Physicochemical Kinetics and Determination of Methyl and Ethyl Alcohols in Own-Manufactured Craft Beer and Comparison with Commercial Mexican Craft Beers

Abstract

Beer is a fermented beverage part of socio-cultural traditions, and recently, craft beer (small and industrial manufacturers) has been covering interest in many regions, such as México. The objective of this work was to analyze physicochemical kinetics and determine the ethyl and methyl content in our own-manufactured (OM) craft beer and compare it with commercial craft beer. The experiment was divided into two sections: (1) three types of Ale craft beer (Blonde, American Pale, and Stout) were manufactured, and during the fermentation process, physicochemical kinetics (pH, specific gravity, lactic acid, ethanol, and sugars) were analyzed, and (2) both the OM and six commercial craft beers were studied to analyze the content of methyl and ethyl alcohols via HPLC. Statistical analysis: (1) A completely randomized design with repeated measurements and a Pearson’s correlation test (p < 0.05) were conducted. (2) A complete block randomized design to compare alcohol production in OM and commercial beers was conducted. Results: (1) The pH, specific gravity, and maltose tended to decrease during the first 24–48 h of fermentation; however, no changes were observed during the remaining 120 h. In contrast, lactic acid production started after 48 h in Blond and American Pale beers and after 72 h in Stout. Maltose, glucose, and fructose were consumed immediately during ethanol production. (2) The HPLC analysis showed a retention time of 25.9 min for ethanol, with the lowest levels for Blond (6.2%) and the highest for Pale (7.5%) in OM, while commercial beers ranged from 4.5% to 6.8%. No methanol was detected in any beer analyzed; on the other hand, lactic acid was observed in all beers analyzed. In conclusion, physicochemical changes were observed immediately at the beginning of the fermentation process. Commercial and OM beers comply with the provisions of the Official Mexican Standards.

1. Introduction

Beer is a popular beverage consumed all over the world and is considered one of the oldest and most consumed alcoholic beverages, and México is no exception [1]. Craft beer consumption in México is growing near 35% in contrast with industrial beer (41%) [2].

In recent years, the craft beer industry in México has experienced significant growth, establishing itself as a burgeoning sector in the global beer market. This rise is driven by a growing consumer preference for diverse and high-quality beverages, particularly those reflecting local flavors and traditions. Craft beer production has surged from 15,000 hectoliters in 2012 to over 330,000 hectoliters by 2022, underscoring the increasing demand for artisanal options [3]. The rise in microbreweries and independent producers has strengthened this sector, creating a dynamic and competitive ecosystem that encourages experimentation and raises production standards. Cities like Tijuana, Guadalajara, Monterrey, and México City have emerged as epicenters of this craft beer revolution, hosting festivals, competitions, and events that promote craft beer culture.

The main motivation for drinking craft beer is generated by three important factors: a desire for more knowledge, new taste experiences, and to move away from the mainstream beer consumption [2]. In other regions like Brazil, craft beer consumers show a preference for bitter, exotic beers with a strong and full-bodied flavor and seek high quality beers and authenticity [4].

Based on the style, ingredients, and brewing techniques, the alcohol content in beer varies because, during fermentation, sugars (glucose, fructose, sucrose, and maltose) are transformed into cellular energy, producing carbon dioxide and ethyl alcohol as by-products [5,6]. Generally, standard beers have an alcohol by volume (ABV) ranging from 4% to 6%; however, craft beers can exceed 10%. Some factors that influence the alcohol content can be the fermentation process (strain yeast used), the type and amount of malt affect the sugar concentration, and controlled fermentation (hygiene and temperature) [7]. During the brewing fermentation step, yeast strains (Saccharomyces cerevisiae) can produce many volatile and phenolic compounds, for instance, diacetyl, esters, ethanol, and higher alcohols (fusel alcohols). Esters are the major compound in terms of aroma profile; some beer’s sensory characteristics, like astringency and bitter flavor, may be influenced by the phenolic compound content in the beverage [8]. In some craft beers manufactured with barley malt and blue corn husk (50:50) which settle the soluble solids with sucrose or sweet brown sugar, the ethanol content can be as high as 9.5%, and a low phenol content could contain higher alcohols (such as propanol or butanol), which form when sugar derivatives are used during fermentation. These compounds contribute to the aroma and flavor of beer [9,10].

