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Antibacterial activity of Lactobacillus sake isolated from dry fermented sausages
International Journal of Food MicrobioloD'. 13 (1991 ) I - 10 © 1991 Elsevier Science Publishers B.V. 0168-1605/91/$03.50
1
FOOD 00392
Antibacterial activity of Lactobacillus sake isolated from dry fermented sausages O d 6 n J. S o b r i n o , J u a n M. R o d r i g u e z , W a g n e r L. Moreira,
Mafia F. Fern~indez, Bernab6 Sanz and Pablo E. Hernhndez Departamento de Higwne y Tecnologla de los Aiimento$, Facultad de Vetermaria, Universidad Complutense. Madr~ Spain
(Received 20 September 1990: accepted 31 December 1990)
~ ~ iso~ted from S p m ~ h dry fermented sausages were u='oened for antagonistic activities under conditio~ that climinmed the effects of low pH and hydrogen peroxide. From 720 i.u~tes teated 119 were inhibitory to LactobacilluJ fermentum CEC'T285. The isolates showing the largest inh/bitory activity exhibited an a n t a ~ t i c effect against several other lactobaciifi and the selected foodborne pathoge~t Staphy/ococcus aureus and l~teria monocytogenes. Comparison of the antimimimimimimimimimim~robial spectra of the sopernatants sugl~ted that the inhibitory compounds were not identical. The imlates were tentatively characterized as Lactobaci//us sake. One of the isolates. / . sake 148 was chosen for further study. ~ c o m p o ~ excreted by L. sake 148 was active a oai~t various laotobacilll and several Gram-positive foodborne bacteria, but not against the Oram-nqlative bacteria tested. The antagonistic effects ~ almost elimimtted by treatment with pro~¢~__.~__,whereas they were heat resistant and bcgteriostatic rather than bactmocidal.
Key words: A~tJ_Bo~m; Lactic acid bacteria: Lactobacillus sake; Dry fermented sausage
introduction The lactic acid bacteria have the potential to inhibit the growth of pathogenic and spoilage bacteria and the possibility exists of using them to improve the hygienic quality and to extend the shelf-life of different meat and meat products (Raccach et al., 1979; Rodriguez et al., 1989; Schillinger and Liicke, 1989a). Lactic acid bacteria isolated from meat and meat products are probably the best candidates for improving the microbiological safety of these foods, because they are well adapted to the conditions in meats and should therefore be more competitive than lactic acid bacteria from other sources (Shillinger and Lticke, 1989b). C o m ~ w m d e n ~ address: P.E. Hernkndez, Dpto. Higiene y Tecnolosia Alirmmto~ Facultad de Veterinaria, U~iver~bd Complute~, 28040 Madrid, Spmn.
In the meat industry, lactic acid bacteria are widely used as starter cultures for sausage fermentation (Liepe, 1983: Adams, 1986), where they contribute to flavor development as well as to the preservation of fermented sausages (Schillinger and LOcke, 1989b). Reduction of pH and removal of carbohydrates are the pnmar3.' effects exerted by the lactic acid bacteria (Daeschel, 1989). It has also been recognized that lactic acid bacteria are capable of producing inhibitory substances other than organic acids that are antagonistic toward other microorganisms. These substances are produced in smaller amounts and include hydrogen peroxide, diacetyl, bacteriocins and other secondary metabolites (Klaenhammer, 1988: Daeschel. 1989). Bacteriocinogenicity within the lactic acid bacteria has been recognized and has been the subject of recent investigations as reported by K l a e n h a m m e r (1988). Bacteriocins form a heterogeneous group with regard to producing bacteria, antibacterial spectrum, mode of action, and chemical properties (Daeschel, 1989; Daeschel et al., 1990). In this study, a number of lactic acid bacteria isolated from Spanish dry fermented sausages were screened for antagonistic activities. Some were found to produce antimicrobial effects other than low p H and hydrogen peroxide into the broth.
Materials and Methods
Materials and organisms Spanish dry fermented sausages manufactured with no added starter cultures were used as the source of lactic acid bacteria. Composition of the sausages and ripening conditions have been already described (Sanz et al., 1988). Samples were taken at different times of the ripening period. The samples weighing 20 g each were added to 180 ml of 15[ sterile ( w / w ) p e p t o n e / w a t e r and thoroughly homogenized during 20 min in a Colworth Stomacher 400 blender (A.J. Seward, U.K.); 0.1 ml of the appropriate dilutions were spread onto the surface of MRS (De Man et al., 1960) agar plates (Oxoid) and covered with a double layer of 6 ml of the same medium. Plates were incubated for 2 days at 32"C, when colonies were large enough to be isolated. A total of 720 colonies were isolated from the MRS plates following a previously described procedure (Ordohez, 1979). The lactic acid bacteria were propagated and maintained in MRS broth, unless stated otherwise. Bacterial strains used as test organisms are given in Tables I and II. Other bacteria than lactic acid bacteria were cultivated in APT broth (Difco) or brain heart infusion broth (BHI, Oxoid) at 32°C. Detection of antagonistic activities A two-step procedure was used. First, the selected lactic acid bacteria on MRS were replicated on two MRS plates with 24 isolates per plate. The inoculated plates were incubated for 6 h at 3 2 ° C to allow isolates to reinitiate growth. Then,
3 TABLE I Inhibitory activity o f concenmued culture s u ~ e4~iaMectedlactic acid bacteria against Lactobacil/us spp. and strains o f L. mon~vtogene~ and SmpiL ~mram • Producer
Inhibition b
Strain
i..actobacillus spp. CECT 285
L monoc.vtogenes
IMTH Lb 726
FRIB LV 13
FVM Scott A
+ + +
(+)
(+) (+) (+)
. + (+)
148
+
+
+
177
180
(+) +
-
209
+
231
+
241 345
31 77 90
NCTC 5105
.
.
Staph. aureus NCTC 7973
FRI 137
(+) (+)
. . (+)
.
