Supplementation of Chlorella vulgaris Extracts During Brewing: The Effects on Fermentation Properties, Phytochemical Activity and the Abundance of Volatile Organic Compounds

Abstract

Chlorella vulgaris, a microalga rich in secondary metabolites and nutrients, offers a promising alternative for promoting microbial growth in food fermentation processes. This study aimed to evaluate the effects of C. vulgaris extracts on fermentation kinetics, sensory characteristics, phytochemical composition, antioxidant activity, and the abundance of volatile organic compounds (VOCs) in treated versus control beers. The bioactive compounds from C. vulgaris were extracted using an ultrasound-assisted method with water as the solvent. A German Pilsner-style lager beer (GPB) was brewed and supplemented with 0.5, 1, and 5 g/L of C. vulgaris extracts prior to primary fermentation. Yeast viability, °Brix, and pH levels were monitored to assess fermentation progress. Phytochemical composition was analyzed by quantifying total polyphenols and flavonoids. The antioxidant activity of the beer was evaluated using both the 2,2-diphenyl-1-picrylhydrazyl (DPPH^●) and hydrogen peroxide assays. The addition of C. vulgaris extracts resulted in increased yeast viability and slight variations in gravity during the 7-day fermentation period. Moreover, the beers supplemented with C. vulgaris extracts demonstrated higher levels of total polyphenols, flavonoids, and antioxidant activity compared to the GPB. Specific volatile organic compounds, including 2-methyl-1-propanol, 1-hexanol, isopentyl hexanoate, 2-methylpropyl octanoate, β-myrcene, and geranyl acetate, were significantly more abundant (p < 0.05) in the treated beers than in the control. Sensory evaluations revealed a favorable impact of the treatment on aroma scores compared to the GPB. Overall, the findings indicate that C. vulgaris extracts could be a valuable ingredient for developing functional beers with enhanced health benefits, particularly regarding antioxidant activity. Additionally, the results underscore the importance of exploring innovative approaches that utilize natural sources like Chlorella to enrich the nutritional profile and sensory qualities of fermented products.

1. Introduction

In recent years, there has been a growing consumer interest in bioactive compounds such as polyphenols and carotenoids, recognized for their health-enhancing properties. These compounds, collectively known as phytochemicals, exhibit a diverse array of biological activities, including antioxidant and antimicrobial effects, attributed to their unique molecular structures [1]. Numerous studies have highlighted that dietary polyphenols, prominent secondary metabolites found in plants, and widely present in nature, offer a multitude of health benefits [2,3]. Plants and algae stand out as rich sources of polyphenols, which possess potent antioxidant properties. These compounds can be extracted and incorporated into various food products to enhance their nutritional profiles and health benefits [4]. Consumption of foods enriched with bioactive components like phenolics has been linked to a reduced risk of cancers and cardiovascular diseases [2,3,5]. Hops and malts, essential ingredients in beer production, contain ~30% and 70% to 80% polyphenols, respectively, contributing significantly to the phenolic content of the final beer [3,6]. It has been speculated that low-to-moderate beer consumption, typically 10–42 g for women and 10–56 g for men, may lower the risk of cardiovascular diseases, dementia, and help regulate cholesterol and glucose metabolism [2,7].

Despite the rich polyphenol composition in beer, the brewing processes, such as mashing and boiling, can lead to a reduction in their concentration. Reports suggest that wort clarification or filtration methods employed during brewing could potentially eliminate these beneficial compounds [2,8,9]. Efforts to preserve and enhance the bioactive compounds present in beer throughout the brewing process are crucial to harnessing the potential health benefits associated with moderate beer consumption. Strategies that maintain the polyphenol content in beer could pave the way for developing beverages that not only offer refreshment but also contribute positively to consumer health and wellness.

Furthermore, the processes of fermentation, maturation, bottling, and storage have been observed to potentially reduce or biotransform phenolic compounds into less beneficial forms [2,9]. Studies suggest that interactions and biotransformations between polyphenols, proteins, and polysaccharides during beer storage can lead to the formation of colloidal haze, ultimately diminishing both the concentration and the potential health benefits of these compounds [2,9,10]. In contrast, recent research has indicated that fermentation can significantly enhance the concentration of specific polyphenols, such as rutin, kaempferol, sinapinic, and protocatechuic acids, leading to an increase in antioxidant activity [11]. Similarly, positive effects of fermentation on various polyphenol concentrations have been reported, while some studies have shown a decrease in flavonoid and certain hydroxycinnamic acid derivatives post-fermentation [12,13,14]. These conflicting findings underscore the necessity of conducting a comprehensive investigation into the impact of incorporating C. vulgaris extracts during the brewing process.

Recent studies have highlighted the potential of leveraging microalgae to enhance the phytochemical and antioxidant properties of beer [15,16,17]. For instance, Taiti et al. supplemented beer with powdered Arthrospira platensis during the carbonation phase and observed that a 0.25% w/v addition increased cytoprotective capabilities and decreased intracellular oxidative stress against tBOOH-induced oxidative damage in H69 cells [18]. Additionally, wort enriched with varying concentrations of C. vulgaris biomass exhibited higher phytochemical and antioxidant potential compared to control ferments, with no significant differences noted in the abundance of volatile organic compounds (VOCs) and sensory properties between treated and control final beers [17].

Given that the addition of C. vulgaris biomass did not significantly impact sensory attributes and VOCs composition, a decision was made to explore the utilization of C. vulgaris extract in beer brewing, with a focus on assessing its influence on fermentation kinetics, yeast growth, and phytochemical concentration. Introducing a high-phenolic extract into wort presents an innovative avenue to enhance the presence of these beneficial compounds in the resulting beer, provided that it does not compromise sensory qualities. Furthermore, there is a lack of published research on the incorporation of bioactive extracts from C. vulgaris into wort during fermentation. Consequently, this study aims to examine the potential outcomes of wort supplementation with a water extract of C. vulgaris throughout the beer fermentation process, including an evaluation of its impact on phenolic compounds, antioxidant profile and the abundance of VOCs in the final product.

