Optimizing brewing beer production using Aspergillus oryzae solid-state fermentation of sorghum koji as an adjunct

ABSTRACT

This study employed solid-state fermentation (SSF) to substitute 30% barley malt with Kinmen waxy sorghum and Australian non-waxy sorghum inoculated with Aspergillus oryzae for koji preparation in the production of sorghum beer. Optimal conditions for koji preparation were determined, and various beer formulations were developed based on Kinmen sorghum koji. The optimal soaking time for Kinmen waxy and Australian non-waxy sorghum was 2.5 h at room temperature with a water absorption rate of 35%. After steaming, Kinmen sorghum and Australian sorghum exhibited blowup rates of 75.33% and 85.67%, respectively. Five beer types were fermented for 12 days using different mashing methods. Glucose content ranged from 9.93 to 17.6 g/L on the first day, decreasing to 0.03 g/L by the third day. On the 12th day, the alcohol content peaked at 3.03–4.39%, with a pH range of 4.3–5.7. Aroma analysis identified higher alcohols, such as ethanol, 3-methyl-1-butanol, and 2-phenyl ethanol, as the main contributors, with 2-phenyl ethanol imparting a solid rose flavor and ethanol providing a hop aroma and mild taste. Given the global competitiveness of the beer industry, our research emphasizes the use of Kinmen sorghum-based malt, offering unique flavors and a rich aroma, presenting significant growth potential in the market.        

KEYWORDS:

• Solid-state fermentation
• Aspergillus oryzae
• sorghum bicolor
• beer
• aroma components

Introduction

Beer is one of the most popular alcoholic beverages among consumers worldwide. Several studies have shown that beer has been produced and consumed worldwide for thousands of years^[1–3]. Various flavored craft beers are created using multiple non-barley malt materials, rapidly developing the craft beer market. Aspergillus oryzae is a fungus widely used in food. The glycine released from the protease of Aspergillus oryzae can enhance the palatability of food. In Asia, it has been widely used in applications such as fermentation and processed food.^[4] At present, beer brewing is based on barley malt. In some regions, the beer has been brewed from unconventional malt grains for years. For example, paddy rice is used in Asia, corn is used in the United States, and sorghum is used in Africa. Manufacturers look for various strategies to reduce the cost of raw materials. They supplement different carbohydrate adjuvants for the unique taste or aroma of beer. Therefore, various auxiliary materials can be used for brewing as long as it complies with the proportion of barley malt regulations.^[5]

Sorghum is one of the main grains in the world, next only to wheat, rice, corn, and barley. It is a drought-tolerant grain that can grow in severe drought.^[6] Due to its drought resistance, it mainly grows in Africa and India. It is also used as a raw material in traditional foods in Africa and India, such as oatmeal, fermented food, rice substitutes, baked food, and alcoholic and nonalcoholic beverages. It is mainly used as animal feed and for making distillate spirits in China.^[7] Sorghum is mainly used to brew distilled spirits. Sorghum has a high gelatinization temperature (>70°C), which means it needs to be heated to a high temperature to gelatinize the starch fully.

Afterward, exogenous glucoamylase must be added to break the starch into sugars.^[8,9] Sorghum is rarely used in Taiwan for brewing beer due to its complexity. However, sorghum in koji can provide aroma and increase β-amylase and protease, making it a potential raw material for beer brewing compared to other grains.^[8,10] Sorghum beer is a traditional African beer known for its turbidity. However, sorghum and corn from Africa are often contaminated by aflatoxin, posing a potential risk.^[11,12] Researchers and experts are paying attention to industrial crops, including essential brewing grains, as climate change aggravates these concerns.^[13] New ingredients in the brewing industry, such as corn and sorghum, need to be studied since the biochemistry of fermentation is complex and requires clarification in many aspects.^[8] Sorghum bicolor has a relatively high gelatinization temperature due to the presence of tannin, and its dense starch structure leads to low starch utilization. As a result, directly using raw sorghum to brew beer is uncommon, and it is usually malted first. This study uses Kinmen waxy sorghum and Australian non-waxy sorghum as adjuncts inoculated with Aspergillus oryzae to make koji and brew beer. This helps to address the issue of utilizing starch in raw sorghum. Instead of using sour sorghum beer, this method relies on koji fermentation to bring out the sweetness and unique flavors in the beer. The result is a craft beer with distinct local characteristics of Kinmen.

Materials and methods

Materials, microorganisms, and cultivation

The Aspergillus oryzae strain used in this study was purchased from the Bioresource Collection and Research Centre (BCRC No. 31653, Hsinchu, Taiwan). To activate the strain, it was thawed from the freezer and added to 0.1 mL to 0.9 mL of fresh potato dextrose broth (PDB, Maharashtra, India), which was then incubated at 25°C for 4–5 days. This culture medium was used for the first activation. For the second activation and expansion, the activated bacterial solution was added to a fresh PDB culture medium in a volume nine times larger than the bacterial solution and incubated at 25°C for 72 h. The non-waxy sorghum from Australia and waxy sorghum from Kinmen were obtained from Kinmen Royal Liquor Co., Ltd. (Kinmen, Taiwan) and Kinmen Kaoliang Liquor Inc. (Kinmen, Taiwan), respectively. After the steam boiling explosion, the sorghum was cooled to 25°C and added Aspergillus oryzae (The ratio of Aspergillus oryzae to sorghum is 1:500 by weight).

