Imernalirmal Joe rnal of From Microhioh~,, 16 ( I~)2 ) 183- 195 t~.) 1992 El~"vier Science Publishers B.V. All rights reserved tll6g-lht)5/tl2/$(}5.110
183
FOOD 00511
Characterization and partial purification of a bacteriocin produced by L e u c o n o s t o c c a r n o s u m LA44A Rigtte L.J.M. van Laack a, Ulrich Schillinger h and W.H, Holzapfel b " Ih'lntrtment of The &'if'rice of F~anl of Animal Origia. Fucuhy o[ Vcterina~, Medicine. Uith'e~ity of Utrecht. Utrecht, The Netherlamls, and t. bt~tituW of Iiygien¢ and To.ffctdo&,v. Federal Research C('ntrt'for Nutrition, Karhnthe, (h'rmmtl'
{Received 17 February I~q2: accepted 16 April ITS2)
Twenty Leuconostm' strains i,'~dut,:d from vacuum packaged Vienna-type sausages were screened for antagonistic activity against various Gram*ptrsitive organisms (including Listeriu spp.). One of the three strains exhibiting inhibitory activity was chosen fi~r further investigation. This strain was identified as Leuc. camosum and the inhibitory substance produced w~s named carnosin. Carnosin was inactivated by trypsin but not by catalase or other non-proteolytic enzymes tested. Camnsin retained activity after heating at IIIi)°C fi)r 21) rain, whereas heating at 121°C for 15 min resulted in complete Io~ of activity. Carnosin was active at pH values ranging from 2 to 9. Carnosin activity wa,; nnl detectable until cells were in Ihe late log-phase of growth. At low temperatures (4°C), higher cell densities were required before carnosin activity could be detected. (:arnosin was active against various lactic acid bacteria, Enler~'ococs [aecafis and Entcroco¢('lls faecium and against Liswria spp. Difficulties in purification were reduced by growing Leuc. carnosum in a m~ified MRS medium, having 51)~ of Ihe normal peptone concentration and no Tween or meat extract,
Sodium d~ecyl sulphate l~)lyacwlamide gel electrophoresis of partially purified carnosin indicated that it has a molecular ma~'~ between 2510 and 61.MI0Da. Yet, retention of activity after exhaustive dialysis suggested a molecular mass > 14kDa. h is hyl~)thesized that carnnsin forms large activv complexes which can be dissociated to small (active) components. Key wolds: Bacteriocin; Listcrm: F ~ I safety; Starter culture
Introduction Lactic acid bacteria are commonly isolated from meat and meat products, These o r g a n i s m s c a n i n h i b i t t h e m i c r o f l o r a o f a p r o d u c t , t h e r e b y e x t e n d i n g shelf-life a n d i m p r o v i n g p r o d u c t s a f e t y . T h i s a n t i m i c r o b i a l a c t i o n o f lactic a c i d b a c t e r i a h a s b e e n
Correspomlence address: R.L.J.M. van Laack, Department of The Science nf Rxn:l of Animal Origin, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands
184
attributed to major metabolic end products, such as organic acids (lactic acid, acetic aeid~,, diaeetyl and hydrogen peroxide. Other compounds, such as antibiotics and bacteriocins, produced in smaller quantities, may also be involved. Baeteriocins are, by definition, proteinaceous compounds that are gcneraUy active against closely related species (Tagg et al., 1976). Such compounds may be produced by both Gram-negative and Gram-positive organisms, including lactic acid bacteria. Bacteriocin production has been observed among the lactobacilli (Ahn and Stiles, 1990a; Daeschel et al., 1990; Giraffa et al., 1989; Klaenhammer, 1988; Muriana and Klaenhammer, 1991; Rammelsberg et al., 1990; Schillinger and Liicke, 1989; West and Warner, 1988), lactococci (Davey and Richardson, 1981; Geis et al., 1983; Kojic et al., 1991), pediococci (Bhunia et al., 1988; Daeschel and Klaenhammer, 1985; Pucci et al., 1988; Spelhaug and Harlander, 1989) and carnobacteria (Ahn and Stiles, 1990b; Schillinger and Holzapfel, 1990). Recently Hastings and Stiles (1991) reported bacteriocin production by Leuconostoc gelidum; this was the first record of bacteriocin production by a Lc, zconostoc sp. of meat origin. Bacteriocins produced by lactic acid bacteria inhibit a variety of food-borne pathogens including Bacillus cereus, CIostridium perfringens, Listeria spp. and Staphylococcus aureus (Anderson, 1986; Bhunia et al., 1988; Harris et al., 1989; Hastings and Stiles, 1991; Lewus et al., 1991). This suggests that bacteriocin-producing lactic acid bacteria may be useful as natural food preservatives. The large number of reports on the occurrence of bacteriocin-producing lactic acid bacteria indicates great interest in this possibility. Not only may bacteriocins be useful in novel approaches for food preservation, they may also provide information on the strains that are sensitive to these inhibitory compounds. Information on the mechanisms by which pathogens are inhibited and on the determinants of sensitivity to a bacteriocin may prove useful in the development of more species-specific artificial preservatives. This study examined a bacteriocin produced by a Leuconostoc sp. isolated from vacuum packaged Vienna-type sausage. The characteristics of this baeteriocin and conditions for its production were investigated. Further, we report on the partial purification of this bacteriocin and discuss difficulties encountered during this process.
