Hypotheses in Food Science

Concise Reviews &

A Mini-Review on Brewer’s Spent Grain
Protein: Isolation, Physicochemical
Properties, Application of Protein, and Functional
Properties of Hydrolysates
Chaoting Wen, Jixian Zhang, Yuqing Duan , Haihui Zhang, and Haile Ma

Abstract: There is a growing demand in reusing agro-industrial waste to enrich food proteins and develop new
protein-related products. Brewer’s spent grain (BSG), the byproducts generated from the processing of beer-brewing
industry, is rich in abundant protein (26 to 30%) with good physicochemical properties and nutritional benefits. BSG is
mainly composed of four proteins including hordein, gluten, globulin, and albumin. The methods for extracting protein
from BSG mainly include alkali extraction, ultrasonic-assisted extraction, and organic solvent extraction, among others.
However, little researches have described the functional properties of Brewer’s spent grain protein (BSGP) and how it can
be improved by enzymatic modification. Additionally, BSG protein hydrolysates (BSGPHs) have good bioactivities, mainly
including antioxidant, antiinflammatory, and angiotensin-I–converting enzyme inhibitory activities, among others. Based
on the above situation, this review provides a comprehensive overview about the isolation, physicochemical features,
application of the BSGP. In addition, the functional properties and biological activities of BSGPHs are also emphasized.
The purpose of this review is to provide an up-to-date summary of BSGP research, and to broaden the market potential of
this emerging protein in food industry. Besides, this review provides a reference to study the use of BSGP or hydrolysates
for a variety of purposes in-depth, including pharmaceutical, food, and industrial applications.

Keywords: bioactivities, Brewer’s spent grain protein, protein hydrolysates

Introduction paste could be obtained. Malt wort can be used as a fermentation

According to statistics, with the rapid development of the brew-
ing industry, the European Union produces about 3.4 million tons
of brewing waste per year, of which Brewer’s spent grain (BSG)
accounts for about 85% (Stojceska, 2019). Generally, BSG has
been mainly used for animal feed or directly discarded, which is a
huge waste of resources and causes serious environmental pollution
(Silbir & Goksungur, 2019). In developed countries, the reuse of
beer by products has received a high degree of attention due to
the strict restrictions of environmental protection laws (Giacobbe
et al., 2019). In China, people have gradually realized the impor-
tance of deep development and reuse of BSG. Interestingly, the
reuse of BSG not only follows green, sustainable, and environmen-
tally friendly principles, but also achieves great economic benefits
in industrial applications (Wen et al., 2018a).
There is an increasing awareness of the enormous potential of
acquiring proteins from BSG. In general, the acquisition of BSG
may go through several stages. As shown in Figure 1, the barley ker-
nel powder was steeped for 7 days in order to obtain the malt. After
saccharification, the main components of malt such as starch could
be converted into maltose and dextrin (Dragone, Almeida e Silva,
Silva, & Santos, 2002), and the malt wort and insoluble precipitated
JFDS-2019-0936 Submitted 6/15/2019, Accepted 10/1/2019. Authors Wen,
Zhang, Duan, Zhang, and Ma are with School of Food and Biological Engineering,
Jiangsu Univ., Zhenjiang 212013, China. Authors Duan and Ma are also with Inst.
of Food Physical Processing, Jiangsu Univ., Zhenjiang 212013, China. Direct inquiries
to authors Duan and Zhang (E-mail: dyq101@ujs.edu.cn; zhanghh@ujs.edu.cn).
Chaoting Wen and Jixian Zhang contributed equally to this work and should
be regarded as co-first authors.
medium to produce beer, and the insoluble solid substances are
known as BSG. In recent years, the recovery of protein from
BSG have received extensive attention (Lynch, Steffen, & Arendt,
2016). Celus, Brijs, and Delcour (2006) reported that BSGP was
mainly composed of hordeins and glutelins (Figure 2). They also
highlighted that hordeins belong to the storage proteins, and the
glutelins are the structural proteins of barley. In addition, the amino
acid compositions of BSGP have been studied and reported by
several researchers (Aliyu & Bala, 2011; Kissell, Prentice, & Lind-
say, 1979). Connolly, Piggott, and FitzGerald (2013) compared the
amino acid composition of the pale BSGP with that of the black
BSGP (extraction temperature: 50 and 20 °C). As shown in Ta-
ble 1, compared with the pale BSGP extracted at 20 °C, the black
BSGP extracted at 50 °C contained the highest level of amino acids
(except cysteine), of which glutamine/glutamic acid, proline, and
leucine were 24.73 ± 1.31, 7.19 ± 0.23, and 9.71 ± 0.77 g/100 g
protein. However, black BSGP (extraction temperature: 50 and
20 °C) contained the lower levels of amino acids than that of pale
BSGP. Moreover, the hydrolysate of BSGP is a potential functional
component, which could be applied in the food industry. Many
sources in the literature have suggested that BSGPHs has an-
tioxidant, antiinflammatory, and angiotensin-converting enzyme
(ACE) inhibitory activities, among others (Connolly, O’Keeffe,
Piggott, Nongonierma, & FitzGerald, 2015; Connolly, O’keeffe,
Nongonierma, Piggott, & FitzGerald, 2017; Connolly, Piggott,
& FitzGerald, 2014a; McCarthy et al., 2013b; Zong, Li, Zhang,
Luo, & Liu, 2012a). Therefore, the sustainable recycling and
reusing of BSG will contribute to broaden the variety of proteins
and accelerate the process of industrial protein production.

