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Fermentation with Lactobacillus strains for elimination of gluten in wheat (Triticum Aestivum) by-products

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Recently there is an increase in the number of consumers with gluten intolerance that causes expanding of the demand for gluten-free products. Gluten-free diet is unbalanced and usually has a higher percentage of calories from fat, less of carbohydrates, as well as low intake of non-starch polysaccharides. To solve this problem, new strategies are looked for to eliminate immunogenicity of gluten in products of wheat and other cereals and to make them more balanced. Fermentation with lactic cultures and/or enzymes enables to reduce the gluten content in wheat flour. However, this process takes a long time, is complicated to control, and hydrolysed gluten looses its technological properties. The purpose of this work is to find another way of removing gluten residues: at first remove gluten from wheat by wet fractionation, then hydrolyse gluten residues in the remaining fractions by using biotechnological measures. The fractions of starch, fibers and bran had an initial gluten concentration of 85-33750 mg kg-1. For eliminating of gluten residues they were fermented with four probiotic strains separately: Lactobacillus plantarum P-1, Lactobacillus brevis R-1, Lactobacillus acidophilus 308, Lactobacillus acidophilus 336. Short (12 hours) and long fermentation (24 hours) at 30 and 37°C was used. Gluten was degraded in wheat starch to below 20 mg kg-1 using Lactobacillus plantarum in short time, other strains performed better using long fermentation. In conclusion, it could be stated that sourdough-based biotechnology could eliminate the immunogenicity of wheat by-products and to improve the quality of life of celiac patients.
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FOODBALT 2019 and NEEFood 2019
FERMENTATION WITH
LACTOBACILLUS
STRAINS FOR
ELIMINATION OF GLUTEN IN WHEAT
(TRITICUM AESTIVUM)
BY-
PRODUCTS
Vijole Bradauskiene1,3, Lina Vaiciulyte-Funk1, Edita Mazoniene2, Darius Cernauskas1
1Food Institute, Kaunas University of Technology, Radvilenu road 19, Kaunas, Lithuania
2Polymer Science and Technology department, Kaunas University of Technology, Radvilenu road 19, Kaunas, Lithuania
3Food Technology department, Faculty of Technology, Klaipeda State University of Applied Sciences, Bijunu street 10,
Klaipeda, Lithuania
Abstract
Recently there is an increase in the number of consumers with gluten intolerance that causes expanding of the demand for gluten-free
products. Gluten-free diet is unbalanced and usually has a higher percentage of calories from fat, less of carbohydrates, as well as low
intake of non-starch polysaccharides. To solve this problem, new strategies are looked for to eliminate immunogenicity of gluten in
products of wheat and other cereals and to make them more balanced. Fermentation with lactic cultures and/or enzymes enables to
reduce the gluten content in wheat flour. However, this process takes a long time, is complicated to control, and hydrolysed gluten
looses its technological properties. The purpose of this work is to find another way of removing gluten residues: at first remove
gluten from wheat by wet fractionation, then hydrolyse gluten residues in the remaining fractions by using biotechnological
measures. The fractions of starch, fibers and bran had an initial gluten concentration of 85-33750 mg kg-1. For eliminating of gluten
residues they were fermented with four probiotic strains separately: Lactobacillus plantarum P-1, Lactobacillus brevis R-1,
Lactobacillus acidophilus 308, Lactobacillus acidophilus 336. Short (12 hours) and long fermentation (24 hours) at 30 and 37°C was
used. Gluten was degraded in wheat starch to below 20 mg kg-1 using Lactobacillus plantarum in short time, other strains performed
better using long fermentation. In conclusion, it could be stated that sourdough-based biotechnology could eliminate the
immunogenicity of wheat by-products and to improve the quality of life of celiac patients.
Keywords: Wheat, Gluten, Hydrolysis, Sourdough, Lactobacillus
Introduction
Wheat is one of the most popular cereals in the world,
however, gluten proteins of wheat are responsible for
very common allergic reactions in populations, leading
to immune disorder and non-celiac gluten sensitivity
(Gujral et al., 2012; Kang et al., 2013; Catassi et al.,
2014). Currently, the only therapy is a strict, lifelong
gluten-free diet (GFD). Compliance with a GFD is an
extremely challenging task, given a number of
problems related to poor quality of gluten-free products
compared to their gluten-rich counterpart (Do
Nascimento et al., 2017) as well as these products are
more expensive (Stevens, Rashid, 2008). Patients with
celiac disease are looking for alternatives and are using
products from gluten free materials such as corn, rice,
millet, buckwheat, amaranths and potatoes. The diet of
these patients is unbalanced and had a higher
percentage of calories from fat and less from
carbohydrates, also in GFD was obtained low intakes
of non-starch polysaccharides (Thompson et al., 2005;
Wild et al., 2010). Products made from naturally
gluten-free raw materials resulted in breads often have
inferior textural and sensory properties compared to the
corresponding gluten-containing products (Hager et al.,
2012; Miranda et al., 2014; Pellegrini, Agostoni, 2015).
