The Effects of Pasteurization and Beer Type on the Functional Compounds and Flavor Substances in Beer

Abstract

The study of functional compounds is of great importance for the production of nutrient-rich and flavorful beer. In this study, the effects of the pasteurization process and beer type (lager and ale, draft beer, and ripe beer) on the types and contents of functional compounds were investigated, including the derivatives of amino acids (γ-aminobutyric acid and glutathione), β-glucan, phenolic acids, vitamin B, and volatile compounds. Among the six different types of beers tested, it was found that the concentration of functional compounds in ale was higher than that in lager. In addition, the process of pasteurization resulted in the loss of B vitamins and ferulic acid and an increase in some off-flavors such as aldehydes. The results of this study can aid in the development of novel functional beer and new strategies to improve beer quality.

1. Introduction

With the continuous improvement of consumers’ health consciousness, functional food is now the first choice for an increasing number of consumers [1,2]. Beer is a popular beverage due to its unique flavor, pleasing organoleptic properties, and lower cost. Consumers pay simultaneous attention to the stability of beer flavor, quality, and functionality [3]. Beer is the most widely consumed alcoholic beverage in the world [4]; its alcohol content is low, and it contains essential compounds produced by malt and hops and through microbial fermentation [5]. Malt and auxiliary materials in the raw materials are the main sources of carbohydrates, vitamins (vitamin B complex), and dietary fiber, and hops contain many nutrients such as polyphenols and flavonoids. When beer is consumed in moderate amounts, these active ingredients may regulate human functions by activating specific enzymes [6]. As an important neurotransmitter, GABA is naturally produced during barley germination, and research results have confirmed its physiological functions, such as regulating blood pressure, relieving anxiety, and improving water and salt metabolism [7]. GSH, as a key antioxidant (1–20 mg/L), is not only effective in scavenging free radicals but also plays an important role in maintaining the stability of beer flavor [8]. B vitamins in beer exist in various forms, among which nicotinic acid (NA), nicotinamide (Nam), and nicotinamide ribose (NR) can be converted to the coenzyme NAD+, and this conversion pathway has been demonstrated during yeast fermentation [9]. β-Glucan, as a barley cell wall degradation product (0–1152 mg/L), is classified as an important functional ingredient due to its efficacy in immune regulation, intestinal flora balance, and metabolic regulation [10]. In addition, polyphenols in beer excel in free radical scavenging by virtue of their high bioavailability and strong antioxidant activity [11]. However, due to the diversity of raw material selection and differences in brewing techniques, the chemical composition of different beers will show evident differences [12].

A variety of analytical techniques are employed for the detection of functional compounds in liquor. High-performance liquid chromatography (HPLC) is suitable for the separation and determination of amino acids, flavonoids, phenolic acids, and so forth. The γ-aminobutyric acid (GABA) and glutamic acid content in fermented foods can be determined through pre-column derivatization with o-phthalaldehyde (OPA) in combination with HPLC [13,14], and glutathione and γ-glutamylcysteine in wine can be determined simultaneously through the use of high-performance liquid chromatography with a fluorescence detector (FLD) [15]. The phenolics found in beer can be extracted using ethyl acetate, and the main hydroxycinnamic acids (HCAs) and their derivatives, monophenols and flavonoids, and phenolic acids can be identified using high-performance liquid chromatography (HPLC) with a diode array detector [16,17]. The binding of Congo red to β-glucan is highly specific, and the determination of β-glucan in beer can be achieved readily and rapidly through Congo red spectrophotometry [18]. Mass spectrometry (MS) is often coupled with chromatography to achieve high sensitivity, and liquid chromatography–mass spectrometry (HPLC-MS) is suitable for the qualitative and quantitative analysis of non-volatile substances, with the quantitative and simultaneous determination of seven B vitamins in nutritional products using LC-MS/MS after enzymatic digestion [19,20]. The type and content of flavor compounds are important indicators of beer quality, and the use of gas chromatography–mass spectrometry (GC-MS) facilitates the simultaneous measurement of a variety of volatile compounds such as alcohols, esters, acids, aldehydes, phenols, and heterocyclic compounds in conjunction with an effective sample pretreatment method [21], and the main extraction techniques include liquid–liquid extraction (LLE) [22], solid-phase microextraction (SPME) [23], and stir bar sorptive extraction (SBSE) [24].