+
+
+
(+)
(+) (+)
(+) (+)
(+)
. (+) (+) (+) -
+
+
+
+
+
+
+
+
+
+
+
+
(+)
+
+
346 438
+ + +
+ + +
(+) + +
+ + +
449
+
+
+
525
+
+
648
+
+
.
FRI 361
FRI 196E
. . (+) -
-
(+)
-
(+)
(+)
-
(+)
+
(+)
-
-
+ + +
+ + +
+
+
+
+
+
-
+
+
+
+ (+)
(+) -
-
+
(+) (+ ) (+) (+ ) (+)
-
-
-
-
-
• For origin of cultures see Table !1. b Symboh for alpurdiffusion assay: +, large inhibition zone ( > 4 mm): ( + ), small inhibition zone ( < 4 nun); - , no inhibitioQ z o n e . c Strain numbers as in Table II.
a p p r o x i m a t e l y 3 . 1 0 5 cells of Lactobaciilus fermentum C E C T 2 8 5 were a d d e d to 6 ml of semisolid M R S agar (0.8~ agar) a n d p o u r e d over o n e of the two plates o n which the isolates were inoculated. After i n c u b a t i o n for 24 h at 3 2 ° C , the plates were checked for zones of inhibition. T h e isolates displaying the largest halos of i n h i b i tion were recovered from the template plates, grown in 5 ml of M R S b r o t h a n d stored in 1 5 ~ glycerol at - 2 0 ° C until use in disc agar diffusion assay for detection of a n t a g o n i s m against the test organisms i n c l u d e d in T a b l e s I a n d II. F o r this assay, sterile W h a t m a n No. 3 filter p a p e r discs of 7 m m d i a m e t e r c o n t a i n i n g 0.030 mi of c o n c e n t r a t e d c u l t u r e s u p e r n a t a n t s (see below) were placed o n p r e p o u r e d a f a r plates overlaid with a b o u t 3 • 105 cells of the various organisms investigated in 6 ml of soft M R S , A P T or B H I agar. Plates were i n c u b a t e d at 3 2 ° C , a n d the a n t i m i c r o b i a l activity of the s u p e r n a t a n t s was q u a n t i f i e d b y m e a s u r i n g the d i a m e t e r of the clear zones of i n h i b i t i o n a r o u n d the discs. F o r p r e p a r a t i o n of c o n c e n t r a t e d culture s u p e r n a t a n t s the selected isolates were g r o w n in M R S b r o t h for 16 h at 3 2 ° C . A cell-free solution was o b t a i n e d b y c e n t r i f u g i n g the c u l t u r e at 12000 g for 10 rain. This was followed b y n e u t r a l i z a t i o n of the s u p e r n a u m t to p H 6.2 with 1 N N a O H a n d filtration through a 0 . 2 2 / a m pore size filter (Millipore). I n h i b i t o r y activity from h y d r o g e n peroxide was e l i m i n a t e d b y
4 TABLE
Ii
Inhibitory spectrum demonstrated
for
Lactobactllus sake 148
Organisms
Ongzn •
I n h i b i t i o n b~ the culture supernatant
from
L sake 148 b Gram-positwe
bactena
Lactobacillus acidophtlus Lactobacillus brems Lactobacillus carnts Lactobacillus caset Lactobacillus curvatus Carnobactermm dwergens Lactobactllus ferment um Lactobacillus mesentertcus Lactobacillus plantarum Lactobacillus sake Leuconastoc mesenterotdes Mtcrococcus rattans Staphylococcus xilosus Streptococcus faecahs Streptococcus faecalls ( v a t . liquefactens ) Streptococcus faecium Bacillus steaeothermophilus Bacillus cereus Brochothrtx thermosphacta Staphylococcus aureus Staphylococcus aureus ldsterta monocytogenes Ltsterta monoc..vtogenes Lzsteria monocytogenes I.~sterla monocvtogenes Lzsterta monocytogenes Clostrtdlum botuhnum Clostrtdlum perfrmgens Gram-negative
289
CECT
-
Lb289
! MTH
+
LV61
FRI B
-
475
CECT
-
Lb726
1MTH
+
LVI 3
FRIB
+
285
C ECT
+
394
CECT
-
221
CECT
-
Lb68
1MTH
-
394
CECT
+
230
CECT
-
237
CECT
-
481
CECT
-
184
CECT
-
410
CECT
-
49
CECT
-
148
CECT
-
1001 g
NCI B
137
FR1
(+ )
361
FRI
(+ )
5105
NCTC
+
7 973
NCTC
+
-
LI5 sv 1/2
FVM
+
L! 1 sv 4
FVM
+
Scott A
FVM
+
551
CECT
(+ )
376
CECT
(+ )
194
C ECT
-
BW545
M1T
-
bacteria
Enterobacter cloacae Eschertchta colt Escherwhia colt e n t e r o p a t h o g e n i c Salmonella typht Salmonella typhimurium Pseudomonas fluoresccns Pseudomonas fluorescens Pseudomonas fluorescens Yersmta enterocolitica Yerstnta enterocohtica
B41
IEKC
-
409
CECT
-
"I"91
CENAN
-
DC5
FR 1B
-
DC7
F R 1B
-
NTI 9 F..20
FRIB Our collection
-
14405
IPP
-
the addition of catalase Boehringer Mannheim, F.R.G., 130 U l / m l . The culture supernatant was lyophilized and afterward~ |~ was resuspended in 4 mM phosphate buffer, pH 7.0 to a concentration correslJonc[ing to twenty-fold the original concentration. The lactic acid bacteria of particular interest were identified as described by Schillinger and Liicke (1987a).
