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.     

Evaluation of microbial survival post-incidence on fresh Mozzarella cheese

Commercial fresh Mozzarella cheese is made by direct acidification and is stored dry or in water without salt addition. The cheese has a shelf life of 6 wk, but usually develops an off-flavor and loses textural integrity by 4 wk, potentially due to the lack of salt and high moisture that allow the outgrowth of undesirable bacteria. To understand how microbial incidence affects cheese quality and how incident pathogen-related bacteria are limited by salt level during refrigerated storage, we made fresh Mozzarella cheese with high (2%) and low (0.5%) salt. The high-salt cheese was packaged and stored dry. The low-salt cheese was packaged and stored either dry or in 0.5% salt brine. One portion of cheeses was evaluated for surviving incident microbes by aerobic plate counts, coliform counts, and psychrophilic bacterial counts, of which coliforms and psychrophiles were not detected over 9 wk. Aerobic plate counts remained at 100 to 300 cfu/g up to 2 wk but increased by 1,000- to 10,000-fold between 4 and 6 wk at all salt levels and storage conditions. Other portions of cheeses were inoculated with either Escherichia coli or Enterococcus faecalis, both of which increased by 100-fold over 90 d of storage. Interestingly, E. coli added to the cheese brine first grew in the brine by 100-fold before attaching to the cheese, whereas Ent. faecalis attached to the cheese within 24 h and grew only on the cheese. We conclude that incident bacteria, even from similar environments, may attach to cheese curd and survive differently in fresh Mozzarella cheese than in brine. Overall, 2% salt was insufficient to control bacterial growth, and slow-growing, cold- and salt-tolerant bacteria may survive and spoil fresh Mozzarella cheese.     

Virus inactivation by salt (NaCl) and phosphate supplemented salt in a 3D collagen matrix model for natural sausage casings

Due to possible presence and spread of contagious animal viruses via natural sausage casings the international trade in these food products is subject to veterinary and public health requirements. In order to manage these restrictions we determined the effect of casing preservation on four highly contagious viruses for livestock: foot-and-mouth-disease virus (FMDV), classical swine fever virus (CSFV), swine vesicular disease virus (SVDV) and African swine fever virus (ASFV). We used an in vitro 3D collagen matrix model in which cells, infected with the four different viruses were embedded in a bovine collagen type I gel matrix and treated with either saturated salt (NaCl) or phosphate supplemented saturated salt at four different temperatures (4, 12, 20 and 25 °C) during a period of 30 days. The results showed that all viruses were faster inactivated at higher temperatures, but that stability of the various viruses at 4 °C differed. Inactivation of FMDV in the 3D collagen matrix model showed a clear temperature and treatment effect on the reduction of FMDV titres. At 4 and 12 °C phosphate supplemented salt showed a very strong FMDV inactivation during the first hour of incubation. Salt (NaCl) only had a minor effect on FMDV inactivation. Phosphate supplemented salt treatment increased the effect temperature had on inactivation of CSFV. In contrast, the salt (NaCl) treatment only increased CSFV inactivation at the higher temperatures (20 °C and 25 °C). Also SVDV inactivation was increased by phosphate supplemented salt, but salt (NaCl) treatment only resulted in a significant decrease of SVDV titre at a few time points. The ASFV results showed that both salt (NaCl) and phosphate supplemented salt were capable to inactivate ASFV within 48 h. In contrast to the other viruses (FMDV, CSFV and SVDV), ASFV was the most stable virus even at higher temperatures. The results obtained in this in vitro model underline the efficacy of a combined treatment using phosphate supplemented salt and storage at 20 °C or higher for a period of 30 days. This treatment may therefore be useful in reducing the animal health risks posed by spread of contagious animal viruses by international trade of natural sausage casings.     

