Effect of Talc as a Dry-Inoculation Carrier on Thermal Resistance of Enterococcus faecium NRRL B-2354 in Almond Meal

ABSTRACTDry inoculation (DI) methods using a dry carrier have gained considerable interest for assessing thermal inactivation of Salmonella and other microorganisms in low-moisture foods. However, the effect of carrier residues on microbial resistance to heat remains largely unknown. This study aimed to determine the effect of talc powder on thermal resistance of Enterococcus faecium NRRL-B2354 (a Salmonella surrogate) in almond meal at 0.45 water activity (aw). Whole almonds were either immersed in an E. faecium suspension for wet inoculation (WI) or mixed with inoculated talc powder for DI. Two additional experimental conditions, inoculation of WI almond meal with added uninoculated talc (WT) and inoculated talc powder alone, were conducted. After WI, DI, and WT, the almonds were equilibrated to 0.45 aw, ground into a meal, and reequilibrated to 0.45 aw. Isothermal treatments were performed by heating almond meal (about 1 g) in aluminum test cells in a water bath at 80°C, with samples collected at more than five sequential time points from triplicate isothermal runs. E. faecium was enumerated by immediately cooling, diluting, and plating the samples on a nonselective or differential medium. E. faecium was more thermally resistant in DI (D80°C: 63.5 ± 1.9 min) compared with WI almond meal (D80°C: 40.5 ± 1.0 min; P < 0.05), but the resistance in WT almond meal (46.9 ± 0.9 min) was between and different from (P < 0.05) both DI and WI. E. faecium was less resistant in talc powder alone (20.6 ± 1.1 min) compared with all other almond meal samples. Overall, residual talc affected the thermal resistance of E. faecium. Therefore, when determining thermal resistance or validating commercial processes, carriers such as talc should not be used for inoculation of low-moisture foods without first knowing their impact on the target organism.HIGHLIGHTS

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Development of a Dry Inoculation Method for Thermal Challenge Studies in Low-Moisture Foods by Using Talc as a Carrier for Salmonella and a Surrogate (Enterococcus faecium)

Journal of Food Protection ◽

10.4315/0362-028x.jfp-14-396 ◽

2015 ◽

Vol 78 (6) ◽

pp. 1106-1112 ◽

Cited By ~ 27

Author(s):

ELENA ENACHE ◽

AI KATAOKA ◽

D. GLENN BLACK ◽

CARLA D. NAPIER ◽

RICHARD PODOLAK ◽

...

Keyword(s):

Thermal Resistance ◽

Heat Resistance ◽

Water Activity ◽

Enterococcus Faecium ◽

Thermal Inactivation ◽

Death Time ◽

Significant Difference ◽

Growth Method ◽

Low Moisture Foods ◽

Inoculum Preparation

The objective of this study was to obtain dry inocula of Salmonella Tennessee and Enterococcus faecium, a surrogate for thermal inactivation of Salmonella in low-moisture foods, and to compare their thermal resistance and stability over time in terms of survival. Two methods of cell growth were compared: cells harvested from a lawn on tryptic soy agar (TSA-cells) and from tryptic soy broth (TSB-cells). Concentrated cultures of each organism were inoculated onto talc powder, incubated at 35°C for 24 h, and dried for additional 24 h at room temperature (23 ± 2°C) to achieve a final water activity of ≤0.55 before sieving. Cell reductions of Salmonella and E. faecium during the drying process were between 0.14 and 0.96 log CFU/g, depending on growth method used. There was no difference between microbial counts at days 1 and 30. Heat resistance of the dry inoculum on talc inoculated into a model peanut paste (50% fat and 0.6 water activity) was determined after 1 and 30 days of preparation, using thermal death time tests conducted at 85°C. For Salmonella, there was no significant difference between the thermal resistance (D85°C) for the TSB-cells and TSA-cells (e.g. day 1 cells D85°C = 1.05 and 1.07 min, respectively), and there was no significant difference in D85°C between dry inocula on talc used either 1 or 30 days after preparation (P > 0.05). However, the use the dry inocula of E. faecium yielded different results: the TSB-grown cells had a significantly (P < 0.05) greater heat resistance than TSA-grown cells (e.g. D85°C for TSB-cells = 3.42 min versus 2.60 min for TSA-cells). E. faecium had significantly (P < 0.05) greater heat resistance than Salmonella Tennessee regardless what cell type was used for dry inoculum preparation; therefore, it proved to be a conservative but appropriate surrogate for thermal inactivation of Salmonella in low-moisture food matrices under the tested conditions.