Examinations of antogonistic activities The concentrated culture supernatant from Lactobacillus sake 148 was heated at 100°C for 20 rain, and the remaining activity was determined by the disc agar diffusion assay. To test its sensitivity to proteases the concentrated supernatant was treated with papain, protease II, protease XIV, trypsin and pepsin, all enzymes from Sigma (U.S.A.), each at the supplier's recommended level of 1 mg/ml. Samples with and without proteases were incubated at 37°C for 12 h. Residual activity was determined by the agar diffusion assay. Initial studies showed that none of the enzymes themselves exerted any inhibitory effect against the primary test organism 1- fermentwn CECT 285. To study in more detail the effect of the antibacterial compound present in the cell-free cultures of L sake 148, 0.5 ml of the 20-fold concentrated supernatants were added to 5 ml of a culture of the sensitive bacteria (approx. l0 s cells/ml) in MRS, APT or BHI broth. Tubes were incubated at 32°C and, when required, in anarobic jars. At appropriate intervals, the number of viable cells in the culture tubes was determined by pour plating MRS, APT or BHI agar and incubating for 2 d at 32°C. In the control tubes, the sensitive bacteria were tested for the effect of 0.5 ml of a 20-fold concentrated supernatant of L. sake 23, a lactobacilli not displaying a detectable antimicrobial activity, pH measurements were carried out at the end of incubation.
Results
A total of 720 lactic acid bacteria isolated from Spanish dry fermented sausages were tested for their antimicrobial activity against Lactobacillus fermentum CECT285. This organism was used as the primary test strain based on previous
Notes to Table II: • Abbreviations: CECT, Coleco6n Espai~ola de Cultivos Tipo (Valencia, Spain), C E N A N , Ce~ro Nacional de Nutricibn y Alimentaci6n (Madrid, Spain); FRI. Food Research Institute (Madison, USA); FRIB, AFRC Institute of Food Research (Bristol. UK), FVM, Facultad de Veterimu~ (Madrid, Spain); IEKC, International Escher/chm and Kieb~iella Centre, (Copenhq~ DK); IMTH, Institute fUr Mikrobiology, Toxicology und Histology (Kuimbach, FRG); IPP, institute Pasteur (Paris, France): MIT, Masachnssetts Institute of Technology (Boston, USA): NCIB, Natiomd Collection of Industrial and Marine Bacteria, (Aberdeen. UK): NCTC, National ~ of Type Cultur~
0.zed¢~ UK). b Symbols for altar diffusion assay: +, Large inhibition zone ( > 4 nun); ( + ), small inhibition zone ( < 4 ram); - . no inhibition zone.
e x p e r i m e n t s ( u n p u b l i s h e d d a t a ) i n d i c a t i n g t h a t it is very, s e n s i t i v e t o w a r d s a n t i m l c r o b i a l a c t i v i t i e s o f l a c t i c a c i d b a c t e r i a . A t o t a l o f 119 i s o l a t e s w e r e f o u n d t o i n h i b i t this organism. T h e c o n c e n t r a t e d c u l t u r e s u p e r n a t a n t s o f 15 i s o l a t e s s h o w i n g t h e l a r g e s t i n h i b i tion against /- fermentum CECT285, were checked by the disc agar diffusion assay f o r t h e i r a n t a g o n i s t i c a c t i v i t y a g a i n s t Lactobacillus s p p . a n d s t r a i n s o f Staph.vlococcus aureus a n d L i s t e r i a m o n o c y t o g e n e s ( T a b l e 1). It a p p e a r s t h a t t h e l a c t o b a c i l l i a n d t h e s t r a i n s o f L. m o n o c y t o g e n e s a r e m o r e s e n s i t i v e t h a n t h e S t a p h y l o c o c c i . It a l s o a p p e a r s t h a t t h e i n h i b i t o r y s p e c t r a o f t h e l a c t o b a c i l l i a r e v a r y i n g s i g n i f i c a n t l y . All selected isolates w i t h a n t a g o n i s t i c activities were f o u n d to be Lactobacillus sake
TABLE llI Effect of the concentrated culture supematant of the non-antagonistic (C) and the antagonistic (T) Lactobacillus sake strains 23 and 148, respectively, on growth of various organisms " Organisms b
log C F U / m l at 32"C
Lactobacillus fermentum
285
Lactobacillus curvatus
726
Carnobacterium dwcrgen~
LV13
I.,euoconoxtoc mesenteroides
394
l.,isteria monocytogenes
5105
l.~steria monocytogenes
7 973
l.~stena monocytogenes
LI5 sv 1 / 2
Listerta monocytogenes
Scott A
Salmonella typhtmurtum
T91
Yersima enterocolmca
E20
Staphylococcus aureus
137
Staphylococcus aureus
361
Clostridium botuhnum
551
Clostridium perfringens
376
Oh
4h
8h
16h
C T C T C T C T C
5.5 5.3 5.6 5.6 5.6 5.7 5.7 5.7 5.7
6.8 5.3 6.9 5.6 6.9 5.7 6.9 5.8 6.9
8.7 5.4 8.5 5.7 8.7 5.3 8.9 5.9 8.9
9.8 5.5 9.8 6.1 9.5 5.4 9.5 6.4 9.3
T
5.8
5.8
5.7
5.7
C T C T C
5.3 5.3 5.8 5.8 5.8
T
5.8
C T C T C T C T C T C T
5.4 5.5 5.8 5.7 5.2 5.3 5.3 5.2 4.9 4.9 5.1 5.1
6.8 5.2 6.9 5.7 6.9 5.7 6.8 6.9 6.3 6.1 6.9 5.1 6.7 5.3 5.0 4.8 5.3 5.2
8.7 5.7 8.9 5.9 8.8 5.9 8.7 8.6 7.1 6.9 8.9 5.6 8.7 5.3 5.7 4.9 7.7 5.3
9.1 5.7 9.5 6.2 9.5 6.1 9.5 9.4 8.9 8.2 9.2 6.3 9.8 6.5 6.3 4.9 9.3 6.4
• 0.5 ml of 20-fold concentrated supernatants of L. sake 148 were added to tubes containing 5.0 ml of MRS, APT or BH! broth with approximately l0 s celis/ml of the microorgamsms indicated. b For origin of orgamsms see Table I1.