The incidence of methanol contamination of traditionally fermented beverages is increasing globally, resulting in the death of several persons; it may be produced by contaminating microbes during traditional ethanol fermentation, which is often inoculated spontaneously through mixed microbes, with the potential to produce mixed alcohols [11]. A study indicates that methanol can be found in craft beers but not in industrial ones, as it was not detected or quantified in the latter [12].

Based on the hypothesis that methyl and ethyl alcohols are produced in fermented beverages, this study aimed to analyze physicochemical kinetics and to determine the ethyl and methyl content via high-performance liquid chromatography, HPLC, in Mexican craft beers of own and commercial manufacture.

2. Materials and Methods

This work was conducted at the food process laboratory of the Coordinación Académica Región Altiplano Oeste and Instituto de Investigación de Zonas Desérticas of the Universidad Autónoma de San Luis Potosí, México. The experiment was divided into two sections, described as follows.

Section (1): Three styles of ale-type craft beers were manufactured by the authors (Blonde, American Pale, and Stout, five batches of 15 L of each style) according to Palmer’s [13] directions. The beer process was performed under sanitation conditions, with a detergent solution and spreading alcohol 70°, which were applied onto materials, equipment, and recipients; at the same time, the Star San (Five Star Chemicals & Supply Inc. Arvada, CO, USA) solution (2 mL/L of distilled water) was spread onto fermentors, kegs, and bottles.

The manufacturing process was the same for each beer style; some variations were due to the amount of malts and hops according to the beer style (Table 1).

For the three beer styles, the mashing process consisted of mixing the malt with purified water at 67 °C for 60 min. After filtering, the wort was obtained and subsequently pasteurized, boiled for 60 min (hops were added in this step, Table 1), and cooled to 19 °C. The pasteurized wort was introduced into a previously sanitized fermentor, and yeast was added. The fermentor was hermetically sealed, and an airlock was placed on the top to allow the output of CO₂ produced.

After a fermentation period of 7 days in a dark, closed room adapted with an air conditioner to maintain the temperature at 19–20 °C, the beer was placed in an aluminum keg. Carbonation was performed via forced gasification with CO₂ at 10 PSI to obtain 2.5 volumes of CO₂, which is a typical level for many ales [13]. Then, kegs were placed in a refrigerator at 4 °C for a week, with the pressure measured daily and topped up until equilibrium was achieved. Finally, the beer was bottled in amber glass bottles of 355 mL.

2.1. Physicochemical Kinetics

During the fermentation step, a 100 mL sample was taken (n = 5 fermentors), and physicochemical parameters like pH (Oakton pH 700, Vernon Hills, IL, USA) and specific gravity (hydrometer triple scale, Alla France, Chemillé en Anjou, France) were measured. Before fermentation started, the °Brix (Atago PAL-1, Minato-ku, Tokyo, Japan) was measured in the worts.

2.2. Beer Sample Preparation and HPLC Analysis

Beer samples were taken every 24 h during the seven days of fermentation. Each beer subsample was subjected to a sonication bath (Branson 1510, St. Louis, MO, USA) before taking the volume for analysis to remove CO₂. The samples were diluted with 5 mM H₂SO₄ (1:10) and filtered through 0.45 µm pore size membranes (Millipore, USA) before being injected into the HPLC. The analysis of lactic acid, ethanol, methanol, and individual sugars (maltose, glucose, and fructose) was performed in an HPLC system according to Ewen’s method [14] with an external standard. Beer samples were injected (20 µL) into an HP 1100 series HPLC system (Hewlett-Packard, Waldbronn, Germany) equipped with a refractive index detector. The separation of alcohols and sugars was performed using a Hi-Plex H column (Agilent, Santa Clara, CA, USA) and 5 mM H₂SO₄ as the mobile phase, with an isocratic gradient at a flow rate of 0.6 mL/min for 30 min. The temperature of the detector and column compartment was set to 55 °C to optimize the separation. The quantification of lactic acid, ethanol, glucose, fructose, and maltose was performed using calibration curves (r² > 0.99) at different concentrations of high purity standards. The HP ChemStation for LC Rev A. 09.03 software from Agilent (Palo Alto, CA, USA) was used for system control and data analysis.

