Technological and Engineering Trends for Production Gluten-Free Beers

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REVIEW ARTICLE
Technological and Engineering Trends for Production
of Gluten-Free Beers
Monica Rubio-Flores1 • Sergio O. Serna-Saldivar1
Received: 24 November 2015 / Accepted: 28 March 2016 / Published online: 11 April 2016
� Springer Science+Business Media New York 2016
Abstract Beer is the main alcoholic beverage consumed
worldwide. It is essentially composed of malt, adjuncts or
grist, hops, water and metabolites produced by yeast.
Produced regularly with barley, it is unsuitable for patients
with celiac disease due to the presence of storage proteins
known as hordeins. A recent boom in the production of
gluten-free beers has been attributed to the awareness of
celiac disease and the significant boost in the gluten-free
market size. Several strategies have been recognized as
suitable gluten-free brewing procedures: by using non-
gluten grains, non-grain sources such as sugars, syrups and
honey and enzymatic and microbial treatments of barley
worts to reduce to non-significant levels (\20 ppm/L), the
peptides or epitopes which trigger celiac disease. Since the
traditional brewing process uses barley, the use of other
grains, such as rice, maize, sorghum, buckwheat or dif-
ferent types of millets, involves differences in both malting
and mashing procedures especially in terms of process
times and temperatures. Sorghum has been especially
recognized for its potential to substitute barley since it is
widely used in many countries of Africa to create tradi-
tional opaque beers, but compared to barley malt lacks of
sufficient diastatic power due to the lower synthesis of both
a- and b-amylases, and additionally, the starch requires
higher temperatures for gelatinization. Likewise, rice,
maize, millets and buckwheat are being used as source of
both malt and grist with similar problems as sorghum.
Potential future approaches involve the genetic engineering
of barley in order to remove the peptides of the storage
proteins that cause the allergic reaction on celiac patients
and the employment of recombinant yeasts or other fer-
menting microorganisms that express prolyl endopro-
teinases which hydrolyze celiac immunoreactive epitopes
into non-harmful peptides.
Keywords Celiac disease � Gluten-free beer � Malt �
Prolyl endoproteinase � Sorghum � Yeast fermentation
Introduction
Beer is the leading alcoholic beverage consumed world-
wide and has been enjoyed for more than 8000 years. In
2013, approximately 1.89 billion hectoliters of barley
(Hordeum vulgare) beer were brewed around the globe
[26]. About half of the world beer is brewed in China
(26.7 %), USA (11.9 %), Brazil (7.2 %) and Russian
Federation (4.7 %). In 2011, the Irish, Czechs, Lithuanians,
Austrians, Estonians and Germans had the highest per
capita consumption with an estimated intake of 142, 136,
108, 107, 98.5 and 97.9 L beer, respectively [26].
The use of cereals as raw materials by the brewing
industry can be divided into two main categories: malt and
adjuncts. Malt is usually produced from barley because this
husked cereal produces high diastatic or amylase activity
and aids during the critical step of lautering. However,
barley contains gluten proteins, named hordeins, which
trigger celiac disease. Brewing adjuncts are produced from
most cereals and generally consist of refined milled frac-
tions or starches that upon mashing are hydrolyzed into
dextrins and fermentable carbohydrates. Dextrins impart
the typical beer body, and the fermentable sugars are
transformed during yeast (Saccharomyces cerevisiae)
& Sergio O. Serna-Saldivar
sserna@itesm.mx
1 Centro de Biotecnologı́a-FEMSA, Escuela de Ingenierı́a y
Ciencias, Tecnológico de Monterrey, Av. Eugenio Garza
Sada 2501 Sur, CP 64849 Monterrey, NL, Mexico
123
Food Eng Rev (2016) 8:468–482
DOI 10.1007/s12393-016-9142-6
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fermentation into ethanol and other organic and flavorful
compounds. The hordein, gliadin, secalin and avenins from
barley, wheat (Triticum aestivum), rye (Secale cereale) and
oats (Avena sativa), respectively, can trigger celiac disease
[81].
Guerdrum and Bamforth [31] evaluated levels of gliadin
contents of twenty-eight commercially available beers
using a competitive enzyme immunoassay. As expected,
beers containing wheat malt or grist such as Dunkelweizen
and American Pale Wheat beers contained the highest
amounts (98.2–145.8 mg/L). On the other hand, the gliadin
levels ranged from less than 3 mg/L for gluten-free beers to
3.9–12.3 mg/L in the most common American light and
regular lagers.
There has been a recent boom of production of new
gluten-free foods and beverages developed for celiac dis-
ease patients and customers that want to avoid gluten-
containing foods. The brewing industry has not lagged
behind, and some major and specialty breweries launched
new beers labeled as gluten-free. The production of gluten-
free beers without barley malt is challenging because it is
difficult to find replacement malts with adequate diastatic
activity, especially in terms of b-amylase. Furthermore, the
production technology of gluten-free beers usually is
modified particularly in terms of mashing, lautering and
even fermentation.
The standards of ‘‘gluten-free beers’’ differ around the
globe. For example, in the European Union and USA, a
beer with less than 20 parts per million of gluten is con-
sidered ‘‘gluten-free,’’ while in Australia only beers with
no detectable gluten can be labeled as gluten-free. Gluten-
free beers are mainly brewed from cereals such as rice
(Oryza sativa), maize (Zea mays), sorghum (Sorghum
bicolor) and pearl and finger millets (Pennisetum ameri-
canum and Eleusine coracana) and pseudocereals such
as buckwheat (Fagopyrum esculentum). Alternatively,
these beers can be manufactured from gluten-containing
cereals by reducing the level of gluten to below 20 ppm/L
[31, 33].
The aim of this review paper was to review recent
developments and investigations aimed toward the indus-
trial production of gluten-free beers and especially discuss
technologies and raw materials that are alternatively used
for the generation of these sorts of beers for celiacs.
Celiac Disease
Celiac disease is an inflammatory disorder of the upper
small intestine triggered by the ingestion of wheat gliadins,
rye secalins and barley hordeins [81]. Triticale resulting
from the cross of wheat and rye is also considered allergic.
These prolamins plus glutelins conform the gluten and are
responsible for the gastrointestinal and immunological
reactions on celiac patients [32]. Patients usually need to
follow a strictly gluten-free diet for the rest of their lives.
Oats are not toxic in over 95 % of patients with celiac
disease; however, there is a small subgroup in whom oats
are not safe and therefore not recommended [2, 61].