2. Materials and Methods

2.1. Materials and Chemicals

Food-grade C. vulgaris biomass was procured from Zhengzhou Sigma Chemical Co., Limited (Zhengzhou, China), while the dried M54 Californian lager yeast (Saccharomyces cerevisiae) was sourced from Bevie Handcraft NZ Limited (Albany, Auckland, New Zealand). The malts, including Château Pilsen 2rs (3EBC) and Château Cara blond (20EBC), were obtained from Castle Malting Limited (Beloeil, Belgium). German Perle hops (alpha acids (aa); 8%) and German Hersbrucker hops (aa; 3%) were generously provided by Beersfan in Yekaterinburg, Russia. Ethanol and methanol reagents were purchased from Rosbio (Saint Petersburg, Russia), while various chemicals such as ethyl acetate (>99% purity), ethyl hexanoate (>99% purity), isopentyl hexanoate (>99% purity), ethyl caprylate (>99% purity), citronellol (>99% purity), linalool (>99% purity), geranyl acetate (>99% purity), 1-propanol (>99% purity), 2-methyl-1-propanol (>99% purity), 1-hexanol (>99% purity), and phenylethyl alcohol (>99% purity) were acquired from Sigma Aldrich (St. Louis, MO, USA). Gallic acid (anhydrous), Folin–Ciocâlteu solution, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were sourced from Sigma-Aldrich (Darmstadt, Germany). Sodium carbonate crystal (Na₂CO₃), sodium hydroxide (NaOH), aluminum nitrite Al (NO₃)₃, and sodium nitrite (NaNO₂) were obtained from Bashkir Soda Company (Ufa, Bashkortostan, Russia). Quercetin was purchased from Conscientia Industrial Co., Ltd. (Zhejiang, China). It is important to note that all chemicals utilized in the study were of analytical grade.

2.2. Water Extraction of Chlorella vulgaris

Ultrasonic extraction was used in this study due to its efficiency and effectiveness in obtaining valuable bioactive compounds from C. vulgaris, making it a popular technique in food science, nutraceuticals, and pharmaceuticals. The extraction of C. vulgaris bioactive compounds was carried out using an ultrasonic extractor with distilled water as the solvent, following the method described in a previous study [19,20]. Specifically, Chlorella biomass (1 g) was measured into a beaker containing water (10 mL). The mixture was subjected to ultrasonication at 30 °C, 37 kHz, and 60% power for 30 min using an Ultrasonic-Assisted Extractor (UAE) (Elma Schmidbauer GmbH, Singen, Germany). Subsequently, the samples were centrifuged at 6000 rpm for 10 min in an IEC-CL Multispeed centrifuge (Rotoflox 32A, Hettich, Tuttlingen, Germany) and the supernatant was then transferred to a clean sterilized beaker. The water solvent was then removed by evaporation at 50 °C using an IKA Rv8 rotary evaporator (IKA Werke GmbH & Co. KG, Staufen, Germany) resulting with a viscous C. vulgaris water extract (CWE), which was then stored at −18 °C until incorporated into the beer and further analyses.

Extract Characterization

The extract was characterized to determine yield percentage (see Equation (1)), total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity (please refer to Section 2.6 and Section 2.7 for details).

$$\mathrm{E}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{y}\mathrm{i}\mathrm{e}\mathrm{l}\mathrm{d}\left(\mathrm{\%}\right)=\left[{\displaystyle \frac{\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{e}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t} \mathrm{a}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{r} \mathrm{e}\mathrm{v}\mathrm{a}\mathrm{p}\mathrm{o}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}}{\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{d}\mathrm{r}\mathrm{y} \mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}}\right]\times 100\mathrm{\%}$$

(1)

2.3. Brewing, Fermentation, and Carbonation of Beer

The single infusion mashing technique was chosen for its balance of efficiency, simplicity, and effectiveness, making it a popular option among brewers for producing a wide variety of beer styles. Prior to brewing, all equipment was cleaned and sanitized with 75% ethanol. In the brewing process, milled Château Pilsen 2rs malt (6 kg) and Château Cara blond malt (0.5 kg) were measured and added to a 30 L Easy Brew mash tun (Microbrewery, Guangdong, China) containing distilled water (25 L) heated to 55 °C. The mashing process followed specific temperature steps: 10 min at 55 °C, 60 min at 63 °C, 20 min at 72 °C, and a final 5 min step at 78 °C to halt enzymatic activity, followed by mashing off. Lautering was performed using a false bottom, with sparging conducted using 15 L of water at 80 °C. A brief recirculation of turbid runoff was performed until a clear wort was obtained. The resulting wort was then boiled for 90 min at 100 °C, with German Perle (30 g) and Hersbrucker hops (30 g) added to the wort at 60 min and 15 min before the end of the boil, respectively. The copper wort chiller was sanitized by immersion in the wort before boiling to ensure sterilization. After boiling, the chiller effectively cooled the wort to 20 ± 1 °C. A sterilized siphon was then used to transfer the clarified cold wort into 5 L fermentation containers equipped with airlocks, each containing 75% ethanol (5 mL). Different doses of 0.5, 1, and 5 g/L of C. vulgaris water extract (CWE) were added to separate fermenters in duplicate, while one fermenter without CWE served as the control. The dried Californian lager yeast was sanitized externally with 75% alcohol prior to opening. The yeast (10 g) was rehydrated in physiological saline solution (0.9% w/v, (200 mL)) from Solopharm (Saint Petersburg, Russia) for 1.5 h at 25 ± 1 °C before being pitched into the separate wort batches (5 L). Fermentation occurred at 22 ± 1 °C for seven days until a consistent °Brix (°Bx) reading was achieved [17,21,22,23]. Following the fermentation period, amber polyethylene terephthalate (PET) bottles were washed, sanitized with 75% alcohol, and allowed to dry for 2 h before being filled with the green beers (CEB1, 2, and 3 at concentrations of 0.5, 1, and 5 g/L, respectively, and GPB at 1 L). Dextrose sugar (3 g) was added to each bottle, which was then manually agitated. The bottles were incubated at 22 ± 1 °C for 2 days, followed by a temperature reduction to 16 ± 1 °C for an additional 5 days. Subsequently, the temperature was lowered to −4 °C for one week [17,23]. Beer and wort samples (50 mL) were stored at −4 °C for further analyses.