The sorghum was then placed in a constant temperature and humidity incubator and cultured at 99% humidity and 25°C for 5 days. The sorghum koji was turned over every 12 h to ensure timely and effective aeration of the thalli and enzyme formation. Samples were collected daily for 5 days and stored at −4°C for future use. Hops (Bravo and Cascade, the α-acid were 14.6% and 4.1%, respectively) were purchased from PB Craft of Taichung City, and Saccharomyces cerevisiae was purchased from I-Chan Food and Chemistry of Taipei City. The reagents and chromatography standards from Sigma-Aldrich Chemical Co. (St. Louis, Missouri) had a purity of at least 98%.

Determination of the Aspergillus oryzae bacterial cell count of sorghum koji

The Aspergillus oryzae bacterial cell count was performed after the modification.^[14] The sample solution (1 mL) was mixed with 9 mL of 0.9% NaCl aseptic water to perform serial dilution. The resulting dilute solution was added to a sterile dish with a PDA culture medium and mixed thoroughly in an 8-shaped path. After solidification, the dish was cultured upside down at 25°C for 2 days. Using a colony counter, counted the number of colonies and expressed the viable cells as Log colony-forming units (CFU) per gram.

Aspergillus growth pattern and explosion rate of sorghum koji

We observed the change in sorghum koji preparation days after steam explosion pre-treatment using a hand-held electronic digital microscope (Dino-Lite, AM413ZTA, New Taipei, Taiwan) at a working distance of 3 cm with a magnification of 50 over several days. To determine the explosion rate of sorghum bicolor, we randomly selected 100 granules after steam boiling and counted the number of exploded sorghum seeds. The process was repeated three. times to calculate the explosion rate.

Determination of the proximate compositions, and pH value of sorghum koji

The pH value determination was modified according to Coda et al. (2010).^[15] A. desktop pH meter (MP220, Metter Toledo, UK, Switzerland) was calibrated with calibration solutions with pH values of 4.01 and 7.00. Next, 5 g of sourdough was mixed with 45 mL of distilled water and measured the pH value. Moisture, ash and crude fat contents were assayed by the methods of the Association of the Official Analytical Chemists (AOAC, 2006)^[16]; the methods used were 934.01, 942.05 and 920.39, respectively. Crude protein content (N × 6.25) was determined by the AOAC Kjeldahl method 984.13. The determination of crude fiber was modified based on Rühmkorf et al. (2012).^[17]

Brewing of sorghum beer

This experiment involved two groups, each adding a different type of sorghum koji. One group had Australian sorghum koji (Group AF), and the other had Kinmen sorghum koji (Group KF). Red sorghum koji was milled and mixed with potable water at a 5:1 ratio to create the sorghum liquefaction solution. It was then liquefied at 50°C for 1 h. The gelatinized red sorghum solution was made by milling red sorghum and mixing it with water at a 5:1 ratio. It was then gelatinized at 80°C for 30 min. These two solutions were then mixed in a 1:1 ratio.

The sorghum saccharification solution was made by subjecting Australian sorghum koji to saccharification at 70°C, and Kinmen sorghum koji to saccharification at 50°C for 1 h. Barley malt was also used to make a saccharification solution. It was milled and mixed with potable water at a 5:1 ratio and underwent saccharification at 55°C for an hour and then at 65°C for another hour. The sorghum and barley malt saccharification solutions were mixed in a 3:7 ratio, boiled, and had bravo (bittertype) hops added. After boiling for 40 min, cascade (aroma-type) hops were added and boiled for another 10 min. The mixture was cooled to 30°C, beer yeast was added, and fermentation occurred at 20°C for 12 days.

The control group (Group ML) used only barley malt. Barley malt was milled and mixed with potable water at a 5:1 ratio and underwent saccharification at 55°C for 1 h and then at 65°C for another hour. The resulting solution was filtered, boiled for 10 min, and had bitter-type hops added. After boiling for 40 min, aroma-type hops were added and boiled for another 10 min. The mixture was cooled to 30°C, beer yeast was added, and fermentation took place at 20°C for 12 days.

Determination of amylose gelatinization temperature

The method described by Maniñgat and Juliano (1977)^[18] was used to determine the amylose gelatinization temperature. A 0.5% (w/v) starch suspension of 0.3 mL was taken and placed in a 15 mL test tube. The tube was then heated for 30 minutes in a water bath at temperatures ranging from 30°C to 100°C. After cooling to room temperature, the mixture was combined with 4.7 mL of distilled water and 0.5 mL of I-KI solution. The absorbance value was then measured at a wavelength of 660 nm using a spectrophotometer (Waltham, America).

Preparation of crude enzyme solution

A 5 gram sample was weighed and mixed with 50 milliliters of 1% NaCl solution in a 250 mL flask to prepare the crude enzyme solution. The mixture was stirred at 25°C and 150 rpm for 1 h before being centrifuged at 4°C and 1200 rpm for 10 min to separate the supernatant. The resulting supernatant is the crude enzyme solution. A 0.05 mL sample of the enzyme solution was placed in a boiling water bath for 5 min in a blank solution to inactivate the enzyme.

