acceptance of beer. The choice of substrates, yeast strains, and the design of fermentation processes will influence the composition of aroma compounds in beer production [1,2]. Recently, consumers have exhibited a significant preference for the fruity and floral attributes that are discernible in certain beers. These specific beer varieties are commonly obtained through mixed fermentation by Saccharomyces cerevisiae (S. cerevisiae) with non-S. cerevisiae yeasts. The varieties of non-S. cerevisiae yeast strains profoundly impact the biosynthesis of volatile compounds and are crucial for reshaping the overall aromatic profile [3,4,5,6]. The Pichia kluyveri (P. kluyveri) has been applied to beverage fermentation under the above background while licensed with Generally Recognized As Safe (GRAS, GRN No. 938). Remarkably, the low ethanol and high acetate production properties have been used to improve the aroma quality of various low-alcohol beverages. The typical case included that P. kluyveri strains exhibited an enhanced fruity aroma profile in fermented beverages through co-inoculation with S. cerevisiae [7,8] or Torulaspora delbrueckii [9]. However, the predominant focus of the present research was mainly on wine and fruit wine fermentation by using must and fruit juice. The limited applicability of P. kluyveri is due to its restricted fermentation capacity with disaccharides and trisaccharides [10]. The latest research has unveiled its application in beer brewing [11]. By leveraging its poor ability to metabolize maltose through mixed fermentation, it is possible to produce aroma-enhanced low-alcohol beer. However, the weak capacity of metabolizing maltose in the mixed fermentation system is a double-edged sword, it retains a higher residual sugar content in beer [12], which can make a beer that is excessively sweet, unbalanced, heavy, or prone to spoilage. Maintaining a high fermentation attenuation while imparting the aroma characteristics of non-S. cerevisiae yeasts represents an intriguing endeavor. S. cerevisiae var. diastaticus is recognized for its high fermentative capabilities, which are primarily attributed to its ability to secrete glucoamylase. This enzyme allows the yeast to break down complex carbohydrates into fermentable sugars, resulting in a high fermentation attenuation, and it may compensate for the fermentation deficiencies of non-S. cerevisiae species [13,14]. However, there is a scarcity of research investigating the innovative combination of P. kluyveri and S. cerevisiae var. diastaticus in beer brewing.

In the present study, one P. kluyveri (PKL) strain with good aroma performance was screened out from the orchard in Ningdong (Ningxia, China) and the comprehensive impact of PKL on beer quality was further studied in the sequential and simultaneous fermentation with S. cerevisiae var. diastaticus. The effects of P. kluyveri on the fermentation process, the consumption of fermentable sugars, and the volatile characteristics were analyzed. The symbiotic relationship enhances fermentation efficiency by enabling the complete utilization of available sugars. Additionally, the interaction between P. kluyveri and S. cerevisiae var. diastaticus throughout mixed fermentation can improve the ultimate fragrance characteristics of the fermentation broth. The aroma profile of beer brewing was also evaluated by a sensory test. This work provided useful information about the pairwise combination of P. kluyveri yeast applied in the production of fermented beverages.

2. Materials and Methods

2.1. Yeast Strains

The non-S. cerevisiae strain P. kluyveri (PKL) was isolated from the orchard in Ningdong (38.47° N, 106.23° E, in the north part of Ningxia), China. Identified by 5.8S-Internal Transcribed Spacer (ITS) and DNA sequence analysis (Figure S1), it has been preserved in the China General Microbiological Culture Collection Centre (CGMCC No. 27659). The S. cerevisiae strain used in this study is the commercial yeast S. cerevisiae var. diastaticus, Belle Saison (Belgium) yeast. The percent solids were 93%–97%, living yeast cells ≥5 × 10⁹ per gram of dry yeast. Before inoculation, yeast cells were cultivated at 25 °C with agitation at 220 rpm for 24 h in YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose), collected by centrifugation at 5000 rpm for 5 min, and washed twice with 0.85% sterile NaCl solution.