Material and Methods
Bacterial cultures and media Twenty strains isolated from vacuum packaged Vienna-type sausages, preliminarily identified as Leuconostoc, were obtained from Dr A. yon Holy (University of the Witwatersrand, Johannesburg, South Africa). Bacterial strains used as indicator organisms and their origins are listed in Tables I and II. Unless stated otherwise, lactic acid bacteria were propagated and maintained in MRS medium without acetate. The non-lactic acid bacteria were cultivated in standard 1 broth (E, Merck AG, Darmstadt, Germany). Carnobac-
185
terium strains were cultivated in MRS medium, modified by omitting acetate, substituting sucrose for glucose and adjusting pH to 8.5 with 10 N NaOH (D-MRS, de Bruyn, 1987).
Identification of the isolate The range of fermented sugars was determined by using the API 50 CH system (API bioMerieux GmbH, Niirtingen, Germany). Tests were incubated at 25°C and read after 24 and 48 h. DNA was isolated and purified according to a modification of the method of Marmur (1961). DNA base composition was estimated from the DNA melting temperature (Marmur and Doty, 1961). DNA-DNA hybridization was determined spectrophotometrically from renaturation rates using the modified (Huss et al., 1983) optical method of De Ley et al. (1970).
Preparation of culture supernatants Bacteriocin-producing strains were grown in MRS broth for 18 h at 25°C. A cell-free solution was obtained by centrifugation followed by filtration of the supernatant through a 0.2-/~m poresize cellulose acetate filter. Alternatively, the supernatant was heated for 10 min at 100°C. To eliminate any pH effect, the pH of the supernatant was adjusted to 6.5-7.0 with 10 N NaOH. Supernatants were stored at - 2 0 or - 8 0 ° C until analysed.
Detection of antagonistic actirity For detection of antagonistic activity, an agar spot test and a well diffusion assay, as described by Schillinger and Liicke (1989), were used. In routine tests, Lister& innoctoa WS 2258 and Lenc. mesenteroides DSM 20343 were used as indicator strains. For quantitative estimation of the inhibitory activity, two-fold serial dilutions of the supernatant (10 /~l) were spotted on an indicator lawn as described by Barefoot and Klaenhammcr (1983). One activity unit (AU) was defined as the reciprocal of the highest dilution showing an inhibition zone on the indicator lawn after 24 h at 25°C.
Lffect of heat treatment, pH and enzymes Temperature sensitivity was assessed by hcating the supernatant at 100°C for 10 or 20 min or at 121°C for 15 rain, with suhsequcnt assay of activity. Stability at pH values between 2 and I 1 was tested by adjusting the pH of the supernatant with 10 N NaOH or 10 N HCI. As a control, to correct for inhibition due to pH, a trypsin-inactivated supernatant of similar pH was tested against the indicator strain Leuc. mesenteroides DSM 20343. Inhibition by the bacteriocin was substracted by inhibition due to pH. To test enzymatic inactivation (enzymes listed in Table 111), supernatants were incubated with the enzyme at final concentrations of I m g / m l for 2 h at 37°C. Samples with and without enzyme were heated 10 min at 100°C and remaining activity was assayed. All enzyme-supernatant incubations were at pH 7.0, except incubation with pepsin which was at pH 3.0.
