INTRODUCTIONAccording to statistics, atherosclerosis and itscomplications remain the main cause of death worldwide[1]. The mechanisms of atherogenesis are complexand multiple. Its main causes include hyperlipidemia,oxidative stress, thrombosis, and inflammation [2].Modern medicine is striving to find a way tocurb this trend. Various therapeutic approaches arebeing introduced to combat atherosclerosis. However,many of them remain expensive and have variouscontraindications and side effects, which limits theirclinical use [3]. As a result, more and more attentionis given to functional food products with medicinalproperties and minimal side effects. Scientists arelooking for biologically active raw materials that couldmodify human metabolism and prevent the developmentand progression of atherosclerosis [4, 5].In this regard, the oyster mushroom (Pleurotusostreatus L.) is considered advantageous. Its fruitbody has a high nutritional value, natural statin, and awhole complex of other biologically active substances(BAS) [6, 7]. Recent researches proved that the oystermushroom possesses hypolipidemic, antioxidant, antiinflammatory,and thrombolytic properties [8–11],which makes it a valuable raw material. Thus, oystermushrooms can help to improve the existing antiatherogenicfunctional 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 InternationalLicense (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. 2E-ISSN 2310-9599ISSN 2308-4057Research Article DOI: http://doi.org/10.21603/2308-4057-2019-2-375-386Open Access Available online at http:jfrm.ruEffect of pre-treatment conditions on the antiatherogenic potentialof freeze-dried oyster mushroomsSergey I. Piskov1,* , Lyudmila D. Timchenko1 , Igor V. Rzhepakovsky1 , SvetlanaS. Avanesyan1 , Nadezhda I. Bondareva1 , Marina N. Sizonenko1 , David A. Areshidze21 North-Caucasus Federal University, Stavropol, Russia2 Moscow State Regional University, Moscow, Russia* e-mail: piskovsi77@mail.ruReceived June 06, 2018; Accepted in revised form December 04, 2018; Published October 21, 2019Abstract: Oyster mushroom (Pleurotus ostreatus L.) is a valuable food product. It possesses an antiatherogenic potential, which hasto be preserved during processing. The paper features the production of oyster mushroom sublimates. It focuses on such pre-treatmentconditions as grinding, disinfection, and cryostabilisation, and their effect on the antiatherogenic potential of oyster mushrooms. A setof in vitro experiments was performed to measure the levels of lovastatin and antioxidant, catalase, anti-inflammatory, and thrombolyticproperties. Various pre-treatment conditions proved to produce different effects on the biological activity of the freeze-dried oystermushroom 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 propertiesincluded ozonation, UV disinfection, and cryoprotection with pectin. The critical conditions for the antioxidant properties includedhomogenisation, blanching, and cryostabilisation with 10% solutions of sucrose and lactose. The catalase properties did not dependon the degree of grinding and were most pronounced after ozonation. The optimal conditions for the anti-inflammatory propertiesincluded UV disinfection and cryostabilisation with lactose. Ozonation proved to be critical for anti-inflammatory properties. Theoptimal conditions for thrombolytic properties included ozonation and cryoprotection with a 5% sorbitol solution, while hot airdisinfection proved critical. Therefore, the research provided an experimental substantiation for individual pre-treatment conditionsor 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, catalaseproperties, anti-inflammatory properties, thrombolytic effectPlease 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/10.21603/2308-4057-2019-2-375-386.376Piskov S.I. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 375–386However, the concentration and effectiveness ofbiologically active compounds depend not only oncultivation conditions and age of the mushrooms, butalso on the processing methods [12, 13].Some of the existing food processing technologiesmake it impossible to preserve the entire complex ofbiologically active substances [14, 15]. Today, freezedryingis considered the least harsh and the most reliabletreatment method of BAS production. It ensures stabilityof thermolabile and hydrolytically unstable substances,increases shelf life, and optimises storage conditions[16–18]. However, even when all the necessaryregulations for freeze-drying have been observed, theproperties of the product depend on the pre-processingconditions. An appropriate use of various