Section (2): Six commercial craft beers (7Barrios, Minerva, 8Reales, Corona, Alpa Imperial, and Wasumara, n = 3) were studied to compare our results; these beers were selected according to their availability in supermarkets in San Luis Potosi City, Mexico, and the styles that were common among the brands. Both craft beers (commercial and own manufactured) were studied for the production of ethyl and methyl alcohols as well as lactic acid using HPLC.

2.3. Statistical Analysis

For Section (1), a completely randomized design with repeated measures in a specific time period (8 measurements, every 24 h) (p < 0.05) was conducted to analyze physicochemical parameters (pH, lactic acid, maltose, glucose, fructose, and specific gravity) and methyl and ethyl alcohol production between craft beer styles (n = 5 fermentors). Also, for the physicochemical kinetics, a Pearson’s correlation test was carried out (p < 0.05). Section (2): A completely block randomized design was conducted to analyze the alcohols production and lactic acid in own-manufactured and commercial craft beers, where the factor block was the beer style and the brands were considered as treatments; a Tukey mean comparison test (p < 0.05) was performed using the statistical software R ver. 4.1.0. (Vienna, Austria) [15].

3. Results

3.1. Section 1, Physicochemical Kinetics

3.1.1. pH and Lactic Acid

Changes in the pH of Blonde, American Pale, and Stout craft beers for 7 days (168 h) of fermentation are shown in Figure 1a. Statistical differences (p < 0.001) were found due to the factor treatment (beer style), the factor time (p < 0.0001), as well as the interaction between treatment and time (p < 0.0001). A drastic drop in pH of the three OM was observed immediately after 24 h, and then minimal changes were detected. At the end of the fermentation, the lowest pH value was obtained with the American Pale (pH = 4.4). In contrast, the highest pH value was found in the Blonde beer (pH = 4.8).

Lactic acid production is shown in Figure 1b. The main statistical differences were due to the treatment (p < 0.0001) and time (p < 0.0001) factors, as well as the interaction between treatment and time (p < 0.0001). Lactic acid production was evident after 24 h of fermentation, with a moderate increase until the end of this process, with American Pale being the beer with the highest values (0.21%).

3.1.2. Sugars

Figure 2 shows the changes in sugars levels for Blonde, American Pale, and Stout craft beers before and during 168 h of the fermentation process. Before fermentation, the °Brix levels of the pasteurized wort in the three beer styles were 13.5, 12, and 11.6 for the American Pale, Stout, and Blonde, respectively.

Maltose levels are observed in Figure 2a, with American Pale having the highest levels (8.6% w/v). The prompt decrease (up to 0% on average) within 24 to 72 h highlighted the active fermentation in the first three days. In fact, statistical differences were attributed to the treatment and time (p < 0.0001), as well as the interaction of these same factors (p < 0.0001). At the end of the 72 h of fermentation, all the maltose had been consumed in the three beer styles; therefore, no significant differences were observed.

Figure 2b shows the glucose results in Blonde, American Pale, and Stout craft beers before and during fermentation. All three beer types started with low glucose concentrations (~1.3–1.7% w/v), with no significant difference (p = 0.796); however, at the end of 48 h, glucose was completely consumed and remained so until the end of the fermentation. The statistical analysis showed significant differences attributed to the beer style (p < 0.001) and fermentation time (p < 0.0001), as well as to the interaction of these two factors (p < 0.001).

Regarding fructose, Blonde, American Pale, and Stout exhibited 1.2%, 0.7%, and 0.4% w/v, respectively (see Figure 2c). However, like glucose, fructose concentration dropped sharply within the first 24 h and was completely consumed after 48 h in all beer types. Statistical differences were attributed to the treatment (p < 0.0001), time (p < 0.0001), and the interaction between treatment and time (p < 0.0001).

Figure 3 shows the typical HPLC chromatograms of a craft beer sample (American Pale) before and after fermentation. Maltose (t_R = 10.6 min), glucose (t_R = 12.1 min), and fructose (t_R = 13.0 min) were detected before starting fermentation; however, the breakdown of such sugars and fermentation by-product synthesis (lactic acid and ethanol at min 16.8 and 25.9, respectively) were observed over time.

