Clostridium Perfringens Spore Germination Research Articles

Divalent Cation Signaling in Clostridium perfringens Spore Germination

Spore germination plays an essential role in the pathogenesis of Clostridium perfringens-associated food poisoning. Germination is initiated when bacterial spores sense various stimuli, including chemicals and enzymes. A previous study showed that dipicolinic acid (DPA) chelated with calcium (Ca-DPA) significantly stimulated spore germination in C. perfringens. However, whether Ca2+ or DPA alone can induce germination is unknown. Therefore, we aimed to evaluate the possible roles of Ca2+ and other divalent cations present in the spore core, such as Mn2+ and Mg2+, in C. perfringens spore germination. Our study demonstrated that (i) Ca-DPA, but not DPA alone, induced C. perfringens spore germination, suggesting that Ca2+ might play a signaling role; (ii) all tested calcium salts induced spore germination, indicating that Ca2+ is critical for germination; (iii) the spore-specific divalent cations Mn2+ and Mg2+, but not Zn2+, induced spore germination, suggesting that spore core-specific divalent cations are involved in C. perfringens spore germination; and (iv) endogenous Ca2+ and Mg2+ are not required for induction of C. perfringens spore germination, whereas exogenous and partly endogenous Mn2+ are required. Collectively, our results suggest that exogenous spore core-specific divalent cation signals are more important than endogenous signals for the induction of spore germination.

Effect of vacuum cooling followed by ozone repressurization on Clostridium perfringens germination and outgrowth in cooked pork meat under temperature-abuse conditions

The effect of combining vacuum cooling with an ozone-based inhibition process (InhVac) on Clostridium perfringens spore germination and outgrowth in cooked pork meat after exponential chilling (from 54.4 to 7.2 °C in 12, 15, 18, or 21 h) and isothermal storage (20, 25, 30, 36, or 45 °C) was evaluated. Ice cooling (IC) and vacuum cooling (VC) were used to compare the effects with InhVac. The samples were inoculated with a three-strain mixture of C. perfringens spores to obtain concentration of ca. 3 log10 CFU/g. C. perfringens growth in samples treated by InhVac were 0.1, 0.37 and 0.9 log10 CFU/g after 15, 18 and 21 h of cooling from 54.4 to 7.2 °C respectively, significantly lower (P<0.05) than those in samples subjected to IC (1.01, 2.10 and 2.8 log10 CFU/g) and VC (0.56, 1.01 and 2.13 log10 CFU/g). Compared to VC and IC, InhVac treatment increased the lag phase (λ), decreased the growth rates (μmax), and extended the sample shelf-life (the time until a 1 log10 CFU/g increase in C. perfringes from the initial concentration value) at all storage temperatures. InhVac-treated samples not only had a longer shelf-life than those treated by VC, but also exhibited almost two times longer shelf-life compared to those subjected to IC regardless of storage temperatures. Additionally, statistical indexes showed that a primary modified Gompertz model and a secondary Square Root model could fit the data well. Industrial relevanceIn this study, an innovative inhibition approach (InhVac) was found to show a better antimicrobial effect on C. perfringens germination and outgrowth in cooked pork meat compared to ice cooling and vacuum cooling under temperature-abuse conditions. A primary modified Gompertz model and a secondary Square Root model could be used to predict the C. perfringens growth in samples subjected to InhVac treatment.

Control of Clostridium perfringens spore germination and outgrowth by potassium lactate and sodium diacetate in ham containing reduced sodium chloride