To resolve this socioeconomic problem, new strategies
are looking for to eliminate harmful gluten from wheat
and other cereals and to produce balanced products
with good sensory properties (Greco et al., 2011;
Nionelli, Rizzello, 2016).
Wheat gluten fragments (peptides) remain intact during
digestion. They penetrate through the small intestine
wall and initiate antigenic cellular immune responses.
There is no immune response if the gluten hydrolysed
to peptides, which contain less than nine amino acid
residues. Research on the use of biological measures in
wheat products to eliminate or reduce the immune
toxicity of gluten proteins is being actively pursued in
the last decade. Numerous studies (Di Cagno et al.,
2008; Giuliani et al., 2016; Gerez et al., 2012; Loponen
et al., 2007; Romano, Urminská, 2017; De Palma et
al., 2010; Stefanska et al., 2016) were carried out using
lactic cultures - their individual strains or various
combinations. They focus on probiotic strains
possibilities to decrease the toxicity of wheat flour, but
there is a lack of informationon on biological measures
to completely eliminate gluten from wheat processing
products.
Sourdough fermentation with lactic acid bacteria
(LAB) can improve the texture, palatability, aroma,
shelf life and nutritional value of wheat breads
(Guerzoni et al., 2011), texture and palatability of
whole grain, fibre-rich or gluten-free products, stabilise
or increase levels of various bioactive compounds,
retard starch bioavailability and improve mineral
bioavailability (Katina et al., 2005; Moroni et al.,
2009). LAB degrade celiac active gluten peptides,
because some species of LAB produce specific
peptidases during growth, which are capable to
hydrolyse hardly cleavable bonds between amino acids
in proline-rich peptides (Vukotić et al., 2016).
Selecting strains of LAB with targeted proteolytic
effects is vital important (Stefańska et al, 2016).
The use of sourdough LAB was at first proposed with
the aim of eliminating traces of gluten epitopes in
2002. Di Cagno et al. (2002) showed that selected
LAB, possessing proteolytic activities, could efficiently
hydrolyse the toxic peptides of gliadin in wheat
sourdough. The pool of L. alimentarius 15M, L. brevis
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14G, L. sanfranciscensis 7A, and L. hilgardii 51B has a
pattern of specialized peptidases capable of
hydrolysing different peptide bonds that potentially
include the proline (Di Cagno et al., 2004). Study of Di
Cagno et al. (2008) highlighted the use of selected
LAB consisted of Lactobacillus sanfranciscensis LS40
and LS41 and Lactobacillus plantarum CF1 to
eliminate risks of contamination by gluten and to
enhance the nutritional properties of GF bread.
Several studies were carried out using individual
strains of LAB also. Sourdough fermentation using
Lactobacillus sanfranciscensis (Thiele et al, 2004;
Vermeulen et al, 2006) or Lactobacillus plantarum
(Yin et al, 2015) showed a decrease in pH and resulted
hydrolysis and solubilization of wheat proteins.
De Angelis et al. (2006) showed the capacity of
probiotic VSL#3 preparation to hydrolyse extensively
wheat flour. Probiotic product VSL#3 including
Streptococcus thermophilus, L. plantarum, L.
acidophilus, L. casei, L. delbrueckii spp. bulgaricus,
Bifidobacterium breve, B. longum and B. infantis
strains was used in the fermentation of a mass with
wheat flour in order to hydrolyse gliadin peptides and
promoted almost complete hydrolysis of gliadin
peptides. Patent Application WO2006/097415 (2006)
describes a process for gluten degradation by means of
the use of a complex mixture consisting of at least six
lactic acid bacteria and/or bifidobacteria and long
fermentation times (24-31 hours) also. After hydrolysis
some gliadins are partially hydrolysed, but others are
not susceptible to hydrolysis process. Lactobacillus
plantarum CRL 775 and Pediococcus pentosaceus CRL
792 also hydrolysed gliadins during wheat dough
fermentation (Gerez et al, 2012).
