Optimization and evaluation of heat-shock condition for spore enumeration being used in thermal-process verification: Differential responses of spores and vegetative cells of Clostridium sporogenes to heat shock

Byun, Bo Young; Cho, Hong-Yon; Hwang, Han‐Joon; Mah, Jae-Hyung; Liu, Yanhong; Tang, Juming; Kang, Dong‐Hyun

doi:10.1007/s10068-011-0105-7

Cited by 18 publications

(10 citation statements)

References 23 publications

(15 reference statements)

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“…The activation of dormant spores by heat shock is a process that progresses with time and involves an abrupt change in individual spores rather than a gradual change in all spores, as indicated by increased colony counts with time on conventional plating media (41). On the other hand, if heat-shock treatment is too severe, the damage will be so strong that the spores cannot repair themselves and the viable spore counts will be smaller, compared with the conventional and mild heat-shock conditions (27). Our results revealed that heat shock of 68°C for 20 min was above the critical minimum heat activation temperature/time combinations for the spores examined (Fig.…”

Section: Discussionmentioning

confidence: 61%

“…Dormancy phase may be broken by heat treatment at a time and sublethal temperature appropriate to the organism concerned (24,25). Further, there has been a revival of interest in validating and improving the procedure for enumeration of Bacillus spores by using different heat shock (or heat-activation) treatments (23,26,27). The heat shock step as an effective germinant has usually two functions: 1) activate the spores and so start or enhance of germination for all spores; 2) killing or destructing all kinds of vegetative cells in the sample, thereby to provide reliable counts of spores with no viability loss (24,28,29).…”

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confidence: 99%

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Influence of Heat Shock Temperatures and Fast Freezing on Viability of Probiotic Sporeformers and the Issue of Spore Plate Count Versus True Numbers

Jafari¹,

Alebouyeh

Mortazavian

et al. 2016

Nutr Food Sci Res

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Background and Objectives: The purpose of the present study was to investigate effects of various heat shock conditions and fast freezing and subsequent thawing on the viability and recovery of Bacillus coagulans and Bacillus subtilis as probiotic sporeformers, and also to compare spore plate and microscopic counts. Materials and Methods: After preparing the final suspensions of B. coagulans and Bacillus subtilis subsp. Natto spores, they were spread-plated before and after fast freezing treatment (-70°C for about 1 min). Heat shock treatments of the spores were carried out at 68 o C for 15, 20, and 30 min as well as at 80 o C for 10 and 15 min. Concentrations of the examined probiotic sporeformers were determined simultaneously by plate enumerations and microscopically determined counts. Student's t-test and one-way analysis of variance (ANOVA) of SPSS were used for statistical analysis of the data. Analysis of DoE results was carried out using Minitab. Results: The results presented here show that the highest recovery rates for B. coagulans (14.75 log CFU/mL) and B. subtilis spores (14.80 log CFU/mL) were under a heat shock condition of 68°C for 20 min in nutrient agar (p<0.05). In addition, the survival rates of B. coagulans and B. subtilis spores under the fast freezing and subsequent thawing condition were about 90% and 88%, respectively. Plate counts differed significantly from counts determined microscopically, with differences of almost 0.5 and 0.8 log for B. coagulans and B. subtilis spores, respectively (p<0.05). In addition, DoE results of the study revealed that both factors of spore count method and only freezing factor in fast freezing treatment have a significant effect on concentrations of the spores examined (p<0.05). Conclusions: Heat shock conditions, freezing and subsequent thawing circumstances, and plate counts or enumerations determined microscopically have significant influences on the viability of probiotic sporeformers and should be considered in determining of their accurate concentrations.

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Section: Discussionmentioning

confidence: 61%

mentioning

confidence: 99%

Influence of Heat Shock Temperatures and Fast Freezing on Viability of Probiotic Sporeformers and the Issue of Spore Plate Count Versus True Numbers

Jafari¹,

Alebouyeh

Mortazavian

et al. 2016

Nutr Food Sci Res

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“…Notably, while the average values of times for fast release of CaDPA (Δ T release ) in the L -alanine germination of multiple individual spores were similar with spores with and without a heat shock, optimally heat-shocked spores showed the lowest values of T 1 , T lag1 , T release , and T lys , ( Table 1 ). These results suggest that the primary effect of an optimal heat shock on C. sporogenes spore germination is to shorten the T 1 values for individual spores, as seen with spores of other species ( Yi and Setlow, 2010 ; Zhang et al, 2010a ; Byun et al, 2011 ; Setlow et al, 2017 ).…”

