Received: 2 April 2019 | Revised: 9 July 2019 | Accepted: 24 July 2019

DOI: 10.1002/fsn3.1223

ORIGINAL RESEARCH

Black rice addition prompted the beer quality by the extrusion

as pretreatment

Tianyu Zhang1 | Haijing Zhang1 | Zhe Yang1 | Yiran Wang2 | Hongjun Li1

1
School of Agricultural Engineering and
Food Science, Shandong University of Abstract
Technology, Zibo, China Flavor compounds, including total polyphenols, amino acids, and protein, in beer with
2
Shandong Drug and Food Vocational
extruded black rice as adjunct were detected and analyzed. Beer brewing technique
College, Zibo, China
has been intensively investigated in the past century. The chase of beer quality, in-
Correspondence
cluding the color, flavor, foam, nutrition, and functionality, attracted considerable at-
Hongjun Li, School of Agricultural
Engineering and Food Science, Shandong tention. Hence, headspace solid‐phase microextraction in combination with a gas
University of Technology, Zibo, China.
chromatography coupled to mass spectrometry (HS‐SPME‐GC–MS) was used to ana-
Email: Hongjunli1351@hotmail.com
lyze flavor compounds qualitatively and quantitatively. A total of one organic acid,
one aromatic, ten alcohols and 23 esters were present in extruded black rice adjunct
beer. Protein components and molecular weight were analyzed, and the results were
consistent with those of traditional beer in terms of content of foam‐stabilizing pro-
tein. The contents of essential amino acid which is an important nutritive index were
higher than those in traditional rice adjunct beer, especially valine (70.9 mg/L) and
threonine (42.8 mg/L). The representative ingredients of extruded black rice adjunct
beer were polyphenols, nerolidol, geraniol, and geranylgeraniol which affected the
functionality and antioxidant ability.

KEYWORDS
amino acid, extruded black rice adjunct beer, HS‐SPME‐GC–MS, nitrogenous substances

1 | I NTRO D U C TI O N 2013; Zhang, He, Cao, Ma, & Li, 2017). During extrusion, starch ge-
latinization is performed at higher pressures and temperatures and
Beer is the second most consumed (accounting for 37%) alcoholic bev- also contributes to the breakdown of starch by mechanical shear
erage in Europe, with the average per capita consumption of 72.8 L in force (Singh & Smith, 1997). This method used in raw material pre-
Europe in 2009–2011, according to the European Spirits Organization treatment can shorten the production cycle and increase the using
(Quiferrada et al., 2015). At present, up to 85%–90% of beer in the rate of material (Dale et al., 2013).
world is produced with adjuncts (Mallawarachchi, Bandara, Dilshan, The use of special rice malts can enhance the flavor, taste, color,
Ariyadasa, & Gunawardena, 2016). Indigenous cereals are generally and body of rice malt beer (Ceccaroni et al., 2019). Black rice which is
used as adjuncts, thereby supporting local agriculture. Sorghum is the a special type of rice with color pigments has been widely cultivated
most common adjunction in Africa, while it was rice in Asia, corn in and consumed in Southeast Asian countries for decades because
America, and barley and corn in Europe (Mallawarachchi et al., 2016). black rice is characterized by the high content of polyphenols, partic-
Extrusion processing is used widely for the improvement or ularly anthocyanins (Jin, 2010), other antioxidant compounds, such
modification of the food material qualities (Dale, Young, & Makinde, as flavones, proanthocyanidins, and phenolic acids which contribute

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2019 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc.

Food Sci Nutr. 2019;00:1–11. 

www.foodscience-nutrition.com | 1
2 | ZHANG et al.