C 2019 Institute of Food Technologists

 R

3330 Journal of Food Science r Vol. 84, Iss. 12, 2019 doi: 10.1111/1750-3841.14906
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Brewer’s spent grain protein . . .

Figure 1–Schematic representation of the

process to obtain BSG from natural barley
(Mussatto et al., 2006).

Table 1–Amino acid composition of pale and black BSGP from

Connolly et al. (2013).

Pale BSG Black BSG

Amino acid 50 °C 20 °C 50 °C 20 °C
Cysteine 1.37 ± 0.24 1.56 ± 0.00 2.51 ± 0.41 1.51 ± 1.48
Methionine 1.36 ± 0.08 1.07 ± 0.04 0.46 ± 0.00 0.43 ± 0.05
Asparagineb 6.57 ± 0.32 5.98 ± 0.02 2.50 ± 0.00 2.24 ± 0.25
Threonine 3.16 ± 0.14 2.75 ± 0.01 0.24 ± 0.29 0.13 ± 0.19
Serine 4.09 ± 0.22 3.44 ± 0.03 – –
Glutaminec 24.73 ± 1.31 22.33 ± 0.01 14.56 ± 0.33 14.33 ± 1.92
Glycine 3.75 ± 0.15 3.31 ± 0.04 2.48 ± 0.17 2.22 ± 0.38
Alanine 4.29 ± 0.08 3.76 ± 0.11 3.06 ± 0.08 2.88 ± 0.25
Valine 5.97 ± 0.70 4.91 ± 0.04 4.40 ± 0.21 3.87 ± 0.75
Isoleucine 4.19 ± 0.32 3.56 ± 0.10 2.53 ± 0.07 2.37 ± 0.13
Leucine 7.19 ± 0.23 5.84 ± 0.07 4.91 ± 0.13 4.57 ± 0.53
Tyrosine 3.49 ± 0.30 3.21 ± 0.11 2.43 ± 0.01 2.12 ± 0.06
Phenylalanine 6.23 ± 0.31 6.09 ± 0.05 4.26 ± 0.25 4.01 ± 0.35
Hisitidine 3.63 ± 0.25 2.70 ± 0.03 2.00 ± 0.11 2.35 ± 0.33
Lysine 3.15 ± 0.06 2.92 ± 0.06 0.90 ± 0.05 0.78 ± 0.08
Arginine 5.95 ± 0.73 4.67 ± 0.01 – –
Proline 9.71 ± 0.77 9.50 ± 0.54 5.59 ± 0.90 5.61 ± 0.83
Note: “-” represents not determined.

esting and updated perspective on BSGP for the development of

Figure 2–SDS-PAGE of hordeins and glutelins fractions of BSGP (Celus et al.,
2006). Note: A represents barley alcohol soluble albumin or globulin (MW functional and nutritional food products.
< 15 kDa); B represents 70% to 80% of the hordein fraction (B1, B2, and
B3 are three subcomponents of B); C and D represents 10% to 20% of the
hordein fraction.
Agro-Industrial Crop Waste: Brewer’s Spent Grain
Reuse Strategy
As Cherubin et al. (2018) reported that the agro-industrial
crop residues were about 3.331 billion tons by 2003 year around
At present, some literatures provided a good overview of the the world, and the wastes yields were increased by 33% in
potential applications of BSG (Aliyu & Bala, 2011; Lynch et al., comparison with that in 1993 year. Therefore, it was vital to
2016; McCarthy, O’Callaghan, Piggott, FitzGerald, & O’Brien, make reasonable use of such huge agro-agricultural crop waste.
2013c; Mussatto, 2014; Mussatto, Dragone, & Roberto, 2006). Increasing evidence has disclosed that BSG is a promising by
However, there is no systematic summary, discussion, and updat- product of the agro-industry crop. As shown in Figure 3A, BSG
ing of related studies on BSGP. Besides, no comprehensive outlook contains large amounts of cellulose (12 to 25%), hemicellulose
is reviewed about the application and the bioactivities of BSGP. (20 to 25%), and lignin (12 to 28%) (Lynch et al., 2016; Steiner,
Therefore, it is expected that this review could provide an inter- Procopio, & Becker, 2015), which were consistent with the results