pre-treatmentmethods significantly increases the efficiency of drying,improves the quality of the product, and preserves itsproperties [19–21]. A careless use of pre-treatmentmethods can lead to a decrease in the content of certainBAS in sublimates [22–24]. Thus, each raw materialrequires its own freeze-drying technology based onexperimental data about the effect that pre-treatmentconditions produce on the specific properties of thefinished sublimates.The antiatherogenic effects of freeze-dried oystermushrooms have already become focus of scientificstudies [25]. However, there have been no studiesconnected with the effect of pre-treatment methods onthe preservation of BAS and natural antiatherogenicpotential of sublimated oyster mushrooms, which addsto the relevance of the present research.STUDY OBJECTS AND METHODSThe present research used the following chemicals:chloroform (CHCl3), hydroxyamine hydrochloride(NH2OH·HCl), reduced iron, perchloric acid (HClO4),ethanol (C2H5OH) (Sigma-Aldrich), microbiologicallypure lovastatin (C24H36O5) (TEVA, Hungary), reagent(chromogen containing an ABTS•+ radical) (Institute ofBioorganic Chemistry, National Academy of Sciences ofBelarus), trolox 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, ammonium thiocinate (NH4NCS),ferrous chloride (FeCl2), oleic acid (C18H34O2), hydrogenperoxide (H2O2), monosubstituted potassium phosphate(KH2PO4), disubstituted sodium phosphate (sodiumhydrogen phosphate 12-water, Na2HPO4·12H2O) (Sigma-Aldrich), dextrose (C6H12O6·H2O), sucrose (C12H22O11)(Sigma), monohydric citric acid (C6H8O7), sodiumcitrate (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 (Pleurotusostreatus L.), strain NK35 (SYLVAN, Hungary). It washarvested in 2018 and cultivated under the standardmushroom production conditions in the StavropolRegion. The fruit bodies were of the same size andmaturity, undamaged. During the experiments, thefresh mushrooms were stored in a refrigerator at 5–7°C.Before the experiments, the fruit bodies were thoroughlywashed under running water.The antiatherogenic potential of the freeze-driedoyster mushroom product was evaluated in vitro basedon the concentration of lovastatin, as well as antioxidant,catalase, anti-inflammatory, and thrombolytic properties.The first stage featured the effect of the degree ofpreliminary grinding on the antiatherogenic properties ofthe sublimates. The grinding was conducted by reducingthe fruit bodies into pieces with the side sizes of 2.0–2.5 cm and 0.5–1.0 cm. The pieces were homogenisedusing a laboratory Sterilmixer 12 (PBI, Italy) at No. 9high-speed mode. Whole mushrooms served as controlsample. The oyster mushroom samples were spread in onelayer on separate stainless steel trays. The homogenisedsubstance was poured into the trays to form an even layerwith a thickness of ≤ 0.8–1 cm. All samples were frozenin a SE-45 refrigerator (TEFCOLD, Denmark) at –40°Сfor 72 h and subsequently freeze-dried.The second stage tested the effect of preliminarydisinfection methods on the preservation ofantiatherogenic properties in the sublimates. Themushrooms were subjected to blanching, UV disinfection,ozonation, and hot air treatment [26, 27].Blanching is one of the most common pre-treatmentmethods. It reduces microbial challenge and inactivatesthe enzymes that reduce the quality of the freeze-driedproduct. According to Galoburda et al., the optimalblanching temperature regime is 70–80°C, since itprovides the best drying performance for mushrooms[28]. Hence, the oyster mushrooms were blanched inwater 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 wasperformed using an Azov portable ultraviolet irradiator,modification OBN-35-01 UHL 4.2 (Russia). The fruitbodies were put on plastic trays in one layer, placed ata distance of 60 cm from the irradiator and treated for15 min.The ozonation was performed using a universalozoniser of air and water Ozone OViV (Ukraine).The ozonation was carried out in a ventilation hoodat 22°C in a 10-litre chamber improvised from PVCfilm. The ozonation mode was based on [29] and theoperation manual: ozonator power, 100%; gas flow rate,2.0 dm3/min; ozone concentration, 8 mg/dm3; exposuretime, 20 min.The hot air treatment was performed using aTS-1/80 SPU dry-air thermostat (Smolensk SpecialDesign-Technological Bureau of Software ManagementSystems, Russia). The mushrooms were placed on a wireshelf and kept in the thermostat under forced ventilationat 60°C for 60 min.The third stage assessed the effect of variouscryoprotectors on the atherogenic potential of the oystermushroom sublimates. The experiment involved natural377Piskov S.I. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 375–386substrates that are widely used in food industry: a 10%sucrose solution, a 10% lactose solution, a 5% sorbitolsolution, and a 1.5% pectin solution. In all cases, thewhole fruit bodies were soaked in aqueous solutions ofthe cryoprotectors (volume ratio = 1:20) for 30 min. Theuntreated oyster mushroom fruit bodies acted as controlsample.After the disinfections and cryostabilisations, thefruit bodies were placed on separate sheets, frozen, andfreeze-dried.All the samples were dried in an LS-500 freeze dryer(Prointech, Russia), which included a freeze dryer and avacuum station. The glass lid of the drying chamber wascovered with an opaque material to prevent degradationof antioxidants by photo-oxidation. The workingpressure in the drying chamber was 80–90 Pa; thecondenser temperature was 48–49°С. The temperatureof the samples did not exceed 29–30°С during the entiredrying process. The average drying time was 26–27 h.The mushrooms were dehydrated until the residualmoisture content was 6–8%. The moisture content inthe dried oyster mushroom samples was measured usingan MB 25 moisture content analyzer (Ohaus, China).The indicators were measured using the followingautomatic measurement mode: heating temperature= 75°C, measurement time = 5 min. The resultingoyster 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 wasestimated according to the authentic method using thehydroxam method after lovastatin had been extractedwith chloroform and concentrated [30, 31]. The grounddried mushrooms were weighed into portions of 0.1–0.2 g, extracted with 5.0–10.0 cm3 of chloroform, andfiltered. The filtrate was evaporated using a RV 10 BasicV rotary vacuum evaporator (IKA, Germany). Theremaining filtrate was diluted with 1.0 cm3 of a 0.9 Malcohol alkaline solution of hydroxylamine and 5.0 cm3of a 5.73 mM solution of ferric (III) chloride. After that,pH was adjusted to 1.2 ± 0.2 with a 2M hydrochloricacid solution. The extinction of the resultingmagenta solution was measured using an SF-102spectrophotometer (Research and Development CentreNPO INTEROFOTOFIKA, Russia) at a wavelength ofλ = 513 nm. The calculation was performed according tothe calibration curve.To assess the antioxidant activity of the sublimates,we measured the radical absorption and the degree ofinhibition of lipid peroxidation (LPO). To assess theradical absorption, the dry oyster mushrooms weremade into powder. Then the powder was extracted withbidistilled water in a shaker at 50–60°C for 3 h. Therotation speed was 190 rpm. After that, the material wasfiltered as described in [32]. The antioxidant activity ofthe extract was determined in vitro using the OxiStattest system (Institute of Bioorganic Chemistry, NationalAcademy of Sciences of Belarus). It was a one-stageassessment 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•+, theoptical density of the solution of the cation radical felldown to 600–800 nm in proportion to the concentrationand activity of the antioxidant. The optical density wasmeasured using a spectrophotometer at a wavelength of675 nm. The optical path length of the cuvettes was 1.0 cm.To provide a quantitative assessment of theantioxidant activity, we used trolox, i.e. a standardantioxidant, which is a water-soluble analogue ofvitamin 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 mgof trolox equivalent per gram of dry matter (mg TE/g)To evaluate the LPO inhibition activity, 0.1 g ofpowdered dry mushrooms was added to 2.0 cm3 ofbidistilled water. After 24 h of maceration at roomtemperature, the extract was filtered and centrifuged at1300 rpm for 10 min. The LPO inhibition activity of theobtained extract was measured in an oleic acid emulsionsystem according to the slightly modified proceduredescribed in [34]. 0.1 cm3 of the extract was added to4.0 cm3 of phosphate buffer (50 mM, pH 7.0), and0.1 cm3 of oleic acid was added to 4.0 cm3 of ethanol(95 wt%, aqueous solution). The total volume wasbrought to 10.0 cm3 with distilled water, mixed in asealed conical tube with a screw cap, and incubated at40°C in the dark for 7 days. The oxidation state wasevaluated using iron thiocyanate at 24 h intervals. Thereaction solution (100 μL) was mixed with 4.7 cm3of ethanol (75 wt%, aqueous solution), 0.1 cm3 of anammonium thiocyanate aqueous solution (30% w/v), and0.1 cm3 of an iron chloride (II) solution (20 mM in 3.5%(v/v) HCl). After 3 min, the absorbance was measured ata wavelength of 500 nm using a UV spectrophotometer.An increase in optical density meant an increase in thelevel of oleic acid oxidation. Trolox (0.95 mmol/dm3)was used as a reference. The blank sample containeddeionised water instead of the extract.When calculating both the antiradical activity and theactivity of LPO inhibition, we took into account the factthat the extracts had their own colour, which absorbed aparticular wavelength in the visible spectrum.The catalase activity of oyster mushroom sublimateswas measured using a modified technique based onthe Beers and Sizer spectrophotometric method [35].378Piskov S.I. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 375–386This fast and accurate analysis presupposes a hydrogenperoxide dehydrogenation and determination of its lossat λ = 240 nm. The powdered oyster mushrooms wereweighed into portions of 0.020–0.025 g and extractedwith 5.0 cm3 of chilled 50 mM phosphate buffer withpH = 7.0 for 15 min in the cold with periodic stirring.The extract was filtered and centrifuged at 4°C for20 min and acceleration of 1200 g. The experimentalsolution contained 1.0 cm3 of 50 mM phosphate bufferwith pH=7.0 and 1.0 cm3 of 0.1% hydrogen peroxide. Itsoptical density (D1) was measured at 240 nm relativeto the control solution, which contained 2.0 cm3 of50 mM phosphate buffer. After filtration, 0.1 cm3 ofthe sublimate sample extract was introduced into theexperimental sample. In addition, 0.1 cm3 of extract wasadded to the control solution. The optical density of theexperimental solution was determined after 30 s (D2).The enzymatic activity was calculated for 1 mmolof substrate (Н2О2) split in 1 min with 1 g of sublimatesample 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 peroxidesolution before the extract was introduced;D2 – optical density of the hydrogen peroxidesolution after incubation with the extract;V1 – total extract volume, cm3;n – amount of hydrogen peroxide introduced, mmol;m – weight of oyster mushroom sublimate in theextract, g;V2 – volume of extract for analysis, cm3;t – incubation time, min.The anti-inflammatory activity of the oystermushroom sublimates was determined in vitro. Itemployed the method used for assessing the osmoticresistance of erythrocyte membranes [36]. The drymushroom sublimate was made into powder, suspendedin distilled water at a concentration of 5.0 mg/cm3,and incubated at 4°C for 12 h. The suspension wascentrifuged at 7000 rpm for 10 min, after which thesupernatant was filtered. Blood was obtained fromhealthy white laboratory Wistar rats and mixed in a 1:1ratio with Alsever solution. The latter contained equalvolumes of aqueous solutions of 2% dextrose, 0.8%sodium citrate, 0.5% citric acid, and 0.42% sodiumchloride. The resulting solution was centrifuged at4000 rpm for 10 min. The precipitated cells werewashed with physiological saline and centrifuged threetime until the red blood cells were 10% by suspensionvolume in physiological saline. The extracts of oystermushroom sublimates were separately mixed with1.0 cm3 of phosphate buffer, 2.0 cm3 of hypotonicsodium chloride solution (0.42%), and 0.5 cm3 of redblood cell suspension. The control sample contained2.0 cm3 of distilled water instead of the hypotonicsolution. The mixes were incubated at 37°C for 20 minand centrifuged at 3000 rpm.After that, the supernatant liquid was decanted,and the haemoglobin content was estimated using aspectrophotometer at λ = 560 nm. The percentage ofresistance of red cell membranes was assessed basedon the fact that the haemolysis obtained in the controlsample was 100%. It was calculated by the formula:Percentage of resistance = 100 – (optical densityof the experimental sample/optical densityof the control sample) × 100To assess the thrombolytic activity of the sublimates,blood obtained from white Wistar rats was distributedinto 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 completelyremoved without disturbing the clot, and each tube wasagain weighed to calculate the weight of the clot. 100 μLof sublimate extract was added into each tube with a preweighedclot. All tubes were incubated at 37°C for 90 min.After incubation, the released liquid was removed, andthe tubes were weighed again. The difference in weightbefore and after clot dissolution was expressed as apercentage [37].The content of substances and their activity wereexpressed in terms of absolute dry raw materials. Allquantitative parameters were triplicated. The resultswere recorded as arithmetic mean ± standard error ofthe arithmetic mean (M ± m) and subjected to statisticalprocessing using the method of one-way ANOVA testand the Biostat software (version 4.03). The significanceof the differences was measured at P ≤ 0.05.RESULTS AND DISCUSSIONA single-phase ANOVA was conducted to comparethe quantitative