to its healthy nutritional profile (Abdel‐Aal, Christopher, & Iwona, poured into a 150 L saccharifying tank with 90 L of water, and then,
2006). Black rice has beneficial effects on human health and reduces pH was adjusted to 5.35 with acetic acid. Stirring and heating were
the risk of developing chronic disease (Dipti et al., 2012; Samyor, performed as follows: 50°C for 60 min, 63°C for 50 min, and 70°C
Das, & Deka, 2017). A review has shown that headspace solid‐phase for 30 min. The brewer's grains were washed with 80 L water (80°C)
microextraction coupled with gas chromatography–mass spectrom- and filtrated at 78°C. Then, these grains were boiled for 90 min, dur-
etry (HS‐SPME‐GC–MS) has been proved as an important tool for ing this process ,11, 31, and 28 g hop were added at 10, 20, and
the rapid, accurate analysis of liquid foods in many instances (Tian, 30 min, respectively, and then precipitated 30 min. The wort was
2010). Headspace solid‐phase microextraction coupled with gas cooled by the heat exchanger and fed into a fermentation tank, set
chromatography–mass spectrometry is a well‐established proce- to 18°C and added with 3 L of wort (DAB yeast cultured in wort,
dure for volatile flavor analysis in beverages (da Silva et al., 2015). the quantitative of yeast is 4.2 × 105/ml). Naturally heated to 20 de-
Nitrogenous compounds which exist in nucleic, proteins, polypep- grees and sealed the fermentation tank when the sugar content was
tides amino acids, and biogenic amine are extremely important in the dropped to 4.0%. After 5 days, the wort was cooled down to 4°C and
aspect of beer nutrition and stability of the finished product (Marta maintained 1 day. Yeast was drained and fermentation temperature
Fontana, 2009). These compounds are in endless variety in different slowly drops to 2°C to save beer.
beers due to different materials and processing. Some specific amino
acids and proteins are reported as off‐flavors and result in instability
2.4 | Standard and beer sample preparation
of product; in combination with other beer compounds, these factors
contribute to the taste and drinkability of beer (Poveda, 2019). A total of 50 μl of acetaldehyde, 25 μl of isobutyl alcohol, 40 μl of
To the best of our knowledge, no research has been performed N‐propanol, 16 μl of ethyl acetate, 100 μl of isoamyl alcohol, 0.5 μl
on the use of extruded black rice as adjunct for specialty malt pro- of ethyl hexanoate, 5 μl of isoamyl acetate, 0.5 μl of ethyl octanoate,
duction. In addition to the stability and the nutritional value of the 60 μl of N‐butylalcohol, and 40 ml of ethyl alcohol were added to a
beer, the use of the black rice malts enhanced the flavor and color volumetric flask. The total volume was determined to be 1,000 ml
due to their high polyphenol content and good antioxidant capacity with ultrapure water.
which can attract new customers. A total of 500 ml of beer from the fermentation were moved to a
conical flask, and the beer was cooled at 4°C to prevent volatile loss.

2 | M ATE R I A L S A N D M E TH O DS
2.5 | HS‐SPME procedure and GC–MS analysis
2.1 | Materials and chemicals
headspace solid‐phase microextraction procedure and GC–MS analy-
Australian malt was obtained from Jinan Shuangmai Beer Supplies sis were based on Li, Liu, Kun‐Farkas, and Kiss (2015) with some mod-
Co., Ltd.Rice, and black rice was acquired from a local market in ification. Analysis was performed using an Agilent 6890N capillary
Zibo, Shandong Province, China. Ethanol, acetaldehyde, n‐propyl al- gas chromatograph (GC, Agilent Technologies Co., Ltd.), and a Combi
cohol, n‐butanol, isobutanol, isoamyl alcohol, isoamyl acetate, ethyl PAL autosampler (Supelco Co. Ltd, Commonwealth of Pennsylvania)
acetate, and ethyl caprylate were of chromatographic grade; ethyl was assembled with a 65 μm polydimethylsiloxane–divinylbenzene
acetate was of analytical grade. All chemicals were produced from (Supelco Co., Ltd., Commonwealth of Pennsylvania) extraction fiber.
Tianjin Shield Fine Chemicals Co., Ltd. A total of 5 ml of the cooled beer mentioned above were placed
in 20 ml glass vials and contained 3 g of NaCl and magnetic stirrers.
Then, the vial was tightly capped with butyl rubber stopper finally
2.2 | Raw material processing
wrapped with an aluminum cap. A fiber was allowed to puncture the
Rice and black rice were adjusted to 21% moisture level and ex- cap seal and exposed in the headspace of the vial for 30 min at 40°C
truded using a homemade extruder with automatic control system. with continuous stirring at 250 rpm. After extraction, the fiber was re-
The extruder possessed a 79 mm barrel bore, 16.4:1 length‐to‐di- moved from the sample vial and immediately inserted into the heated
ameter ratio, 77 mm outer diameter screw and 8 mm diameter bore. injector of the gas chromatograph (250°C) for 5 min desorption time.
Feed rate (100 kg/h), screw speed (200 r/min), and extrusion tem- A GC system (Agilent 6890 N) with HS‐SPME‐suitable injector,
perature (70°C) were kept constant in all experiments. an Agilent 5973 mass spectrometry (MS) detector and a HP‐5 cap-
After extrusion, the materials were allowed to cool at room tem- illary column 30 m × 0.25 mm, with 0.25 μm film thickness (Agilent
perature, and then, we adjusted the moisture to 13% and reduced the Technologies Co., Ltd.) and N (3.5 ml/min) as the carrier gas, were
particle size to 0.9 mm by crushing. The malt was crushed by a crusher. used to identify the flavor compounds of beer after the HS‐SPME
procedure. The injector temperature was programmed to increase
from 40°C (held for 2 min) to 250°C in the rate of 7°C/min. The final
2.3 | Beer production
temperature of 250°C was held for 3 min.
The extruded black rice flour was homogeneous mixture with Mass spectrometry was performed in an electron impact ion
crushed malt. Black rice flour (6.80 kg) and malt (15.20 kg) were source mode at 70 eV electron beam, and the scanning range was
ZHANG et al. | 3