(Schillinger and Liicke, 1987a). One of these strains, /., sake 148 was chosen for further study. The antagonistic effect of the concentrated culture supernatant of L. sake 148 was tested on various Gram-positive and Gram-negative bacteria (Table II). It is seen that the supernatant was active against strains of L brevLs, I.. ozrvatus, Carnobacterium divergens, Leuconostoc mesenteroides, Stapiz aureus, Ciostridium botulinum, CI. perfringens and I.. monocywgenes, but none of the Gram-negative bacteria tested were inhibited; these included, among others, the foodborne pathogens Salmonella typhimurium and Yersinia enterocolitica. The antagonistic activity produced by L sake 148 was destroyed completely by treatment with the papain and protease XIV, and severely inhibited by pepsin, protease II and trypsin (data not shown). However, the activity of the concentrated culture supernatants was resistant to heat; about 60~$ of the antibacterial activity remained after heating for 20 min at IO0°C (data not shown). The antagonistic activity remained during frozen storage (results not shown). The addition of concentrated culture supematants of L. sake 148 to freshly inoculated bacteria in MRS, APT or BHI broth (Table III), resulted in a cessation of their growth. In contrast, the growth of the cultures was unaffected by a concentrated supernatant of the non-antagonistic isolate L sake 23. No significant differences in pH for cultures with L. sake 148 and 23 were observed. These results indicate a bacteriostatic mode of action. As expected the growth of S. typhimurium and Y. enterocolitica was unaffected by the presence of the concentrated supernatant of L sake 148.
Dtlg~l~[on A number of lactic acid isolates were found to be inhibitory against L. fermentum CECT285, the primary test organism used. Similar results have bcen obtained by GeLs et al. (1983) with streptococci and by Schillinger and Liicke (1989b) with lactobaciUi. Concentrated culture supernatants produced from 15 isolates showing the largest antagonistic activity asainst I_ fermentum CECr285 were found to be inhibitory against several strains of Stap~ aureus and L monocytogenes. The inhibitory spectra of these strains differed significantly (Table I), sugsesting that the antibacterial substances produced may be heterogeneous. All identified isolates with antagonistic activities were tentatively identified as L sake. The presence of large numbers of this microorganism in dry fermented sausages is not surprising since it has been observed (Liicke, 1986: Schillinger and Liicke, 198To) that this species may become dominant at the low ripening temperatures often used in Europe for fermented sausages. The inhibitory activity of L sake 148 (Table II), was not restricted to other closely related bacteria, although it did not display activity a~alnst Gram-negative organisms. Inhibition was observed against Gram-positive foodbome pathogens of interest in the meat industry e.g., listeriae, staphylococci and clostridia. The activity seems to be strain-specific rather than species-specific. The sensitivity of fisteriae to
an antagonistic compound produced by a Lactobacillus species is not surprising as they are closely associated with the genus Lactobaciilus (Wilkinson and Jones, 1977; Seeliger and Jones, 1986; Ruhiand and Fiedler, 1987). Several reports have confirmed the antimicrobial activity of Lactobaciilus species against L monocytogenes (Harris et at., 1989; Raccach et ai., 1989, Schillinger and Liicke, 1989b), but interestingly enough, information on their activity against other Gram-positive pathogens is scarce (Andersson, 1986; Talarico et al., 1988). It could happen that in analogy with some pediococci (Bhunia et al., 1988), there are isolates more active than others against foodborne pathogens such as Staph. aureus. C. botulinum and C. perfringens. According to preliminary studies included in this work, the antibacterial compound produced by L. sake 148 is proteinaceus in nature, heat-resistant and bacteriostatic. The proteinaceus nature of this substance is a typical characteristic of bacteriocins (Tagg et at., 1976). Similar to the bacteriocins of Lactobaciilus acidophi/us (Barefoot and Klaenhammer, 1983; Murina and Klaenhammer, 1987), Lactoba. ciilus heiveticus (Upreti and Hinsdill, 1975; Joerger and Klaenhammer, 1986), Lactobaciilus plantarum (West and Warner, 1988; Daeschel et al., 1990) and Lactobacillus sake (SchiUinger and Liicke,, 19891>) it is also heat-stable. However, it is bacteriostatic and does not comply with the definition of bacteriocins being protein-containing macromolecules which exert a bacteriocidal mode of action (Tagg et at., 1976). This investigation, together with the work of Raccach et al. (1989), which reported on the bacteriostatic and, in some cases, bacteriocidal effects of L. acidophilus toward L monocytogenes, and those of Buchanan et at. (1987), Johnson et al. (1988) and Shelef (1989), which reported survival but no growth of /_. monocytogenes in raw meat, ground beef and poultry products, may indicate that the presence in meat and meat products of lactic acid bacteria with a bacteriostatic activity is more common than realiTed at present.
Acknowledgements We express our appreciation to all cited research institutions for their generous gifts of bacterial strains. O.J.S. and J.M.R. are recipients of Fellowships from the Mimsterio de Educacibn y Ciencia, Spain. W.L.M. holds a Fellowship from the CNPq, Brasil.
References
Adams, M.R. (1986) Fermented flesh foods. In: M.R. Adams (Ed.), Progressin Industrial Microbiology. Vol. 23, Elsevier, Amsterdam, pp. 159-198.
Andersson. R. (1986) Inhibition of Staphylococcusaureus and spheroplasts of Gram*negativebacteria by an antaBonisticcompound prod,__,,':e~__by • strain of Lactobacillusplanlarum. Int. J. Food Microbiol.3, 149-160.