Stability of α-tocotrienol and α-tocopherol in salami-type sausages and curing brine depending on nitrite and pH

We studied the stability of the valuable vitamer nutrients α-tocotrienol and α-tocopherol and options for their protection in salami-type sausages (blended with α-tocotrienol-rich barley oil) and curing brine. Four different sausage formulations were produced containing nitrite curing salt; nitrite curing salt and ascorbic acid (300 mg/kg); nitrite curing salt and carnosic acid (45 mg/kg); or sodium chloride. Initial vitamer contents (100 mg/kg) did not decrease significantly during ripening and decreased only slightly during storage. Ascorbic acid and carnosic acid were found to be effective in preserving the vitamers in fresh sausages. Freeze-drying of sausages resulted in a significant loss of vitamers (97%), particularly after 14-day storage at room temperature, even in the presence of shielding gases. The vitamer content in the curing brine decreased with decreasing pH in the presence of nitrite. A nitrite concentration of 136 mg/L at pH 4 resulted in significant loss (90%) of the vitamers. Sufficient stability of the vitamers in salami-type sausage and curing brine can be achieved by processing, formulation, and storage conditions.     

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.     

Inhibition of Clostridium tyrobutyricum by Streptococcus macedonicus ACA-DC 198 under conditions mimicking Kasseri cheese production and ripening

Three fermentations in skim milk were used to study the effectiveness of the bacteriocin-producing Streptococcus macedonicus ACA-DC 198 strain to inhibit Clostridium tyrobutyricum LMG 1285T spore outgrowth under conditions prevailing during Kasseri cheese production and ripening. In fermentation A, Clostridium spores were used solely; in fermentation B, S. macedonicus ACA-DC 198 and Clostridium spores were used; in fermentation C, a commercial starter culture and Clostridium spores were used. The temperature program applied was similar to that of Kasseri cheese production and ripening. The presence of macedocin, the bacteriocin produced by S. macedonicus ACA-DC 198, was confirmed in fermentation B. The results showed that macedocin was able to inhibit the outgrowth of Clostridium spores, since significantly higher inhibition in spore outgrowth was detected in fermentation B than in fermentation C.     

Involvement of Clostridium gasigenes and C. algidicarnis in 'blown pack' spoilage of Brazilian vacuum-packed beef

The objectives of this study were to isolate psychrotrophic clostridia from Brazilian vacuum-packed beef cuts (spoiled or not) and to identify the isolates by using 16S rRNA gene sequencing. Anaerobic psychrotrophic microorganisms were also enumerated and samples were collected to verify the incidence of psychrotrophic clostridia in the abattoir environment. Vacuum-packed beef cuts (n = 8 grossly distended and n = 5 non-spoiled) and environmental samples were obtained from a beef packing plant located in the state of São Paulo, Brazil. Each sample was divided in three subsamples (exudate, beef surface and beef core) that were analyzed for vegetative forms, total spore-forming, and sulfide reducing spore-forming, both activated by alcohol and heat. Biochemical profiles of the isolates were obtained using API20A, with further identification using 16S rRNA gene sequencing. The growth temperature and the pH range were also assessed. Populations of psychrotrophic anaerobic vegetative microorganisms of up to 10¹⁰ CFU/(g, mL or 100 cm²) were found in ‘blown pack’ samples, while in non-spoiled samples populations of 10⁵ CFU/(g, CFU/mL or CFU/100cm²) was found. Overall, a higher population of total spores and sulfide reducing spores activated by heat in spoiled samples was found. Clostridium gasigenes (n = 10) and C. algidicarnis (n = 2) were identified using 16S rRNA gene sequencing. Among the ten C. gasigenes isolates, six were from spoiled samples (C1, C2 and C9), two were isolated from non-spoiled samples (C4 and C5) and two were isolated from the hide and the abattoir corridor/beef cut conveyor belt. C. algidicarnis was recovered from spoiled beef packs (C2). Although some samples (C3, C7, C10 and C14) presented signs of ‘blown pack’ spoilage, Clostridium was not recovered. C. algidicarnis (n = 1) and C. gasigenes (n = 9) isolates have shown a psychrotrophic behavior, grew in the range 6.2-8.2. This is the first report on the isolation of psychrotrophic Clostridium (C. gasigenes and C. algidicarnis) in Brazil. This study shows that psychrotrophic Clostridium may pose a risk for the stability of vacuum-packed beef produced in tropical countries during shelf-life and highlights the need of adopting control measures to reduce their incidence in abattoir and the occurrence of ‘blown pack’ spoilage.     