3.1.3. Specific Gravity and Alcohol

Figure 4a shows the change in specific gravity over time (hours) for the three types of beer: Blonde, American Pale, and Stout. The statistical analysis found differences due to the time (p < 0.0001) and the interaction between the treatment and time (p < 0.001). All three types of beer started with higher specific gravity values (around 1.06 to 1.07), indicating a higher sugar content at the beginning. The American Pale (Orange line) had the highest initial specific gravity, followed by Blonde and Stout. Statistical differences between treatments were observed at 0 h (p < 0.01), 24 h (p < 0.0001), and 48 h (p < 0.05); after that (72 h), the specific gravity values were stabilized close to 1.024 in the three treatments.

Methanol results were not presented as they were not detected in the three treatments, possibly because the amount was lower than the limit of detection (LOD) of 0.0016 mg/mL and the limit of quantification (LOQ) of 0.0056 mg/mL. Ethanol results are shown in Figure 4b; the graph shows the progression of ethanol content (% v/v) over time (hours) for the three types of beer: Blonde, American Pale, and Stout. Statistical differences were detected due to the treatments (p < 0.0001), time (p < 0.0001), and the interaction between the treatment and time (p < 0.0001). Ethanol content increases for all three types of beer as time progresses, starting from 0% at time zero. At the end of the fermentation period, the American Pale style had the highest final ethanol concentration (7.5%), followed by Stout (6.5%), and then by Blonde (6.2%).

Table 2 shows the results of the Pearson’s correlation test between the physicochemical kinetic variables. The pH showed a strong positive correlation with glucose (r = 0.8422) and fructose (r = 0.8116), both highly significant (p < 0.0001), In contrast, pH showed a negative correlation with ethanol (r = −0.7383), also significant. This suggests that as ethanol production increases (likely during fermentation), the pH decreases.

Lactic acid presented a negative correlation with pH (r = −0.5882), indicating that an increase in lactic acid lowers pH, which is consistent with acidic fermentation processes. Ethanol showed a negative correlation with the three sugars: maltose, glucose, and fructose (r = −0.7704, r = −0.9421, and r = −0.7022, respectively). The specific gravity had a strong positive correlation with maltose (r = 0.9067), while in contrast with ethanol, the correlation was negative (r = −0.7574).

3.2. Section 2, Alcohol Comparison Between Own-Manufactured and Commercial Beers

Methanol results were not presented because it was not detected in the OM or in commercial craft beers, possibly because the amount was lower than the limit of detection (LOD) of 0.0016 mg/mL and the limit of quantification (LOQ) of 0.0056 mg/mL.

Figure 5 compares the ethanol content (% v/v) of different types of beer (Blonde, Stout, and American Pale) across various commercial brands and the own-manufactured (OM) craft beer. The statistical analysis detected differences due to the treatments (p < 0.0001) and the block factor (p < 0.0001). American Pale (orange bars): OM and 7barrios presented the highest ethanol content (7.5 and 6.5%, respectively), significantly different from others American Pale brands, while 8Reales showed the lowest ethanol content (4.5%). Stout (brown bars): 7Barrios and OM showed the higher ethanol content (6.5 and 6.3%, respectively) with significant differences in contrast with the other three Stout beers; 8Reales presented the lowest ethanol content (4.5%). Blonde (yellow bars): OM had the highest ethanol content (6.2%). The beer 7Barrios had the lowest ethanol content (4.3%) in this category and also compared with the other two beer styles.

During this study, lactic acid was detected in OM and commercial craft beers; values are shown in Figure 6. The statistical analysis showed differences due to the treatments (p < 0.0001) and the block factor (p < 0.01). American Pale beers (Orange bars) generally had a higher lactic acid concentration than Blonde beers but lower than the 7Barrios Stout. Stout beers (Brown bars) tend to have low lactic acid content, except for 7Barrios being the highest in the entire dataset. Finally, the Blonde beers (Yellow bars) exhibit more variability in lactic acid content, with some brands showing low levels (Corona and Minerva) while others are significantly higher (Watsumara). In general, lactic acid content by beer style (block) was as follows: American Pale 0.23, Stout 0.16, and Blonde 0.15% w/v.

