Fermentation properties of beer produced from Korean two-row barley or malt (Gwangmaek) supplemented with Korean red ginseng extracts and Bokbunja (Rubus coreanus Miquel) juice

Abstract

This study involved the production of specialty lager beers supplemented with Korean red ginseng extracts or Bokbunja (Korean black raspberry, Rubus coreanus Miquel) juice. The effects of the Korean red ginseng extracts or Bokbunja juice on the specific gravity, pH, yeast viability, free amino nitrogen content, reducing sugar content, color, alcohol content, turbidity, and sensory evaluation were evaluated. The alcohol content of the beers containing the extracts or juice were within the standard alcohol amounts (3.63-4.0%, v/v). The pH values of the three samples containing Bokbunja juice were lower than that of the control values. The sensory evaluation showed that the addition of Bokbunja juice was superior to the ginseng extracts, and the optimal addition time was before or after the secondary fermentation. These data indicate that the flavor and odor of the Bokbunja juice are more persistent than that of the ginseng extracts.

Introduction

Beer is a well-known alcoholic beverage that is classified based on the nature of the raw materials and microorganisms used in fermentation and flavoring (Boulton and Quain, 2008). Brewers add supplementary materials during the brewing process to provide additional fermentable ingredients, particularly carbohydrates for yeast growth. In the mashing process, materials such as maize, rice, corn, and wheat may be mixed with barley malt. The use of each material affects the character of the wort or beer (Briggs et al., 2004). In particular, fruits are used to produce a variety of beers, and their juices can be added during brewing. The introduction of whole fruit into beer was originally practiced in Belgium (De Keersmaecker, 1996). Young lambic beer is blended with various fruits including cherries (Prunus cerasus L.) and raspberries (Rubus idaeus L.) (Daenen et al., 2008; Prasad, 2014).

As discretionary income and an interest in personal health has risen, the consumption of healthy foods has been promoted. To meet increased consumer demand, the brewing industry has invested in the development of new technologies and innovations for specialty beers (Lee et al., 2013b; Yeo and Liu, 2014).

Ginseng has been used as a tonic for 2,000 years in Asian countries (Jang et al., 2011; Jeon, 2013). Ginseng and red ginseng products are representative health-functional foods and are wellknown for their anti-carcinogenesis and immune system-boosting properties (Lee et al., 2013b). Ginseng products consumed in Korea include ginseng tea, liquid extracts, capsules, soft drinks, and ginseng wine (Jang et al., 2011; Jee et al., 2006). However, beers with added ginseng or red ginseng have not been developed or commercialized, even in Korea where consumers prefer purchasing healthier products.

Bokbunja, Korean black raspberry (Rubus coreanus Miquel) fruit, is native to Korea, Japan, and China (Lee, 2015). Bokbunja fruits are mainly used to produce traditional wine in Korea, and they contain anthocyanins, which function as antioxidants and possess anti-inflammatory properties. The consumption of Bokbunja wine has increased because of its therapeutic effects on asthma, enuresis, and allergic diseases, etc. (Jin et al., 2008; Lee, 2013a). The development of Bokbunja beers has also been lacking. Thus, a beer with Korean red ginseng extract or Bokbunja juice is expected to be the premium specialty beer of Korea.

In this study, Korean red ginseng extract and Korean black raspberry (Bokbunja) juice were added at various fermentation stages. Several properties of each beer were analyzed during the fermentation process, and sensory analysis was performed after secondary fermentation.

