Open AccessArticle

Alcoholic Fermentation Activators: Bee Pollen Extracts as a New Alternative

Juan Manuel Pérez-González

José Manuel Igartuburu

Víctor Palacios

Pau Sancho-Galán

Ana Jiménez-Cantizano

^1,*

and

Antonio Amores-Arrocha

Department of Chemical Engineering and Food Technology, Vegetal Production Area, Institute of Wine and Agri-Food Research (IVAGRO), University of Cadiz, Agrifood Campus of International Excellence (ceiA3), 11510 Puerto Real, Spain

Allelopathy Group, Department of Organic Chemistry, Institute of Biomolecules (INBIO), University of Cadiz, Agrifood Campus of International Excellence (ceiA3), 11510 Puerto Real, Spain

Department of Chemical Engineering and Food Technology, Food Technology Area, Institute of Wine and Agri-Food Research (IVAGRO), University of Cadiz, Agrifood Campus of International Excellence (ceiA3), 11510 Puerto Real, Spain

Author to whom correspondence should be addressed.

Agronomy 2024, 14(12), 2802; https://doi.org/10.3390/agronomy14122802

Submission received: 28 October 2024 / Revised: 20 November 2024 / Accepted: 22 November 2024 / Published: 25 November 2024

(This article belongs to the Special Issue Extraction and Analysis of Bioactive Compounds in Crops—2nd Edition)

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Abstract

Searching for natural alternatives to synthetic fermentation activators has led to the study of bee pollen as a natural alcoholic fermentation activator. This study evaluated the potential of different bee pollen extracts (0.25 g/L) as activators in a Palomino Fino grape must. By analysing the composition of each extract, it was possible to identify the specific bee pollen fractions with the highest efficacy for activating alcoholic fermentation. Four extracts were obtained through sequential extraction using various organic solvents of increasing polarity (hexane, acetone, ethanol, and water), and their compositions were characterised. The effect of each extract was evaluated by monitoring the viable yeast populations and fermentation kinetics throughout the alcoholic fermentation process, along with the physicochemical and colour characterisation of the white wines obtained. The bee pollen fraction extracted with hexane, which was rich in long-chain fatty acids, significantly increased the maximum yeast populations and improved the fermentation kinetics. However, the extracts rich in polyphenolic compounds exhibited slower fermentation rates. Based on the obtained results, the lipid fraction of bee pollen extracted with hexane may be responsible for its ability to activate alcoholic fermentation.

Keywords:

bee pollen; grape must; alcoholic fermentation; natural fermentative activator; palomino fino

1. Introduction

Wine is the result of the partial or total alcoholic fermentation of grape must [1]. Grape must has to contain all the nutrients essential for yeast growth, so that alcoholic fermentation can develop correctly [2,3]. These nutrients consist of a high sugar concentration, an adequate level of yeast-assimilable nitrogen (YAN), fatty acids, and vitamins, among others [4,5,6]. Insufficient YAN levels in grape musts can lead to sluggish or stuck fermentations, reduced yeast populations, an increased microbial contamination risk, and the formation of undesirable compounds [7,8]. However, an adequate concentration of YAN promotes an increase in yeast biomass and can improve the fermentation kinetics, as well as the formation of the volatile compounds responsible for floral and fruity aromas [7]. According to some authors, 140 mg/L of YAN is necessary for the successful alcoholic fermentation of grape must [7,9].

Several factors influence grape must composition, including the grape variety, soil type, vineyard management practices, grape health, and climate conditions [10]. Climatology is currently one of the main problems affecting winemaking [11]. The increase in temperature in recent years has had a negative impact on the growth and development of vines. These aspects could affect grape must composition, and as a result, winemaking is becoming increasingly challenging [12]. Under these temperature conditions, grape musts present a higher concentration of sugars, a lower acidity, high potential alcoholic strength, and a lower concentration of nutritional compounds for yeasts [13].

To address these challenges, the use of fermentation activators has become a common practice in winemaking to improve alcoholic fermentation [8]. These types of synthetic oenological products are mainly composed of fatty acids, diammonium phosphate (DAP), vitamins, amino acids, etc. [14]. However, no alternative products with a natural origin have yet been found that fulfil the same functions. For this reason, several studies have been carried out to investigate the potential use of bee pollen as a fermentation activator in winemaking [15]. Bee pollen is a natural source of proteins, essential amino acids, lipids, fatty acids, sterols, phospholipids, carbohydrates, carotenoids, and polyphenols [16,17,18]. Studies have reported notable improvements in fermentation kinetics, including an increase in the yeast population and enhanced survival rates at the end of alcoholic fermentation [10,19]. Furthermore, it has been observed that the addition of bee pollen at low doses leads to significant improvements in the sensory profile of the resulting wines [10]. The sequential extraction of bee pollen with various organic solvents of increasing polarity enables the isolation of extracts with distinct compositions. This process facilitates the individual concentration of the various nutrients present in bee pollen into separate extracts. Accordingly, the objective of this study was to evaluate the potential of each bee pollen extract as an activator of alcoholic fermentation. The influence of these extracts was assessed by analysing the yeast growth and fermentation kinetics, focusing on the evolution of the relative density and the consumption of yeast-assimilable nitrogen (YAN) throughout the fermentation process. This methodology allowed for the identification of the specific bee pollen fraction, and thus the compound type, with the greatest potential as an alcoholic fermentation activator.

2. Materials and Methods

2.1. Winemaking Conditions

The Palomino Fino variety was selected for this experiment. It is a white grape variety widely cultivated in southern Spain, specifically in the Marco de Jerez region, located in Andalusia. It is primarily used for the production of sherry wines under the designation of origin <<Jerez-Xérès-Sherry>> [20].