Gluten proteins have several unique features that con-
tribute to their immunogenic properties. They are extre-
mely rich in the amino acids proline and glutamine. Gluten
is highly resistant to proteolytic degradation because gas-
tric and pancreatic enzymes lack post-proline cleaving
activity. The proline-rich gluten peptides are relatively
resistant to gastrointestinal digestion and therefore persist
in the intestinal lumen to elicit an immune reaction in
genetically susceptible individuals [12, 28]. The degraded
gluten proteins reach the small intestine and trigger an
inappropriate T-cell-mediated immune response, which can
result in intestinal mucosal inflammation, chronic diarrhea,
steatorrhea, vomiting and abdominal distension. Further-
more, celiac disease patients commonly have malabsorp-
tion of vital macro- and micronutrients resulting in loss of
weight and failure to thrive. To date, no pharmacological
treatment is available to gluten-intolerant patients,and a
strict, lifelong gluten-free diet is the only safe and efficient
treatment available. Inevitably, this may produce consid-
erable psychological, emotional and economic stresses.
Novel forms of therapy include strategies to eliminate
gluten peptides from the celiac diet so that the immuno-
genic effect of the gluten epitopes can be neutralized [11].
Non-celiac disease gluten sensitivity is a related disorder
in which both allergic and autoimmune mechanisms have
been ruled out. Patients with non-celiac disease gluten
sensitivity have an apparently normal duodenal histology
and a lack of celiac disease-specific autoantibodies [66].
The prevalence of celiac disease in the adult population
varies between roughly one in 100 and one in 300 in most
parts of the world [62]. According to scientists of the
University of Chicago, it affects at least 3 million Ameri-
cans, an average of 1 in 133. This prevalence is signifi-
cantly higher than that recognized 20 years ago. Recent
studies have demonstrated that the number of new cases of
celiac disease found in a specific period in a given popu-
lation (the incidence) is increasing especially in North
America and Europe [13, 46]. The epidemiology of celiac
disease has iceberg characteristics—there are far more
undiagnosed cases (below the waterline) than diagnosed
ones (above the waterline) [27].
In the United States, the average length of time it takes
for a symptomatic person to be diagnosed with celiac
disease is 4 years. This type of delay dramatically increases
an individual’s risk of developing autoimmune disorders,
neurological problems, osteoporosis and even cancer [30].
In adults, celiac disease is diagnosed on average more than
Food Eng Rev (2016) 8:468–482 469
123
10 years after the first symptoms appear. Approximately
70 % of patients report an improvement in symptoms
within 2 weeks after starting the gluten-free diet [49]. With
strict dietary control, antibody levels may decrease very
soon after the diet has been instituted. In contrast, complete
histological resolution is not always achieved, or may take
years [72].
Gluten-Free Food Market
There are currently thought to be over 300 million people
managing special diets worldwide, as a result of food
allergies or intolerances, and the number is rising each
year. The increased diagnosis of food sensitivities has
given rise to a new and growing market for food intoler-
ance—or ‘‘free-from’’—products. Remarkably, gluten-free
products represented the fastest growing sector of the food
intolerance products market over the review period, with
sales up by 126 % overall. In 2012, the Western Europe
accounted for almost half of the sales with North America
coming second. The global sales of gluten-free products are
led by USA, Italy, Germany, United Kingdom and Sweden.
United States showed a 217.7 % growth between 2003 and
2008. In second place, Italy held 235 million USD in sales
in 2008, whereas the Czech Republic showed a 202 %
growth even though its sales only grew from 1 to 3 million
USD [25].
The gluten-free food trend is now replicating in the
alcoholic drinks sector. The ‘‘gluten-free’’ spin has become
a key selling point within the brewing industry. World-
wide, different breweries have started gluten-free beer
production. In 2006, Anheuser-Busch became the first
major brewer to launch a wheat- and gluten-free beer,
Redbridge Sorghum Beer, with 4 ABV and an $8.00 retail
price [80]. In 2010, Carlsberg launched Daura gluten-free
from Estrella Damm, at 5.4 % ABV that is retailed in four
packs at a suggested retail price of US$7.99 [48]. Unlike
other gluten-free beers, Daura has been made with barley
malt and the company claims to have a much wider target
audience than consumers allergic to gluten. Desnoes &
Geddes, brewer of the Jamaican lager Red Stripe, has
announced that cassava (Manihot esculenta) will be used as
the main brewing ingredient for its brand in 2014. In Ire-
land, SuperValu has announced details of a new
$218,000.00 contract with a local brewery which includes
exclusive rights to the microbrewery’s new gluten-free
lager [53].
Commercial gluten-free beers from millet malt (Sch-
nitzer Brau) or buckwheat grist (Belgian style Green Quest
or Green Discovery) are marketed in Germany and United
Kingdom, respectively. The first is produced similarly as
sorghum beer, whereas the second is manufactured from
buckwheat which is used as source of malt (buckwheat
malt ale) and in the grist formulation [3].
Beer Consumption
Beer is the main alcoholic beverage consumed in the
world, with an average of 5.11 million hectoliters per day.
In over 50 years, beer consumption has more than
quadrupled, from 43 million tonnes in 1960 to over 189
million tonnes in 2013 [26]. It is expected to continue to
grow by around 1.2 % in the next several years, fueled by
consumers’ increasing purchasing power as well as the
consumer’s changing drinking habits [25]. Global beer
value and volume sales grew by 7 and 2 %, respectively, in
2013. In 2014, the growth in global volumes was not
reflected in the performance of the top four breweries, as
much of the growth was derived from smaller brewers and
growing demand in China.
Production of Gluten-Free Beers
Regular beers are typically produced from five major
ingredients: water, barley malt, adjuncts, hops and yeast
and four main operations: malting, mashing/lautering,
boiling/hopping and pitching or fermentation [68].
In order to produce gluten-free beers, the malt and
adjuncts should be constituted of gluten-free materials and
the production line should be located in enclosed estab-
lishments and devoted for the production of these special
beers in line to avoid cross contamination.
The aim of malting is to achieve the maximum per-
centage of germination and diastatic activity and conclude
the process with the lowest possible dry matter loss.
Malting is divided into three suboperations: steeping, ger-
mination and kilning. The aim of steeping is to hydrate the
kernel under aerobic conditions in order to activate the
synthesis of gibberellic acid that will control the whole
germination process. Generally, the operation is simply
performed by immersing or by spraying water onto the
grains for 24–80 h to increase the grain moisture to
42–48 %. Generally, barley is stepped and germinated at
15 �C, whereas gluten-free cereals such as sorghum, maize,
millets or even rice at temperature around 25 �C [1, 18, 22–
24, 34, 40, 47, 50, 52, 59, 76]. For this reason, gluten-free
grains are more prone to microbial contamination during
the critical step of malting [68].