2.4. Monitoring of Yeast Growth/Viability During Fermentation

Samples were collected at specific time points (0, 24, 48, 72, 110, 120, 144, and 168 h) to monitor yeast growth by measuring the optical density at 600 nm (OD₆₀₀) using a Shimadzu ultraviolet (UV-1800) spectrophotometer (Kyoto, Japan).

2.5. Physicochemical Analysis of Ferments and Green Beer

Prior to analysis, the ferments were degassed in an ultrasonic bath (VWR, Dietikon, Switzerland) for 15 min at 45 kHz and 180 W, followed by centrifugation at 2000× g for 10 min. The resulting supernatant (20 mL) was frozen for the analysis of VOCs. Sugar levels in °Bx were measured using a refractometer RSG-100ATC (COMINHKPR124469, Xindacheng, Jiaozhou, China), and the alcohol by volume (% v/v) content was extrapolated from the measured °Bx using the Equation (2).

$$\mathrm{A}\mathrm{B}\mathrm{V}=\left(\mathrm{O}\mathrm{r}\mathrm{i}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l} \mathrm{g}\mathrm{r}\mathrm{a}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}-\mathrm{F}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l} \mathrm{g}\mathrm{r}\mathrm{a}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\right)\times 131.25$$

(2)

pH was determined using a digital pH meter (Mettler Toledo ^TM, Leicester, UK). The Free Amino Nitrogen (FAN), color, and bitterness were assessed according to the standards set by the American Society of Brewing Chemists [24]. FAN was evaluated using the ninhydrin method. Color was determined by measuring the absorbance of the beer at 430 nm and 700 nm. Bitterness was analyzed by mixing beer samples with 3 N HCl (1 mL) and 2,2,4-trimethylpentane (20 mL), followed by vortexing for 15 min. The absorbance of the resulting supernatant (the upper transparent layer) was then measured at 275 nm using a Shimadzu UV-1800 spectrophotometer (Kyoto, Japan).

2.6. Phytochemical Analyses

2.6.1. Determination of Total Phenolic Content

The total phenolic content of the beer samples/extracts was determined using a method previously described [25], with minor modifications. Briefly, 0.25 mL of 10-fold diluted samples were pipetted into test tubes containing distilled water (5.5 mL) and Folin–Ciocâlteu solution (0.5 mL). The mixture was homogenized and incubated for 5 min, after which 20% Na₂CO₃ solution (1 mL) was added. The assay tubes were incubated at 20 °C for 2 h, and the absorbance was measured at 765 nm against distilled water using a Shimadzu UV-1800 spectrophotometer. The total phenolic content was calculated using a gallic acid standard curve (y = 0.0038x + 0.0487, R² = 0.9982) and expressed as milligrams of gallic acid equivalents per liter of beer (GAE mg/L).

2.6.2. Determination of Total Flavonoid Content

The total flavonoid content (TFC) of the samples was determined using the NaNO₂–Al(NO₃)₃–NaOH colorimetric procedure [26]. Aliquots of the samples (0.5 mL) were pipetted into separate assay tubes containing 30% ethanol (2 mL) and 5% NaNO₂ (0.15 mL), followed by a 5 min incubation. After incubation, 10% Al(NO₃)₃ (0.15 mL) was added, and the mixture was allowed to stand for 6 min. Subsequently, 1 M NaOH (2 mL) was added, and the volume was adjusted to 5 mL 30% ethanol (0.2 mL). The mixture was incubated at room temperature for 10 min. Absorbance was measured at 510 nm using a Shimadzu UV-1800 spectrophotometer (Kyoto, Japan). In the blank, the amounts of Al(NO₃)₃ and NaOH solutions were replaced with the same volume of 30% ethanol. Aliquots of a standard solution of quercetin (0, 10, 20, 40, and 120 mg/L) were treated similarly. The TFC was calculated as quercetin equivalents per liter of beer (QE mg/L) using the quercetin standard curve (y = 0.0007x + 0.0086, R² = 0.9954).

2.7. Antioxidant Activity

2.7.1. In Vitro DPPH^● Antioxidant Activity

In vitro Antioxidant Activity (AOA) was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH^●) method [21,22,23]. Briefly, 1:10 diluted beer (2 mL) was pipetted into vials containing 0.1 mM DPPH methanol solution (2 mL). The mixture was incubated in a dark cupboard for 30 min at room temperature, and the absorbance was measured at 515 nm using a Shimadzu UV-1800 spectrophotometer. The control sample was prepared by mixing ethanol (2 mL) and 0.1 mM DPPH^● methanol solution (2 mL). DPPH^● antioxidant activity was calculated using Equation (3):

$$\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e}\left(\mathrm{\%}\right)\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}={\displaystyle \frac{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}\times 100\mathrm{\%}$$

(3)

2.7.2. Hydrogen Peroxide (H₂O₂) Scavenging Activity

The hydrogen peroxide (H₂O₂) scavenging capacity of the beer samples was evaluated following a modified method [27]. A 43 mM H₂O₂ solution was prepared in a 50 mM phosphate buffer at pH 7.4, and its absorbance was measured at 230 nm. Subsequently, 3 mL aliquots of the diluted beer samples (50-fold dilution) were added to separate test tubes containing H₂O₂ solution (1 mL). The reaction mixtures were incubated for 10 min at room temperature, with absorbance measured at 230 nm using the same Shimadzu UV-1800 spectrophotometer (Kyoto, Japan). Phosphate buffer served as the blank. The percentage of H₂O₂ scavenging by the beer samples was calculated using Equation (4).

$$\% {\mathrm{H}}_2{\mathrm{O}}_2 \mathrm{scavenging} \mathrm{activity}= [{\displaystyle \frac{\mathrm{Absorbance} \mathrm{of} \mathrm{control}-\mathrm{Absorbance} \mathrm{of} \mathrm{sample}}{\mathrm{Absorbance} \mathrm{of} \mathrm{control} \mathrm{sample}}}] \times 100$$

(4)