Vol. 84, Iss. 12, 2019 r Journal of Food Science 3331

Hypotheses in Food Science
Concise Reviews &
Brewer’s spent grain protein . . .
Figure 4–Bread baked with BSG (Brewery A & Brewery B, 1980). Note:
control represents that bread baked with wheat flour; ∗ , represents that
bread baked with 6% BSG.
of Mussatto et al. (2006), who demonstrated that BSG contained
amount of fibrous tissue, and these tissues mainly include lignin,
arabinoxylan, and cellulose (Figure 3B). These special constituents
of BSG have a great influence on its application in various fields
(Mussatto, 2014; Xiros & Christakopoulos, 2012). For example,
BSG flour (6%) could be added in the wheat flour to bake
bread, which had accepted flavor and texture in comparison
with control (Figure 4; Brewery & Brewery, 1980). In addition,
BSG is also widely used to produce biofuels and ethanol (Xiros,
Katapodis, & Christakopoulos, 2011; Xiros, Topakas, Katapodis,
& Christakopoulos, 2008). Furthermore, some literatures have
also reported the application of BSG for the production of biogas,
building materials, papermaking materials, and adsorbents, among
others (Buffington, 2014; Mussatto & Roberto, 2006; Mussatto
et al., 2006; Xiros & Christakopoulos, 2012). In addition to
the above-mentioned application, the bioactive substances (for
example, protein) from BSG have captured high interest of
consumers and researchers (Day, 2013; Lynch et al., 2016). BSG
can provide green, innovative and high-quality protein resource,
which could meet health needs of human beings. Therefore, in
order to make BSGP safer and more environmentally friendly
in food industry, it is necessary to clarify its extraction methods,
physicochemical and functional properties.
3332 Journal of Food Science r Vol. 84, Iss. 12, 2019
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Figure 3–Schematic diagram of the basic
composition of BSG. Note: (A) Composition of
BSG; (B) Image SEM of BSG (300×) (Mussatto
et al., 2006).
Fundamentals of BSG Protein
Isolation, physicochemical properties of protein from BSG
In general, the methods used for extracting protein from
BSG mainly include alkali-soluble acid precipitation, organic sol-
vent extraction, and ultrasonic-assisted extraction, among others
(Diptee, Smith, Alli, & Khanizadeh, 1989; Tang et al., 2010; Wen
et al., 2018b,c, 2019b; Zhang et al., 2018). The different methods
for extracting BSGP are listed in Table 2. Connolly et al. (2013)
reported that pale and black BSGP (recovery: 96.58 ± 0.86 and
49.03 ± 2.65 mg/g BSG dw) were extracted by using the contin-
uous alkali-soluble acid precipitation method (110 mM NaOH,
pH 3.8). The results of amino acid composition analysis indi-
cated that pale BSGP was rich in glutamine/glutamic acid, va-
line, and leucine, whereas the content of sulfur-containing amino
acids (cysteine and methionine) was low. Interestingly, the amino
acid component of BSGP was comparable to germinated barley
food, suggesting that BSGP has the same utilization value as barley
protein.
In addition, this result was similar to the report of Treimo,
Aspmo, Eijsink, and Horn (2008), who demonstrated that BSGP
had a considerable amino acid balance with barley grain, which
was also rich in higher levels of glutamine/glutamate, valine, and
leucine. Besides, Vieira et al. (2014) have innovatively utilized a
recyclable reagent for the continuous extraction of proteins and
arabinoxylans, indicating that the recycled reagent (citric acid,
ethanol) significantly increased the total protein content to 82%
to 85%. It is worth noting that this method can greatly reduce
energy consumption and reagent usage. Additionally, the differ-
ent pretreatment methods including alkaline, acids, hydrothermal,
and its combinations were also used to assist the extraction of
proteins from BSG. Qin, Johansen, and Mussatto (2018) evalu-
ated the effect of different pretreatments on the extraction rate
of BSGP, the results showed that the highest protein extraction
rate (95%) was obtained by continuous alkaline and dilute acid
pretreatment, followed by dilute acid pretreatment (90%) and hy-
drothermal pretreatment (64 to 66%). It was worth noting that
hydrothermal pretreatment had the advantages of environmental
protection, low temperature, and no chemical addition, compared
to chemical extraction. Furthermore, some studies reported that
ultrasound extraction as an ideal, nonthermal physics processing
technology could significantly improve the yield and properties
of proteins (Fukase, Ohdaira, Masuzawa, & Ide, 1994; Kadam,
Tiwari, Álvarez, & O’Donnell, 2015; Karki et al., 2010; Vilkhu,
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Brewer’s spent grain protein . . .

Table 2–Summary of the extraction of protein from BSG.

Source Extraction method Condition Extraction yield Structural analysis References

BSG Ethanol precipitation Substrate : extractant = 1:20 49% Amino acid analysis; (Ervin et al.,
(m/v); T = 27, 45, 60, 75, 90, SDS electrophoresis 1989)
100 °C; Ethanol precipitation
time = 16 hr
BSG Ultrasound-assisted Substrate : extractant = 1:10 20.09 ± 1.40% Not studied (Tang et al., 2009)
extraction and (m/v); t = 1 hr; Membranes of
ultrafiltration MWCO : 5, 30 kDa
treatment
Pale and black Alkali-soluble acid Substrate : extractant = 96.58 ± 0.86 and Amino acid analysis; (Connolly et al.,
BSG precipitation 1:20 (m/v); HCl solution 49.03 ± 2.65 SDS electrophoresis; 2013)
precipitation time = 15 min mg/g BSG dw molecular weight
distribution;
antioxidant activities
assay
BSG Alkali extraction Substrate : extractant = 79–83% Chemical composition (Vieira et al.,
1:2 (w/v); Room analysis 2014)
temperature, overnight
BSG Alkaline, acid, Substrate : alkaline extractant = 95 % Amino acid analysis; (Qin et al., 2018)
hydrothermal, and 1:20 (w/v); T = 50 °C; chemical composition
their combinations Dilute acid : BSG = 16.15:1 analysis; particles
extraction (w/w); hydrothermal
pretreatment condition: solid
to liquidratio of 2.5% (w/v); T
(35–135 °C)
BSG Ultrasonic-assisted Extraction time = 81.4 min; 104.2 mg/g BSG Not studied (Tang et al., 2010)
extraction Ultrasonic power = 88.2 W/
100 mL; Solid-liquid ratio =
2.0 g/100 mL
BSG Salt solution extraction BSG: extractant = 2.5:100 60 % Not studied (Diptee et al.,
(w/w); T = 90 °C; t = 95 min 1989)