Romanová, Urminská, 2017 described growth
characteristics and intracellular aminopeptidases
activities of Lactobacillus plantarum CCM 3627 and
Lactobacillus brevis CCM 1815. The results confirm
production of active proline aminopeptidase, which is
important for cleavage of proline rich-peptides. Two
strains: Enterococcus mundtii and Wickerhamomyces
anomalus exhibited the potential to be used as
probiotic for sourdough fermentation: they have shown
the ability to tolerate low pH, bile salt properties and
hydrophobicity compared to other gluten-degrading
yeast and bacterial strains (Sakandar et al., 2018).
Stefańska et al. (2016) have selected 11 LAB strains
capable of hydrolysing gliadin in bakery sourdoughs.
However, in all sourdoughs were found some
polypeptides with IgE‐reactive epitopes. Previous
research has shown that fermentation with LAB
reduces the amount of reactive gluten fragments, but
does not reach the safe limit for gluten free products,
which is 20 mg kg-1 (Standard 118-1979, 2015).
Fermentation with mixtures of selected lactic acid
cultures in combination with fungal enzymes enable to
reduce the gluten content in wheat flour to gluten free
limit (Rizzello et al., 2007; 2014). However, this
process takes a long time, it is necessary to control it in
several stages, but hydrolysed gluten still loses its
technological properties: enzymatic hydrolysis destroys
the gluten network, reduces the elasticity of the dough
and baked goods (Van Den Broeck et al., 2009).
In this work, it would be advisable to combine physical
and biotechnological measures for the preparation of
raw materials for the production of gluten-free wheat
products: at first remove gluten from wheat by wet
fractionation, then hydrolyse gluten residues in the
remaining fractions by using LAB fermentation.
Whereas the levels of gluten are low in the wheat by-
products, it can be expected that the effect of LAB will
be sufficient to eliminate it.
Wet fractionation of wheat could be done by
centrifuging of the flour-water mixture in the
laboratory according to Czuchajowska, Pomeranz,
1993, as well as at industrial plants producing gluten
and starch from wheat that are widely used in the food
industry, meanwhile the fractions of fibers and bran are
diverted to feed production.
Eliminating of gluten residue in wheat processing
products allows produce gluten free starch and gluten
free fraction of arabinoxylan and other non-starch
polysaccharides, suitables for flour mixtures or bakery
production for users intolerant to gluten or celiac
sufferers.
The aim of the research - to use fermentation with
Lactobacillus strains for elimination of the
immunogenicity of gluten in wheat by-products.
Materials and Methods
Materials
Investigations were carried out at the Kaunas
University of Technology, Food Institute, Lithuania.
Samples of wheat fractions after dry and wet
fractionation: starch, fibers and bran were provided by
Roquette Amilina, AB, Lithuania.
A fraction of wheat bran is obtained as a by-product
during the dry milling of wheat grain and is composed
of outer layers of wheat kernel, mainly pericarp. A
fraction of fibers is obtained as a by-product in the wet
processing of the flour for starch and gluten separation,
and is composed mainly of seed coat and aleurone
residues.
Physical-chemical analysis of composition of wheat by-
products
Moisture content of wheat by-products was measured
by humidity measuring device Kern MLS 50-3HA
160N;
pH was measured by pH-meter ORION 3STAR;
Determination of protein content by Kjeldahl method
(LST EN ISO 20483).
Research on the selection of Lactobacillus strains
Research to removal of the immunogenicity of wheat
was carried out experimenting with microorganisms of
the collection from the KTU Food Institute. Four
probiotic strains: Lactobacillus plantarum P-1,
Lactobacillus brevis R-1, Lactobacillus acidophilus
308, Lactobacillus acidophilus 336 were used.
The LAB cultures were stored for the study at -72-
74°C in the VIABANK (MWE medical wire) system.
Cultures revived in MRS broth (Biolife, Italy): an
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initial LAB suspension was prepared by seeding of the
initial culture on a MRS agar (Biolife, Italy) and
incubating at 30°C (L. plantarum P-1, L. brevis R-1)
and at 37°C (L. acidophilus 308, L. acidophilus 336)
for 24h. Each LAB culture was transformed then into
sterile milk and incubated at an appropriate
temperature for 72h under anaerobic conditions.
The number of lactic acid bacteria was determined by
the method of seeding in Petri dishes by incubation on
a MRS agar under anaerobic conditions for 72 h at 30
or 3C.