2.8. Quantification of Volatile Organic Compounds

The abundance of VOCs was determined using established methods [21,22,23,28]. Thawed beer samples (5 mL) were placed directly into headspace vials containing NaCl (1.5 g). A 5 µL aliquot of 12-bromo-1-dodecanol was added as an internal standard, and the mixture was agitated for one min. Subsequently, a 1 µL sample was manually injected into a gas chromatograph coupled with a mass spectrometer (GC-MS, 7890B; Agilent Technologies, Santa Clara, CA, USA). The VOCs were separated using a DB-5 ms capillary column (30 m × 0.25 mm, 0.25 µm, Agilent Technologies, Santa Clara, CA, USA) with helium as the carrier gas at a flow rate of 1.5 mL/min. The column oven temperature was initially set at 40 °C for 5 min, then increased to 190 °C and held for an additional 5 min. The temperatures of the injection port, ion source, and quadrupole were maintained at 240 °C, 230 °C, and 150 °C, respectively. Mass spectra were acquired in electron ionization (EI) mode at 70 eV, covering a mass range of 30–300 m/z. A mixed standard solution containing the target VOCs was prepared according to the method described [29]. This solution included ethyl acetate (0.625 ppm), ethyl hexanoate (0.375 ppm), isopentyl hexanoate (0.375 ppm), ethyl caprylate (0.375 ppm), citronellol (0.375 ppm), linalool (0.375 ppm), geranyl acetate (0.375 ppm), 1-propanol (0.625 ppm), 2-methyl-1-propanol (0.625 ppm), 1-hexanol (0.625 ppm), and phenylethyl alcohol (0.625 ppm), all prepared in an approximately 5% ethanol solution to simulate beer samples. This standard mixture was analyzed separately under the same conditions.

VOCs were identified by comparing the mass spectra of each compound against the National Institute of Standards and Technology library. A match factor (M), reverse match factor (R. Match), and a probability threshold (>80%) were used for confident identification. Retention indices (RIs) were calculated (https://database.pherobase.com/kovats/kovats-calculator, accessed on 8 March 2023) using the retention times of a C₉–C₃₀ n-alkane series analyzed under the same conditions, further validating the tentative identification of the selected VOCs.

2.9. Sensory Evaluation of Beer Samples

Sensory evaluation of the beer was conducted using the descriptive free sorting technique [21,22,23]. Twenty panelists (10 male and 10 female) from the Department of Technology for Organic Synthesis at Ural Federal University in Yekaterinburg, Russia, participated in the evaluation. The 9-point hedonic scale was used, where 9 indicated extremely like and 1 indicated extremely dislike. The assessment criteria included appearance, transparency, taste, bitterness, color, foam height, aroma, and overall acceptability of the beer samples (CEB1, CEB2, CEB3, and GPB), which were served in labeled 100 mL glasses. Water was provided for panelists to cleanse their palates between samples. The assessment criteria for evaluating beer samples focus on various sensory and visual attributes. The appearance refers to the visual appeal of the beer, including clarity and color. Transparency indicates how clear the beer is; a clear beer is generally preferred. The taste assesses the flavor profile, including sweetness, sourness, and other taste elements whereas bitterness measures the level of bitterness, often derived from hops, which can affect the overall flavor. Color evaluates the hue of the beer, which can range from pale to dark, influencing perceptions of style and quality. The foam height refers to the amount and stability of the foam (head) produced, which can enhance aroma and visual appeal. The aroma considers the smell of the beer, which can greatly impact the drinking experience. The overall acceptability is the general impression of the beer, combining all the criteria into a single assessment of enjoyment. Together, these criteria help judges or tasters provide a comprehensive evaluation of the beer’s quality and appeal.

2.10. Statistical Analysis

For statistical analyses, the data were analyzed using a one-way analysis of variance (ANOVA) in Minitab 18 (Minitab, LLC, Centre County, PA, USA). Post hoc Tukey tests were conducted to determine significant differences between the means at a significance level of p < 0.05. The relative areas of VOCs were normalized through autoscaling (mean-centered and divided by the standard deviation of each variable). Furthermore, Partial Least-Squares Discriminant Analysis (PLS-DA), variance in projection (VIP), and heatmap analysis were performed using MetaboAnalyst 6.0, an online web-based tool (https://www.metaboanalyst.ca/, accessed on 8 March 2024) [30].

3. Results and Discussions

3.1. Effect of Chlorella vulgaris Water Extracts on Wort Fermentation

Extraction Yield, Yeast Cells in Suspension, Wort Gravity, and pH

The extraction yield achieved with UAE was 67%, influenced by factors such as ultrasonic frequency, intensity, and duration [31]. This yield was significantly higher than that reported in a previous study that utilized novel buoyant beads in conjunction with UAE [32]. UAE is known for its advantages, including time efficiency, reduced solvent usage, and enhanced extraction efficiency, making it a preferred method over traditional chemical extraction techniques [33].

During fermentation, samples were taken periodically, and the optical density at 600 nm (OD₆₀₀) was measured to evaluate the effect of supplementing C. vulgaris water extracts on yeast growth over a 168 h fermentation period (Figure 1). Initially, at 0 h, the OD₆₀₀ readings averaged approximately 0.92 ± 0.01 for all samples, with no significant differences observed (p > 0.05). However, after 24 h of fermentation, yeast cell densities in suspension increased to 1.74 ± 0.01 (GPW), 1.81 ± 0.07 (CEW1), 1.97 ± 0.02 (CEW2), and 2.40 ± 0.04 (CEW3). In addition, CEW3 exhibited a significantly higher yeast cell density compared to GPW, CEW1, and CEW2 (p < 0.05). By the 42 h mark, CEW2 and CEW3 also displayed significant differences in yeast cell density when compared to GPW and CEW1. Interestingly, between 72 and 168 h, CEW3 consistently maintained a higher yeast cell density compared to the other treatments. The maximum yeast cell densities were observed at 24 and 48 h post-fermentation. These results suggest that C. vulgaris water extracts promote yeast growth during beer fermentation. The yeast cells in suspension serve as a reliable indicator for evaluating yeast performance and monitoring overall fermentation, as non-viable yeast cells settle at the bottom of the fermentation vessel and do not contribute to the fermentation process [29]. A recent study demonstrated that the growth and viability of brewer’s yeast were significantly enhanced when Sabouraud media was supplemented with 0.1% C. vulgaris water extract [19]. Additionally, the incorporation of C. vulgaris at varying concentrations has been shown to improve the growth and viability of lactic acid bacteria (LAB), while also shortening the logarithmic growth phase during the fermentation of non-alcoholic beverages [34,35,36]. Furthermore, enriching fermented dairy products such as yogurt, cheese, and kefir with C. vulgaris has been found to increase LAB concentrations [37,38,39]. The mechanisms by which C. vulgaris extract enhances yeast growth during beer fermentation are multifaceted. First, these extracts are rich in nutrients, including vitamins, minerals, amino acids, and growth factors, which can serve as supplementary nutrition for yeast cells, supporting their growth and metabolic functions. Moreover, the antioxidant properties of C. vulgaris extracts, particularly from chlorophyll and carotenoids, play a crucial role in protecting yeast cells from oxidative stress, ultimately improving yeast viability and fermentation efficiency [19,40]. Additionally, certain compounds in C. vulgaris extracts have been shown to enhance yeast stress tolerance, enabling yeast cells to better withstand environmental stressors such as high ethanol concentrations and temperature fluctuations [4,16,18,19,34,40]. These extracts may also act as metabolic stimulants by activating key metabolic pathways in yeast cells, thereby boosting growth rates. Furthermore, bioactive compounds present in the extracts can interact with yeast cells, influencing their physiology and gene expression to optimize fermentation performance [34,40]. The combined effects of nutrient supplementation, antioxidant protection, stress tolerance enhancement, metabolic stimulation, and bioactive compound interactions significantly enhance yeast growth during beer fermentation.

