Effect of Nisin on the Outgrowth of Clostridium botulinum Spores

Nisin, an antibiotic produced by certain strains of Streptococcus lactis, is effective in preventing the outgrowth of Clostridium botulinum spores. Type A C. botulinum spores were the most resistant to the inhibitory action of nisin requiring 1000-2000 I.U. of nisin/ml for a 50% inhibition of outgrowth on TPYG agar plates. Type E spores were more sensitive requiring only 50-100 I.U./ml for 50% inhibition of outgrowth on TPYG agar plates. Type B spores displayed an intermediate level of sensitivity requiring 500-1000 I.U. of nisin/ml for 50% inhibition of outgrowth on TPYG agar plates. Similar levels of nisin were necessary to prevent spore outgrowth in TPYG broth and BHI broth over a 7-day incubation period. With prolonged incubation periods of up to 65 days in TPYG broth, spore outgrowth was observed sporadically at higher nisin levels with the type A and B spores which may indicate some decomposition of nisin with storage. Nisin levels of 5000 I.U./ml for the type A spores and 2000 I.U./ml for the type B spores and the Minnesota E spores were insufficient to prevent spore outgrowth by C. botulinurn in cooked meat medium. For the Beluga E spores, a nisin level of 2000 I.U./ml was necessary to prevent spore outgrowth in cooked meat medium. The need for higher levels of nisin in cooked medium to prevent spore outgrowth may be due to the binding of the nisin by meat particles.     

Further Studies on the Antibotulinal Effectiveness of Nisin in Acidic Media

The antibotulinal effectiveness of nisin in TPYG broth was increased somewhat by lowering the pH to pH 5.5. The ability of nisin to inhibit the outgrowth of strain 56A spores was markedly increase at pH 5.5 by comparison to its effectiveness at higher pHs observed in previous studies. The increased effectiveness of nisin at pH 5.5 was less notable for the strain 69A, 113B, and 213B spores. The nisin sensitivity of the type E spores was essentially unchanged from that observed in earlier studies at higher pHs. At pH 6, nisin levels of 5000 I.U./ml were insufficient to prevent spore outgrowth by C. botulinum in cooked meat medium. Comparatively, much lower levels of nisin were effective in preventing botulinal outgrowth in TPYG broth at pH 6. The decreased effectiveness of nisin in cooked meat medium may be due to the binding of nisin to meat particles, and this binding is apparently not affected by lowering the pH to pH 6.0.     

Germination, Growth, and Toxin Production of Nonproteolytic Clostridium botulinum as Affected by Multiple Barriers

ABSTRACT The effect of combinations of pH (6.5, 5.75), NaCl (0.25,1.75%), and incubation temperatures (7,13 °C) on spore germination, outgrowth, and time to toxicity of nonproteolytic Clostridium botulinum was examined in a broth system. Spores of four toxin type E and nonproteolytic type B were inoculated (10⁴/ml) into Tryptone Peptone Glucose Yeast Extract (TPGY) broth. Cultures were monitored for three weeks, or until toxin was detected. A modified FSIS‐amplified ELISA (comparable in sensitivity to the mouse bioassay) was used to screen cultures for neurotoxin. Combinations of the most inhibitory level for each barrier reduced the degree and rate of germination, the lag and growth rate of vegetative cells, and the time to toxicity.     

Growth and Toxin Production by Clostridium botulinum in Model Acldified Systems

TPGY medium was acidified to pH 4.20, 4.60, 5.00 and 5.40 with acetic or citric acid. The media were inoculated with spores from three strains of C. botulinum type A, B or E. Growth, pH, and toxin production under anaerobic conditions were monitored for 8 wk. Spores of C. botulinum types A and B were incapable of outgrowth and toxin production at pH 4.60 or below when incubated at 35°C. Spores of C. botulinum type E were capable of growth and toxin production at 26°C in citric acid acidified systems at pH 4.20. Growth and toxin production were not detected below pH 5.00 when acetic acid was used.     