Palomino Fino grape must was obtained from the winery Cooperativa Andaluza, Unión de Viticultores Chiclaneros of Chiclana de la Frontera (Cádiz, Spain) and transported in 25 L food-grade plastic bottles from the winery’s cellar, where the grapes had been destemmed, crushed, and pressed. Potassium metabisulfite (80–90 mg/L) (Sigma-Aldrich Chemical S.A., Madrid, Spain) was added, and the pH was adjusted to 3.2–3.3 by adding tartaric acid (Sigma-Aldrich Chemical S.A., Madrid, Spain). The sulfited grape must was then deflated by gravity in methacrylate reservoirs at 10 °C for 24 h. Finally, the grape must was homogenised and distributed into glass fermenters (V = 5 L) with cooling jackets to maintain a controlled temperature of 18 °C during the alcoholic fermentation process.

2.2. Bee Pollen Extracts

Commercial multifloral bee pollen was chosen (Valencia, Spain). Prior to use, the pollen was crushed in a mill (Vorwerk’s Thermomix TM31, Wuppertal, Germany) and then stored in a dark glass bottle under desiccator conditions.

The bee pollen extracts were elaborated in a Soxhlet by sequential solid–liquid extractions using organic solvents in an increasing order of polarity: hexane, acetone, ethanol, and water. In the hexane extract, neutral lipids were obtained; in the acetone extract, compounds of an intermediate polarity, such as polar lipids, were obtained; in ethanol, polar compounds such as polyphenols and some proteins were obtained; and in water, sugars, complex carbohydrates, proteins, and other more polar components were obtained. The organic solvents were removed using a rotary evaporator, ensuring that their concentration in the extracts was less than 1 ppm [21]. In the case of the aqueous extract, a lyophilizer was used to remove the humidity and avoid the degradation of its components under the following conditions: initial freezing at −50 °C, followed by primary drying at −20 °C under a vacuum pressure of 0.1 mbar for 36 h. Secondary drying was performed at 20 °C for an additional 24 h. The organic solvent extracts were frozen until use.

2.3. Experimental Layout

The bee pollen extracts were added individually at a dosage of 0.25 g/L to each glass fermenter and homogenised using the ultrasonic equipment P-Selecta Digit Cool (P-Selecta, Barcelona, Spain) until achieving complete dissolution in grape must. In addition, a trial using untreated bee pollen (without extraction) in a dosage of 0.25 g/L was added to compare the performance with respect to the bee pollen extracts, as well as a control trial (without any additions). All the trials were performed in triplicate (n = 3) to ensure statistical significance. The strain of Saccharomyces cerevisiae Lalvin QA23B^TM (Lallemand, Barcelona, Spain) was used to induce the alcoholic fermentation, with an inoculum of 20 g/hL. Once the density measure was unchanged and the residual sugar had a concentration lower than 2 g/L, the alcoholic fermentation was considered complete. The resulting wines were subsequently treated with gelatine (4 g/hL, over 24 h) (Agrovin, Ciudad Real, Spain) and bentonite (40 g/hL, over 5 days) (Agrovin, Ciudad Real, Spain) to remove solid particles in suspension. Finally, the wines were filtered (0.45 µm) and bottled in opaque bottles under a nitrogen flow. A cork was employed to close the bottles.

2.4. Analytical Measurements

The characterisation of the bee pollen extracts was based on the determination of their content of fatty acids, protein amino acids, free amino acids, and sugars as alditol acetates, as shown in Table A1 in Appendix A. In addition, the extracts were characterised in terms of their total polyphenol content using a 13% v/v hydroalcoholic mixture, where the extracts were added at a concentration of 1.00 g/L until completely dissolved. The fatty acid composition was determined using margaric acid (C_17:0) (Sigma-Aldrich Chemical S.A., Madrid, Spain) as an internal standard and a subsequent gas chromatography–mass spectrometry analysis (GS-MS) according to the Official Standard Methods approved by the IUPAC for the analysis of oils, fats, and derivatives [22,23]. The amino acid analysis was carried out by microwave derivatization. L(+)-norleucine (Thermo Scientific, Waltham, MA, USA) was used as an internal standard and the analysis was performed by GS-MS according to the procedures described by Pinto et al. [24]. The composition of the sugars was based on a chromatographic analysis of carbohydrates in the form of alditol acetates, using myo-inositol (Sigma-Aldrich Chemical S.A., Madrid, Spain) as an internal standard, following the Pierce–Pourtallier method [25].

In the white grape must, the following physicochemical parameters were measured: the Baumé degree (°Be), density, pH, total acidity, and YAN content, and, in the white wines, the pH, total acidity, volatile acidity, alcoholic strength, residual sugars (RSs), YAN content, total polyphenols, and total polyphenol index (TPI). The °Be was determined using a calibrated Dujardin–Salleron hydrometer (Laboratories Dujardin–Salleron, Arcueil Cedex, France). The pH determinations were carried out using a portable pH meter (pH7 + DHS, XS instruments, Barcelona, Spain). The total acidity, volatile acidity, and alcohol strength were determined by following the officially approved methods of wine analysis by the OIV [26]. The total acidity was based on the determination of the main acids present in the must or wine using titration by adding a 0.1 M NaOH solution in the presence of bromothymol blue as an indicator [26]. The volatile acidity was based on the determination of volatile acids, mainly acetic acid. The wine distillate was titrated using 0.1 M NaOH in the presence of phenolphthalein as an indicator. The alcohol strength was defined as the number of litres of ethanol present in 100 L of a hydroalcoholic solution [26].

The YAN content was based on the determination of the ammonia nitrogen and amine nitrogen content by an enzymatic chemical analysis and colorimetric quantification [26] using the chemical analyser Micro Miura^TM (TDI, Barcelona, Spain). The RSs (glucose and fructose) were determined by ion chromatography (930 Compact IC Flex, Metrohm, Herisau, Switzerland) containing a pulse-amperometric detector and a gold working electrode. The elution was carried out isocratically at a 0.5 mL/min flow rate with 300 mM sodium hydroxide (NaOH) and 1 mM sodium acetate (NaOAc). The separation was achieved on a Metrosep Carb 2–150/4.0 column (Metrohm). The determination of the total polyphenols was carried out by an enzymatic chemical analysis and colorimetric quantification using the chemical analyser Micro Miura^TM (TDI, Barcelona, Spain). The total polyphenol index (TPI) values were determined through an absorbance measurement at 280 nm [26] using an UV–visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA).