186
Effect o f p H and temperature on production o f inhibitory substance M R S - b r o t h was p r e p a r e d a n d the p H adjusted to 5.1, 5.5 or 6.0. Latconostoc L A 4 4 A i n o c u l u m f r o m a n overnight culture was a d d e d at a c o n c e n t r a t i o n o f I % o f the volume of M R S . Samples w e r e r e m o v e d at specified time intervals a n d assayed for bacterial g r o w t h ( O D at 600 nm a n d C F U / m l ) a n d b a c t e r i o c i n activity. T o test the effect o f t e m p e r a t u r e , M R S b r o t h was t e m p e r e d a n d i n c u b a t e d at indicated t e m p e r a t u r e s before I % o f a n overnight culture w a s a d d e d . S a m p l i n g a n d f u r t h e r d e t e r m i n a t i o n s w e r e p e r f o r m e d as d e s c r i b e d for p H effect. Preliminary characterization F o r purification, 500 ml o f modified M R S ( M M R S ; 5.0 g / I universal p e p t o n e , without m e a t extract o r T w c e n - 8 0 ) was i n o c u l a t e d with 1 ml of an overnight M M R S culture. A f t e r ca. 18 h at 25°C, cells w e r e removed by c e n t r i f u g a t i o n , p H w a s a d j u s t e d a n d the s u p c r n a t a n t w a s c o n c e n t r a t e d to ca. 10% o f the original volume by m e m b r a n e filtrati0n. T h e m e m b r a n e h a d a n exclusion limit o f 5000 Da, Proteins in the c o n c e n t r a t e w e r e p r e c i p i t a t e d by f r a c t i o n a t e d precipitation at 40, 60 a n d 8 5 % a m m o n i u m s u l p h a t e (AS). O n c e A S w a s a d d e d , the s u p e r n a t a n t w a s stirred for 1 h at ca. 20°C. T h e p r e c i p i t a t e s w e r e recovered by c e n t r i f u g a t i o n ,
TABLE
I
Inhibitory spectrum of the culture supernatant f r o m Leuc. carnrx~um LA44A Species
Strain
Lactobacillns plantarum Laclobacillus curcatus Lactobacillus sake Lactobacillusfermentum Let~ctmostoc carm)szon Lellconostoc gelidum Leuconostoc mc~olteroides Let~conostoc paramt'senteroide.~ Camobacterimn piscicola Enterococcusfaecalis Emerococcusfaeci~mt Pediococcus acidilactici Listeria innr~'aa Listeria monocytogenes Bacillus cereus Staphylococcus aurens Salmonella enteriditis Yersinia enterocolitica
DSM 20174 DSM 2U019 DSM 2(X)17 DSM 2lH)52 NCFB 2776 DSM 5578 DSM 20343 DSM 20288 DSM 20730 DSM 20409 DSM 20477 DSM 21)333 WS 2258 DSM 20600 CM 2010 ATCC 14458 MLS 5271 ATCC 27729
~'
Sensitivity to the supematant + + + + + + + + + + + -
a DSM. Deutsche Sammlung van Mikroorganismen, Braunschweig, Germany; ATCC, American Type Culture Collection, Rochville, USA: CCM. Czechoslovak Collection of Microorganisms, Brno, Czechoslowakia; NCFB, National Collection of Food Bacteria. Reading, UK: MLS, Medizinisches Landesuntersuchungsamt, Stuttgart. Germany: WS, Teehnische Unlversir;it, Miinchen-Weihenstephan, Germany
187 TABLE II Listeria spp. tested for inhibition by the eulturc supernatant fro~i'* Lt'nt'. carno~nm LAOAA Species Listeria monocyrogenes Listeria monocytogent,~ Listeria uleulo(.),togent'.5 Listeria monocytogenes Listerla seeh~'ri Listeria weL~hhneri Listeria il'anorii Listeria inn,~'na
Strain WS 2247 WS 2249 WS 2250 WS 2251 WS 2253 WS 2254 WS 2255 WS 2257
Set, type I/2C 4b 4b I/2a I/2b ha 5 6a
I~lhtles supplied and semlyped by Prof. Dr Busse, Technisehc Universit'3t, Miinchen-Weihenstephan, Germany. dissolved in 50 m M N a - p h o s p h a t e buffer, p H %0, a n d exhaustively dialysed a g a i n s t N a - p h o s p h a t e buffer. A f t e r 3 m o n | h s s t o r a g e at -800(7, the 6 0 % fraction w a s t h a w e d a n d dialysed a g a i n s t 1000 volumes o f 50 m M a m m o n i u m h y d r o g e n c a r b o n a t e for 48 h. Subsequently the dialysate w a s f r e e z e - d r i e d a n d then s t o r e d at r o o m t e m p e r a t u r e . Fifteen /~g of the f r e e z e - d r i e d s a m p l e was mixed with 50 /ti s a m p l e b u f f e r (4.6% SDS, 1 0 % / ] - m e r c a p t o e t h a n o l , 2 0 % glycerol, 1.5% Tris a n d 1% b r o m o p h e nol blue) a n d h e a t e d at 60°C for 5 rain. Eight p.I aliquots w e r e e x a m i n e d by SDS-PAGE. Polyacrylamide gel e l e c t r o p h o r e s i s in the p r e s e n c e o f S D S w a s c o n d u c t e d in a vertical slab gel by the m c t h o d o f L a c m m l i 0 9 7 0 ) . Polyacrylamide a n d N ' , N ' -
TABLE III The effect of different enzymes (incubadntt 2 h at 37°C. I mg/ml) and heat treatment on the inhibitory effect tlf carnusin 44A against Left('. mesenteroides DSM 20343 (+ indicates no inhibition of the bacleriocin activity. -indicates complete inhibition of bactcriotin activity Treatment ¢-Chymotrypsia (Scrva, 17100) Trypsin (Merck, no 8367) Pepsin (Merck, no 7189) a-Amylase (Pluka, 10070" ) a-Amylase (Sigma, type V I I - A ) Lipase (Serva, no 27930)
Phospholipase (Sigma, P7633) DNA-ase ( Fluka. 31135) Cat alase (Sigma. C- 10) Lyso~me l0 rain at 100°C 20 rain at 100°C 15 rain at 121°C
Activity
+ +
+ + + + + + +
188 methylene-bisacrylamide concentrations in the stacking gel were 5 and 1.5%, respectively. In the separating gel concentrations were 20% for polyacrylamide and 2% for bisaerylamide. After 3 h electrophoresis the gel was removed and divided in two. One half was silver stained and the other part was assayed for antimicrobial activity by the direct detection system described by Bhunia et al. (1987). To estimate molecular weight, low molecular weight peptide standards (Standard 1, Merck: myoglobin 111, M r 2512; Myoglobin 11, M r 6214; Myoglobin 1, M r 8159, Myoglobin 1 + V, M r 14404; Myoglobin, M r 16949) were used.