The effect of potassium lactate and sodium diacetate mixture (Opti.Form PD4®) on Clostridium perfringens spore germination and outgrowth during abusive cooling of reduced sodium ham was evaluated. A ham formulation containing NaNO2 (0 or 100 ppm), NaCl (1 or 2%) and Opti.Form PD4® (0, 1.5 or 2.5%) was prepared. Meat (10 g) was vacuum-packaged, stored under refrigeration (5 °C) for 3 or 24 h and inoculated with a three-strain C. perfringens spore cocktail. Samples were vacuum-packaged, heat treated (75 °C, 20 min) and cooled from 54.4 °C to 7.2 °C within 9, 15 or 21 h. C. perfringens population increases of 5.18 and 4.26 log CFU/g were observed in non-cured ham (control; stored for 3 h) formulated with 1 or 2% of NaCl, respectively and cooled exponentially within 21 h. Incorporation of Opti.Form PD4® (1.5%) inhibited C. perfringens spore germination and outgrowth regardless of the cooling rate (9, 15 or 21 h). Addition of NaNO2 (100 ppm) enhanced antimicrobial activity of Opti.Form PD4®. In general, abusive cooling (≥15 h) of ham after 24 h of storage resulted in greater populations of C. perfringens. Addition of NaNO2, Opti.Form PD4® and processing immediately resulted in greater inhibition of C. perfringens spore germination and outgrowth.

Inhibition of germination and outgrowth of Clostridium perfringens spores by buffered calcium, potassium and sodium citrates in cured and non-cured injected pork during cooling

Inhibition of Clostridium perfringens spore germination and outgrowth by buffered calcium, potassium, and sodium citrates (Ca-Cit, K-Cit, and Na-Cit, respectively) during exponential cooling of non-cured and cured pork was evaluated. Antimicrobials were added to a base formulation of a pork product at a final concentration of 0.5, 1.0 or 1.5% w/w along with a control (without citrates). A three strain cocktail of C. perfringens spores was added to achieve a final spore population of 2.5–3.0 log CFU/g. Heat treatment and exponential cooling of inoculated, non-cured and cured control pork from 54.4 °C to 7.2 °C in 6.5, 9, 12, 15, 18, and 21 h resulted in C. perfringens spore germination and outgrowth of 0.52, 1.53, 4.45, 4.81, 5.83, 6.98 log CFU/g and 0.52, 1.55, 3.45, 4.59, 5.65 and 6.04 log CFU/g, respectively. Incorporation of K- or Na-citrates (>1.0%) inhibited germination and outgrowth of C. perfringens spores (<1.0 log CFU/g) compared to the control product. Ca-Cit was more effective in inhibiting C. perfringens spore germination and outgrowth at 0.5% compared to sodium and potassium citrates. Incorporation of Ca, K or Na citrates into pork product formulations can inhibit C. perfringens spores during extended cooling or during cooling process deviations.

New amino acid germinants for spores of the enterotoxigenic Clostridium perfringens type A isolates

Clostridium perfringens spore germination plays a critical role in the pathogenesis of C. perfringens-associated food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases. Germination is initiated when bacterial spores sense specific nutrient germinants (such as amino acids) through germinant receptors (GRs). In this study, we aimed to identify and characterize amino acid germinants for spores of enterotoxigenic C. perfringens type A. The polar, uncharged amino acids at pH 6.0 efficiently induced germination of C. perfringens spores; l-asparagine, l-cysteine, l-serine, and l-threonine triggered germination of spores of most FP and NFB isolates; whereas, l-glutamine was a unique germinant for FP spores. For cysteine- or glutamine-induced germination, gerKC spores (spores of a gerKC mutant derivative of FP strain SM101) germinated to a significantly lower extent and released less DPA than wild type spores; however, a less defective germination phenotype was observed in gerAA or gerKB spores. The germination defects in gerKC spores were partially restored by complementing the gerKC mutant with a recombinant plasmid carrying wild-type gerKA-KC, indicating that GerKC is an essential GR protein. The gerKA, gerKC, and gerKB spores germinated significantly slower with l-serine and l-threonine than their parental strain, suggesting the requirement for these GR proteins for normal germination of C. perfringens spores. In summary, these results indicate that the polar, uncharged amino acids at pH 6.0 are effective germinants for spores of C. perfringens type A and that GerKC is the main GR protein for germination of spores of FP strain SM101 with l-cysteine, l-glutamine, and l-asparagine.

