INTRODUCTION
These days, biochemists and food industry workers
are facing an important task: they have to provide
population with high-quality protein. Introducing dairy
products into bakery formulae can solve the problem,
since milk proteins are biologically valuable according
to the content and ratio of essential amino acids. The
amino acid composition of whey proteins is closest to
that of human muscle tissue. Whey proteins are superior
to all other animal or plant proteins in terms of essential
amino and branched-chain acids, i.e. valine, leucine, and
isoleucine [1–3].
However, there is the problem of people with lactose
intolerance. According to the Institute of Immunology
(Ministry of Health of the Russian Federation), 65% of
allergic patients demonstrate intolerance to some kind
of food, e.g. dairy products. This problem is especially
common among children [4–7]. Therefore, dairy
products as additives require a thorough research [8].
Although people of any age can digest unaltered
milk proteins, cow’s milk remains one of the strongest
and most common allergen [6–8]. It contains about
20 proteins with different degrees of antigenicity,
including those with the highest clinical relevance,
such as β-lactoglobulin, α-lactalbumin, bovine serum
albumin (BSA), γ-globulin, and α- and β-caseins [9–11].
β-lactoglobulin is the predominant whey protein in
cow’s milk: 50% of whey protein and about 10% of total
protein. It is considered one of the main milk allergens,
while α-lactalbumin and BSA have a lower immune
reactivity [12]. Sensitization to β-lactoglobulin is caused
by numerous continuous epitopes located along the
entire length of its molecule [2, 12, 13].
A β-lactoglobulin molecule consists of 162 amino acid
residues and has a molecular weight of about 18300 Da.
Copyright © 2019, Savkina et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International
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Foods and Raw Materials, 2019, vol. 7, no. 2
E-ISSN 2310-9599
ISSN 2308-4057
Research Article DOI: http://doi.org/10.21603/2308-4057-2019-2-X-X
Open Access Available online at http:jfrm.ru
Degradation of β-Lactoglobulin during sourdough bread production
Olesya A. Savkina1,* , Olga I. Parakhina1, Marina N. Lokachuk1, Elena N. Pavlovskaya1,
and Vadim K. Khlestkin1,2
1 St. Petersburg Branch of the State Research Institute of Baking Industry, St. Petersburg, Russia
2 Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
* e-mail: o.savkina@gosniihp.ru
Received December 11, 2018; Accepted in revised form December 28, 2018; Published X X, 2019
Abstract: The research featured various types and strains of lactic acid bacteria (LAB) and yeast. The research objective was to
study their ability to utilize β-lactoglobulin during sourdough fermentation. The present paper also described the effect of sourdough
fermentation and baking on β-lactoglobulin degradation. A set of experiments with various types and strains of LAB showed that
β-lactoglobulin decreased in gluten-free sourdough with 30%, 60%, and 90% of skimmed milk powder (SMP). L.plantarum E36
demonstrated the highest biodegradation of β-lactoglobulin (by 53%) with SMP = 30%. L.helveticus ATCC8018T showed the lowest
content of β-lactoglobulin with SMP = 60% and 90%: the content fell by 48% and 40%, respectively. The largest decrease in the
content of β-lactoglobulin was observed in the sourdough with Saccharomyces cerevisiae 17 (by 28–42%) and Candida milleri
Pushkinsky (by 25–41%). The content of total protein increased, which was not associated with yeast biomass growth. The content
was determined after fermentation in sourdoughs with SMP = 60% and 90% using a bicinchoninic acid reagent kit. The content of
β-lactoglobulin in the control and experimental samples did not exceed 1 μg/g in the finished bakery products. This fact indicated a
significant effect of thermal treatment on β-lactoglobulin degradation in baking. Thus, temperature processing (baking) had a greater
impact on the destruction of β-lactoglobulin than enzymatic processing (fermentation).
