Abstract (English):
Oyster mushroom (Pleurotus ostreatus L.) is a valuable food product. It possesses an antiatherogenic potential, which has to be preserved during processing. The paper features the production of oyster mushroom sublimates. It focuses on such pre-treatment conditions as grinding, disinfection, and cryostabilisation, and their effect on the antiatherogenic potential of oyster mushrooms. A set of in vitro experiments was performed to measure the levels of lovastatin and antioxidant, catalase, anti-inflammatory, and thrombolytic properties. Various pre-treatment conditions proved to produce different effects on the biological activity of the freeze-dried oyster mushroom product. The best results were obtained after the mushrooms were reduced to pieces of 0.5 cm, underwent UV disinfection, blanched, treated with hot air, and cryostabilised with a 1.5% apple pectin solution. The best conditions for the antioxidant properties included ozonation, UV disinfection, and cryoprotection with pectin. The critical conditions for the antioxidant properties included homogenisation, blanching, and cryostabilisation with 10% solutions of sucrose and lactose. The catalase properties did not depend on the degree of grinding and were most pronounced after ozonation. The optimal conditions for the anti-inflammatory properties included UV disinfection and cryostabilisation with lactose. Ozonation proved to be critical for anti-inflammatory properties. The optimal conditions for thrombolytic properties included ozonation and cryoprotection with a 5% sorbitol solution, while hot air disinfection proved critical. Therefore, the research provided an experimental substantiation for individual pre-treatment conditions or their combinations that turn sublimated oyster mushrooms into a valuable functional product with antiatherogenic properties.

Keywords:
Oyster mushroom, freeze-drying, functional food, antiatherogenic potential, lovastatin, antioxidant properties, catalase properties, anti-inflammatory properties, thrombolytic effect

INTRODUCTION
According to statistics, atherosclerosis and its
complications remain the main cause of death worldwide
[1]. The mechanisms of atherogenesis are complex
and multiple. Its main causes include hyperlipidemia,
oxidative stress, thrombosis, and inflammation [2].
Modern medicine is striving to find a way to
curb this trend. Various therapeutic approaches are
being introduced to combat atherosclerosis. However,
many of them remain expensive and have various
contraindications and side effects, which limits their
clinical use [3]. As a result, more and more attention
is given to functional food products with medicinal
properties and minimal side effects. Scientists are
looking for biologically active raw materials that could
modify human metabolism and prevent the development
and progression of atherosclerosis [4, 5].
In this regard, the oyster mushroom (Pleurotus
ostreatus L.) is considered advantageous. Its fruit
body has a high nutritional value, natural statin, and a
whole complex of other biologically active substances
(BAS) [6, 7]. Recent researches proved that the oyster
mushroom possesses hypolipidemic, antioxidant, antiinflammatory,
and thrombolytic properties [8–11],
which makes it a valuable raw material. Thus, oyster
mushrooms can help to improve the existing antiatherogenic
functional foods and develop new ones.
Copyright © 2019, Piskov et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International
License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix,
transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.
Foods and Raw Materials, 2019, vol. 7, no. 2
E-ISSN 2310-9599
ISSN 2308-4057
Research Article DOI: http://doi.org/10.21603/2308-4057-2019-2-375-386
Open Access Available online at http:jfrm.ru
Effect of pre-treatment conditions on the antiatherogenic potential
of freeze-dried oyster mushrooms
Sergey I. Piskov1,* , Lyudmila D. Timchenko1 , Igor V. Rzhepakovsky1 , Svetlana
S. Avanesyan1 , Nadezhda I. Bondareva1 , Marina N. Sizonenko1 , David A. Areshidze2
1 North-Caucasus Federal University, Stavropol, Russia
2 Moscow State Regional University, Moscow, Russia
* e-mail: piskovsi77@mail.ru
Received June 06, 2018; Accepted in revised form December 04, 2018; Published October 21, 2019
Abstract: Oyster mushroom (Pleurotus ostreatus L.) is a valuable food product. It possesses an antiatherogenic potential, which has
to be preserved during processing. The paper features the production of oyster mushroom sublimates. It focuses on such pre-treatment
conditions as grinding, disinfection, and cryostabilisation, and their effect on the antiatherogenic potential of oyster mushrooms. A set
of in vitro experiments was performed to measure the levels of lovastatin and antioxidant, catalase, anti-inflammatory, and thrombolytic
properties. Various pre-treatment conditions proved to produce different effects on the biological activity of the freeze-dried oyster
mushroom product. The best results were obtained after the mushrooms were reduced to pieces of 0.5 cm, underwent UV disinfection,
blanched, treated with hot air, and cryostabilised with a 1.5% apple pectin solution. The best conditions for the antioxidant properties
included ozonation, UV disinfection, and cryoprotection with pectin. The critical conditions for the antioxidant properties included
homogenisation, blanching, and cryostabilisation with 10% solutions of sucrose and lactose. The catalase properties did not depend
on the degree of grinding and were most pronounced after ozonation. The optimal conditions for the anti-inflammatory properties
included UV disinfection and cryostabilisation with lactose. Ozonation proved to be critical for anti-inflammatory properties. The
optimal conditions for thrombolytic properties included ozonation and cryoprotection with a 5% sorbitol solution, while hot air
disinfection proved critical. Therefore, the research provided an experimental substantiation for individual pre-treatment conditions
or their combinations that turn sublimated oyster mushrooms into a valuable functional product with antiatherogenic properties.