35–350 μm. The test voltage, pressure, and transfer line tempera- chlorogenic acid equivalent (GAE) standard curve and expressed
ture were 0.8 kV, 49.5 kPa, and 250°C, respectively. as μg GAE/ml beer.
The total anthocyanins content was determined following
method described by (Ivanova et al., 2011) with some modifica-
2.6 | Quantitative analysis of volatile compounds
tion. Beer was degassed through ultrasonic oscillation method,
Firstly, the quantitative calibration factor fi was calculated according and 1 g Nylon 66 powder added into 10 ml beer. The sample was
to Equation (1), as follows: oscillated and extracted for 10 min and centrifuged (2000 r/min,
10 min).Discarded the supernatant, washed the precipitation with
As × Wi
fi = (1) water, and centrifuged again, repeat three times. The precipitation
Ai × Ws
was dissolved in 12 ml n‐butanol‐hydrochloric (5:1) solution on
boiling water bath for 30 min, and the absorbance was measured
where As is the peak area of the internal standard, Wi is the iden-
at the wavelength of 550 nm (1 cm optical path in the cuvette) with
tified concentration of a particular standard, Ai is the peak area
a UV‐2102PCS spectrophotometer (Unico instrument Co., Ltd.). The
of particular standard, and Ws is the concentration of the internal
anthocyanins content was calculated using the following equation
standard. Headspace chromatographic profiles were compared with
proposed by Di Stefano & Cravero (1991):
the known reference flavor standard compounds for identification
(Table 1). Total anthocyanins content = A550nm × 16.7 × d.
Volatile compounds were determined and quantified with a
where A550 nm is absorbance at 540 nm, d is dilution factor; total an-
flame ionization detector, and signals were stored and integrated by
thocyanins content was expressed in mg/L as malvidin‐3‐glucoside
a computer software. The concentration of each volatile component
equivalents.
(Ci ) was calculated using Equation (2), as follows:

Ai × Cs × fi
Ci = (2) 2.8 | Protein component and molecular weight
As
determination
where Cs is the internal standard concentration. The nitrogenous substance content was measured by Lundin frac-
tion. In acidic solution, high‐molecule nitrogenous substances are
easily precipitated by tannin, and high‐molecule nitrogenous sub-
2.7 | Determination of the functional
stances and medium‐molecule substances are precipitated by
components of beer
phosphomolybdate. Briefly, 25 ml of beer was acidified with 1 ml
The Folin phenol reagent method (Prior, Wu, & Schaich, 2005) was of H2SO 4 (0.5832 mol/L) and 20 ml of distilled water in 20°C water
used to measure the total polyphenols content. Briefly, 0.5 ml of bath for 20 min, added 16% (16 g tannin:100 g distilled water) tanning
beer sample was mixed with 1.0 ml of Folin phenol reagent and solution, constant volume of 50 ml and then filtered immediately.
then stood for 5 min, added with 2.0 ml saturated sodium car- Automatic Kjeldahl Apparatus (k‐370 BUCHI Labortechnik AG) was
bonate solution and then placed for 1 hr under 30°C water bath. applied to measure soluble N content (C1) in filtrate. The acidifica-
The absorbance was measured at the wavelength of 747 nm by tion of beer was conducted as follows: 25 ml of beer was mixed with
using a UV‐2102PCS spectrophotometer (Unico Instrument Co., sodium molybdate (2.5 ml, 2.43 mol/L) and distilled water (15.0 ml)
Ltd.) after cooling. Total polyphenols content was calculated using to incubate in water bath for 20 min at 20°C. After that, 1 ml of

TA B L E 1 Quantitation calibration factors (fi) of flavor composition

Peak area
Concentration quantitative calibration
Volatile compound 1 2 3 4 5 Average (mg/L) factor fi

Acetaldehyde 21.70 21.00 21.20 21.90 21.10 21.40 15.60 0.86

N‐Propanol 6.40 6.60 6.00 6.30 6.80 6.37 32.14 5.96
Ethyl acetate 113.80 110.20 114.20 115.80 108.90 112.97 14.4 0.15
Isobutyl alcohol 25.60 26.40 26.10 26.50 24.80 25.80 20.05 0.92
N‐butanol 58.10 59.00 58.70 56.70 56.80 57.40 48.59 1.00
Isoamyl alcohol 82.30 84.30 77.20 86.10 79.40 80.90 81.00 1.18
Isoamyl acetate 52.90 55.50 52.10 55.10 51.10 52.77 4.38 0.10
Ethyl hexanoate 2.80 2.90 2.60 2.80 2.80 2.73 0.44 0.19
Ethyl octanoate 6.30 6.70 6.20 6.30 6.90 6.47 0.43 0.08
4 | ZHANG et al.