Keywords: β-lactoglobulin, enzyme-linked immunosorbent assay, lactic acid bacteria, milk, sourdough, bread
Please cite this article in press as: Savkina OA, Parakhina OI, Lokachuk MN, Pavlovskaya EN, Khlestkin VK. Degradation of
β-Lactoglobulin during sourdough bread production. Foods and Raw Materials. 2019;7(2):X–X. DOI: http://doi.org/10.21603/2308-
4057-2019-2-X-X.
66
Savkina O.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
At pH 6.8–7, β-lactoglobulin can be found in milk as a
dimer [14].
β-Lactoglobulin is relatively resistant to acid
hydrolysis and intestinal proteases. As a result, when
consumed with food, part of the protein remains intact
in the gastrointestinal tract and can penetrate through
the intestinal wall. Heat treatment reduces the IgEbinding
ability in proportion to the degree of heating.
However, new antigenic sites may form in denatured
proteins. These sites were unavailable for binding in the
native molecule or appeared during a chemical reaction
with other food molecules. IgE obtained from patients
with an allergy to β-lactoglobulin was found specific to
both native and denatured proteins [2, 10].
Like any proteins, milk proteins are exposed to
temperature, pressure, and enzymes. The following
scheme is generally accepted for the thermal
denaturation of β-lactoglobulin: deployment of protein
molecules – dissociation of dimer – aggregation of
denatured protein. Dimeric β-lactoglobulin reversibly
dissociates into monomers at 30–55°C. At 80°C, the
molecule is almost completely unfolded [11, 13]. The
reversibility of β-lactoglobulin denaturation depends on
the heating degree and time. After a low temperature
heating, a small part of the denatured (unfolded)
β-lactoglobulin molecules can restore their native
structure. However, an hour at 95–97 °C leads to an
active aggregation of β-lactoglobulin molecules. As a
result, protein denatures irreversibly. After denaturation
at ≥ 70°C, the β-lactoglobulin structure can partially
stabilize as the chains re-clot and disulfide bridges are
formed. At 130–140°C, the disulfide bonds break, and
the protein polypeptide chains deploy completely and
irreversibly [2, 15–17]. Denaturation and hydrolysis of
β-lactoglobulin is possible when exposed to microwave
radiation [18, 19]. Denatured or hydrolyzed milk
proteins used in dairy mixes are known to be less
allergenic [20, 21]
During baking, the temperature of the crust can
reach 180–230°C, while the core crumb warms up to no
more than 95°C for several minutes [22]. In this regard,
the effect of the baking process on the β-lactoglobulin
content in bread with dairy products remains
understudied.
In fermented milk products, most milk proteins
are destroyed by various microorganisms, including
LAB. Prebiotic cultures of LAB are known to reduce
the allergenicity of cow’s milk due to the partial
denaturation of allergenic proteins [24, 25].
Microorganisms play an important part in baking.
For instance, fermentation process takes place in
sourdough and dough. Various types of LAB are widely
used in sourdough [22, 23]. Hence, it is necessary
to study the effect of LAB sourdough and dough
fermentation on the destruction of cow milk allergen
protein. The research can result in a method of reducing
the allergenicity of dairy products and creating new,
safer bakery products.
Thus, the research objective was to study the effect
of LAB and yeast on the destruction of β-lactoglobulin
during baking.
STUDY OBJECTS AND METHODS
Effect of LAB on the β-lactoglobulin content
and acidity of the sourdough. The research featured
sourdough of 8 LAB strains: Lactobacillus plantarum
E36, Lactobacillus plantarum E4, Lactobacillus
plantarum E1, Lactobacillus parabuchneri E7,
Lactobacillus paracasei/casei E31, Lactobacillus
paracasei E3, Lactobacillus acidophilus 22n2, and
Lactobacillus helveticus ATCC 8018T. As for the
yeast strains, 8 types were employed: Saccharomyces
cerevisiae – strains L-1, 90, 512, 17, XII, and
Krasnodarsky; Candida milleri Chernorechensky; and
Kluyveromyces marxianus Pushkinsky. The samples
were obtained from the Collection of the St. Petersburg
Branch of the State Research Institute of Baking
Industry (St. Petersburg, Russia) [26].