Keywords: Oyster mushroom, freeze-drying, functional food, antiatherogenic potential, lovastatin, antioxidant properties, catalase
properties, anti-inflammatory properties, thrombolytic effect
Please cite this article in press as: Piskov SI, Timchenko LD, Rzhepakovsky IV, Avanesyan SS, Bondareva NI, Sizonenko MN,
et al. Effect of pre-treatment conditions on the antiatherogenic potential of freeze-dried oyster mushrooms. Foods and Raw Materials.
2019;7(2):375–386. DOI: http://doi.org/10.21603/2308-4057-2019-2-375-386.
376
Piskov S.I. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 375–386
However, the concentration and effectiveness of
biologically active compounds depend not only on
cultivation conditions and age of the mushrooms, but
also on the processing methods [12, 13].
Some of the existing food processing technologies
make it impossible to preserve the entire complex of
biologically active substances [14, 15]. Today, freezedrying
is considered the least harsh and the most reliable
treatment method of BAS production. It ensures stability
of thermolabile and hydrolytically unstable substances,
increases shelf life, and optimises storage conditions
[16–18]. However, even when all the necessary
regulations for freeze-drying have been observed, the
properties of the product depend on the pre-processing
conditions. An appropriate use of various pre-treatment
methods significantly increases the efficiency of drying,
improves the quality of the product, and preserves its
properties [19–21]. A careless use of pre-treatment
methods can lead to a decrease in the content of certain
BAS in sublimates [22–24]. Thus, each raw material
requires its own freeze-drying technology based on
experimental data about the effect that pre-treatment
conditions produce on the specific properties of the
finished sublimates.
The antiatherogenic effects of freeze-dried oyster
mushrooms have already become focus of scientific
studies [25]. However, there have been no studies
connected with the effect of pre-treatment methods on
the preservation of BAS and natural antiatherogenic
potential of sublimated oyster mushrooms, which adds
to the relevance of the present research.
STUDY OBJECTS AND METHODS
The present research used the following chemicals:
chloroform (CHCl3), hydroxyamine hydrochloride
(NH2OH·HCl), reduced iron, perchloric acid (HClO4),
ethanol (C2H5OH) (Sigma-Aldrich), microbiologically
pure lovastatin (C24H36O5) (TEVA, Hungary), reagent
(chromogen containing an ABTS•+ radical) (Institute of
Bioorganic Chemistry, National Academy of Sciences of
Belarus), trolox 6-hydroxy-2,5,7,8-tetramethylchroman-
2-carboxylic acid, ammonium thiocinate (NH4NCS),
ferrous chloride (FeCl2), oleic acid (C18H34O2), hydrogen
peroxide (H2O2), monosubstituted potassium phosphate
(KH2PO4), disubstituted sodium phosphate (sodium
hydrogen phosphate 12-water, Na2HPO4·12H2O) (Sigma-
Aldrich), dextrose (C6H12O6·H2O), sucrose (C12H22O11)
(Sigma), monohydric citric acid (C6H8O7), sodium
citrate (C6H5Na3O5), sodium chloride (NaCl), lactose
(C12H22O11·H2O), and sorbitol (C6H14O6) (Sigma-Aldrich).
All the substances were purchased from Diaem (Russia).
The study featured oyster mushroom (Pleurotus
ostreatus L.), strain NK35 (SYLVAN, Hungary). It was
harvested in 2018 and cultivated under the standard
mushroom production conditions in the Stavropol
Region. The fruit bodies were of the same size and
maturity, undamaged. During the experiments, the
fresh mushrooms were stored in a refrigerator at 5–7°C.
Before the experiments, the fruit bodies were thoroughly
washed under running water.
The antiatherogenic potential of the freeze-dried
oyster mushroom product was evaluated in vitro based
on the concentration of lovastatin, as well as antioxidant,
catalase, anti-inflammatory, and thrombolytic properties.
The first stage featured the effect of the degree of
preliminary grinding on the antiatherogenic properties of
the sublimates. The grinding was conducted by reducing
the fruit bodies into pieces with the side sizes of 2.0–
2.5 cm and 0.5–1.0 cm. The pieces were homogenised
using a laboratory Sterilmixer 12 (PBI, Italy) at No. 9
high-speed mode. Whole mushrooms served as control
sample. The oyster mushroom samples were spread in one
layer on separate stainless steel trays. The homogenised
substance was poured into the trays to form an even layer
with a thickness of ≤ 0.8–1 cm. All samples were frozen
in a SE-45 refrigerator (TEFCOLD, Denmark) at –40°С
for 72 h and subsequently freeze-dried.
The second stage tested the effect of preliminary
disinfection methods on the preservation of
antiatherogenic properties in the sublimates. The
mushrooms were subjected to blanching, UV disinfection,
ozonation, and hot air treatment [26, 27].