Effect of meat ingredients (sodium nitrite and erythorbate) and processing (vacuum storage and packaging atmosphere) on germination and outgrowth of Clostridium perfringens spores in ham during abusive cooling

The effect of nitrite and erythorbate on Clostridium perfringens spore germination and outgrowth in ham during abusive cooling (15 h) was evaluated. Ham was formulated with ground pork, NaNO2 (0, 50, 100, 150 or 200 ppm) and sodium erythorbate (0 or 547 ppm). Ten grams of meat (stored at 5 °C for 3 or 24 h after preparation) were transferred to a vacuum bag and inoculated with a three-strain C. perfringens spore cocktail to obtain an inoculum of ca. 2.5 log spores/g. The bags were vacuum-sealed, and the meat was heat treated (75 °C, 20 min) and cooled within 15 h from 54.4 to 7.2 °C. Residual nitrite was determined before and after heat treatment using ion chromatography with colorimetric detection. Cooling of ham (control) stored for 3 and 24 h, resulted in C. perfringens population increases of 1.46 and 4.20 log CFU/g, respectively. For samples that contained low NaNO2 concentrations and were stored for 3 h, C. perfringens populations of 5.22 and 2.83 log CFU/g were observed with or without sodium erythorbate, respectively. Residual nitrite was stable (p > 0.05) for both storage times. Meat processing ingredients (sodium nitrite and sodium erythorbate) and their concentrations, and storage time subsequent to preparation of meat (oxygen content) affect C. perfringens spore germination and outgrowth during abusive cooling of ham.

Inhibition of Clostridium perfringens Spore Germination and Outgrowth by Lemon Juice and Vinegar Product in Reduced NaCl Roast Beef

Inhibition of Clostridium perfringens spore germination and outgrowth in reduced sodium roast beef by a blend of buffered lemon juice concentrate and vinegar (MoStatin LV1) during abusive exponential cooling was evaluated. Roast beef containing salt (NaCl; 1%, 1.5%, or 2%, w/w), blend of sodium pyro- and poly-phosphates (0.3%), and MoStatin LV1 (0%, 2%, or 2.5%) was inoculated with a 3-strain C. perfringens spore cocktail to achieve final spore population of 2.5 to 3.0 log CFU/g. The inoculated products were heat treated and cooled exponentially from 54.4 to 4.4 °C within 6.5, 9, 12, 15, 18, or 21 h. Cooling of roast beef (2.0% NaCl) within 6.5 and 9 h resulted in <1.0 log CFU/g increase in C. perfringens spore germination and outgrowth, whereas reducing the salt concentration to 1.5% and 1.0% resulted in >1.0 log CFU/g increase for cooling times longer than 9 h (1.1 and 2.2 log CFU/g, respectively). Incorporation of MoStatin LV1 into the roast beef formulation minimized the C. perfringens spore germination and outgrowth to <1.0 log CFU/g, regardless of the salt concentration and the cooling time. Cooked, ready-to-eat meat products should be cooled rapidly to reduce the risk of Clostridium perfringens spore germination and outgrowth. Meat processors are reducing the sodium chloride content of the processed meats as a consequence of the dietary recommendations. Sodium chloride reduces the risk of C. perfringens spore germination and outgrowth in meat products. Antimicrobials that contribute minimally to the sodium content of the product should be incorporated into processed meats to assure food safety. Buffered lemon juice and vinegar can be incorporated into meat product formulations to reduce the risk of C. perfringens spore germination and outgrowth during abusive cooling.

Predictive model for growth of Clostridium perfringens during cooling of cooked uncured meat and poultry