Preparing the sourdough: The nutritional mixture
consisted of rice flour and SMP (30%, 60%, and 90%
per 100 kg of mixture). The moisture content w as 75%.
The LAB culture fluid had a cell content of 108 CFU/ml
cultivated in SMP for 48 h. During the first phase, it
was added to a mixture of raw materials and water,
stirred, and placed in a thermostat for 24 h at 30°C. The
fermented sourdough was then added to the nutrient
mixture in the ratio of 1:3 and allowed to ferment for 24 h
at 30°C. Table 1 shows the formulae for sourdough of the
propagating and production cycles. A nutritional mixture
devoid of any LAB served as a control sample.
The quality of the sourdoughs was assessed
according to their acidity. The acidity was determined
by the common method used in baking industry.
The sourdough suspension was titrated in water at
H = 0.1 with NaOH solution and phenolphthalein [27].
Table 1 Formulae for sourdough with SMP and pure LAB
cultures in the propagating and production cycles
Material Raw materials in the sourdough with the
content of SMP, % to dry solids
30 60 90 30 60 90
Phase I of the
propagating cycle
Production cycle
LAB culture
fluid, ml
10.0 10.0 10.0 – – –
Sourdough
(Phase
I of the
propagating
cycle), g
– – – 50.0 50.0 50.0
Rice flour, g 35.0 20.0 5.0 29.0 16.6 4.1
SMP, g 15.0 30.0 45.0 12.4 24.9 37.3
Water, g 121.0 121.0 121.0 108.6 108.6 108.6
Total: 181.0 181.0 181.0 200.0 200.0 200.0
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Savkina O.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
Effect of the yeast on the content of
β-lactoglobulin in the sourdough. Preparing the
sourdough: Yeast strains grown on malt wort slant agar
(8% DS) were used to screen the allergen reducing
activity of the yeast. 10 ml of yeast culture were
introduced into an aqueous suspension with 10 CFU/ml
cell content in the nutritional mixture (Table 2). The
mixture consisted of rice flour, SMP (30%, 60%,
and 90% per 100 kg of the mixture), and water. The
moisture content of the mixture was 75%. To prevent
the development of extraneous microflora, L.helveticus
ATCC 8018T was added to the nutrient mixtures. The
strain had been selected during the first stage of the
experiment. It demonstrated the highest allergenreducing
activity.
The sourdoughs were fermented for 24 h at 30°C and
then examined for acidity, temperature, and moisture
content.
The effect of sourdough and dough fermentation
and baking on the content of β-lactoglobulin.
Laboratory baking was used to study of the effect of the
technological process (fermentation and baking) on the
content of β-lactoglobulin in dough and gluten-free bread.
Preparing the sourdough: The nutritional mixture
consisted of rice flour and SMP (0%, 30%, 60%,
90%, and 100%). The moisture content was 75%.
LAB of L.helveticus ATCC 8018T strain and yeast of
S.cerevisiae 17 and C.milleri Pushkinsky were added to
the mixture in the quantities indicated in Table 3.
Preparing the dough: The dough for the control
sample was kneaded from corn starch, extrusion starch,
soy protein isolate, rice flour, and SMP in the amount of
3%, 6%, 9%, and 10% to the weight of the mixture. The
mixture contained sugar, salt, pressed baking yeast, and
vegetable oil. The moisture content was 53.5%.
The dough for the samples was prepared from the
sourdough obtained at phase II of the propagating cycle
(10% of the mixture in the intermediate product), corn
starch, extrusion starch, rice flour, sugar, salt, pressed
baking yeast, vegetable oil, and water. Table 4 shows the
formulae of the dough.