During alcoholic fermentation, the viable yeast population, relative density, and YAN content were determined. Viable yeast population counts were performed by the methylene blue staining method [19], using an optical Nikon microscope with 40× magnification and a Neubauer chamber (Brand^TM, Merck, Madrid, Spain). The density values were determined by direct measurement with a DMA 5000 M density meter (ANTOM-PAAR, Net InterLab SAL, Madrid, Spain). The relative density of the samples was expressed as the ratio of the sample density compared to the initial sample density.

Finally, the chromatic characters of the wines were also evaluated according to the CIELab parameters (L*, a*, b* and C*) [27,28], colour intensity (CI), and tone (N). The CIELab parameters were carried out by following the recommendations of the International Commission of L’Eclairage and were used to evaluate the wine colour. The CI and N were determined by the methods recommended by the OIV [26]. Also, the Euclidean distance between two points in a three-dimensional space defined by L*, a*, and b* was used to calculate the colour differences perceived by the human eye (∆E*ab) [29].

2.5. Sensory Evaluation

A sensory evaluation of the wines was performed to assess differences in the organoleptic properties of the trials. The analysis compared the wines produced with bee pollen extracts, bee pollen, and the control sample. The sensory analysis was performed in individual booths with controlled illumination, located in the tasting room of the University Institute of Viticulture and Agri-Food Research (IVAGRO, Puerto Real, Cádiz, Spain). The sensory evaluations were carried out by a trained panel of 10 experts (aged 30–55 years, both men and women) with experience and training in wine evaluation. Each taster was presented with 50 mL of wine in standard tasting glasses [30], covered with watch glasses to minimize the evaporation of volatile compounds. The tasters assessed the trials based on specific sensory attributes, including olfactory (floral, fruity, dried fruits, etc.) and gustatory (saltiness, sweetness, etc.) characteristics, as well as an overall evaluation of the wine. All the attributes were scored on a 10-point scale, selected according to Jackson (2002) [31]. The wines were served at room temperature (20 ± 2 °C), and the evaluations were conducted between 11:30 am and 2:00 pm.

2.6. Statistical Analysis

The means and standard deviations were calculated, and significant differences were evaluated by a two-way ANOVA and Bonferroni’s multiple range (BSD) test; p < 0.05 was considered significant (GraphPad Prims version 8.0.1 for Windows, GraphPad Software, San Diego, CA, USA).

3. Results and Discussion

3.1. Influence of Bee Pollen Extract Addition on Grape Must Composition

In previous research, Amores-Arrocha et al. [10,15,32] carried out a study on the application of bee pollen at different concentrations, ranging from 0.1 g/L to 20 g/L, in the alcoholic fermentation. The aim was to analyse how this variability affected the vinification process of two types of white grape must: Palomino Fino and Riesling. The pollen doses that provided the most favourable results in terms of the alcoholic fermentation kinetics and physicochemical parameters at the end of the winemaking process ranged from 0.25 g/L to 1.00 g/L. However, in the sensory analysis, the wines produced with a bee pollen dose of 0.25 g/L received the highest ratings for aromatic and gustatory quality, as well as the highest scores in the overall judgement. In this sense, a bee pollen extract concentration of 0.25 g/L was selected for the development of the experiment [10].

The physicochemical composition of the Palomino Fino grape musts after the addition of bee pollen or bee pollen extracts and the control sample (without any additions) are shown (Table 1). In general, the °Be, total acidity, and pH parameters showed very similar results, with no significant differences. The additions of bee pollen or its extracts did not seem to significantly affect these physicochemical properties. In a previous study [32], the use of bee pollen at a concentration of 0.25 g/L in grape must also showed no significant difference compared to the control. Therefore, the same trend was expected to be observed when using bee pollen extracts at the same concentration in these oenological parameters.

Regarding the YAN content, in general, no significant differences were found between the extracts compared to the control, except for the water extract (ANOVA, p < 0.05), for which there was a slight increase of 3% after the extract addition to the grape must. This may have been due to the fact that the water extract had the highest concentration of protein amino acids (Table A1 in Appendix A). As a result, there was an increase in the amino nitrogen content (α-NH₂) of the grape must. Several authors have reported that amino acids are a good nitrogen source and contribute to an increase in YAN levels, as Hernández-Ortez et al. indicate [8,33]. Nevertheless, this increase is less than the 6% increase obtained with the 0.25 g/L dose of bee pollen, which also showed significant differences compared to the control (ANOVA, p < 0.05). Amores-Arrocha et al. [32] observed a similar behaviour in their study, where different white grape musts were enriched with various doses of bee pollen. In this sense, it was to be expected that the most significant differences between the trials would be found in the nitrogen content.

3.2. Bee Pollen Extract Influence on Yeast Growth Kinetics of Alcoholic Fermentation

The development of viable Saccharomyces cerevisiae during the alcoholic fermentation of Palomino Fino grape must, using different bee pollen extracts, bee pollen (without extraction), and a control, was monitored (Figure 1). The addition of bee pollen or its extracts did not reduce the yeast lag phase compared to the control. From the fourth day onwards, the yeast populations began to develop exponential growth until the sixth day, where, in all cases, the maximum values of the yeast populations were reached. At this point, significant differences were observed between the trials (ANOVA, p < 0.05).

The maximum yeast populations were observed in the samples where bee pollen or the hexane extract were added, with 1.08 × 10⁸ and 1.05 × 10⁸ CFU/mL, resulting in a 15% and 12% increase over the control, respectively. These values were not significantly different from each other, but they were significantly different from the rest of the samples and the control (ANOVA, p < 0.05). The yeast populations were also significantly larger for the sample where the water extract was added compared to the remaining trials (acetone and ethanol) (ANOVA, p < 0.05). On the other hand, the acetone and ethanol extract trials showed a very similar behaviour to each other, with the smallest yeast population values below the control.