Results
A total of 20 Leuconostoc strains isolated from vacuum-packaged Vienna sausages (Holy et al., 1991) was screened for bacteriocin production using an agar spot test and a well diffusion assay. Three of these strains showed antagonistic activity to Lister& innocua and Leuconostoc mesenteroides. One of these three strains, designated Leuconostoc LA44A, was chosen for further experiments. Identification o f Leuconostoc LA44A Leuconostoc LA44A was identified using the diagnostic characteristics proposed by Shaw and Harding (1989) for leuconostocs. The carbohydrate fermentation pattern of strain LA44A was identical to that given for Leuconostoc carnosum (Shaw and Harding, 1989). Growth occurred at 1 and 30°C, but not at 37°C. The G + C content of the DNA was 37.7 mol%, and DNA-DNA hybridization resulted in an 99% DNA homology of strain LA44A with the type strain of Leuconostoc carnosum (NCFB 2776). The inhibitory substance produced by this strain of Leuconostoc carnosum was named carnosin 44A. Characterization of the inhibitory substance Leuconostoc carnosum LA44A showed inhibitory activity against a variety of lactic acid bacteria and against Listeria monocytogenes (Table I). However, no inhibitory zones were evident with Staphylococcus aureus, Sablionella enteritidis, Brochothrix thermosphacta or Bacillus cereus. All tested serotypes of Listeria spp. (listed in Table II) were inhibited by the carnosin. As illustrated by Table Ill, the inhibitory substance was inactivated by two proteases. Of the other, non-proteolytie, enzymes only the a-amylase preparation from Fluka affected bacteriocin activity. Catalase did not affect the inhibitory activity, indicating that the inhibition was not due to hydrogen peroxide. Carnosin activity was not affected by heating at 100*C, whereas 15 min at 121°C completely inactivated it. Data on the stability at different pH values are presented in Table IV. The bacteriocin was hardly affected by low pH. At pH above 10, however, activity appeared to decrease.
189 TABLE IV Effect o f p H on the activity of carnosin 44A and trypsin-inactivated carnosln 44A (control) against Leuc. mesenteroides D S M 20.343 pH
Carnosin 44A activity a
Control
2,0 4.0 5.0 6.0 7.0 9.0 I 1.0
512 1024 2048 1024 21148 512 4
2 0 0 0 0 (I I
;' Activity expressed in AU; one A U is defined as the reciprocal o f the highest dilution showing a definite inhibition zone on the indicator lawn (agar diffusion as.say)
As can be seen in Fig. 1, production of the inhibitory substance was detected in the late growth cycle. Carnosin production was not only dependent on bacterial growth phase but also on temperature and initial pH. Leuc. carnosum LA44A was able to grow and to produce baeteriocin in the 4-10°C range. At 10°C, maximum cell density (log 10 9.6/ml) was achieved after 72 h. 8acteriocin concentration increased until 148 h, when it was 128 AU. At 6oc, Leuc. carnosum LA44A reached a maximum cell density of about log 9 / m l after about 100 h and a maximum bacteriocin activity of 128 A U after about 200 h (Fig. 2). At 4°C there was only slow growth and bacteriocin production was extremely poor. After 200 h, only a very low level of bacteriocin activity was detectable.
10 9
LOGCFU/rnl
AU
~ E L ~ - ~ .
250
'~- . . . ~
tl
300
200
100
_2
20
40
60
80
100
120
140
100
Time (hi Fig. I.
