Inhibitory Action of Lactobacillusbulgaricus Toward Psychrotrophic Bacteria from Raw Milk

Transcription

1 Inhibitry Actin f Lactbacillusbulgaricus Tward Psychrtrphic Bacteria frm Raw Milk S.E. Gilliland and D.R. Martin Stry in Brief Frzen cncentrated cultures were prepared frm selected lactbacilli islated frm three cmmercial samples f ygurt. Prtins f the cncentrated cultures were added t milk cntaining a psychrtrphic bacteria which had been islated frm raw milk. The milk was stred at 5.5 C. A significant inhibitin f grwth f the psychrtrphic culture ccurred when at least 2.5 x 107 lactbacilli were added per m!. Higher numbers flactbacilli resulted in greater inhibitin fpsychrtrph. The intensity f inhibitin f a psychrtrphic culture varied amng the cultures flactbacilli tested. In mst cases the mre active inhibitry cultures prduced mre perxide in refrigerated milk than less inhibitry nes. Intrductin Psychrtrphic bacteria can grw readily in refrigerated raw milk and seriusly limit the keeping quality f the milk. While mst psychrtrphs are easily inactivated by pasteurizatin, sme prduce prtelytic enzymes and liplytic enzymes that are nt inactivated by pasteurizatin (Law et at., 1976, and Speck and Adams, 1976). These heat resistant enzymes can cause spilage in heat prcessed milk and in sme prducts made frm it. It wuld be desirable t prevent the grwth fpsychrtrphic micrrganisins in raw milk, thus decreasing the amunt f such enzymes. Cncentrated suspensins f certain starter culture bacteria, when added t sme refrigerated fds, have been shwn t suppress grwth at refrigeratin temperatures f spilage bacteria (Gilliland and Speck, 1975). Hydrgen perxide was identified as the antagnistic agent prduced by the lactbacilli included in the starter cultures tested. Cmmercial starters cntaining mixed species flactic cultures have als been used t restrict grwth fpsychrtrphs in raw milk and in autclaved milk inculated with a psychrtrphic culture stred at 3.5 and 7 C (juffs and Babel, 1975). The purpse f this study was t islate lactbacilli frm cmmercial ygurt which, when added t refrigerated milk, wuld cntrl the grwth f psychrtrphic bacteria. Experimental Prcedure Cmmercial brands f plain ygurt were purchased frm lcal supermarkets and health fd stres. All samples f ygurt were subcultured twice in sterile recnstituted 10 percent NFMS using I percent incula and incubatin at 37 C fr 24 hr. With the aid f a sterile glass hckey stick, 0.1 ml f an apprpriate dilutin f a milk culture was spread nt the surface f a milk base agar medium cntaining perxidase ( Animal Science Research Reprt 45