Throughout the fermentation process, a gradual decrease in wort gravity was observed, dropping from 12 to approximately 5.47 ± 0.04 °Bx over a 168 h period (Figure 2). The treated groups demonstrated significantly lower gravity readings compared to the control group (p < 0.05). Although the gravity remained stable with no significant differences (p > 0.05) between 42 and 120 h, CEW3 recorded a lower gravity of 5.90 ± 0.05 at 110 h, indicating a significant deviation from the other groups. Following this, the gravity continued to decline, with no significant variations (p > 0.05) among all groups at subsequent time points. By the 168 h mark, CEW3 exhibited the lowest gravity reading at 5.47 ± 0.19, demonstrating a statistically significant difference (p < 0.05) from the other treatments.

Wort gravity serves as an indicator of the fermentable sugar content in the wort and is essential for assessing yeast performance during fermentation [21,22,28]. As fermentation progresses, yeast metabolizes the fermentable sugars present in the wort, leading to a decrease in gravity as sugars are converted into metabolites and carbon dioxide by the yeast [28]. The uptake and utilization of sugars are crucial for brewery fermentation, as yeast relies on the fermentable sugars abundant in C. vulgaris biomass to produce ethanol [41,42]. In the context of this study, assessing whether the addition of C. vulgaris extracts influenced the availability of fermentable sugars as a substrate for yeast during fermentation presents challenges. This uncertainty stems from the potential effects of the extraction process on the quantity of fermentable sugars available. Previous research has shown that Tetraselmis chui SAG 8–6 biomass does not inhibit the release of fermentable sugars during micro-mashing [16]. While algae have primarily been utilized in the biofuel industry for alcoholic fermentation, their application in alcoholic beverage fermentation remains limited [43].

During the fermentation process, a decline in pH was observed, decreasing from 5.66 ± 0.01 to 4.33 ± 0.01 (CEW2), 4.34 ± 0.03 (GPW), 4.35 ± 0.01 (CEW1), and 4.35 ± 0.02 (CEW3). No significant differences were noted (p > 0.05) (Figure 3). A similar trend was reported during beer fermentation under audible sound treatment [28]. Throughout fermentation, yeast excretes acids, alcohol, and other metabolites to maintain metabolic balance. An imbalance in metabolic flux can stress yeast, potentially compromising fermentation efficiency, the synthesis of volatile organic compounds (VOCs), and the overall quality of the final products [28,34].

3.2. Physicochemical and Sensory Properties of the Final Beer

The final gravity is a crucial indicator for evaluating the maturation of green beer. As shown in

Table 1. Physicochemical properties of Chlorella extract beers.

Samples	Final Gravity (°Bx)	Alcohol (ABV%)	Bitterness (IBU)	Color (EBC Unit)	pH	FAN
						B/F	A/F
GPB	5.34 ± 0.09 b	3.69 ± 0.05 a	6.00 ± 0.00 a	5.91 ± 0.09 b	4.29 ± 0.01 a	87.96 ± 6.38 a	86.87 ± 12.29 a
CEB1	5.34 ± 009 b	3.69 ± 0.05 a	4.33 ± 0.46 b	6.10 ± 0.18 b	4.26 ± 0.00 a	81.82 ± 1.24 a	86.41 ± 0.89 a
CEB2	5.37 ± 0.05 b	3.67 ± 0.03 a	5.08 ± 0.11 ab	6.42 ± 0.09 b	4.26 ± 0.00 a	79.31 ± 14.37 a	86.91 ± 0.53 a
CEB3	5.84 ± 0.05 a	3.42 ± 0.03 b	5.65 ± 0.00 a	7.88 ± 0.36 a	4.29 ± 0.02 a	86.08 ± 4.79 a	94.56 ± 2.48 a

The results represent the means ± SD of the measurements made in triplicate. Means with different letters in each column denote significant differences (p < 0.05) using Tukey’s test. Where, GPB, German pilsner beer; CEB = Chlorella extract beer (CEB1 = 0.5 g/L, CEB2 = 1 g/L, CEB3 = 5 g/L), GPB = control beer without added Chlorella extract. B/F = before fermentation, A/F = after fermentation.

, the beer treated with CEW3 exhibited significantly higher gravity compared to the other treatments, including the control group. This difference may be attributed to the higher concentration of CEW3 used in the supplementation. Previous studies have indicated that yeast can metabolize C. vulgaris extract into simple sugars, potentially leading to increased final beer gravities [16,17].