The observed trend in the hexane extract may be attributable to the presence of organic compounds in its composition. As can be seen in the extract characterisation (Table A1 in Appendix A), the hexane extract had the highest fatty acid content. Fatty acids are fundamental to the yeast life cycle, as they are involved in different cellular processes. One of the most important functions is the maintenance of the cell structure during alcoholic fermentation as a consequence of ethanol formation, intervening in the fluidity and permeability of the yeast lipid membrane [34]. Moreover, several studies have reported that long-chain fatty acids, such as stearic acid (C₁₈), oleic acid (C_18:1), linoleic acid (C_18:2), and alpha-linolenic acid (C_18:3), could act as growth activators when metabolised by yeast to increase the proportion of living cells during alcoholic fermentation [35,36]. In addition to fatty acids, hexane was able to extract other lipid-based organic compounds from bee pollen, such as sterols and phospholipids [37,38]. These compounds are involved in various cellular processes, such as cell multiplication [39], but they were not investigated in this work. According to these results, it is likely that the lipid fraction of bee pollen extracted in hexane is responsible for the alcoholic-fermentation-activating properties. On the other hand, the yeast populations for the acetone and ethanol extract trials remained below that of the control. These extracts could be providing some kind of compounds that inhibit the correct development of yeasts in grape must. As can be observed (Table A1 in Appendix A, total polyphenols), the acetone and ethanol bee pollen extracts were rich in polyphenolic compounds. One of the most important characteristics of bee pollen is its antioxidant capacity due to its richness in polyphenolic compounds [40,41]. Some authors have demonstrated that a higher concentration of polyphenols in the medium can lead to a decrease in yeast growth during alcoholic fermentation [42]. In view of this fact, the contribution of polyphenolic compounds by the acetone and ethanol extracts to the grape must could have had a negative effect on the correct development of the yeast biomass.

From the 7th day on, as might be expected, a decrease in the yeast populations was observed due to the beginning of the cell lysis phase [15] in all cases. The use of bee pollen and the hexane extract also had an effect on the death phase of the yeast. The surviving yeast populations at the end of alcoholic fermentation in these trials were higher than the control and the remaining samples, showing significant differences (ANOVA, p < 0.05). Therefore, in view of the obtained results, the bee pollen fractions richer in lipid compounds could also establish a positive response in the yeast survival rate, acting as a protector against the hostile environmental conditions generated at the end of alcoholic fermentation [43].

3.3. Bee Pollen Extract Influence on Fermentation Progress

The influence of the bee pollen and extracts on the fermentation kinetics through the evolution of the relative density during the alcoholic fermentation process for Palomino Fino grape must was determined (Figure 2). The results obtained were in line with the evolution of the yeast populations observed in Figure 1. It was observed that, from the beginning of fermentation until the 4th day (yeast latency phase), no significant differences were observed.

From the 4th day on, coinciding with the yeast growth exponential phase (Figure 1, 4th day), the relative density values began to decrease in all cases due to the consumption of the sugars present in the grape must. Previous research has shown that the addition of bee pollen at concentrations above 0.1 g/L significantly accelerates the reduction in the initial grape must density, thereby improving the fermentation kinetics and the overall alcoholic fermentation efficiency [15,32]. In this sense, it should be noted that an improvement in the alcoholic fermentation kinetics was produced by the 0.25 g/L dose of bee pollen, followed by the hexane and water extracts, compared to the control sample. Significant differences were observed between them as well as between the control and the rest of the trials (acetone and ethanol extracts) (ANOVA, p < 0.05). As expected, the bee pollen and hexane extract trials showed the highest fermentation kinetics during the exponential phase. This was likely due to the fact that they showed greater cell multiplication than the other samples (Figure 1). However, although no significant differences were found between the water extract and the control during the exponential phase with respect to the yeast populations (Figure 1), significant differences were observed in terms of the fermentation kinetics (ANOVA, p < 0.05). The aqueous extract showed better fermentation kinetics than the control sample, as it had a more pronounced slope.

This decrease in the relative density values was observed until the 8th day. At this point, the behaviour of all the trials began to homogenise gradually. From the 11th day on, the bee pollen and hexane trials showed constant values of relative density. These trials had the highest yeast populations at the end of the process, and therefore, they completed alcoholic fermentation earlier than the other samples. This finding supports the idea that the provision of lipid compounds, such as long-chain fatty acids, through bee pollen or a hexane extract may improve the fermentation kinetics due to the increased cell multiplication shown in Figure 1. Nevertheless, it was not until the 14th day that the remaining trials achieved a constant relative density. This delay was due to a lower survival rate of the yeast populations, which required an additional 3 days to complete alcoholic fermentation. From the 14th on, all the trials showed similar trends up to the end of alcoholic fermentation and no significant differences were observed.

3.4. Bee Pollen Extract Influence on YAN Evolution During Alcoholic Fermentation

Figure 3 shows the influence of the bee pollen and its extracts on the YAN content during the alcoholic fermentation process of Palomino Fino grape must. It should be noted that the starting raw material was a grape must enriched in YAN content in all cases, with values above 230 mg/L. According to some authors, the minimum YAN content should not be less than 140 mg/L in the starting grape must [8,9]. Therefore, it was expected that all the trials would experience correct alcoholic fermentation.

From the beginning of alcoholic fermentation until the second day, no significant YAN consumption was observed. Coinciding with the beginning of the exponential phase (Figure 1), on the 4th day, the YAN content values showed significant differences in all cases. Nevertheless, the acetone and ethanol extracts did not show significant differences between them, but did show significant differences with respect to the remaining trials (ANOVA, p < 0.05).