Saccharifying enzyme activity

First, 0.05 mL of the crude enzyme and blank solution were added sequentially to 0.45 mL of a bath at varying temperatures for 30 minutes. Following the reaction, 1 mL of DNS reagent was added to each test tube, mixed thoroughly, placed in a thermostatic water bath at 90°C, and left undisturbed for 5 minutes. The absorbance was then measured at 540 nm. To create a calibration curve, a glucose solution ranging from 0 to 1.0 mg/mL was used and substituted into the equation below: Calculation of enzyme activity

$\mathrm{E}\mathrm{n}\mathrm{z}\mathrm{y}\mathrm{m}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\left(\mathrm{U}/\mathrm{g}\right)=\left\{\mathrm{A}-\mathrm{A}0\left(\mathrm{m}\mathrm{g}\right)\hspace{0.17em}\right\}\ast 1/30\ast 1/0.05\left(\mathrm{m}\mathrm{L}\right)\ast \mathrm{N}$

N: sample dilution factor, 0.05 (mL): volume of enzyme solution, A0 (mg): Glucose content after inactivation (blank group) (mg), A: Glucose increment after enzyme action (mg).

Liquifying enzyme activity

The 0.05 mL of the enzyme solution and a blank solution were added to 0.45 mL of 1% starch and 0.5 mL of 0.1 M pH 6.0 disodium hydrogen phosphate-citric acid buffer solution to test the enzyme solution. This mixture was then incubated at different temperatures for 30 minutes in a water bath. Once the reaction was complete, we transferred 0.2 mL of the mixture to a 15 mL test tube and mixed it thoroughly with 1 mL of dilute iodine reagent. We measured the absorbance at 660 nm and used a calibration curve with 0–1.0 mg/mL of starch solution, which we substituted into the following equation:

$\mathrm{E}\mathrm{n}\mathrm{z}\mathrm{y}\mathrm{m}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\left(\mathrm{U}/\mathrm{g}\right)=\left\{\mathrm{A}-\mathrm{A}0\left(\mathrm{m}\mathrm{g}\right)\right\}\ast 1/30\ast 1/0.05\left(\mathrm{m}\mathrm{L}\right)\ast \mathrm{N}$

N: Sample dilution factor, 0.05: Volume of enzyme solution (mL), A0: Starch content after inactivation (blank group) (mg), A: Residual starch content after enzyme action (mg).

Proteinase activity

The 1 mL crude enzyme solution and 1.0 mL 2% casein solution were placed in a test tube. They react in a circulating water bath at 40°C for 30 min. To neutralize the effect of protease on casein, 2.0 mL of 0.4 M TCA was quickly added. The mixture was then left still for 20 minutes to allow for the precipitation of casein. Afterward, 1.5 mL of the reactant liquor was placed in a small centrifuge tube and spun at 12,000 rpm for 10 minutes. From this, 1 mL of the supernatant was extracted. Next, 5 mL of 0.5 M aqueous sodium carbonate solution was added to each tube, followed by 1 mL of Folin-ciocalteu’s phenol reagent (FCPR) diluted three times. The tubes were then thoroughly mixed and placed in a 40°C water bath for reaction and coloration for 20 min. Finally, the absorbance was read at 660 nm, and the calibration curve was created using 0 ~ 200 μg/mL of tyrosine solution and substituted into the following equation:

$\mathrm{E}\mathrm{n}\mathrm{z}\mathrm{y}\mathrm{m}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\left(\mathrm{U}/\mathrm{g}\right)=\mathrm{A}\left(\mu \mathrm{g}\right)\ast 1/30\ast 4/1\left(\mathrm{m}\mathrm{L}\right)\ast \mathrm{N}$

N: Sample dilution factor, 4 (mL): Total reaction volume (1 mL of enzyme solution +2 mL of TCA +1 mL of 2% casein), 1(mL): Reaction volume of FCPR coloration, A(µg): Tyrosine content (μg) produced after enzyme action.

Determination of turbidity, chromaticity, and titratable acid of beer

Chiang et al. (2022)^[19] described the method to measure and modify beer turbidity. The turbidity was measured using a turbidimeter calibrated with formalin standards of 0.1, 1, 10, 100, and 1000 NTU. Before analysis, the sample was allowed to reach room temperature (25°C). The average value of 10 consecutive measurements taken at 1-sec intervals was used to determine the turbidity. The colorimeter (NE4000, Nippon Denshoku, Tokyo, Japan) was used to measure. Hunter L, a, and b values to determine the color of five kinds of sorghum beer. The comprehensive color difference was represented by △E* using the following formula:

$\mathrm{\Delta }\mathrm{E}\ast =[(\mathrm{\Delta }\mathrm{L}\ast )\text{^}2+(\mathrm{\Delta }\mathrm{a}\ast )\text{^}2+(\mathrm{\Delta }\mathrm{b}\ast )\text{^}{2]}^{\text{^}1/2}$

The titratable acid was measured using the method described by Chiang et al. (2022).^[19] Specifically, 10 mL of the sample with 45 mL of distilled water in a triangular flask and added 0.2 mL of 1% phenolphthalein indicator. The mixture was titrated with 0.1 N NaOH solution until the solution turned pink and did not fade within 0.5 min (pH = 8.1 ~ 8.3). The volume of NaOH solution consumed to reach the endpoint was recorded to calculate the titration acid content.

Glucose and alcohol content

The glucose and alcohol content were measured by high-performance liquid chromatography (HPLC; Shimadzu LC-10ATVP system, Kyoto, Japan). The analytical column was a Coregel 87H3 of Transgenomic (7.8 × 300 mm) (ANPEL, Shanghai, China). The mobile phase is 0.01N H2SO4 filtered through 0.45 μm filter paper, the flow velocity is 0.6 mL/min, and a column temperature of 65°C. RI detector (Waters 2414, 45°C) and SISC software are used for analysis. Glucose and alcohol retention times are 10.1 and 23.1 min, respectively.