Materials and Methods

Raw materials

Korean two-row barley malt (Gwangmaek malt) was supplied by the National Institute of Crop Science (Iksan, Jeollabuk-do, Korea). Czech Saaz pellet-type hops (Hopunion, Czech) and Saccharomyces cerevisiae Saflager W-34/70 (Fermentis, Marcq-enBaroeul, France) were used for brewing. Alpha-amylase (from Bacillus licheniformis, Almylex BT2), glucoamylase (from Aspergillus niger, GA-L New), and β-glucanase (from Trichoderma reesei, Laminex BG2) were provided by Vision Biochem Co. Ltd. (Seongnam, Korea). Fermented red ginseng extract G21 (Myeongjang^®, Basan Co., Anseong, Korea) or Bokbunja juice (Berrywell, Gochang, Korea) was added during each fermentation step. Beers supplemented with Korean red ginseng extract at the start of primary fermentation, at the end of primary fermentation, and at the end of secondary fermentation are indicated as S1, P1, and SF1, respectively. Beers supplemented with Bokbunja juice at the start of primary fermentation, at the end of primary fermentation, and at the end of secondary fermentation are indicated as S2, P2, and SF2, respectively.

Mashing and wort boiling

Mashing was performed according to the European Brewery Convention (EBC) mash method (Convention EB, 1998). Seventyfive (75) grams of malt were mixed with 600 mL of distilled water and preheated to 52°C in an amber bottle (Schott Duran, Mainz, Germany). Three enzymes (α-amylase, glucoamylase, and βglucanase) were then added (0.1% of barley malt, v/w). The mashing-in process began with a 20 min incubation at 52°C. The temperature was then increased at a rate of 1°C/min. The bottle was cooled down to 20°C, and filtration was conducted using filter paper (No. 597 1/2, Whatman, Dassel, Germany). The clarified wort was boiled with the hops intermittently for 20 min and cooled to 15°C until the hops settled.

Fermentation

The upper layer of the hopped wort was collected for beer fermentation. Korean red ginseng extract and Bokbunja juice were added to the hopped wort (0.05% of wort, w/v and 3% of wort, v/v, respectively) at the start of primary fermentation (S). S. cerevisiae Saflager W-34/70 (Fermentis) was inoculated at a ratio of 1 g/L, and the initial concentration was 5.0×10⁶ cells/mL. Red ginseng extract or Bokbunja juice was also added at the end of primary fermentation (P) and secondary fermentation (SF). Primary fermentation and secondary fermentation were conducted at 14°C for seven days and at 4°C for 21 days, respectively.

Yeast viability

Yeast viability was assessed according to methods established by the American Society of Brewing Chemists (ASBC) (Crumplen, 1997). Yeast cells were counted using yeast extract-peptone-dextrose (YPD) agar plates during primary and secondary fermentation.

Beer quality analysis

To determine the differences between samples, several beer quality properties were analyzed. All measurements were conducted in triplicate. The pH of the wort and beer was measured by a pH meter (ORION 3 STAR, Thermo, Singapore).

The specific gravity (SG) of the wort and beer was measured using a hydrometer (200-DK-6, Daekwang, Seoul, Korea). The SG of the hopped wort was measured before the yeast was pitched and at the end of each fermentation stage.

Reducing sugar (RS) was measured using the 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959). The absorbance of each sample was determined at 550 nm, and the RS content was calculated based on a standard curve.

The color of all samples was measured throughout the fermentation process with a portable spectrophotometer (CR-2500d, Minolta Co., Ltd., Osaka, Japan). One (1) mL of each sample was deposited into a specially produced vial. The data were recorded as L, a, and b values, which represent lightness, redness, and yellowness, respectively (Han et al., 2016).

The free amino nitrogen (FAN) content of the wort and beer were measured during fermentation in accordance with the official analysis method of the Association of Official Agricultural Chemists (AOAC) (Horwitz and Latimer, 2011). The absorbance of each sample was determined at 570 nm using a spectrophotometer (UVmini-1240, Shimadzu, Kyoto, Japan). FAN content was calculated as follows:

Free amino nitrogen (mg/L)=net absorbance value of the test solution 2 (dilution/net absorbance value of the standard solution)

Alcohol content and turbidity

After secondary fermentation, the alcohol content of each sample was determined using a vinometer (211-DK-12, Daekwang, Seoul, Korea). All values were calculated according to the method described in the Korean Food Standard Codex (Korea Food K. and Drug Administration, 2012). Turbidity was analyzed using a UV/Vis-spectrophotometer (UVmini-1240, Shimadzu, Kyoto, Japan). The absorbance of each sample was determined at 430 and 700 nm. If the absorbance at 700 nm was 0.039 times higher than at 430 nm, the beer was deemed “turbid.”