Blanching is one of the most common pre-treatment
methods. It reduces microbial challenge and inactivates
the enzymes that reduce the quality of the freeze-dried
product. According to Galoburda et al., the optimal
blanching temperature regime is 70–80°C, since it
provides the best drying performance for mushrooms
[28]. Hence, the oyster mushrooms were blanched in
water at 70 °C for 3 min, cooled under running water,
and drained in a sieve for several minutes.
The UV disinfection of the oyster mushrooms was
performed using an Azov portable ultraviolet irradiator,
modification OBN-35-01 UHL 4.2 (Russia). The fruit
bodies were put on plastic trays in one layer, placed at
a distance of 60 cm from the irradiator and treated for
15 min.
The ozonation was performed using a universal
ozoniser of air and water Ozone OViV (Ukraine).
The ozonation was carried out in a ventilation hood
at 22°C in a 10-litre chamber improvised from PVC
film. The ozonation mode was based on [29] and the
operation manual: ozonator power, 100%; gas flow rate,
2.0 dm3/min; ozone concentration, 8 mg/dm3; exposure
time, 20 min.
The hot air treatment was performed using a
TS-1/80 SPU dry-air thermostat (Smolensk Special
Design-Technological Bureau of Software Management
Systems, Russia). The mushrooms were placed on a wire
shelf and kept in the thermostat under forced ventilation
at 60°C for 60 min.
The third stage assessed the effect of various
cryoprotectors on the atherogenic potential of the oyster
mushroom sublimates. The experiment involved natural
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substrates that are widely used in food industry: a 10%
sucrose solution, a 10% lactose solution, a 5% sorbitol
solution, and a 1.5% pectin solution. In all cases, the
whole fruit bodies were soaked in aqueous solutions of
the cryoprotectors (volume ratio = 1:20) for 30 min. The
untreated oyster mushroom fruit bodies acted as control
sample.
After the disinfections and cryostabilisations, the
fruit bodies were placed on separate sheets, frozen, and
freeze-dried.
All the samples were dried in an LS-500 freeze dryer
(Prointech, Russia), which included a freeze dryer and a
vacuum station. The glass lid of the drying chamber was
covered with an opaque material to prevent degradation
of antioxidants by photo-oxidation. The working
pressure in the drying chamber was 80–90 Pa; the
condenser temperature was 48–49°С. The temperature
of the samples did not exceed 29–30°С during the entire
drying process. The average drying time was 26–27 h.
The mushrooms were dehydrated until the residual
moisture content was 6–8%. The moisture content in
the dried oyster mushroom samples was measured using
an MB 25 moisture content analyzer (Ohaus, China).
The indicators were measured using the following
automatic measurement mode: heating temperature
= 75°C, measurement time = 5 min. The resulting
oyster mushrooms sublimates were placed into a dry,
hermetically sealed container and stored in dark at
≤ 25°C for further analysis.
The amount of lovastatin in the sublimates was
estimated according to the authentic method using the
hydroxam method after lovastatin had been extracted
with chloroform and concentrated [30, 31]. The ground
dried mushrooms were weighed into portions of 0.1–
0.2 g, extracted with 5.0–10.0 cm3 of chloroform, and
filtered. The filtrate was evaporated using a RV 10 Basic
V rotary vacuum evaporator (IKA, Germany). The
remaining filtrate was diluted with 1.0 cm3 of a 0.9 M
alcohol alkaline solution of hydroxylamine and 5.0 cm3
of a 5.73 mM solution of ferric (III) chloride. After that,
pH was adjusted to 1.2 ± 0.2 with a 2M hydrochloric
acid solution. The extinction of the resulting
magenta solution was measured using an SF-102
spectrophotometer (Research and Development Centre
NPO INTEROFOTOFIKA, Russia) at a wavelength of
λ = 513 nm. The calculation was performed according to
the calibration curve.
To assess the antioxidant activity of the sublimates,
we measured the radical absorption and the degree of
inhibition of lipid peroxidation (LPO). To assess the
radical absorption, the dry oyster mushrooms were
made into powder. Then the powder was extracted with
bidistilled water in a shaker at 50–60°C for 3 h. The
rotation speed was 190 rpm. After that, the material was
filtered as described in [32]. The antioxidant activity of
the extract was determined in vitro using the OxiStat
test system (Institute of Bioorganic Chemistry, National
Academy of Sciences of Belarus). It was a one-stage
assessment of reduction value of the resulting ABTS•+
radical by antioxidants. The scheme is described in [33]
as follows: ABTS•+ + AO → ABTS + AO•+.
When antioxidants interacted with ABTS•+, the
optical density of the solution of the cation radical fell
down to 600–800 nm in proportion to the concentration
and activity of the antioxidant. The optical density was
measured using a spectrophotometer at a wavelength of
675 nm. The optical path length of the cuvettes was 1.0 cm.
To provide a quantitative assessment of the
antioxidant activity, we used trolox, i.e. a standard
antioxidant, which is a water-soluble analogue of
vitamin E:
% inhibition = 100(1–ΔАо/ΔАc) (1)
АА = [Сst]/% standard inhibition ×
× % sample inhibition (2)
where:
АА – antioxidant activity;
ΔАо – optical density of the experimental sample;
ΔАc – optical density of the control sample (buffer);
Сst – standard concentration (trolox).