H2SO 4 (0.58 mol/L) was added to the resultant mixture and adjusted bank 1.6 of the US National Institute of Standards and Technology.
to 50 ml and filtered for further use. Then, we measured the soluble The areas of the peaks were calculated manually (Zhang, He, Ma,
N content (C2) in the filtrate. A total of 25 ml of beer total soluble N & Li, 2017). A total of 34 volatile flavor compounds were identified
content (Ct) were measured. High‐molecule nitrogenous substances in extruded black rice adjunct beer (Figure 1a and Table 2, match-
content: Ct‐C1; medium‐molecule nitrogenous substance content: ing degree ≥80%), including one organic acid, one aromatic, ten al-
C1‐C2; low‐molecule nitrogenous substances content: C2. cohols, and 22 esters. And there are 37 volatile flavor compounds
A total of 2 ml of beer and 2 ml of acetone were mixed and cen- were identified in rice adjunct beer (Figure 1b and Table 2, matching
trifuged at 8,000 r/min for 10 min. The sediment was solved with degree ≥80%), including one aromatic, two organic acids, seven al-
40 μl of distilled water and mixed using a XH‐C vortex mixer (Jintan cohols, and 27 esters. All volatile flavor compounds accounted for
City Medical Instrument Factory) for 2 min. A total of 20 μl of sample 89.04% and 86.29% of the total peak area, respectively.
and 20 μl of loading buffer were placed in a 1 ml centrifuge tube and Esters, the main carrier of aroma is the dominant flavor com-
boiled for 3 min. pounds in the extruded black rice adjunct beer and constitute an
Protein component molecular weight was analyzed by sodium important group of aromatic compounds in beer (Geroyiannaki et al.,
dodecyl sulfate‐polyacrylamide gel electrophoresis by using a 2007).The mainly flavor‐active esters in extruded black rice adjunct
DYY‐8C electrophoresis system (Beijing 61 Biotechnology Co., Ltd.) beer are ethyl acetate which has a fruity or solvent‐like aroma, iso-
(Yoon et al., 2010). The 5% stacking and 15% separating gels were amyl acetate which has a fruity or banana aroma, ethyl caproate, and
used throughout, with a 29:1 (w/w) acrylamide: bisacrylamide ratio. ethyl caprylate which have a sour apple and fruity aroma, phenethyl
Gelpro32 software was used to treat the decolorized adhesive. acetate which has an apple or rose aroma, and ethyl caprate which
has a fruity aroma (Verstrepen et al., 2003). Ethyl ester principal is
predominant in extruded black rice adjunct beer. The sum of ethyl
2.9 | Amino acid analysis
esters accounted for >66% of the total esters in beer. Isoamyl ace-
Amino acid constitute and content were analyzed by L‐8900 auto- tate accounted for 26% of the total esters which suggested that the
matic amino acid analyzer (Hitachi Limited). The chromatographic threshold value effect on flavor was significant.
conditions are as follows: column temperature of 57°C, detection The alcohols in beer are mainly higher alcohols, mainly 3‐
wavelengths of 570 and 440 nm, injection volume of 20 μl, constant methyl‐1‐butanol, 1‐propanol, 2‐methyl‐1‐propanol, and phenylethyl
flow rate of 0.40 (pump1) and 0.35 ml/min (pump2) for 35 min. alcohol. Higher alcohols are the main components of the fermen-
tation by‐products of beer. These alcohols came from ketonic acid
which is related to the synthesis of cellular white matter by yeast.
3 | R E S U LT S A N D D I S CU S S I O N S
Appropriate amounts of 3‐methyl‐1‐butanol and 1‐propanol gave the
beer a feeling of mellowness, and phenethyl alcohol has a pleasant
3.1 | GC‐MS analysis of the flavor compounds with
rose aroma (Luigi & Peter, 2008). However, excess higher alcohol gave
HS‐SPME
the beer unpleasant bitter taste and wine essence stimulation effect.
The volatile flavor compounds of beer were analyzed by GC‐MS and Nerolidol, geraniol, and geranylgeraniol are substances that are
matched with the experimental spectra that referred to the 1998 data undetected in rice adjunct beer. Geranylgeraniol has antitumour,