The dough was poured into 250-gram moulds and
allowed to rise at 35–40°C at an average humidity of
Table 2 Sourdough formulae with SMP and pure cultures
of yeast and LAB
Material Raw materials in the sourdough with
the content of SMP, % to dry solids
30 60 90
Yeast suspension, ml 10.0 10.0 10.0
Culture fluid of
L.helveticus
ATCC 8018T, ml
10.0 10.0 10.0
Rice flour, g 35.0 20.0 5.0
SMP, g 15.0 30.0 45.0
Water, g 111.0 111.0 111.0
Total: 181.0 181.0 181.0
Table 3 Sourdough formulae with SMP in propagating
and production cycles
Material Raw materials in the sourdough with the
content of SMP, % to dry solids
30 60 90 30 60 90
Phase I of the
propagating cycle
Production cycle
Culture fluid
of L.helveticus
ATCC 8018T, ml
10.0 10.0 10.0 – – –
Yeast suspension,
ml: S.cerevisiae 17
5.0 5.0 5.0
C.milleri
Pushknsky
5.0 5.0 5.0
Sourdough,g – – – 50.0 50.0 50.0
Rice flour, g 35.0 20.0 5.0 29.0 16.6 4.1
SMP, g 15.0 30.0 45.0 12.4 24.9 37.3
Water, g 111.0 111.0 111.0 98.6 98.6 98.6
Total: 181.0 181.0 181.0 200.0 200.0 200.0
Table 4 Dough formulae
Material Consumption of raw materials per 100 kg of the mixture with the SMP content, % to the weight of the
mixture in the dough
Control sample Experimental sample
3 6 9 10 3 6 9 10
Corn starch, g 64.2 61.2 58.2 57.2 64.2 61.2 58.2 57.2
Extrusion starch, g 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0
Rice flour, g 20.0 20.0 20.0 20.0 13.0 16.0 19.0 20.0
SMP, g 3.0 6.0 9.0 10.0 – – – –
Sourdough, g – 36.0
Pressed baking yeast, g 2.5
Vegetable oil, g 3.8
Salt, g 0.8
Sugar, g 2.0
Water, g 110.6 84.7
Total 217.0
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Savkina O.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
80 ± 2%. After that, the samples were baked in an oven
at 210°C for 18 min with a 5-second steam supply.
Preparing samples for the immunoassay and gel
electrophoresis. Preceding the analysis, the samples
underwent the following procedures. 9 ml of phosphatesaline
buffer (PBS, pH = 7.4) was added to 1g of the
test sample (sourdough, dough, or bread). The buffer
contained sodium azide to protect the samples from
microorganisms. After that, a 12-hour extraction
was performed using a shaker at 20 ± 1°C. After the
extraction, the samples were centrifuged at 40°C and
14000 rpm in an Eppendorf Centrifuge 5417R to remove
microorganisms and undissolved components. After the
centrifugation, the samples were diluted 10 thousand
times in a phosphate-buffered saline (20 mM phosphate,
150 mM NaCl, pH 7.2). The dilution was adapted to the
concentration range defined by the test system.
The method of enzyme-linked immunosorbent assay
(ELISA method) was used to measure the content of
β-lactoglobulin in the sourdoughs at the onset and at the
end of fermentation. The process involved antibody No.
362-beta-lactoglobulin – a set of reagents provided by
OOO Hema (St. Petersburg, Russia).
Electrophoresis in a sodium dodecyl sulphate
polyacrylamide denaturing gel was employed to confirm
the presence of β-lactoglobulin in the sourdoughs at the
onset and at the end of fermentation, as well as in the bread.
A bicinchoninic acid reagent kit (BCA, Pierce) was
used to define the total protein in the sourdoughs at the
onset and at the end of fermentation and in the produced
bread. The disc electrophoresis was conducted in nonreducing
conditions according to Laemmli method. The
samples were diluted to a protein concentration of 1 mg/
ml before they were applied to a 13% separating gel.
Statistics. The statistical analysis was performed
using Excel software. The method of two-way ANOVA
was used to compare the effects of the SMP amount
and the type of strain on the content of β-lactoglobulin
in the sourdoughs, dough, and bread. The research also
assessed the correlation and covariance between the
β-lactoglobulin content and the sourdough acidity.
The data show the confidence intervals, which prove
the accuracy of the methods for determining protein
content and acidity.