4. Discussion

Beer is a fermented beverage that contains a broad range of different components like water, ethanol, dextrin, carbon dioxide, and traces of organic acids that give its character, and it is made with a mixture of water, malt, hops, and yeast. During the fermentation process, yeast can convert relatively high concentrations of sugars into ethanol, carbon dioxide, and a wide range of secondary metabolites [16,17].

In the present study, the initial approach to quantifying methanol in craft beer samples was undertaken because methanol serves as an indicator of poor quality in both fermented and distilled alcoholic beverages, being a toxic compound for human consumption [18]. Attention was focused on the physicochemical changes during the fermentation step of Blonde, American Pale, and Stout beers, as well as the identification and quantification of possible undesirable methyl alcohol, and how ethanol content varies between beer styles. In fact, according to the Mexican regulation (NOM-199), it points out that an alcoholic fermented beverage is allowed to have between 2 and 20% ethanol content; on the other hand, regarding methanol, the same standard specifies that the maximum allowable limit is 300 mg/100 mL [19]. In this regard, all the beers analyzed comply with the provisions of the Official Mexican Standard. In this trial, methanol was not detected with the HPLC technique. Methanol is an alcohol that is produced naturally in the fermentation of fruit juices. To be produced, it requires the presence of soluble and insoluble fibers from fruits, that is, pectins, cellulose, and hemicellulose, because methanol is produced from the demethylation of these substrates and by the action of pectin methyl esterase enzymes that are generated by bacteria present in fruit juices [11,20]

The Pearson’s correlation test is an analysis that provides a detailed view of how variables interact during processes like fermentation, highlighting critical relationships for understanding system behavior.

All three beer types exhibit a significant pH decrease during the first 24 h; after the first 48 h, the pH of each beer type stabilizes, showing minor fluctuations over time (Figure 1a). The standard values [19] indicate that pH values ranged between 2.5 and 5.0, while pH of own-manufactured beers showed values that ranged between 4.4 and 4.8. A drop in pH values is likely due to fermentation activity, where yeast produces organic acids that acidify the medium, causing a decrease in pH. In our study, we found that these changes in pH were attributed to the production of organic acids like lactic acid during the fermentation process. The presence of lactic acid in beer can be due to the fact that under anaerobic conditions, yeast cells need to regenerate the NAD+ concentration through two possible routes: homolactic and heterolactic. The first of these routes involves the production of two molecules of lactic acid from one molecule of glucose; however, in the second route, which is the most likely to have occurred during our experiment, from one molecule of glucose, one molecule of lactic acid, one molecule of ethanol, and one molecule of CO₂ are produced [21].

The pyruvate resulting from glycolysis and its subsequent breakdown/oxidation to generate ATP and NADH in the citric acid cycle and oxidative phosphorylation could not be the case, since this process only occurs in the presence of oxygen [22], and also, a possible spoiling through lactic acid bacteria (LAB) cannot be ruled out, which is also the most frequent bacteria of beer spoilage [23]. It is important to point out that all craft beers, OM and commercial manufacturers (Figure 6), showed a small content of lactic acid. The variation among brands may indicate differences in brewing techniques or microbial activity. Higher lactic acid levels in stouts could be related to longer fermentation times or specific yeast and bacterial activity.

Maltose was the predominant sugar, while glucose and fructose were the minor ones; this also aligns with what was stated by Stewart [24]. A very strong positive correlation with specific gravity was observed, suggesting that maltose is a key component affecting overall sugar concentration and density. Maltose, glucose, and fructose presented a negative correlation with ethanol, implying that the three sugars are consumed during ethanol production, decreasing sugar concentration and density. Palmer [13] pointed out that a typical sugar spectrum of a wort ranged as follows: maltose (50–60%), dextrins (20–30%), maltotriose (15–20%), glucose (10–15%), and, finally, fructose and sucrose ranged between 1 and 2%. These values were similar to those obtained in this study.

The rapid depletion of sugars during fermentation occurs because yeast consumes glucose and fructose almost immediately (Figure 2b,c), within 24 to 48 h; this is also reported by Stewart [24], where glucose and fructose are consumed by brewer’s yeast cultures between 0 and 48 h of fermentation. The Stout beer style showed the fastest glucose consumption, followed closely by Blonde and American Pale. Stabilization at zero glucose and fructose confirms that fermentation progresses to more complex sugars after the initial glucose is exhausted. This indicates that glucose and fructose, being simple sugars, are quickly metabolized by yeast during the early stages of fermentation.