Malting is usually carried on beds placed inside special
rooms with strict temperature and relative humidity con-
trols. This process lasts 4–6 days and is greatly affected by
the malting house temperature. The processes are adjusted
in order to achieve the desired diastatic activity, cell divi-
sion and the development of the rootlets and acrospires
470 Food Eng Rev (2016) 8:468–482
123
with the lowest dry matter loss. The malting process and
the botanical growth of the rootlets and acrospires are
stopped by kilning. Drying conditions are critical to pro-
duce different sorts of malts with different enzyme activi-
ties, colors and flavors. Malts destined for lagers are
generally less extensively modified (higher soluble sugars
and FAN) than those aimed for ales that are kilned to a
relatively mild regime. Lager malts therefore develop less
color and produce pale or straw amber-colored beers. Malts
destined for ale production are kilned to higher temperature
and thus have darker colorations. A high kilning temper-
ature yields melanoidins from soluble sugars and amino
acids and complex flavors that eventually affect the flavor,
color and aroma of the wort and beer. Barley and gluten-
free malts are generally dehydrated to a final moisture
content of about 5 %. Afterkilning, the malt is cooled
down with fresh air and the rootlets stripped or deculmed.
The culms are separated because they impart bitter flavors,
are high in nitrogenous compounds and contain high levels
of nitrosamines [5, 10, 60, 68]. In the specific case of
sorghum, the culms are rich in cyanogenic compounds
mainly dhurrin [34, 78]. Generally, sorghum, rice, maize,
buckwheat and millets are commonly steeped and germi-
nated at a temperatures between 20 and 30 �C [1, 10, 18,
22–24, 34, 40, 47, 50, 52, 59, 71, 76].
Figure 1 compares malting procedures of conventional
barley and sorghum suited for gluten-free beers. The major
differences between procedures are the malting tempera-
tures. Both processes yield similar amounts of malts
(around 86 kg malt/100 grain). A notable difference
throughout both processes is the grains’ moisture content.
Compared with sorghum, barley grain requires a higher
amount of water (45 %) before entering germination, but
has a slightly lower one (6 %) after kilning. Another
notable difference is the amount of culms obtained from
each grain, where sorghum grains commonly have a higher
deculming loss [34, 68]. The malting of maize, millet,
buckwheat and rice follows similar steps and temperatures
as sorghum [1, 50, 52, 71].
Figure 2 continues comparing typical lager beer pro-
cesses and beer yields of regular gluten-containing and
gluten-free beers manufactured from sorghum malt and
refined grits. Due to the lower diastatic activity of sorghum
malt and the lack of b-amylase activity, worts contained
less fermentable sugars and thus less alcohol content. A
beer yield comparison clearly favors the barley malt beer
with around 15 % more production compared to the sor-
ghum beer supplemented with amyloglucosidase. This
particular enzyme greatly improves fermentable sugars and
alcohol content and allows the production of light beers
Fig. 1 Comparative flowcharts of typical malting schemes and malt yields of barley and sorghum
Food Eng Rev (2016) 8:468–482 471
123
[70]. The solo use of sorghum malt increases lautering
times [58] and reduces beer yields and alcohol contents
[18, 22–24, 76].
The mashing conditions of gluten-free worts differ from
regular counterparts. In this operation, the ground malt and
brewing adjuncts are placed in mash cooker in the presence
of water in order to hydrolyze the starch and proteins into
fermentable carbohydrates, linear and branched dextrins
and soluble nitrogen. The fermentable carbohydrates and
soluble nitrogen are important yeast substrates, whereas
dextrins impart the typical body or texture associated with
regular beers [5, 37, 60, 68]. In most gluten-free beer
mashing processes, extrinsic enzymes are supplemented to
the reactors in order to enhance conversion and increase the
amounts of fermentable sugars and soluble nitrogen [70].
The temperature program during mashing is commonly
modified in order to produce gluten-free worts. The
mashing time is usually prolonged, whereas the different
temperature stands modified in order to enhance conver-
sion [22–24, 50].
Most lagers in the American Continent are produced by
the double-mashing procedure because the grist formula-
tions usually contain high amount of starchy adjuncts.
These starchy materials require cooking in order to achieve
complete starch gelatinization. This step is considerably
modified when brewing gluten-free sorghum beers because
its starch has higher gelatinization temperature compared
to barley [34, 50, 63, 68, 69, 75]. The mashing consists of
two distinctive stages. In the first, commonly known as
adjunct mash, the brewing adjuncts are mixed with water
and heated to 35 �C. After a 30- to 60-min stand, the
contents are heated first to 70 �C for about 30 min and then
to 100 �C for 30–45 min. The aim is to first hydrate the
adjuncts and malt and then promote starch gelatinization
and conversion since most cereal starches gelatinize at
temperatures higher than 65 �C. Boiling is required to
denature proteins and to inactivate all microbes and
enzymes. Once the adjunct mashing schedule is ongoing,
the second mash is prepared by mixing and heating to
35 �C most of the malt and water. Then, the contents of the
two vessels are mixed. The aim of the second mashing step
is to convert starch and proteins into simpler carbohydrates
and soluble proteins or peptides. This is optimally achieved
by programming a gradual temperature increase which
starts at 35 �C for approximately 30 min. The temperature
is usually ramped 10–15 �C each step until achieving
70 �C. The sequential temperature increase favors the
proteolytic enzymes, followed by b-amylase (optimum
temperature of 60 �C) and a-amylase (optimum tempera-
ture of 70 �C). The high temperature of the last mashing
stage stops most enzymatic activity, reduces viscosity and
improves the fluidity and filtering capacity of the resulting
Fig. 2 Comparison of production of sorghum beer and barley beer [68]
472 Food Eng Rev (2016) 8:468–482
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mash. During this operation, the contents are agitated to
achieve a better malt solubilization and to enhance the
exposure of adjuncts to enzymatic hydrolysis [5, 10, 60,
68]. The second stage of mashing is considerably modified
when brewing gluten-free beers because of the use of malts
with less diastatic activity and the use of adjuncts that
commonly have higher starch gelatinization temperatures
[50]. It is also frequent to add extrinsic enzymes such as
amylases, amyloglucosidases and proteases in order to
improve extraction and reduce mashing times. The differ-
ent sorts of enzymes require different optimum tempera-
tures, so the temperature program should be adjusted
accordingly [70].
After mashing, the mash is transferred to the lauter tun
where the sweet wort is separated from the brewer’s spent
grains. The lauter tun is a relatively shallow apparatus with
a double mesh floor which is equipped with rakes that
loosen the filtrating bed structure minimizing compaction.
The mash contents are allowed to precipitate for 30 min so
as to enhance the formation of the natural filtration bed rich
in husks. The use of gluten-free malts such as sorghum
prolongs lautering times due to the lack of the natural husk
filtering bed and due to the higher viscosity of worts
(generally high in dextrins). The sweet wort is separated by
filtration through the double mesh floor. Generally in the
first stage of lautering, the wort is recirculated through the
bed of spent grains in order to decrease wort turbidity. The
filtering is generally done at temperatures of 65–70 �C and
the spent grains sparged with hot water (75–80 �C) to
remove the remaining extract from spent grains. The aim of
lautering, which is considered the bottleneck of the brew-
ing process, is to obtain as much of the sweet wort or
extract with the minimum insoluble contamination. The
more rapidly the filtrates, the more brews can be daily
performed [37, 60, 68]. One of the major limitations of the
gluten-free beer process is the long lautering times because
non-barley grains do not contain glumes or husks which aid
in the filtration process and the lack of sufficient diastatic
activity that yields worts with more viscosity and dextrin
contents [34, 58].