Barefoot, S.F. and KJaenkammer, T.R. (]983) Detection and activity of lactacm B. a bacterioon produced by Lactobncillus acidophi/us. Appi. F.,nyiron. Microbioi. 45, 1808-1815. Bhunia, A.K., Johnson, M.C. 'and Ray, B. (~988) Pt~flcatiolL Characteci~fion and antimicrobial
spectrum of a bacteriocin p~
10 Shelef, L.A. (1989) Survival of l.~ster~a monocytogenes in ground beef and hver after storage at 4 and 2 5 ° C . J. Food Protect. 52. 379-383. Tagg. J.R.. Dajam, A.S. and Wannamaker. L. (1976) Bactenocins of gram-positive bacteria. Bactenol. Rev. 40, 722-756. Talanco, T.L., Casas, I.A., Chung, T.C. and Dogrogosz, W.J. (1988) Production and isolation of reutenn, a growth inhibitor produced by a Lactobacillus reutert. Antinucrob. Agents Chemother. 32. 1854-1858. Upreti, G.C. and Hinsdill. R.D. (1973) Production and mode of action of lactacm 27: a bacterioctn from a homoferrnentative l.~ctobacillus. Antimicrob. Agents Chemother. 7, 139-145. West, C.A. and P.J. Warner (1988) Plantacin B. a bacteriocin produced by Lactoboctllus plamarum N C D O 1193. FEMS Microbiol. Left. ,*9. 163-165. Wilkinson, B.J. and Jones, D. (1977) A numerical taxonomic survey of LJsteraa and related bacteria. J. Gen. Microbiol. 98, 399-421.
1
FOOD 00392
Antibacterial activity of Lactobacillus sake isolated from dry fermented sausages O d 6 n J. S o b r i n o , J u a n M. R o d r i g u e z , W a g n e r L. Moreira,
Mafia F. Fern~indez, Bernab6 Sanz and Pablo E. Hernhndez Departamento de Higwne y Tecnologla de los Aiimento$, Facultad de Vetermaria, Universidad Complutense. Madr~ Spain
(Received 20 September 1990: accepted 31 December 1990)
~ ~ iso~ted from S p m ~ h dry fermented sausages were u='oened for antagonistic activities under conditio~ that climinmed the effects of low pH and hydrogen peroxide. From 720 i.u~tes teated 119 were inhibitory to LactobacilluJ fermentum CEC'T285. The isolates showing the largest inh/bitory activity exhibited an a n t a ~ t i c effect against several other lactobaciifi and the selected foodborne pathoge~t Staphy/ococcus aureus and l~teria monocytogenes. Comparison of the antimimimimimimimimimim~robial spectra of the sopernatants sugl~ted that the inhibitory compounds were not identical. The imlates were tentatively characterized as Lactobaci//us sake. One of the isolates. / . sake 148 was chosen for further study. ~ c o m p o ~ excreted by L. sake 148 was active a oai~t various laotobacilll and several Gram-positive foodborne bacteria, but not against the Oram-nqlative bacteria tested. The antagonistic effects ~ almost elimimtted by treatment with pro~¢~__.~__,whereas they were heat resistant and bcgteriostatic rather than bactmocidal.
Key words: A~tJ_Bo~m; Lactic acid bacteria: Lactobacillus sake; Dry fermented sausage
introduction The lactic acid bacteria have the potential to inhibit the growth of pathogenic and spoilage bacteria and the possibility exists of using them to improve the hygienic quality and to extend the shelf-life of different meat and meat products (Raccach et al., 1979; Rodriguez et al., 1989; Schillinger and Liicke, 1989a). Lactic acid bacteria isolated from meat and meat products are probably the best candidates for improving the microbiological safety of these foods, because they are well adapted to the conditions in meats and should therefore be more competitive than lactic acid bacteria from other sources (Shillinger and Lticke, 1989b). C o m ~ w m d e n ~ address: P.E. Hernkndez, Dpto. Higiene y Tecnolosia Alirmmto~ Facultad de Veterinaria, U~iver~bd Complute~, 28040 Madrid, Spmn.
In the meat industry, lactic acid bacteria are widely used as starter cultures for sausage fermentation (Liepe, 1983: Adams, 1986), where they contribute to flavor development as well as to the preservation of fermented sausages (Schillinger and LOcke, 1989b). Reduction of pH and removal of carbohydrates are the pnmar3.' effects exerted by the lactic acid bacteria (Daeschel, 1989). It has also been recognized that lactic acid bacteria are capable of producing inhibitory substances other than organic acids that are antagonistic toward other microorganisms. These substances are produced in smaller amounts and include hydrogen peroxide, diacetyl, bacteriocins and other secondary metabolites (Klaenhammer, 1988: Daeschel. 1989). Bacteriocinogenicity within the lactic acid bacteria has been recognized and has been the subject of recent investigations as reported by K l a e n h a m m e r (1988). Bacteriocins form a heterogeneous group with regard to producing bacteria, antibacterial spectrum, mode of action, and chemical properties (Daeschel, 1989; Daeschel et al., 1990). In this study, a number of lactic acid bacteria isolated from Spanish dry fermented sausages were screened for antagonistic activities. Some were found to produce antimicrobial effects other than low p H and hydrogen peroxide into the broth.
Materials and Methods
Materials and organisms Spanish dry fermented sausages manufactured with no added starter cultures were used as the source of lactic acid bacteria. Composition of the sausages and ripening conditions have been already described (Sanz et al., 1988). Samples were taken at different times of the ripening period. The samples weighing 20 g each were added to 180 ml of 15[ sterile ( w / w ) p e p t o n e / w a t e r and thoroughly homogenized during 20 min in a Colworth Stomacher 400 blender (A.J. Seward, U.K.); 0.1 ml of the appropriate dilutions were spread onto the surface of MRS (De Man et al., 1960) agar plates (Oxoid) and covered with a double layer of 6 ml of the same medium. Plates were incubated for 2 days at 32"C, when colonies were large enough to be isolated. A total of 720 colonies were isolated from the MRS plates following a previously described procedure (Ordohez, 1979). The lactic acid bacteria were propagated and maintained in MRS broth, unless stated otherwise. Bacterial strains used as test organisms are given in Tables I and II. Other bacteria than lactic acid bacteria were cultivated in APT broth (Difco) or brain heart infusion broth (BHI, Oxoid) at 32°C. Detection of antagonistic activities A two-step procedure was used. First, the selected lactic acid bacteria on MRS were replicated on two MRS plates with 24 isolates per plate. The inoculated plates were incubated for 6 h at 3 2 ° C to allow isolates to reinitiate growth. Then,
3 TABLE I Inhibitory activity o f concenmued culture s u ~ e4~iaMectedlactic acid bacteria against Lactobacil/us spp. and strains o f L. mon~vtogene~ and SmpiL ~mram • Producer
Inhibition b
Strain
i..actobacillus spp. CECT 285
L monoc.vtogenes
IMTH Lb 726
FRIB LV 13
FVM Scott A
+ + +
(+)
(+) (+) (+)
. + (+)
148
+
+
+
177
180
(+) +
-
209
+
231
+
241 345
31 77 90
NCTC 5105
.