In terms of alcohol by volume (ABV%), GPB, CEB1 (3.69 ± 0.05), and CEB2 (3.67 ± 0.03) showed higher levels compared to CEB3 (3.42 ± 0.03), which aligns with the gravity readings shown in Table 1. Since the ABV% is extrapolated from gravity readings, it is challenging to explain why CEB3 exhibited a lower alcohol percentage compared to ferments supplemented with lower concentrations of C. vulgaris extract. However, a plausible explanation is that the concentration of CEB3 may have been excessive, negatively affecting yeast performance and fermentation efficiency, resulting in a lower alcohol percentage in the final beer. Throughout fermentation, brewing yeast metabolizes wort sugars into alcohol and other metabolites. Beers supplemented with C. vulgaris extract demonstrated higher mean color values in EBC units, correlating with the hue of C. vulgaris. Specifically, the beer supplemented with 5 g/L of C. vulgaris extract (CEB3) exhibited a statistically significant difference (p < 0.05) in color compared to CEB1, CEB2, and the control. Additionally, the control beer (GPB) and CEB3 showed statistical differences (p < 0.05) in bitterness compared to CEB1, which had a bitterness of 4.33 IBU. The pigments present in C. vulgaris extract contribute to the intensified color values and reduced bitterness levels in the beer [16,17]. Furthermore, the use of hops with low alpha acid content may explain the lower IBU values observed. The addition of Chlorella extract did not significantly affect the pH or FAN levels (p > 0.05).

The mean sensory attributes of the C. vulgaris-supplemented beer, including the control samples, are illustrated in Figure 4. The incorporation of C. vulgaris extract significantly influenced the mean aroma score (p < 0.05), with CEB3 beer (7.29 ± 0.08) receiving the highest rating compared to the other treatments. C. vulgaris is a rich source of nutrients known to enhance the taste and aroma of beer, thereby improving its overall flavor profile through its unique chemical properties [44]. The study found no significant effects (p > 0.05) on various attributes such as appearance, transparency, taste, bitterness, color, foam height, or overall quality. However, Beisler and Sandmann [15] reported that beer supplemented with spirulina powder during various brewing stages exhibited an algae-like taste and a deep blue color, negatively impacting consumer acceptance due to its distinct flavor profile, as noted in previous studies [17]. To address these sensory concerns, optimizing the dosage of Chlorella extract added to the wort using surface response methodology may help identify the optimal amount that could significantly enhance all assessed sensory parameters.

3.3. Phytochemical Composition and Antioxidant Activity of Final Beer Samples

The total phenolic content (TPC) and total flavonoid content (TFC) in beer samples enriched with CWE ranged from 480.61 to 333.68 mg GAE/L and 1359.29 to 1150.95 mg QE/L, respectively (

Table 2. Phytochemical and antioxidant activities of beers.

Samples	Total Polyphenol Content (mg GAE/L)	Total Flavonoid Content (mg QE/L)	DPPH● Antioxidant Activity (%)	H2O2 Scavenging Activity (%)
GPB	245.75 ± 22.64 c	97.36 ± 13.64 c	16.52 ± 3.40 c	94.71 ± 0.23 b
CEB1	333.68 ± 13.03 b	1150.95 ± 57.24 b	60.66 ± 2.12 b	95.54 ± 1.17 ab
CEB2	411.32 ± 55.82 ab	1227.14 ± 40.41 ab	69.07 ± 3.40 ab	96.70 ± 0.00 ab
CEB3	480.61 ± 53.34 a	1359.29 ± 75.76 a	77.63 ± 1.27 a	97.32 ± 0.20 a

The results represented are the means ± SD of triplicate measurements. Means with different letters in each column denote significant differences (p < 0.05) using Tukey’s test, where GPB, German pilsner beer; CEB, Chlorella extract beer (CEB1 = 0.5 g/L Chlorella extract, CEB2 = 1 g/L Chlorella extract, CEB3 = 5 g/L Chlorella extract).

). Beers supplemented with C. vulgaris extract (CEB3 and CEB2) exhibited higher TPC and TFC values compared to the control and CEB1. Additionally, the incorporation of C. vulgaris extracts significantly (p < 0.05) enhanced antioxidant activity compared to the control group (Table 2).

In extracts obtained using UAE, TPC and TFC ranged from 400 to 4800 mg GAE/L and 3000 to 22,000 mg QE/L, respectively. Furthermore, the TPC and TFC of CWE3 extracts were significantly higher (p < 0.05) compared to CWE1 and CWE2 (Table S1).

Beer supplemented with 5 g/L of Chlorella extract (CEB3) demonstrated significantly greater (p < 0.05) DPPH^● and H₂O₂ scavenging activities compared to the control. Interestingly, the DPPH^● assay results revealed a significant difference (p < 0.05) between CEB3 and CEB1, which contradicted the findings from the H₂O₂ scavenging assay. The incorporation of Chlorella extracts enhanced the phytochemical composition and antioxidant activities of the beers, surpassing the results of a previous study [17] that utilized Chlorella biomass. Previous research has highlighted the abundance of phytochemicals, such as phenols and flavonoids, in C. vulgaris biomass, with concentrations ranging from 220 mg/g GAE to 547.023 mg/g RE [45,46,47]. The TPC and TFC of C. vulgaris water extracts have been shown to be higher than those obtained from other solvents [48], as evidenced by earlier research [49]. Studies indicate that beers rich in phenolic and flavonoid content tend to exhibit extended shelf life and improved sensory properties, including enhanced flavor and foam stability [25,50].

The CEB3 exhibited significantly higher (p < 0.05) DPPH^● scavenging activities, compared to CEB1 and GPB. Similarly, CEB3 showed higher H₂O₂ scavenging activity than the GPB (Table 2). Previous research indicates that Chlorella extracts, when prepared with water as a solvent, can achieve DPPH^● scavenging activities of up to 68.5%, outperforming extracts from other solvents [48]. However, ethanolic extracts of C. vulgaris cultivated in various media exhibited even higher inhibition rates, measuring 81.29% and 97.9% [47].

Statistical analysis of the extracts revealed that the DPPH^● antioxidant activity of the CWE3 extract was significantly higher (p < 0.05) than that of CWE1 and CWE2 (Table S2). In contrast, the H₂O₂ scavenging activity of the CWE3 extract exceeded that of CWE2, while CWE2 demonstrated higher activity than CWE1 (Table S2). An earlier study has reported a comparatively low DPPH^● radical scavenging activity (19.5%) for a methanolic extract (1 mg/mL) of C. vulgaris [44]. Additionally, the incorporation of CWE enhanced H₂O₂ scavenging activity in the final beer, which contradicted a prior study [17] that utilized only Chlorella biomass. The antioxidant properties of plants and microalgae are largely attributed to their flavonoid composition, with phenolic compounds enhancing their activities in beverage [3,44]. Chlorella is particularly rich in flavonoids and phenols. Various compounds, including flavonols, flavanones, flavones, phenolic acids, and others, have been shown to enhance the antioxidant activity of both plants and microalgae [3]. Chlorella extracts are especially abundant in these beneficial compounds, further contributing to their antioxidant properties.