2 mg/ml) and O-tlidine (0.2 mg/ml). The plates were incubated 24 hr at 37 C. Varius clny types were selected frm each ygurt culture, including thse with n clr znes surrunding them, and thse with intermediate and large brwn znes. The brwn clr was due t hydrgen perxide prduced by the lactbacilli. Cells frm selected clnies were inculated with the aid f a sterile inculating needle int lactbacilli MRS brth (Difc) and incubated at 37 C until grwth was evident. The islates were identified using prcedures described by Gilliland and Speck (1977). Gram psitive, catalase negative rd-shaped bacteria were assumed t be lactbacilli. Each culture f lactbacilli was inculated (I percent) int MRS brth and incubated at 37 C until the culture reached the late expnential phase f grwth. The cells were harvested by centrifugatin, resuspended in small vlumes f cld sterile 10 percent NFMS and dispensed int sterile plastic freezing vials (2 g each). The vials were immediately submerged in liquid nitrgen (-196 C) fr strage. Psychrtrph RM was a gram negative rd-shaped bacterium islated frm raw milk btained frm the Oklahma State University Dairy Farm. The milk was held 5 days at 5.5 C then plated n Plate Cunt Agar (Difc). Cells frm a predminating type f clny were inculated int sterile 10 percent NFMS and incubated 24 hr at 21 C. Fr the interactin studies, the psychrtrph was subcultured twice n successive days in 10 percent NFMS, using I percent incula and incubatin fr 19 hr at 21 C just prir t use. Desired ppulatins f lactbacilli were prepared by aseptically adding the required amunt f thawed and apprpriately diluted cultures (diluted in cld sterile 10 percent NFMS) int sterile bttles cntained in an ice bath. The vlume in each bttle was adjusted t 10 ml with cld sterile 10 percent NFMS. Ten ml f autclaved 10 percent NFMS, cntaining the desired number f cells f a freshly prepared psychrtrphic culture, were added t each bttle. Frm each test sample, 5 ml were remved fr day 0 bacterial cunts (facultative lactbacilli and nn-lactbacilli). The remainder f each sample was stred fr 6 days in a 5.5 C water bath, and the numbers f nn-lactbacilli were then determined by plate cunt. The ph f each sample was measured n the initial and final day f the experiment. Lactbacilli were enumerated by plating the apprpriate dilutins n MRS agar. and incubating the plates 48 hr at 37 C. Nn-lactbacilli were enumerated by plating required dilutins n Plate Cunt Agar (PCA) (Difc) and incubating the plates 5 days at 21 C. The lactbacilli used in this study did nt frm clnies n this medium at 21 C. All clnies visible with the aid f a Quebec Clny Cunter were cunted. The fllwing frmula was used fr cmparing the inhibitin fpsychrtrphs by cultures f lactbacilli: [ A -B % inhibitin = xa x] x 100 Ax = number f nn-lactbacilli in cntrl n day x. Bx = number f nn-lactbacilli in sample cntaining added lactbacilli n day x. Duncan's new multiple range test was used t cmpare the means f percent inhibitin prduced by cultures fiactbacilli. It was als used t cmpare the effect f different numbers f lactbacilli n the grwth f the psychrtrph. T test fr perxide prductin by cells frm the frzen cncentrated cultures, the required amunt f each thawed culture was added t 25 ml vlumes f cld 10 percent 46 Oklahma Agricultural Experiment Statin

3 NFMS cntained in 50 ml Erlenmeyer flasks t yield a ppulatin f I x IOB/m!. The samples were incubated fr 22 hr at 5.5 C n a platfrm shaker (76 strkes per min). Hydrgen perxide was measured using the methd f Gilliland (1969). Fr the experiments in this study, the relative amunts f perxide frmed by the cultures are represented as absrbance at 400 nm. Results and Discussin Thse cultures which exhibited differences in their ability t prduce hydrgen perxide in refrigerated milk in preliminary experiments were selected fr further study. They were identified as Lactbacillus bulgaricus. After strage fr I mnth, the frzen cncentrated cultures were thawed and tested fr hydrgen perxide prductin in milk at 5.5 C. The relative amunt f perxide prduced by the cultures \'aried; culture Fl prduced the lwest level, cultures E I and E2 prduced intermediate amunts f perxide and cultures DI, D3 and F2 prduced higher levels f perxide (Table I). The number f lactbacilli in all samples was I x IOB/m!. T determine a reasnable ppulatin fr cmparing different cultures f lactbacilli fr the ability t inhibit the grwth f a psychrtrphic culture in refrigerated milk, increasing numbers f L. bulgaricus E2 were added t autclaved 10 percent NFMS inculated with 2 x 103 psychrtrph RM per ml (Figure I). The data frm three trials are presented graphically as the lg 10f the numbers f nn-lactbacilli per ml after 6 days f strage at 5.5 C pltted against the numbers flactbacilli added per m!. The grwth f the psychrtrph was nt significantly affected (P>.05) by ppulatins f lactbacilli up t I x 107/ml (increase nted in Trial I), but at 2.5 x 107 lactbacilli per ml there was significant inhibitin (P<.05) f the psychrtrphic culture. This antagnism against the psychrtrph increased significantly (P<.05) as higher numbers f lactbacilli were added. A cmparisn was made f the inhibitin fpsychrtrph RM by five cultures f L. bulgaricus in autclaved 10 percent NFMS (Table 2). The number flactbacilli added t the milk was 2.5 x 107/ml fr all cultures tested. There was cnsiderable \'ariatin in the percentages f inhibitin prduced by cultures flactbacilli in the three trials. A cmparisn f the average percentages f inhibitin indicated that L. bulgaricus D I and D3 were the mst active inhibitrs; L. bulgaricus E2 and F2 were intermediate inhibitrs and L. bulgaricus F I was the least active inhibitr. Statistical analysis f the data Table 1. Hydrgen perxide prductin in 10% NFMSby cells frm cncentrated cultures f Lactbacillus bulgaricus". Ygurt islates L. bulgaricus D1 L. bulgaricus D3 L. bulgaricus E1 L. bulgaricus E2 L. bulgaricus F1 L. bulgaricus F astred 1 mnth in liquid nitrgen; tested at 1 x 1()8celis/mi. baiter 22 hr incubatin at SOCwith cntinuus agitatin; the higher the absrbance reading, the mre perxide is present Animal Science Research Reprt 47