Beer yeast is one of the important raw materials used for brewing beer, and it is responsible for converting the saccharides in wort into alcohol and carbon dioxide through fermentation. In this process, beer yeast will also produce esters, higher alcohols, and acids, giving beer a unique aroma and taste, such as fruity or floral. Volatile components in beer play an important role in shaping the taste of beer [25]. The flavor of beer is primarily derived from the synergistic effect of compounds, including alcohols, esters, acids, and aldehydes, which are generally produced during the fermentation stage and consist of intermediate metabolites or yeast by-products [26]. The main types of beer include lager and ale. In their study, Daniel Granato and colleagues discovered evidently different metabolites between lager and ale using GC/MS metabolomics [27]. Yeast used in beer brewing is generally divided into two categories: top-fermenting beer yeast and bottom-fermenting beer yeast, which are used to produce ale and lager, respectively. The top-fermenting beer yeast is fermented at a higher temperature (16–25 °C) to produce ale, and the bottom-fermenting beer yeast is fermented at a low temperature (8–15 °C), with floc forming at the bottom of the container after fermentation, which is widely used in the production of industrialized lager.

In addition, beer is generally pasteurized to maintain quality stability, and pasteurization can induce slight increases in beer protein content. Generally speaking, ripe beer is pasteurized beer, whereas draft beer is packaged beer that has not been subjected to pasteurization. Draft beer is incredibly popular among consumers because it retains its original flavor and nutritional value [28]. In previous studies, researchers have primarily focused on the types and contents of nutrients in different types of beer [29,30]. Therefore, in this study, we aimed to mainly compare different functional compounds in lager beer and ale beer, draft beer, and ripe beer, including amino acid derivatives (GABA and GSH), β-glucan, phenolic acid, vitamin B, and volatile substances, and to explore the changes in functional compounds in beer. The research results presented herein lay a foundation for the development of functional beer in the future.

2. Materials and Methods

2.1. Beer

In this study, we focus on two types of beer, lager and ale, with detailed sample information provided below. Pasteurization is carried out by gradually increasing the temperature in a tunnel sterilizer. The temperature is generally controlled within the range of 60–65 °C, and the products are maintained at this temperature for 15–20 min, achieving a pasteurization intensity (PU value) of 19 (PU value is the unit of pasteurization strength, and 1 PU is equal to the cumulative sterilization strength at 60 °C within 1 min). The experimental samples included two lagers, one ale, and their respective unpasteurized draft beers. The two lagers contained different types of malt and hops, the same rice auxiliary materials, and lager yeast and were subjected to the same fermentation processes. The ale beers use whole malt and ale yeast. Three parallel sets of each sample were used. The lager contained malt 1 and malt 2, which were of the same cultivar but were derived from different regions, whereas the ale contained malt 3, which was a different cultivar to malt 1 and malt 2. Similarly, hops 1 and hops 2 were of the same cultivar but of different origins. Ale yeast ferments at higher temperatures (16–25 °C), whereas lager yeast ferments at lower temperatures (8–15 °C). The lager (classic ripe beer, classic draft beer, pure ripe beer, and pure draft beer) and ale (ale ripe beer and ale draft beer) were provided by the Tsingtao Beer Co., Ltd. (Qingdao, China). The specific characteristics of the beers are detailed below (

Table 1. Beer sample information.

Beer Type	Beer Sample	Pasteurization Treatment	Raw Wort Concentration	Alcoholic Strength	Raw Materials
Classic beer	CRB	Pasteurized	8°P	3.10%vol	69.99% malt 1, 29.99% rice, 0.2‰ hops 1, 107 CFU/mL lager yeast
	CDB	Unpasteurized	8°P	3.10%vol
Pure beer	PRB	Pasteurized	8°P	3.10%vol	69.99% malt 2, 29.99% rice, 0.2‰ hops 2, 107 CFU/mL lager yeast
	PDB	Unpasteurized	8°P	3.10%vol
Ale beer	ARB	Pasteurized	14°P	5.20%vol	99.99% malt 3, 0.2‰ hops 1, 107 CFU/mL ale yeast
	ADB	Unpasteurized	14°P	5.20%vol

).

2.2. Determination of Amino Acid Derivatives (GABA and GSH)

2.2.1. Detection of γ-Aminobutyric Acid (GABA)

Pretreatment of the beer samples involved the following procedures: degas 1 mL of sample solution; add 1 mL of 10% trichloroacetic acid (TCA) solution; mix well; leave overnight at a low temperature to remove macromolecular substances, such as insoluble protein and sugar in the sample; centrifuge at 10,000× g to obtain the supernatant; and filter it with a 0.45 μm membrane.