The process of hop (Humulus lupulus) addition to glu-
ten-free beers is practically identical to regular beers. Hops
are added to the sweet wort in order to impart the dis-
tinctive European beer flavor and odor. The type and
amount used will greatly impact the final flavor and texture
of beer. The process simply consists of adding the hops to
the wort in order to promote the extraction of solubles by
boiling for 1.5–2.5 h. Generally, half to two-thirds of the
hops are added at the beginning of the program and the rest
at the end of the process with the objective of keeping key
volatiles that enhances beer flavor and aroma. During
boiling, the enzymes are inactivated,the wort becomes
darker due to intrinsic wort compounds and caramelization
reactions, and the hopped wort becomes sterile. During this
thermal treatment, from 4 to 12 % of water is lost due to
evaporation. In addition, some soluble proteins will bind to
tannins and precipitate improving clarity. Spent hops are
removed from the hopped wort using the whirlpool prin-
ciple. The boiling wort is passed tangentially into a large
whirlpool equipped with a conical trub collector located in
the central part of the base of apparatus. The hopped wort
is cooled in a heat exchanger to about 6 �C for traditional
lagers and as high as 15–20 �C for ales. The wort is also
aerated with sterile air in order to increase its oxygen
content which is critically important for yeast growth and
budding especially during the early phase of fermentation.
During cooling, more proteins become insoluble and
removed by centrifugation [5, 37, 60].
The wort is fermented into beer in special tanks or
reactors equipped with cooling coils or jackets. The wort is
pitched or inoculated commonly with fresh yeast from
previous batches or less frequently with dry yeast. The
inoculum generally added is 4.5–7.5 or 1.5–2.5 g fresh or
dry yeast/L (typically 5–20 million yeast cells/mL of wort
or 10 million cells/mL wort containing 12� Plato) [5, 60,
68]. During the first stage of fermentation, the yeast
reproduces asexually by budding increasing the biomass
from 4 to 5 times and utilizes the available oxygen. Thus,
the conditions gradually switch from aerobic to anaerobic.
It is considered that after 12- to 24-h fermentation, the
reactor conditions are anaerobic. During this phase, the
yeast metabolizes fermentable carbohydrates and free
amino nitrogen producing ethanol and fusel alcohols,
respectively. The main fusel alcohols are isopropanol,
amylic, isoamylic and butanol [8]. During this stage, car-
bon dioxide is also produced and intermediate organic
products that help to impart the characteristic beer flavor.
The use of gluten-free malts and adjuncts generally change
the types and amounts of fusel alcohols and other important
volatiles. For production of regular and gluten-free lagers,
the fermentation process is carried at 8–13 �C during
several days followed by cold storage at less than 0 �C and
for Ales at approximately 14–17 �C for 3–5 days. Lagers
are almost always fermented with bottom cropping yeast
whereas Ales with top cropping yeast [60]. Most beers are
kept in closed tanks at a temperature of 0 �C for 4–6
additional weeks in order to further reduce oxygen to a
level of less than 0.2 ppm and enhance bouquet and aroma
due to chemical changes such as the generation of diacetyl,
dimethylsulfide and hydrogen sulfide. During fermentation,
yeast cells transform maltose, and maltotriose into glucose
that is further metabolized into carbon dioxide, energy,
ethanol and other organic metabolites such as organic acids
and volatile compounds. Approximately one-third of the
fermentable sugars are transformed into carbon dioxide
[39]. The progress of fermentation is usually followed
Food Eng Rev (2016) 8:468–482 473
123
using a refractometer that measures beer density. The ini-
tial wort density is about 1.040 and the finished product
between 1.008 and 1.010 g/cm3. The wort with an initial
pH of 5.5 lowers its pH to 5.2 after boiling and then
gradually decreases to a level of 3.8–4.2 during beer fer-
mentation due to production of organic acids and direct
excretion of H ? ions by yeast [37, 60]. The change in the
acidity coagulates some proteins and decreases even more
the solubility of some acidic hop resins. Also during fer-
mentation, important quantities of soluble nitrogen are
metabolized into fusel alcohols that affect the organoleptic
properties of beer. The sweetness of beers is due to residual
sugars that have not been fermented into alcohol [5, 37,
60].
Many different types of chemical compounds besides
ethanol, fusel alcohols and those derived from hops also
influence beer flavor, aroma and stability. A wide array of
esters present in concentrations of ppm affects sensory
properties. The most relevant are ethyl acetate, butyl
acetate, isoamyl acetate, ethyl valerate, isoamyl propionate,
phenylethyl acetate, methyl caprate and methyl and iso-
amyl caprate, which impart different fruit flavors. The
same applies for sulfur compounds such as dimethyl sulfide
or disulfide, ethyl or amyl mercaptan and methional. These
sulfur-containing volatiles affect flavor (onion, garlic, egg,
rotting leek) at even lower concentrations, generally in ppb.
One of the major concerns in the industry is the control of
levels of dimethyl sulfide and diacetyl which change when
producing gluten-free beers [5].
Silica gel has been effectively used to stabilize beers
especially against chill haze. Additionally, it quantitatively
removed gluten cross-reactive material at doses similar to
those used to provide beer colloidal stability. This filtering
aid effectively reduced the levels of gluten analyzed with
the ELISA test and reduced in most beer levels to less than
20 mg gluten/kg [9].
There are currently two main strategies for producing
gluten-free beers. The first approach consists of substitut-
ing barley malt and adjuncts with gluten-free cereals,
pseudocereals or non-grain materials such as fer-
mentable sugars, yeast extract or proteins from non-cereal
sources. Due to the substitution of barley malt, exogenous
enzymes are normally supplemented to counteract the
weaker diastatic activity of other sorts of malts [18, 22–24,
70, 76]. The other alternative is the use of barley which is
first subjected to proteolytic treatment with prolyl endo-
proteinase, and then, gluten reactive proteins were removed
with regular filtering and with the use of filtering aids such
as silica [9, 45].
Beer contains approximately 500 mg/L protein
depending on the brewing process employed. This protein
is in the form of polypeptides and is commonly quantified
with the free amino nitrogen assay. The majority of which
lie within the 10–40 kD size range. Some of these
polypeptides are responsible for causing colloidal haze,
some enhance foam stability, and the remainder appear to
have no function in beer except to contribute to mouth-feel.
Silica preferentially adsorbs polypeptides rich in proline
such as the ones associated with gluten proteins that trigger
celiac disease [45].
In order to produce gluten-free beers with comparable
technological and sensory characteristics to regular beers,
a combination of different gluten-free raw materials and
exogenous enzymes is commonly used. The enzymes
improve wort fermentability, foam stability, alcohol
content and final organoleptic properties [70]. Lactic acid
bacteria and/or yeast fermentation represent promising
tools to produce flavor enhancing compounds which will
improve the poor sensory quality of gluten-free bever-
ages. A notable example is foam stability since it influ-
ences consumer preference. Beer foam stability is
affected by malting and brewing conditions and also by
barley’s hordeins, which are known as foam-active pro-
teins [6, 32, 45].