Nitrogen compound consumption by yeasts during alcoholic fermentation is essential for their development and metabolism [44]. In studies conducted by Amores-Arrocha et al. [32] using bee pollen as a fermentation activator, it was observed that a larger yeast population was associated with a higher YAN consumption during alcoholic fermentation. As can be seen, this trend was observed from the 4th to the 6th days. The results obtained are in line with those in Figure 1 and Figure 2. The bee pollen, hexane, and water extracts exhibited a higher decrease in YAN content, consuming 86%, 81%, and 79% of the initial total YAN, respectively. In contrast, the control consumed only 76%. Below the control, the ethanol and acetone extracts showed 75% and 71% consumption, respectively (Figure 3, 6th day). This performance would be related to the lower yeast population, possibly due to the contribution of polyphenolic compounds from these extracts. On the 8th day, all the trials reached the minimum YAN values. This coincided with the decrease in the yeast populations (Figure 1, 8th day) and the stabilization of the relative density values (Figure 2, 8th day). At this point, no significant differences were found between the trials with respect to the YAN consumption. From the 8th day onwards, a slight increase in the YAN content was observed in all cases, coinciding with the yeast death phase (Figure 1). As expected, this increase may have been due to the release of nitrogen compounds by the yeasts during the cell lysis phase [45]. No significant differences were observed between the samples at this point. In previous studies, a 0.25 g/L dose of bee pollen resulted in an approximate 26% increase in the YAN during the yeast death phase (lasting 8 days) at the end of the alcoholic fermentation of Palomino Fino grape must [32]. However, in the present study, using the same bee pollen concentration, the increase was limited to 5% (from 32 mg/L to 45 mg/L) over the 9-day death phase.

Although the grape musts had similar YAN values, the bee pollen and hexane extract led to a faster consumption of YAN during alcoholic fermentation. This is consistent with the results obtained for the biomass development (Figure 1) and relative density evolution (Figure 2) observed in both trials.

3.5. Final Wine Physicochemical and Colour Characterisation

The results of the physicochemical and colour analysis of the white wines elaborated with the addition of bee pollen or bee pollen extracts, as well as the control sample, are shown in Table 2. Regarding the alcohol content, pH, total acidity, volatile acidity, and residual sugars, all the trials showed very similar results, with no significant differences. From an oenological point of view, this could mean that the addition of the extracts to the initial grape must did not have any influence on these parameters in the final wines. It should be noted that the YAN values presented in Table 2 are slightly lower than those measured at the end of alcoholic fermentation and shown in Figure 3 (17th day). This difference was attributed to the fact that the YAN characterisation was performed after the filtration and clarification of the wines. Concerning the polyphenolic content, significant differences were observed between the different extracts, the bee pollen, and the control (ANOVA, p < 0.05). The highest levels of total polyphenols were found in the acetone and ethanol extract trials. This is in accordance with the fact that these were the extracts with the highest polyphenolic content in their composition, as can be seen in Table A1 in Appendix A.

The mean values and standard deviations of the CIELab, L* (lightness), a* (red/green), b* (yellow/blue), H* (hue), C* (chroma), and absorbance at the 420 nm wavelength for both experiments are also shown in Table 2. It was observed that all the wines showed similar results in their L*, a*, and H* coordinates, as well as the absorbance at 420 nm, the colour intensity, and the tone. No significant differences were observed between the samples. On the other hand, there was a significant decrease in the values of the b* and C* coordinates for the wine made with the hexane bee pollen extract, showing significant differences with respect to the rest of the trials (ANOVA, p < 0.05). Despite this fact, all the wines remained within the yellow tones that are typical of young white wines. However, previous studies have shown that bee pollen concentrations exceeding 1.00 g/L in white wines increased the CIELab parameter values, shifting them from yellow toward dark orange tones [32].

To evaluate the colorimetric implications of adding bee pollen or its extracts, the mean colour difference (ΔE*ab) among pairs of wines (control and experimental samples) was calculated. A value of ΔE*ab of more than 3 CIELab units indicated colour differences visible to the human eye. In all cases, the results obtained were ΔE*ab < 3. These results would indicate that the bee pollen (0.25 g/L), and its extracts proportional to this dose, would not have a negative effect on the visual quality of the white wines obtained.

3.6. Descriptive Sensory Analysis

The results of the sensory analysis of the white wines produced with the addition of bee pollen or bee pollen extracts and the control sample are presented in Figure 4. Spider web diagrams show the mean values for the attributes analysed.

No significant differences were observed among the wines for the attributes sweetness, acidity, bitterness, saltiness, or body/structure. The use of bee pollen or its extracts did not appear to influence these sensory attributes in the final wines. However, significant differences were detected for the “fruitiness” attribute. The hexane extract showed no significant differences compared to the acetone extract, but differed significantly from the other treatments (ANOVA, p < 0.05). Bee pollen and the extracts from ethanol and water exhibited no significant differences among themselves, but were distinct from the other treatments (ANOVA, p < 0.05). The control wine showed significant differences compared to all the other samples (ANOVA, p < 0.05). These findings suggest that bee pollen and its extracts may enhance the fruity aromas in wine.

The fatty acids in bee pollen, beyond promoting yeast growth during fermentation, significantly impact the physicochemical and sensory properties of grape must [46]. Long-chain unsaturated fatty acids (e.g., oleic, linoleic, and linolenic acids) contribute to an improved wine sensory quality by promoting the formation of desirable fruity and floral aromas and enhancing the mouthfeel structure [47]. These compounds are also critical in producing esters that generate complex and appealing fruity notes, resulting in a more balanced sensory profile. On the other hand, the presence of medium- to short-chain saturated fatty acids (e.g., caprylic, capric, and lauric acids) affects the sensory profile, depending on their concentration [46]. At low concentrations, these fatty acids facilitate the formation of medium-chain esters, which impart tropical fruit and spice notes [48]. However, high concentrations can lead to the development of undesirable aromatic compounds [49].

As shown in Table A1 in Appendix A, the hexane and acetone extracts were composed mainly of long-chain unsaturated fatty acids and contained lower concentrations of short- to medium-chain fatty acids. The fruity and floral aromas identified by the tasters in the wines treated with the hexane and acetone extracts could be attributed to these fatty acids’ contributions during alcoholic fermentation.