2.2. Fermentation Conditions

The fermentation of the single and multi-strain inoculation with P. kluyveri followed the method described, advanced with minor modifications [7]. A lab-scale fermentation was carried out in 500 mL Erlenmeyer flasks containing artisanal wort at 25 °C with an “S” shape airlock which can let CO₂ out. The wort was prepared by our laboratory as follows: each 80 g ground barley malt and 420 mL water were combined in a beaker and agitated for 3 h at 68 °C using a Mashing bath (Bioer Technology, Tianjin, China); the combined barley malt solutions were then filtered through a Buchner funnel to obtain wort. The wort was boiled for 10 min, while 0.3 g of hop powder was added 5 min to the end of the boiling time. Finally, the wort was characterized by pH 4.7 and a specific gravity of 8.8°Bx. The single and mixed fermentations of SY and PKL were carried out as follows: (1) single inoculation with SY, the initial viable cell population was 1 × 10⁶ CFU/mL; (2) simultaneous inoculation of PKL (2 × 10⁷ CFU/mL) and SY (1 × 10⁶ CFU/mL) (SI-PKL/SY); and (3) sequential inoculation of PKL (2 × 10⁷ CFU/mL) followed by SY (1 × 10⁶ CFU/mL) after 3 days (SE-3-PKL/SY), the inoculation ratio of PKL with SY is 20:1 in both inoculation methods. Each fermentation with different inoculation strategies was set up in triplicate. Progress of the fermentation was monitored daily by measuring CO₂ evaporation and pH. The weight loss of the entire device was measured by a ten-thousandth balance and recorded as the amount of CO₂ released due to the function of the “S” shape airlock. The pH was measured by using a portable pH meter. Five milliliters of fermenting wort were sampled at 12 h, 24 h, 3 d, 5 d, 7 d, 9 d, and 11 d, and the cell-free supernatants were stored at −20 °C to analyze physical and chemical indicators. The fermentation was considered completed when the CO₂ evaporation remained unchanged. The ethanol content was measured in the final beer.

2.3. Determination of Sugar Residue Content

The residual sugar content of the samples, including glucose, fructose, sucrose, maltose, and maltotriose, as well as ethanol in fermentation broth, were analyzed by high-performance liquid chromatography (Agilent 1100 HPLC, Agilent Technologies, Santa Clara, CA, USA) using an Aminex HPX-87H ion exchange column (300 × 7.8 mm, BioRad, Richmond, CA, USA) coupled to a refractive-index detector and eluted with 0.5 mM H₂SO₄ at 0.6 mL/min and at 60 °C. Each sample was analyzed for 30 min [15]. The total fermentable sugar content was determined as the sum of all residual fermentable sugars. The concentrations of individual fermentable sugar in all samples were quantified using external calibration curves established with standard solutions ranging from 10 to 100 ppm. The samples before HPLC analysis were filtered through a 0.22 μm membrane filter (Dikma Technologies, Lake Forest, CA, USA). All samples underwent triple evaluation.

2.4. Volatile Compound Analysis

Volatile compounds were determined by headspace solid-phase micro-extraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS) according to the method described advanced with minor modifications [16]. Briefly, sodium chloride (1.5 g) was placed in the headspace bottle, followed by the addition of a 5 mL fermentation sample and 5 μL of 2-octanol as the internal standard (the final concentration was 1.71 mg/L). The sample was incubated at 60 °C for 10 min and then extracted for 0.5 h using the ageing 50/30 μm car/PDMS/DVB. The ageing 50/30 μm car/PDMS/DVB was transferred into a gas chromatographic injector and desorbed at 250 °C for 5 min. The analyses were carried out on a GC-MS system (Thermo, CA, USA) equipped with the TR-5MS column (30 m × 0.25 mm, 0.25 μm, J&W Scientific, CA, USA) and quadrupole DSQ II MS. The oven temperature process was maintained at 50 °C for 3 min, after which it was incrementally raised to 240 °C at a gradient of 5 °C/min and maintained for 10 min. The total analysis time was 51 min. Mass spectra were carried out in the electron impact (EI) mode at 70 eV ionization energy by the full scan mode (45–400 amu). The compounds were identified by matching the mass spectra with the NIST02 mass spectral database. For the aroma compounds detected, a modified semi-quantitative method described advanced was utilized in this study to monitor the changes in the concentration of various aroma compounds in fermentations [17,18]. The relative concentration of each compound in fermentation liquor was calculated by comparing its area with the internal standard, 2-octanol. Furthermore, amongst the aroma compounds, 8 vital individual components including 3-methyl-1-butanol, isoamyl acetate, 2-phenylethanol, ethyl octanoate, 2-phenethyl acetate, linalool, geraniol, and ethyl laurate were quantified using the corresponding calibration curves. NIST databases were utilized for spectral identifications with a match factors threshold of >700 by comparing the retention indexes and the mass spectra with the standards. All experiments were performed in three replicates.