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Exponentially Increased Thermal Resistance ofSalmonellaspp. andEnterococcus faeciumat Reduced Water Activity

Applied and Environmental Microbiology ◽

10.1128/aem.02742-17 ◽

2018 ◽

Vol 84 (8) ◽

pp. e02742-17 ◽

Cited By ~ 25

Author(s):

Shuxiang Liu ◽

Juming Tang ◽

Ravi Kiran Tadapaneni ◽

Ren Yang ◽

Mei-Jun Zhu

Keyword(s):

Relative Humidity ◽

Thermal Resistance ◽

Silicon Dioxide ◽

Linear Relationship ◽

Water Activity ◽

Enterococcus Faecium ◽

Content Type ◽

Low Moisture Foods ◽

Log Linear ◽

Insight Into

ABSTRACTSalmonellaspp. exhibit prolonged survivability and high tolerance to heat in low-moisture foods. The reported thermal resistance parameters ofSalmonellaspp. in low-moisture foods appear to be unpredictable due to various unknown factors. We report here that temperature-dependent water activity (aw, treatment temperature) plays an important role in the sharply increased thermal resistance ofSalmonella entericaserovar Enteritidis PT 30 and its potential surrogateEnterococcus faeciumNRRL B-2354. In our study, silicon dioxide granules, as carriers, were separately inoculated with these two microorganisms and were heated at 80°C with controlled relative humidity between 18 and 72% (resulting in corresponding aw,80°Cvalues for bacteria between 0.18 and 0.72) in custom-designed test cells. The inactivation kinetics of both microorganisms fitted a log-linear model (R2, 0.83 to 0.97). Reductions in the aw,80°Cvalues of bacterial cells exponentially increased theD80°C(the time needed to achieve a 1-log reduction in a bacterial population at 80°C) values forS. Enteritidis andE. faeciumon silicon dioxide. The log-linear relationship between theD80°Cvalues for each strain in silicon dioxide and its aw,80°Cvalues was also verified for organic wheat flour.E. faeciumshowed consistently higherD80°Cvalues thanS. Enteritidis over the aw,80°Crange tested. The estimated zaw(the change in aw,80°Cneeded to changeD80°Cby 1 log) values ofS. Enteritidis andE. faeciumwere 0.31 and 0.28, respectively. This study provides insight into the interpretation ofSalmonellathermal resistance that could guide the development and validation of thermal processing of low-moisture foods.IMPORTANCEIn this paper, we established that the thermal resistance of the pathogenS. Enteritidis and its surrogateEnterococcus faecium, as reflected byDvalues at 80°C, increases sharply with decreasing relative humidity in the environment. The log-linear relationship between theD80°Cvalues of each strain in silicon dioxide and its aw,80°Cvalues was also verified for organic wheat flour. The results provide new quantitative insight into the way in which the thermal resistance of microorganisms changes in low-moisture systems, and they should aid in the development of effective thermal treatment strategies for pathogen control in low-moisture foods.

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Effects of Inoculation Procedures on Variability and Repeatability of Salmonella Thermal Resistance in Wheat Flour

Journal of Food Protection ◽

10.4315/0362-028x.jfp-16-057 ◽

2016 ◽

Vol 79 (11) ◽

pp. 1833-1839 ◽

Cited By ~ 31

Author(s):

IAN M. HILDEBRANDT ◽

BRADLEY P. MARKS ◽

ELLIOT T. RYSER ◽

ROSSANA VILLA-ROJAS ◽

JUMING TANG ◽

...

