PSZCZELNICZE ZESZYTY NAUKOWERok XXIX 1985

THE EFFECT OF PROPOLIS EXTRACTS CONTAININGFLAVONOID COMPOUNDS ON ACID-RESISTANT

SAPROPHYTIC BACILLI

Z b ig n li e 'w J ó ź w i k, and J o l a n t a T r y t e kDepartment of PlantPhysiology,

M a r i a C u r i e - S k ł o d o w s k a U n i ve r s i t y, L u b li n

INTRODUCTION

Water and alcoholextracts 0If propolis and its various productsshowbacteriostatic action on tubercle bacilli (K i wał k i n a 1959, Kar m i o-wa 1963, Lindenfelster 1968, Jóźwiket al. 1976). F'lavonoidsand their derivatives eontained in propolis constitute a large group ofbiologically activecompou:nds {P o p r a w k o et al. 1969). These sub-stances are also characterized by antibacterial and antiviral action (W i 1-s k a - Jesz k a 1959).

The effect of flavonoid eompounds present in propolis extraets on thegrowth of tubercle bacilli has not been studied so far. The action ofextraets from propolis, obtained by using various organie solvents onthis group of bacteria was studied i:n this paper. Speclal attention wasgiven to. fractions containing flavonoid compounds.

MATERIAL AND METHODS

1. Propolis samplos were obtained direetly from bee-keepers in theLublin province and were also purehased in an apiarian shop in Lublin.

2. Saprophytie strains of aeid-resistant baeillli; Mycobacterium sme-gmatis, Mycobacterium butyricum, Mycobacterium phlei, Mycobacteriumsp. 607 and Mycobacterium sp. 279 were from the Museum of Strainsof the Department of Plant Physiology, Maria Curie-Skłodowska Uni-versity, Lublin.

3. Preparatlon of ethanol propolis extraet (EPE). Propolis was pum-fied from aecidental impurities cuch as wood, straw fragments, insectand wax remains. Then 10% solutlon was made in 96% ethanol. It wasshaken for half an hour and left in a shaded place over 24 h. Thiisprocedurę was repeated three times. After three days the extract was

47

Iiltred on filter paper Whatman No 4. The obtained ethanol propolisextract (EPE) was eondensed to a thiek mass on water Ibath in vacuum,which hardened after cooling.

From thus prepared extract 10% propolis solutions were made in96% ethanol. The activity of 10010 EPE was examined in relation to My-cobacterium sp. by the cylinder method. All propolis samples used inthe studies inhibited the growth of 'tubercle bacilli.

4. Obtaining a uniform bacterlal mass. Saprophytdc acid-resistantbacilli were cultivated on Sauton's medium for 4 days. Then the bac-teria were collected with on ooze into previously sterihzed and weighedbacteriological mortars, The mortars with the bacteria were weighedagain, Frive ml of bacilli suspenslon at a eoncentration of 1 mg/mi pre-pared in Sauton's medium was introduced into 200 ml of Sauton mediumwith agar (1,4010). Heated medium with bacilli was poured into Petridishes on which glass cylinders had .been set up.

5. Examination of propolis activity by the cylinder-płat e method.Three-five glass cylinders 7 mm in outer diameter were set up on Petridishes into each of thern i15 ml Santon medium with 1,4% agar andsuspended saprophytic tubercle bacilli were poured, When the mediumhardened the cylinders were r emoved and the formed wells were dis-pensed each 0,1 ml of 10% propolis extracts in 96010 ethylene alkohol.The plates were kept for 6 h' at room temperature for the substancestudied to diffuse into the medium. Next, the plates were put into atherrnostate (37°C).

The results were ehecked after four days. In the case of active pre-parations zones free of bacteria, the so-called growth inhibition zones,were formed round the wells, which were measured in mm.

6. Separatlon of flavonoid compounds, F'lavonoid eompounds wereseparated from EPE by the methods used to isolate these compoundsfrom alcehol plant extracts (Jerzmanowska 1967, Strzeleckaet al. 1978). The author's own modifications were alsoused.

The separatdon was carried out aceording to three schemes,Scheme I: lOg of EPE condensed ot soild mass was leached three

times with distilled water heated to 50°C, u:sing ,250 rnl of water eachtime. A milky white ernulsion and dark brown sedimant were obtained.The sedirnent contained waxes, resins and tars and did not show anti-bacterlal activity.