The GABA content was analyzed using an Agilent HPLC system equipped with a variable wavelength detector (VWD) (Agilent Technologies, Santa Clara, CA, USA) [31]. The specific methods employed were as follows: chromatographic conditions: Agilent 1200 HPLC; chromatographic column: Agilent ODS 250 × 4.6 μm, 5 μm liquid chromatographic column; detection wavelength: 338 nm; column temperature box: 40 °C; sample volume: 10 μL; mobile phase A: aqueous phase with A: pH 7.2 (2 M sodium acetate aqueous solution, 0.02% triethylamine, and 0.5% tetrahydrofuran); mobile phase B: organic phase (2 M pH 7.2 sodium acetate aqueous solution–methanol–acetonitrile = 1:2:2). The sample introduction procedure was as follows: using the pre-column derivatization pretreatment method, extract 7 μL of 0.4 m boric acid solution with pH 10.2 at 200 μL/min and mix it with 1 μL of sample solution and then add 2 μL o-phthalaldehyde (OPA) solution at 200 μL/min and mix it evenly and finally extract 30 μL water at 200 μL/min and inject 10 μL. The gradient elution procedure was as follows: 0 min, 8.0% B, 0.7 mL/min; 31.50 min, 100.0% B, 0.7 mL/min; 32.0 min, 100.0% B, 0.9 mL/min; 35.0 min, 100.0% B, 0.9 mL/min; 35.50 min, 8.0% B, 0.7 mL/min; 40 min, 8.0% B, 0.7 mL/min.

2.2.2. Detection of Glutathione (GSH)

Pretreatment of the beer samples involved the following procedures: degas 1 mL of sample solution; add 1 mL of 10% TCA solution and mix well; centrifuge 10,000× g for 8 min; take 1 mL of supernatant (standard) solution; add 0.5 mL of 500 μm Tris-HCl solution with a pH of 8.0 and mix well, and then add 200 μL of pure water. Thereafter, 0.5 mL of 0.01 M DTNB was shaken evenly and reacted in the dark at room temperature for 5 min. Lastly, 0.1 mL of 7 mL phosphoric acid was added and centrifuged at 10,000× g for 8 min, and the supernatant was taken and passed through the membrane at 0.45 μm.

GSH was analyzed using the Agilent HPLC system equipped with a variable wavelength detector (VWD) (Agilent Technologies, USA) [31]. The chromatographic conditions were as follows: Agilent high-performance liquid chromatograph; chromatographic column: Ai chrom C18 250 × 4.6 μm, 5 μm reverse chromatographic column; detection wavelength: 327 nm; column temperature box: 25 °C; flow rate: 0.8 mL/min; sample volume: 10 μL; mobile phase A: water phase (1% formic acid); mobile phase B: acetonitrile (10% tetrahydrofuran). The gradient elution procedure was as follows: 0 min, 10.0% B; 12 min, 14.0% B; 22 min, 28.0% B; 35 min, 80.0% B; 40 min, 90.0% B.

2.3. Determination of β-Glucan

The determination of β-glucan was performed using a method outlined in a previous study [32]. β-Glucan was analyzed using a UV-550 spectrophotometer (Shanghai Metash Instruents Co., Ltd., Shanghai, China). To perform spectrophotometry analysis using Congo Red, the following procedure was employed: take 2 mL degassed beer sample (standard); add 4 mL 100 mg/L Congo Red solution; shake evenly; react in the dark at 20 °C for 10 min; and detect its absorbance at 550 nm.

2.4. Determination of Phenolic Acids and Vitamin B

2.4.1. Detection of Phenolic Acids

Pretreatment of the beer sample involved the following procedure: take 25 mL of degassed beer; adjust the pH to 2 with 37% phosphoric acid (H₃PO₄); add 5 g of sodium chloride (NaCl); and perform triple extraction with 25 mL of ethyl acetate. Thereafter, combine the organic phases and concentrate to dryness through rotary evaporation at 40 °C under reduced pressure; dissolve the residue with 2 mL V(methanol):V(water) = 1:1; and filter with a 0.45 μm organic membrane.