Potential production and preservation of dahi by Lactococcus lactis W8, a nisin-producing strain

Lactococcus lactis W8 produced nisin concomitantly while fermenting milk to “dahi”, a traditional Indian fermented milk. The activity of nisin was detected at 3 h of fermentation, which increased in parallel to growth of the organism and reached its maximum at 6 h. The activity remained essentially stable thereafter. At 7 h of fermentation of milk with the strain L. lactis W8 the pH of the medium dropped to 4.2, when the milk became converted to dahi. The produced dahi displayed antibacterial property against spoilage and pathogenic bacteria including Listeria monocytogenes. When L. monocytogenes was mixed with dahi at 5.2 log CFU/ml and stored at 4 °C, the number of L. monocytogenes gradually decreased and became undetectable at 10 h. L. lactis W8 appeared to be a suitable starter culture for production of dahi from milk and preservation of the dahi.     

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.     

A survey of dry-salted natural casings for the presence of Salmonella spp., Listeria monocytogenes and sulphite-reducing Clostridium spores

A monitoring study was performed for the presence of Salmonella spp., Listeria monocytogenes and sulphite-reducing Clostridium spores in (internationally) traded dry-salted natural hog and sheep casings. Two hundred and fourteen consignments were examined for Salmonella spp. and L. monocytogenes, and 138 for the Clostridium spores.None of the 214 sampled consignments (25 g amounts investigated) yielded Salmonella spp., or L. monocytogenes.Differential reinforced clostridial medium was effective in detecting the presence of sulphite-reducing clostridia. Iron sulphite agar (ISA) overall showed higher clostridia counts as compared to differential reinforced clostridial agar (DRCA). The maximum spore counts obtained on DRCA and ISA were 17.5 and 2500 cfu g⁻¹, respectively. From the casings from China, 3 (of 35 hog consignments) and 7 (of 22 sheep) showed spore counts above 100 cfu g⁻¹. From the remaining 81 samples, originating from Netherlands, New Zealand and UK, none showed a count above 100 cfu g⁻¹.The relevance of the presence of sulphite-reducing Clostridium spores for the manufacture of various meat products is discussed. It is recommended that determination of the Clostridium strains present is carried out and their properties investigated in relation to the manufacture of meat products, since some of the strains may be potentially pathogenic and/or able to spoil products.     

Antimicrobial properties of salt (NaCl) used for the preservation of natural casings

The antimicrobial properties of salt (NaCl) used for the preservation of natural casings were studied by investigating the survival of six bacterial species in natural casings at different water activity (aw) levels. Individual sheep casings were inoculated with ca. 10(5) colony-forming units (cfu) g(-1) of Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens and 10(2)cfu g(-1) of E. coli O157:H7. The casings were stored at 20+/-1.5 degrees C in different brines and dry salt, giving aw-levels of 0.90 aw, 0.87aw, 0.85 aw, 0.83 aw and 0.75 aw. Samples were taken at day 1, 3, 6, 8, 13, 20, 27 and 30 after inoculation and the number of bacteria present was determined. Based on survival curves, death rates (day(-1)) were calculated to quantify the reduction in log10 cfu g(-1) per day. The influence of aw on death rates was higher for Gram-negative bacteria than for Gram-positive bacteria. The death rates were overall higher for Gram-negatives than for Gram-positives. No clear reduction in the survival of C. perfringens in relation to any aw level was observed in this study. These results indicate that the antimicrobial properties of salt used for the preservation of natural casings are sufficient to reduce the bacterial contamination (except for Clostridium spores) well below acceptable levels at a water activity level of 0.85 or lower during a 30-day storage period.     