The radical absorption results were expressed in mg
of trolox equivalent per gram of dry matter (mg TE/g)
To evaluate the LPO inhibition activity, 0.1 g of
powdered dry mushrooms was added to 2.0 cm3 of
bidistilled water. After 24 h of maceration at room
temperature, the extract was filtered and centrifuged at
1300 rpm for 10 min. The LPO inhibition activity of the
obtained extract was measured in an oleic acid emulsion
system according to the slightly modified procedure
described in [34]. 0.1 cm3 of the extract was added to
4.0 cm3 of phosphate buffer (50 mM, pH 7.0), and
0.1 cm3 of oleic acid was added to 4.0 cm3 of ethanol
(95 wt%, aqueous solution). The total volume was
brought to 10.0 cm3 with distilled water, mixed in a
sealed conical tube with a screw cap, and incubated at
40°C in the dark for 7 days. The oxidation state was
evaluated using iron thiocyanate at 24 h intervals. The
reaction solution (100 μL) was mixed with 4.7 cm3
of ethanol (75 wt%, aqueous solution), 0.1 cm3 of an
ammonium thiocyanate aqueous solution (30% w/v), and
0.1 cm3 of an iron chloride (II) solution (20 mM in 3.5%
(v/v) HCl). After 3 min, the absorbance was measured at
a wavelength of 500 nm using a UV spectrophotometer.
An increase in optical density meant an increase in the
level of oleic acid oxidation. Trolox (0.95 mmol/dm3)
was used as a reference. The blank sample contained
deionised water instead of the extract.
When calculating both the antiradical activity and the
activity of LPO inhibition, we took into account the fact
that the extracts had their own colour, which absorbed a
particular wavelength in the visible spectrum.
The catalase activity of oyster mushroom sublimates
was measured using a modified technique based on
the Beers and Sizer spectrophotometric method [35].
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This fast and accurate analysis presupposes a hydrogen
peroxide dehydrogenation and determination of its loss
at λ = 240 nm. The powdered oyster mushrooms were
weighed into portions of 0.020–0.025 g and extracted
with 5.0 cm3 of chilled 50 mM phosphate buffer with
pH = 7.0 for 15 min in the cold with periodic stirring.
The extract was filtered and centrifuged at 4°C for
20 min and acceleration of 1200 g. The experimental
solution contained 1.0 cm3 of 50 mM phosphate buffer
with pH=7.0 and 1.0 cm3 of 0.1% hydrogen peroxide. Its
optical density (D1) was measured at 240 nm relative
to the control solution, which contained 2.0 cm3 of
50 mM phosphate buffer. After filtration, 0.1 cm3 of
the sublimate sample extract was introduced into the
experimental sample. In addition, 0.1 cm3 of extract was
added to the control solution. The optical density of the
experimental solution was determined after 30 s (D2).
The enzymatic activity was calculated for 1 mmol
of substrate (Н2О2) split in 1 min with 1 g of sublimate
sample according to the following formula:
А= ((D1-D2)V1·n)/D1·m·V2·t) (3)
where:
А – enzymatic activity, mmol/g·min;
D1 – optical density of the hydrogen peroxide
solution before the extract was introduced;
D2 – optical density of the hydrogen peroxide
solution after incubation with the extract;
V1 – total extract volume, cm3;
n – amount of hydrogen peroxide introduced, mmol;
m – weight of oyster mushroom sublimate in the
extract, g;
V2 – volume of extract for analysis, cm3;
t – incubation time, min.
The anti-inflammatory activity of the oyster
mushroom sublimates was determined in vitro. It
employed the method used for assessing the osmotic
resistance of erythrocyte membranes [36]. The dry
mushroom sublimate was made into powder, suspended
in distilled water at a concentration of 5.0 mg/cm3,
and incubated at 4°C for 12 h. The suspension was
centrifuged at 7000 rpm for 10 min, after which the
supernatant was filtered. Blood was obtained from
healthy white laboratory Wistar rats and mixed in a 1:1
ratio with Alsever solution. The latter contained equal
volumes of aqueous solutions of 2% dextrose, 0.8%
sodium citrate, 0.5% citric acid, and 0.42% sodium
chloride. The resulting solution was centrifuged at
4000 rpm for 10 min. The precipitated cells were
washed with physiological saline and centrifuged three
time until the red blood cells were 10% by suspension
volume in physiological saline. The extracts of oyster
mushroom sublimates were separately mixed with
1.0 cm3 of phosphate buffer, 2.0 cm3 of hypotonic
sodium chloride solution (0.42%), and 0.5 cm3 of red
blood cell suspension. The control sample contained
2.0 cm3 of distilled water instead of the hypotonic
solution. The mixes were incubated at 37°C for 20 min
and centrifuged at 3000 rpm.
After that, the supernatant liquid was decanted,
and the haemoglobin content was estimated using a
spectrophotometer at λ = 560 nm. The percentage of
resistance of red cell membranes was assessed based
on the fact that the haemolysis obtained in the control
sample was 100%. It was calculated by the formula:
Percentage of resistance = 100 – (optical density
of the experimental sample/optical density
of the control sample) × 100
To assess the thrombolytic activity of the sublimates,
blood obtained from white Wistar rats was distributed
into different pre-weighed sterile microcentrifuge tubes
(0.5 cm3 in each) and incubated at 37°C for 45 min.