The ernulsion was poured into a flask and left in a ,ref~jgeratO'l'{4°C)for 48 h. A Iight-yellow sediment (O)precipitated after this time. 10%solutions in 96% ethanol were made from the sediment. The supernatantwas extracted with ethylacetate in 100 rnl portions. This extraction wasperformed several tirnes until the ethyl aeetate layer eoloured. The wa-ter layer being inactive was discarded, The solvent was separared fromeombined ethyl-acetate extraets under reduced pressure, From the dryresidue 10% solutlon was made in 96% ethanol. The activity of the

48

sediment (O) dissolved in ethanol as well as ofethyl aeetate fractionwas examined for tubercle bacilli (Table I).

Scheme II: 10 g of condensed EPE was dissolved in 100 ml of 960/0ethanol and iextrected with ethyl acetate mixed at 1: 2 ratio (200 mlof ethyl aeetate + 400 ml water). Two fractions were obtained: lightyellow water-alcehol and brown ethyl acetate (top layer). A yellowsediment (Oj) precipitated then from which 100/0solutions were madein 960/0ethanol.

The water-alcehol layer was evaporated to dryness. A sediment (02)

was obtained from which 10% solutions were also made in 96% ethanol.Both sedirnents were tested for biological activity in the presence ofMycobacterium sp. (Table I).

Scheme III: 10 g of condensed EPE was subsequently extracted withorganie solvents of increasing polarity: 15 ml of etherbenzene (1: 2),150 ml of benzene, 150 ml of carbon tetrachloride, 150 ml of chloroform

Table 1The activity of propolis fractions in relation to acid-resistant saprophytic bacilli

(cylinder-plate method)Aktywność frakcji propolis owych w stosunku do saprofitycznych prątków kwaso-

opornych (metoda cylinderkowo-płytkowa)Strains

SzczepyGrowth inhibition zones in mm

Strefy zahamowania wzrostu w mm

Myc. Myc. Myc. Myc. Myc.Fractions smeg- buty- phlei sp. sp.Frakcje matis ricum 279 607Sediment (O) 16 16Osad (O) 17 25 17Ethyl acetateOctan etylu 19 22 22 32 24

Ethyl acetateOctan etylu 19 20 18 29 23Sediment (Oj)

Osad (Oj) 16 15 16 20 18Sediment (02)

Osad (02) 18 17 19 25 26Ether-benzeneEther-benzen 18 21 20 31 20BenzeneBenzen 16 19 18 28 22Carbon tetrachloride 16 18Czterochlorek węgla 17 20 18Chloroform 17 17Chloroform 16 21 17Acetone 9 13Aceton 8

The wella were dispensed each 0,1 ml ol 10'/, solution of the particular fractions.Do iltudzienek wprowadzono 0,1 rnl roztworów 10'/, poszczególnych frakcji.

4 - Plzczelnicze zeszyty Naukowe - r. XXIX 49

and 150 mI of acetone. Five fracctions were obtained from whieh 001-

vents were evaporated under reduced pressure, and from the obtainedsediments 10°/0 solutions were made in 96°/0 ethanol. The activity 01these fractions was examined for tubercle bacilli (Table I).

From all fractions obtained 'On the Ibasis of the above sehemes 100/0solutions were rnade in 96°/0 ethyl aleohol, carrying 'Out the Iollowingdeterminations for these Iractions: the activity in relation to saprophyticacid-resistant bacilli, the amount of flavonoids by the method ofC h r i s t - M li 11 e r, and measurements of Iight absorption in the range250-450 nm on a spectrophotometer. UV VIS Carl Zeiss, Jena, GDR.Moreover, qualitative tests were made for the presence of eompoundsfrom the group of flawo:noids, and fractions were separated using thin--Iayer chromatography.

7. Qualitative tests for the presenee of flawonoids, They were carriedout :in ald fractions obtained kom the schernes I, II and III (J e r z m a-n o w s k a et al. 1969).

8. Quantitative determinations of flavonoids. F'lawonoids were de-termined.by C h r is t - M li 11 e r''s method (Strzelecka et al. 1978) orni-titing the initial stage of material preparatlon because of the fact thatpropolis was purified earlier.