Sensitivity of spores of Bacillus subtilis and Clostridium sporogenes PA 3679 to combinations of high hydrostatic pressure and other processing parameters

The combined effects of pressure, temperature, pH and the presence of nisin or sucrose laurate on the survival of spores of Bacillus subtilis 168 and Clostridium sporogenes PA 3679 were investigated. Spore populations of PA 3679 were reduced by 2.5-log₁₀ when exposed to 404 megapascals (MPa) at 25°C, pH 4.0 for 30 min, but the same treatment at pH 7.0 resulted in a <0.5-log₁₀ reduction in spore counts. Pressurization of B. subtilis spores at 70°C, pH 6.0 or 7.0 for 15 min at 404 MPa resulted in a 5-log₁₀ reduction as compared to a <0.5-log₁₀ reduction for the same pressurization treatment at 25°C. For the inactivation of spores of B. subtilis and PA 3679, the addition of nisin to the plating medium appeared to be synergistic in some instances when combined with pressurization at elevated temperatures and reduced pH. B. subtilis 168 was resistant to 0.1% sucrose laurate, but when combined at ≤6.0 pH with a 15-min treatment of 404 MPa at 45°C, a dramatic synergistic effect eliminated spore suspensions of 1×10⁶/ml.     

Proteolytic Clostridium botulinum growth at 12–48°C simulating the cooling of cooked meat: development of a predictive model

The objective of this study was to develop a model to predict the germination, outgrowth and lag (GOL), and exponential growth rates of Clostridium botulinum from spores at temperatures (12–48°C) applicable to the cooling of cooked meat products. The growth medium, Reinforced Clostridial medium (RCM) supplemented with oxyrase enzyme to create suitable anaerobic conditions, was inoculated with approximately 4 log₁₀spores ml⁻¹. Clostridium botulinum populations were determined at appropriate intervals by plating onto RCM. Clostridium botulinum growth from spores was not observed at temperatures <12°C or >48°C for up to 3 weeks. Growth curves were determined by fitting Gompertz functions to the data. From the parameters of the Gompertz function the growth characteristics, GOL times and exponential growth rates were calculated. These growth characteristics were subsequently described by Ratkowsky functions using temperature as the independent variable. Closed form equations were developed that allow for predicting relative growth for a general cooling scenario. By applying multivariate statistical procedures, the standard errors and confidence intervals were computed on the predictions of the amount of relative growth for a cooling scenario. The predictive model is capable of predicting spore outgrowth and multiplication for general cooling scenarios, for suitable but un-verified mathematical assumptions, and should aid in evaluating the safety of cooked products after cooling.     

NISIN: A BACTERIOCIN WITH A POTENTIAL USE IN BREWING

Depending on the amount used and the strain of bacteria involved, nisin either kills lactic acid bacteria or inhibits their growth. In medium inoculated with approximately 10⁵ cells ml^?1 of a sensitive strain of Lactobacillus (BSO 375) nisin, added at levels recommended for commercial use (100 International Units ml^?1, killed all the cells in less than 6 h. In the absence of nisin this inoculum grew to a concentration of 10¹⁰ cells ml^?1 in about 50 h. Lower nisin concentrations killed fewer cells but inhibited the growth of those still viable. For the more resistant strain Lactobacillus (BSO 343) growth was only inhibited at the higher nisin concentrations. Nisin maintained its activity against lactic acid bacteria in brewing fermentations. It had no effect on the growth and fermentative performance of the 9 brewing yeast strains tested, and, in a pilot brewery fermentation, had no deleterious affect on the taste of the beer produced. Nisin could be used either as a preventative measure by regular addition to fermentations, or as a remedial measure once contamination by lactic acid bacteria had been detected.     

Reduction of Clostridium sporogenes spore outgrowth in natural sausage casings using nisin

Preservation of natural sausage casings using dry salt or saturated brine is regarded as sufficient to inactivate vegetative pathogenic non-spore-forming bacteria present on the casings. Although the outgrowth of bacterial spores is prevented by salt or saturated brine preservation, these spores will remain present and develop into vegetative cells when conditions are more favourable. To prevent subsequent outgrowth additional preservation measures should be implemented. In the experiments described the use of nisin was evaluated to reduce outgrowth of spores in desalinated casings. The bacteriocin nisin was chosen because of its known efficacy against spore-forming bacteria and their spores in various foodstuffs. Clostridium spore suspensions (Clostridium sporogenes, ATCC 3584) were used in two concentrations to inoculate three nisin concentrations (10, 50, 100 μg/mL) in water containing gamma-irradiated casings. Additionally, the binding of nisin to casings, using ¹⁴C-labeled nisin Z and subsequent availability of nisin were evaluated. Results demonstrate that nisin is partly reversibly bound to casings and can reduce the outgrowth of Clostridium spores in the model used by approximately 1 log₁₀ (90%). However, the biological relevance of these results needs to be determined further by conducting industrial trials before any recommendation can be made on the practical implementation of nisin in the preservation of natural sausage casings.     