Volatile organic compounds analysis

In a recent study by Adidi et al. (2021),^[20] a method described to measure and modify the volatile organic compounds of beer was described. The process involves adding 5 mL of each sample to a bottle, then diluting a standard internal cinnamaldehyde with n-hexane and combining it with the sample. The mixture is then heated to 50°C in a water bath for 30 min, while the air above the sample is absorbed using solid phase microextraction (SPME) and analyzed using GC-MS (QP2010 SE-SHIMADZU, Kyoto, Japan). The analytical column used is an Rtx-5 MS (30 m, 0.25 mm ID, 0.25 μM (He)) with an inlet temperature of 280°C, an oven temperature of 40°C, and a total flow velocity of 1.11 mL/min. The MS ion source and inlet temperatures are set at 260°C and 280°C, respectively. The heating process begins at 40°C and gradually increases to 140°C at a rate of 10°C/min, before reaching 280°C at 7°C/min and maintaining that temperature for 3 min.

Sensory analysis of five different beers

The sensory analysis of the samples was carried out using a 5-point Likert scale by a panel of 50 semi-trained members (25 males and 25 females, 22–50 years old) from the Department of Food Science, National Quemoy University, Kinmen, Taiwan. Sensory evaluation was carried out at 27 ± 2°C, and the overall acceptability was calculated as the average of all the attributes.

Statistical analysis

SPSS18.0 for Windows is used for statistical analysis. Descriptive statistics, one-way analysis of variance (ANOVA), and t-test multiple comparisons are used.

Results and discussion

Explosion rate and growing state of sorghum bicolor

The seed coat of sorghum bicolor treated by a steam explosion (SE) is ruptured, and the aspergilli are likely to enter the rent in the seed coat to grow and obtain nutrients.^[21] Kinmen sorghum’s and Australian sorghum’s explosion rates are 75.33 ± 1.63% and 85.67 ± 3.09%, respectively (Figure 1). The explosion rate of Australian sorghum is 10.34% higher than that of Kinmen red sorghum due to the seed coat thickness. The variety with a thicker seed coat performs better in an explosion because the cellulose and moisture contents can influence the explosion of sorghum bicolor,^[22] matching this study. The crude fiber content in Australian sorghum is higher than Kinmen sorghum in this study (Table 1). In addition, the changes in the growth of Australian and Kinmen sorghum koji are observed through a hand-held electronic digital microscope during 0–5 days of koji preparation, as shown in Figure 1. There is no change in the surface of Australian and Kinmen koji after inoculation (Figure 1a, b). After one day’s culture, white hyphae appeared at the sorghum rupture. It is the initial appearance of aspergilli growth (Figure 1b - h). The white part turns green on Day 2, and the surface begins to grow (Figure 1c - i). On the third day, the sorghum koji surface and rupture are covered with green aspergillus hyphae (Figure 1d - j). There are scarce differences between the sorghum varieties on Day 4 and Day 5.

Figure 1. Growth status of sorghum koji (a~f: the order is from the 0th day to the 5th day of Australian sorghum koji making; g~l: the order is from the 0th day to the 5th day of Kinmen sorghum koji making).

Table 1. The proximate compositions, pH value, and the number of colonies for different making process days of Australian and Kinmen sorghum koji.

Sorghum koji making process time	Proximate compositions (%)				pH value	Colony count (CFU/g)
	Moisture	Crude protein	Crude Fat	Crude Fiber
Australian sorghum koji
Day 0	36.01 ± 0.4^aA	10.69 ± 0.42^bA	3.49 ± 0.2^aA	5.17 ± 0.68^aA	6.16 ± 0.04^aA	3.8×10^3dA
Day 1	33.83 ± 0.89^abA	11.31 ± 0.09^abA	3.98 ± 0.01^aA	4.74 ± 0.26^bA	6.00 ± 0.05^aA	7.95×10^5cB
Day 2	28.66 ± 0.93^bA	11.16 ± 0.17^abA	3.98 ± 0.41^aA	3.56 ± 0.21^cA	5.01 ± 0.05^bA	8.5×10^5cB
Day 3	22.24 ± 0.85^cB	11.84 ± 0.08^abA	3.74 ± 0.05^aA	3.27 ± 1.7^cA	4.41 ± 0.05^cA	1.63×10^7abA
Day 4	22.22 ± 1.03^cB	12.14 ± 0.53^aA	3.28 ± 0.11^aA	2.74 ± 1.82^cA	4.62 ± 0.01^cA	1.76×10^7aA
Day 5	21.72 ± 0.59^cB	12.41 ± 0.51^aA	3.34 ± 0.25^aB	1.89 ± 0.00^dA	4.60 ± 0.04^cA	1.45×10^7bA
Kinmen sorghum koji
Day 0	38.64 ± 0.59^aA	8.98 ± 0.65^bB	4.64 ± 0.19^aA	1.97 ± 0.10^abB	6.49 ± 0.08^aA	3.4×10^3bA
Day 1	36.05 ± 0.98^aA	9.79 ± 0.08^bB	3.27 ± 0.78^bA	1.54 ± 0.07^bB	6.38 ± 0.13^aA	1.35×10^6aA
Day 2	29.39 ± 0.59^bA	9.99 ± 0.03^bB	4.01 ± 0.12^abA	1.35 ± 0.02^bB	5.04 ± 0.1^bA	1.46×10^7aA
Day 3	28.09 ± 1.10^bA	10.21 ± 0.33^aB	3.77 ± 0.45^bA	1.36 ± 0.02^bB	4.39 ± 0.04^cA	1.5×10^7aA
Day 4	27.82 ± 0.88^bA	10.26 ± 0.27^aB	3.67 ± 0.42^bA	2.04 ± 0.02^aA	4.77 ± 0.06^cA	1.4×10^7aB
Day 5	26.92 ± 0.99^bA	10.62 ± 0.17^aB	4.26 ± 0.03^aA	2.17 ± 0.21^aA	4.72 ± 0.03^cA	1.24×10^7aA