.
Staph. aureus NCTC 7973
FRI 137
(+) (+)
. . (+)
.
+
+
+
(+)
(+) (+)
(+) (+)
(+)
. (+) (+) (+) -
+
+
+
+
+
+
+
+
+
+
+
+
(+)
+
+
346 438
+ + +
+ + +
(+) + +
+ + +
449
+
+
+
525
+
+
648
+
+
.
FRI 361
FRI 196E
. . (+) -
-
(+)
-
(+)
(+)
-
(+)
+
(+)
-
-
+ + +
+ + +
+
+
+
+
+
-
+
+
+
+ (+)
(+) -
-
+
(+) (+ ) (+) (+ ) (+)
-
-
-
-
-
• For origin of cultures see Table !1. b Symboh for alpurdiffusion assay: +, large inhibition zone ( > 4 mm): ( + ), small inhibition zone ( < 4 nun); - , no inhibitioQ z o n e . c Strain numbers as in Table II.
a p p r o x i m a t e l y 3 . 1 0 5 cells of Lactobaciilus fermentum C E C T 2 8 5 were a d d e d to 6 ml of semisolid M R S agar (0.8~ agar) a n d p o u r e d over o n e of the two plates o n which the isolates were inoculated. After i n c u b a t i o n for 24 h at 3 2 ° C , the plates were checked for zones of inhibition. T h e isolates displaying the largest halos of i n h i b i tion were recovered from the template plates, grown in 5 ml of M R S b r o t h a n d stored in 1 5 ~ glycerol at - 2 0 ° C until use in disc agar diffusion assay for detection of a n t a g o n i s m against the test organisms i n c l u d e d in T a b l e s I a n d II. F o r this assay, sterile W h a t m a n No. 3 filter p a p e r discs of 7 m m d i a m e t e r c o n t a i n i n g 0.030 mi of c o n c e n t r a t e d c u l t u r e s u p e r n a t a n t s (see below) were placed o n p r e p o u r e d a f a r plates overlaid with a b o u t 3 • 105 cells of the various organisms investigated in 6 ml of soft M R S , A P T or B H I agar. Plates were i n c u b a t e d at 3 2 ° C , a n d the a n t i m i c r o b i a l activity of the s u p e r n a t a n t s was q u a n t i f i e d b y m e a s u r i n g the d i a m e t e r of the clear zones of i n h i b i t i o n a r o u n d the discs. F o r p r e p a r a t i o n of c o n c e n t r a t e d culture s u p e r n a t a n t s the selected isolates were g r o w n in M R S b r o t h for 16 h at 3 2 ° C . A cell-free solution was o b t a i n e d b y c e n t r i f u g i n g the c u l t u r e at 12000 g for 10 rain. This was followed b y n e u t r a l i z a t i o n of the s u p e r n a u m t to p H 6.2 with 1 N N a O H a n d filtration through a 0 . 2 2 / a m pore size filter (Millipore). I n h i b i t o r y activity from h y d r o g e n peroxide was e l i m i n a t e d b y
4 TABLE
Ii
Inhibitory spectrum demonstrated
for
Lactobactllus sake 148
Organisms
Ongzn •
I n h i b i t i o n b~ the culture supernatant
from
L sake 148 b Gram-positwe
bactena
Lactobacillus acidophtlus Lactobacillus brems Lactobacillus carnts Lactobacillus caset Lactobacillus curvatus Carnobactermm dwergens Lactobactllus ferment um Lactobacillus mesentertcus Lactobacillus plantarum Lactobacillus sake Leuconastoc mesenterotdes Mtcrococcus rattans Staphylococcus xilosus Streptococcus faecahs Streptococcus faecalls ( v a t . liquefactens ) Streptococcus faecium Bacillus steaeothermophilus Bacillus cereus Brochothrtx thermosphacta Staphylococcus aureus Staphylococcus aureus ldsterta monocytogenes Ltsterta monoc..vtogenes Lzsteria monocytogenes I.~sterla monocvtogenes Lzsterta monocytogenes Clostrtdlum botuhnum Clostrtdlum perfrmgens Gram-negative
289
CECT
-
Lb289
! MTH
+
LV61
FRI B
-
475
CECT
-
Lb726
1MTH
+
LVI 3
FRIB
+
285
C ECT
+
394
CECT
-
221
CECT
-
Lb68
1MTH
-
394
CECT
+
230
CECT
-
237
CECT
-
481
CECT
-
184
CECT
-
410
CECT
-
49
CECT
-
148
CECT
-
1001 g
NCI B
137
FR1
(+ )
361
FRI
(+ )
5105
NCTC
+
7 973
NCTC
+
-
LI5 sv 1/2
FVM
+
L! 1 sv 4
FVM
+
Scott A
FVM
+
551
CECT
(+ )
376
CECT
(+ )
194
C ECT
-
BW545
M1T
-
bacteria
Enterobacter cloacae Eschertchta colt Escherwhia colt e n t e r o p a t h o g e n i c Salmonella typht Salmonella typhimurium Pseudomonas fluoresccns Pseudomonas fluorescens Pseudomonas fluorescens Yersmta enterocolitica Yerstnta enterocohtica
B41
IEKC
-
409
CECT
-
"I"91
CENAN
-
DC5
FR 1B
-
DC7
F R 1B
-
NTI 9 F..20
FRIB Our collection
-
14405
IPP
-
the addition of catalase Boehringer Mannheim, F.R.G., 130 U l / m l . The culture supernatant was lyophilized and afterward~ |~ was resuspended in 4 mM phosphate buffer, pH 7.0 to a concentration correslJonc[ing to twenty-fold the original concentration. The lactic acid bacteria of particular interest were identified as described by Schillinger and Liicke (1987a).