values of the properties responsible forthe antiatherogenic potential of the freeze-dried oystermushroom product. It also made it possible to checkwhether there was any significant difference in theseproperties after various pre-treatment methods.As a potential antiatherogenic product, the oystermushrooms were checked for the concentration oflovastatin. This natural statin reduces the productionof endogenous cholesterol as it inhibits the activity ofhydroxyl-3-methyluracil-coenzyme reductase [38]. Theexperiment took into account the level of antioxidant andcatalase activities that resist the accumulation of excessreactive oxygen. Together with excessive lipids in theblood, reactive oxygen is known to cause atherosclerosis[39]. We tested the abilities of freeze-dried oystermushrooms to inhibit the inflammation and thrombosis.They are considered the key pathogenetic mechanismsof atherosclerosis as they facilitate the transformation ofrisk factors into morphological changes [40].A set of experiments was performed to define theeffect of disinfection, cryoprotection, and variousdegrees of preliminary grinding on the safety andactivity of the abovementioned properties.379Piskov S.I. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 375–386Thickness, shape, and volume ratio of samplesare known to affect the drying rate and quality of thefinished product [41].The whole dried oyster mushroom fruit bodiesselected as control sample were tested for theabovementioned properties. The obtained quantitativeindicators appeared to be comparable with the data forPleurotus mushrooms presented in previous studies(Table 1). When the fruit bodies were ground to pieceswith side sizes of 2.0–2.5 cm and 0.5–1.0 cm at the pretreatmentstage, it did not affect the concentration oflovastatin in the freeze-dried product. However, thecontent of lovastatin in the homogenised sublimatesturned out to be 45% less than in the whole-driedsamples. The technological process of homogenisationprobably reduced the degree of heterogeneity of thedistribution of chemicals and phases by volume. Itmight have changed the sensitivity of lovastatin to theconditions of the subsequent stages of lyophilisation.In addition, homogenisation is known to cause a shiftin the pH of raw materials. According to Piecha, whenpH of the medium increases, the lactone structures ofstatins can be partially or completely converted to thecorresponding forms of hydroxyacids [42].The assessment of the effect of grinding size onthe preservation of antioxidant properties had a similarresult. Homogenization reduced the activity of radicalabsorption by almost 46%. The homogenised samples alsodemonstrated minimal LPO inhibition activity (Fig. 1).The data were consistent with some studies thatfeatured the effect of homogenisation during freezedryingof berries [19]. According to Paciulli et al., theresults may be explained by the fact that large tissuedamage caused a loss of antioxidant substances [48].However, the preliminary grinding of oystermushroom affected neither catalase nor antiinflammatoryproperties of its sublimates.Pleurotus mushrooms owe their thrombolyticproperties to the high level of biosynthesis of theprotease enzyme complex. Proteases have an affinityfor fibrin and cause its lysis [49]. The thrombolyticproperties of freeze-dried oyster mushrooms dependedon the degree of grinding at the pre-treatment stage: theexperiment showed a statistically significant decreaseas the fruit bodies were ground into smaller pieces.The samples subjected to preliminary homogenisationdemonstrated the lowest thrombolytic activity. Suchresult might be connected with the fact that cellulardisruption facilitates interaction between proteolyticenzymes and extracellular protease inhibitors.Although pre-treatment grinding may facilitatethe drying process, it proved irrational in terms ofpreservation of lovastatin and other antioxidant andthrombolytic 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 absorptionactivity, mgTE/gCatalase activity,mmol/g·minAnti-inflammatoryeffect, %Thrombolyticactivity, %1 Whole fruit bodies(control sample)316.2 ± 8.3a 9.6 ± 0.3a 17.9 ± 0.6a 34.6 ± 0.8a 15.2 ± 0.4a3 Pieces with sidesize 2.0–2.5 cm305.6 ± 7.6a 9.1 ± 0.3a 18.7 ± 0.6a 35.1 ± 0.7a 14.1 ± 0.3a4 Pieces with sidesize 0.5–1.0 cm298.4 ± 6.1a 9.4 ± 0.4a 17.3 ± 0.5a 36.4 ± 0.9a 12.4 ± 0.3b5 Homogeneous state 174.8 ± 4.2b 5.2 ± 0.2b 16.9 ± 0.5a 34.1 ± 0.8 a 9.9 ± 0.2c6 Sources 50.0–505.0 (Pleurotusostreatus) Gunde-Cimerman et al. [43];165,3–606,5Chen et al. [44]0.61–14.07(Pleurotuscitrinopileatu)Nattoh et al. [45]14.66 (PleurotusOstreatus)Susmithaet al. [46]18.66–43.50 (Pleurotusflorida) Pandimeenaet al. [47];54,33–85,12 (Pleurotusflorida) Varghese et al. [36]18.62(Pleurotusostreatus)Islam et al. [8]Mean values with different letters in the same column are statistically different (Р < 0.05). mgTE/gFigure 1 Effect of preliminary grinding on the LPO inhibitionof the freeze-dried product. (Note: in Figs. 1–3, a lower opticaldensity at 500 nm corresponds to a higher LPO inhibition)0.00.51.01.52.02.50 1 2 3 4 5 6 7Optical density at λ = 500 nmIncubation period, daysNo treatment 2.0–2.5 cm pieces0.5–1.0 cm pieces Homogeneous sampleTrolox Blank sample0.51.01.52.02.5Optical 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)380Piskov S.I. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 375–386Food security is as important as its nutritional andbiological value. Microbiological contamination is anindicator of food security. Therefore, disinfection isa necessary pre-treatment stage. Blanching and hotair treatment had no statistically significant effect onthe content of lovastatin in the finished sublimatesif compared with the control samples (Table 2).UV disinfection may cause photodegradation of statins.However, it also demonstrated no significant changesin the concentration of lovastatin in the sublimates. Theonly difference was a slight decrease in the content oflovastain, which is consistent with the results obtainedby [42], according to which lovastatin proved to be themost UV resistant statin.The ozonation resulted in a significant loss oflovastatin. Its concentration in the sublimates decreasedby 31.4% compared to the control samples, whichconfirmed the data published in [50], according to whichoxygen makes lovastatin instable.UV disinfection and ozonation resulted in a higherradical absorption and LPO inhibition (Fig. 2). Suchresults are consistent with other studies [51–53] thatproved a better preservation, and sometimes even anincrease, of antioxidant substances in mushrooms andfruits after ozonation and UV disinfection. According toSudheer et al., ozone can trigger the formation of suchsecondary metabolites as phenols and flavonoids [54].Hot air disinfection caused no statistically significantchanges in the antiradical activity and LPO inhibition(Fig. 2). These results contradicted with those describedin [55]. On the one hand, the effect might be explainedby the thermally induced extraction of previouslybound or polymerized molecules of antioxidants, inparticular, phenols. On the other hand, it may be due tothe inactivation of enzymes involved in their catabolism,as demonstrated by recent studies of vegetable dryingprocesses [56]. In addition, the obtained results might beexplained by the fact that hot air treatment can triggerthe formation of new compounds, e.g. Maillard reactionproducts, which possess good antioxidant properties [57].The blanching produced a significant decrease inthe antiradical activity of the sublimates. Its valuewas 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., leachingand a low ability to absorb oxygen radicals resultedin a lower concentration of antioxidant substancesafter preliminary blanching. In addition, the blanchedsublimates showed a minimal LPO inhibition [58, 59].Various disinfection methods produced differentresults 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], accordingto which a higher drying temperature reduced the residualactivity 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 offruits after ozonation. The increase was explained bythe fact that ozone came into contact with the biologicaltissue of mushrooms and caused oxidative stress, whichwas accompanied by activation of various antioxidantenzyme systems, e.g. catalase.Contrary to previous assumptions [63], theblanching caused no changes in the level of catalaseactivity. According to Egbebi et al., blanching ofmushrooms inactivated catalase. In our opinion, it canbe explained by the relatively short blanching time [63].This presumption corresponds with the observationspublished in [64], which described catalase inactivationonly in cases when blanching lasted > 10 min.Various disinfection methods produced variouseffects on the anti-inflammatory activity of the product0.00.51.01.52.02.50 1 2 3 4 5 6 7Optical density at λ = 500 nmIncubation period, daysNo treatment 2.0–2.5 cm pieces0.5–1.0 cm pieces Homogeneous sampleTrolox Blank sample0.00.51.01.52.02.50 1 2 3 4 5 6 7Optical density at λ = 500 nmIncubation period, daysNo treatment UV Ozonation BlanchingNo treatment Trolox Blank sampleTable 2 Effect of various methods of preliminary disinfection on the bioactive properties of freeze-dried