Comparison of Clostridium perfringens spore germination and outgrowth in cooked uncured products during cooling for different meat species is presented. Cooked, uncured product was inoculated with C. perfringens spores and vacuum packaged. For the isothermal experiments, all samples were incubated in a water bath stabilized at selected temperatures between 10 and 51 °C and sampled periodically. For dynamic experiments, the samples were cooled from 54.4 to 27 °C and subsequently from 27 to 4 °C for different time periods, designated as x and y hours, respectively. The growth models used were based on a model developed by Baranyi and Roberts (1994. A dynamic approach to predicting bacterial growth in food. Int. J. Food Micro. 23, 277–294), which incorporates a constant, referred to as the physiological state constant, q 0. The value of this constant captures the cells’ history before the cooling begins. To estimate specific growth rates, data from isothermal experiments were used, from which a secondary model was developed, based on a form of Ratkowsky’s 4-parameter equation. The estimated growth kinetics associated with pork and chicken were similar, but growth appeared to be slightly greater in beef; for beef, the maximum specific growth rates estimated from the Ratkowsky curve was about 2.7 log 10 cfu/h, while for the other two species, chicken and pork, the estimate was about 2.2 log 10 cfu/h. Physiological state constants were estimated by minimizing the mean square error of predictions of the log 10 of the relative increase versus the corresponding observed quantities for the dynamic experiments: for beef the estimate was 0.007, while those for pork and chicken the estimates were about 0.014 and 0.011, respectively. For a hypothetical 1.5 h cooling from 54 °C to 27° and 5 h to 4 °C, corresponding to USDA-FSIS cooling compliance guidelines, the predicted growth (log 10 of the relative increase) for each species was: 1.29 for beef; 1.07 for chicken and 0.95 log 10 for pork. However, it was noticed that for pork in particular, the model using the derived q 0 had a tendency to over-predict relative growth when the observed amount of relative growth was small, and under-predict the relative growth when the observed amount of relative growth was large. To provide more fail-safe estimate, rather than using the derived value of q 0, a value of 0.04 is recommended for pork.

Inhibition of Clostridium perfringens Spore Germination and Outgrowth by Buffered Vinegar and Lemon Juice Concentrate during Chilling of Ground Turkey Roast Containing Minimal Ingredients

Inhibition of Clostridium perfringens spore germination and outgrowth in ground turkey roast containing minimal ingredients (salt and sugar), by buffered vinegar (MOstatin V) and a blend (buffered) of lemon juice concentrate and vinegar (MOstatin LV) was evaluated. Ground turkey roast was formulated to contain sea salt (1.5%), turbinado sugar (0.5%), and various concentrations of MOstatin V (0.75, 1.25, or 2.5%) or MOstatin LV (1.5, 2.5, or 3.5%), along with a control (without MOstatins). The product was inoculated with a three-strain spore cocktail of C. perfringens to obtain initial spore levels of ca. 2.0 to 0.5 log CFU/g. Inoculated products were vacuum packaged, heat shocked for 20 min at 75 degrees C, and cooled exponentially from 54.4 to 4.0 degrees C in 6.5, 9, 12, 15, 18, or 21 h. In control samples without MOstatin V or MOstatin LV, C. perfringens populations reached 2.98, 4.50, 5.78, 7.05, 7.88, and 8.19 log CFU/g (corresponding increases of 0.51, 2.29, 3.51, 4.79, 5.55, and 5.93 log CFU/g) in 6.5, 9, 12, 15, 18, and 21 h of chilling, respectively. MOstatin V (2.5%) and MOstatin LV (3.5%) were effective in inhibiting C. perfringens spore germination and outgrowth in ground turkey roast to <1.0 log CFU/g during abusive chilling of the product within 21 h. Buffered vinegar and a blend (buffered) of lemon juice concentrate and vinegar were effective in controlling germination and outgrowth of C. perfringens spores in turkey roast containing minimal ingredients.

Predictive model for growth of Clostridium perfringens during cooling of cooked ground chicken

Traditional methodologies for development of microbial growth models under dynamic temperature conditions do not take into account the organism's history. Such models have been shown to be inadequate in predicting growth of the organisms under dynamic conditions commonly encountered in the food industry. The objective of the current research was to develop a predictive model for Clostridium perfringens spore germination and outgrowth in cooked chicken products during cooling by incorporating a function to describe the prior history of the microbial cell in the secondary model. Incorporating an assumption that growth kinetics depends in an explicit way on the cells' history could provide accurate estimates of growth or inactivation. Cooked, ground uncured chicken was inoculated with C. perfringens spores, and from this chicken, samples were formed and vacuum packaged. For the isothermal experiments, all samples were incubated in a constant temperature water baths stabilized at selected temperatures between 10 and 51 °C and sampled periodically. The samples were cooled from 54.4 to 27 °C and subsequently from 27 to 4 °C at different time periods (cooling rates) for dynamic cooling experiments. The standard model provided predictions that varied from the observed mean log10 growth values by magnitudes up to about 0.65 log10. However, for a selected memory model, estimates of log10 relative growth provided predictions within 0.3 log10 of the mean observed log10 growth values. These findings point to an improvement of predictions obtained by memory models over those obtained by the standard model. More study though is needed to validate the selected model. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