Section: Resultsmentioning

confidence: 78%

Analysis of the Germination of Individual Clostridium sporogenes Spores with and without Germinant Receptors and Cortex-Lytic Enzymes

et al. 2017

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The Gram-positive spore-forming anaerobe Clostridium sporogenes is a significant cause of food spoilage, and it is also used as a surrogate for C. botulinum spores for testing the efficacy of commercial sterilization. C. sporogenes spores have also been proposed as a vector to deliver drugs to tumor cells for cancer treatments. Such an application of C. sporogenes spores requires their germination and return to life. In this study, Raman spectroscopy and differential interference contrast (DIC) microscopy were used to analyze the germination kinetics of multiple individual C. sporogenes wild-type and germination mutant spores. Most individual C. sporogenes spores germinated with L-alanine began slow leakage of ∼5% of their large Ca-dipicolinic acid (CaDPA) depot at T1, all transitioned to rapid CaDPA release at Tlag1, completed CaDPA release at Trelease, and finished peptidoglycan cortex hydrolysis at Tlys. T1, Tlag1, Trelease, and Tlys times for individual spores were heterogeneous, but ΔTrelease (Trelease – Tlag1) periods were relatively constant. However, variability in T1 (or Tlag1) times appeared to be the major reason for the heterogeneity between individual spores in their germination times. After Trelease, some spores also displayed another lag in rate of change in DIC image intensity before the start of a second obvious DIC image intensity decline of 25–30% at Tlag2 prior to Tlys. This has not been seen with spores of other species. Almost all C. sporogenes spores lacking the cortex-lytic enzyme (CLE) CwlJ spores exhibited a Tlag2 in L-alanine germination. Sublethal heat treatment potentiated C. sporogenes spore germination with L-alanine, primarily by shortening T1 times. Spores without the CLEs SleB or CwlJ exhibited greatly slowed germination with L-alanine, but spores lacking all germinant receptor proteins did not germinate with L-alanine. The absence of these various germination proteins also decreased but did not abolish germination with the non-GR-dependent germinants dodecylamine and CaDPA, but spores without CwlJ exhibited no germination with CaDPA. Finally, C. sporogenes spores displayed commitment in germination, but memory in GR-dependent germination was small, and less than the memory in Bacillus spore germination.

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“…As expected, the thermal treatment (70 °C for 10 min) determined a reduction in absorbance (approximately 15%), as it is well known that a slight heat shock can activate spores (Byun et al ., ). A promoting effect on spore germination was also achieved by l ‐alanine (absorbance was reduced by 25%), but this was an expected result as it is well known that l ‐alanine induces spore germination (Barlass et al ., ).…”

Section: Resultsmentioning

confidence: 99%

Effects of lysozyme on Alicyclobacillus acidoterrestris under laboratory conditions

Bevilacqua

Ciuffreda

Sinigaglia

et al. 2013

Int J of Food Sci Tech

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This research was aimed at investigating the bioactivity of lysozyme towards Alicyclobacillus acidoterrestris. Lysozyme (0.1-20 ppm) was tested towards five different strains; the experiments were performed on both spores and vegetative cells using a microdilution approach to assess the minimal inhibitory concentration (MIC). Thereafter, spore viability of two selected strains was evaluated through the traditional plate count. Finally, an experiment was run to assess the role of lysozyme on the complex phenomenon of spore germination. The MIC of lysozyme towards vegetative cells varied from 0.1 to 6 ppm, while for the spores, it was from 0.1 to 3 ppm. Concerning spore viability, the effect of lysozyme relied upon its amount on the broth, as at MIC it caused a slight reduction in spores (approximately 1 log cfu ml À1 ) after 24 h; otherwise, higher concentrations caused a decrease in spore level below the detection limit. Concerning spore germination, lysozyme exerted a promoting effect on this phenomenon and reduced the optical density by 66%.

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Optimization and evaluation of heat-shock condition for spore enumeration being used in thermal-process verification: Differential responses of spores and vegetative cells of Clostridium sporogenes to heat shock

Abstract: To evaluate a heat-shock condition for the enumeration of Clostridium sporogenes spores, a surrogate for C. botulinum spores, we examined the heat tolerance of C. sporogenes spores and vegetative cells exposed to a heat shock at 90

Cited by 18 publications

References 23 publications

Influence of Heat Shock Temperatures and Fast Freezing on Viability of Probiotic Sporeformers and the Issue of Spore Plate Count Versus True Numbers

Influence of Heat Shock Temperatures and Fast Freezing on Viability of Probiotic Sporeformers and the Issue of Spore Plate Count Versus True Numbers

Analysis of the Germination of Individual Clostridium sporogenes Spores with and without Germinant Receptors and Cortex-Lytic Enzymes

Effects of lysozyme on Alicyclobacillus acidoterrestris under laboratory conditions

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