Thermal resistance of bacterial spores in milk-based beverages supplemented with nisin.

The effect of nisin, added in the form of Nisaplin, on the thermal resistance of bacterial spores and the effects of medium composition, exposure time, and pH on nisin enhancement of heat sensitivity were evaluated. Nisin apparently required specific nutrients to sensitize spores to heat. For example, D130 degrees C values of approximately 10 s were observed in sodium phosphate buffer with and without 6% sucrose with no significant (P> or =0.05) differences detected as a result of increased nisin concentration. In a nutrient-rich chocolate milk model system (CMMS), increasing either the time of exposure to nisin (5, 15, or 24 h) before heating or nisin concentration (0, 2,000, or 4,000 IU/ml) increased the sensitivity of Bacillus stearothermophilus spores to heat. In the CMMS with 10 to 12% fat cocoa powder, increasing nisin concentration (at 5 h of exposure) significantly (P< or =0.05) reduced D130 degrees C values; D130 degrees C values were 21.7, 17.2, and 17.8 s, respectively, for the 0-, 2,000-, and 4,000-IU/ ml nisin treatments. Fifteen and 24 h of exposure further reduced D130 degrees C values in the nisin-containing treatments compared to the control (0 IU of nisin per ml). A lower-fat CMMS (0 to 1% fat cocoa powder) had lower D130 degrees C values (19.3, 15.8, and 14.7 s for the 0-, 2,000-, and 4,000-IU/ml nisin treatments, respectively). Nisin activity was enhanced by lowering pH in the CMMS (10 to 12% fat cocoa powder), with reductions in D130 degrees C values across all pH values (ranging from 18.0% at pH 6.4 to 41.9% at pH 5.0). zD values were 9.6, 9.0, and 8.4 degrees C for the 0-, 2,000-, and 4,000-IU/ml nisin treatments, respectively. Spores of B. licheniformis yielded results similar to those obtained with B. stearothermophilus. For example, decreasing CMMS (10 to 12% fat cocoa powder) pH values from 6.4 to 5.0 produced D100 degrees C values of 3.3, 2.8, and 2.8 min (pH 6.4) and 1.0, 0.8, and 0.8 min (pH 5.0) for the 0-, 2,000-, and 4,000-IU/ml nisin treatments. This study clearly verified that the addition of Nisaplin to dairy-based beverages, such as a chocolate milk drink, or other foods intended to be heated reduces the thermal resistance of selected bacterial spores. Increased spore sensitivity to heat may provide food processors with an opportunity to reduce their thermal processes and expenses while maintaining product quality, functionality, and shelf stability.     

Low Temperature Growth of Type E Clostridium botulinum Spores. 1. Effects of Sodium Chloride, Sodium Nitrite and pH

SUMMARY— The effects of pH and the addition of sodium chloride (NaCl) or sodium nitrite (NaNO₂) to trypticase-peptone-glucose (TPGI and trypticase-peptone-sucrose-yeast extract (TPSY) media upon spore outgrowth were investigated using seven Type E Clostridium botulinum strains. An inoculum of 1.0 × 10⁵ spores/ml was used and the pH was adjusted with hydrochloric acid to cover the range of 5.2 to 6.6. To define salt tolerance, NaCl was added to the media at intervals of 0.5% in the range of 2.0 to 5.0% and at 0.1% intervals in the 4.5 to 5.0% range. The effect of NaNO₂ was investigated with the addition of 100 and 200 ppm NaNO₂ to the media. Samples were incubated at 30, 15.6, 10, 7.2, 5.0 and 3.4°C. Outgrowth of all strains tested was inhibited at pH 5.2 at 15.6°C. Inhibition occurred at higher pH at lower temperatures. None of the strains showed outgrowth with 4.87% NaCl in the media at any of the incubation temperatures used. Addition of 100 and 200 ppm NaNO₂ to the media inhibited the outgrowth of the Minneapolis, 517, 26080 and A6247 strains but not the Kalamazoo and Seattle Forks strains.     