Predictive model for growth of Clostridium perfringens during cooling of cooked ground pork

Abstract A predictive dynamic model for Clostridium perfringens spore germination and outgrowth in cooked pork products during cooling is presented. Cooked, ground pork was inoculated with C. perfringens spores and vacuum packaged. For the isothermal experiments, all samples were incubated in a water bath stabilized at selected temperatures between 10 and 51 °C and sampled periodically. For dynamic experiments, the samples were cooled from 54.4 to 27 °C and subsequently from 27 to 4 °C for different time periods, designated as x and y hours, respectively. The growth models used were based on a model developed by Baranyi and Roberts (1994), which incorporates a constant, referred to as the physiological state constant, q 0 . The value of this constant captures the cells' history before the cooling begins. To estimate specific growth rates, data from isothermal experiments were used, from which a secondary model was developed, based on a particular form of Ratkowsky's 4-parameter equation. Using the data from dynamic experiments and the Ratkowsky model, an optimal value of q 0 (=0.01375) was derived minimizing the mean square error of predictions. However, using this estimate, the model had a tendency to over-predict relative growth when there was observed small amounts of relative growth, and under-predict relative growth when there was observed large relative growth. To provide more fail-safe estimates, rather than using the derived value of q 0 , a value of 0.04 is recommended. The predictive model with this value of q 0 would provide more fail-safe estimates of relative growth and could aid producers and regulatory agencies with determining disposition of products that were subjected to cooling deviations. Industrial relevance Safe time/temperature for cooling of cooked pork is very important to guard against the pathogen in cooked products. Predictive model will assist industry to determine compliance with regulatory performance standards and to ensure microbiological safety of cooked products.

Clostridium perfringens spore germination: characterization of germinants and their receptors.

Clostridium perfringens food poisoning is caused by type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe), while C. perfringens-associated non-food-borne gastrointestinal (GI) diseases are caused by isolates carrying a plasmid-borne cpe gene (P-cpe). C. perfringens spores are thought to be the important infectious cell morphotype, and after inoculation into a suitable host, these spores must germinate and return to active growth to cause GI disease. We have found differences in the germination of spores of C-cpe and P-cpe isolates in that (i) while a mixture of L-asparagine and KCl was a good germinant for spores of C-cpe and P-cpe isolates, KCl and, to a lesser extent, L-asparagine triggered spore germination in C-cpe isolates only; and (ii) L-alanine or L-valine induced significant germination of spores of P-cpe but not C-cpe isolates. Spores of a gerK mutant of a C-cpe isolate in which two of the proteins of a spore nutrient germinant receptor were absent germinated slower than wild-type spores with KCl, did not germinate with L-asparagine, and germinated poorly compared to wild-type spores with the nonnutrient germinants dodecylamine and a 1:1 chelate of Ca2+ and dipicolinic acid. In contrast, spores of a gerAA mutant of a C-cpe isolate that lacked another component of a nutrient germinant receptor germinated at the same rate as that of wild-type spores with high concentrations of KCl, although they germinated slightly slower with a lower KCl concentration, suggesting an auxiliary role for GerAA in C. perfringens spore germination. In sum, this study identified nutrient germinants for spores of both C-cpe and P-cpe isolates of C. perfringens and provided evidence that proteins encoded by the gerK operon are required for both nutrient-induced and non-nutrient-induced spore germination.