RESULTS AND DISCUSSION
The experiment measured the acidity in the
sourdoughs based on various strains with different
content of SMP. Acidity reflects the development of
microorganisms in the environment. A high level of
acidity improves the absorption of nutrients from the
environment. High acidity values accelerate proteolysis,
which is important for the destruction of protein and
its constituents, including the allergenic ones. During
phases I and II of fermentation, L.acidophilus 22n2 and
L.helveticus ATCC 8018T showed the highest titrated
acidity indicators at the end of phase II (Table 5). These
strains demonstrated the maximum titratable acidity
with SMP = 60%.
All the LAB strains had different effects on
β-lactoglobulin (Fig. 1). The degree of β-lactoglobulin
degradation decreased with the increase in the SMP
concentration in the nutritional mixture, while different
strains reacted differently to the increase in the SMP
concentration. At SMP = 30%, the sourdough sample
with L.plantarum E36 showed the biggest drop in
β-lactoglobulin content in the fermentation process –
by 53%. However, at SMP = 60% and 90%, it was the
L.helveticus ATCC8018T sample that showed the biggest
drop in the content of the allergen – by 48 and 40%,
respectively. In the sourdoughs, the SMP amount might
have a different effect on the vital activity of lactic acid
bacteria, since they normally live in silage and flour,
except L.acidophilus 22n2 and L.helveticus ATCC8018T.
The two-way ANOVA method gave the following
results. The SMP amount had a significant effect on
the β-lactoglobulin content in the sourdough after
fermentation: alpha = 0.05, P < 0 .001, F = 2 7.78,
Fcritical = 3.63. However, the type of LAB strain
factor produced no effect: alpha = 0.05, P = 0 .25,
F = 1.46, Fc
ritical = 2.59. A strong positive correlation
and covariance was revealed between the final
β-lactoglobulin content and the final acidity level of
the sourdough for L.plantarum E4 and L.acidophilus
22n2. The correlation coefficients were 0.99 and 0.91,
Table 5 Effect of various LAB strains on the sourdough
acidity
LAB strain in the
sourdough at different
SMP amounts
Titrated acidity of the sourdough, degree
Phase I Phase II
onset final onset final
SMP = 30%
L.paracasei E3
L.paracasei E31
L.plantarum E36
L.plantarum E4
L.parabuchneri E7
L.acidophilus 22n2
L.helveticus ATCC 8018T
L.plantarum E1
3.0 ± 0.3
3.0 ± 0.3
2.5 ± 0.3
3.0 ± 0.3
3.0 ± 0.3
3.0 ± 0.3
3.0 ± 0.3
2.7 ± 0.3
12.9 ± 1.3
12.3 ± 1.2
7.7 ± 0.8
7.5 ± 0.8
6.8 ± 0.7
15.8 ± 1.6
12.8 ± 1.3
9.5 ± 1.0
4.5 ± 0.5
4.2 ± 0.4
2.9 ± 0.3