The effects on vegetative cells and spores of three bacteriocins from lactic acid bacteria

The sensitivities of vegetative cells of strains ofListeria, Clostridium, Staphylococcus, Lactococcus, Lactobacillus, MicrococcusandPediococcus, and of spores ofClostridiumandBacillusto three broad spectrum bacteriocins (nisin A, nisin Z and pediocin) from lactic acid bacteria were determined by a critical dilution micro-assay. The minimal inhibitory concentrations (MIC) of partially purified bacteriocins, prepared by a pH-dependent adsorption/desorption process, were determined and expressed in arbitrary units ml⁻¹and in μ g ml⁻¹of pure bacteriocin. The MICs of bacteriocins varied considerably between species and even between strains of the same species, as clearly shown for nine strains ofListeria monocytogenes. When bacteriocin activity was expressed in μ g ml⁻¹, pediocin was more effective againstListeria monocytogenesthan nisin A or nisin Z. The latter bacteriocins, in concentrations between 23 and 69 μ g ml⁻¹, prevented outgrowth ofClostridiumandBacillusspores for at least 10 days. Although pediocin at 17 μ g ml⁻¹prevented outgrowth ofB. stearothermophilusandC. butyricumspores for up to 7 days, it apparently activated the germination ofB. subtilisspores.     

Inhibition of Bacillus anthracis and potential surrogate bacilli growth from spore inocula by nisin and other antimicrobial peptides

The ability of nisin, synthetic temporin analogs, magainins, defensins, and cecropins to inhibit Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, and Bacillus subtilis growth from spore inocula was determined using well diffusion assays. Nisin, magainin II amide, and defensins were inhibitory in screening against B. anthracis Sterne or B. cereus ATCC 7004, but only nisin inhibited virulent B. anthracis strains. The MICs of nisin against the 10 Bacillus strains examined were 0.70 to 13.51 microg/ml. Synthetic temporin analogs also inhibited B. anthracis but were not as potent as nisin. None of the strains examined were appropriate B. anthracis surrogates for testing sensitivity to antimicrobial peptides.     

Combined effects of heat, nisin and acidification on the inactivation of Clostridium sporogenes spores in carrot-alginate particles: from kinetics to process validation

Combined effects of mild temperatures, acidification and nisin on the thermal resistance of Clostridium sporogenes ATCC 11437 spores were assessed. Inoculated carrot-alginate particles were used as a solid-food model for the validation of the spore inactivation during the flow of a solid-liquid food system through the holding tube of an aseptic processing unit. Inactivation kinetics was studied in a water bath with the spores inoculated into carrot-alginate particles and in Sorensen's phosphate buffer. For temperatures of 70-90 degrees C, D-values in the buffer were 24.9-5.7 min, much lower than those evaluated for the particles (115.1-22.2 min). Statistical analyses showed significant synergistic effects of temperature and pH on spore inactivation for both media. Acidification reduced the heat resistance of the spores by reducing the D-values. Nisin was not significantly effective at the lower concentrations (up to 750 IU/g). The combination of 90 degrees C, pH: 4.5 and 500IU/g nisin resulted in a ten-fold decrease of the D-value for spores inoculated in the particles (from 111.1 to 10.6 min). Microbial validation tests were conducted using a pilot-scale aseptic processing unit with a mixture of carrot cubes (10%) and carrier liquid of 2%-carboxymethylcellulose solution (90%). Spore-inoculated carrot-alginate particles (initial counts of 106 CFU/g, obtained after come-up-time pre-heat) with pH 3.5 and 2000 IU/g nisin were processed at 90 degrees C in the aseptic processing unit. Microbial analysis showed no spore survivors in the particles after passing through the holding tube (5.2-6.0 min of residence time). The proposed combination of these hurdles significantly enhanced the spore inactivation rate (D(90)=1.17 min) as compared to that for thermal treatment only (D(90)=19.6 min).     