Keyword(s):

Thermal Resistance ◽

Water Activity ◽

Wheat Flour ◽

Thermal Inactivation ◽

Culture Method ◽

Isothermal Treatment ◽

Inactivation Kinetics ◽

Inoculation Methods ◽

Low Moisture Foods ◽

D Values

ABSTRACT Limited prior research has shown that inoculation methods affect thermal resistance of Salmonella in low-moisture foods; however, these effects and their repeatability have not been systematically quantified. Consequently, method variability across studies limits utility of individual data sets and cross-study comparisons. Therefore, the objective was to evaluate the effects of inoculation methodologies on stability and thermal resistance of Salmonella in a low-moisture food (wheat flour), and the repeatability of those results, based on data generated by two independent laboratories. The experimental design consisted of a cross-laboratory comparison, both conducting isothermal Salmonella inactivation studies in wheat flour (~0.45 water activity, 80°C), utilizing five different inoculation methods: (i) broth-based liquid inoculum, (ii) lawn-based liquid inoculum, (iii) lawn-based pelletized inoculum, (iv) direct harvest of lawn culture with wheat flour, and (v) fomite transfer of a lawn culture. Inoculated wheat flour was equilibrated ~5 days to ~0.45 water activity and then was subjected to isothermal treatment (80°C) in aluminum test cells. Results indicated that inoculation method impacted repeatability, population stability, and inactivation kinetics (α = 0.05), regardless of laboratory. Salmonella inoculated with the broth-based liquid inoculum method and the fomite transfer of a lawn culture method exhibited instability during equilibration. Lawn-based cultures resulted in stable populations prior to thermal treatment; however, the method using direct harvest of lawn culture with wheat flour yielded different D-values across the laboratories (α = 0.05), which was attributed to larger potential impact of operator variability. The lawn-based liquid inoculum and the lawn-based pelletized inoculum methods yielded stable inoculation levels and repeatable D-values (~250 and ~285 s, respectively). Also, inoculation level (3 to 8 log CFU/g) did not affect D-values (using the lawn-based liquid inoculum method). Overall, the results demonstrate that inoculation methods significantly affect Salmonella population kinetics and subsequent interpretation of thermal inactivation data for low-moisture foods.

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Influence of Water Activity on Thermal Resistance of Microorganisms in Low-Moisture Foods: A Review

Comprehensive Reviews in Food Science and Food Safety ◽

10.1111/1541-4337.12190 ◽

2016 ◽

Vol 15 (2) ◽

pp. 353-370 ◽

Cited By ~ 95

Author(s):

Roopesh M. Syamaladevi ◽

Juming Tang ◽

Rossana Villa-Rojas ◽

Shyam Sablani ◽

Brady Carter ◽

...

Keyword(s):

Thermal Resistance ◽

Water Activity ◽

Influence Of Water ◽

Low Moisture Foods

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Influence of low water activity on the thermal resistance of Salmonella Enteritidis PT30 and Enterococcus faecium as its surrogate in egg powders

Food Science and Technology International ◽

10.1177/1082013220937872 ◽

2020 ◽

pp. 108201322093787

Author(s):

Marco E Pérez-Reyes ◽

Xu Jie ◽

Mei-Jun Zhu ◽

Juming Tang ◽

Gustavo V Barbosa-Cánovas

Keyword(s):

Thermal Resistance ◽

Linear Relationship ◽

Water Activity ◽

Enterococcus Faecium ◽

Salmonella Enteritidis ◽

Thermal Treatments ◽

Thermal Death ◽

Industrial Food Production ◽

Log Linear ◽

The Impact

Egg powders are increasingly popular ingredients, due to their functionality and compactness, in industrial food production and preparation at homes. However, there is a lack of studies that evaluate the thermal resistance of Salmonella Enteritidis PT30 and its potential surrogate Enterococcus faecium NRRL B-2354 in egg powders. This study examined the log-linear relationship between the thermal resistance of Salmonella Enteritidis (D-value) and the water activity (aw) of egg powders. The changes of aw in the egg powders with temperature were measured using a Vapor Sorption Analyzer and a high-temperature cell. The D80 ℃-value of S. Enteritidis PT30 and E. faecium inoculated in the egg powders preconditioned to three aw levels (0.3, 0.45, and 0.6) at 20 ℃ were determined using aluminum thermal death test cells. The aw values increased (P < 0.05) in all three egg powders when the temperature of the samples was raised from room temperature to 80 ℃. The D80 ℃-values ranged from 5.3 ± 0.1 to 25.9 ± 0.2 min for S. Enteritidis while 10.4 ± 0.4 to 43.8 ± 0.4 for E. faecium in samples of the three different aw levels. S. Enteritidis PT30 showed a log-linear relationship between D80 ℃-values and aw80 ℃ for the egg powders. This study contributes to our understanding of the impact of aw on the development of thermal treatments for low-moisture foods.