2.9 ± 0.3
2.8 ± 0.3
3.5 ± 0.4
3.3 ± 0.3
5.0 ± 0.5
13.5 ± 1.4
15.3 ± 1.5
9.8 ± 1.0
8.6 ± 0.9
11.0 ± 1.1
18.5 ± 1.9
16.5 ± 1.7
12.2 ± 1.2
SMP = 60%
L.paracasei E3
L.paracasei E31
L.plantarum E36
L.plantarum E4
L.parabuchneri E7
L.acidophilus 22n2
L.helveticus ATCC 8018T
L.plantarum E1
3.5 ± 0.4
3.5 ± 0.3
3.0 ± 0.3
3.4 ± 0.3
3.8 ± 0.4
4.5 ± 0.5
4.5 ± 0.5
4.1 ± 0.4
13.7 ± 1.4
14.2 ± 1.4
9.5 ± 1.0
8.2 ± 0.8
8.5 ± 0.9
21.0 ± 2.1
17.2 ± 1.7
10.5 ± 1.1
5.3 ± 0.5
6.5 ± 0.7
3.6 ± 0.4
5.0 ± 0.5
4.0 ± 0.4
6.0 ± 0.6
5.5 ± 0.6
4.5 ± 0.5
19.8 ± 2.0
20.4 ± 2.0
11.9 ± 1.2
11.2 ± 1.1
11.8 ± 1.3
28.0 ± 2.8
22.5 ± 2.3
10.2 ± 1.0
SMP = 90%
L.paracasei E3
L.paracasei E31
L.plantarum E36
L.plantarum E4
L.parabuchneri E7
L.acidophilus 22n2
L.helveticus ATCC 8018T
L.plantarum E1
4.7 ± 0.5
4.2 ± 0.4
4.5 ± 0.5
4.5 ± 0.5
5.2 ± 0.5
5.7 ± 0.6
5.5 ± 0.6
5.0 ± 0.5
13.2 ± 1.3
12.5 ± 1.3
9.5 ± 1.0
11.0 ± 1.1
9.1 ± 0.9
23.9 ± 2.4
20.0 ± 2.0
10.5 ± 1.1
6.0 ± 0.6
5.6 ± 0.6
5.5 ± 0.6
5.3 ± 0.5
5.7 ± 0.6
7.0 ± 0.7
6.0 ± 0.6
5.8 ± 0.6
16.2 ± 1.6
19.6 ± 2.0
17.4 ± 1.7
16.3 ± 1.6
17.0 ± 1.7
27.5 ± 2.8
22.5 ± 2.3
15.3 ± 1.5
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Savkina O.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
respectively. The covariance coefficients were 3270
and 2449, respectively. L.paracasei E31 demonstrated a
weak inverse correlation (coefficient = 0.25).
The screening of the allergen-reducing activity of
various yeast strains (Fig. 2) showed that the strains
produced a different effect. As for Saccharomyces
cerevisiae, strain 17 demonstrated the highest allergenreducing
activity: the β-lactoglobulin content fell by
28–42%. As for the Candida milleri, it was Pushkinsky
strain: the β-lactoglobulin content fell by 25–41%.
The two-way ANOVA method gave the following
results. The SMP amount had a significant effect on
the β-lactoglobulin content in the sourdough after
fermentation: alpha = 0.05, P < 0 .001, F = 9 3.60,
Fcritical = 3.56. However, the type of yeast strain factor
produced no effect: alpha = 0.05, P = 0 .37, F = 1 .17,
Fcritical = 2.46.
Lactic acid bacteria strain L.helveticus ATCC 8081T
and two yeast strains, S.cerevisiae 17 and C. milleri
Pushkinsky, were selected for further research, which
featured the effect of fermentation and baking on the
β-lactoglobulin content in sourdough, dough, and bread.
The enzyme immunoassay showed a decrease
in β-lactoglobulin at the end of phases I and II by
1.4–1.8 times, if compared with its content in the
nutrient mixture immediately after mixing (Fig. 3).
Thus, the allergen was destroyed by the LAB enzymes.