3.4. Abundance of Volatile Organic Compounds

A total of 34 VOCs were carefully selected based on criteria such as the National Institute of Standards and Technology library match factor (M), probability (>80%), and their impact on beer flavor [29] (

Table 3. Volatile compounds identified in green beers.

Codes	Volatile Compounds	Classes	RI (Lit)	RI (Cal)	Method of ID
V1	1-propanol	Higher alcohols	1036	1030	NIST, IS
V2	2-methyl-1-propanol	Higher alcohols	1092	1085	NIST, IS
V3	1-hexanol	Higher alcohols	1340	1355	NIST, IS
V4	2-methyl-1-butanol	Higher alcohols	1208	1203	NIST
V5	3-ethoxy-1-propanol	Higher alcohols	1373	1370	NIST
V6	Phenylethyl alcohol	Higher alcohols	1906	1901	NIST, IS
V7	1-decanol	Higher alcohols	1760	1957	NIST
V8	2-nonen-1-ol	Higher alcohols	1105	1109	NIST
V9	3-methyl-1-butanol	Higher alcohols	1209	1206	NIST
V10	Isopentyl hexanoate	Esters	1452	1450	NIST, IS
V11	2-methylpropyl octanoate	Esters	1548	1530	NIST
V12	Propyl octanoate	Esters	1510	1503	NIST
V13	Ethyl acetate	Esters	888	881	NIST, IS
V14	Ethyl hexanoate	Esters	1233	1210	NIST, IS
V15	Ethyl caprylate	Esters	1429	1411	NIST, IS
V16	2-phenylethyl hexanoate	Esters	1185	1181	NIST
V17	Ethyl undecanoate	Esters	1739	1740	NIST
V18	Methyl octanoate	Esters	1386	1390	NIST
V19	Heptyl acetate	Esters	1377	1370	NIST
V20	Ethyl 3-hexenoate	Esters	1294	1280	NIST
V21	Acetic acid	Organic acids	1449	1440	NIST
V22	Octanoic acid	Organic acids	2060	2058	NIST
V23	Heptanoic acid	Organic acids	1950	1940	NIST
V24	2-ethyl-hexanoic acid	Organic acids	1910	1907	NIST
V25	β-Myrcene	Monoterpenes	1161	1150	NIST, IS
V26	Geranyl acetate	Monoterpenes	1752	1750	NIST
V27	2-eethylbutyl isobutyrate	Monoterpenes	1185	1181	NIST
V28	Linalool	Monoterpenes	1547	1530	NIST, IS
V29	Citronellol	Monoterpenes	1765	1758	NIST, IS
V30	Humulene	Monoterpenes	1667	1670	NIST
V31	(E)-2-octenal	Aldehydes	1430		NIST
V32	3-methyl-2(5H)-furanone	Ketone			NIST
V33	4-cyclopentene-1,3-dione	Ketone			NIST
V34	2,5-dimethyl-4-hydroxy-3(2H)-furanone	Ketone			NIST

RI (Cal)—calculated retention index; RI (Lit)—literature retention index (NIST12 database); NIST—National Institute of Standards and Technology (https://webbook.nist.gov, accessed on 8 March 2023); IS—internal standard.

). These selected VOCs encompassed a variety of compounds, including nine higher alcohols (HAs), eleven esters, four organic acids, six monoterpenes, three ketones, and one aldehyde.

To understand the impact of treatment on the abundance of these VOCs, a multivariate analysis was conducted using the VOCs dataset (Figure 5, Figure 6, Figure 7 and Figure 8). The unsupervised principal component analysis (PCA) biplot revealed that PC 1 and PC 2 collectively accounted for 31.1% and 17.9% of the total variance, respectively (Figure 5). The separation along PC 1 was notably influenced by the abundance of specific compounds such as humulene, ethyl undecanoate, 2-phenylethyl hexanoate, isopentyl hexanoate, 3-ethoxy-1-propanol, phenylethyl alcohol, 1-decanol, and 1-hexanol, all known to impact beer flavor. Conversely, the abundance of compounds like propyl octanoate, 3-methyl-1-butanol, 2-methyl-1-propanol, β-myrcene, linalool, 2-methylpropyl octanoate, 4-cyclopentene-1,3-dione, and 2-methylbutyl isobutyrate positively influenced PC 2. The data suggested that esters and higher alcohols played a significant role in influencing both PC 1 and PC 2. Furthermore, Partial Least Squares Discriminant Analysis (PLS-DA) accounted for 38.8% of the total variance, although there was minimal separation observed between CEB1, CEB2, CEB3, and GPB (Figure 6). A VIP plot highlighted the 15 most crucial VOCs (VIP score > 1) contributing to the beer’s flavor profile (Figure 7). Interestingly, all monoterpenes, with the exception of 2-methylbutyl isobutyrate, significantly contributed to the PLS-DA, potentially attributed to the treatment process.

Through analysis of variance (ANOVA), it was determined that treatment significantly impacted 19 out of the 34 VOCs under consideration (Figure 8). Specifically, CEB2-treated beer exhibited a significantly higher abundance (p < 0.05) of compounds such as 2-methyl-1-propanol, 1-hexanol, and 3-methyl-1-butanol compared to GPB and CEB3-treated beer.

Significant differences were observed in the abundance of various compounds in the different treatments. For instance, 3-ethoxy-1-propanol (V5) was found to be significantly higher (p < 0.05) in CEB2-treated beers compared to GPB, CEB3-, and CEB1-treated beers. Among the esters, CEB-treated beer exhibited a significantly higher (p < 0.05) abundance of isopentyl hexanoate (V10) compared to GPB and CEB3. Similarly, the abundance of 2-methylpropyl octanoate (V11) was significantly higher (p < 0.05) in CEB2 than in CEB3, but not compared to GPB or CEB1-treated beer. Additionally, ethyl caprylate (V15) was significantly higher (p < 0.05) in CEB1 than in CEB3-treated beer. Furthermore, CEB2-treated beer showed a significantly higher (p < 0.05) abundance of 2-phenylethyl hexanoate (V16) and heptyl acetate (V19) compared to GPB and CEB3-treated beer. Moreover, CEB2-treated beer displayed a significant abundance (p < 0.05) of methyl octanoate (V18) compared to CEB3. Notably, treatment had a significant impact on 2-ethyl-hexanoic acid (V24), with higher abundances observed in CEB1- and CEB2-treated beers compared to GPB.