2.5. Sensory Evaluation

The sensory evaluation was performed as described previously with minor modifications [19]. Descriptive sensory analysis was performed by 20 trained tasters (10 females and 10 males, aged between 20 and 30 years old, with experience in drinking and consumption). Volunteers had a detailed understanding of the purpose of sensory evaluation and all ingredient samples (all edible, without any food safety risks). They were aware and agreed to become a part of sensory evaluation research before participating. Before the sensory evaluation, each fermentation broth was placed in a sample bottle and left undisturbed at room temperature for 1 h with the lid closed. In all cases, samples (20 mL) were poured into glasses and presented in a random order. Each sample was repeated three times. Potable water was provided for rinsing the palate during the test. The sensory description encompasses two intensity indexes (“intensity”, “balance”) and four generic words (“fruit”, “honey”, “chemical”, and “yeast”). The descriptors were scored on a five-point scale (1 = extremely low, 3 = moderate intensity, and 5 = extremely high).

2.6. Statistical Analysis

Origin Pro 2017 (Origin Lab Cooperation, Northampton, MA, USA) was used for the statistical calculations. Data were analyzed by a one-way analysis of variance using SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). Duncan’s multiple range test was carried out to analyze significant differences among the mean values. Partial Least Squares Discriminant Analysis (PLS-DA), and clustering analysis were performed using MetaboAnalyst 5.0 https://www.metaboanalyst.ca/ (accessed on 10 January 2025).

3. Results and Discussion

3.1. Fermentation Process Monitoring

The development of CO₂ evaporation and pH were monitored during all the single and co-inoculation fermentations (Figure 1). All fermentation could be completed on the ninth day (the CO₂ evaporation remained unchanged). During the initial four days of fermentation, the rate of carbon dioxide released by the monoculture of Saccharomyces cerevisiae var. diastaticus yeast (SY) surpassed that observed in co-fermentations. (Figure 1A), and the pH of SY monoculture decreased significantly (Figure 1B). The mixed fermentation of simultaneous (SI-PKL/SY) coculture started immediately on the first day. However, the change in CO₂ evaporation and pH was at a relatively slow rate compared with SY, and the final pH was slightly lower than the SY monoculture. For the co-fermentation of the sequential approach (SE-3-PKL/SY), the variation of the CO₂ evaporation and pH value in the initial three days of fermentation is relatively small and started accelerating after inoculation with S. cerevisiae SY after three days. In the early stage of fermentation with SE-3-PKL/SY, it was indicated that PKL exhibited a poor fermentation capacity, resulting in a longer lag time. The observable change in the fermentation may mainly originate from the metabolism of monosaccharides. However, after the SY strain was later inoculated in the wort, the fermentation was accelerated because the SY could use the nutrients that remained in the wort [20].