Inhibitory effects of nisin against Clostridium perfringens food poisoning and nonfood-borne isolates

The enterotoxigenic Clostridium perfringens type A is the causative agent of C. perfringens type A food poisoning (FP) and nonfood-borne (NFB) human gastrointestinal diseases. Due to its ability to form highly resistant endospores, it has become a great concern to the meat industry to produce meat free of C. perfringens. In this study, we evaluated the antimicrobial effect of nisin against C. perfringens FP and NFB isolates. No inhibitory effect of nisin was observed against germination of spores of both FP and NFB isolates in laboratory medium. However, nisin effectively arrested outgrowth of germinated spores of C. perfringens in rich medium. Interestingly, germinated spores of NFB isolates possessed higher resistance to nisin than that of FP isolates. Furthermore, nisin exhibited inhibitory effect against vegetative growth of both FP and NFB isolates in laboratory medium, with vegetative cells of NFB isolates showing higher resistance than that of FP isolates. However, the antimicrobial activity of nisin against C. perfringens was significantly decreased in a meat model system. In conclusion, although nisin showed inhibitory effect against spore outgrowth and vegetative cells of C. perfringens FP and NFB isolates in laboratory conditions, no such effect was observed against C. perfringens spores inoculated into a meat model system.     

Inactivation of classical swine fever virus in porcine casing preserved in salt

Pig intestines used for the production of natural sausage casings may carry classical swine fever (CSF) virus. Feeding pigs with human food waste that contains pig casings may then spread the virus to CSF-free animals. Casings derived from a pig experimentally infected with CSF by dosing with 10(6) tissue culture infectious doses (TCID50) of the highly virulent CSF virus strain "Koslov", were treated with phosphate supplemented or citrate supplemented NaCl, instead of with NaCl alone, which is the standard preservation treatment for casings. Treated casings were stored for 30 days at either 4 degrees C or 20 degrees C. After storage the casings were fed to 16 susceptible pigs. CSF infection was confirmed in the four animals that had been fed casings treated with citrate supplemented salt and stored at 4 degrees C. All other animals remained healthy. It is therefore possible to avoid the inadvertent spread of CSF virus via porcine sausage casings by treating casings with phosphate supplemented salt and storing them for 30 days at temperatures over 4 degrees C.     

Effect of exopolysaccharides (EPSs) produced by Lactobacillus delbrueckii subsp. bulgaricus strains to bacteriophage and nisin sensitivity of the bacteria

Lactobacillus strains used in this study were isolated from village-type yogurt and raw milk. The isolates were identified as Lactobacillus delbrueckii subsp. bulgaricus by 16 s rDNA sequence analysis and API 50 CHL identification systems. The exopolysaccharide (EPS) production of the strains growth in skim milk were investigated. In addition sensitivity and insensitivity of these strains against domestic bacteriophages and nisin were examined. It was deduced that those strains which had relatively high EPS-producing capacity were insensitive against phages and nisin. Linear relationships were determined between EPS production of the bacteria and bacteriophage and nisin insensitivity of the bacteria.There was a negative correlation between EPS production quantity and phage and nisin sensitivity of the bacteria. Of all the strains, L. delbrueckii subsp bulgaricus B3 produced the highest EPS quantity, and it was insensitive against phages and nisin. Based on these results, it is suggested that L. delbrueckii subsp bulgaricus B3 can be used with the starter culture in dairy industry for stable and high-quality yogurt production.     