In terms of monoterpenes, CEB2-treated beer exhibited a significant abundance (p < 0.05) of β-myrcene (V25) compared to CEB1, but not to GPB and CEB3. Conversely, CEB1-treated beer had a significantly higher (p < 0.05) abundance of geranyl acetate (V26) than CEB2. The esters and HAs identified in this study are consistent with a previous study involving the addition of Spirulina to beer [18].

Previous reports have highlighted that 3-methyl-1-butanol (60–80%), 2-methyl-1-propanol (15–25%), and 1-propanol (4–7%) are the main higher alcohols found in beer, synthesized from amino acid metabolism [51,52]. The higher abundance of higher alcohols in the treated samples compared to the control group may be attributed to increased wort nutrition, leading to a higher fermentation rate. Previous research indicated a high protein content in Chlorella, which could enhance the nutritional profile of wort when supplemented [53].

Volatile esters play a crucial role in imparting fruity, candy, and perfume-like flavor characteristics to beer. The synthesis rate of these esters is influenced by factors such as the concentration of acyl-coenzyme A (CoA), alcohols, the activity of enzymes (e.g., AATase activity) involved in their synthesis, and the extent of hydrolysis [54]. Volatile organic compounds (VOCs) are generated during yeast metabolism in beer fermentation and are vital components that impact consumer perceptions of beer [29]. Their levels are affected by various factors, including the choice of raw materials (e.g., malt, hops), mashing techniques, yeast strains, and fermentation conditions [21,22,28,29].

The supplementation of wort with C. vulgaris extract has the potential to enhance the production of volatile organic compounds in beer by inducing metabolic changes related to the tricarboxylic acid (TCA) cycle, amino acid metabolism, and fatty acid metabolism [29,51,54,55]. C. vulgaris extract contains bioactive compounds such as vitamins, minerals, amino acids, and fatty acids [56] that can act as precursors or cofactors for various metabolic pathways in yeast, thereby stimulating the production of volatile organic compounds during fermentation. Additionally, the bioactive compounds present in C. vulgaris extract can activate key metabolic pathways in yeast, including the TCA cycle, amino acid metabolism, and fatty acid metabolism [51,54,55]. The stimulation induced by supplementing wort with C. vulgaris extracts can enhance metabolic pathways, providing an increased pool of precursors for the biosynthesis of VOCs. The tricarboxylic acid (TCA) cycle, also known as the citric acid cycle, serves as a fundamental metabolic pathway for energy generation in yeast. Therefore, the supplementation of wort with C. vulgaris extracts may boost TCA cycle activity, leading to the production of more precursors for VOC biosynthesis. Additionally, bioactive compounds present in C. vulgaris extract could modulate gene expression in yeast, potentially upregulating genes (e.g., ATF1, ATF2) involved in essential metabolic pathways related to VOC production [51,54,55]. This gene upregulation can enhance substrate and precursor utilization for the efficient synthesis of desired VOCs. Overall, incorporating C. vulgaris extract during brewing can establish a synergistic relationship that augments metabolic activities associated with the TCA cycle, amino acid metabolism, and fatty acid metabolism. These metabolic enhancements can contribute to increased VOCs production in beer, thereby influencing its aroma and flavor profile [57,58].

Monoterpenes present in beer primarily originate from hop essential oils and play a significant role in shaping consumer perception. Studies have revealed that these monoterpenes can undergo biotransformation from one form to another due to specific genes (e.g., old yellow enzyme (OYE), alcohol acetyltransferase (ATF)) present in brewing yeast [55,59,60,61]. For example, fermentation of wort with different yeast strains (e.g., S. cerevisiae, S. cerevisiae var. Diastaticus, and S. pastorianus) has been shown to decrease geraniol while increasing the abundance of monoterpenes like citronellol, citronellyl acetate, and geranyl acetate [62]. A study has shown the absence of β-Citronellol in wort, with its levels increasing during beer fermentation, attributed to flavor precursors and a glucoside hydrolase activity in the lager yeast [60]. Recent research has shown that the Chlorella–Saccharomyces co-culture system significantly increased the concentration of protein, lipids, and carotenoids compared to single cultures, potentially contributing to the elevated abundance of VOCs [53]. Previous studies have also identified a range of VOCs, such as alcohols, ketones, aldehydes, esters, acids, terpenes, and furans, in Chlorella extract [63,64,65,66]. Therefore, leveraging the synergistic effects of Chlorella and Saccharomyces, breweries may enhance product quality while promoting sustainability and health benefits.

4. Conclusions

In conclusion, the supplementation of C. vulgaris extracts during brewing has significant effects on fermentation properties, phytochemical activity, and the abundance of volatile organic compounds. The findings of this study underscore the potential of C. vulgaris as a functional ingredient in brewing, providing opportunities to enhance both the nutritional value and sensory characteristics of the final product. The addition of C. vulgaris extract improved fermentation kinetics by enhancing yeast cell viability. Furthermore, incorporating C. vulgaris increased the levels of phytochemicals and antioxidant potential in the treated samples compared to the controls, with a concentration of 1 g/L (CEB2) identified as optimal for maximizing these benefits. The supplementation significantly affected the mean aroma score (p < 0.05), while taste, bitterness, and overall impression received lower ratings. Further research, particularly employing response surface methodology, is necessary to determine the optimal dosage of C. vulgaris extract that could influence the overall sensory attributes of the treated beer. Additionally, measuring individual phenolics and examining the effects of fermentation on their concentrations during brewing will be valuable for future studies. Overall, this research contributes to the growing body of knowledge on the use of algae-derived ingredients in food and beverage applications, highlighting the potential of C. vulgaris as a valuable resource in the brewing industry.