3.2. Analysis of Fermentable Sugars

To better understand the metabolism differences among the fermentation caused by PKL and SY strain, we monitored the utilization of maltose, sucrose, maltotriose, glucose, and fructose (Figure 2). The initial contents of these sugars were 35.71, 6.7, 14.49, 1.38, and 0.85 g/L, respectively. Maltose, sucrose, and maltotriose are the main components of the wort medium, accounting for 96% of the total fermentable sugars. During the fermentation process, it was discovered that the maltose and sucrose with SY monoculture were almost consumed during the first three days of fermentation, reaching a final concentration of 1.82 g/L and 0.83 g/L, respectively (Figure 2A,B). However, the co-fermentation of SE-3-PKL/SY had an extremely low maltose and sucrose metabolism rate till day four, and it went into a rapid fermentation phase immediately after the SY was inoculated, corroborating previous findings which indicate that strain PKL exhibits a significant impediment in fermenting maltose and sucrose [21,22]. In the co-fermentation of SI-PKL/SY, the rate of maltose and sucrose consumption was observed to be intermediate between that of the SY monoculture and the SE-3-PKL/SY coculture. Unlike the rapid metabolism of maltose and sucrose, the maltotriose content in SY monoculture showed an even decline during the whole fermentation and decreased more slowly in SI-PKL/SY (Figure 2C). It can be inferred that PKL may exert a mild inhibitory effect on SY’s metabolism of maltotriose in the SI-PKL/SY coculture. In the SE-3-PKL/SY coculture, the concentration of maltotriose remained relatively constant for the initial three days of the fermentation process, a noticeable decline in maltotriose levels was only registered following the introduction of SY into the fermentation broth on the fourth day, attributable to the poor capacity of PKL to metabolize maltotriose [23]. The glucose was nearly completely depleted at the initial stage of fermentation, with comparable consumption rates observed across all fermentation broths (Figure 2D). This finding is consistent with previous observations [24], suggesting that simultaneous inoculation did not significantly influence glucose metabolism. Nonetheless, the significantly faster decrease in fructose concentration on day 1 for the SE-3-PKL/SY coculture (p < 0.05) compared to the other beers indicates a prioritized and more efficient metabolism of fructose by PKL compared to SY (Figure 2E). This indicates that a stronger metabolic capacity for fructose may be a characteristic feature of some non-S. cerevisiae yeasts, including PKL [25,26].

At the termination of fermentation, the concentrations of the primary sugar component including maltose, sucrose, and maltotriose and the whole fermentable sugars (Figure 2F) in all mixed fermentation exhibited no statistically significant divergence compared to SY monoculture. This significantly ameliorates the drawback of high residual sugar commonly associated with traditional mixed fermentation [12]. It can be deduced that the enhanced fermentative capacity of S. cerevisiae var. diastaticus yeast plays a pivotal role in improving fermentation attenuation in mixed-culture fermentation systems.

3.3. Key Aroma Compounds of Co-Fermentation of P. kluyveri and S. cerevisiae

To elucidate the influence of PKL yeast on the final beer product’s aroma characteristics, the primary aromatic components in beer under various co-fermentation schemes were measured. Based on changes in CO₂ evaporation in the fermentation broth (Figure 1), samples at day nine were selected as the endpoints of fermentation, and these samples were subsequently analyzed. In this work, thirty-one aroma compounds were identified in the final beer, including six higher alcohols, eight fatty acids, thirteen esters, three polyphenols, and one ketone. To evaluate the distinguished aroma profile of these volatile compounds in beer, the regulation cut-offs to identify differentially abundant aroma compounds between the SY group and SI-PKL/SY, and between SY and SE-3-PKL/SY were first established. Aroma compounds with a fold change > 1.2 (or fold change < 0.83, p < 0.05) were considered to be significantly differentially altered (Table S1).

Amongst the volatiles, alcohols make up the largest relative proportion, which are predominantly contributed by ethanol. Although the non-S. cerevisiae has been considered with relatively poor fermentation ability [27], the mixed culture broth with non-S. cerevisiae PKL yields comparable levels of ethanol to the SY monoculture (2.1%) due to the strong fermentation capacity of S. cerevisiae var. diastaticus. Comparing the higher alcohol yields of these three mixed fermentation strategies, the concentrations of 3-methyl-1-butanol in SY pure fermentation at the end of fermentation were 24.3% and 91.9% higher than those of the mixed fermentation (SI-PKL/SY and SE-3-PKL/SY), respectively, and the content of 2-phenylethanol in SY monoculture was higher by 23.5% and 20.0%, respectively (

Table 1. The content of the main aroma compounds responsible for the separation of the three fermentation schemes.