Mawson, Simons, & Bates, 2008; Zhu, Sun, & Zhou, 2009). For
example, Tang et al. (2010) studied that the effects of ultrasonic
power, extraction time, and solid–liquid ratio on the extraction
yield of BSGP, the results showed that the maximum protein
yield (104.2 mg/g BSG) was obtained under ultrasonic power
88.2 W/100 mL, extraction time 81.4 min, and solid–liquid ratio
2.0 g/100 mL.
From a critical point of view, these extraction methods have
both advantages and disadvantages, as shown in Figure 5. The alkali
dissolution acid precipitation method (Cameron & Myers, 1983;
Cortés-Ruiz, Pacheco-Aguilar, Lugo-Sánchez, Carvallo-Ruiz, &
Garcı́a-Sánchez, 2008; Liu, Zhao, Ren, Zhao, & Yang, 2011) is
the most commonly used in plant-derived protein extraction. The
principle of this method is that plant-derived proteins are easily
soluble in an alkaline environment and precipitates under acidic
isoelectric conditions. The advantages of the alkali dissolution
acid precipitation method include high extraction rate, ease of
operation, and low cost. However, high alkali concentration also
affects the nutritional properties of the protein through causing the
Maillard reaction. Besides, the organic solvent extraction method
(Diptee et al., 1989; Ervin, Alli, Smith, & Li, 1989; Vieira et al.,
2014) is mainly suitable for the insoluble protein in water, alkali,
acid, and dilute salt solutions. These proteins are only soluble in a
lipophilic organic solvent such as ethanol or acetone, and so on.
It is noted that this extraction method must be operated at low
temperatures to prevent protein denaturation. Moreover, some
other innovative extraction techniques, such as ultrasound-assisted
extraction (Chandrapala, Oliver, Kentish, & Ashokkumar, 2013;
De-Song, Gang-Mming, Bing, & Lin, 2010; Tang et al., 2010;
Wen et al., 2018c; Wen, Zhang, Zhang, Duan, & Ma, 2019a)
and the combination of multiple extraction methods (Qin et al.,
2018), aim to significantly increase protein extraction rate and
reduce energy consumption. However, the research on extraction
methods is relatively simple, and the investigation about extraction
mechanism is not deep enough.
In fact, the physicochemical properties and amino acid compo-
sition of the protein largely depend on the difference in extraction
conditions. Connolly et al. (2013) showed that high extraction
temperature would lead to the loss of amino acid content in the
black BSGP, which may cause the components to participate in
the Maillard reaction. This result was agreement with (Ervin et al.,
1989) who reported that the extraction temperature could signif-
icantly affect the amino acid composition of BSGP (extraction
yield: 49%). In which protein (extraction temperature: 25 °C)
contained higher levels of proline and glutamic acid than that ex-
tracted at 100 °C. Notably, the authors also stated that BSGP had
high levels of essential amino acids, including phenylalanine, me-
thionine, and valine, compared to the amino acid composition of
soy protein.
Although spectroscopy (Khan & Yu, 2013; Pelton & McLean,
2000), microscopy (Topf & Sali, 2005; Topf, Baker, John,
Chiu, & Sali, 2005), nuclear magnetic resonance (Jaroniec,
2015; Lipsitz, Sharma, And, & Tjandra, 2002), and mass spec-
trometry (Chavez & Bruce, 2019; Politis et al., 2014) tech-
niques are known to effectively evaluate the molecular struc-
ture of protein, there are few studies on the physicochemical
properties of BSGP. Therefore, the detailed structure informa-
tion of BSGP is still lacking. Future researches need to ob-
tain new insights into the structure of BSGP by using these
techniques.

Vol. 84, Iss. 12, 2019 r Journal of Food Science 3333

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Brewer’s spent grain protein . . .

Brewer's spent grain (BSG)

Protein extraction

Advantages Methods Disadvantages

Low cost; easy to operate Alkali dissolution acid precipitation High solvent

Low cost; increased yield Organic solvent extraction High solvent

High yield; low energy Ultrasound-assisted extraction Swelling material

Acid precipitation

Isoelectric precipitation

Application Protein concentration

Purification via membranes

Others

Figure 5–Flow chart of protein extracted from BSG.

Application of BSGP in food industry
In view of the physiochemical and functional properties of
BSGP, the utilization value and application range of BSGP are
worth being explored and popularized. In recent years, the ap-
plications of BSGP mainly include cookies, edible films, protein
complexes, high protein feeds, fermented beverages, nutrient hy-
drolysates, feed enzymes, and edible protein products, among oth-
ers (Ding, Li, Fang, & Yan, 2012; Lin, Li, Tian, Zhang, & Li, 2012;
Quan-Yi et al., 2010; Wahlström et al., 2017; Zong, Liu, Li, &
Liu, 2010).