Results from three separate experiments are expressed as mean ± SD. a-c: Data with identical letters in the row of the same samples are not significantly different (p< .05). A–B Data with identical letters in the row of different samples are not significantly different (p< .05).

Sorghum aspergillus colony variation

The changes during koji preparation after steam boiling both sorghums are shown in Table 1. The results show that after steam boiling, aspergillus spores, which is 2% of total weight, are added into both sorghums, and the colony counts (Day 0) are 3.4 × 10³ and 3.8 × 10³ CFU/g after addition. The colony count of Australian sorghum koji is 7.95 × 10⁵ ~ 8.5 × 10⁵ CFU/g during Day 1~Day 2. From Day 2 to Day 4, Aspergilli began multiplying, and the bacterial count reached the maximum of 1.63 × 10⁷ ~ 1.76 × 10⁷.The colony count decreased to 1.45 × 10⁷ CFU/g after Day 4. Kinmen sorghum koji aspergilli proliferate after Day 1. The colony count increased from 1.35 × 10⁶ to 1.46 × 10⁷ CFU/g from Day 1 to Day 2. The bacterial count reaches its maximum from Day 2 to Day 4, and the colony count reaches 1.4 × 10⁷ ~ 1.5 × 10⁷ CFU/g. It decreases to 1.24 × 10⁷ CFU/g after Day 4.

General component analysis

Table 1 shows the contents of crude ash, water, protein, fiber, and fat. The crude protein content of Sorghum bicolor in this study ranges from 8.98% to 10.69%, compared to 7.24% to 18.92% in other studies,^[7,23] indicating variations due to different varieties. The protein content slightly increases with the duration of koji preparation, consistent with previous findings. Fermentation increases protein content due to concentration effects.^[7] The water content decreases gradually during fermentation as water is not added. The ash content in Sorghum bicolor primarily comes from the seed coat, resulting in minimal changes during fermentation. The crude fiber content in Australian sorghum koji significantly decreases due to enzymatic activity, leading to the decomposition of the outer bran cell wall and reduced nutrients and minerals.^[24] Kinmen sorghum koji has lower crude fiber content compared to Australian sorghum koji.

During growth, aspergilli metabolize acid, resulting in a gradual decrease in pH during koji preparation. On Day 0, the pH values of Australian and Kinmen sorghum koji are 6.16 and 6.49, respectively. By Day 5, the pH values of both varieties decrease to 4.6 and 4.72. This acidification improves saccharification conditions, enhances saccharifying enzyme activity, and contributes to flavor development, including amino acids and carbohydrates.^[25]

Changes in gelatinization state and enzyme activity of Australian and Kinmen sorghum

Australian and Kinmen sorghum starches are gelatinized at 30 ~ 100°C for 30 min. OD660 measures the appearance of blue iodine solution as the basis of gelatinization. Figure 2(a) shows that the starch structure is not gelatinized at 30 ~ 50°C, and the iodine molecules cannot be combined with the starch. However, the two varieties of sorghum bicolor begin to gelatinize at 60°C. The gelatinization at 80°C is the maximum (0.36 ~ 0.49). It is the highest degree of gelatinization. It begins to decline after 80°C, and the gelatinization is completed after 30 min. The decline may be induced by pyrohydrolysis or enzyme action. The gelatinization temperature of Australian and Kinmen sorghum starches is 80°C, and the starch is hydrolyzed at 90°C.

Figure 2. Gelatinization conditions and enzyme activity of Australian and Jinmen sorghum (a) Gelatinization degree of Australian and Jinmen sorghum starch; (b) α-amylase activity of Australian sorghum in response to different koji-making times and temperature (c) α-amylase activity of Kinmen sorghum in response to different koji-making times and temperature (d) β-amylase activity of Australian sorghum in response to different koji-making times and temperature (e) β-amylase activity of Kinmen sorghum in response to different koji-making times and temperature (f) Protease activity of Australian and Kinmen sorghum fermented at 40°C with different koji-making time.

The liquifying enzyme (α–amylase) activities of Australian sorghum koji and Kinmen sorghum koji during 0 ~ 5 days of koji preparation are shown in Figure 2(b,c). Australian sorghum koji has low enzyme activity during 0 ~ 1 days of koji preparation. The enzyme activity occurs after Day 2, reaches its maximum on Day 4, and decreases on Day 5. Figure 2(b) shows that Australian sorghum koji’s maximum liquifying enzyme activity occurs on Day 4 of koji preparation. The activity can be 38.43 U/g at 50°C, followed by 38.18 U/g at 60°C. There is no significant difference between 50°C and 60°C. The enzyme activity is reduced by more than 50% under 80°C. It is significantly different from the other temperatures. Figure 2(c) shows that the optimal activity of Kinmen sorghum koji occurs on Day 2. The activity can be 31.36 U/g at 50°C, followed by 40°C, but no significant difference exists between 40 and 50°C. In addition, there is no significant change in the activity from Day 2 to Day 3 under 40°C. The liquifying enzyme declines gradually with the number of days of koji preparation after Day 2 at 40 ~ 60°C, and the activity does not increase after 80°C.