Phenolic acids (including ferulic acid, catechin, gallic acid, vanillic acid, and protocatechuic acid) were analyzed with an Agilent HPLC system equipped with a variable wavelength detector (VWD) (Agilent Technologies, USA). The chromatographic conditions were as follows: Agilent high-performance liquid chromatograph; chromatographic column: Ai chrome C18 reverse chromatographic column (250 × 4.6 μm, 5 μm); detection wavelength: 280 nm; column temperature: 25 °C; flow rate: 0.7 mL/min; injection volume: 10 μL; mobile phase A: water phase (0.1% glacial acetic acid); mobile phase B: methanol (0.1% glacial acetic acid). The gradient elution procedure was as follows: 0 min, 5.0% B; 15 min, 20.0% B; 30 min, 60.0% B; 34 min, 5.0% B; 40 min, 5.0% B.

2.4.2. Detection of B Vitamins

Pretreatment of the beer and wort samples involved the following procedures: degas 1 mL of sample solution; centrifuge at 10,000× g for 10 min; take 200 μL of supernatant; add 800 μL of methanol; place it in a refrigerator at 4 °C for 6 h; centrifuge at 10,000× g for 10 min; and acquire the supernatant and spin-steam it until it becomes dry. Lastly, add 200 μL of 20% methanol to allow for re-dissolution; centrifuge at 10,000× g for 10 min; and filter with a 0.45 μm membrane.

The chromatographic conditions were as follows: QTRAP 5500 triple quadrupole ion trap liquid mass spectrometer; chromatographic column: HSS T3 column (1.8 μm, 2.1× 100 mm); column temperature: 50 °C; flow rate: 0.35 mL/min; sample volume: 2 μL; mobile phase: mobile phase A: water (2 mM ammonium formate, 0.01% formic acid); mobile phase B: methanol (2 mM ammonium formate, 0.01% formic acid). The gradient elution procedure was as follows: 0 min, 2% B; 1 min, 2% B; 6 min, 98% B; 7.1 min, 2% B; 10 min, 2% B.

The mass spectrometry conditions were as follows: ion source: electrospray ionization; ion mode: positive+negative ion mode. The parameters of each substance are listed in the table presented below (

Table 2. The parameters of positive+negative ion mode for five B vitamins.

Q1	Q3	RT	ID	DP	CE
218	88	3.95	VB5_1	−54	−19
218	146	3.95	VB5_2	−54	−22
204	168	2.40	VB6_1	−52	−15
204	150	2.40	VB6_2	−52	−25
124	80	1.66	VB3_1	80	30
124	80	1.66	VB3_2	80	31
124	53	1.66	VB3_3	80	39
265	122	1.74	VB1_1	90	22
265	144	1.74	VB1_2	90	24
377	243	4.80	VB2_1	29	34
377	172	4.80	VB2_3	33	48

Q1—parent ion (m/z); Q2—daughter ion (m/z); RT—retention time (ms); ID—vitamin ID, multiple groups of test results of the same vitamin (such as VB5) under different Q3 and CE conditions will be distinguished by suffix numbers (_1, _2, etc.); DP—declustering potential (V); CE—collision energy (V).

).

2.5. Determination of Volatile Compounds

The volatile compounds were determined using a method described in a previous study [33]. The volatile substances present in the beer were determined using headspace solid-phase microextraction gas chromatography (HS-SPME-GC-MS) (Thermo Fisher Scientific Shier Technology Company, Waltham, MA, USA). First, 5 mL of degassed beer was poured into a 20 mL brown headspace bottle; 2 g of sodium chloride and 10 μL of 50 mg/L sec-caprylic alcohol internal standard solution were added, and the samples were extracted with a SPEM fiber extraction head of 50 μm/30 μm CAR/DVB/PDMS, incubated at 55 °C for 15 min, and extracted and adsorbed for 30 min. Following extraction, the extraction head was quickly inserted into the GC inlet and desorbed at 250 °C for 0.5 min. GC-MS was used to identify the volatile substances in the beer.

The chromatographic conditions were as follows: carrier gas: high-purity helium; column flow: 1.2 mL/min; sample inlet temperature: 250 °C. The initial temperature was 40 °C, maintained for 1 min, and then increased to 180 °C at 3 °C /min and lastly increased to 230 °C at 20 °C /min and maintained for 15 min; split mode, split ratio: 4.2:1.

The mass spectrometry conditions were as follows: ionization mode: electron ionization (EI); single four-stage rod mass analyzer; transmission line temperature: 230 °C; ion source temperature: 260 °C; mass range: 29–350 amu; scanning time: 0.2 s.