F I G U R E 1 (a) GC–MS analysis of the flavor compounds of extruded black rice adjunct beer. (b) GC–MS analysis of the flavor compounds
of rice adjunct beer
ZHANG et al. | 5

TA B L E 2 Volatile compounds of extruded black rice adjunct beer

Peak area/%

Extruded black rice

No. Compound CAS KI adjunct beer Rice adjunct beer

1 Ethanol 64‐17‐5 463 4.29 5.70

2 3‐Methyl‐1‐butanol 123‐51‐3 697 3.24 3.44
3 1‐Propanol 71‐23‐8 562 2.10 –
4 Ethyl Acetate 141‐78‐6 586 3.30 3.00
5 2‐Methyl‐1‐propanol 78‐83‐1 597 1.07 0.28
6 Octanoic acid 124‐07‐2 1,173 1.56 1.88
7 2‐Methylbutylacetat 624‐41‐9 820 2.24 2.29
8 Isobutyl acetate 110‐19‐0 721 0.19 0.14
9 Ethyl butyrate 105‐54‐4 785 0.22 0.23
10 Isoamyl acetate 123‐92‐2 820 17.97 13.87
11 Ethyl hexanoate 123‐66‐0 984 4.52 5.38
12 Phenylethyl alcohol 60‐12‐8 1,136 4.77 3.98
13 Ethyl caprylate 106‐32‐1 1,183 15.86 14.92
14 Octyl acetate 112‐14‐1 1,183 1.65 1.41
15 Phenethyl acetate 103‐45‐7 1,259 10.43 9.08
16 Ethyl nonanoate 123‐29‐5 1,282 0.09 0.12
17 Ethyl trans‐4‐decenoate 76649‐16‐6 1,389 0.26 0.01
18 Ethyl caprate 110‐38‐3 1,381 7.91 12.35
19 3‐Methylbutyl octanoate 2035‐99‐6 1,417 0.14 –
20 2‐Phenylethyl octanoate 5457‐70‐5 1,856 0.09 0.23
21 Dimethyl phthalate 131‐11‐3 1,440 0.22 –
22 1‐Undecanol 112‐42‐5 1,357 0.12 –
23 2,4‐Di‐tert‐butylphenol 96‐76‐4 1,555 0.03 0.02
24 Ethyl laurate 106‐33‐2 1,580 1.31 3.47
25 Diethyl phthalate 84‐66‐2 1,639 4.69 2.26
26 (+)‐Cedrol 77‐53‐2 1,543 0.13 0.05
27 Decanoic acid, 3‐methylbutyl ester 2306‐91‐4 1,615 0.03 0.07
28 Nerolidol 7212‐44‐4 1,564 0.05 –
29 Arachidic acid ethyl ester 18281‐05‐5 2,375 0.02 0.04
30 Geranylgeraniol 24034‐73‐9 2,192 0.03 –
31 Diisobutyl phthalate 84‐69‐5 1,908 0.21 0.34
32 Dibutyl phthalate 84‐74‐2 2,037 0.09 0.07
33 Pentadecanoic acid ethyl ester 41114‐00‐5 1,878 0.04 0.02
34 Geraniol 106‐24‐1 1,861 0.12 –
35 Hexyl acetate 142‐92‐7 984 – 0.16
36 1‐Octanol 111‐87‐5 1,059 – 0.05
37 Enanthylic ether 106‐30‐9 1,083 – 0.03
38 Isopentyl hexanoate 2198‐61‐0 1,218 – 0.04
39 Ethyl dihydrocinnamate 2021‐28‐5 1,359 – 0.02
40 Decanoic acid 334‐48‐5 1,372 – 0.77
41 1‐Dodecanol acetate 112‐66‐3 1,580 – 0.08
42 Phenylethyl octanoate 5457‐70‐5 1,856 – 0.14
43 1‐Dodecanol 112‐53‐8 1,457 – 0.09
44 Phenylethyl caproate 6290‐37‐5 1,657 – 0.26

Note: KI is the retention index calculated by the Kovats method.