Control of Clostridium perfringens Spores by Green Tea Leaf Extracts during Cooling of Cooked Ground Beef, Chicken, and Pork

We investigated the inhibition of Clostridium perfringens spore germination and outgrowth by two green tea extracts with low (green tea leaf powder [GTL]; 141 mg of total catechins per g of green tea extract) and high (green tea leaf extract [GTE]; 697 mg of total catechins per g of extract) catechin levels during abusive chilling of retail cooked ground beef, chicken, and pork. Green tea extracts were mixed into the thawed beef, chicken, and pork at concentrations of 0.5, 1.0, and 2.0% (wt/wt), along with a heat-activated (75°C for 20 min) three-strain spore cocktail to obtain a final concentration of ∼3 log spores per g. Samples (5 g) of the ground beef, chicken, and pork were then vacuum packaged and cooked to 71°Cfor1hina temperature-controlled water bath. Thereafter, the products were cooled from 54.4 to 7.2°C in 12, 15, 18, or 21 h, resulting in significant increases (P < 0.05) in the germination and outgrowth of C. perfringens populations in the ground beef, chicken, and pork control samples without GTL or GTE. Supplementation with 0.5 to 2% levels of GTL did not inhibit C. perfringens growth from spores. In contrast, the addition of 0.5 to 2% levels of GTE to beef, chicken, and pork resulted in a concentration-and time-dependent inhibition of C. perfringens growth from spores. At a 2% level of GTE, a significant (P < 0.05) inhibition of growth occurred at all chill rates for cooked ground beef, chicken, and pork. These results suggest that widely consumed catechins from green tea can reduce the potential risk of C. perfringens spore germination and outgrowth during abusive cooling from 54.4 to 7.2°C in 12, 15, 18, or 21 h of cooling for ground beef, chicken, and pork.

Use of calcium, potassium, and sodium lactates to control germination and outgrowth of Clostridium perfringens spores during chilling of injected pork

Inhibition of Clostridium perfringens spore germination and outgrowth during abusive chilling regimes was investigated by the incorporation of lactates of calcium (CaL), potassium (KL) and sodium (NaL) in injected pork. Lactates (Ca, K, or Na) were incorporated into injected pork samples at four different concentrations (1.0%, 2.0%, 3.0%, and 4.8%), along with a no-lactate control. A three-strain cocktail of C. perfringens spores was inoculated into the product (injected pork) to obtain a final spore population of ca. 2.0–2.5 log 10 CFU/g. Chilling of injected pork (control) from 54.4 to 7.2 °C within 6.5, 9, 12, 15, 18, and 21 h exponential chill rates resulted in C. perfringens population increases of 0.49, 2.40, 4.02, 5.03, 6.24, and 6.30 log 10 CFU/g, respectively. Addition of CaL at 1.0% or KL and NaL ⩾2.0% to injected pork was able to control C. perfringens germination and outgrowth to <1 log CFU/g, meeting the USDA-FSIS performance standard. However, extension of chilling rates beyond 9.0 h (up to 21 h) required addition of CaL (⩾2.0%), KL or NaL (⩾3.0%) to meet the stabilization performance standard. In general, CaL was more effective compared to KL or NaL for all the chilling regimes, in reducing the potential risk of C. perfringens germination and outgrowth.

Mode of Action of a Germination-Specific Cortex-Lytic Enzyme, SleC, ofClostridium perfringensS40

The hydrolysis of the bacterial spore peptidoglycan (cortex) is a crucial event in spore germination. It has been suggested that SleC and SleM, which are conserved among clostridia, are to be considered putative cortex-lytic enzymes in Clostridium perfringens. However, little is known about the details of the hydrolytic process by these enzymes during germination, except that SleM functions as a muramidase. Muropeptides derived from SleC-digested decoated spores of a Bacillus subtilis mutant that lacks the enzymes, SleB, YaaH and CwlJ, related to cortex hydrolysis were identified by amino acid analysis and mass spectrometry. The results suggest that SleC is most likely a bifunctional enzyme possessing lytic transglycosylase activity and N-acetylmuramoyl-L-alanine amidase activity confined to cross-linked tetrapeptide-tetrapeptide moieties of the cortex structure. Furthermore, it appears that during germination of Clostridium perfringens spores, SleC causes merely small and local changes in the cortex structure, which are necessary before SleM can function.