3334 Journal of Food Science r Vol. 84, Iss. 12, 2019


Brewer’s spent grain protein . . .
BSGP is characterized by interesting physiochemical properties,
which could be added into the wheat flour to prepare cookies.
Zong, Bian, Li, Qiang, and Luo (2012b) studied that the effect
of BSGP on the sensory and texture of cookies, and obtained the
optimum formula conditions (15% of BSGP, 50% of eggs, 45%
of butter, and 20% of sugar). The results showed that BSG not
only improved the flavor, nutrition, and quality of cookies, but
also increased the utilization rate of BSG. At the same time, it also
met the diversified needs of consumers for cookies.
The demand growth of protein coprecipitates product is driven
by increasing demand for plant by product proteins with abundant
nutrients and high functional properties (Alu’Datt et al., 2013).
Protein coprecipitates is a valuable food ingredient, which could
significantly enhance the functional, physicochemical, nutritional
properties, and biological activities of the protein compared to sin-
gle protein component (Haast, Morressey, & Fox, 2010). BSGP has
attracted more and more attention of researchers, which could be
used as source for preparation of protein coprecipitates. Alu’Datt
et al. (2018) prepared protein coprecipitates by using BSG and soy-
bean flour as raw materials and investigated the effect of ultrasonic
treatment on the structure and biological properties. In this study,
ultrasound treatment could significantly reduce the particle size
of protein, and improve its ACE inhibitory activity in compari-
son with the non-ultrasound samples. The protein coprecipitates
prepared with BSG not only has good nutrition and functional
properties, but also follows the principle of waste recycling, is
economical and environmentally friendly.
BSGP is a potential source of biodegradable film. In recent years,
large amounts of studies have shown that composite film prepared
by adding polysaccharide into protein possess good mechanical
properties and functional properties (Chen, 1995; Chen, Remon-
detto, & Subirade, 2006; Shit & Shah, 2014; Yoo & Krochta,
2011). Lee et al. (2015) obtained biodegradable film by adding chi-
tosan into the BSGP. The tensile elongation of the composite film
and the added amount of protein had concentration-dose effect.
However, as the added amount of chitosan increasing, the water
vapor permeability of the composite film gradually decreased. It
is worth noting that the composite film could significantly inhibit
the growth of pathogenic bacteria, including Staphylococcus aureus,
Listeria monocytogenes, Escherichia coli, and Salmonella typhimurium.
In addition, the composite film is capable of a strong ability to
scavenge DPPH radicals, indicating that BSGP composite film has
great potential application in food antioxidant and antibacterial
packaging materials.
In general, the BSGP could be applied as a functional ingredient
in the food industry, which could contribute to the efficient and
comprehensive utilization of Brewer’s waste resources. It greatly
increases the added value of the products, and brings good eco-
nomic and society benefits.
Functional Properties and Bioactivities of BSG
Protein Hydrolysates
Functional properties of the protein hydrolysates
from BSGP
It is necessary to further understand its functional properties to
better expand the application range of BSGP in food industry.
However, the solubility of BSGP is very poor and there were little
studies about the functional properties of BSGP (Rommi, Niemi,
Kemppainen, & Kruus, 2018). Besides, most of studies focused
on the enzymatic treatment BSGP to obtain hydrolysates with
better functional properties (Celus, Brijs, & Delcour, 2007, 2009;
Hypotheses in Food Science
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Jin, 2008; Rommi et al., 2018). This work reviews the functional
properties of BSGPHs, including emulsifying properties, foaming
properties, solubility, turbidity, oil, and water absorption capacity.
The studies on the functional properties of BSGP from 2007 to
2018 are listed in Table 3.
Celus et al. (2007) reported that the poor solution of BSGP (ex-
tracted by alkaline) limited its application in food industry. How-
ever, Connolly, Piggott, and FitzGerald (2014b) also highlighted
the enzymatic treatment of BSGP by using Alcalase, Corolase
PP, Flavourzyme, and Promod 144MG, which could significantly
improve their emulsification, solubility, foaming, and foaming sta-
bility. In addition, the BSGPHs still has strong thermal stability at
140 °C. Besides, large amounts of literatures have shown that en-
zymatic modification could significantly improve the emulsifying
ability and foaming capacity of plant-derived proteins (Clemente,
2000; Don, Pilosof, & Bartholomai, 1991; Panyam & Kilara,
1996). These results might be mainly attributed with that enzy-
matic hydrolysis could reduce the size of the protein hydrolysates
and increase its hydrophobicity (Yin et al., 2008). Therefore, all
these results demonstrated that enzymatic modification is an ef-
fective method to improve the functional properties of BSGPHs.
However, long-time enzymatic hydrolysis may also result in the
production of excessive small peptides, which could reduce the
foaming and emulsifying properties of the protein (Chen, Chen,
Ren, & Zhao, 2011). In addition, shorter enzymatic hydrolysis
time could produce a smaller proportion of small peptides which
produce some bad bitterness and odor (Day, 2013). Therefore,
it is necessary to select a specific protease and suitable hydrolysis
conditions to effectively control the enzymatic hydrolysis process,
thereby achieving the purpose of improving the functional prop-
erties of the protein. Furthermore, future research should pay at-
tention to use some new, ideal, combined modification techniques
to modify the functional properties of BSGP.
Bioactivities of the protein hydrolysates from BSGP
Bioactive peptides have attracted widespread attention because
they could simultaneously regulate, improve, or inhibit a variety
of physiological pathways (Udenigwe & Aluko, 2012). BSGP is
deeming as the potential resources of bioactive peptide. Recently,
some studies have focused on producing bioactive peptides from
BSGP with alkaline extraction and enzymatic hydrolysis meth-
ods (Connolly et al., 2015, 2017). Antioxidant activity, ACE
inhibitory activity, and anti-inflammatory activity, among oth-
ers, have been identified as bioactive characteristics of BSGPHs
(Table 4).
The production of excessive free radicals in the human body
could cause oxidative stress, which in turn induces some chronic
diseases, including diabetes, cancer, inflammation, and cardiovas-
cular diseases, among others (Siti, Kamisah, & Kamsiah, 2015).
In addition, oxidation is considered as the main cause of food
deterioration, which could trigger rancidity, unacceptable flavor,
texture, and shortening shelf life (Vaclavik & Christian, 2014).
Many synthetic antioxidants (BHA, BHT, and n-propyl gallate)
have exhibited good antioxidant capacity. However, some coun-
tries have resisted or banned the use of synthetic antioxidants due
to their potential risks in human health (Lobo, Patil, Phatak, &
Chandra, 2010). Therefore, some antioxidant peptides from natu-
ral products have been utilized to scavenge free radicals and inhibit
the oxidation process in living organisms or food stuffs (Kim &
Wijesekara, 2010). Antioxidant peptides or protein hydrolysates
eliminate the free radicals mainly through inhibiting lipid per-
oxidation, directly scavenging reactive oxygen radical, scavenging
Vol. 84, Iss. 12, 2019 r Journal of Food Science 3335
Hypotheses in Food Science
Concise Reviews &