6 | ZHANG et al.

antibacterial, and other physiological activities and maintains ce- ethyl hexanoate, and ethyl octanoate, present in extruded black rice
rebral cholesterol (Kotti, Ramirez, Pfeiffer, Huber, & Russell, 2006). adjunct and rice adjunct beers (Figure 2a,b) were identified by de-
Meanwhile, geraniol is an important intermediate in the synthesis tecting the standard solution (Figure 2c). The analyzes were meas-
of coenzyme Qn (I), vitamin A, vitamin E, and vitamin K 2, and it is an ured in triplicate. The recovery ratios of volatile compounds from
important precursor for terpenoid synthesis paclitaxel and steroids the beers ranged from 95.3% to 99.7%. The volatile composition and
in organisms. The content of nerolidol and geraniol which may con- contents of the extruded black rice adjunct beer were obtained.
tribute to the aroma by synergized Linalool and citronellol were low. Volatile compounds increase the complex flavor more than non-
Ethanol, acetaldehyde, ethyl butanoate, ethyl 2‐methylpropa- volatile compounds. Acetaldehyde was the only aldehyde identified
noate and ethyl 4‐methylpentanoate showed the highest odor ac- among the major volatile compounds, and increased acetaldehyde
tivity values (Kishimoto, Noba, Yako, Kobayashi, & Watanabe, 2018). provides a pungent odor to the beverage and it also leads to health
The 2‐phenylethyl octanoate, geraniol, arachidic acid ethyl ester, hazards (Geroyiannaki et al., 2007). The acetaldehyde content
and pentadecanoic acid ethyl ester contents were low. However, the (14.16 mg/L in extruded black rice adjunct beer and 13.52 mg/L in
presence of different esters can have a synergistic effect on the indi- rice adjunct beer) was lower than those of several kinds of beer on
vidual flavors which indicated that esters can also affect beer flavor the market (Ceccaroni et al., 2019; Tian, 2010). At low concentra-
well below their individual threshold concentrations. tions, the sensory of acetaldehyde is described as “classic,” “nutty,”
and “sherry‐like” (Dragone, Mussatto, Oliveira, & Teixeira, 2009).
The ester content in the extruded rice adjunct beer (ethylac-
3.2 | Volatile component concentrations
etate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate at
Nine flavor compounds, namely acetaldehyde, N‐propanol, ethyl ac- 12.60, 3.85, 0.40, and 0.37 mg/L, respectively) was higher than that
etate, isobutyl alcohol, N‐butanol, isoamyl alcohol, isoamyl acetate, in rice adjunct beer (Table 3). Esters are the main carrier of aroma

F I G U R E 2 (a) The GC of the extruded

black rice adjunct beer. The corresponding
compounds and their retention times for
the peaks are acetaldehyde (0.704 min),
ethanol (0.735 min), N‐propanol
(0.825 min), ethyl acetate (0.923 min),
isobutyl alcohol (0.953 min), isoamyl
alcohol (1.405 min), isoamyl acetate
(3.280 min), ethyl hexanoate (5.840 min),
and ethyl octanoate (9.362 min). (b)
The GC of the rice adjunct beer. The
corresponding compounds and their
retention times for the peaks are
acetaldehyde (0.705 min), ethanol
(0.735 min), N‐propanol (0.826 min),
ethyl acetate (0.924 min), isobutyl
alcohol (0.955 min), isoamyl alcohol
(1.407 min), isoamyl acetate (3.228 min),
ethyl hexanoate (5.842 min), and ethyl
octanoate (9.361 min). (c) The GC
chromatogram of the mixed stand sample.
The peaks correspond to acetaldehyde
(0.704 min), ethanol (0.734 min), N‐
propanol (0.825 min), ethyl acetate
(0.923 min), isobutyl alcohol (0.953 min),
N‐butanol (1.061 min), isoamyl alcohol
(1.406 min), isoamyl acetate (3.282 min),
ethyl hexanoate (5.838 min), and ethyl
octanoate (9.364 min)
ZHANG et al. | 7

TA B L E 3 Concentrations of the volatile beer components

N‐Butyl alcohol Extruded black rice adjunct beer Rice adjunct beer

Average peak area Content (mg/L) Volatile compound Average peak area Content (mg/L) Average peak area Content (mg/L)

53.20 48.59 Acetaldehyde 18.00 14.16 17.20 13.52

N‐Propanol 5.20 28.32 5.80 32.45
Ethyl acetate 91.60 12.60 78.40 9.86
Isobutyl alcohol 20.10 16.85 15.60 12.56
Isoamyl alcohol 65.10 70.33 56.50 61.55
Isoamyl acetate 43.00 3.85 42.10 3.60
Ethyl hexanoate 2.30 0.40 3.10 0.75
Ethyl octanoate 5.20 0.37 5.00 0.30