After the clot was formed, the serum was completely
removed without disturbing the clot, and each tube was
again weighed to calculate the weight of the clot. 100 μL
of sublimate extract was added into each tube with a preweighed
clot. All tubes were incubated at 37°C for 90 min.
After incubation, the released liquid was removed, and
the tubes were weighed again. The difference in weight
before and after clot dissolution was expressed as a
percentage [37].
The content of substances and their activity were
expressed in terms of absolute dry raw materials. All
quantitative parameters were triplicated. The results
were recorded as arithmetic mean ± standard error of
the arithmetic mean (M ± m) and subjected to statistical
processing using the method of one-way ANOVA test
and the Biostat software (version 4.03). The significance
of the differences was measured at P ≤ 0.05.
RESULTS AND DISCUSSION
A single-phase ANOVA was conducted to compare
the quantitative values of the properties responsible for
the antiatherogenic potential of the freeze-dried oyster
mushroom product. It also made it possible to check
whether there was any significant difference in these
properties after various pre-treatment methods.
As a potential antiatherogenic product, the oyster
mushrooms were checked for the concentration of
lovastatin. This natural statin reduces the production
of endogenous cholesterol as it inhibits the activity of
hydroxyl-3-methyluracil-coenzyme reductase [38]. The
experiment took into account the level of antioxidant and
catalase activities that resist the accumulation of excess
reactive oxygen. Together with excessive lipids in the
blood, reactive oxygen is known to cause atherosclerosis
[39]. We tested the abilities of freeze-dried oyster
mushrooms to inhibit the inflammation and thrombosis.
They are considered the key pathogenetic mechanisms
of atherosclerosis as they facilitate the transformation of
risk factors into morphological changes [40].
A set of experiments was performed to define the
effect of disinfection, cryoprotection, and various
degrees of preliminary grinding on the safety and
activity of the abovementioned properties.
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Thickness, shape, and volume ratio of samples
are known to affect the drying rate and quality of the
finished product [41].
The whole dried oyster mushroom fruit bodies
selected as control sample were tested for the
abovementioned properties. The obtained quantitative
indicators appeared to be comparable with the data for
Pleurotus mushrooms presented in previous studies
(Table 1). When the fruit bodies were ground to pieces
with side sizes of 2.0–2.5 cm and 0.5–1.0 cm at the pretreatment
stage, it did not affect the concentration of
lovastatin in the freeze-dried product. However, the
content of lovastatin in the homogenised sublimates
turned out to be 45% less than in the whole-dried
samples. The technological process of homogenisation
probably reduced the degree of heterogeneity of the
distribution of chemicals and phases by volume. It
might have changed the sensitivity of lovastatin to the
conditions of the subsequent stages of lyophilisation.
In addition, homogenisation is known to cause a shift
in the pH of raw materials. According to Piecha, when
pH of the medium increases, the lactone structures of
statins can be partially or completely converted to the
corresponding forms of hydroxyacids [42].
The assessment of the effect of grinding size on
the preservation of antioxidant properties had a similar
result. Homogenization reduced the activity of radical
absorption by almost 46%. The homogenised samples also
demonstrated minimal LPO inhibition activity (Fig. 1).
The data were consistent with some studies that
featured the effect of homogenisation during freezedrying
of berries [19]. According to Paciulli et al., the
results may be explained by the fact that large tissue
damage caused a loss of antioxidant substances [48].
However, the preliminary grinding of oyster
mushroom affected neither catalase nor antiinflammatory
properties of its sublimates.
Pleurotus mushrooms owe their thrombolytic
properties to the high level of biosynthesis of the
protease enzyme complex. Proteases have an affinity
for fibrin and cause its lysis [49]. The thrombolytic
properties of freeze-dried oyster mushrooms depended
on the degree of grinding at the pre-treatment stage: the
experiment showed a statistically significant decrease
as the fruit bodies were ground into smaller pieces.
The samples subjected to preliminary homogenisation
demonstrated the lowest thrombolytic activity. Such
result might be connected with the fact that cellular
disruption facilitates interaction between proteolytic
enzymes and extracellular protease inhibitors.
Although pre-treatment grinding may facilitate
the drying process, it proved irrational in terms of
preservation of lovastatin and other antioxidant and
thrombolytic substances [41].