Growth and production of carnosin44a by Leuc. carnosum LA44A in M R S 2.5~C. { ~, = growthCFU/ml, [] = bacteritx:inactivityALl).
broth, p H 6.5
at
lg0
10
LOG CFUIml
7
AU
//
//
6
~'//0 . .m
183
FOOD 00511
Characterization and partial purification of a bacteriocin produced by L e u c o n o s t o c c a r n o s u m LA44A Rigtte L.J.M. van Laack a, Ulrich Schillinger h and W.H, Holzapfel b " Ih'lntrtment of The &'if'rice of F~anl of Animal Origia. Fucuhy o[ Vcterina~, Medicine. Uith'e~ity of Utrecht. Utrecht, The Netherlamls, and t. bt~tituW of Iiygien¢ and To.ffctdo&,v. Federal Research C('ntrt'for Nutrition, Karhnthe, (h'rmmtl'
{Received 17 February I~q2: accepted 16 April ITS2)
Twenty Leuconostm' strains i,'~dut,:d from vacuum packaged Vienna-type sausages were screened for antagonistic activity against various Gram*ptrsitive organisms (including Listeriu spp.). One of the three strains exhibiting inhibitory activity was chosen fi~r further investigation. This strain was identified as Leuc. camosum and the inhibitory substance produced w~s named carnosin. Carnosin was inactivated by trypsin but not by catalase or other non-proteolytic enzymes tested. Camnsin retained activity after heating at IIIi)°C fi)r 21) rain, whereas heating at 121°C for 15 min resulted in complete Io~ of activity. Carnosin was active at pH values ranging from 2 to 9. Carnosin activity wa,; nnl detectable until cells were in Ihe late log-phase of growth. At low temperatures (4°C), higher cell densities were required before carnosin activity could be detected. (:arnosin was active against various lactic acid bacteria, Enler~'ococs [aecafis and Entcroco¢('lls faecium and against Liswria spp. Difficulties in purification were reduced by growing Leuc. carnosum in a m~ified MRS medium, having 51)~ of Ihe normal peptone concentration and no Tween or meat extract,
Sodium d~ecyl sulphate l~)lyacwlamide gel electrophoresis of partially purified carnosin indicated that it has a molecular ma~'~ between 2510 and 61.MI0Da. Yet, retention of activity after exhaustive dialysis suggested a molecular mass > 14kDa. h is hyl~)thesized that carnnsin forms large activv complexes which can be dissociated to small (active) components. Key wolds: Bacteriocin; Listcrm: F ~ I safety; Starter culture
Introduction Lactic acid bacteria are commonly isolated from meat and meat products, These o r g a n i s m s c a n i n h i b i t t h e m i c r o f l o r a o f a p r o d u c t , t h e r e b y e x t e n d i n g shelf-life a n d i m p r o v i n g p r o d u c t s a f e t y . T h i s a n t i m i c r o b i a l a c t i o n o f lactic a c i d b a c t e r i a h a s b e e n
Correspomlence address: R.L.J.M. van Laack, Department of The Science nf Rxn:l of Animal Origin, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands
184
attributed to major metabolic end products, such as organic acids (lactic acid, acetic aeid~,, diaeetyl and hydrogen peroxide. Other compounds, such as antibiotics and bacteriocins, produced in smaller quantities, may also be involved. Baeteriocins are, by definition, proteinaceous compounds that are gcneraUy active against closely related species (Tagg et al., 1976). Such compounds may be produced by both Gram-negative and Gram-positive organisms, including lactic acid bacteria. Bacteriocin production has been observed among the lactobacilli (Ahn and Stiles, 1990a; Daeschel et al., 1990; Giraffa et al., 1989; Klaenhammer, 1988; Muriana and Klaenhammer, 1991; Rammelsberg et al., 1990; Schillinger and Liicke, 1989; West and Warner, 1988), lactococci (Davey and Richardson, 1981; Geis et al., 1983; Kojic et al., 1991), pediococci (Bhunia et al., 1988; Daeschel and Klaenhammer, 1985; Pucci et al., 1988; Spelhaug and Harlander, 1989) and carnobacteria (Ahn and Stiles, 1990b; Schillinger and Holzapfel, 1990). Recently Hastings and Stiles (1991) reported bacteriocin production by Leuconostoc gelidum; this was the first record of bacteriocin production by a Lc, zconostoc sp. of meat origin. Bacteriocins produced by lactic acid bacteria inhibit a variety of food-borne pathogens including Bacillus cereus, CIostridium perfringens, Listeria spp. and Staphylococcus aureus (Anderson, 1986; Bhunia et al., 1988; Harris et al., 1989; Hastings and Stiles, 1991; Lewus et al., 1991). This suggests that bacteriocin-producing lactic acid bacteria may be useful as natural food preservatives. The large number of reports on the occurrence of bacteriocin-producing lactic acid bacteria indicates great interest in this possibility. Not only may bacteriocins be useful in novel approaches for food preservation, they may also provide information on the strains that are sensitive to these inhibitory compounds. Information on the mechanisms by which pathogens are inhibited and on the determinants of sensitivity to a bacteriocin may prove useful in the development of more species-specific artificial preservatives. This study examined a bacteriocin produced by a Leuconostoc sp. isolated from vacuum packaged Vienna-type sausage. The characteristics of this baeteriocin and conditions for its production were investigated. Further, we report on the partial purification of this bacteriocin and discuss difficulties encountered during this process.