Producing Gluten-Free Beer with Non-gluten
Grains
Currently, sorghum, maize, pearl and finger millets,
buckwheat and rice appear to be successful gluten-free beer
ingredients [1, 34, 50–52, 71].
In Africa, sorghum has been traditionally malted for the
production of opaque beers that are quite different from
European beers [64, 68]. However, sorghum has also been
recognized for its potential to substitute barley malt in
lager beer production [34, 59] and it is now widely used in
European-type lager beer brewing in many developing
countries of the tropics [51]. Some brewers purposely add
exogenous enzymes, such as amylases and proteases, to
increase the sugar and amino acid contents in worts so as to
facilitate yeast growth and performance during fermenta-tion [18, 22–24, 54, 76].
Sorghum
The most promising and researched cereal for production
of gluten-free beers is sorghum [34, 54, 55]. Sorghum is the
fifth most important cereal worldwide with an annual
production exceeding 61 million tons in year 2013 [26].
This cereal crop widely adapted to arid and subtropical
ecosystems around the globe can play a dual role as
brewing ingredient: as refined brewing adjuncts and as a
source of diastatic malt. Therefore, it is totally feasible to
manufacture European sorghum beer types using hops to
impart the characteristic and desired flavor and yeast as
fermenting agent. An advantage of sorghum is that it can
474 Food Eng Rev (2016) 8:468–482
123
yield a crop under harsh environmental stress, such as
drought, where temperate cereals fail to grow [34, 64, 69].
For centuries, the indigenous sorghum beers have
occupied an important place in the diets of many African
people, and the industrialization of this kind of beer started
more than 70 years ago. Due to an increasing demand for
Western clear beer, research work on the use of locally
grown sorghum and exogenous enzymes was carried out in
Nigeria in the 1980s, after barley was banned in this
country. As a consequence, Nigeria’s 23 breweries reached
an annual production of around 10 million hectoliters [34,
54, 55].
Osorio-Morales et al. [58] produced refined brewing
grits from regular and waxy sorghums that were first sub-
jected to mechanical decortication followed by dry milling
and classification that met the expected particle size dis-
tribution, and percentages of fat and fiber. The waxy
brewing grits obtained from white sorghum containing in
the starch more than 95 % amylopectin favored the double-
mashing procedure and lautering. These grits were as
effectively converted or extracted during mashing com-
pared to refined maize grits. Interestingly, the resulting
mash of regular sorghum grits had a lower filtration rate
during the critical step of lautering. Barredo-Moguel et al.
[7] further studied the substrate and product profiles of
Lager worts and beers produced from waxy sorghum and
barley malt. Results demonstrated that the kinetic profiles
of worts produced from waxy sorghum grits were compa-
rable to the control wort produced from maize grits and
barley malt. Worts produced from waxy sorghum grits had
comparable pH, reducing sugars and number of yeast cells.
Production of ethanol followed the same trend during
144 h of fermentation. Thus, the refined waxy sorghum
grits were suitable brewing adjuncts for production of
Lagers. The same authors [8] also determined the levels of
free amino nitrogen (FAN) and fusel alcohols during fer-
mentations of lager worts produced from waxy sorghum
grits. Worts produced from waxy sorghum grits had com-
parable FAN compared to the commercial wort. The uti-
lization of FAN for production of propanol, isobutanol and
amyl-isoamyl alcohols from waxy sorghum grits was also
comparable to the control wort. Isobutanol was produced in
the lowest amount. The final concentrations of ethanol and
fusel alcohols were within the expected range found in
commercial beers. These studies clearly indicated that
worts produced from barley malt and waxy sorghum grits
were an adequate substrate for Saccharomyces cerevisiae
and were comparable to commercial wort and beer.
Several research papers documented the production of
lager beers from gluten-free adjuncts including sorghum
grits and diastatic sorghum malt. These investigations
clearly documented that sorghum malts are deficient in
total diastatic activity particularly in b-amylase activity
which is critical in converting dextrins into fer-
mentable carbohydrates. Therefore, sorghum malts yielded
lower extracts and beer when compared to mashes and
beers produced from barley malt. The addition of extrinsic
b-amylase or microbial amyloglucosidase during mashing
of sorghum worts significantly increased extract and starch
conversion and favored the ratio between fermentable sug-
ars and dextrins. Consequently, the most favorable wort
composition increased the alcohol content and yield of the
gluten-free beers. Thus, the best technology to produce
gluten-free sorghum beers is with the use of white sorghum
genotypes adequate for the production of malt and refined
grits supplemented with amyloglucosidase during the crit-
ical step of mashing [16–18, 22–24, 57, 76]. The main
problems when brewing with sorghum are the lower dia-
static power of its malt, especially deficient in b-amylase
activity, and the comparatively higher gelatinization tem-
perature of sorghum starch compared with barley starch
[59, 68]. Compared with barley malt, mashing with sor-
ghum requires an adjusted procedure during the tempera-
ture ramping to increase the solubility and hydrolysis of the
starch by the amylolytic enzymes [50, 51, 55].
As with all cereal grains, starch is the major component
of sorghum kernels. On a weight basis, 50–75 % of the
sorghum grain is starch. Refined sorghum grits generally
contain about 5 % more starch than their respective whole
kernel. Gelatinization temperatures of sorghum starch have
been reported to vary from 71 to 80 �C, with starch isolated
from corneous endosperm having a higher gelatinization
temperature than that from the floury endosperm. Corneous
endosperm starch also imparts a higher intrinsic viscosity,
and lower iodine-binding activity than that of the floury
endosperm [64].
Starch from normal kernels contains 23–30 % amylose,
whereas starch from waxy endosperm types has less than
5 % amylose. Waxy sorghum starch differs in its properties
when compared to normal starch and has higher vis-
coamylograph peak viscosity as well as water-binding
capacity [69]. Interestingly, the structure and morphology
of the peripheral endosperm of waxy sorghums contain a
weaker protein matrix that favors starch hydrolysis during
mashing. In fact, waxy sorghum brewing adjuncts had
similar extraction and filtration rates during lautering
compared to refined maize grits [58]. The digestibility of
waxy sorghum starch is also reported to be higher than that
of normal sorghum starch [63].
In sorghum, recent studies based on improved extraction
procedures show that the prolamins or kafirins account for
roughly 70–90 % of the total grain protein. Kafirins have
been subclassed into a, b and c based on their solubility,
structure and amino acid sequence. The major kafirin is the
a subclass which represents about 65–85 % of the total
kafirins, while the b and c subclasses account for
Food Eng Rev (2016) 8:468–482 475
123
approximately 7–8 and 9–12 % of the prolamins, respec-
tively. The protein bodies of sorghum are highly resistant
to enzymatic digestion and to disruption by processing
such as extrusion. It is currently thought that b- and c-
kafirins form a highly cross-linked shell around the more
easily digested a-kafirins [36].