Aroma Compounds	Content (mg/L)			OAVs			Odor Descriptors
	SY	SE-3-PKL/SY	SI-PKL/SY	SY	SE-3-PKL/SY	SI-PKL/SY
ethanol	2.10% ± 0.35a	2.07% ± 0.39a	2.08% ± 0.28a
3-Methyl-1-butanol	95.94 ± 3.35a	49.99 ± 3.17c	77.19 ± 3.37b	>1	>1	>1	Fruity, apple, banana
Isoamyl acetate	1.16 ± 0.28 c	14.35 ± 0.19a	8.15 ± 0. 23b	>1	>1	>1	Banana, fruity, sweet
2-Phenylethanol	37.48 ± 0.92a	31.24 ± 2.13b	30.33 ± 0.58b	>1	>1	>1	Rose, pollen, perfume
Ethyl octanoate	1.96 ± 0.23b	2.52 ± 0.20b	3.41 ± 0.25a	>1	>1	>1	Floral, apple, beer, burnt
2-Phenethyl acetate	0.18 ± 0.06c	2.16 ± 0.13a	1.42 ± 0.04b	>1	>1	>1	Fruity, floral, pleasant
Linalool	0.064 ± 0.003b	0.075 ± 0.002a	0.066 ± 0.002b	>1	>1	>1	Citrus, floral, fruity
Geraniol	0.045 ± 0.001b	0.022 ± 0.004c	0.070 ± 0.007a	>1	>1	>1	Citrus, floral, rose
Ethyl laurate	0.019 ± 0.002c	0.552 ± 0.012a	0.201 ± 0.024b	>1	>1	>1	Fruity, strawberry, pleasant

Different letters within the same row indicate mean values significantly different (Tukey test at 95% confidence level).

). These results are consistent with previous reports that P. kluyveri could decrease the content of higher alcohols and result in more appropriate sensory characteristics in the wine [10]. A certain concentration of higher alcohols had a positive contribution to the aroma form of fermentation broth and could increase the fruity and floral aroma and complexity. However, they may also be one of the causes of headaches associated with the general discomfort of hangovers that follow excessive fermentation liquor consumption [28,29,30], so keeping low concentrations of higher alcohols increases the flavor quality. The adjustment effect of mixed brewing strategies on the level of higher alcohols in beer in this study provided an alternative approach to mitigating headaches of beer production in the future. In addition to the higher alcohols produced by yeasts during fermentation, terpenoid alcohol compounds from hops will also impart a distinctive flavor and enhance the overall harmony of beer. Notably, linalool in hops was believed to improve the aromatic properties of beer [31], and it was partially generated from the biotransformation of geraniol [32]. In this study, the linalool content in SE-3-PKL/SY was 1.17 folds greater than the fermentation schemes of SY, and it increased by 13.39% over SI-PKL/SY while the content of geraniol in SE-3-PKL/SY was the least among the three co-fermentation (Table 1). It can be inferred that geraniol in the SE-3-PKL/SY coculture underwent more efficient bioconversion to linalool.

Fatty acids play a diverse role in the development of fermented aroma, contributing significantly to the formation of specific aroma compounds and overall sensory characteristics [33]. Octanoic acid, 9-decenoic acid, decanoic acid, and dodecanoic acids were produced more significantly by both coculture groups than SY monoculture (p < 0.05). Further analysis of the differences between the two cocultures revealed that levels of octanoic acid and dodecanoic acid in SE-3-PKL/SY coculture were 1.25 and 1.82 times higher than those in the SI-PKL/SY coculture, respectively. Conversely, the content of 9-decenoic acid in the SI-PKL/SY coculture was 1.83 times higher than that in the SE-3-PKL/SY coculture (Table 1). These findings were consistent with previous research results involving coculture fermentation [34].