The 25 cm" measuring flasks were dispensed 10 ml of the solutioneach, adding ,2 ml of .2°/0aluminium chloride (2 g of AlOls' 6 HzO wasdissolved in 10 rnl of acetie acid- ethanol mixture 5: 10), and toppędup to the scalę mark with acetic acid ~ ethanol mixture (5 : 100). Atter45 min the solutlon extinction was measured agamst the blank (aceticacid: ethanol, 5: 100) at the wave length 425 nrn 'Ona photocolorimeterSpecol. The amount of flavonoids in the particular fractions was calcu-lated from the analytical curve of quercetin. The eontent of flavonoidsin the particular fractions and the bićlogical activity of these fractionsin relatiori to Mycobacterium sp. 279 are presented in Table II and inthe diagram.

9. Separatlon of propolis extraets by thin-layer chromatography,The compounds oecurring in propolis extracts were separated 'Onplatescover ed with cellulose and silica gel. A better separatlon was obtainedusing plates with DC-Fertigplatten Plśl-Cellulose and a developer whichwas 60°/0acetic acid.

One ml of 10oio ethanol extracts IQfpropolis fractions obtained accor-ding to the schemes I, II, and III was deposited at each starting point.The separatlon time was 6-7 h. The separated Ilavonoid compoundsseen in UV Iight as coloured spots were transferred together with theadsorbent into test tubes nad flooded with 2 rnl of 960f0ethan'Ol. Theseparated compounds passed into ethanol, and the adsorbent was remo-ved by filtering the solutions on filter paper Whatman N'O4.

In thus obtained subfractions the arnount of flavonoids was deterrni-ned by the method described above, as well as light absorption in the

50

spectrum range 250-450 nm on a spectrophotometer UV VIS. Furtber-more, the activity of the partioułar subfractions wasexamined in rela-tion to Myc. sp. 279 (Table III).

RESULTS AND DISCUSSION

Basing on the data from literaturę, purification and separatlon ofoompounds from the group of flavonoids in plant material as well asusing the modyfications according to the schemes I, II and III, separa-tion of ethanol propolis extract into individual fractions was carried out.As solvents were used: water of 50°C, ethyl acetate, benzene, ether,chloroform,carhon tetrochloride and acetone. The active substancespassed Into these solvents, and the largest zones of growth Inhibition oftuberc1e bacilli were obtained, treating with ethyl acetate Iractions(scheme I and II) and with ether-benzene a:nd ,benzene Iraction- scheme

Table 2The amount of flavonoids and biological activity of propolis fractions in relation

to Mycobacterium sp. 279Ilość flawonoidów i aktywność biologiczna frakcji propolis owych w stosunku do

Mycobacterium sp. 279

Frac- Scheme I Scheme II Scheme IIItions

Frak_~--s-c-h-e7m-a-t-I--'~-----s7ch-e-m-a-t~II----I---~~--S-c7h_e_m_a_t~I~I_I__~ 1cje

Amount offlavonoidsin mg/rnlIlość fla- 1,6132 2,0551 1,7328 1,4797 1,8051 1,9461 1,9328 1,4671 1,4797 0,6799 1,6830wonoidóww mg/miGrowthinhibitionzonesin mmStrefy za-hamowaniawzrostu

TestsPróby -

25 32 20 25 3120

w mm

EPEEEP

28 20 21 l3 30

The wells were dlspensed each 0,1 ,mI ol 10'1. solution ot the parttcular fractions andalso 0,1 mI ol EPE.

Do stuózienek wprowadzano po 0,1 mI 10'1. roztworów poszczególnych frakcji a także0,1 mI 10'1. EEP.

4* 51

III. The most sensitive strain to the action of flavonoid compounds inpropolis Iractions was Myc. sp. 279. This strain was used in subsequenttests.

Propolis fractions undergomg qualitative tests for the eontent of fla-vonoids contained f1avones, flavanones and flavonoles. This was the ba-sis forcarrying out quantitative determinations for the eontent of thesecompounds in the particular fractions, The results are summarized intables.

In furtber experiments the growth inhibition zones of Myc. sp. 279were eompared with the amount of flavonoids in mg/mI of the particularfractions. JIt appeared that a proportional relationship existed betweenthe eoncentratlen of these eompounds and the biological activity measu-red by the size of the growth inhibition zones of bacteria (Table II anddiagram).

The use of the methods for separatlon of flavonoid compounds accor-ding to the schemes I, II and III made it possible to obtain propolisfractions active in relation to. saprophytic acid-resistant bacilli,

Scheme III, in which solvents of increasing polacity were used, seernsto be the simplest separatlon method of EPE into active fractions (U s z-kał o w a et al. 1973).Ethyl acetate used in scheme I and II is also a goodsolvent for these compounds. The growth irihibition zone of Myc. sp. 279for ethyl acetate fraction (scheme I) was 32 mm.