Antimicrobial effects of a bioactive glycolipid on spore-forming spoilage bacteria in milk

The growth of psychrotolerant aerobic spore-forming bacteria during refrigerated storage often results in the spoilage of fluid milk, leading to off-flavors and curdling. Because of their low toxicity, biodegradability, selectivity, and antimicrobial activity over a range of conditions, glycolipids are a novel and promising intervention to control undesirable microbes. The objective of this study was to determine the efficacy of a commercial glycolipid product to inhibit spore germination, spore outgrowth, and the growth of vegetative cells of Paenibacillus odorifer, Bacillus weihenstephanensis, and Viridibacillus arenosi, which are the predominant spore-forming spoilage bacteria in milk. For spore germination and outgrowth assays, varying concentrations (25–400 mg/L) of the glycolipid product were added to commercial UHT whole and skim milk inoculated with ～4 log₁₀ spores/mL of each bacteria and incubated at 30°C for 5 d. Inhibition of spore germination in inoculated UHT whole milk was only observed for V. arenosi, and only when glycolipid was added at 400 mg/L. However, concentrations of 400 and 200 mg/L markedly inhibited the outgrowth of vegetative cells from spores of P. odorifer and B. weihenstephanensis, respectively. No inhibition of spore germination or outgrowth was observed in inoculated UHT skim milk for any strain at the concentrations tested (25 and 50 mg/L). The effect of glycolipid addition on vegetative cell growth in UHT whole and skim milk when inoculated with ～4 log₁₀ cfu/mL of each bacteria was also determined over 21 d of storage at 7°C. Glycolipid addition at 50 mg/L was bactericidal against P. odorifer and B. weihenstephanensis in inoculated UHT skim milk through 21 d of storage, whereas 100 mg/L was needed for similar control of V. arenosi. Concentrations of 100 and 200 mg/L inhibited the growth of vegetative cells of B. weihenstephanensis and P. odorifer, respectively, in inoculated UHT whole milk, whereas 200 mg/L was also bactericidal to B. weihenstephanensis. Additional studies are necessary to identify effective concentrations for the inhibition of Viridibacillus spp. growth in whole milk beyond 7 d. Findings from this study demonstrate that natural glycolipids have the potential to inhibit the growth of dairy-spoilage bacteria and extend the shelf life of milk.     

The wet-heat resistance of Bacillus weihenstephanensis KBAB4 spores produced in a two-step sporulation process depends on sporulation temperature but not on previous cell history

While bacterial spores are mostly produced in a continuous process, this study reports a two-step sporulation methodology. Even though spore heat resistance of numerous spore-forming bacteria is known to be dependent on sporulation conditions, this approach enables the distinction between the vegetative cell growth phase in nutrient broth and the sporulation phase in specific buffer. This study aims at investigating whether the conditions of growth of the vegetative cells, prior to sporulation, could affect spore heat resistance. For that purpose, wet-heat resistance of Bacillus weihenstephanensis KBAB4 spores, produced via a two-step sporulation process, was determined from vegetative cells harvested at four different stages of the growth kinetics, i.e. early exponential phase, late exponential phase, transition phase or early stationary phase. To assess the impact of the temperature on spore heat resistance, sporulation was performed at 10 °C, 20 °C and 30 °C from cells grown during a continuous or a discontinuous temperature process, differentiating or not the growth and sporulation temperatures. Induction of sporulation seems possible for a large range of growth stages. Final spore concentration was not significantly affected by the vegetative cell growth stage while it was by the temperature during growing and sporulation steps. The sporulation temperature influences the heat resistance of B. weihenstephanensis KBAB4 spores much more than growth temperature prior to sporulation. Spores produced at 10 °C were up to 3 times less heat resistant than spores produced at 30 °C.     