Inhibition of Germination and Outgrowth of Clostridium perfringens Spores by Lactic Acid Salts during Cooling of Injected Turkey

Inhibition of Clostridium perfringens spore germination and outgrowth by lactic acid salts (calcium, potassium, and sodium) during exponential cooling of injected turkey product was evaluated. Injected turkey samples containing calcium lactate, potassium lactate, or sodium lactate (1.0, 2.0, 3.0, or 4.8% [w/w]), along with a control (product without lactate), were inoculated with a three-strain cocktail of C. perfringens spores to achieve a final spore population of 2.5 to 3.0 log CFU/g. The inoculated product was heat treated and exponentially cooled from 54.5 to 7.2°C within 21, 18, 15, 12, 9, or 6.5 h. Cooling of injected turkey (containing no antimicrobials) resulted in C. perfringens germination and an outgrowth of 0.5, 2.4, 3.4, 5.1, 5.8, and 5.8 log CFU/g when exponentially cooled from 54.4 to 7.2°C in 6.5, 12, 15, 18, and 21 h, respectively. The incorporation of antimicrobials (lactates), regardless of the type (Ca, Na, or K salts), inhibited the germination and outgrowth of C. perfringens spores at all the concentrations evaluated (1.0, 2.0, 3.0, and 4.8%) compared to the injected turkey without acetate (control). Increasing the concentrations of the antimicrobials resulted in a greater inhibition of the spore germination and outgrowth in the products. In general, calcium lactate was more effective in inhibiting the germination and outgrowth of C. perfringens spores at ≥1.0% concentration than were sodium and potassium lactates. Incorporation of these antimicrobials in cooked, ready-to-eat turkey products can provide additional protection in controlling the germination and outgrowth of C. perfringens spores during cooling (stabilization).

Carvacrol, Cinnamaldehyde, Oregano Oil, and Thymol Inhibit Clostridium perfringens Spore Germination and Outgrowth in Ground Turkey during Chilling†

Inhibition of Clostridium perfringens by plant-derived carvacrol, cinnamaldehyde, thymol, and oregano oil was evaluated during abusive chilling of cooked ground turkey. Test substances were mixed into thawed turkey product at concentrations of 0.1, 0.5, 1.0, or 2.0% (wt/wt) along with a heat-activated three-strain C. perfringens spore cocktail to obtain final spore concentrations of ca. 2.2 to 2.8 log CFU spores per g of turkey meat. Aliquots (5 g) of the ground turkey mixtures were vacuum packaged and then cooked in a water bath, where the temperature was raised to 60°C in 1 h. The products were cooled from 54.4 to 7.2°C in 12, 15, 18, or 21 h, resulting in 2.9-, 5.5-, 4.9-, and 4.2-log CFU/g increases, respectively, in C. perfringens populations in samples without antimicrobials. Incorporation of test compounds (0.1 to 0.5%) into the turkey completely inhibited C. perfringens spore germination and outgrowth (P ≤ 0.05) during exponential cooling in 12 h. Longer chilling times (15, 18, and 21 h) required greater concentrations (0.5 to 2.0%) to inhibit spore germination and outgrowth. Cinnamaldehyde was significantly (P < 0.05) more effective (<1.0-log CFU/g growth) than the other compounds at a lower concentration (0.5%) at the most abusive chilling rate evaluated (21 h). These findings establish the value of the plant-derived antimicrobials for inhibiting C. perfringens in commercial ground turkey products.

Chitosan Protects Cooked Ground Beef and Turkey Against Clostridium perfringens Spores During Chilling