One important feature of sorghum is that its protein
digestibility decreases upon cooking, apparently through
the formation of more protein cross-links during the ther-
mal process [20]. In agreement with this finding, Hamaker
and Bugusu [35] observed by laser scanning confocal
microscopy that cooking causes sorghum proteins to form
extended, web- and sheet-like structures. Both formation of
oligomers and formation of web-like protein structures
occurred to a lesser extent in maize [20].
In a previous research, Espinosa-Ramirez et al. [23, 24]
successfully produced lager beers from different types of
sorghum malts and adjuncts, supplemented with b-amylase
or amyloglucosidase. After conducting the different treat-
ments, the best 100 % sorghum beer yields were obtained
in treatments produced with decorticated waxy adjuncts
and amyloglucosidase, regardless of the sorghum malt
used. This is because white regular and waxy sorghum
varieties with intermediate endosperm texture yieldedhigher amounts of refined grits and fermentable carbohy-
drates. The waxy grits also produced worts with enhanced
lautering or filtration performance. Amyloglucosidase
addition during mashing proved to be an excellent option to
produce beers with a low content of dextrins, relatively
higher ethanol content, and therefore, it was an excellent
alternative to manufacture gluten-free beers. Sorghum
lager beers generally have a different flavor profile com-
pared with barley beer counterparts. This is due to changes
in availability of fermentable sugars in the wort, amount
and composition of peptides conforming the free amino
nitrogen (FAN) ethanol and fusel alcohol concentrations
and flavor and aromatic compounds generated during fer-
mentation and during beer maturation [7, 8, 18, 22–24, 54,
76].
Maize
The overall starch content and starch composition of maize
are similar to those of sorghum. Both maize and sorghum
contain waxy genotypes [68]. However, only maize contain
high-amylose genotypes with 50–80 % amylose. The rel-
atively higher gelatinization temperature of maize starch
indicates that it cannot be converted between 63 and 67 �C,
like the starches of wheat and barley, but must be heated to
temperatures which approach or exceed 100 �C in order to
ensure proper endosperm disruption and starch gelatiniza-
tion [40]. In addition, maize must be invariably processed
to remove the oil-laden germ and the high fiber bran in
order to produce good-quality brewing adjuncts [51].
However, since the gelatinization temperatures of maize
starches are reported to be high, it is likely that, as with
sorghum malts, improved extract recovery would be
obtained if modified mashing regimens are used [38].
Singh and Bains [71] recommend a malting regime
whereby the grains are pre-dried for 12 h at 36 �C before
steeping to 40 % moisture at 25 �C, germinated for 168 h
at 25 �C and finally kilned at 45 �C for 24 h. Therefore, as
with sorghum, maize needs to be malted ‘‘wet and warm,’’
resulting in the extreme likelihood of mold infestation.
This could be prevented by soaking kernels in the presence
of chlorine or other approved antimicrobial disinfectants.
Rice
Rice is widely used for the production of alcoholic bev-
erages such as sake in Japan, Shoshinshu in China and
miscellaneous alcoholic drinks in southeastern Asia [82].
In addition, rice is used as a popular brewing adjunct in
the production of lager beers [15]. Unfortunately, the
potential of rice as a source of diastatic malt is very
limited. Agu et al. [1] examined the performance of rice
when malted under various temperature conditions and
lengths of time and concluded that it adequately germi-
nated at 20, 25 and 30 �C. However, the rice malt had
lower a-amylase activity when compared to buckwheat.
Likewise, early studies suggested that beer can be brewed
from malted rice [4, 47, 56]. In these trials, the Califor-
nian paddy rice was steeped for 48–60 h at 15 �C, ger-
minated for 72 h at 17.8–18.9 �C and finally kilned for
48 h, with temperatures rising from 32.2 to 65.5 �C.
These low temperatures were used to prevent vitrification
of the kernel. However, the resulting malt was poor in
diastatic activity which resulted in deficient modification
of the starchy endosperm and expensive to produce.
Okafor and Iwouno [56] reported that malted rice is not
satisfactory as a brewing material as malting losses are
often quite high. When rice malts are finely ground and
conventionally mashed, saccharification is incomplete and
wort runoff is slow. In addition, rice malt was found to be
bitter [47]. The incongruence of the studies on rice indi-
cates that there is a strong need to optimize the mashing
regimes in order to increase the extract recoveries and
improve flavor.
Nonetheless, in contrast to unmalted rice, liquefaction in
the cooker was faster and occurred at a comparatively
lower temperature. Furthermore, the utilization of both rice
malt and refined brewing grits were a good choice because
of the high extract yield and advantages in terms of beer
flavor and aroma [10].
476 Food Eng Rev (2016) 8:468–482
123
Production of Gluten-Free Beers with Non-grain
Sources
Beers derived from fermentable sugars and non-grain-
derived materials are also becoming important in some
places around the globe. According to Hager et al. [33],
several Japanese products are based on fermented sugar
syrups and proteins from peas (Pisum sativum) or soybeans
(Glycine max), with yeast extract as source of amino acids,
hops for flavoring and caramel for coloring.
A patent by Scott et al. [67] claimed the production of
gluten-free beers from a liquid mixture which includes a
base of filtered water and two sources of fermentable sug-
ars: honey and molasses. The liquid mixture also includes a
bittering agent, obtained from a plurality of hops (Cen-
tennial, Perle, Saaz and Hallertauer hops). Finally, the
protein coagulant, yeast nutrient, and a plurality of yeast
cells are added to the liquid mixture. Figure 3 depicts the
flowchart of the production of gluten-free beer using these
ingredients and technology.
Producing Gluten-Free Beer with Enzymatic
and Microbial Treatments
Enzymatic treatments have been effective on protein
hydrolysis in the medical industry for potential celiac
disease oral treatments. Recent research has shown that
different proline-specific endopeptidases can be used for
gluten degradation [32], not only in oral therapy but also
for the elimination of gluten in raw materials and foods
[81].
Germinated cereal grains as well as bacteria and fungi
are known to be suitable sources for proline-specific pep-
tidases. A common enzyme used in these treatments is
prolyl endoproteinase (PEP), which is commonly derived
from Aspergillus niger.