2.6. Statistical Analysis

GraphPad Prism 8.4 was used for data analysis. The OPLS-DA model was constructed using SIMCA14.1, and the variables for each component were calculated. The weight value (VIP > 1.0) was used to screen the different compounds. The data were analyzed to determine significant differences using IBM SPSS Statistics 27 software, and letters are used to indicate significant differences in the different groups of data; i.e., the same letters indicate that there is no significant difference between the two groups of data, and different letters indicate that there is a significant difference between them (p < 0.05).

3. Results and Discussion

3.1. Establishment of Screening Scheme for Functional Compounds in Beer

To establish a functional compound detection scheme for the system, the bioactive ingredients related to the health benefits of beer were identified through literature research and analysis. Based on the existing research data, a systematic classification of functional compounds in beer was conducted. In the industrial beer production process, besides ethanol, beer primarily contains the following functional components: (1) amino acids and derivatives (GABA and GSH); (2) polyphenolic compounds (including tannic acid and flavonoids); (3) B vitamins (B1, B2, B3, B5, and B6); (4) polysaccharide substances. The content of functional compounds in industrial beer is generally low, and these functional compounds not only contribute significantly to the flavor characteristics of beer but also exhibit health benefits such as antioxidant, anti-tumor, metabolic promotion, and immune enhancement properties.

3.2. Effects of Different Types on Amino Acid Derivatives in Beers (GABA and GSH)

GABA performs a variety of physiological functions and is known to have anti-hypertensive and anti-stress effects on human health [7,34,35]. Barley, as one of the raw materials used in beer, is rich in GABA, and yeast exhibits glutamic acid decarboxylase activity, which can aid in the catalysis of glutamic acid decarboxylation to produce GABA. It can be used as an antidepressant, diuretic, and antioxidant. Our results showed that the concentrations of GABA in the different types of beers ranged from 60 to 110 mg/L, and there was no significant difference in GABA content between the draft and ripe beer (Figure 1A), indicating that the process of pasteurization does not lead to a loss of GABA content in beer. Although it has been reported that GABA undergoes the Maillard reaction when it reacts with sugar, resulting in a 10% loss in GABA content at high temperatures [36], pasteurization at a temperature of 60 °C does not affect the stability of GABA. The content of GABA in pure draft beer (PDB) is lower than that in classic ripe beer (CDB), with there being no significant difference among the lager mature beers. The content of GABA in the ale draft beer (ADB) (105.79 mg/L) was found to be 26.40% higher than the value in classic ripe beer (CDB) (77.90 mg/L). Rice adjunct in lager beers is high in starch and low in protein and is added as an additional carbon source during saccharification, but this addition also reduces the proportion of soluble nitrogen and amino acids in the wort. In addition, as the brewing yeast generates GABA throughout the process of beer fermentation [37], the different synthetic capacities of amino acids from various types of yeast can activate and/or inhibit the formation of GABA in wine [38], which may be caused by the higher GABA synthetic capacity of top-fermenting yeast used in ale than the capacity of bottom-fermenting yeast used in lager. This difference has also been noted in previous studies.

Regarding GSH, glutathione (GSH) is widely used in functional foods and has been found to delay aging, enhance immunity, and prevent tumors [8,39]. GSH in beer not only plays an antioxidant role but also maintains the flavor stability of beer. It can be seen from Figure 1B that the GSH content in the beers studied was incredibly low, and its content in beer was between 1 and 2 mg/L (Figure 1B). No significant difference in the GSH content was found between the two types of lager (classic beer and draft beer); however, the content of GSH in ale draft beer (ADB) (1.82 mg/L) was 43.96% higher than the content in classic draft beer (CDB) (1.02 mg/L). This difference is mainly determined by the metabolism in yeast under varied raw material conditions and manufacturing techniques. Similar to the synthetic capacity of GABA, the GSH synthetic ability of top-fermenting beer yeast is stronger than that of lower beer yeast. Our results are consistent with those of previous studies in that the abilities of GSH synthesis significantly differ among the different industrial strains for beer production [40]. Despite the fact that GSH is readily oxidized to oxiglutathione (GSSG) [41], no significant difference in GSH content was found before and after pasteurization, indicating that the GSH content in beer is not affected by pasteurization.