Sensory evaluation

A descriptive analysis was used to test sensory evaluation. The panels were comprised of graduate and undergraduate students from Dongguk University. Panel members were selected based on their accuracy and ability to reproducibly detect bitterness. Each candidate was tested four times with five reference materials (20, 40, 60, 80, and 100 mg/L of an aqueous iso-humulone solution). Candidates showing insignificant differences (p>0.05) between the five means of four replicates per reference material (poor reproducibility) and who did not show an increasing order that correlated with the iso-humulone concentrations (low accuracy) were excluded (Kim et al., 1993). Twenty panels were composed of men and women at a ratio of ten to ten. They were trained through several orientation and discussion sessions so that they became familiar with the descriptors, which can be found in Table 1.

Table 1. Recommended descriptors in the sensory evaluation of beer and their reference materials

Descriptive words were adopted from the descriptive terminologies listed in the ASBC standard (Crumplen, 1997). They include odor, taste, mouthfeel, and aftertaste. The odor was sub-categorized as fruity (O1) or alcoholic (O2); the taste was sub-categorized as bitter (T1), sweet (T2) or sour (T3); and mouthfeel was subcategorized as mouth coating (M1), carbonation (M2), or astringent (M3). The duration of aftertaste was denoted as A1, and the overall preference was denoted as P1. The intensity of each attribute was scored on a 1-9 linear interval scale (1: not present at all; 2: very weak; 3, 4: weak; 5, 6: moderate; 7, 8: strong; 9:very strong) (Kim, 1998).

All samples were stored at 4°C for one week before use, and the tests were performed in tasting booths at 20°C. Ten (10) mL of each beer sample was given to the panels in glass cups labeled with random three-digit codes. The panels answered questions for each sample, and further questions were provided for another sample. Tasting panels were required to test each sample within 2 min on average and to take 30 s rests between each sample to cleanse their palates with water and crackers (King and Heymann, 2014).

Statistical analysis

Statistical analyses were performed using GraphPad PRISM software (GraphPad Software, Inc.). All experiments were performed in triplicate. The data were analyzed using analysis of variance (ANOVA) at a significance level of α=0.05 followed by Tukey’s post-test.

Results and Discussion

Yeast viability

Figure 1 shows the yeast viability during fermentation. Cell numbers for all samples steadily increased during primary fermentation from 6.8 to 7.6 log cells/mL, and yeast growth was maintained at 6.2 log cells/mL during secondary fermentation. The yeast viability of beers supplemented with Korean red ginseng extract or Bokbunja juice before primary fermentation (S1 and S2) was higher by 0.2 log cells/mL when compared to the control during primary fermentation (Fig. 1A). Fig. 1B shows the changes in yeast viability during secondary fermentation. The number of initial yeast cells was higher in beers with extract or juice added after primary fermentation (P1 and P2) when compared to other samples. The yeast viability of P1 and P2 changed similarly during secondary fermentation, and the differences were statistically significant compared to the control (p<0.05). There were also significant differences between S1 and P1 during secondary fermentation (p<0.05).

Fig. 1. Change in yeast viability of the control and extract- or juice-supplemented samples during primary and secondary fermentation.

Specific gravity

Figure 2 shows the changes in SG throughout the fermentation process. The SGs of all samples were similar, and there were no significant differences (p>0.05). The SGs for all samples before fermentation were close to 1.040, and it decreased in all samples to about 1.010 after primary fermentation (Fig. 2). The SG of S2 was slightly higher than other samples at the start of primary fermentation, and there was no significant difference (p>0.05). The SG of SF2 (1.011±0.000) was higher than that of other samples, but it was not significantly different (p>0.05). This could indicate that the red ginseng extract and Bokbunja juice did not affect the SG of the wort or beers due to their small quantities.