The results of an arialysis of the subfractions obtained by thin-layerchromatography, using cellulose as absorbent (DC-Ferti,gplatten PEI--Cellulose F), are suanmarized in Ta'ble III. The concentration of flavo-noids, biologreal activity in relation to Myc. sp. 279 as well as the

.----------------------_.,I

10.• Scheme I• Scheme II• Soheme III

EE 40.~II)Q)coN

Co

30

:t::.os:.~s:~oL-

U

20

•

0.5 1.0 1.5 2.0 2.5

Amounł of flavonoids in mg/ml

Fig. 1. Relationship between the concentration of flavonoids and growth inhibitionzones of Mycobacterium sp. 279

52

Table 3The effect of propolis subfractions obtained by thin-Iayer chrornatography on the

growth of Mycobacterium sp. 279Wpływ subfrakcji propolis owych uzyskanych na drodze chromatografii cienkowar-

stwowej na wzrost Mycobacterium sp. 279

Max. of light Growth inhi

Amount of flavonids absorption bition zones

rngjml (in 96 % C;,H.OH) in mm.Fraction Rf Strefy zahaFrakcje Ilość flawonidów w Max. absorpcji

mg/rnl światła mowania

(w 96 % C.H.OH) wzrostumm

Sediments (O) 0,59 0,01494 270,364 11•..... Osad (O) 0,72 0,03154 271, 324, 418 10•.....

0,82 0,06660 273, 290, 418 16....,Q) ctS

E E 0,92 0,10000 293, 19Q) Q)

Ethyl 273.<::.<:: 0,45 0,01162 9(.) (.)CIlCll acetate 0,61 0,00996 273 9

Octan 0,81 0,02990 274,288 11etylu 0,93 0,05000 290 13

Ethyl acetate 0,58 0,00996 279, 9•..... Octan etylu 0,83 0,02158 271 11•.....

0,95 0,04502 282,313, 418 13....,ctS Sediment (01) 0,54 0,01494 287, 418 9EQ) Osad (Ol) 0,68 0,01498 287, 418 10.<::o 0,76 0,01669 287, 10CIl

•..... 0,98 0,01162 272,312 9•..... 0,96 0,03800 294 12Q)

Sediment (02) 0,44 0,02155 271E 9Q) Osad (Oz) 0,58 0,02300 272 10.<::(.)

0,77 0,02820 290, 310CIl 110,89 0,02492 290,310 110,94 0,09672 287 13

Ether- 0,63 0,01494 290 9benzene 0,81 0,01660 272, 425 10

0,87 0,02820 270, 418 13Benzene 0,65 0,01162 288 10

•..... Benzen 0,81 0,02158 288 11•.....•.....0,95 0,08994 277 14

+>CIS Car bon tetra 0,61 0,01660 285 10EQ) chloride 0,73 0,03800 274 11.<::(.) Czterochlorek 0,86 0,02820 271, 289, 418 12CIl

węgla•..... Chloroform 0,55 0,00996 278 9•.....•.....

Chloroform 0,79 0,01660 286, 418 11Q)E 0,88 0,01162 289 9Q)

.<:: 0,95 0,03800 292, 314 12(.)

CIl Acetone 0,33 0,00498 2820,45 0,00664 2880,64 0,00664 280, 4180,68 0,00996 285, 418 90,72 0,00996 288, 418 11

53

.'_._-_._---

adsorption spectrum in the rangę 250--450 nm were determined in theparticular subfractions. For indlvidual subfractions absorption maximawere found, corresponding to compounds from the group of flavonoidsdescribed in the monograplues of H a rb o n e (1967) and M ark h a fi

(19&2).Compounds were also found the maxima of which deviated from

those de.scribed ID the literaturę. These compounds showed maximaat293 and 294 !!1ID. It was also found that the Iowest eoncentration offlavonoids at which inhibitiori of bacteria growth was obtained amourrted,0,00996 mg/rnl,

Basing on the obtaimed results it can be found that the presentedschemes of propolis purification allow the obtaining of biologically activefractions containing compounds from the group of flavonoids. Furtherseparation by using thin-layer chromatography results in obtaining indi-vidual compounds of this groupcharacterized by absorption maxima.