(Geroyiannaki et al., 2007) and responsible for the fruity character molecule with at least two binding sites attaches to two proteins,
(Verstrepen et al., 2003). and then, bridges are formed between the polyphenol and two
The higher alcohols which are a source of fragrance in beer are proteins to yield a three‐membered structure. The precipitation
the by‐products of fermentation process, although it has additional was insignificant in the extruded black rice adjunct beer which in-
side effects (Strubelt, Deters, Pentz, Siegers, & Younes, 1999). dicated that the interaction between proteins and polyphenols was
Beer has a stale feeling when it is higher alcohol content >50 mg/L. related to the polyphenol content and the polyphenol types (e.g.,
Isoamyl alcohol was the most abundant alcohol in both samples monophenolic compounds, nonflavonoid polyphenols, flavonoids,
which accounted for more than two‐thirds of the total alcohol and condensed tannins) (Callemien & Collin, 2010), protein concen-
(Table 3), and it is the main cause of drunkenness (Luigi & Peter, trations, polyphenols, polysaccharides, alcohol content, oxygen, pH,
2008). The N‐propanol and isobutyl alcohol contents were low in temperature, ionic strength, and the presence of metal ions(Siebert,
both beers. Troukhanova, & Lynn, 1996).

3.3 | Functional components of beer 3.4 | Soluble nitrogen and protein

component analysis
The polyphenol content was 79.98 μg GAE/ml (Table 4). The total
anthocyanin content and antioxidant activity were positively corre- The total soluble nitrogen content in extruded black adjunct beer
lated (Yang, Wei, Mengjie, & Guixing, 2010). Oladokun et al. (2016) (3,220.54 mg/L) was lower than the wort (4,750.7 mg/L), and the
analyzed the polyphenols of 34 commercial lager beers, and their high‐molecule, medium‐molecule, and low‐molecular N contents
results showed that these beers were produced using a range of dif- in extruded black rice adjunct beer were 632.13, 770.38, and
ferent raw materials which result in the polyphenol content ranging 1,817.6 mg/L, respectively (Table 5). The soluble nitrogen provided a
from 3.91 mg/L to 21.17 mg/L. nitrogen source for yeast in fermentation, which has been reported
The main anthocyanins were cyperus japonicas‐3‐O‐glucoside in previous research (Silva et al., 2008). The high‐molecule nitrog-
(C3G) and paeoniflorin‐3‐0‐glucoside (P3G). Direct sunlight will enous substances ratio is 19.63% in beer, which mainly includes
damage the structure of anthocyanins. Under acidic conditions (pH nucleic, proteins and their degradation products, medium‐molecule
of 4–7), anthocyanins were stable for light scattering and thermal nitrogenous substances (23.92%) mainly includes polypeptides and
stability. The fermentation process was carried out in a low‐tem- protein, and low‐molecule nitrogenous substances (56.45%) mainly
perature (2°C) and light‐tight tank to obtain beer with high antho- includes amino acids and biogenic amine. Nitrogenous compounds
cyanin content. are considered extremely important in beer because it affects flavor,
Furthermore, some polyphenols should be able to effectively foam stability, haze formation, color, yeast nutrition, and biological
crosslink proteins to form a stable network causing nonbiological stability (Marta Fontana, 2009).
precipitation (Siebert, 2006). The basic mechanism underlying the The relative molecular weight distribution of protein in extruded
interaction between proteins and polyphenols is that a polyphenol black rice adjunct beer is shown in Figure 3. The relative protein

TA B L E 4 The contents of total

Light absorption value
polyphenols and anthocyanin in beer
Compound 1 2 3 Average Content

Polyphenols 0.801 0.803 0.803 0.803 79.98 μg GAE/ml

Anthocyanins 0.129 0.128 0.128 0.128 23.80 mg/L
8 | ZHANG et al.

TA B L E 5 The soluble nitrogen content of extruded black rice adjunct beer

Total nitrogenous High‐molecule nitrogenous Medium‐molecule nitrog‐ Low‐molecule nitrog‐

substances substances enous substances enous substances

Wort
Content (mg/L) 4,750.7 1,085.1 100.24 2,663.2
Proportion (%) 22.84 21.10 56.06
Extruded black rice adjunct beer
Content (mg/L) 3,220.5 632.13 770.38 1817.90
Proportion (%) 19.63 23.92 56.45