4 U 10 Iri-!'IS (/) >.!'IS CD... Q) ~ 6. Trial 1. Trial 2. Trial3 5 E:-:.- '0 4!'IS.D.9 J2 c: I Ẕ f!? 3 Q).D E:J Ẕ d).3 2 I 1"]) Numbers f Lactbacilli(X 107)Added Per ml Figure 1. Effect n numbers f lactbacillus bulgarlcus E2 n the grwth f psychrtrphlc culture In 10% NFMS. revealed that the differences amng the cultures DI, D3, 2, and F2 were nt significant (P>.05). Hwever, culture DI was significantly (P<.05) mre inhibitry than culture F I. With the exceptin f strain F2, the ranking f the lactbacilli with respect t the relative percentages f inhibitin was similar t that bserved fr perxide frmatin as shwn in Table I. Strain F2, which prduced the highest level f perxide, did nt cause the greatest amunt finhibitin f the psychrtrph. In related experiments, we have bserved that the amunt f detectable perxide frmed by sme strains f 48 Oklahma Agricultural Experiment Statin

5 Table 2. Inhibitin f grwth f psychrtrph RMIn autclaved 10% NFMSby cells f different cultures f lactbacillus bulgarlcus. PercentInhibitin Culture Trial 1 Trial 2 Trial 3 Ava L. bulgaricus D L. bulgaricus D L. bulgaricus E L. bulgaricus F L. bulaaricus F a2.5 x 107 lactbacilli addedlml fr each culture; initial ppulatin f psychrtrph - 1 x 10"/mi. lactbacilli in refrigerated milk reached a peak after 12 t 14 hr f strage, then dissipated slwly during subsequent strage. Fr ther cultures flactbacilli, similar amunts were prduced in 12 t 14 hr, but the amunt did nt appear t dissipate during subsequent strage (unpublished data). This might explain sme f the differences bserved amng the strains f L. bulgaricus with respect t the intensity f antagnism tward the psychrtrph in refrigerated milk. The lactbacilli islated frm ygurt did nt grw at refrigeratin temperature and thus caused very little change in the ph f the milk during refrigerated strage. N samples exhibited decreases in ph f mre than 0.1 t 0.2 units. Such cultures wuld be useful in aiding in the preservatin f certain refrigerated fds since they wuld be expected t cause little r n change in the rganleptic quality f the fd. In rder fr the lactbacilli t be used advantageusly t inhibit psychrtrphs in raw milk, they must prduce sufficient hydrgen perxide either t vercme perxidase r t activate the lactperxidase-thicyanate system that is present in raw milk. This system has been shwn t inhibit psychrtrphs in raw milk if sufficient hydrgen perxide is present (Reiter, 1978). Preliminary experiments indicated that the strains f lactbacilli used in the present study did nt prduce sufficient perxide at the ppulatins tested t inhibit psychrtrphs. If the lactbacilli are t be used in raw milk t inhibit the grwth fpsychrtrphs, then strains must be selected t prduce sufficient perxide r the strage cnditins mdified t inhance perxide prductin. Literature Cited Gilliland, S. E J. Dairy Sci. 52:321. Gilliland, S. E. and M. L. Speck J. f Fd Sci. 40:903. Gilliland, S. E. and M. L. Speck Appl. and Envirn. Micrbil. 33:1289. Juffs, H. S. and F. J. Babel J. f Dairy Sci. 58:1612. Law, B. A. et al J. f Dairy Research. 43:459. Reiter, B f Dairy Research. 45:131. Speck, M. L. and D. M. Adams f Dairy Sci. 59: Animal Science Research Reprt 49