Toxin producing Bacillus cereus persist in ready-to-reheat spaghetti Bolognese mainly in vegetative state

The potential of Bacillus cereus to cause a diarrheal toxico-infection is related to its ability to perform de novo enterotoxin production in the small intestine. A prerequisite for this is presence of sufficient numbers of B. cereus that have survived gastro-intestinal passage. It is known that the percentage of survival is much smaller for vegetative cells in comparison to spores and it is therefore important to know the state in which B. cereus is ingested. The results of the current study performed on twelve B. cereus strains, comprising both diarrheal and emetic type, indicate that exposure via contaminated foods mainly concerns vegetative cells. Inoculated vegetative cells grew to high counts, with the growth dynamic depending on the storage temperature. At 28 °C growth to high counts resulted in spore formation, in general, after 1 day of storage. One strain was an exception, producing spores only after 16 days. At 12 °C obtained high counts did not result in spore formation for 11 of 12 tested strains after two weeks of storage. The highest counts and time to sporulation were different between strains, but no difference was observed on the group level of diarrheal and emetic strains. The spore counts were always lower than vegetative cell counts and occurred only when food was obviously sensory spoiled (visual and odor evaluation). Similar observations were made with food inoculated with B. cereus spores instead of vegetative cells. Although the prospect of consuming spores was found very weak, the numbers of vegetative B. cereus cells were high enough, without obvious sensory deviation, to survive in sufficient level to cause diarrheal toxico-infection.     

Relevant factors affecting microbial surface decontamination by pulsed light

Pulsed Light (PL) uses intense flashes of white light rich in ultraviolet (UV) light for decontamination. A log-reduction higher than 5 was obtained in one flash and at fluences lower than 1.8 J/cm² on spores of a range of spore-forming bacteria, of vegetative cells of non-spore-forming bacteria and on yeasts spread on agar media. Vegetative cells were more sensitive than spores. The inactivation by PL of Bacillus subtilis, B. atrophaeus, B. cereus, Geobacillus stearothermophilus, and Aspergillus niger spores sprayed on polystyrene was similar. The inactivation by PL of B. subtilis and A. niger spores sprayed on glass was slightly lower than on polystyrene. No alteration of the spore structures was detected by scanning electron microscopy for both PL treated B. subtilis and A. niger spores. The inactivation of spores of B. subtilis, B. atrophaeus, B. cereus and B. pumilus by PL or by continuous UV-C at identical fluences was not different, and was much higher by PL for A. niger spores. The increase in the input voltage of the lamps (which also increases the UV-C %) resulted in a higher inactivation. There was no correlation between the resistance to heat and the resistance to PL. The relative effect of UV-C radiations and light thermal energy on PL inactivation was discussed.     

Effects of nisin and reutericyclin on resistance of endospores of Clostridium spp. to heat and high pressure

The effects of high pressure, temperature, and antimicrobial compounds on endospores of Clostridium spp. were examined. Minimal inhibitory concentrations (MIC) of nisin and reutericyclin were determined for vegetative cells and endospores of Clostridium sporogenes ATCC 7955, Clostridium beijerinckii ATCC 8260, and Clostridium difficile 3195. Endospores of C. sporogenes ATCC 7955 and C. beijerinckii ATCC 8260 were exposed to 90 °C and 90 °C/600 MPa in the presence of 16 mg L⁻¹ nisin or 6.4 mg L⁻¹ reutericyclin for 0–60 min in a 0.9% saline solution. Dipicolinic acid (DPA) release was measured using a terbium-DPA fluorescence assay, and endospore permeability was assessed using 4′,6-diamidino-2-phenylindole (DAPI) fluorescence. Vegetative cells of C. sporogenes ATCC 7955 exhibited higher sensitivity to nisin relative to endospores, with MIC values 0.23 ± 0.084 mg L⁻¹ and 1.11 ± 0.48 mg L⁻¹, respectively. Nisin increased DPA release when endospores were treated at 90 °C; however, only C. sporogenes ATCC 7955 exhibited higher inactivation, suggesting strain or species specific effects. Reutericyclin did not enhance spore inactivation or DPA release. Use of nisin in combination with high pressure, thermal treatments enhanced inactivation of endospores of Clostridium spp. and may have application in foods.