ABSTRACT: We investigated the inhibition of Clostridium perfringens spore germination and outgrowth by the biopolymer chitosan during abusive chilling of cooked ground beef (25% fat) and turkey (7% fat) obtained from a retail store. Chitosan was mixed into the thawed beef or turkey at concentrations of 0.5%, 1.0%, 2.0%, or 3.0% (w/w) along with a heat‐activated 3‐strain spore cocktail to obtain a final spore concentration of 2 to 3 log10 CFU/g. Samples (5 g) of the ground beef or turkey mixtures were then vacuum‐packaged and cooked to 60 °C in 1 h in a temperature‐controlled water bath. Thereafter, the products were cooled from 54.4 to 7.2 °C in 12, 15, 18, or 21 h, resulting in 4.21, 4.51, 5.03, and 4.70 log10 CFU/g increases, respectively, in C. perfringens populations in the ground beef control samples without chitosan. The corresponding increases for ground turkey were 5.27, 4.52, 5.11, and 5.38 log10 CFU/g. Addition of chitosan to beef or turkey resulted in concentration‐ and time‐dependent inhibition in the C. perfringens spore germination and outgrowth. At 3%, chitosan reduced by 4 to 5 log10 CFU/g C. perfringens spore germination and outgrowth (P≤ 0.05) during exponential cooling of the cooked beef or turkey in 12, 15, or 18 h. The reduction was significantly lower (P &lt; 0.05) at a chilling time of 21 h, about 2 log10 CFU/g, that is, 7.56 log10 CFU/g (unsupplemented) compared with 5.59 log10 CFU/g (3% chitosan). The results suggest that incorporation of 3% chitosan into ground beef or turkey may reduce the potential risk of C. perfringens spore germination and outgrowth during abusive cooling from 54.4 to 7.2 °C in 12, 15, or 18 h.

Control of Clostridium perfringens in Cooked Ground Beef by Carvacrol, Cinnamaldehyde, Thymol, or Oregano Oil during Chilling

Inhibition of Clostridium perfringens spore germination and outgrowth by carvacrol, cinnamaldehyde, thymol, and oregano oil was evaluated during abusive chilling of cooked ground beef (75% lean) obtained from a local grocery store. Test substances were mixed into thawed ground beef at concentrations of 0.1, 0.5, 1.0, or 2.0% (wt/wt) along with a heat-activated three-strain C. perfringens spore cocktail to obtain final spore concentrations of ca. 2.8 log spores per g. Aliquots (5 g) of the ground beef mixtures were vacuum-packaged and then cooked in a water bath, the temperature of which was raised to 60°C in 1 h. The products were cooled from 54.4 to 7.2°C in 12, 15, 18, or 21 h, resulting in 3.18, 4.64, 4.76, and 5.04 log CFU/g increases, respectively, in C. perfringens populations. Incorporation of test compounds (≥0.1%) into the beef completely inhibited C. perfringens spore germination and outgrowth (P ≤ 0.05) during exponential cooling of the cooked beef in 12 h. Longer chilling times (15, 18, and 21 h) required greater concentrations to inhibit spore germination and outgrowth. Cinnamaldehyde was significantly (P < 0.05) more effective (<1.0 log CFU/g growth) at a lower concentration (0.5%) at the most abusive chilling rate evaluated (21 h) than the other compounds. Incorporation of lower levels of these test compounds with other antimicrobials used in meat product formulations may reduce the potential risk of C. perfringens germination and outgrowth during abusive cooling regimes.

Mechanism of nitrite-induced germination of Clostridium perfringens spores.

A study has been undertaken to understand the mechanism(s) of the nitrite‐induced germination of Clostridium perfringens S40 spores. An increase in germination rates of the spores in response to increasing NaNO2 concentrations was entirely dependent on both pH and temperature of incubation. Low pH and high temperature were effective in accelerating the germination rate, the maximal germination level being reached at pH 4.0 and 60°C in the presence of 0.5 M NaNO2. On the basis of germination rate, the activation energy (μ) for the nitrite‐induced germination calculated was approximately 9.9 kcal/mol. Germination was greatly stimulated after pretreatment of spores with DTT at pH 10.5 to remove the coats. Furthermore, cortical fragments prepared from spores of the same organism were lysed not only by lysozyme but also by NaNO2. Hexosamine‐containing material was also solubilized by these reagents. However, nitrite, unlike lysozyme, released a considerable amount of free hexosamine as well. These results suggest that nitrite‐induced germination may involve an interaction of sodium nitrite as nitrous acid with some component of the cortex. A possible mechanism of nitrite‐induced germination is discussed.