Significant differences were observed in the control sample compared to all the other treatments for the attribute “spiciness” (ANOVA, p < 0.05). The use of bee pollen and its extracts appeared to reduce the intensity of the aromas associated with this attribute. Based on the results for the global judgement, the hexane and acetone extracts, along with bee pollen, demonstrated improvements in the sensory profile, showing significant differences with respect to the control and the rest of the trials.

4. Conclusions

The sequential extraction of bee pollen using various solvents of increasing polarity enabled the production of extracts that were rich in specific organic compounds. The aqueous extract, which was rich in protein amino acid compounds, increased the initial nitrogen compounds in grape must by 3% (241 ± 3, Table 1). As the extract with the highest nitrogen content (YAN), significant differences were expected compared to the control. However, both assays yielded remarkably similar values (9.69 × 10⁷ and 9.40 × 10⁷ CFU/mL, respectively). The hexane extract, in addition to producing the largest yeast populations (1.08 × 10⁸ CFU/mL, Figure 1), managed to maintain their survival at the end of alcoholic fermentation (increasing by 8% compared to the control). Furthermore, an enhancement in the fermentation kinetics and a faster consumption of YAN (86% of the total, 6th day, Figure 3) were observed compared to the remaining extracts. The lipid fraction of the bee pollen extracted with hexane is responsible for the pollen’s ability to activate alcoholic fermentation, especially long-chain unsaturated fatty acids (e.g., oleic, linoleic, and linolenic acids). The extraction of bee pollen with solvents such as acetone or ethanol generates fractions rich in polyphenolic compounds (27.00 ± 0.00 and 19.50 ± 1.00 mg/L present in 1.00 g/L of extract). This leads to a decrease in the development of viable yeast populations and the alcoholic fermentation kinetics. The use of bee pollen and its extracts at low concentrations did not appear to have a negative impact on the physicochemical properties of the wines, nor on their visual quality. The descriptive sensory analysis determined that the hexane and acetone extracts improved the fruity and floral attributes of the wines compared to the control. The presence of long-chain unsaturated fatty acids in the extracts facilitated the formation of volatile esters during alcoholic fermentation, which are associated with these sensory attributes. These compounds were highly appreciated by the tasting panel.

Bee pollen extracts may serve as an alternative to conventional fermentation activators for several reasons: (1) they are derived from a natural raw material, bee pollen; (2) their intrinsic properties allow for diverse outcomes in alcoholic fermentation, depending on the desired result; and (3) bee pollen extracts, particularly those obtained using organic solvents such as hexane and acetone, contribute to an enhanced aromatic and flavour complexity.

Author Contributions

Investigation, methodology, data curation, formal analysis, and writing—original draft, J.M.P.-G.; conceptualization, methodology, and supervision, J.M.I.; data curation and writing—review and editing, V.P.; software and writing—review and editing, P.S.-G.; supervision, resources, and validation, A.J.-C.; conceptualization, methodology, data curation, supervision, and writing—review and editing, A.A.-A. All authors have read and agreed to the published version of the manuscript.

Funding

This publication has been funded by the Dipulnnova Plus Program 2024—VitiLab.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Appendix A

Table A1. Organic compound composition (mg/L) present in 0.25 g/L of each extract after sequential extraction of bee pollen.