Growth and toxigenesis of C. botulinum type E in fishes packaged under modified atmospheres

Modified atmosphere packaging of fresh fish is used to market high quality products in some European countries. The potential risk of C. botulinum growth in these extended shelf-life foods is still a concern; especially since toxigenesis may precede organoleptic spoilage. This paper will present toxigenic data from rockfish, salmon and sole muscle tissues which were inoculated with a pool of non-proteolytic C. botulinum type E at seven levels (10⁻² −10⁴ spores/sample), and stored under vacuum and 100% CO₂, at incubation temperatures between 30 and 4°C, for up to 60 days. Factorial experimental design allowed predictive formulae to be developed able to describe the lag time prior to C. botulinum toxigenesis and the probability of one spore to initiate toxigenesis based upon the storage conditions. Accurate characterization of the microbial ecology of C. botulinum in modified atmosphere-packaged fish, will support safe exploitation of these packaging systems in the market place, and identify critical control points for potential product or process abuses.     

Predictive model for growth of Clostridium botulinum from spores at temperatures applicable to cooling of cooked ground pork

Cooling deviations and temperature abuse are two main reasons leading to the risk of Clostridium botulinum outgrowth in cooked pork. The aim of this research was to create a model that could be used to estimate C. botulinum growth from spores in cooked pork at temperatures similar to those used to chill cooked pork in processing facilities and food establishments. A cocktail of proteolytic C. botulinum types A and B consisting of five strains per type were used to inoculate pork to a final spore concentration of approximately 2 log CFU/g and cooked to 71 °C to heat shock the spores and kill vegetative microbes. The growth of C. botulinum was established at constant storage temperatures from 10 to 46 °C. C. botulinum growth was also studied under dynamic temperature conditions with cooling set to start at 54.4 °C and end at 4.4 °C or 7.2 °C in monophasic or biphasic cooling profiles, respectively. Growth parameters were estimated using the Baranyi model as a primary model and growth rates were fitted using the modified Ratkowsky secondary model with respect to temperature. The R² values ranged from 0.7653 to 0.9995 indicating that the Baranyi primary model was well suited to the growth data. The modified Ratkowsky secondary model's R² was 0.9653 and its root mean square error (RMSE) was 0.0687. All 11 prediction error values computed were within the limit of acceptable prediction zone (−1.0 to 0.5) suggesting a good fit of the model. The predictive model can provide information for the safety of cooked pork exposed to longer chilling times or for customized process schedule development as cooling of larger diameter products presents a processing challenge in the meat process operations.     

Diversity Analysis,Batch Fermentation and Characterization of Nisin in Identified Strains of Lactococcus lactis spp. lactis

Thirty-one strains of Lactococcus lactis spp. lactis were identified out of 89 isolates of lactic acid bacteria (LAB) from dairy and nondairy sources. Of the 31 strains, 24 (46.1%) were obtained from dairy and seven (18.9%) from non-dairy sources. The cluster analysis of rep-PCR showed that (GTG)₅-PCR followed by ERIC-PCR exhibited more discriminating potential than BOX-PCR. The obtained banding patterns characterized the polymorphism among strains. The strain level polymorphism was also obtained by the combined cluster analysis of (GTG)_5, ERIC and BOX-PCR which exhibited a level of heterogeneity among strains but not with the sources of isolation. Among 31 strains, 17 strains were able to produce zones of inhibition against Lactobacillus acidophilus NCDC 015 and therefore considered as nisin producers. Nisin production by strains was further confirmed by PCR amplification of nisA/Z of 174 bp size. The nisin activity and cell growth observed to be higher in pH controlled batch fermentation than in uncontrolled fermentation. The nisin activity, cell concentration, and acidity were high in immobilized cell system than free cell batch fermentation. The hot acid and chloroform extraction method was found to be the efficient way for the partial purification of nisin from fermented broth.     