Table 1 Effect of preliminary grinding of oyster mushroom fruit bodies on the bioactive properties of freeze-dried product (M ± m)
№ Grinding size Lovastatin, mg/kg Radical absorption
activity, mgTE/g
Catalase activity,
mmol/g·min
Anti-inflammatory
effect, %
Thrombolytic
activity, %
1 Whole fruit bodies
(control sample)
316.2 ± 8.3a 9.6 ± 0.3a 17.9 ± 0.6a 34.6 ± 0.8a 15.2 ± 0.4a
3 Pieces with side
size 2.0–2.5 cm
305.6 ± 7.6a 9.1 ± 0.3a 18.7 ± 0.6a 35.1 ± 0.7a 14.1 ± 0.3a
4 Pieces with side
size 0.5–1.0 cm
298.4 ± 6.1a 9.4 ± 0.4a 17.3 ± 0.5a 36.4 ± 0.9a 12.4 ± 0.3b
5 Homogeneous state 174.8 ± 4.2b 5.2 ± 0.2b 16.9 ± 0.5a 34.1 ± 0.8 a 9.9 ± 0.2c
6 Sources 50.0–505.0 (Pleurotus
ostreatus) Gunde-
Cimerman et al. [43];
165,3–606,5
Chen et al. [44]
0.61–14.07
(Pleurotus
citrinopileatu)
Nattoh et al. [45]
14.66 (Pleurotus
Ostreatus)
Susmitha
et al. [46]
18.66–43.50 (Pleurotus
florida) Pandimeena
et al. [47];
54,33–85,12 (Pleurotus
florida) Varghese et al. [36]
18.62
(Pleurotus
ostreatus)
Islam et al. [8]
Mean values with different letters in the same column are statistically different (Р < 0.05). mgTE/g
Figure 1 Effect of preliminary grinding on the LPO inhibition
of the freeze-dried product. (Note: in Figs. 1–3, a lower optical
density at 500 nm corresponds to a higher LPO inhibition)
0.0
0.5
1.0
1.5
2.0
2.5
0 1 2 3 4 5 6 7
Optical density at λ = 500 nm
Incubation period, days
No treatment 2.0–2.5 cm pieces
0.5–1.0 cm pieces Homogeneous sample
Trolox Blank sample
0.5
1.0
1.5
2.0
2.5
Optical density at λ = 500 nm
(1) No treatment
(2) 0.5–1.0 cm pieces
(3) Trolox
(1)
(2)
(3)
(4) 2.0–2.5 cm pieces
(5) Homogeneous sample
(6) Blank sample
(5)
(6)
(4)
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Food security is as important as its nutritional and
biological value. Microbiological contamination is an
indicator of food security. Therefore, disinfection is
a necessary pre-treatment stage. Blanching and hot
air treatment had no statistically significant effect on
the content of lovastatin in the finished sublimates
if compared with the control samples (Table 2).
UV disinfection may cause photodegradation of statins.
However, it also demonstrated no significant changes
in the concentration of lovastatin in the sublimates. The
only difference was a slight decrease in the content of
lovastain, which is consistent with the results obtained
by [42], according to which lovastatin proved to be the
most UV resistant statin.
The ozonation resulted in a significant loss of
lovastatin. Its concentration in the sublimates decreased
by 31.4% compared to the control samples, which
confirmed the data published in [50], according to which
oxygen makes lovastatin instable.
UV disinfection and ozonation resulted in a higher
radical absorption and LPO inhibition (Fig. 2). Such
results are consistent with other studies [51–53] that
proved a better preservation, and sometimes even an
increase, of antioxidant substances in mushrooms and
fruits after ozonation and UV disinfection. According to
Sudheer et al., ozone can trigger the formation of such
secondary metabolites as phenols and flavonoids [54].
Hot air disinfection caused no statistically significant
changes in the antiradical activity and LPO inhibition
(Fig. 2). These results contradicted with those described
in [55]. On the one hand, the effect might be explained
by the thermally induced extraction of previously
bound or polymerized molecules of antioxidants, in
particular, phenols. On the other hand, it may be due to
the inactivation of enzymes involved in their catabolism,
as demonstrated by recent studies of vegetable drying
processes [56]. In addition, the obtained results might be
explained by the fact that hot air treatment can trigger
the formation of new compounds, e.g. Maillard reaction
products, which possess good antioxidant properties [57].
The blanching produced a significant decrease in
the antiradical activity of the sublimates. Its value
was 47.7% lower than that of the control sample.
The results confirmed the data described in [58, 59].
According to Lam et al. and Radzki et al., leaching
and a low ability to absorb oxygen radicals resulted
in a lower concentration of antioxidant substances
after preliminary blanching. In addition, the blanched
sublimates showed a minimal LPO inhibition [58, 59].
Various disinfection methods produced different
results on the level of catalase activity of the sublimates.
Hot-air treatment resulted in the lowest catalase activity.
These results confirmed those described in [60], according
to which a higher drying temperature reduced the residual
activity of the oyster mushroom catalase enzyme.
The ozonation produced the highest catalase activity.
These results confirmed those described in [61, 62],
which showed an increase in the catalase activity of
fruits after ozonation. The increase was explained by
the fact that ozone came into contact with the biological
tissue of mushrooms and caused oxidative stress, which
was accompanied by activation of various antioxidant
enzyme systems, e.g. catalase.
Contrary to previous assumptions [63], the
blanching caused no changes in the level of catalase
activity. According to Egbebi et al., blanching of
mushrooms inactivated catalase. In our opinion, it can
be explained by the relatively short blanching time [63].
This presumption corresponds with the observations
published in [64], which described catalase inactivation
only in cases when blanching lasted > 10 min.