Total plate count of samples performed according to
standard procedures LST EN ISO 4833:2003, found
2,1 *102 CFU / g, count of of yeast and mold 1.2 * 101
cfu / g, aerobic and anaerobic spore forming bacteria is
not found. The amounts of these microorganisms in the
samples are small and the samples are suitable for
consumption and biotechnological work without
sterilization from the microbiological safety point of
view.
Wheat starch or fiber samples (5-10 g) was weighed in
into glass tubes (30 mL), distilled water (10 mL) and
LAB suspension (7 mL) at an active concentration of
1,7-2,8*106 was added (see Table 1) and mixed well.
Table 1
Preparing of samples of wheat product with
different LAB strains
Sample
Code
Wheat product
LAB strains
Active
concen-
tration
Starch
Fibers
g
g
CFU/g
SLA1
5
-
L. acidophilus 308
2,0*106
SLA2
5
-
L. acidophilus 336
2,1*106
SLB
5
-
L.brevis R-1
1,7*106
SLP
5
-
L.plantarum P-1
2,8*106
FLA1
-
10
L. acidophilus 308
2,0*106
FLA2
-
10
L. acidophilus 336
2,1*106
FLB
-
10
L.brevis R-1
1,7*106
FLP
-
10
L.plantarum P-1
2,8*106
Samples were incubated at 30 and 37°C, pH and gluten
content in sourdough were measured after 12 and 24
hours.
Gluten quantitation by ELISA
Gluten residues in wheat products were quantitated by
competitive ELISA using G12 antibody AACCI 38-
52.01 (Romer Labs, UK Ltd) according to the
manufacturer’s instructions. Gluten concentrations
were established based on calibration function
provided by Romer Labs. Multiscan EX microplate
reader with a 450 nm filter was used for the reading the
strips.
Results and Discussion
The fractions of starch, fibers and bran had different
moisture content (10,78-72,01%), different amount of
total protein (0,32-16,9%) an initial gluten
concentration of 85-33750 mg kg-1 (see Table 2).
Table 2
Characteristics of samples
Wheat
by-
products
Total
protein
content
Gluten
content
%
mg kg-1
Fibers
Bran
Starch
3,80±0,02
16,90±0,02
0,32±0,02
7800,00±218,00
33750,00±945,00
85,00±2,00
For eliminating of gluten residues starch and fiber were
fermented with four probiotic strains separately:
Lactobacillus plantarum P-1, Lactobacillus brevis R-1,
Lactobacillus acidophilus 308, Lactobacillus
acidophilus 336. Short (12 hours) and long
fermentation (24 hours) at 30 and 37°C was used.
The reducing of gluten content depending on the
decreasing of pH was observed. Gerez et al. (2008),
Rollan et al. (2016) demonstrated also that protein
hydrolysis in sourdough were partially caused by pH-
dependent activation of cereal enzymes according to
change in proteolytic activity. Di Cagno et al. (2002)
proved also, that primary proteolysis is exerted by
wheat endogenous enzymes, which are activated by the
low pH. Among the selected LAB cultures, the lowest
pH was achieved by using L. acidophilus 308, at least
acidic sourdough was obtained with L.brevis R-1. The
pH of the fermentation of starch was also lower than
sourdough of fiber (see Figure1).
Figure 1. Decreasing of pH in different sourdough
LA1 L. acidophilus 308; LA2 L. acidophilus 336;
LB L. brevis R-1; LP L . plantarum P-16
Further hydrolysis of peptides were exerted by
intracellular peptidases of LAB in a strain-specific
manner: the type and amount of released amino acids
depend on the fermenting strain (Di Cagno et al.,
2002).
The selected strains exhibited different proteolytic
activity in this research, which leads to a reduction of
gluten content in wheat sourdoughs. The amount of
non-digestible gluten peptides decreased in all
fermented starch samples after 12h, but the most
pronounced proteolytic effect was observed in
3,62
3,45
3,77
3,64
3,72
3,56
3,77
3,56
4,65
4,44 4,39
3,82
4,6
4,35
4,39
3,79
3
3,2
3,4
3,6
3,8
4
4,2
4,4
4,6
4,8
After 12h
Starch After 24h
Sratch After 12h
Fiber After 24h
Fiber
PH
TIME
LA1
LA2
LB
LP
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sourdough with L. Plantarum P-1 (see Table 3).