Fig. 2. Kinetic changes in the specific gravity of the control and extract- or juice-supplemented samples.

pH

Figure 3 shows the changes in pH for all samples during the fermentation process. As fermentation progressed, the pH values of all samples decreased. In particular, the pH values of samples containing Bokbunja (S2, P2, and SF2) were lower than thecontrol and red ginseng samples. The initial pH values of the wort were between 5.3-5.6 depending on the materials used during mashing (Martínez et al., 2017). At the start of primary fermentation, all samples, except for S2, exhibited pH values of 5.83±0.03 and 5.86±0.03. The pH of S2 was 5.04±0.00, and there was a statistically significant difference between S2 and the other samples (p<0.05). This difference was likely caused by the low pH range of the Bokbunja juice (3.42-3.56) (Lee and Ahn, 2009; Ra and Kim, 2016). The pH values of the three samples supplemented with Bokbunja juice (S2, P2, and SF2) were 4.55±0.01, 4.46±0.02, and 4.33±0.20 at the end of secondary fermentation (p<0.05).While the pH value of S2 was constant before (4.55±0.05) and after secondary fermentation (4.55±0.01), the pH values of the control, P2, and SF2 decreased after secondary fermentation. The values changed according to the stage at which the juice was added. At the end of secondary fermentation, the pH values of P2 and SF2 decreased when compared to the control (p<0.05).The addition of red ginseng extract to the wort or beer did not affect the pH value of any sample (p>0.05).

Fig. 3. Kinetic changes in the pH of the control and extract- or juice-supplemented samples.

Free amino nitrogen (FAN) content

Free amino nitrogen (FAN) content indicates the amount of small peptides, amino acids, ammonium ions, etc. in beer. If the FAN content is too high, it forms a haze that makes the beer cloudy. In contrast, low FAN content leads to insufficient yeast growth and low alcohol production (Briggs et al., 2004). FAN is regarded as an index for predicting yeast viability and fermentation efficiency because it is used by yeast as a nutrient source and influences beer quality and stability (Han et al., 2016; Lekkas et al., 2009).

Figure 4 presents the changes in FAN values during primary and secondary fermentation. The FAN content of all samples gradually decreased during primary fermentation (Fig. 4A). Over that period, the initial FAN value of S2 was higher than that of the control and S1 (p>0.05). There was no change between the initial and final FAN values for all samples during secondary fermentation (Fig. 4B). In addition, there was a statistically significant difference between the changes in the FAN values of P1, P2, and the control over the same period (p<0.05).

Fig. 4. Change in the free amino nitrogen content of the control and extract- or juice-supplemented samples during primary and secondary fermentation.

Reducing sugar (RS) content

Changes in RS content during primary fermentation are presented in Fig. 5. The RS content decreased as the primary fermentation proceeded. The initial RS content was highest in S2. The results indicate that the Bokbunja juice had a higher saccharide content than the red ginseng extract. It is known that Bokbunja juice is high in sugar and can likely be used as a carbon source by yeast. The RS content of the control, S1, and S2 did not show anysignificant differences at the start of primary fermentation (p>0.05). The initial RS content of P1 showed no difference when compared with the control or S1. The RS reduction was similar in all samples during primary fermentation. The initial RS content of P2 was slightly higher than that of the control and S2. The change in RS content during secondary fermentation was not significantly different for any sample (data not shown).

Fig. 5. Change in the reducing sugar content of the control and extract- or juice-supplemented samples during primary fermentation.

Color

Changes in the color of the control and extract- or juicesupplemented samples during fermentation are presented in Table 2 and 3, respectively. The L values of the control and red ginseng samples tend to increase as fermentation progress (Table 2). This is due to decreasing amounts of nitrogen compounds such as FAN caused by the Maillard reaction (Poreda et al., 2014). Compared to the control, S1 had a lower lightness (L=33.00±0.38), redness (a= 1.18±0.21), and yellowness (b=12.89±0.27) at the start of primary fermentation (p<0.05). The b value of S1 was higher after primary fermentation (13.24±0.26) and then decreased after secondary fermentation (12.54±0.94). In addition, the b value of P1 (14.62± 0.31) was highest among red ginseng-supplemented samples. For the Bokbunja juice-supplemented samples, the L and b values tended to decrease, and the a value increased after adding the Bokbunja juice.