Figure 1 Content of β-lactoglobulin in the sourdoughs with various LAB strains after fermentation
Figure 2 Content of β-lactoglobulin in the sourdoughs various yeast strains after fermentation
0
800
1600
2400
3200
4000
control L.paracasei E3 L.paracasei
E31
L.plantarum
E36
L.plantarum
E4
L.parabuchneri
E7
L.acidophilus
22n2
L.helveticus
ATCC 8018T
L.plantarum
E1
Content of β-lactoglobulin, μg/g
30% SMP 60% SMP 90% SMP
0
1500
3000
4500
6000
without yeast,
S.cerevisiae L-1
S.cerevisiae L-1 S.cerevisiae 17 S.cerevisiae 512 S.cerevisiae 90 S.cerevisiae
Krasnodarsky
S.cerevisiae XII C.milleri
Pushkinsk
C.milleri
Chernorechensky
Content of β-lactoglobulin, μg/g
30% SMP 60% SMP 90% SMP
0
800
1600
2400
3200
4000
control L.paracasei E3 L.paracasei
E31
L.plantarum
E36
L.plantarum
E4
L.parabuchneri
E7
L.acidophilus
22n2
L.helveticus
ATCC 8018T
L.plantarum
E1
Content of β-lactoglobulin, μg/g
30% SMP 60% SMP 90% SMP
0
1500
3000
4500
6000
without yeast,
S.cerevisiae L-1
S.cerevisiae L-1 S.cerevisiae 17 S.cerevisiae 512 S.cerevisiae 90 S.cerevisiae
Krasnodarsky
S.cerevisiae XII C.milleri
Pushkinsk
C.milleri
Chernorechensky
Kluyveromyces
marxianus
Content of β-lactoglobulin, μg/g
30% SMP 60% SMP 90% SMP
Figure 3 Content of β-lactoglobulin in the sourdough before
and after fermentation at the end of phases I and II
0
2500
5000
7500
10000
30% SMP 60% SMP 90% SMP
Content of β-lactoglobulin,
μg/g
Sourdough before fermentation End of phase I End of phase II
0
15
30
45
60
30% Total protein content, mg/g
Sourdough before 0
300
600
900
1200
1500
30% SMP 60% SMP 90% SMP
Content of β-lactoglobulin, μg/g
before fermentation after fermentation
Figure 4 Total protein in the sourdough before fermentation
and after phases I and II
0
2500
5000
7500
10000
30% SMP 60% SMP 90% SMP
Content of β-lactoglobulin,
μg/g
Sourdough before fermentation End of phase I End of phase II
0
15
30
45
60
30% SMP 60% SMP 90% SMP
Total protein content, mg/g
Sourdough before fermentation End of phase I End of phase II
0
300
600
900
1200
1500
Content of β-lactoglobulin, μg/g
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Savkina O.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
Despite the destruction of β-lactoglobulin, the total
protein content in samples with SMP = 60% and 90%
increased in the fermentation process, if compared with
the initial amount (Fig. 4). The total protein content
was determined using a bicinchoninic acid reagent
kit. Presumably, there are two ways additional protein
could appear during the experiment. First, it could
increase during fermentation due to the accumulation
of yeast biomass. Second, it could be released from
any supramolecular or covalent complexes with other
macromolecules – proteins or polysaccharides. To
understand how the increase in the microbial biomass
affected the increase in total protein, an experiment was
conducted with sourdough based on rice flour, without
SMP. In this case, the amount of total protein in the
sourdough without SMP remained virtually unchanged
during the fermentation. It was 3.8 mg/g before
fermentation and 4.0 mg/g at the end of phase I. The
increase in the total protein in the sourdoughs with SMP
might have been caused by the release of the previously
bound protein. It happened under the influence of yeast
and LAB enzymes, not because their biomass increased.
The experiment revealed a decrease in
β-lactoglobulin in the dough after fermentation,
compared with its content immediately after kneading
(Fig. 5). Due to the fact that the kneading involved
pressed yeast, the decrease in β-lactoglobulin could
be explained by the combined effect of fermenting
microflora enzymes and industrial yeast.
As for the finished products, the content of
β-lactoglobulin in the control and experimental bread
samples did not exceed 1 μg/g. Hence, the temperature
degradation of β-lactoglobulin proved highly efficient
for bakery products.
The electrophoresis was conducted according
to Laemmli’s method in sodium dodecyl sulphate
polyacrylamide gel with non-reducing conditions. It also
confirmed a decrease in the content of β-lactoglobulin
(Fig. 6 and 7 ). N either b lotting o f p olyacrylamide g el
proteins to nitrocellulose, nor detection of β-lactoglobulin
by antibodies from the ELISA test system gave any
results. Neither of the antibodies was able to identify the
antigen after electrophoresis in such conditions. That
proved that the content of β-lactoglobulin in the finished
products was extremely low.
Thus, the research proved that thermal treatment has
a greater impact on the destruction of β-lactoglobulin
than enzymatic treatment.