Esters mainly show fruit and flower fragrance in the fermentation broth and contribute positively to the aroma of the fermentation broth [35]. In this study, acetyl esters from both coculture fermentations were detected at higher levels than the SY monoculture, the yield of isoamyl acetate fermented with the SE-3-PKL/SY and SI-PKL/SY was approximately 12.37 and 7.02 folds higher than the SY monoculture. For the content of the 2-phenethyl acetate, the increasing folds were 12.00 and 7.88. This indicated the mixed-fermentation involved with the non-S. cerevisiae PKL was more valuable to product isoamyl acetate and 2-phenethyl acetate than the SY monoculture (p < 0.05), supporting earlier studies which also fermented with P. kluyveri [36,37], displaying the yeast PKL was good at producing acetyl esters. Except for the acetyl esters, co-inoculation of PKL with SY significantly improved the production of ethyl esters, the SE-3-PKL/SY produced the highest concentration of ethyl octanoate, ethyl nonanoate, ethyl trans-4-decenoate, ethyl decanoate, ethyl laurate, ethyl tetradecanoate, and ethyl hexadecanoate, followed by SI-PKL/SY. For instance, the yield of ethyl laurate fermented with the SE-3-PKL/SY and SI-PKL/SY was approximately 29.05 and 10.58 folds higher than the SY monoculture. This result suggested that coculture may improve the efficiency of biosynthesis of these esters and their corresponding fatty acid substrates compared to monoculture. The content distribution of main acetyl esters among the three fermentation groups showed an opposite trend to the content distribution of corresponding higher alcohols. For instance, the fermentation broth of the SE-3-PKL/SY group had the largest quantity of isoamyl acetate and 2-phenethyl acetate but the lowest amount of 3-methyl-1-butanol. We noted that 3-methyl-1-butanol and 2-phenylethanol were diverted toward esterification to generate isoamyl acetate and 2-phenethyl acetate more extensively in co-fermentation than in the SY monoculture [38]. The content distribution of some ethyl esters among the three fermentation schemes presented a similar trend to the content distribution of corresponding fatty acids. The relative content of decanoic acid, octanoic acid, and dodecanoic acid as well as the corresponding esters were both higher in the co-fermentation broth. It implied volatile fatty acids played an extremely important role in fermentation, not only in directly determining the flavor feature of the produced fermentation products, but also in influencing the biosynthesis of ethyl esters [39]. In addition, the content of geranyl acetate fermented with SE-3-PKL/SY was 4.71 and 3.99 times higher than SY and SI-PKL/SY, respectively (Table 1). Many studies have indicated that geranyl acetate was produced by esterifying from geraniol, and was considered vital to enhance the fruity characteristic profile of the beer [40,41]. In this study, the outstanding performance in producing geranyl acetate in SE-3-PKL/SY indicated its potential to generate more geranyl acetate to ultimately improve the aromatic profile of beer.

3.4. The Influence of Mixed-Fermentation on the Sensory Evaluation

To evaluate the fermentation differences along with the sensory profile of the three fermentation schemes, the partial least-squares discriminant analysis (PLS-DA) method was applied as a multivariate data analysis technique to display the aroma compounds and sensory scores. All the main aroma compounds and sensory scores were designated as dependent variables and samples as the independent variables. The biplot of PLS-DA revealed a clear separation between the different fermentation schemes based on the first two latent variables with good coefficient fractions (Figure 3). The first two principal components (PCs) explained most of the variation with PC1 and PC2, explaining 76.5% and 12.3% for three fermentation schemes, respectively.

In the biplot result, many volatile compounds (isoamyl acetate, 2-phenethyl acetate, and ethyl laurate, etc.) and sensory scores (intensity, balance, and fruity) in the negative part of PC1 were mapped close to SE-3-PKL/SY coculture. The high loadings of main higher alcohols (2-phenylethanol and 3-methyl-1-butanol) and nonanoic acid were detected in the positive part of PC1, mainly related to SY monoculture. However, the whole aroma profile was inferior to coculture. The SI-PKL/SY was mapped in the middle position of PC1 between SY monoculture and SE-3-PKL/SY coculture. SE-3-PKL/SY co-inoculation significantly increased the aroma features of “intensity”, “balance”, and “fruity”, compared to SY monoculture (Figure 4). Regarding the results of the box chart for the inoculation methods, SE-3-PKL/SY scored best for aroma “intensity” (p < 0.05), “balance” (p < 0.05), and “fruit” (p < 0.05). The “intensity” scores of SE-3-PKL/SY, SI-PKL/SY, and SY were 3.76, 3.38, and 3.14; the value for the “balance” was 3.90, 3.38, and 3.33; and the “fruity” scores were 3.95, 3.14, and 2.71, respectively (Figure 4). These data denoted that the aromatic property of SY can be further enhanced by co-fermentation with PKL. In comparison with SE-3-PKL/SY co-inoculation, SI-PKL/SY co-inoculation had less ability to diversify the aromatic profiles, although the fermentation made by SI-PKL/SY was separated from SY monoculture. To further elucidate the correlation of the aroma compounds with the sensory characteristics, several aroma compounds were obtained that were responsible for this separation (Variable Importance in Projection>1) and have Odor Activity Values (OAVs) over one including 3-methyl-1-butanol, isoamyl acetate, 2-phenylethanol, ethyl octanoate, 2-phenethyl acetate, linalool, geraniol and ethyl laurate (Table 1). These volatile compounds were considered key in remodeling the aroma profile. The Pearson correlation was analyzed for the aroma compounds content and sensory scores of the fermented broth in three fermentation schemes (

Table 2. The Pearson correlation of sensory description with corresponding aroma components in fermentation broth.