Material and Methods
Bacterial cultures and media Twenty strains isolated from vacuum packaged Vienna-type sausages, preliminarily identified as Leuconostoc, were obtained from Dr A. yon Holy (University of the Witwatersrand, Johannesburg, South Africa). Bacterial strains used as indicator organisms and their origins are listed in Tables I and II. Unless stated otherwise, lactic acid bacteria were propagated and maintained in MRS medium without acetate. The non-lactic acid bacteria were cultivated in standard 1 broth (E, Merck AG, Darmstadt, Germany). Carnobac-
185
terium strains were cultivated in MRS medium, modified by omitting acetate, substituting sucrose for glucose and adjusting pH to 8.5 with 10 N NaOH (D-MRS, de Bruyn, 1987).
Identification of the isolate The range of fermented sugars was determined by using the API 50 CH system (API bioMerieux GmbH, Niirtingen, Germany). Tests were incubated at 25°C and read after 24 and 48 h. DNA was isolated and purified according to a modification of the method of Marmur (1961). DNA base composition was estimated from the DNA melting temperature (Marmur and Doty, 1961). DNA-DNA hybridization was determined spectrophotometrically from renaturation rates using the modified (Huss et al., 1983) optical method of De Ley et al. (1970).
Preparation of culture supernatants Bacteriocin-producing strains were grown in MRS broth for 18 h at 25°C. A cell-free solution was obtained by centrifugation followed by filtration of the supernatant through a 0.2-/~m poresize cellulose acetate filter. Alternatively, the supernatant was heated for 10 min at 100°C. To eliminate any pH effect, the pH of the supernatant was adjusted to 6.5-7.0 with 10 N NaOH. Supernatants were stored at - 2 0 or - 8 0 ° C until analysed.
Detection of antagonistic actirity For detection of antagonistic activity, an agar spot test and a well diffusion assay, as described by Schillinger and Liicke (1989), were used. In routine tests, Lister& innoctoa WS 2258 and Lenc. mesenteroides DSM 20343 were used as indicator strains. For quantitative estimation of the inhibitory activity, two-fold serial dilutions of the supernatant (10 /~l) were spotted on an indicator lawn as described by Barefoot and Klaenhammcr (1983). One activity unit (AU) was defined as the reciprocal of the highest dilution showing an inhibition zone on the indicator lawn after 24 h at 25°C.
Lffect of heat treatment, pH and enzymes Temperature sensitivity was assessed by hcating the supernatant at 100°C for 10 or 20 min or at 121°C for 15 rain, with suhsequcnt assay of activity. Stability at pH values between 2 and I 1 was tested by adjusting the pH of the supernatant with 10 N NaOH or 10 N HCI. As a control, to correct for inhibition due to pH, a trypsin-inactivated supernatant of similar pH was tested against the indicator strain Leuc. mesenteroides DSM 20343. Inhibition by the bacteriocin was substracted by inhibition due to pH. To test enzymatic inactivation (enzymes listed in Table 111), supernatants were incubated with the enzyme at final concentrations of I m g / m l for 2 h at 37°C. Samples with and without enzyme were heated 10 min at 100°C and remaining activity was assayed. All enzyme-supernatant incubations were at pH 7.0, except incubation with pepsin which was at pH 3.0.