Specific gravity decreases significantly within the first 48 h for all three beers, indicating active fermentation and sugar conversion to ethanol. The final specific gravity values are similar for all three types (around 1.024), indicating similar residual sugar content. The decline in specific gravity correlates with the increase in ethanol content.

It is important to recognize that the beer-making process relies on the complete fermentation of simple sugars (i.e., they are fully consumed and converted into ethanol). According to Palmer [13], he pointed out that fermentation has three phases: adaptative (12–24 h), attenuative (1–3 days), and maturation–flocculation (day 3 to 7), with the last one being a reduction of acetaldehyde into alcohols; when the enzymatic breakdown of diacetyl occurs and when yeast becomes quiescent, the beer clears due to cells flocculating and settling to the fermentor bottom. In our case, flocculation was observed after 7 days of fermentation, when the beer was moved into the kegs.

Therefore, the alcohol content depends on the amount of malt specified in the formulation; the more malt used, the higher the alcohol content expected at the end of fermentation. This is reflected in the alcohol content obtained in the Blonde beer, which, with 4.25 kg of malt, had the lowest alcohol content. In contrast, the American Pale and Stout, with 5.2 kg and 4.85 kg of malt, respectively, had higher alcohol contents than the Blonde. Thus, the formulation is critical for estimating the potential alcohol content of our beer. In a study conducted by Giovenzana [25], the alcohol content of ale and lager beers was measured via spectrophotometry, and their results are consistent with those obtained in our work, where the ethanol content ranges between 4.9 and 7.1% alcohol by volume. In various studies, beers from different countries were analyzed, and it was identified using gas chromatography that ethanol is the main component of fermentation. Additionally, they mention that accelerated fermentation, due to high temperature, abundant inoculum, and high oxygenation, can increase ethanol production by up to 14% (v/v). They also point out that gas chromatography can identify more than 50 compounds in beer [26]. In another trial, 34 international commercial beer brands were analyzed, and the alcohol content values (ranging from 3.0 to 8.0% v/v) are also consistent with our values. They further note that variations in ethanol content between beers can be due to the raw materials and their quality, specifically the sources of the sugars. For example, in China, some beers, in addition to using malt and hops, also include rice and corn in their formulations [27]. In another work, they pointed out that fermenting beer in wooden barrels for long periods (3 months) increases ethanol production (11% v/v) and other volatile compounds with higher molecular weight, such as 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-phenylethanol [28].

Finally, as well as consumption, research concerned about craft beer grew exponentially; a total of 90% of published articles are concentrated in the 5-year period from 2015 to 2019, led by US, Italy, and France in the fields of marketing, sociology, food technology, information, etc. [29]. Future research should focus on the analysis of quality control of small brewers (also called homebrewers) and the analysis of fusel alcohols that can be produced during the fermentation process, as well as phenolic compounds and bitterness.

5. Conclusions

Physicochemical changes are observed almost immediately at the beginning of the fermentation process (0–48 h); then, they tend to stabilize. Once the fermentation process started, the maltose, glucose, and fructose were consumed totally. Methanol was not detected in any of the beers, OM or commercial ones. The highest alcohol content detected in all the beers analyzed was ethanol. The American Pale and Stout styles were the ones with the highest ethanol content, in contrast to the Blonde style, which is related to the amount of malt used. Both the own and commercial beers have an ethanol content of no more than 7.5%. All craft beers, OM and commercial beers, had a low lactic acid content. Even so, they all comply with the quality control standards outlined in the Official Mexican Standard for consumer safety

Author Contributions

Conceptualization, J.A.R.-H. and C.I.G.-H.; methodology, J.A.R.-H., T.d.J.R.-C. and C.I.G.-H.; investigation, J.A.R.-H., T.d.J.R.-C. and C.I.G.-H.; data curation, C.M.-C. and J.A.R.-H.; writing—original draft preparation, J.A.R.-H. and C.I.G.-H.; writing—review and editing, B.C.-P., C.I.G.-H., C.M.-C. and J.A.R.-H. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The data presented in this study are available on request from the corresponding author (accurately indicate status).