Organic Compound	Hexane Extract	Acetone Extract	Ethanol Extract	Water Extract
Fatty Acids
Capric acid (C₁₀)	0.08 ± 0.01	-	-	-
Lauric acid (C₁₂)	0.30 ± 0.11	0.14 ± 0.06	-	-
Myristic acid (C₁₄)	0.17 ± 0.02	0.34 ± 0.02	-	-
Palmitoleic acid (C_16:1)	1.01 ± 0.18	0.53 ± 0.12
Palmitic acid (C_16:0)	3.06 ± 0.28	1.43 ± 0.07	-	-
alpha-Linolenic acid (C_18:3)	6.61 ± 1.75	3.97 ± 0.40	-	-
Linoleic acid (C_18:2)	7.80 ± 1.36	1.81 ± 0.39	-	-
Oleic acid (C_18:1)	1.31 ± 0.19	4.31 ± 0.20	-	-
Stearic acid (C₁₈)	1.09 ± 0.16	1.38 ± 0.19	-	-
Arachidic acid (C₂₀)	0.80 ± 0.02	1.25 ± 0.17	-	-
Erucic acid (C_22:1)	0.88 ± 0.19	0.91 ± 0.15
Behenic acid (C₂₂)	0.69 ± 0.02	1.13 ± 0.29	-	-
Lignoceric acid (C₂₄)	0.47 ± 0.07	0.72 ± 0.21	-	-
Total fatty acids	24.27 ± 4.36	17.92 ± 2.27	-	-
% fatty acids	9.71%	7.17%	0.00%	0.00%
Protein Amino Acids
Alanine	-	-	0.87 ± 0.11	3.02 ± 0.18
Glycine	-	-	-	3.09 ± 0.39
Valine	-	0.39 ± 0.02	-	1.35 ± 0.22
Leucine	0.11 ± 0.01	0.08 ± 0.02	-	2.73 ± 0.28
Isoleucine	0.06 ± 0.01	0.24 ± 0.03	1.78 ± 0.62	9.24 ± 1.91
Proline	0.48 ± 0.19	-	0.05 ± 0.00	1.83 ± 0.28
Methionine	-	0.37 ± 0.14	-	5.03 ± 0.38
Serine	-	-	-	0.89 ± 0.29
Threonine	-	-	-	0.50 ± 0.04
Phenylalanine	-	-	-	1.18 ± 0.11
Aspartic acid	-	-	-	2.28 ± 0.57
Glutamic acid	-	-	-	0.16 ± 0.03
Total amino acids	0.65 ± 0.21	1.08 ± 0.21	2.70 ± 0.73	31.30 ± 4.68
% amino acids	0.26%	0.43%	1.08%	12.52%
Free Amino Acids
Alanine	-	0.02 ± 0.00	0.11 ± 0.03	0.03 ± 0.00
Glycine	-	0.03 ± 0.01	0.01 ± 0.00	-
Valine	-	0.05 ± 0.02	0.09 ± 0.00	-
Leucine	0.11 ± 0.01	0.03 ± 0.01	0.07 ± 0.01	0.02 ± 0.00
Isoleucine	-	0.04 ± 0.01	0.13 ± 0.02	-
Proline	0.06 ± 0.00	0.04 ± 0.00	0.21 ± 0.01	0.02 ± 0.00
Methionine	-	0.08 ± 0.01	0.05 ± 0.00	0.41 ± 0.09
Serine	-	0.12 ± 0.02	0.14 ± 0.03	-
Threonine	-	-	0.05 ± 0.01	-
Phenylalanine	-	-	0.16 ± 0.02	0.03 ± 0.00
Aspartic acid	-	-	0.03 ± 0.00	0.05 ± 0.01
Glutamic acid	-	0.11 ± 0.04	0.02 ± 0.00	-
Lysine	-	-	0.17 ± 0.05	-
Histidine	-	-	0.21 ± 0.04	-
Tyrosine	-	-	-	-
Total amino acids	0.17 ± 0.01	0.52 ± 0.12	1.45 ± 0.22	0.56 ± 0.10
% amino acids	0.01%	0.21%	0.58%	0.22%
Carbohydrates
Rhamnose	-	0.04 ± 0.00	0.17 ± 0.03	0.09 ± 0.00
Fucose	-	0.01 ± 0.00	-	0.09 ± 0.01
Arabinose	-	0.02 ± 0.00	0.05 ± 0.01	1.34 ± 0.29
Xylose	-	0.01 ± 0.00	0.03 ± 0.00	0.21 ± 0.03
Mannose	-	0.06 ± 0.01	0.24 ± 0.03	0.82 ± 0.07
Glucose	0.03 ± 0.00	0.85 ± 0.16	7.40 ± 0.83	11.04 ± 1.93
Galactose	0.17 ± 0.01	0.02 ± 0.00	0.07 ± 0.02	0.63 ± 0.12
Total carbohydrates	0.20 ± 0.01	1.01 ± 0.17	7.95 ± 0.92	14.23 ± 2.45
% carbohydrate	0.01%	0.40%	3.18%	5.69%
Total polyphenols (mg/L) *	0.00 ± 0.0	27.00 ± 0.0	19.50 ± 1.0	1.00 ± 0.0

The total value is the sum of all the compounds in each family present in 0.25 g/L of extract. The total percentage indicates the total concentration of each family of compounds present in 0.25 g/L of extract. * The total polyphenol content was determined in a 13% v/v hydroalcoholic solution to which the extracts were added at a concentration of 1.00 g/L.

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Figure 1. Development of viable Saccharomyces cerevisiae during the process of alcoholic fermentation of the Palomino Fino grape must using bee pollen extracts, bee pollen, or a control.

Figure 2. Relative density evolution during alcoholic fermentation of Palomino Fino grape must using doses of bee pollen and extracts.

Figure 3. Evolution of YAN in Palomino Fino grape must using doses of bee pollen extracts, bee pollen, and control during alcoholic fermentation.

Figure 4. Olfactory and taste evaluation of Palomino Fino wines made with bee pollen or bee pollen extracts and control. Stars (*) indicate significant differences between trials for the respective attributes (ANOVA, p < 0.05), as determined by a two-way ANOVA and applying a Bonferroni multiple range (BSD) test.

Table 1. Physicochemical composition of the must of Palomino Fino after the addition of bee pollen extracts or bee pollen and the control.

Parameters	Palomino Fino Grape Must with Bee Pollen Extracts
Parameters	Hexane Extract 0.25 g/L	Acetone Extract 0.25 g/L	Ethanol Extract 0.25 g/L	Water Extract 0.25 g/L	Bee Pollen 0.25 g/L	Control
°Be	10.28 ± 0 ^a	10.25 ± 0.01 ^a	10.24 ± 0.01 ^a	10.23 ± 0.02 ^a	10.25 ± 0.05 ^a	10.24 ± 0.01 ^a
Total acidity (g/L TH₂)	5.64 ± 0.07 ^a	5.63 ± 0.04 ^a	5.69 ± 0.04 ^a	5.69 ± 0.07 ^a	5.68 ± 0.00 ^a	5.67 ± 0.00 ^a
pH	3.32 ± 0.00 ^a	3.36 ± 0.01 ^a	3.34 ± 0.00 ^a	3.34 ± 0.03 ^a	3.33 ± 0.03 ^a	3.30 ± 0.03 ^a
Density (g/cm³)	1.067 ± 0.005 ^a	1.066 ± 0.003 ^a	1.063 ± 0.001 ^a	1.065 ± 0.004 ^a	1.073 ± 0.002 ^a	1.066 ± 0.000 ^a
Amine nitrogen (α-NH₂) (mg/L)	188 ± 2 ^a,b	190 ± 1 ^a	187 ± 1 ^b	194 ± 2 ^c	198 ± 1 ^d	187 ± 1 ^b
Ammonia nitrogen (NH₄) (mg/L)	44 ± 1 ^a	45 ± 3 ^a,b,c	47 ± 1 ^b,c,d	48 ± 1 ^d	49 ± 1 ^d	47 ± 1 ^c,d
YAN (mg/L)	232 ± 3 ^a	235 ± 3 ^a	234 ± 2 ^a	241 ± 3 ^b	247 ± 2 ^c	233 ± 1 ^a

Different superscript letters mean a significant difference between the extracts, bee pollen, and control (ANOVA, p < 0.05), determined by a two-way ANOVA and applying a Bonferroni multiple range (BSD) test.