The optimal number of days of koji preparation of Australian sorghum koji is 4 days with an optimal operative temperature between 50 ~ 60°C. Kinmen sorghum koji has the highest activity on Day 2 with optimal operative temperature between 40 ~ 60°C. There is no difference between the two varieties of sorghum bicolor in liquifying enzyme activity at 40 ~ 60°C. This might be because the liquifying enzyme has higher temperature stability.^[23,24] Kinmen sorghum generates enzymes on Day 1, which differs from the Australian sorghum liquifying enzyme. This difference is supposed to be induced by the difference between non-waxy and waxy sorghum starch structures.

The saccharifying enzyme (β–amylase) activities of Australian sorghum koji and Kinmen sorghum koji during 0 ~ 5 days of koji preparation are shown in Figure 2(d,e). In this study, the Australian sorghum koji and Kinmen sorghum koji saccharifying enzymes are tested at 40 ~ 80°C at intervals of 10°C.

The activity of Australian sorghum koji increases significantly after Day 1. Optimal activity occurs under 70°C. The activity can be 4.27 U/g on Day 3 of koji preparation, followed by 4.17 U/g on Day 2. Regarding temperature, the liquifying enzyme activity under 50°C is only lower than 70°C. The enzyme activity does not increase under 80°C.

The optimal activity of Kinmen sorghum koji is 4.49 U/g on Day 3 at the operative temperature of 40°C, followed by 50°C (4.33 U/g). There is no significant difference between 40 and 50°C. In addition, the enzyme activity degrades significantly as the number of days increases after Day 3.

The optimal operative temperatures of Australian sorghum koji and Kinmen sorghum koji are 70°C and 40 ~ 50°C, respectively. Australian and Kinmen sorghum koji begin to produce enzymes on Day 2. Both have the highest activity on Day 3, and then the activity declines. The Australian sorghum koji has higher liquifying enzyme activity than Kinmen sorghum, without any significant difference. As barley sprouts, the saccharifying enzyme decreases, and the saccharifying enzyme activity of sorghum bicolor is insufficient.^[10,25,26] However, the saccharifying enzyme activity can be enhanced after koji preparation and compared with the sorghum saccharifying enzyme (1.2 & 0.5 U/g) in other studies.^[8] The sorghum koji produced in this experiment can significantly improve the saccharifying enzyme activity of sorghum bicolor (p < .05). Figure 2(f) shows the protease activity of Australian and Kinmen sorghum koji at 40°C during 0 ~ 5 days of koji preparation. Australian and Kinmen sorghum koji generate protease on Day 2, and the proteinase activity reaches its peak on Day 3. The proteinase activity of Australian sorghum is the highest at 187.9 U/g, while that of the Kinmen sorghum koji is slightly lower at 174.2 U/g. There is no significant difference (p > .05) between the two kinds of sorghum koji in the protease activity. The proteinase activity variation matches the Aspergillus growth curve.^[27,28] The proteolytic enzyme of Aspergillus oryzae plays a vital role in decomposing protein into various peptides and amino acids. Thus, the palatability of fermented food is enhanced.^[29]

Changes in turbidity, pH value, and titratable acid of different sorghum beers during fermentation

The turbidity of beer results from the interaction of carbohydrates, polyphenols, and proteins.^[29] Figure 3(a) shows the turbidity variation of different sorghum beers during fermentation. The KE and AF groups have lower turbidity values on Day 0 of fermentation, which are 5.2 and 5.0. The turbidity of the other groups is higher than 10, and the turbidity values on the other days are different. It is supposed that after saccharification and liquefaction on Day 0, different groups have different carbohydrates and proteolysis degrees. The turbidity is high if many small molecular substances are left after filtration. During 3 ~ 12 days of fermentation, the turbidity increases. This might have been caused by the fermented liquid being kept still for clarification or due to gas production and agglomeration sedimentation. As a result, the turbidity is irregular during fermentation. The ML group is pure barley malt with a higher protein content than Sorghum bicolor, so the turbidity is higher than sorghum beer.^[30]

Figure 3. (a) Beer turbidity (b) pH value and (c) organic acidity changes during fermentation of different sorghum beers During a 12-day fermentation period, five types of beer were produced using different methods. These included pure malt (ML), separate mashing of Australian sorghum/sorghum koji and malt (AF), joint mashing of Australian sorghum koji and malt (AE), separate mashing of Kinmen sorghum/sorghum koji and malt (KF), and joint mashing of Kinmen sorghum koji and malt (KE).

The samples in this study are filtered only through filter cloth without clarifying agents. In comparison, the distiller’s wort is filtered with clarifying agents and filters in industrial brewing, so turbidity is not a problem.^[9,31] Figure 3(b) shows the pH variation of different beers. It is observed that the pH of various groups of sorghum beer decreases significantly during 0 ~ 3 days of fermentation with no apparent variation. There is no significant difference in pH between the ML group and the beer with sorghum koji or Sorghum bicolor. The pH value of sorghum beer studied by Coulibaly et al. (2020)^[11] (is 3.47 ~ 3.8), and the pH value of available commercial beer is about 4.03 ~ 4.32,^[31] which is lower than the pH in this study. This might be because this study uses sorghum koji as an adjuvant to brew beer. Aspergillus oryzae influences the growth of Saccharomyces cerevisiae, leading to the difference in pH during fermentation. Figure 3(c) shows the organic acid variation of different sorghum beers. The changes from Day 0 to Day 12 are 0.30 ~ 0.38 of AF, 0.37 ~ 0.46 of AE, 0.28 ~ 0.39 of KF, and 0.26 ~ 0.34 of KE. There is a slight increase until Day 12 of fermentation. The ML group has lower titratable acid than sorghum beer.