Conflicts of Interest

The companies (7 barrios, Minerva, 8 Reales, Corona, Alpa Imperial, and Wasumara) were not involved in the study design, execution, data collection, analysis, interpretation, and writing of this article or the decision to submit it for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Footnotes

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Figures and Tables

Figure 1. Physicochemical changes in the three styles of OM craft beers, Blonde ([Image omitted. Please see PDF.]), American Pale ([Image omitted. Please see PDF.]), and Stout ([Image omitted. Please see PDF.]) during 168 h of fermentation: (a) pH values and (b) lactic acid production.

Figure 2. Physicochemical changes: (a) maltose, (b) glucose, and (c) fructose in Blonde ([Image omitted. Please see PDF.]), American Pale ([Image omitted. Please see PDF.]), and Stout ([Image omitted. Please see PDF.]) craft beers during the fermentation process.

Figure 2. Physicochemical changes: (a) maltose, (b) glucose, and (c) fructose in Blonde ([Image omitted. Please see PDF.]), American Pale ([Image omitted. Please see PDF.]), and Stout ([Image omitted. Please see PDF.]) craft beers during the fermentation process.

Figure 3. Representative HPLC chromatograms of an American Pale beer. The chromatogram in dotted lines corresponds to the wort, and the chromatogram in solid line corresponds to the same sample but after fermentation. The retention time of sugars and fermentation by-products was as follows: maltose (10.6 min), glucose (12.1 min), fructose (13.0 min), lactic acid (16.8 min), and ethanol (25.9 min). Methanol was not detected in any of the beers.

Figure 4. Physicochemical changes in the three craft beer styles; Blonde ([Image omitted. Please see PDF.]), American Pale ([Image omitted. Please see PDF.]), and Stout ([Image omitted. Please see PDF.]); (a) represents specific gravity values, and (b) represents ethanol concentration during the fermentation process.

Figure 5. Ethanol content between brands and the own-manufactured (OM) craft beer; beer style is the block factor. * Significant statistical difference within the blocks at p [less than] 0.05.

Figure 6. Lactic acid content between brands and the own-manufactured (OM) craft beer; beer style is the block factor. * Significant statistical difference at p [less than] 0.05.

Table 1

Formulations and materials for the manufacture of batches (15 L) of Blonde, American Pale, and Stout craft beers.

	Beer Styles
Ingredients	Blonde	American Pale	Stout
Malt € (kg)	4.10 Base0.15 Roasted caramel	4.40 Base0.30 Munich0.20 Wheat0.30 Victory	4.00 Base0.40 Chocolate0.25 Caramel 120 °L0.20 Black
Yeast £ (g)	11.00 Safale US-05	11.00 Safale US-05	11.00 Safale S-04
Hops (g)	28.00 * Willamette	14.00 * Columbus	32.00 * East Kent Golding
Other hops		7.00 ** Cascade7.00 ** Centennial14.00 *** Cascade14.00 *** Centennial

Hops addition times: * 60 min boiling, ** last 10 min boiling, *** after 60 min boiling. °L = Lovibond degrees. ^€ Briess malt & ingredients Co. (Chilton, WI, USA), ^£ Fermentis by Lesaffre (Marcq-en-Barœul cedex, France).

Table 2

Pearson correlation test between the physicochemical kinetic variables in the three craft beer styles.

Variables	pH	Lactic Acid	Maltose	Glucose	Fructose	Sp. Gr. 1	Ethanol
pH	1.000
Lactic acid	−0.5882(<0.0001)	1.000
Maltose	0.6217(<0.0001)	0.0182(0.8729)	1.000
Glucose	0.8422(<0.0001)	−0.1889(0.8790)	0.5809(0.0008)	1.000
Fructose	0.8116(<0.0001)	−0.2832(0.3986)	0.6458(<0.0001)	0.6360(0.0002)	1.000
Sp. Gr. 1	0.6089(<0.0001)	0.1905(0.0926)	0.9067(<0.0001)	0.5731(0.0009)	0.6496(<0.0001)	1.000
Ethanol	−0.7383(<0.0001)	0.8149(<0.0001)	−0.7704(<0.0001)	−0.9421(<0.0001)	−0.7022(<0.0001)	−0.7574(<0.0001)	1.000

¹ Specific gravity. The parentheses express p-values.

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