Examinations of antogonistic activities The concentrated culture supernatant from Lactobacillus sake 148 was heated at 100°C for 20 rain, and the remaining activity was determined by the disc agar diffusion assay. To test its sensitivity to proteases the concentrated supernatant was treated with papain, protease II, protease XIV, trypsin and pepsin, all enzymes from Sigma (U.S.A.), each at the supplier's recommended level of 1 mg/ml. Samples with and without proteases were incubated at 37°C for 12 h. Residual activity was determined by the agar diffusion assay. Initial studies showed that none of the enzymes themselves exerted any inhibitory effect against the primary test organism 1- fermentwn CECT 285. To study in more detail the effect of the antibacterial compound present in the cell-free cultures of L sake 148, 0.5 ml of the 20-fold concentrated supernatants were added to 5 ml of a culture of the sensitive bacteria (approx. l0 s cells/ml) in MRS, APT or BHI broth. Tubes were incubated at 32°C and, when required, in anarobic jars. At appropriate intervals, the number of viable cells in the culture tubes was determined by pour plating MRS, APT or BHI agar and incubating for 2 d at 32°C. In the control tubes, the sensitive bacteria were tested for the effect of 0.5 ml of a 20-fold concentrated supernatant of L. sake 23, a lactobacilli not displaying a detectable antimicrobial activity, pH measurements were carried out at the end of incubation.
Results
A total of 720 lactic acid bacteria isolated from Spanish dry fermented sausages were tested for their antimicrobial activity against Lactobacillus fermentum CECT285. This organism was used as the primary test strain based on previous
Notes to Table II: • Abbreviations: CECT, Coleco6n Espai~ola de Cultivos Tipo (Valencia, Spain), C E N A N , Ce~ro Nacional de Nutricibn y Alimentaci6n (Madrid, Spain); FRI. Food Research Institute (Madison, USA); FRIB, AFRC Institute of Food Research (Bristol. UK), FVM, Facultad de Veterimu~ (Madrid, Spain); IEKC, International Escher/chm and Kieb~iella Centre, (Copenhq~ DK); IMTH, Institute fUr Mikrobiology, Toxicology und Histology (Kuimbach, FRG); IPP, institute Pasteur (Paris, France): MIT, Masachnssetts Institute of Technology (Boston, USA): NCIB, Natiomd Collection of Industrial and Marine Bacteria, (Aberdeen. UK): NCTC, National ~ of Type Cultur~
0.zed¢~ UK). b Symbols for altar diffusion assay: +, Large inhibition zone ( > 4 nun); ( + ), small inhibition zone ( < 4 ram); - . no inhibition zone.
e x p e r i m e n t s ( u n p u b l i s h e d d a t a ) i n d i c a t i n g t h a t it is very, s e n s i t i v e t o w a r d s a n t i m l c r o b i a l a c t i v i t i e s o f l a c t i c a c i d b a c t e r i a . A t o t a l o f 119 i s o l a t e s w e r e f o u n d t o i n h i b i t this organism. T h e c o n c e n t r a t e d c u l t u r e s u p e r n a t a n t s o f 15 i s o l a t e s s h o w i n g t h e l a r g e s t i n h i b i tion against /- fermentum CECT285, were checked by the disc agar diffusion assay f o r t h e i r a n t a g o n i s t i c a c t i v i t y a g a i n s t Lactobacillus s p p . a n d s t r a i n s o f Staph.vlococcus aureus a n d L i s t e r i a m o n o c y t o g e n e s ( T a b l e 1). It a p p e a r s t h a t t h e l a c t o b a c i l l i a n d t h e s t r a i n s o f L. m o n o c y t o g e n e s a r e m o r e s e n s i t i v e t h a n t h e S t a p h y l o c o c c i . It a l s o a p p e a r s t h a t t h e i n h i b i t o r y s p e c t r a o f t h e l a c t o b a c i l l i a r e v a r y i n g s i g n i f i c a n t l y . All selected isolates w i t h a n t a g o n i s t i c activities were f o u n d to be Lactobacillus sake
TABLE llI Effect of the concentrated culture supematant of the non-antagonistic (C) and the antagonistic (T) Lactobacillus sake strains 23 and 148, respectively, on growth of various organisms " Organisms b
log C F U / m l at 32"C
Lactobacillus fermentum
285
Lactobacillus curvatus
726
Carnobacterium dwcrgen~
LV13
I.,euoconoxtoc mesenteroides
394
l.,isteria monocytogenes
5105
l.~steria monocytogenes
7 973
l.~stena monocytogenes
LI5 sv 1 / 2
Listerta monocytogenes
Scott A
Salmonella typhtmurtum
T91
Yersima enterocolmca
E20
Staphylococcus aureus
137
Staphylococcus aureus
361
Clostridium botuhnum
551
Clostridium perfringens
376
Oh
4h
8h
16h
C T C T C T C T C
5.5 5.3 5.6 5.6 5.6 5.7 5.7 5.7 5.7
6.8 5.3 6.9 5.6 6.9 5.7 6.9 5.8 6.9
8.7 5.4 8.5 5.7 8.7 5.3 8.9 5.9 8.9
9.8 5.5 9.8 6.1 9.5 5.4 9.5 6.4 9.3
T
5.8
5.8
5.7
5.7
C T C T C
5.3 5.3 5.8 5.8 5.8
T
5.8
C T C T C T C T C T C T
5.4 5.5 5.8 5.7 5.2 5.3 5.3 5.2 4.9 4.9 5.1 5.1
6.8 5.2 6.9 5.7 6.9 5.7 6.8 6.9 6.3 6.1 6.9 5.1 6.7 5.3 5.0 4.8 5.3 5.2
8.7 5.7 8.9 5.9 8.8 5.9 8.7 8.6 7.1 6.9 8.9 5.6 8.7 5.3 5.7 4.9 7.7 5.3
9.1 5.7 9.5 6.2 9.5 6.1 9.5 9.4 8.9 8.2 9.2 6.3 9.8 6.5 6.3 4.9 9.3 6.4
• 0.5 ml of 20-fold concentrated supernatants of L. sake 148 were added to tubes containing 5.0 ml of MRS, APT or BH! broth with approximately l0 s celis/ml of the microorgamsms indicated. b For origin of orgamsms see Table I1.