CONCL USIONS

1. F'lavonoids preserit in propolis inhibit the growth of saprophyticacid-resistant bacilli.

2. The strain Mycobacterium sp. 279 is the most sensitive to theaction of these compounds.

3. A proportional relational relationship was found between the eon-centratlon of flavonoids ,and the anhibition zones of Mycobacterium sp.279 growth.

4. The 1000estconcentration of flavonoids at which inhibition growthof bacteria was obtained arnounted 0,00996 mg/ml.

LITERATURA

1. H a r b o n e J. B., 1967: Comparative Biochemistry of the Flavonoids. AcademicPress, London, New York.

2. Jer z m a n o w s k a Z., 1967: Substancje roślinne - metody wyodrębniania.PWN, Warszawa.

3. J6źwik Z., Baraniecka-Włoszycka A., 1976: The Effect of Propolison Mycobacterium sp. Annales UMCS, sec. C, XXXI, 9, 127-134.

4. Kar i m o waZ. N., 1963: Tuberkulez legkich i propolis, Pczelowodstwo, 1,36-37.

5. K i wał k i n a W. P., 1959: Prottiwmikrobnoje dejstwije propolisa. Pczeio-wodstwo, 10, 50-51.

6. L i n d e n f e l s t er L. A., 1968: Antimicrobial Activity of Propolis. Am. Bee J.,3,90-92.

7. M ark h a m K. R., 1982: Techniques of Flavonoid Identification. AcademicPress, London, New York, Paris, San Diego, San Francisco, Sao Paulo, Sydney,Tokyo, Toronto.

8. P o p r a w koS. A., G u r e w i c z A. J., Koł o s o w H. N., 1969: Fławonoj-dny je komponenty propolisa. Chimija Prir. Sojedin. 5/6, 476-482.

9. S t r z e 1e c k a H., K a m iń s k a J., K o wal s k i J., Wal e w s k a E., 1978:.

54

Chemiczne metody badań roślinnych surowców leczniczych. PZWL, Warszawa.lO. U s z kał o w a W. H., M u r y c h n i c z T. P., 1973: Antikislitielnyje swoistwa

propolisa. Pczelowodstwo, 2, 15---16.11. W i l s k a - J e s z k a J., 1959: Chemia i własności fizjologiczne flawonoidów.

Wiad. Chem., 6 (144), XIII, 289-327.

WPŁYW EKSTRAKTOW PROPOLISOWYCH ZAWIERAJĄCYCH FLA WONOIDYNA SAPROFITYCZNE PRĄTKI KWASOOPORNE

Z. J ó ź w ik, J. T r y t e k

Streszczenie

Flawonoidy i ich pochodne wchodzą w skład propolisu jako liczna grupa bio-logicznie czynnych związków. Substancje te wykazują własności antybakteryjnei przeciwwirusowe. W pracy tej przedstawiono wpływ ekstraktów propolis owychzawierających związki z grupy flawonoidów na wzrost saprofitycznych prątków'kwasoopornych (Mycobacterium smegmatis, Mycobacterium 607, Mycobacterium279, Mycobacterium phlei i Mycobacterium butyricum). Propolis otrzymywano odpszczelarzy z makroregionu lubelskiego. Ekstrakt etanolowy propolisu (EEP) o zna-nej aktywności przeciwprątkowej poddawano ekstrakcji przy użyciu szeregu roz-puszczalników organicznych stosując trzy schematy rozdziału. EEP poddawanoekstrakcji: octanem etylu, octanem etylu z gorącą wodą oraz stosowano rozpusz-czalniki o wzrastającej polarności (eter, benzen, chloroform czterochlorek węglai aceton). W uzyskanych frakcjach oznaczono ilość flawonoidów stosując w mody-fikacji własnej, metodę Christa-Miillera. Frakcje zawierające flawonoidy poddanochromatografii cienkowarstwowej na celulozie PEI-Cellulose F stosując jako roz-wijacz 60% CHsCOOH. W otrzymanych subfrakcjach oznaczono także poziom fla-wonoidów a także sprawdzono działanie tych subfrakcji na wzrost prątków gruź-licy. Okazało się, że istnieje proporcjonalna zależność między ilością flawonoidówa wielkością stref zahamowania wzrostu saprofitycznych prątków kwascepornych.Najbardziej wrażliwym szczepem na działanie poszczególnych frakcji był Myco-bacterium sp. 279. Octan etylu jest dobrym rozpuszczalnikiem ekstrahującym zwią-zki flawonoidowe. Związki te przechodziły również do eteru, benzenu i cztero-chlorku węgla.