Chromaticity variation of different sorghum beers during fermentation

Table 2 shows the chromaticity variation of different sorghum beers during fermentation. The L-value of ML is lower than that of the other sorghum beers without any significant difference, and the a and b values are higher than sorghum beers. Regarding a-value, sorghum beer is greener than the control group ML. It may have resulted from the addition of sorghum koji. The b-value of ML inclines to yellow compared with sorghum beer.

Table 2. The change in color of five different kinds of sorghum beer during different fermentation times.

	Fermentation time (Day)
	0				3				6				9				12
Sample	L^*	a^*	b^*	ΔE	L^*	a^*	b^*	ΔE	L^*	a^*	b^*	ΔE	L^*	a^*	b^*	ΔE	L^*	a^*	b^*	ΔE
AF	92.06	−0.92	27.13	95.98	93.91	−1.26	24.29	97.01	94.72	−1.43	24.08	135.59	92.21	−0.56	25.38	95.64	93.58	−1.01	23.96	96.60
AE	92.33	0.38	38.98	100.22	89.66	0.48	34.77	96.17	88.99	0.92	34.59	131.03	87.68	1.63	35.58	94.64	88.66	1.76	36.62	95.94
KF	94.34	−1.30	24.55	97.49	95.30	−1.75	22.11	97.85	95.07	−1.11	17.83	136.43	95.29	−1.4	23.65	98.19	95.55	−1.56	22.87	98.26
KE	96.3	−1.18	16.93	97.78	93.65	−0.94	24.11	96.71	92.35	−0.29	24.24	133.72	91.58	−0.03	24.60	94.38	90.27	0.81	24.54	93.55
ML	77.48	8.53	57.75	97.01	87.34	3.45	51.42	101.41	86.56	4.11	52.21	133.39	84.68	4.26	53.48	100.24	84.53	4.23	52.12	99.40

During a 12-day fermentation period, five types of beer were produced using different methods. These included pure.malt (ML), separate mashing of Australian sorghum/sorghum koji and malt (AF), joint mashing of Australian sorghum koji and malt (AE), separate mashing of Kinmen sorghum/sorghum koji and malt (KF), and joint mashing of Kinmen sorghum koji and malt (KE).

The L*, a*, and b* values of the E series of sorghum beer are relatively regular. The L-value decreases slightly with the fermentation time, and the brightness decreases. The a-values are positive, increasing gradually with fermentation time, showing red color. There is no significant change in the b-value. AE has the highest a and b values, 1.76 and 36.62 shown in orange, but AE has the lowest L-value, which is 88.66, shown as less bright. The L-value of the F series is relatively smooth, the a-values are negative and relatively green, and there is no significant change in the b-value.

Alcohol and glucose content variations of different sorghum beers during fermentation

Figure 4 shows that the alcohol content in four kinds of sorghum beer increases significantly during Day 0~Day 3 of fermentation and then becomes smooth. The range is 3.03 ~ 4.39% on Day 12. The maximum value is 4.39% of AE, followed by 3.86% of KF. The alcohol content in sorghum beer in the other studies is 4.3 ~ 4.45%,^[32] which is lower in this study. KE has the highest glucose content on Day 0, 17.6 g/L, followed by 16.67 g/L of AE. Overall, the glucose and alcohol content in the four kinds of beer became smooth after Day 3. This is because glucose is used, and the alcohol content has not changed. Therefore, the four kinds of beer were fermented on Day 3.

Figure 4. The levels of glucose and alcohol in four different sorghum beers on different fermentation days Separate mashing of Australian sorghum/sorghum koji and malt (AF), joint mashing of Australian sorghum koji and malt. (AE), separate mashing of Kinmen sorghum/sorghum koji and malt (KF), and joint mashing of Kinmen sorghum koji and malt (KE).

Analysis of aroma components of different sorghum beers

According to Figure 5, the aroma components of the five kinds of beer are approximately similar. KF has the highest, followed by AE, and KE has the lowest total aromatic content. KF, AE, and AF have the highest proportion of Ethanol, while KE and ML have the highest 3-methyl-1-butanol. Compared with other sorghum beers, ML has higher proportions of 3-methyl-1-butanol and phenylethyl alcohol and lower ethanol.

Figure 5. A ratio of five different sorghum beers volatile organic compounds Pure malt (ML), separate mashing of Australian sorghum/sorghum koji and malt (AF), joint mashing of Australian sorghum koji and malt (AE), separate mashing of Kinmen sorghum/sorghum koji and malt (KF), and joint mashing of Kinmen sorghum koji and malt (KE).