Various disinfection methods produced various
effects on the anti-inflammatory activity of the product
0.0
0.5
1.0
1.5
2.0
2.5
0 1 2 3 4 5 6 7
Optical density at λ = 500 nm
Incubation period, days
No treatment 2.0–2.5 cm pieces
0.5–1.0 cm pieces Homogeneous sample
Trolox Blank sample
0.0
0.5
1.0
1.5
2.0
2.5
0 1 2 3 4 5 6 7
Optical density at λ = 500 nm
Incubation period, days
No treatment UV Ozonation Blanching
No treatment Trolox Blank sample
Table 2 Effect of various methods of preliminary disinfection on the bioactive properties of freeze-dried product (M ± m)
№ Disinfection Lovastatin, % Radical absorption
activity, mgTE/g
Catalase activity,
mmol/g×min
Anti-inflammatory
action, %
Thrombolytic
activity, %
1 No treatment (control sample) 316.2 ± 7.9a 8.8 ± 0.3a 16.0 ± 0.4a 35.2 ± 0.8a 15.7 ± 0.4a
2 UV 308.4 ± 8.1a 10.3 ± 0.4b 15.6 ± 0.4a 65.1 ± 1.6b 14.8 ± 0.4а
3 Ozonation 217.4 ± 7.1b 9.4 ± 0.3b 21.6 ± 0.5c 6.3 ± 0.5с 23.9 ± 0.6b
4 Blanching 311.3 ± 8.1a 4.6 ± 0.2c 17.1 ± 0.5а 33.2 ± 0.8a 16.1 ± 0.5a
5 Hot air 299.4 ± 6.5a 8.4 ± 0.3а 1.6 ± 0.1b 36.4 ± 1.0a 10.6 ± 0.3с
Mean values with different letters in the same column are statistically different (P < 0.05)
Figure 2 Effect of various methods of preliminary disinfection
on the LPO inhibition of the freeze-dried product
(1) No treatment
(2) UV
(3) Ozonation
(4) Blanching
(5) Hot air
(1)
(2)
(3)
(5)
(6)
(4)
(7)
(6) Trolox
(7) Blank sample
381
Piskov S.I. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 375–386
that had undergone a spray-freeze drying. The antiinflammatory
properties were determined using the
erythrocyte membrane stabilisation test. The UV-treated
samples showed the highest anti-inflammatory activity.
Mushrooms owe most of their anti-inflammatory
properties to polysaccharides, especially glucans [65,
66]. Thus, UV treatment served as an elicitor that
increases production of extracellular polysaccharides
in mushrooms. In addition, UV treatment might have
produced phenolic compounds that produce a protective
effect on biological membranes [67].
The ozonation resulted in the lowest antiinflammatory
activity. This effect might have
been caused by ozone-induced oxidative reactions.
According to Mzoughi et al., ozone-induced oxidative
reactions lead to the selective depolymerisation of
polysaccharides, followed by a possible increase or,
conversely, a decrease in their biological activity [68].
The thrombolytic properties also proved to depend
on the methods of preliminary disinfection. Thus, the
maximum thrombolytic properties were manifested
in the ozonised samples. This result might have been
caused by the ability of ozone to inactivate protease
inhibitors [69]. The minimal thrombolytic properties
were detected in the samples that had been treated with
hot air. According to Ali et al., protease inhibitors in
mushrooms are thermally stable [70]. According to Rai
et al. and de Castro et al., proteases demonstrate the
maximum activity at 55–60°С and may be wasted on the
autohydrolysis of proteins heating [71, 72].
Freezing is an obligatory stage of freeze-drying.
Freezing can damage the cell structure with ice
crystals. The degree of damage depends on the size of
the crystals and the heat transfer rate. It can affect the
rheological and textural properties of the product, as
well as redox processes in favour of oxidation. As a
result, the number and the biological activity of the
substances in the product may change. Therefore, a
stabilising cryoprotector should be applied before
freezing. Cryoprotectors maintain the quality of the
frozen product by increasing the reversibility of the
process and preserving BAS [73]. In this regard, we
assessed various natural cryoprotectants and their effect
on the anti-atherogenic properties of the freeze-dried
oyster mushrooms (Table 3).
The chemical analysis demonstrated that the
cryoprotectors had a different effect on the amount
of lovastatin in the mushroom sublimates. The
samples pretreated with a 1.5% solution of pectin
showed no statistical changes in the concentration
of oxidation-sensitive natural statin. However, there
was a clear tendency to its increase. It confirmed the
results published in [74], which proved that insoluble
polysaccharides effectively inhibit oxidation processes
in frozen semi-finished products.
The samples pretreated with a 5% sorbitol solution
demonstrated a 47.6% decrease in lovastatin. The
samples pretreated with a 10% sucrose solution and a
10% lactose solution appeared to contain no lovastatine.
It was probably due to the hydrolysis of lovastatine in the
aqueous medium of the cryoprotectors. Thus, freezing
did not prove to be a limiting factor with respect to
lovastatin concentration. Hence, cryoprotectors are not
obligatory in this aspect.
1.5% pectin solution proved to be the best
cryoprotector for oyster mushrooms as it ensured
the maximum preservation of antiradical and LPO
inhibition (Fig. 3). The results were consistent with [75],
according to which pectin has a greater water absorption
capacity compared with sorbitol and monosaccharides.