CIostridium botulinurn Spore Gerrnination, Outgrowth, and Toxin Production Below pH 4.6; Interactions Between pH, Total Acidity, and Buffering Capacity

The effect of soy protein concentration on Clostridium botulinurn spore germination, outgrowth, and toxin production in the presence of varying levels of inorganic (HCI) and organic (citric) acids was investigated. Media were blended using deoxygenated soy concentrate (1–7% protein), acidified with deaerated acids to effect post-autoclaving pH values of 4.1, 4.3 and 4.5, and inoculated with C. botulinum Type A spores. All manipulations were conducted under conditions of strict anaerobiosis (0–2 ppm atmospheric oxygen, media E_h7 values of < — 345 mV). Germination, proliferation, active motility, and elaboration of neurotoxin occurred as low as pH 4.07. Titratable acidity, which accounted for the soy protein buffering capacity, appeared to define toxicity endpoints as pH alone could not.     

Interacting Effects of pH and NaCl on Heat Resistance of Bacterial Spores

Spores of PA3679 (C. sporogenes) and Clostridium botulinum 213B were less heat resistant when heated in a menstruum of decreasing pH. The D value of the spore population decreased by 50% when heated in pH 5.0 buffer compared to pH 7.0 buffer. Addition of NaCl to the recovery medium reduced the number of colony forming units in a population of heated spores. Presence of NaCl at 2.0% in the recovery medium decreased the spore D-value by 20-40% irrespective of pH of the heating menstruum. Combined effects of pH and NaCl could be illustrated by three-dimensional histograms.     

Use of nisin for inhibition of Alicyclobacillus acidoterrestris in acidic drinks

The inhibitory effects of nisin on the growth of the thermoacidophilic spoilage bacteriumAlicyclobacillus acidoterrestris were investigated for the purpose of preventing flat-sour-type spoilage in acidic drinks. Minimum inhibitory concentration values of nisin against the spores were from less than 0·78 to 12·5 IU ml⁻¹and from 25 to 100 IU ml⁻¹on mYPGA plates at pH 3·4 and 4·2, respectively. The levels of nisin inhibition against the vegetative cells were, however, higher than those of the spores. In determining the effects of nisin on the thermal resistance of A. acidoterrestris spores, the addition of nisin contributed to the reduction of the thermal resistance of A. acidoterrestris spores in acidic drinks. Furthermore, the outgrowth of A. acidoterrestris spores was inhibited by the addition of 25–50 IU ml⁻¹nisin in both orange and fruit-mixed drinks, but was not inhibited by the higher level (600 IU ml⁻¹) addition in a clear-apple drink. From these findings, we conclude that it would be useful to add nisin for preventing the spoilage caused by A. acidoterrestris in all but clear-apple acidic drinks.     

Optimization of Nisin Production by Lactococcus lactis UQ2 Using Supplemented Whey as Alternative Culture Medium

Abstract: Lactococcus lactis UQ2 is a nisin A-producing native strain. In the present study, the production of nisin by L. lactis UQ2 in a bioreactor using supplemented sweet whey (SW) was optimized by a statistical design of experiments and response surface methodology (RSM). In a 1st approach, a fractional factorial design (FFD) of the order 2^5-1 with 3 central points was used. The effect on nisin production of air flow, SW, soybean peptone (SP), MgSO₄/MnSO₄ mixture, and Tween 80 was evaluated. From FFD, the most significant factors affecting nisin production were SP (P = 0.011), and SW (P = 0.037). To find optimum conditions, a central composite design (CCD) with 2 central points was used. Three factors were considered, SW (7 to 10 g/L), SP (7 to10 g/L), and small amounts of added nisin as self-inducer (NI 34.4 to 74.4 IU/L). Nisin production was expressed as international units (IU). From RSM, an optimum nisin activity of 180 IU/mL was predicted at 74.4 IU/L NI, 13.8 g/L SP, and 14.9 or 5.11 g/L SW, while confirmatory experiments showed a maximum activity of 178 ± 5.2 IU/mL, verifying the validity of the model. The 2nd-order model showed a coefficient of determination (R²) of 0.828. Optimized conditions were used for constant pH fermentations, where a maximum activity of 575 ± 17 IU/mL was achieved at pH 6.5 after 12 h. The adsorption-desorption technique was used to partially purify nisin, followed by drying. The resulting powder showed an activity of 102150 IU/g. Practical Application : Nisin production was optimized using supplemented whey as alternative culture medium, using a native L. lactis UQ2 strain. Soybean peptone, SW, and subinhibitory amounts of nisin were successfully employed to optimize nisin production by L. lactis UQ2. Dried semipurified nisin showed an activity of 102150 IU/g.