A beer containing too many vicinal diketones is a sign of poor quality. Bacterial contamination and improper fermentation will increase the content of vicinal diketones. Therefore, sorghum beer brewers seek low content of vicinal diketones.^[33,34] The vicinal diketones are not detected from the sorghum beer in this experiment. Particularly, hexadecanoic acid and ethyl ester of KF is not found in the other four kinds of beer. Hexadecanoic acid and ethyl ester have higher fatty acid ethyl ester with high molecular weight, which makes the distillate spirits turbid.^[35] The aroma components of sorghum beer, such as phenylethyl alcohol, ethyl acetate, hexanoic acid, ethyl ester, hexadecanoic acid, and ethyl ester have been found in Kaoliang liquor.^[35]

The main aroma components of sorghum beer are higher alcohols, such as ethanol, 3-methyl-1-butanol, and phenylethyl alcohol. Highly concentrated 3-methyl-1-butanol and phenylethyl alcohol have been detected in other African sorghum beers.^[36] The higher alcohols produced in fermentation are flavor aids.^[37] Phenethyl alcohol is regarded as the primary alcohol of fermentation. It brings a solid rose fragrance to beers and ethanol brings a warm taste to beers.^[38,39]

Regarding esters, ethyl acetate, 3-methyl-1-butanol, acetate, hexanoic acid, ethyl ester, and octanoic acid are found in the four kinds of sorghum beer. These ethyl esters are representative esters in beer, with a fruity and solvent-like flavor.^[40] The sorghum beer has a higher content of 2-methoxy-4-vinylphenol than ML, meaning that the sorghum beer has more noticeable smoky and clove flavors than ML

Sensory analysis-consumer hedonic tests

Table 3 presents the results of the sensory analysis evaluation. AE beer is golden yellow and not turbid but relatively straightforward. It has a pure aroma of wheat and hops, followed by fruity and flowery aromas. The taste is strong, silky, and sweet with an aftertaste. On the other hand, AF has a light yellow, opaque, and milky white appearance, a pure aroma of wheat and hops, and a slightly sour taste that becomes sweet after drinking. KE is light yellow and clear, with a round mouth and wheat fragrance and a sweet taste. KF is light yellow and opaque, with a milky white appearance, hops, wheat, and fruit aroma, and a smooth but sour mouth. The E series beers taste sweet, while the F series beers taste sour. All five types of beer produce less foam and stimulation. According to the testees, AE has the highest degree of preference for smell, followed by KE.

Table 3. Analysis on the preference of five kinds of beer in various aspects.

	AF	AE	KF	KE	F
Foma preference	2.96 ± 0.6^a	2.92 ± 0.74^a	3.3 ± 0.62^ab	3.38 ± 0.70^b	3.25*
Color preference	3.23 ± 0.65^a	4.03 ± 0.66^b	3.38 ± 0.7^a	3.8 ± 0.49^b	9.09***
Clarity preference	2.92 ± 0.74^a	3.96 ± 0.53^b	3.27 ± 0.67^a	3.65 ± 0.75^b	11.60***
Aroma preference	3.42 ± 0.76	3.77 ± 0.76	3.5 ± 0.86	3.42 ± 0.80	1.10
Mouthfeel preference	3.08 ± 0.84^a	3.85 ± 1.08^b	3.5 ± 0.86^ab	3.65 ± 0.69^b	3.58*
Carbonation (stimulation)	3.12 ± 0.82	3.15 ± 0.88	3.30 ± 0.93	3.30 ± 0.68	0.38
Aftertaste preference	3.27 ± 0.72	3.58 ± 0.86	3.46 ± 0.86	3.61 ± 0.80	0.95

*: p<.05; **: p<.01; ***: p<.001

Different letters indicate statistical differences between varieties (p<.05).

Similarly, the highest degree of preference for mouthfeel is AE, followed by KE. Sorghum beer shows highly significant differences in chroma and clarity preference (p < .001). There is also a significant difference in foam and entrance preference (p < .05). The overall preference is higher in the E series, which has a higher proportion of sorghum koji, with AE being the most preferred, followed by KE. There is also a significant correlation between the smell and taste of sorghum beer.

Conclusion

The optimal number of days of koji preparation of Australian non-waxy sorghum koji is 4, and the liquifying enzyme is optimal at 38.44 U/g at 50°C. The saccharifying enzyme is optimal at 4.27 U/g at 70°C. The optimal number of days of koji preparation of Kinmen waxy sorghum koji is 3. The optimal liquifying enzyme is 31.36 U/g at 50°C, and the optimal operative temperature of saccharifying enzymes is 50°C. There are 12 ~ 13 aroma components of sorghum beer, and KF has the highest total aromatic content, followed by AE. The main aroma components are ethanol, 3-methyl-1-butanol, and phenol alcohol. The floral, fruity, and alcoholic aromas are the principal parts. The beer market has been globalized and increasingly competitive, with very high requirements for new and particular products. Researchers have also found a growing demand for unique-tasting beer worldwide, and they are exploring ways to enhance its flavor and scent while simplifying production. One method they have discovered is to change the conditions and methods of beer yeast fermentation. During fermentation, the brewing yeast produces volatile organic compounds (VOCs) that contribute to the aroma and taste of the beer. Adadi et al. (2021)^[20] identified 33 VOCs in beer samples, including fruity esters, higher alcohols, sulfur compounds, and vicinal diketones. Another method is to change the raw materials used for brewing beer. This study uses Kinmen waxy sorghum and Australian non-waxy sorghum as adjuncts, which are inoculated with Aspergillus oryzae to make koji and brew beer. This method brings out the sweetness and unique flavors in the beer, addressing the issue of utilizing starch in raw sorghum to brew beer with rich aroma components and development potential.

Disclosure statement

No potential conflict of interest was reported by the author(s).