The L value was highest for S2 and lowest for SF2. Visually, S2 also seemed to be more transparent than P2 or SF2 at the endof secondary fermentation. Regarding the a value, S2 showed the lowest value (7.90±1.10) while SF2 showed the highest value (10.15±0.66). This indicates that the red color of the sample darkened due to the later addition of the Bokbunja juice during fermentation. Furthermore, there was a statistically significant difference between the control and the Bokbunja juice-supplemented samples according to L, a, and b values (p<0.05),

Table 2. Change in color of the control and three samples with red ginseng extract

Table 3. Change in color of the control and three samples with Bokbunja juice

Alcohol content and turbidity

The alcohol content and turbidity of the samples after secondary fermentation are presented in Table 4. The alcohol content in commercial lager beers is 2.8-8.5% (Zhao et al., 2010). The alcohol content of S2 was 4.2% (p>0.05), which was similar to all other samples. There was no significant difference between the control and beer samples supplemented with extract or juice (p>0.05). Martínez et al. (2017) reported that the initial RS concentration of persimmon-added wort affected the final ethanol content of the beers, and the beer with a higher ratio of added persimmon juice contained more alcohol. All samples showed turbidity, likely because clarification and filtration were not carried out after secondary fermentation.

Table 4. Alcohol content and turbidity of the control and extractor juice-supplemented samples

Sensory evaluation

Tables 5 and 6 present the results of the sensory evaluation. The intensity of the bitter taste (T1) was stronger in S1 and P1 when compared to the control (p<0.05). This could be explained by the bitter taste of the red ginseng, itself. In addition, the intensity of the astringent mouthfeel (M3) was stronger in S1 (4.50±0.33), P1 (4.55±0.38), and SF1 (4.58±0.38) than in the control (3.82±0.11), and there was no significant difference (p>0.05). In terms of overall preference (P1), SF1 had the highest score (p<0.05). Regarding Bokbunja juice-supplemented beers, SF2 had the highest score in terms of fruity odor intensity (O1), sweetness (T2), sourness (T3), aftertaste (A1), and overall preference (P1). The odor and flavor of S2 and P2 were lower than SF2, which may be because the odor and flavor were altered during fermentation. The odor and flavor of the sample supplemented with juice at the end of secondary fermentation almost remained unchanged before the sensory test was conducted. Regarding the fruity odor (O1), all samples supplemented with Bokbunja juice showed a stronger intensity than the control (p<0.05). Consequently, the values of the three samples containing Bokbunja juice were higher than the red ginseng-supplemented samples. The beers with extract or juice added later during fermentation received higher scores in terms of overall preference (P1).

Table 5. Sensory properties of the control and red ginseng extract-supplemented beer

Table 6. Sensory properties of the control and Bokbunja-supplemented beer

Conclusion

Beer with Korean red ginseng extract or Bokbunja juice was maintained with original beer’s own fermentation properties. Yeast viability, specific gravity, free amino nitrogen, reducing sugar, and alcohol content were not significantly different between any samples due to the small amount of extract or juice added. The lower pH of Bokbunja juice affected the pH of the wort or beer, and the beers supplemented with juice received a higher score when compared to others in terms of overall sensory evaluation preference. In addition, beers supplemented with Korean red ginseng extract or Bokbunja juice after secondary fermentation had higher scores than beer with these added before primary fermentation. Therefore, the time at which extract or juice is added must be considered to produce specialty beers such as fruit beer.Adding them after primary fermentation is better than before primary fermentation in maintaining each beer’s flavor and taste until consumption.

Recently, craft breweries have been producing beers with various flavors. Developing Korean red ginseng and Bokbunja beers requires focusing on specific factors such as age, gender, preference between beer and other alcoholic beverages, etc. The optimal amount of extract or juice has to be taken into account, and investigating the consumer's perception of each beer is necessary.