186
Effect o f p H and temperature on production o f inhibitory substance M R S - b r o t h was p r e p a r e d a n d the p H adjusted to 5.1, 5.5 or 6.0. Latconostoc L A 4 4 A i n o c u l u m f r o m a n overnight culture was a d d e d at a c o n c e n t r a t i o n o f I % o f the volume of M R S . Samples w e r e r e m o v e d at specified time intervals a n d assayed for bacterial g r o w t h ( O D at 600 nm a n d C F U / m l ) a n d b a c t e r i o c i n activity. T o test the effect o f t e m p e r a t u r e , M R S b r o t h was t e m p e r e d a n d i n c u b a t e d at indicated t e m p e r a t u r e s before I % o f a n overnight culture w a s a d d e d . S a m p l i n g a n d f u r t h e r d e t e r m i n a t i o n s w e r e p e r f o r m e d as d e s c r i b e d for p H effect. Preliminary characterization F o r purification, 500 ml o f modified M R S ( M M R S ; 5.0 g / I universal p e p t o n e , without m e a t extract o r T w c e n - 8 0 ) was i n o c u l a t e d with 1 ml of an overnight M M R S culture. A f t e r ca. 18 h at 25°C, cells w e r e removed by c e n t r i f u g a t i o n , p H w a s a d j u s t e d a n d the s u p c r n a t a n t w a s c o n c e n t r a t e d to ca. 10% o f the original volume by m e m b r a n e filtrati0n. T h e m e m b r a n e h a d a n exclusion limit o f 5000 Da, Proteins in the c o n c e n t r a t e w e r e p r e c i p i t a t e d by f r a c t i o n a t e d precipitation at 40, 60 a n d 8 5 % a m m o n i u m s u l p h a t e (AS). O n c e A S w a s a d d e d , the s u p e r n a t a n t w a s stirred for 1 h at ca. 20°C. T h e p r e c i p i t a t e s w e r e recovered by c e n t r i f u g a t i o n ,
TABLE
I
Inhibitory spectrum of the culture supernatant f r o m Leuc. carnrx~um LA44A Species
Strain
Lactobacillns plantarum Laclobacillus curcatus Lactobacillus sake Lactobacillusfermentum Let~ctmostoc carm)szon Lellconostoc gelidum Leuconostoc mc~olteroides Let~conostoc paramt'senteroide.~ Camobacterimn piscicola Enterococcusfaecalis Emerococcusfaeci~mt Pediococcus acidilactici Listeria innr~'aa Listeria monocytogenes Bacillus cereus Staphylococcus aurens Salmonella enteriditis Yersinia enterocolitica
DSM 20174 DSM 2U019 DSM 2(X)17 DSM 2lH)52 NCFB 2776 DSM 5578 DSM 20343 DSM 20288 DSM 20730 DSM 20409 DSM 20477 DSM 21)333 WS 2258 DSM 20600 CM 2010 ATCC 14458 MLS 5271 ATCC 27729
~'
Sensitivity to the supematant + + + + + + + + + + + -
a DSM. Deutsche Sammlung van Mikroorganismen, Braunschweig, Germany; ATCC, American Type Culture Collection, Rochville, USA: CCM. Czechoslovak Collection of Microorganisms, Brno, Czechoslowakia; NCFB, National Collection of Food Bacteria. Reading, UK: MLS, Medizinisches Landesuntersuchungsamt, Stuttgart. Germany: WS, Teehnische Unlversir;it, Miinchen-Weihenstephan, Germany
187 TABLE II Listeria spp. tested for inhibition by the eulturc supernatant fro~i'* Lt'nt'. carno~nm LAOAA Species Listeria monocyrogenes Listeria monocytogent,~ Listeria uleulo(.),togent'.5 Listeria monocytogenes Listerla seeh~'ri Listeria weL~hhneri Listeria il'anorii Listeria inn,~'na
Strain WS 2247 WS 2249 WS 2250 WS 2251 WS 2253 WS 2254 WS 2255 WS 2257
Set, type I/2C 4b 4b I/2a I/2b ha 5 6a
I~lhtles supplied and semlyped by Prof. Dr Busse, Technisehc Universit'3t, Miinchen-Weihenstephan, Germany. dissolved in 50 m M N a - p h o s p h a t e buffer, p H %0, a n d exhaustively dialysed a g a i n s t N a - p h o s p h a t e buffer. A f t e r 3 m o n | h s s t o r a g e at -800(7, the 6 0 % fraction w a s t h a w e d a n d dialysed a g a i n s t 1000 volumes o f 50 m M a m m o n i u m h y d r o g e n c a r b o n a t e for 48 h. Subsequently the dialysate w a s f r e e z e - d r i e d a n d then s t o r e d at r o o m t e m p e r a t u r e . Fifteen /~g of the f r e e z e - d r i e d s a m p l e was mixed with 50 /ti s a m p l e b u f f e r (4.6% SDS, 1 0 % / ] - m e r c a p t o e t h a n o l , 2 0 % glycerol, 1.5% Tris a n d 1% b r o m o p h e nol blue) a n d h e a t e d at 60°C for 5 rain. Eight p.I aliquots w e r e e x a m i n e d by SDS-PAGE. Polyacrylamide gel e l e c t r o p h o r e s i s in the p r e s e n c e o f S D S w a s c o n d u c t e d in a vertical slab gel by the m c t h o d o f L a c m m l i 0 9 7 0 ) . Polyacrylamide a n d N ' , N ' -
TABLE III The effect of different enzymes (incubadntt 2 h at 37°C. I mg/ml) and heat treatment on the inhibitory effect tlf carnusin 44A against Left('. mesenteroides DSM 20343 (+ indicates no inhibition of the bacleriocin activity. -indicates complete inhibition of bactcriotin activity Treatment ¢-Chymotrypsia (Scrva, 17100) Trypsin (Merck, no 8367) Pepsin (Merck, no 7189) a-Amylase (Pluka, 10070" ) a-Amylase (Sigma, type V I I - A ) Lipase (Serva, no 27930)
Phospholipase (Sigma, P7633) DNA-ase ( Fluka. 31135) Cat alase (Sigma. C- 10) Lyso~me l0 rain at 100°C 20 rain at 100°C 15 rain at 121°C
Activity
+ +
+ + + + + + +
188 methylene-bisacrylamide concentrations in the stacking gel were 5 and 1.5%, respectively. In the separating gel concentrations were 20% for polyacrylamide and 2% for bisaerylamide. After 3 h electrophoresis the gel was removed and divided in two. One half was silver stained and the other part was assayed for antimicrobial activity by the direct detection system described by Bhunia et al. (1987). To estimate molecular weight, low molecular weight peptide standards (Standard 1, Merck: myoglobin 111, M r 2512; Myoglobin 11, M r 6214; Myoglobin 1, M r 8159, Myoglobin 1 + V, M r 14404; Myoglobin, M r 16949) were used.