and polypeptide molecular masses in extruded black rice beer were beer is the main skeleton component of beer foam. Protein has a
mainly found in the following five ranges: 20.0–22.0, 25.0–28.5, considerable influence on beer foam quality. The water‐soluble pro-
30.0–32.0, 37.0–39.0, and 43.0–46.0 kDa. The range of relative tein in the raw material is the main source of beer foam protein.
molecular weight distribution of protein in wort was same with the Low protein surface hydrophobic property stabilizes beer foam.
beer, but total content was higher. This result was consistent with Beer contains approximately 500 mg/L of proteinaceous material
those of a previous research (Hejgaard & Kaersgaard, 2013). Protein (Hejgaard & Kaersgaard, 2013), typically with the size ranging from
which is as a kind of substance accounting for a large content in 5 kDa to 100 kDa.
Hordein (mainly 10–35 kDa) which is the main protein in barely
(40%–50%) that is weakly soluble in water are responsible for haze
formation in beer. Protein Z is a main protein with a molecular mass
of 43–46 kDa which has high surface viscosity and hydrophobic-
ity. Protein Z is the most important protein to maintain foam sta-
bility. Between 20 and 22 kDa barley dimeric α‐amylase inhibitor‐1
(BDAI‐1) is the major protein. Within this range, high protein con-
tent was detected in >50 samples in the previous research (Takashi
et al., 2008). Lipid transfer proteins (LTPs, 7–14 kDa) are the most
abundant proteins in beer foam. LTPs contribute to foam quality
by reducing the adverse lipid to stabilize beer foam. Proteins, such
as protein Z, LTP‐1, hordein, and BDAI‐1, determine foam stability
(Takashi et al., 2008).

3.5 | Amino acid analysis

Amino acid analyzer method was used to determine amino acid com-
position and content. Amino acids are one of the important com-
ponents in beer. Figure 4 shows that 16 kinds of amino acids were
detected in extruded black rice adjunct beer and rice adjunct beer.
Amino acids have two main sources in beer, that is, one is the ac-
tion of cereal proteolytic enzymes to release amino acids from the
proteins of malt black rice and hops (Poveda, 2019), and the other is
secreted into fermentation fluid by yeast metabolism. Amino acids
from both of these sources were existed in beer in the state of dis-
solution (Redruello et al., 2017). There are 15 kinds of amino acids
in extruded black rice adjunct beer are higher than rice adjunct beer
(Figure 4).
The most abundant amino acids in extruded black rice adjunct
beer were Pro, which constituted the majority of the samples
(35.8% of the total amino acid content). This finding was consis-
tent with those of previous works (Pomilio, Duchowicz, Giraudo, &
Castro, 2010; Poveda, 2019). High amino acid content was caused
F I G U R E 3 (a) The bands after decoloration. (b) The distribution
interval of protein in wort. (c) The distribution interval of protein in by the fact that yeasts do not metabolize Pro (Lucie, Thibault,
extruded black adjunct beer Isabelle, Sylvie, & Carole, 2012). The next most abundant amino
ZHANG et al. | 9

F I G U R E 4 The contents of various

kinds of amino acids in beer 400 Exrtuded black rice adjunct beer
Rice adjunct beer

Content (mg/L)
300

200

100

0
Thr Lys Val Met Ile Leu Tyr Phe Asp Ser Glu Gly Ala His Arg Pro

acids in extruded black rice adjunct beer were Ala (134.0 mg/L), AC K N OW L E D G M E N T S
Glu (88.9 mg/L), Val (70.9 mg/L), Tyr (62.8 mg/L), Phe (52.8 mg/L),
We wish to thank Dr. Dongliang Zhang for advice on interpretation
Arg(44.7 mg/L), Thr (42.8 mg/L), Leu (36.4 mg/L), and Lys
of results.
(31.1 mg/L). These results were consistent with the findings of
previous studies (Poveda, 2019; Redruello et al., 2017). The con-
tents of the two essential amino acids (Val and Thr) were greater C O N FL I C T O F I N T E R E S T
than rice adjunct beer (Figure 4) and other beers (pale dark lager
The authors declared that they have no conflicts of interest to
or ale). Compared with different varieties of rice (Liyanaarachchi,
this work. Written informed consent was obtained from all study
Mahanama, Somasiri, & Punyasiri, 2018), the essential amino acid
participants.
(EAA) content was high in black rice. This phenomenon may be
reason why the EAA content was higher in extruded black rice
adjunct beer. The sample with the EAA proportion close to 40% E T H I C A L A P P R OVA L
in the total free amino acids (FAA) was supposed to good qual-
ity (Liu, Wang, & Zhou, 2007). The EAA/FAA of extruded black This study does not involve any human or animal testing.

rice adjunct beer was 35.8%, and high Pro content resulted in low
EAA/FAA ratio; meanwhile, the EAA/FAA of rice adjunct beer ORCID
was 11.1%.
The amino acid content is a fingerprint for the authenticity of Tianyu Zhang https://orcid.org/0000-0001-7359-6456

beers and can be used as a specific biomarker to identify the end Hongjun Li https://orcid.org/0000-0002-2615-5452
product. Hence, the nature and relative amount of the amino acids
can be related to the wort composition and beer fermentation con-
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