3.3. Effect of Different Types on the Content of β-Glucan in Beers

β-Glucan can improve the intestinal environment, stimulate innate and acquired immunity, adsorb mycotoxins, promote wound healing [10], prevent cancer, and exert antioxidative and blood sugar- and blood lipid-lowering effects. β-Glucan in beer is primarily derived from the degradation of malt grains and yeast cell walls, and its content has an important influence on beer quality [42,43]. A low content of β-glucan can lead to a bad taste, while a high content can make filtration difficult. Therefore, during the brewing process, the β-glucan content will be controlled within a certain range. As shown in Figure 2, the content of β-glucan in the various types of beer ranges between 6 and 45 mg/L. Among the two types of lager brewed with the same beer yeast, the β-glucan content in the pure beer was significantly higher than that in classic beer. This finding indicates that the type of malt is a key compound affecting the level of β-glucan in beer. No significant difference in its content was found between the two types of lager. However, in ale, the content of β-glucan in ADB (42.73 mg/L) was found to be 8.80% higher than the content in ale ripe beer (ARB) (38.96 mg/L). Pasteurization treatment can improve the compatibility and suspension stability of biopolymers [43], which may promote the combination of β-glucan and protein in beer to form a stable soluble complex, thus reducing the content of β-glucan in ale ripe beer. The β-glucan content in ARB is five-fold higher than the value in CDB, with the cultivar of malt affecting the content of β-glucan in beer. Simultaneously, the main difference between the dissolved substance in beer waste yeast (BSY) from top-fermenting yeast (lager yeast) and bottom-fermenting yeast (ale yeast) is that the former is primarily composed of glucan, whereas the latter is primarily composed of mannoglycoprotein [44]. From the above results, it can be seen that different yeast species may have distinct effects on the β-glucan content in beer.

3.4. Effect of Different Types on the Contents of B Vitamins and Phenolic Acids in Beers

Vitamin B is an essential nutrient for basic metabolic activities in the human body and an important index for evaluating the nutritional value of beer. Different forms of vitamin B deficiency can lead to specific health problems [29,45]. For example, vitamin B1 deficiency can lead to beriberi, whereas vitamin B2 and vitamin B6 deficiency are mainly manifested as skin inflammation and gastrointestinal discomfort. Vitamins B1, B2, B3 (nicotinic acid), B5, and B6 (pyridoxine) were detected in the different types of beers (Figure 3A). The content of B vitamins in draft beer is higher than that in ripe beer because heat treatment can lead to the loss of B vitamins during food processing [46]. In lager, the content of vitamin B3 was the highest, ranging from 170 to 251 μg/L, followed by B6, B5, B2, and B1. Excluding vitamin B2, the content of vitamin B in pure ripe beer was higher than that in classic ripe beer, with this same theory applying to draft beers. The content of vitamin B3 in pure draft beer B was the highest (250.10 μg/L). In ale, the content of vitamin B6 was the highest, followed by vitamins B3, B2, B5, and B1. In ale draft beer, the content of vitamin B6 was the highest (453.45 μg/L). Excluding B5, the content of B vitamins in ale was higher than that in lager. Vitamins B6 and B3 are the main vitamins found in beer [29], with the content of vitamin B1 being the lowest among all types of beer [47].

Phenolic acid is one of the main phenolic compounds widely distributed in the diet and is characterized by strong antioxidant activity, anti-inflammation properties, and ease of intestinal absorption. This antioxidant activity is mainly influenced by the polyphenols present in beer [48]. The contents of ferulic acid, catechin, gallic acid, vanillic acid, and protocatechuic acid in the different types of beer are detailed in Figure 3B. Depending on the detection results for phenolic acids, there was a significant difference in ferulic acid between draft beer and ripe beer. The content of phenolic acids in the draft beer was higher than the values in ripe beer. The content of ferulic acid in the draft beer was higher than that in ripe beer, and phenolic acids are readily decomposed by heat, resulting in a decrease in their content [49]. Among the five phenolic acids, the ferulic acid content was the highest, followed by catechin, vanillic acid, and gallic acid, and the protocatechuic acid content was very low. In lager, the contents of ferulic acid and vanillic acid in classic draft beer were higher than those in pure draft beer, and the contents of other phenolic acids are lower than in pure draft beer, which is primarily the result of differences between malt and hops [50]. The ferulic acid content of ale draft beer (ADB) (3.73 mg/L) was the highest, which was 7.33% higher than that of ADB (3.46 mg/L), and the content of catechin ranked second. The above results are consistent with those of previous studies [51].