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Brewer’s spent grain protein . . .

(Connolly et al., 2014b)

oxygen, and chelating metal ions (Esfandi, Walters, & Tsopmo,

(Rommi et al., 2018)

(Treimo et al., 2008)
2019). Another antioxidant mechanism is that peptides or protein

(Celus et al., 2007)

(Celus et al., 2009)

References
hydrolysates could activate the body’s inherent antioxidant defense
system. For example, peptides or protein hydrolysates could target

(Xiao, 2008)
the Nrf2/Keap1 ARE and NF-κB signaling pathways to exert an-
tioxidant effects (Samaranayaka & Li-Chan, 2011). Recently, the
preparation of antioxidant hydrolysates from BSGP has become
a research hotspot. McCarthy et al. (2013a) studied the conver-
sion of BSGP into antioxidant hydrolysates for alleviating H2 O2 -
Oil absorption
induced oxidative stress. The BSGP isolates were hydrolyzed with
— Alcalase 2.4L, Flavourzyme, and Corolase PP, and the BSGPHs
—
—
—
—
—
was further separated into three fractions by ultrafiltration de-
vice (membrane Mw: 3.5 kDa). The results showed that BSGPHs
could significantly increase the activity of SOD in the H2 O2 -
induced U937 cell damage. They also highlighted that BSGPHs
(Mw < 5 kDa) could reduce H2 O2 -induced DNA damage in
Water absorption

U937 cell. In another study, Vieira et al. (2017) reported that BSGP
was hydrolyzed (condition: E/S ratio of 10:100 [v/v], 50 °C,
—
—
—
—
—
—

4 hr) with Neutrase, Brewer’s spent yeast (BSY) proteases, and

Alcalase. The BSGPHs was further separated into three fractions
by ultrafiltration device (membrane Mw: 3.10 kDa). The ferric
ion reducing antioxidant power (FRAP) of the hydrolysates and its
Protein functionality

fractions were assessed, and the results showed that BSGPHs (Al-
calase hydrolysate group) showed higher FRAP (0.101 mg TE/mg
Turbidity

dw) than other hydrolysates groups (Neutrase and BSY). BSGPHs

—
—
—
—

—
↑

(Mw < 10 kDa) could exert significant protection effect to scav-

enge free radicals in Caco-2 and HepG2 cells.
It is known that chronic inflammation could cause some chronic
Note:“—” represent that the data was not shown in the article. “↑” represent that the data was improving compared with control group.

diseases, including diabetes, arthritis, atherosclerosis, and cardio-

Emulsifying

vascular disease, among others (Körner et al., 2019). According

—

—
↑
↑

↑
↑

to epidemiological and clinical studies, 15% to 20% of malignant

tumors are caused by chronic inflammation, which causes a large
number of morbidity and mortality worldwide (Germini et al.,
2019). Large amounts of food-derived bioactive peptides have
Foaming

been investigated due to its anti-inflammatory activity, including

—

milk, soy, fish, and eggs, among others (Martı́nezaugustin,

↑

↑
↑

Riverogutiérrez, Mascaraque, & Sánchez, 2014). Chalamaiah, Yu,

and Wu (2018) had reviewed the anti-inflammatory mechanisms
of protein hydrolysates or peptides by enhancing the induction
Table 3––Summary of the functionality of BSGPHs (2007 to 2019).