product (M ± m)№ Disinfection Lovastatin, % Radical absorptionactivity, mgTE/gCatalase activity,mmol/g×minAnti-inflammatoryaction, %Thrombolyticactivity, %1 No treatment (control sample) 316.2 ± 7.9a 8.8 ± 0.3a 16.0 ± 0.4a 35.2 ± 0.8a 15.7 ± 0.4a2 UV 308.4 ± 8.1a 10.3 ± 0.4b 15.6 ± 0.4a 65.1 ± 1.6b 14.8 ± 0.4а3 Ozonation 217.4 ± 7.1b 9.4 ± 0.3b 21.6 ± 0.5c 6.3 ± 0.5с 23.9 ± 0.6b4 Blanching 311.3 ± 8.1a 4.6 ± 0.2c 17.1 ± 0.5а 33.2 ± 0.8a 16.1 ± 0.5a5 Hot air 299.4 ± 6.5a 8.4 ± 0.3а 1.6 ± 0.1b 36.4 ± 1.0a 10.6 ± 0.3сMean values with different letters in the same column are statistically different (P < 0.05)Figure 2 Effect of various methods of preliminary disinfectionon 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 sample381Piskov S.I. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 375–386that had undergone a spray-freeze drying. The antiinflammatoryproperties were determined using theerythrocyte membrane stabilisation test. The UV-treatedsamples showed the highest anti-inflammatory activity.Mushrooms owe most of their anti-inflammatoryproperties to polysaccharides, especially glucans [65,66]. Thus, UV treatment served as an elicitor thatincreases production of extracellular polysaccharidesin mushrooms. In addition, UV treatment might haveproduced phenolic compounds that produce a protectiveeffect on biological membranes [67].The ozonation resulted in the lowest antiinflammatoryactivity. This effect might havebeen caused by ozone-induced oxidative reactions.According to Mzoughi et al., ozone-induced oxidativereactions lead to the selective depolymerisation ofpolysaccharides, followed by a possible increase or,conversely, a decrease in their biological activity [68].The thrombolytic properties also proved to dependon the methods of preliminary disinfection. Thus, themaximum thrombolytic properties were manifestedin the ozonised samples. This result might have beencaused by the ability of ozone to inactivate proteaseinhibitors [69]. The minimal thrombolytic propertieswere detected in the samples that had been treated withhot air. According to Ali et al., protease inhibitors inmushrooms are thermally stable [70]. According to Raiet al. and de Castro et al., proteases demonstrate themaximum activity at 55–60°С and may be wasted on theautohydrolysis of proteins heating [71, 72].Freezing is an obligatory stage of freeze-drying.Freezing can damage the cell structure with icecrystals. The degree of damage depends on the size ofthe crystals and the heat transfer rate. It can affect therheological and textural properties of the product, aswell as redox processes in favour of oxidation. As aresult, the number and the biological activity of thesubstances in the product may change. Therefore, astabilising cryoprotector should be applied beforefreezing. Cryoprotectors maintain the quality of thefrozen product by increasing the reversibility of theprocess and preserving BAS [73]. In this regard, weassessed various natural cryoprotectants and their effecton the anti-atherogenic properties of the freeze-driedoyster mushrooms (Table 3).The chemical analysis demonstrated that thecryoprotectors had a different effect on the amountof lovastatin in the mushroom sublimates. Thesamples pretreated with a 1.5% solution of pectinshowed no statistical changes in the concentrationof oxidation-sensitive natural statin. However, therewas a clear tendency to its increase. It confirmed theresults published in [74], which proved that insolublepolysaccharides effectively inhibit oxidation processesin frozen semi-finished products.The samples pretreated with a 5% sorbitol solutiondemonstrated a 47.6% decrease in lovastatin. Thesamples pretreated with a 10% sucrose solution and a10% lactose solution appeared to contain no lovastatine.It was probably due to the hydrolysis of lovastatine in theaqueous medium of the cryoprotectors. Thus, freezingdid not prove to be a limiting factor with respect tolovastatin concentration. Hence, cryoprotectors are notobligatory in this aspect.1.5% pectin solution proved to be the bestcryoprotector for oyster mushrooms as it ensuredthe maximum preservation of antiradical and LPOinhibition (Fig. 3). The results were consistent with [75],according to which pectin has a greater water absorptioncapacity compared with sorbitol and monosaccharides.The results may be attributed to the antioxidantproperties of pectin itself, since its diffused part couldenhance the antioxidant properties of the obtaineddry product. According to Kopjar et al., if added tobioactive substances containing phenolic compounds,pectin provides a synergistic effect on their antioxidantproperties [76].10% solutions of lactose and sucrose resulted in asignificant decrease in the level of antiradical activityand LPO inhibition. On the one hand, these results maybe explained by the extraction of antioxidant substancesinto the aqueous solutions of the cryoprotectors. On theother hand, the decrease might have been caused bythe cryoprotective effect itself, since it reduces both icecrystal formation in the mushroom tissue matrix anddamage to the cell structure. According to Yang et al.,cryoprotective effect increases extraction of antioxidantsubstances, e.g. phenolic compounds, from cells [77].Cooling is known to reduce the catalase propertiesof certain substances [78]. However, catalase activitydecreased significantly in all the experimental samples.Its value was minimal in the sublimates pretreated witha 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/kgRadical absorptionactivity, mgTE/gCatalase activity,mmol/g·minAnti-inflammatoryaction, %Thrombolyticactivity, %1 No treatment (control sample) 310.3 ± 7.6a 7.9 ± 0.3a 17.1 ± 0.5a 30.3 ± 0.9a 14.9 ± 0.4a2 10% sucrose solution – 5.4 ± 0.2b 9.5 ± 0.4b 29.4 ± 0.8a 15.4 ± 0.4a3 10% lactose solution – 5.6 ± 0.2b 2.9 ± 0.1c 56.4 ± 1.5b 16.0 ± 0.5a4 1.5% pectin solution 339.2 ± 8.5a 15.5 ± 0.5c 7.0 ± 0.3d 31.8 ± 1.0a 14.6 ± 0.3a5 5% sorbitol solution 152.4 ± 5.9b 7.5 ± 0.3a 3.1 ± 0.1c 28.6 ± 1.1a 25.1 ± 0.6bMean values with different letters in the same column are statistically different (P < 0.05)382Piskov S.I. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 375–386The maximum anti-inflammatory activity wasmanifested in the sublimates pretreated with a 10%lactose solution. The results confirmed those publishedin [79], according to which lactose proved to be a moreadvantageous cryoprotector than sorbitol or sucrosewhen used in freeze-drying of liposome preparations. Itcan be explained by the fact that disaccharides producea greater stabilising effect on cell membranes duringfreezing than other cryoprotectors, thereby preservingpolysaccharides and glycoproteins of cell membranes.As for the thrombolytic properties, sublimatespretreated with sorbitol showed the best results. Unlikemono-, di-, and oligosaccharides, sorbitol can penetrateinto cells [80]. It protects intracellular proteases andtheir fibrinolytic properties from possible denaturationcaused by low temperature.CONCLUSIONA set of biochemical experiments was performedto study the effect of various pre-treatment conditionson the biologically active properties that providethe antiatherogenic potential of freeze-dried oystermushrooms. The antiatherogenic properties under studyincluded the content of natural statin (lovastatin), aswell as antioxidant, catalase, anti-inflammatory, andthrombolytic properties. The results showed that eachpre-treatment method produced a different effect on theabovementioned properties of the freeze-dried product.The experiments demonstrated that the best resultsfor lovastatin were obtained when the raw material wasground to pieces with a side size of ≥ 0.5 cm, subjectedto UV disinfection, blanched, treated with hot air, andcryoprotected with a 1.5% pectin solution.As for the antioxidant properties, such as radicalabsorption and LPO inhibition, the best conditionsincluded UV disinfection, ozonation, and cryoprotectionwith a 1.5% pectin solution. Homogenisation, blanching,and cryostabilisation with 10% solutions of sucrose andlactose were found critical for antioxidant properties.The catalase activity of the product did not depend onthe degree of grinding, blanching, and UV disinfection.It was maximal after ozonation. The list of critical pretreatmentconditions included hot air treatment andexposure to all the cryoprotectors except pectin.The anti-inflammatory properties were bestpreserved after UV disinfection and cryoprotection witha 10% lactose solution. Ozonation appeared to be theonly critical pre-treatment factor.The best results for thrombolytic properties wereobtained when the oyster mushrooms were ozonatedand cryoprotected using a 5% sorbitol solution. Criticalfactors included homogenisation and hot air treatment.Thus, the experiments revealed advantagesof individual pre-treatment conditions and theircombinations. The applied conditions can turn freezedriedoyster mushrooms into a functional food productor ingredient. The new functional product significantlyimproved the properties that affect such pathogeneticfactors of atherogenesis as hyperlipidemia, oxidativestress, inflammatory reaction, and thrombosis.CONFLICT OF INTERESTThe authors declare that there is no conflict ofinterests related to this article.