The results may be attributed to the antioxidant
properties of pectin itself, since its diffused part could
enhance the antioxidant properties of the obtained
dry product. According to Kopjar et al., if added to
bioactive substances containing phenolic compounds,
pectin provides a synergistic effect on their antioxidant
properties [76].
10% solutions of lactose and sucrose resulted in a
significant decrease in the level of antiradical activity
and LPO inhibition. On the one hand, these results may
be explained by the extraction of antioxidant substances
into the aqueous solutions of the cryoprotectors. On the
other hand, the decrease might have been caused by
the cryoprotective effect itself, since it reduces both ice
crystal formation in the mushroom tissue matrix and
damage to the cell structure. According to Yang et al.,
cryoprotective effect increases extraction of antioxidant
substances, e.g. phenolic compounds, from cells [77].
Cooling is known to reduce the catalase properties
of certain substances [78]. However, catalase activity
decreased significantly in all the experimental samples.
Its value was minimal in the sublimates pretreated with
a 10% lactose solution and a 5% sorbitol solution.
Table 3 Effect of pre-treatment with cryoprotectors on the bioactive properties of freeze-dried product (M ± m)
№ Cryoprotector Lovastatin,
mg/kg
Radical absorption
activity, mgTE/g
Catalase activity,
mmol/g·min
Anti-inflammatory
action, %
Thrombolytic
activity, %
1 No treatment (control sample) 310.3 ± 7.6a 7.9 ± 0.3a 17.1 ± 0.5a 30.3 ± 0.9a 14.9 ± 0.4a
2 10% sucrose solution – 5.4 ± 0.2b 9.5 ± 0.4b 29.4 ± 0.8a 15.4 ± 0.4a
3 10% lactose solution – 5.6 ± 0.2b 2.9 ± 0.1c 56.4 ± 1.5b 16.0 ± 0.5a
4 1.5% pectin solution 339.2 ± 8.5a 15.5 ± 0.5c 7.0 ± 0.3d 31.8 ± 1.0a 14.6 ± 0.3a
5 5% sorbitol solution 152.4 ± 5.9b 7.5 ± 0.3a 3.1 ± 0.1c 28.6 ± 1.1a 25.1 ± 0.6b
Mean values with different letters in the same column are statistically different (P < 0.05)
382
Piskov S.I. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 375–386
The maximum anti-inflammatory activity was
manifested in the sublimates pretreated with a 10%
lactose solution. The results confirmed those published
in [79], according to which lactose proved to be a more
advantageous cryoprotector than sorbitol or sucrose
when used in freeze-drying of liposome preparations. It
can be explained by the fact that disaccharides produce
a greater stabilising effect on cell membranes during
freezing than other cryoprotectors, thereby preserving
polysaccharides and glycoproteins of cell membranes.
As for the thrombolytic properties, sublimates
pretreated with sorbitol showed the best results. Unlike
mono-, di-, and oligosaccharides, sorbitol can penetrate
into cells [80]. It protects intracellular proteases and
their fibrinolytic properties from possible denaturation
caused by low temperature.
CONCLUSION
A set of biochemical experiments was performed
to study the effect of various pre-treatment conditions
on the biologically active properties that provide
the antiatherogenic potential of freeze-dried oyster
mushrooms. The antiatherogenic properties under study
included the content of natural statin (lovastatin), as
well as antioxidant, catalase, anti-inflammatory, and
thrombolytic properties. The results showed that each
pre-treatment method produced a different effect on the
abovementioned properties of the freeze-dried product.
The experiments demonstrated that the best results
for lovastatin were obtained when the raw material was
ground to pieces with a side size of ≥ 0.5 cm, subjected
to UV disinfection, blanched, treated with hot air, and
cryoprotected with a 1.5% pectin solution.
As for the antioxidant properties, such as radical
absorption and LPO inhibition, the best conditions
included UV disinfection, ozonation, and cryoprotection
with a 1.5% pectin solution. Homogenisation, blanching,
and cryostabilisation with 10% solutions of sucrose and
lactose were found critical for antioxidant properties.
The catalase activity of the product did not depend on
the degree of grinding, blanching, and UV disinfection.
It was maximal after ozonation. The list of critical pretreatment
conditions included hot air treatment and
exposure to all the cryoprotectors except pectin.
The anti-inflammatory properties were best
preserved after UV disinfection and cryoprotection with
a 10% lactose solution. Ozonation appeared to be the
only critical pre-treatment factor.
The best results for thrombolytic properties were
obtained when the oyster mushrooms were ozonated
and cryoprotected using a 5% sorbitol solution. Critical
factors included homogenisation and hot air treatment.
Thus, the experiments revealed advantages
of individual pre-treatment conditions and their
combinations. The applied conditions can turn freezedried
oyster mushrooms into a functional food product
or ingredient. The new functional product significantly
improved the properties that affect such pathogenetic
factors of atherogenesis as hyperlipidemia, oxidative
stress, inflammatory reaction, and thrombosis.
CONFLICT OF INTEREST
The authors declare that there is no conflict of
interests related to this article.

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