Sensory Descriptors		3-Methyl-1-butanol	Isoamyl Acetate	2-Phenylethanol	Ethyl Octanoate	2-Phenethyl Acetate	Ethyl Laurate	Linalool	Geraniol
Intensity	Pearson cor.	−0.103	0.284 *	−0.332	0.185	0.271 *	0.294 *	0.181	0.199
	Sig.	0.420	0.024	0.008	0.146	0.032	0.019	0.155	0.119
Balance	Pearson cor.	−0.002	0.261 *	−0.221	0.272 *	0.251 *	0.297 *	0.266 *	−0.260 *
	Sig.	0.989	0.039	0.081	0.031	0.047	0.018	0.035	0.040
Yeast	Pearson cor.	0.137	0.033	0.005	0.169	0.033	0.085	0.187	−0.134
	Sig.	0.283	0.797	0.966	0.187	0.795	0.508	0.142	0.294
Fruity	Pearson cor.	−0.127	0.513 **	−0.370	0.422 **	0.503 **	0.521**	0.299 *	−0.350 **
	Sig.	0.321	0.000	0.003	0.001	0.000	0.000	0.017	0.005
Chemical	Pearson cor.	0.113	0.022	−0.048	−0.042	0.012	0.047	0.131	−0.106
	Sig.	0.377	0.866	0.708	0.743	0.929	0.717	0.305	0.410
Honey	Pearson cor.	0.135	−0.057	0.076	−0.099	−0.068	−0.039	0.087	−0.035
	Sig.	0.293	0.657	0.555	0.438	0.596	0.764	0.499	0.786

Significant correlations are shown in * (p < 0.05); ** (p < 0.01).

). The result represented the sensory score for “intensity” was significantly relevant to the content of isoamyl acetate, 2-phenethyl acetate and ethyl laurate. The “fruity” score was significantly relevant to the content of isoamyl acetate, ethyl octanoate, 2-phenethyl acetate, ethyl laurate, linalool, and geraniol. Hence, it was inferred that the high contents of the main ester compounds and linalool were responsible for the enhanced sensory results. These findings agreed with the results of the previous research [42]. Furthermore, it is not only the aroma compounds with odor activity values (OAVs) exceeding one that significantly influences sensory quality, a variety of compounds with OAVs below one will also play a key role in shaping the overall sensory profile [1]. Aside from the main compounds mentioned, other components, such as geranyl acetate, cubenol, and octanoic acid, may contribute synergistically to the development of the aromatic profile in this study.

4. Conclusions

In summary, our findings underscored that the co-fermentation of non-S. cerevisiae strains (PKL) with S. cerevisiae var. diastaticus can lead to positive fermentation attenuation and distinctive aromatic profiles in beer brewing. The simultaneous inoculation of PKL and SY significantly boosted the production of key volatile compounds associated with “fruity” characteristics and resulted in lower residual sugar levels. In particular, SE-3-PKL/SY exhibited the capacity to enhance fruity aromas, notably leading to a 12.00-fold increase in 2-phenethyl acetate, a 12.37-fold increase in isoamyl acetate, and a 1.17-fold increase in linalool compared to SY single inoculation in beer brewing. The elevated levels of these compounds contributed to superior acceptability and pronounced fruity characteristics in the SE-3-PKL/SY coculture. These results underscored the significant role of PKL in refining the aromatic profile of beer brewing, thereby enriching the aromatic diversity of fermented beverages. Furthermore, our study broadened the application scope of PKL yeast beyond low-alcohol beer, indicating its potential to produce novel fermented foods through co-fermentation with other yeast strains exhibiting distinct fermentation characteristics.