In an attempt to produce gluten-free beer, Guerdrum and
Bamforth [32] conducted a study to quantify at what point
in the brewing process the gluten is lost when supple-
menting PEP. Approximately half of the prolamin was lost
during mashing and lautering, presumably by proteolysis or
precipitation. It is noteworthy that much more protein was
lost in sweet wort production than at the boiling stage,
which suggests that protein removal was much more
important during mashing/lautering. Most of the gluten
proteins remain associated with the brewer’s spent grains
after lautering. After fermentation, only 1.9 % of the
original hordein from the malt remained in the unfiltered
beer. It should also be noted that this beer was not cold-
stabilized, during which, presumably, more protein could
have been removed. The brewing and fermentation pro-
cesses serve to eliminate most of the detectable prolamin
Fig. 3 Production of gluten-free beer using non-grain sources [67]
Fig. 4 Production of gluten-free beer from barley supplemented with
prolyl endoproteinases [32]
Food Eng Rev (2016) 8:468–482 477
123
from beer. Figure 4 depicts the fate of the barley prolamins
during the production of beer supplemented with PEP. The
key stages appear to be mashing and yeast fermentation.
These authors concluded that the prolamin in the beer after
fermentation represented less than 2 % of the total pro-
lamin in the malt. The exogenous enzyme prolyl endo-
proteinase added in fermentation or to the finished product
renders beers essentially free of gluten and without nega-
tively impacting foaming capacity.
Analysis of Gluten Epitopes in Beer
In 2004, the Food Allergen Labeling and Consumer Pro-
tection Act became law and went into effect in 2006. It
required manufacturers to label products with any of the
‘‘Big Eight’’ allergens—milk, eggs, fish, shellfish, tree
nuts, peanuts, soybeans and wheat.
FDA allowed manufacturers to begin labeling food
gluten-free in 1993 without definitional consensus about
what it meant to be ‘‘free’’ from gluten [19]. FALCPA
required that the definition of ‘‘gluten-free’’ be set by 2006
and implementationof the regulation governing gluten be
adopted by 2008.
For a product to be considered gluten-free, the current
Codex Alimentarius dictates a maximum presence of
20 mg/kg [14]. Current proposed FDA legislation dictates
that, for a product to be labeled as being suitable for celiac
sufferers, it cannot be made from cereals such as barley and
wheat [77]. Clearly, PEP-treated beers might fulfill the
other criterion of not containing gluten at more than
20 ppm, but the very use of a grist founded on a ‘‘pro-
hibited grain’’ would exclude any barley- or wheat-based
product from qualifying as being gluten-free [32].
In recent times, gluten-free foods and beverages have
attracted much research interest motivated by the growing
market. Therefore, it is necessary to effectively detect the
peptide sequences that are immunotoxic in very small
amounts to celiacs [65]. Brewers who manufacture gluten-
free beers are required to test every batch for gluten and
record levels in ppm. Compared to wheat bakery products,
the analysis of gluten-hordein epitopes in beer is more
complicated because the final alcoholic product resulted
from mashes that are extensively hydrolyzed by proteolitic
enzymes present in the malt during mashing and yeast
during fermentation. Tanner [74] concluded that the
employment of PEP may confound measurement of gluten
concentration in food and beverages by destroying epitopes
that are used to enumerate gluten peptides. The same
author designed an experiment to find out whether the PEP
treatment destroyed celiac reactive epitopes or merely
disguised them from the official ELISA test and concluded
that there is sufficient data to show that treatment of gluten
peptides with bacterial PEP in combination with other
proteolytic enzymes diminished the immunoreactivity of
gluten peptides estimated after determining celiac T cells.
PEP treatment also accompanied the destruction of key
epitopes that are used by antibodies to enumerate gluten
peptides during ELISA reactions. Thus, both immunore-
activity and ELISA measurements are reduced to near zero
after PEP treatment. It is known that the sandwich omega-
gliadin ELISA test severely underestimates gluten from
barley. This test has a cross-reactivity of only 4–8 %. On
the other hand, there is an assay available for testing
hydrolyzed products such as beer. The test is named
competitive R5 ELISA and measures gluten peptides
instead of total gluten.
Guerdrum and Bamforth [32] monitored levels of pro-
lamins throughout the brewing process with the
RIDASCREEN Gliadin competitive R5 enzyme-linked
immunosorbent assay method and found that the barley
malt contained 6832.3 ± 61 mg/kg and beer only
131.1 ± 1 mg/kg of prolamin. Addition of PEP lowered
the level of prolamins further to concentrations below the
reliable limit of detection (\20 mg/kg). Interestingly, the
foam stability of the barley malt beer treated with PEP was
not affected.
Future Trends in the Production of Gluten-Free
Beers
Genetic Modification of Gluten-Containing Kernels
Recent investigations document that it is feasible to
genetically engineer barley, wheat and other cereals that
trigger celiac disease. The use of genetic engineering to
down-regulate gene expression is an attractive opportunity
to reduce the immunotoxic components of gluten and
therefore the incidence of gluten-related allergies related to
beer or consumption of bakery products. The structural
complexity and polymorphism of gliadin or hordein pro-
teins make it difficult to identify varieties naturally devoid
of toxicity or to develop such varieties through breeding.
Simultaneous silencing of the full complement of gliadins
results in effective reduction of the epitopes recognized by
T cells in celiac disease, which shows that it is possible to
eliminate or reduce the toxicity of wheat [65].
Gil-Humanes et al. [29] used RNAi to down-regulate the
expression of gliadins in bread wheat. A set of hairpin
constructs were designed and expressed in the endosperm.
The expression of gliadins was strongly down-regulated in
the transgenic lines. Total gluten protein was extracted
from transgenic lines and tested for ability to stimulate four
different T-cell clones derived from the intestinal lesion of
CD patients and specific for the DQ2-a-II, DQ2-c-VII,
478 Food Eng Rev (2016) 8:468–482
123
DQ8-a-I and DQ8-c-I epitopes. For five of the transgenic
lines, there was a 1.5–2 log reduction in the amount of the
DQ2-a-II and DQ2-c-VII epitopes and at least 1 log
reduction in the amount of the DQ8-a-I and DQ8-c-I epi-
topes. Furthermore, transgenic lines were also tested with
two T-cell lines that are reactive with x-gliadin epitopes.
The total gluten extracts were unable to elicit T-cell
responses for three of the transgenic wheat lines, and there
were reduced responses for six of the transgenic lines. This
work shows that the down-regulation of gliadins by RNAi
can be used to obtain wheat lines with very low levels of
toxicity for celiacs.
A significant amount of genetic engineering research has
been also conducted on barley grain, aimed at improving
the qualities for malting, brewing and distilling. Research
conducted at the University of Washington has eliminated
most of the gluten proteins from wheat and barley kernels
which cause immunological and allergic reactions in sus-
ceptible individuals. Needless to say, the short-term goal is
to completely remove all major immunoreactive gluten
proteins. The key for the production of these genetically
engineered kernels is the silencing of two regulatory genes
that disable the enzyme responsible for the development of
gluten proteins. The researchers have successfully elimi-
nated all major pathogenic gluten proteins from wheat
grains while retaining key high-molecular-weight glutenins
needed for bread baking. Likewise, Wen et al. [79] sup-
pressed the transcription of wheat or barley DEMETER
(DME) homeologs using RNA interference. DME encodes
a 5-methylcytosine DNA glycosylase responsible for
transcriptional derepression of gliadins or hordeins and
low-molecular-weight glutenins (LMWgs) by active
demethylation of their promoters in the endosperm. These
proteins are considered the major source of immunogenic
epitopes. The epigenetic influence of DME silencing on
accumulation of LMWgs and gliadins was determined
using 20 transformants expressing hairpin RNA in their
endosperm. These transformants showed up to 85.6 %
suppression in DME transcript abundance and up to 76.4 %
reduction in the amount of immunogenic prolamins,
demonstrating the possibility of developing wheat or barley
varieties compatible for the celiac patients. With these
advances, it will be feasible to produce straw-colored
wheat-based beers known as Weizenbier.