Results
A total of 20 Leuconostoc strains isolated from vacuum-packaged Vienna sausages (Holy et al., 1991) was screened for bacteriocin production using an agar spot test and a well diffusion assay. Three of these strains showed antagonistic activity to Lister& innocua and Leuconostoc mesenteroides. One of these three strains, designated Leuconostoc LA44A, was chosen for further experiments. Identification o f Leuconostoc LA44A Leuconostoc LA44A was identified using the diagnostic characteristics proposed by Shaw and Harding (1989) for leuconostocs. The carbohydrate fermentation pattern of strain LA44A was identical to that given for Leuconostoc carnosum (Shaw and Harding, 1989). Growth occurred at 1 and 30°C, but not at 37°C. The G + C content of the DNA was 37.7 mol%, and DNA-DNA hybridization resulted in an 99% DNA homology of strain LA44A with the type strain of Leuconostoc carnosum (NCFB 2776). The inhibitory substance produced by this strain of Leuconostoc carnosum was named carnosin 44A. Characterization of the inhibitory substance Leuconostoc carnosum LA44A showed inhibitory activity against a variety of lactic acid bacteria and against Listeria monocytogenes (Table I). However, no inhibitory zones were evident with Staphylococcus aureus, Sablionella enteritidis, Brochothrix thermosphacta or Bacillus cereus. All tested serotypes of Listeria spp. (listed in Table II) were inhibited by the carnosin. As illustrated by Table Ill, the inhibitory substance was inactivated by two proteases. Of the other, non-proteolytie, enzymes only the a-amylase preparation from Fluka affected bacteriocin activity. Catalase did not affect the inhibitory activity, indicating that the inhibition was not due to hydrogen peroxide. Carnosin activity was not affected by heating at 100*C, whereas 15 min at 121°C completely inactivated it. Data on the stability at different pH values are presented in Table IV. The bacteriocin was hardly affected by low pH. At pH above 10, however, activity appeared to decrease.
189 TABLE IV Effect o f p H on the activity of carnosin 44A and trypsin-inactivated carnosln 44A (control) against Leuc. mesenteroides D S M 20.343 pH
Carnosin 44A activity a
Control
2,0 4.0 5.0 6.0 7.0 9.0 I 1.0
512 1024 2048 1024 21148 512 4
2 0 0 0 0 (I I
;' Activity expressed in AU; one A U is defined as the reciprocal o f the highest dilution showing a definite inhibition zone on the indicator lawn (agar diffusion as.say)
As can be seen in Fig. 1, production of the inhibitory substance was detected in the late growth cycle. Carnosin production was not only dependent on bacterial growth phase but also on temperature and initial pH. Leuc. carnosum LA44A was able to grow and to produce baeteriocin in the 4-10°C range. At 10°C, maximum cell density (log 10 9.6/ml) was achieved after 72 h. 8acteriocin concentration increased until 148 h, when it was 128 AU. At 6oc, Leuc. carnosum LA44A reached a maximum cell density of about log 9 / m l after about 100 h and a maximum bacteriocin activity of 128 A U after about 200 h (Fig. 2). At 4°C there was only slow growth and bacteriocin production was extremely poor. After 200 h, only a very low level of bacteriocin activity was detectable.
10 9
LOGCFU/rnl
AU
~ E L ~ - ~ .
250
'~- . . . ~
tl
300
200
100
_2
20
40
60
80
100
120
140
100
Time (hi Fig. I.
Growth and production of carnosin44a by Leuc. carnosum LA44A in M R S 2.5~C. { ~, = growthCFU/ml, [] = bacteritx:inactivityALl).
broth, p H 6.5
at
lg0
10
LOG CFUIml
7
AU
//
//
6
~'//0 . .m