Solubility

of immunomodulators, increasing leukocyte counts, activation of

—

the transcription factor nuclear factor-κB (NF-κB), and mitogen-

↑
↑
↑

↑
↑

activated protein kinase (MAPK)-dependent pathways, and

inhibition of proinflammatory mediators. The BSGPHs also
showed good anti-inflammatory activity. McCarthy et al. (2013a)
Limited Enzymatic Hydrolysis

studied the conversion of BSGP into bioactive hydrolysates for the

Treatment technology

anti-inflammatory related food and drug. Compared with other

protein hydrolysates fractions, BSGPHs (Mw > 5 kDa) exhibited
Enzymatic Hydrolysis
Enzymatic Hydrolysis
Enzymatic Hydrolysis
Enzymatic Hydrolysis
Enzymatic extraction

higher anti-inflammatory potential by reducing the production

of interferon-γ . In another study, the anti-inflammatory capacity
of BSG peptides obtained by Alcalase 2.4 L, Flavourzyme, and
Corolase PP hydrolysis was investigated (McCarthy et al., 2013b).
The authors reported that BSGP isolate and its hydrolysates
possess good anti-inflammatory activity, which could signifi-
cantly decrease the production of pro-inflammatory cytokine
Interferon-γ (IFN-γ ), Interleukin-2 (IL-2), Interleukin-4 (IL-4),
BSG protein concentrate
BSG protein concentrate
BSG protein concentrate

and Interleukin-10 (IL-10). They also highlighted that BSGPHs

might be used as a useful treatment agent to prevent the
BSG protein isolate
Raw materials

inflammatory diseases.
Blood pressure is regulated primarily by the renin–angiotensin
BSG protein

system (RAS) and kallikrein–kinin system (KKS). In RAS, ACE

is capable of cleaving angiotensin I into angiotensin II, it
belongs to a vasoconstricting octapeptide that could raise the

3336 Journal of Food Science r Vol. 84, Iss. 12, 2019

 Table 4–Bioactive peptides derived from BSGP.

Peptide
Activity Characteristic Preparation Index sequence Potential mechanism Reference
Antioxidant Enzyme/substrate ratio: 0.29:1 (w/w); time: Brewers’ spent yeast proteases FRAP assay Not studied Eliminate the free radicals; Activate (Vieira, Teixeira, &
6 hr; temperature: 50 °C; pH 6 the body’s inherent antioxidant Ferreira, 2016)
defense system (Samaranayaka &
Li-Chan, 2011)
Enzyme/water ratio: 2.5%; temperature: Alcalase; Corolase PP; Flavourzyme SOD activity, MTT Assay Not studied (McCarthy et al., 2013b)
50 °C; pH 7; time: 4 hr
Mw < 3 kDa and 5 kDa; enzyme/water Alcalase; Corolase PP; Flavourzyme SOD activity, MTT Assay; Not studied (McCarthy et al., 2013a)
ratio: 1% Comet assay
Enzyme/substrate ratio: 10:100 (v/v); Brewer’s spent yeast proteases; FRAP assay Not studied (Vieira et al., 2017)
temperature: 50 °C; time: 4 hr; Neutrase and Alcalase
Brewer’s spent grain protein . . .

Mw < 10 kDa
Enzyme/substrate ratio: 1 % (w/w);
time: 2 hr
Antiinflammatory Enzyme/water ratio: 1 %; temperature:
60 °C; pH 9; time: 4 hr
Mw > 5 kDa; Enzyme/water ratio: 1 %
Enzyme/substrate ratio: 1 % (w/w);
time: 2 hr
ACE inhibitory Enzyme/substrate ratio: 1.0%, 2.0%, 2.5%
(v/w); temperature: 50 and 60 °C; pH
7and 9
Enzyme/substrate ratio: 1.0%; pH 7; time:
4 hr Simulated gastrointestinal digestion of
BSGPHs: enzyme (pepsin)/substrate ratio:
2.5%; time: 90 min; enzyme (Corolase
PP)/substrate ratio: 1.0%; Time: 150 min
Modulate Enzyme/substrate ratio: 1.0%, 2.0%, 2.5%
glycaemic (v/w); temperature: 50 and 60 °C; pH 7
and 9
Enzyme/substrate ratio: 1.0%; pH 7;
time: 4 hr
Enzyme/substrate ratio: 1 % (w/w);
time : 2 hr
Abbreviations: BSGPI, Brewer’s spent grain protein isolate; SGID, simulated gastrointestinal digestion; DPP-IV, dipeptidyl peptidase-IV; FRAP, ferric ion reducing antioxidant power; ILDL, isoleucine-leucine-asparticacid-leucine; LLPGAQDGL,
leucine–leucine–proline–glycine–alanine–glutarnine–aspartic acid–glycine–glycine.
Alcalase; Flavourzyme ORAC; FRAP and ABTS Not studied (Connolly et al., 2018)
Alcalase; Corolase PP; Flavourzyme Antiinflammatory activity Not studied Increase leukocyte counts; activate (McCarthy et al.,
assay (cytokine the transcription factor nuclear 2013b)
production:IL-2, IL-4, factor-κB (NF-κB) and
IL-10, IFN-γ ) mitogen-activated protein kinase
(MAPK)-dependent pathways;
Inhibit proinflammatory mediators
(Chalamaiah et al., 2018)
Alcalase; Corolase PP; Flavourzyme IFN-γ Not studied (McCarthy et al., 2013a)
Alcalase; Flavourzyme IL-6 Not studied (Connolly et al., 2018)
Alcalase 2.4L; Corolase L10; ACE inhibition assay Not studied Interact with the arginine-nitric oxide (Connolly et al.,
CorolasePP; Trypsin 250; pathway, the AT-II receptor, the 2014a)
Flavourzyme 500 L; Promod endothelin system, RAS-related
144MG; Protex6L; Protamex; 2+
renin and Ca channels
Promod 24P; Promod 439; Prolyve (Udenigwe & Mohan, 2014)
1000
Alcalase; Corolase PP; Flavourzyme ACE inhibition assay ILDL (Connolly et al., 2015)
and Promod 144MG
Alcalase 2.4L; Corolase L10; α-Glucosidase inhibition Not studied Inhibit some enzymes related to (Connolly et al.,
CorolasePP; Trypsin 250; assay; α-amylase glucose absorption (α-glucosidase; 2014a)
Flavourzyme 500 L; Promod inhibition assay; α-amylase; dipeptidyl peptidase
144MG; Protex6L; Protamex; dipeptidyl peptidase IV IV); incretin secretion; regulation
Promod 24P; Promod 439; Prolyve (DPP-IV) inhibition of glucose uptake in peripheral
1000 assay tissue (Oseguera-Toledo, de Mejı́a,
Reynoso-Camacho,
Cardador-Martı́nez, &
Amaya-Llano, 2014)
Alcalase, Corolase PP, Flavourzyme, DPP-IV inhibitory assay ILDL and (Connolly et al., 2017)
promod 144MG; SGID; LLP-
semipreparative RP-HPLC; GAQDGL
UPLC)- MS/MS
Alcalase; Flavourzyme DPP-IV inhibitory assay Not studied (Connolly et al., 2018)