Table 3
pH and gluten content in samples of wheat
starch after fermentation
Sample
Tempe-
rature
°C
Duration of fermentation, h
12
24
pH
Gluten
content
pH
Gluten
content
mg kg-1
mg kg-1
SLA1
37
3,62±0,02
25,00±0,60
3,45±0,02
12,00±0,30
SLA2
37
3,72±0,02
28,00±0,70
3,56±0,02
26,00±0,70
SLB
30
4,65±0,02
36,00±0,90
4,44±0,02
30,00±0,80
SLP
30
4,60±0,02
20,00±0,50
4,35±0,02
15,00±0,40
After 24 hours the lowest amount of immunoreactive
gluten peptides was found in sourdough with L.
acidophilus 308 and L. Plantarum P-1. Sourdough
fermentation decreases the disulphide bonds in gluten
network, which influence its digestibility in people with
gluten sensitivity (Gänzle et al, 2008). Although
fermentation of starch with L. acidophilus 336 most
reduced pH, failed to reduce the gluten content to 20
mg kg-1.
Fibers of wheat (after wet fractionation) had a high
initial content of gluten (up to 33750 mg kg-1). Despite
a significant decrease in pH after fermentation with
LAB the amount of gluten was reduced but remained
still high (see Table 4). Table 4
pH and gluten content in samples of wheat fibers
after fermentation
Sam-
ple
Tempe-
rature
Duration of fermentation, h
12
24
pH
Gluten
content
pH
Gluten
content
°C
mg kg-1
mg kg-1
FLA1
37
3,77±0,02
4500,00±126,00
3,64±0,02
2450,00±67,00
FLA2
37
3,77±0,02
4650,00±130,00
3,56±0,02
2200,00±62,00
FLB
30
4,37±0,02
5100,00±143,00
3,82±0,02
2810,00±79,00
FLP
30
4,39±0,02
4700,00±132,00
3,79±0,02
2500,00±70,00
The results showed that LAB fermentation can only
eliminate small amounts of gluten while content of
gluten at high concentrations still remains high after
hydrolysis.
Similar results were obtained by hydrolysis of
fermentation of wheat flour. In the wheat flour 74590-
80127 mg kg-1 of gluten were found (Greco et al.,
2011). Results achieved in the studies (De Angelis et
al., 2006; Stefańska et al., 2016) demonstrate that the
proteolytic activity of the selected LAB strains is not
high enough to allow their use for the degradation of
allergenic proteins in bakery products made from
wheat flour intended for patients with food allergy to
gluten. However, fermentation with LAB could be used
in production from raw materials with low gluten
content and, at high concentrations, their effects must
be combined with proteolytic effects of the enzymes.
Conclusions
The role of a fermentation process for improving the
quality of GF products and for developing a new
concept of GF products is very important, however,
this method is not suitable in order the gluten to be
completely degraded, when its initial amount in the raw
material is high enough.
Gluten degraded in wheat starch to below 20 mg kg-1
using Lactobacillus plantarum-P1 in short time, and L.
acidophilus-308 after long fermentation. These
selected LAB strains may be applied as specific starter
cultures to prepare bakery products of special
nutritional use from wheat starch, but did not reduce
the amount of gluten in fibers and bran to a safe limit
for gluten-free diet.
Acknowledgment
The authors thank Roquette Amilina, AB for the
samples of wheat processing products: fractions of
starch, fibers and bran.
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... Starch had the lowest gluten content compared to other byproducts; therefore, the fermentation reduced the gluten concentrate to 20 and 12 mg kg À1 after 12 and 24 h fermentation, respectively, while the initial gluten concentration was 85 mg kg À1 , which means reducing by 76.5% and 85.9% respectively. The reduction of gluten concentration of fibre was 42.3% and 71.8% after 12 and 24 h fermentation, respectively, when the initial concentration was 7800 mg kg À1 (Bradauskiene et al., 2019). Rizzello et al. (2007) studied the effect of fermentation by several types of Lactobacillus and two types of proteases extracted from Aspergillus oryzae and Aspergillus nigre on the IRG by using R5 antibody of ELISA, R5 antibody WB, two-dimensional electrophoresis and LC-MS. ...
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... Lacticaseibacillus acidophilus 308, Lactiplantibacillus plantarum P-1 -Enzymatic hydrolysis of toxic peptides derived from gliadin [100] In vitro (Caco-2 ...
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... Several studies had shown that dairy, soymilk, and glutenbased fermented foods decrease the IgE immunoreactivity [38,[55][56][57][58][59]. Since the in vitro IgE binding capacity and the ability to crosslink IgE on mast cell/basophils are not always correlated [60], the gluten fermented by Lc. lactis LLGKC18 was submitted to tests in a functional model of RBL activated with pooled patients sera. ...
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