~JB1JIHHE 3KCTPAKTOB IIPOIIOJlMCA, CO.ll:EP)KAIll;l1X <l>JIABOHM)J;bIHA CAIlPO<I>l1TWłECKl1E Kl1CJIOTOYIIOPHbIE IIAJIOQKl1

3. I03BHK, H. TpbITeK

Pe3IDMe

B COCTaBrrponormca BXO,'\!łTcp.rraB<lHl1):\blH l1X rrponanozmsre, MHOI'O'l'liCJIeHHaHrpynna 5HOJIOrH'leCKHaKTm3HbIX COe)\:J1H€UHW.3TH Be~eCTBa o5JIa,ąaIDT aHTH5aKTe-pvtaJIbHblMHu aHT'liBnpYCHbIMlfcBoMCTBaMlf.

B HacTOII~eM pa60Te pacczrarpemaerca BJIl1.fłHl1e3KCTpaKTOBnponoaxca, conep-:lKaW;HXcoe,ąUHeID1H'li3 rpyrrrrsr cpmmoHU):\OB,Ha pocr canpoqnrrxxecxax Kl'ICJIOTO-yrropsrsrx rranoxex (Mycobacterium smegmatis, Mycobacterium 607, Mycobacteriu'Tll.279, Mycobacterium phlei, Mycobacterium butiricum).

IIPOIIIOJIHC6bIJI ,l{oC'MBJIeHrrsenoaonasor JlID5JIMHCKOroMaKpOper110Ha. Cnapro-BOM3KcTpaKT nponorraca, iH3BecT-HbIMnpoTl1BOTy6epKyJle3HOMaKTl1EHOCTbID,3KcTPa-rrrpoaazrx pJl,l{OMopranaxecxxx paCTBOpHTeJIeM,npJ1MeH'HB npx 3TOM TPH CXeM,hl

55

,lJ;eJleHMH.CIU!pTOBOH 3KCTpaKT 3KCTParJtpoBaJIH 3rnJla~eTaTOM, 3rnJI~eTaTOM c ro-pX'łeH BO,ll;OHH npx nOMO~ paCTBOPłITeJleM C B03paCTalO~eH nOJIJłl)HOCTblO (3<PI1P,6eH30H, XJIOPO<PoPM,rerpaxaopyrorepon H a~eTOH). KOJIH'leCTBO CPJIaBOHJ1,ll;OBB no-nyxenasrx Q:>paKlV1JIX onpenename npx nOMO~ MO,ll;HcpJ1U;J1poBaHHOroaBTOpaMHMeTOJ\a X. MlOJIJIepa. Coaepacanme cpJIaBOHJ1AblcppaKI.J;HHnonaeprcra TOHKOCJIOHHOI1:xposcarorparpnx Ha u;eJlJIlOJI03e PEI-Cellulose F B 600f0-OM CHsCOOH. B nony-ren-HbIX ItO,ll;Q:>paKU;J1HXonpenenaner YP<lBeHb cpJIaBOHJ1,ll;OBJ1 npOBepHJIJ1 ,ll;eHCTBJ1e3T'l1XItO,ll;cppaKu;J1ii:Ha pOICTnanonex Ty6epKYJIe3a. YCTaHOBJleHa nporropmronansnaa 3aBJ1-CJ1MOCTbMe'1K,ll;yKOJIJ1'leCTBOMcpJIaBOHH.n;OBJ1 BeJIH'lJ1HOH 30H 3aTOpMo:a<eHHHpocracanpo<pwrJ1'lecKJ1X KHCJIOTOynopHbIX naJIOfL!eK. HaM60JIee '1yBcTBI1TeJlbHbIM, K .n;e,u1-CTBHlO OT,ll;eJIbHbIX cppaK~,l WTaMMOM OKa3aJICH Mycobacte1ium sp. 279. 3THJlal\e-TaT SłBJISIeTCHxopomsos paCTBOpJ1TeJIeM,::'KcTpanfpylO~HM cpJIaBoHH.n;Hble coezraae-HJ1H. 3TH coe,ll;łfHelIJ1JI npOHHKaJlH TaKJKe B :)cpJ1p, 6eH30H H rerpaxnopyrnepon,