Vol. 84, Iss. 12, 2019 r Journal of Food Science 3337

Concise Reviews &
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Brewer’s spent grain protein . . .

blood pressure (Chen et al., 2009). In KKS, ACE can inacti- meet requirements of the reproducibility, safety and low energy
vate the vasodilator bradykinin, which increases the blood pres- consumption. Third, due to the different origins, the structural
sure (Madureira, Tavares, Gomes, Pintado, & Malcata, 2010). The differences and diversity of the source, the relationship between
excessive angiotensin-converting enzyme may cause vasoconstric- protein modification techniques, the high order structure and
tion and hypertension, and then, which could induce diseases such properties of proteins have not been well established. Finally, a va-
as myocardial infarction, heart and renal failure, among others riety of bioactivities of BSGPHs have been demonstrated mostly
(Eriksson, Danilczyk, & Penninger, 2002). It is worth noting that in vitro or in vivo animal experiments. More clinical studies are
these diseases are closely related to 12.8% of the global death rate still needed to be studied to evaluate safety and bioavailability
(Alwan, 2011). Therefore, inhibition of ACE may be a way to of these BSGPHs before investing in large-scale functional food
reduce blood pressure. It has been reported that the food-derived production.
peptides possess strong ACE inhibitory activity due to its C- This review provides a comprehensive perspective to further un-
terminus of amino acids, such as Tyr, Phe, Trp, and Pro (Ruiz, derstand BSGP and provide a theoretical reference for broadening
Ramos, & Recio, 2004). Udenigwe and Mohan (2014) empha- the application add-value of proteins. The application of BSGP
sized that ACE inhibitory peptides could lower blood pressure has great significance for promoting the health development of
through multiple mechanisms, including bioactive peptides were the brewing beer industry, reducing environmental pollution, and
capable of interacting with the arginine-nitric oxide pathway, the increasing social and economic effects. In addition, future research
AT-II receptor, the endothelin system, RAS-related renin and will focus on overcoming the challenges mentioned above in this
Ca2+ channels. It is worth noting that barley proteins are rich in area.
the known ACE inhibitory peptide sequences (Loponen 2004).
Connolly et al. (2015) reported that BSGPHs retained good ACE Conflicts of Interest
inhibitory activity after simulated gastrointestinal digestion. Be- The authors declare that there are no conflicts of interest.
sides, two peptides (IVY and ILDL) were identified by using
UPLC-MS/MS and their IC50 values of ACE inhibitory activ- Acknowledgement
ity were 80.4 ± 11.9 and 96.4 ± 8.36 µM, respectively. This work was funded by National Key R&D Program, China
In addition to the bioactivities mentioned above, the BSGPHs (2016YFD0400303); Postgraduate Research & Practice Innova-
also has other activities, including dipeptidyl peptidase IV (DPP- tion Program of Jiangsu Province, China (KYCX17 1799); and
IV) inhibitory activity, α-glucosidase inhibitory activity, and an- the Priority Academic Program Development of Jiangsu Higher
tibacterial activity, among others (Connolly et al., 2014a, 2017; Education Institutions (PAPD).
Zong et al., 2012a).
Authors’ Contributions
Chaoting Wen and Jixian Zhang collected the test data and
Conclusion and Future Trends
drafted the manuscript. Yuqing Duan and Haihui Zhang designed
In this overview, a comprehensive summary about the isola-
the study and interpreted the results. Haile Ma assisted to critically
tion and physicochemical properties of BSGP was presented. The
review the manuscript and revise it for important intellectual con-
advantages and disadvantages of the extraction techniques were
tent, and approve it. All of the authors completed and authorized
discussed. In particular, the applications of BSGP have been sum-
the definitive manuscript.
marized, including producing cookie, edible film, protein com-
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