3.5. Effect of Different Types on Volatile Compounds in Beers

To examine the characteristics of volatile compounds in the different types of beer, six types of beer samples were analyzed and identified using gas chromatography–mass spectrometry (GC-MS), and three groups of each sample were examined in parallel. A total of 162 volatile substances were detected (Figure 4), among which the contents of alcohols, esters, and acids were relatively high. Among these compounds, higher alcohols and esters are essential flavor substances in beer [52]. Specifically, the contents of alcohol substances in classic beer and pure beer were relatively high, accounting for 63% of the total volatile substances. In addition, the content of esters in the draft beer was high, accounting for 54% of the total volatile substances, primarily due to the high proportion of ethyl octanoate. Following pasteurization, the content of esters in all types of beer decreased, whereas the content of alcohols in pure beer and ale increased. The content of isoamyl alcohol in classic ripe beer (CRB) was lower than that in classic draft beer (CDB). Our results show that the change in the content of higher alcohols is not closely related to the pasteurization process [53]. Furthermore, aldehydes are the key compounds involved in the aging process of beer, and the levels of aldehydes increase to certain degrees in different types of beer after pasteurization [54]. Following pasteurization, the relative content of aldehydes in classic beer increased from 7.73% to 9.16%, and the relative content of aldehydes in pure beer and ale also increased. Regarding terpenes and other volatile substances, we noted little change in their contents.

The volatile compounds in the lager and ale were identified and analyzed, and 56 common flavor compounds were obtained, including 18 alcohols, 3 aldehydes, 24 esters, 5 organic acids, 2 ketones, 3 phenols, and 1 heterocyclic compound (Table S1). Through principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) using multivariate statistical methods, the total volatile substances in the different beers were statistically verified. In the OPLS-DA model (Figure 5A), the dependent variable was the 56 total volatile substances, and the six different types of beer samples were taken as independent variables. The figure shows that the classic beer and pure beer exhibited obvious aggregation, and there was no significant difference in volatile components between the two lagers. The substances in lager and ale were effectively separated, and there were significant differences between the lager and ale. To verify the validity of the model, the order of Y values was randomly permuted 200 times, and the X matrix remained unchanged. The blue regression line of Q² in Figure 5B intersects below the zero scale line of the vertical axis, indicating that the original model is valid. This result can, therefore, enable us to effectively distinguish the lager from the ale. Among the common substances, potentially functional compounds such as farnesol and caprylic acid are present in all types of beer (Table S1). As a natural sesquiterpene alcohol, farnesol has the potential to strongly alleviate inflammation, oxidative stress, and lung injury [55,56], and its content in all types of beer ranges from 0.82 to 4.69 mg/L. The content of octanoic acid in beer ranges from 0.12 to 1.95 mg/L. Caprylic acid is a medium-chain fatty acid that plays an important role in the taste of beer. Caprylic acid is an essential substance for the normal functioning of the human body [48,57]. It is the main representative substance of yeast flavor in beer, exhibiting antibacterial activity, and is used in dietary food. Caprylic acid can also specifically acylate ghrelin, which is a peptide hormone with an appetizing effect. The content of geraniol in beer ranges from 0 to 62.27 mg/L. Geraniol is a monoterpene alcohol with antioxidant and antibacterial properties [58]. Geraniol not only has antioxidant, anti-inflammatory, anti-apoptosis, and neuroprotective effects but also exhibits antifungal characteristics.

4. Conclusions

In this study, we investigated the changes in known functional compounds in six different types of beer. The known functional compounds included γ-aminobutyric acid (GABA), glutathione (GSH), β-glucan, B vitamins (B1, B2, B3, B5, and B6), and phenolic acids (gallic acid, ferulic acid, catechin, vanillic acid, and protocatechuic acid). We found that the content of functional compounds in ale was higher than that in lager except for vitamin B5. Under the same yeast and brewing conditions, the contents of functional compounds in the two types of lager with different malts and hops varied. The process of pasteurization showed no significant effect on GABA and GSH levels (p > 0.05), but it can lead to the loss of B vitamins (B2, B3, B5, B6) and ferulic acid and an increase in aldehyde odor substances. Among the volatile components, farnesol and caprylic acid were present in all the types of beer. Regarding functional substances, the selection of appropriate pasteurization conditions and the application of top-fermenting yeast in ale beer could aid in the development of functional beers and the improvement of their quality.