Table 2. Physicochemical composition and colour analysis of Palomino Fino final wines with the addition of bee pollen extracts or bee pollen and the control.

Parameters	Palomino Fino Final Wines with Bee Pollen Extracts
Parameters	Hexane Extract 0.25 g/L	Acetone Extract 0.25 g/L	Ethanol Extract 0.25 g/L	Water Extract 0.25 g/L	Bee Pollen 0.25 g/L	Control
% Alc.	10.01 ± 0.00 ^a	10.10 ± 0.12 ^a	10.14 ± 0.18 ^a	10.14 ± 0.18 ^a	10.05 ± 0.06 ^a	10.14 ± 0.17 ^a
Total Acidity (g/L TH₂)	6.34 ± 0.05 ^a	6.11 ± 0.05 ^a	6.15 ± 0.32 ^a	6.19 ± 0.05 ^a	6.64 ± 0.16 ^a	6.53 ± 0.21 ^a
Volatile Acidity (g/L)	0.248 ± 0.080 ^a	0.250 ± 0.090 ^a	0.218 ± 0.020 ^a	0.181 ± 0.000 ^a	0.202 ± 0.010 ^a	0.199 ± 0.000 ^a
pH	3.18 ± 0.16 ^a	3.19 ± 0.02 ^a	3.14 ± 0.11 ^a	3.24 ± 0.02 ^a	3.20 ± 0.02 ^a	3.20 ± 0.02 ^a
YAN (mg/L)	27 ± 1 ^a	26 ± 2 ^a,c	23 ± 1 ^b,d	21 ± 0 ^b	26 ± 4 ^a,c	24 ± 1 ^c,d
Residual Sugars (g/L)	0.53 ± 0.24 ^a	0.73 ± 0.01 ^a	0.84 ± 0.02 ^a	0.96 ± 0.00 ^a	0.86 ± 0.08 ^a	0.82 ± 0.02 ^a
Total Polyphenols (mg/L)	201.50 ± 3.54 ^a	218.50 ± 2.12 ^b	213.00 ± 2.83 ^c	204.50 ± 3.54 ^a	208.00 ± 0.00 ^d	209.00 ± 1.41 ^d
TPI	14.72 ± 0.01 ^a	14.27 ± 0.02 ^a	11.76 ± 0.01 ^b	12.11 ± 1.74 ^b	13.93 ± 0.02 ^a	13.34 ± 0.01 ^a
L*	98.06 ± 0.69 ^a	97.52 ± 0.08 ^a	97.39 ± 0.01 ^a	97.42 ± 0.26 ^a	97.24 ± 0.11 ^a	97.65 ± 0.41 ^a
a*	−1.04 ± 0.01 ^a	−1.10 ± 0.05 ^a	−1.06 ± 0.04 ^a	−1.19 ± 0.04 ^a	−2.47 ± 0.03 ^a	−2.05 ± 0.00 ^a
b*	8.44 ± 2.04 ^a	9.89 ± 0.14 ^a,b	10.19 ± 0.08 ^b	10.34 ± 0.32 ^b	10.81 ± 0.05 ^b	10.31 ± 054 ^b
H*	98.67 ± 3.54 ^a	96.34 ± 0.14 ^a,b	95.97 ± 0.27 ^b	96.56 ± 0.44 ^a,b	101.36 ± 0.19 ^c	101.27 ± 0.60 ^c
C*	7.52 ± 3.00 ^a	9.95 ± 0.19 ^a,b	10.24 ± 0.07 ^b	10.41 ± 0.31 ^b	11.02 ± 0.04 ^b	10.51 ± 0.52 ^b
Colorant Intensity (CI)	0.15 ± 0.06 ^a	0.19 ± 0.02 ^a	0.20 ± 0.00 ^a	0.20 ± 0.01 ^a	0.23 ± 0.00 ^a	0.21 ± 0.02 ^a
Abs 420 nm	0.11 ± 0.05 ^a	0.15 ± 0.01 ^a	0.15 ± 0.00 ^a	0.15 ± 0.01 ^a	0.18 ± 0.00 ^a	0.16 ± 0.01 ^a
Tone (N)	3.87 ± 0.19 ^a	4.03 ± 0.08 ^a	3.98 ± 0.02 ^a	4.12 ± 0.21 ^a	4.78 ± 0.12 ^a	5.10 ± 0.44 ^a

Different superscript letters mean a significant difference between the samples (ANOVA, p < 0.05) determined by a two-way ANOVA and applying a Bonferroni multiple range (BSD) test.

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Pérez-González, J.M.; Igartuburu, J.M.; Palacios, V.; Sancho-Galán, P.; Jiménez-Cantizano, A.; Amores-Arrocha, A. Alcoholic Fermentation Activators: Bee Pollen Extracts as a New Alternative. Agronomy 2024, 14, 2802. https://doi.org/10.3390/agronomy14122802

AMA Style

Pérez-González JM, Igartuburu JM, Palacios V, Sancho-Galán P, Jiménez-Cantizano A, Amores-Arrocha A. Alcoholic Fermentation Activators: Bee Pollen Extracts as a New Alternative. Agronomy. 2024; 14(12):2802. https://doi.org/10.3390/agronomy14122802

Chicago/Turabian Style

Pérez-González, Juan Manuel, José Manuel Igartuburu, Víctor Palacios, Pau Sancho-Galán, Ana Jiménez-Cantizano, and Antonio Amores-Arrocha. 2024. "Alcoholic Fermentation Activators: Bee Pollen Extracts as a New Alternative" Agronomy 14, no. 12: 2802. https://doi.org/10.3390/agronomy14122802

APA Style

Pérez-González, J. M., Igartuburu, J. M., Palacios, V., Sancho-Galán, P., Jiménez-Cantizano, A., & Amores-Arrocha, A. (2024). Alcoholic Fermentation Activators: Bee Pollen Extracts as a New Alternative. Agronomy, 14(12), 2802. https://doi.org/10.3390/agronomy14122802

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