CONCLUSION
The research investigated the effect of various LAB
and yeast strains on the β-lactoglobulin content in
gluten-free sourdough with SMP. Increasing the amount
of SMP had an inhibitory effect on the utilization of
β-lactoglobulin by L.plantarum E36, L.plantarumE1,
and L.helveticus ATCC8018T. The last demonstrated the
highest allergen-reducing activity when SMP equalled
60% and 90% of the solid weight: β-lactoglobulin
decreased by 48% and 40%, respectively. The yeast
strains Saccharomyces cerevisiae 17 and Candida milleri
Pushkinsky showed the biggest decrease in the content of
β-lactoglobulin: by 28–42% and 25–41%, respectively.
Figure 5 Content of β-lactoglobulin in the dough before and
after fermentation
Figure 6 Electrophoregramme samples: sourdough before
fermentation: SMP = 30% (1), SMP = 60% (2), SMP = 90% (3);
sourdough after fermentation: SMP = 30% (4),
SMP = 60% (5), SMP = 90% (6), and the marker (M)
Figure 7 Electrophoregramme samples: control bread sample
with SMP = 30% (1), SMP = 60% (2), SMP = 90% (3);
experiment bread samples with SMP = 30% (4), SMP = 60%
(5), SMP = 90% (6), and the marker (M)
0
2500
5000
30% SMP 60% SMP 90% SMP
Content of Sourdough before fermentation End of phase I End of phase II
0
15
30% SMP 60% SMP 90% SMP
Total protein Sourdough before fermentation End of phase I End of phase II
0
300
600
900
1200
1500
30% SMP 60% SMP 90% SMP
Content of β-lactoglobulin, μg/g
before fermentation after fermentation
1 2 3 4 5 6 M
97 kDa
66 kDa
45 kDa
30 kDa
20 kDa
14 kDa
1 2 3 4 5 6 M 97 kDa
66 kDa
45 kDa
30 kDa
20 kDa
14 kDa
71
Savkina O.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х
S.cerevisiae L-1, S.cerevisiae 512, S.cerevisiae 90,
and S.cerevisiae XII demonstrated an increase in the
content of β-lactoglobulin at SMP concentration of
90%. This might have been connected with a release of
β-lactoglobulin, previously bound to other proteins.
The content of β-lactoglobulin in the control
and experimental samples of bread did not exceed
1μg/g, which proved a high efficiency of temperature
degradation of β-lactoglobulin in the baking process.
Therefore, temperature processing (baking) had a
greater impact on the destruction of β-lactoglobulin than
enzymatic processing (fermentation).
CONFLICT OF INTEREST
The authors declare that there is no conflict of
interest related to this article.
ACKNOWLEDGMENTS
The authors would like to express their deepest
gratitude to Yevgeny Alexandrovich Kozhevnikov
(OOO Hema) for his consultations on the biochemistry
of cow’s milk proteins, Pavel Pavlovich Kornev
(St. Petersburg Branch of the State Research Institute of
Baking Industry) for the baking, and Vasily Mikhailovich
Matveyev (St. Petersburg Branch of the State Research
Institute of Baking Industry) for IT support.
FUNDING
The research was conducted on the premises of
the St. Petersburg branch State Research Institute of a
Baking Industry within the framework of the following
research topic: 0593-2014-0017 ‘Biotechnologies for
sourdoughs based on the microbial composition of
lactic acid bacteria and yeast with an allergen-reducing
abilities to develop technology and assortment of baked
goods with reduced allergenicity’, a basic program of
fundamental scientific researches of the state academies.
The research employed microorganisms from the
Collection of the St. Petersburg branch State Research
Institute of a Baking Industry (St. Petersburg, Russia).
The Collection is on the list of collections that deposit
non-pathogenic microorganisms for government use,
as approved by the Decree of the Government of the
Russian Federation (June 24, 1996 No. 725-47) and the
Order of the Ministry of Agriculture and Food of Russia
(August 15, 1996 No. 14c).

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