(Schillinger and Liicke, 1987a). One of these strains, /., sake 148 was chosen for further study. The antagonistic effect of the concentrated culture supernatant of L. sake 148 was tested on various Gram-positive and Gram-negative bacteria (Table II). It is seen that the supernatant was active against strains of L brevLs, I.. ozrvatus, Carnobacterium divergens, Leuconostoc mesenteroides, Stapiz aureus, Ciostridium botulinum, CI. perfringens and I.. monocywgenes, but none of the Gram-negative bacteria tested were inhibited; these included, among others, the foodborne pathogens Salmonella typhimurium and Yersinia enterocolitica. The antagonistic activity produced by L sake 148 was destroyed completely by treatment with the papain and protease XIV, and severely inhibited by pepsin, protease II and trypsin (data not shown). However, the activity of the concentrated culture supernatants was resistant to heat; about 60~$ of the antibacterial activity remained after heating for 20 min at IO0°C (data not shown). The antagonistic activity remained during frozen storage (results not shown). The addition of concentrated culture supematants of L. sake 148 to freshly inoculated bacteria in MRS, APT or BHI broth (Table III), resulted in a cessation of their growth. In contrast, the growth of the cultures was unaffected by a concentrated supernatant of the non-antagonistic isolate L sake 23. No significant differences in pH for cultures with L. sake 148 and 23 were observed. These results indicate a bacteriostatic mode of action. As expected the growth of S. typhimurium and Y. enterocolitica was unaffected by the presence of the concentrated supernatant of L sake 148.
Dtlg~l~[on A number of lactic acid isolates were found to be inhibitory against L. fermentum CECT285, the primary test organism used. Similar results have bcen obtained by GeLs et al. (1983) with streptococci and by Schillinger and Liicke (1989b) with lactobaciUi. Concentrated culture supernatants produced from 15 isolates showing the largest antagonistic activity asainst I_ fermentum CECr285 were found to be inhibitory against several strains of Stap~ aureus and L monocytogenes. The inhibitory spectra of these strains differed significantly (Table I), sugsesting that the antibacterial substances produced may be heterogeneous. All identified isolates with antagonistic activities were tentatively identified as L sake. The presence of large numbers of this microorganism in dry fermented sausages is not surprising since it has been observed (Liicke, 1986: Schillinger and Liicke, 198To) that this species may become dominant at the low ripening temperatures often used in Europe for fermented sausages. The inhibitory activity of L sake 148 (Table II), was not restricted to other closely related bacteria, although it did not display activity a~alnst Gram-negative organisms. Inhibition was observed against Gram-positive foodbome pathogens of interest in the meat industry e.g., listeriae, staphylococci and clostridia. The activity seems to be strain-specific rather than species-specific. The sensitivity of fisteriae to
an antagonistic compound produced by a Lactobacillus species is not surprising as they are closely associated with the genus Lactobaciilus (Wilkinson and Jones, 1977; Seeliger and Jones, 1986; Ruhiand and Fiedler, 1987). Several reports have confirmed the antimicrobial activity of Lactobaciilus species against L monocytogenes (Harris et at., 1989; Raccach et ai., 1989, Schillinger and Liicke, 1989b), but interestingly enough, information on their activity against other Gram-positive pathogens is scarce (Andersson, 1986; Talarico et al., 1988). It could happen that in analogy with some pediococci (Bhunia et al., 1988), there are isolates more active than others against foodborne pathogens such as Staph. aureus. C. botulinum and C. perfringens. According to preliminary studies included in this work, the antibacterial compound produced by L. sake 148 is proteinaceus in nature, heat-resistant and bacteriostatic. The proteinaceus nature of this substance is a typical characteristic of bacteriocins (Tagg et at., 1976). Similar to the bacteriocins of Lactobaciilus acidophi/us (Barefoot and Klaenhammer, 1983; Murina and Klaenhammer, 1987), Lactoba. ciilus heiveticus (Upreti and Hinsdill, 1975; Joerger and Klaenhammer, 1986), Lactobaciilus plantarum (West and Warner, 1988; Daeschel et al., 1990) and Lactobacillus sake (SchiUinger and Liicke,, 19891>) it is also heat-stable. However, it is bacteriostatic and does not comply with the definition of bacteriocins being protein-containing macromolecules which exert a bacteriocidal mode of action (Tagg et at., 1976). This investigation, together with the work of Raccach et al. (1989), which reported on the bacteriostatic and, in some cases, bacteriocidal effects of L. acidophilus toward L monocytogenes, and those of Buchanan et at. (1987), Johnson et al. (1988) and Shelef (1989), which reported survival but no growth of /_. monocytogenes in raw meat, ground beef and poultry products, may indicate that the presence in meat and meat products of lactic acid bacteria with a bacteriostatic activity is more common than realiTed at present.
Acknowledgements We express our appreciation to all cited research institutions for their generous gifts of bacterial strains. O.J.S. and J.M.R. are recipients of Fellowships from the Mimsterio de Educacibn y Ciencia, Spain. W.L.M. holds a Fellowship from the CNPq, Brasil.
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