Recently, Tanner et al. [73] created the first ultra-low
gluten barley for celiac and gluten-intolerant people in
which the hordein content was reduced to below 5 ppm.
This was achieved using traditional breeding strategies to
combine three recessive alleles, which act independently of
each other to lower the hordein content in the parental
lines. However, the grain of the initial variety was shrun-
ken compared with wild-type counterparts but were suc-
cessfully malted and brewed.
Genetic Modification of Yeast and Other
Microorganisms
There are natural and recently developed recombinant
microorganisms capable of expressing and producing PEP
which hydrolyzes prolamin epitopes that triggers celiac
disease. This particular enzyme has been proposed to
prevent or reduce beer haze [21].
Gass et al. [28] characterized the in vitro gluten
detoxifying properties of a therapeutically PEP from
Myxococcus xanthus (MX PEP). In addition, the authors
developed an enteric-coated capsule containing a phar-
macologically useful dose of this enzyme. A high-cell
density fed-batch fermentation process was developed for
overproduction of recombinantMX PEP in Escherichia
coli, yielding 0.25–0.4 g/L purified protein. A simple,
scalable purification and lyophilization procedure was
established that yielded[95 % pure of highly active and
stable enzyme that was further blended with excipients
and encapsulated in a hard gelatin capsule. Capsules
were enteric coated using Eudragit L30-D55 polymer
coat, which provided sufficient resistance to gastric
conditions and rapid release under duodenal conditions at
pH 6.0 in the presence of pancreatic enzymes trypsin and
chymotrypsin. In conjunction with these enzymes, MX
PEP broke down whole gluten into a product mixture
that was virtually indistinguishable from that generated
by the Flavobacterium meningosepticum. Competitive
studies involving selected immunogenic peptides mixed
with whole gluten revealed that the capsules were
effective for celiac sprue patients.
Kang et al. [42] expressed a novel endoprotease Endo-
Pro-Aspergillus niger at high level in the methylotrophic
yeast Pichia pastoris. The endoprotease is 95 % identity
with proline-specific endopeptidase from Aspergillus
niger CBS513.88 while sharing low identity with those
from other microorganisms. The purified enzyme was a
monomer of 60 kDa. A three-dimensional model
revealed that the active site of the enzyme was located in
Ser(179)–Asp(458)–His(491), based on template 3n2zB with
sequence identity of 17.6 %. The optimum pH and
temperature of the endoprotease were pH 4–5 and 35 �C,
and the stabilities were from pH 3 to 7 and in the tem-
perature range of 15–60 �C. Furthermore, the endopro-
tease had the ability to digest peptides with the
C-terminal of proline as well as alanine and was also
capable of hydrolyzing larger peptides. The properties of
the endoprotease make it a highly promising candidate
for future application in the field of brewing. The same
authors [43, 44] successfully produced a recombinant
Pichia pastoris with significant activity of PEP obtained
from Aspergillus. niger. Amino acid sequence analysis
of showed that this enzyme belongs to a class serine
Food Eng Rev (2016) 8:468–482 479
123
peptide S28 family. It was successfully expressed using
expression vector pPIC9 K with high activity
(620 ± 25 U/l). The recombinant optimized PEP could
work in an extremely broad temperature range and over
40 % relative activity were remained in the temperature
range of 15–70 �C. The optimal pH value and tempera-
ture for activity were 4.0–5.0 and 35–40 �C, respec-
tively. The enzyme was activated and stabilized by metal
ion Ca2? and inhibited by Zn2?, Mn2?, Al3? and Cu2?.
A fed-batch strategy was first developed for high cell
density fermentation and the enzyme activity reached
1890 U/l after cultivation in a pilot plant reactor. The
broad temperature range and efficient expression made
this enzyme possible to apply in the beer industry
directly. This study also investigated the positive effect
of PEP on removing the beer haze protein. However, the
potential use of PEP to reduce peptides that cause celiac
disease is not discussed in these investigations.
Likewise, Kanatani et al. [41] identified and studied
an Aeromonas hydrophila strain which showed good PEP
activity. The enzyme gene was cloned and expressed
in Escherichia coli JM83. A 12-kbp EcoRI fragment
containing the enzyme gene was subcloned at the HincII
site of pUC19 to construct plasmid pAPEP-3 with a 3.5-
kbp insert. E. coli JM83 transformed with this plasmid
showed about 100-fold higher activity than the par-
ent Aeromonas. Analysis of the nucleotide sequence of
the insert revealed that the mature enzyme-encoding
sequence starts just after the ATG initiation codon of the
open reading frame. The enzyme was a single polypep-
tide composed of 689 amino acid residues with a
molecular weight of 76,383. It showed properties very
similar to those of Flavobacterium PEP, except that the
isoelectric point was 5.5. The amino acid sequence was
56 and 41 % homologous to those of Flavobacterium
and porcine brain PEP, respectively. From a survey of
sequence homology with other members of the PEP
family, the amino acid residues involved in the catalytic
triad were deduced to be Ser-537, His-656 and Asp-512
(or Asp-621). More research is needed in order to assess
the use of recombinant yeast with regard to consumer
acceptance of beers.
The previous investigations clearly show that there is
potential to develop gluten-free barley for malting and
wheat as a source of both malt and brewing adjuncts and/or
recombinant yeast or other related microorganisms capable
of hydrolyzing epitopes remaining in the beer after fer-
mentation. More research is needed in order to assess
whether these genetic engineered cereals or recombinant
microorganisms affect yeast performance, ethanol and
fusel alcohol concentrations and the organoleptic proper-
ties of ale and lager beers.
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	Technological and Engineering Trends for Production of Gluten-Free Beers
	Abstract
	Introduction
	Celiac Disease
	Gluten-Free Food Market
	Beer Consumption
	Production of Gluten-Free Beers
	Producing Gluten-Free Beer with Non-gluten Grains
	Sorghum
	Maize
	Rice
	Production of Gluten-Free Beers with Non-grain Sources
	Producing Gluten-Free Beer with Enzymatic and Microbial Treatments
	Analysis of Gluten Epitopes in Beer
	Future Trends in the Production of Gluten-Free Beers
	Genetic Modification of Gluten-Containing Kernels
	Genetic Modification of Yeast and Other Microorganisms
	References