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Improving design of thermal water activity cell to study thermal resistance of Salmonella in low-moisture foods

LWT ◽

10.1016/j.lwt.2018.02.046 ◽

2018 ◽

Vol 92 ◽

pp. 371-379 ◽

Cited By ~ 7

Author(s):

Ravi Kiran Tadapaneni ◽

Jie Xu ◽

Ren Yang ◽

Juming Tang

Keyword(s):

Thermal Resistance ◽

Water Activity ◽

Thermal Water ◽

Low Moisture Foods

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Influence of Water Activity on Thermal Resistance of Microorganisms in Low-Moisture Foods: A Review

Comprehensive Reviews in Food Science and Food Safety ◽

10.1111/1541-4337.12203 ◽

2016 ◽

Vol 15 (3) ◽

pp. 668-668 ◽

Cited By ~ 1

Keyword(s):

Thermal Resistance ◽

Water Activity ◽

Influence Of Water ◽

Low Moisture Foods

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Thermal Resistance of Single Strains of Shiga Toxin-Producing Escherichia coli O121:H19 and O157:H7 Based on Culture Preparation Method and Osmolyte-Reduced Water Activity

Journal of Food Protection ◽

10.4315/jfp-20-122 ◽

2020 ◽

Author(s):

Jennifer C Acuff ◽

Kim Waterman ◽

Jahnavi Ramakrishnan ◽

Monica A Ponder

Keyword(s):

Escherichia Coli ◽

Thermal Resistance ◽

Water Activity ◽

Shiga Toxin ◽

Thermal Inactivation ◽

Preparation Method ◽

High Water ◽

Intermediate Water ◽

D Values ◽

Reduced Water

Pathogen thermal resistance studies on low-water activity foods (LWAF) use a variety of methods to inoculate food, as well as strategies to reduce water activity, which can influence thermal resistance observations. This study investigated effects of culture preparation method and osmolyte-induced water activity on thermal resistance of two Shiga toxin-producing Escherichia coli (STEC; O121:H19, O157:H7) challenged with isothermal conditions, determining D - and z -values for each isolate (56, 59, and 62 ° C). Tryptic Soy Broth (TSB) and Agar (lawn cultures) were compared. D -values of broth cultures were significantly and consistently larger than those of lawn cultures, and O121 was significantly more resistant than O157, but only at 56 ° C ( p < 0.05). To compare potential effects of water activity on STEC thermal resistance, cells were suspended in osmolyte solutions with varying water activity: high (TSB, a w 0.99), intermediate (61% glycerol or 26% NaCl, a w 0.75), and low (82% glycerol, a w 0.5). In most instances, STEC in high-water activity broth exhibited greater heat resistance compared to reduced-water activity solutions, except the glycerol intermediate-water activity solution (a w 0.75). Magnitudes varied with strain and temperature. The z -values of lawn cultures were significantly lower than those of broth cultures ( p < 0.05), but there were only some differences between high-a w and reduced-a w samples. There were no significant differences of z -values based on strain type. These results highlight that thermal resistance can be affected by culture preparation and that osmolyte-induced changes to water activity influence thermal inactivation of STEC by varying magnitudes. These results emphasize the challenges between extrapolating results from lab inactivation kinetic experiments to determine the inactivation of low water activity foods, especially those considered dry in nature.

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Moisture content of bacterial cells determines thermal resistance of Salmonella Enteritidis PT 30

Applied and Environmental Microbiology ◽

10.1128/aem.02194-20 ◽

2020 ◽

Author(s):

Yucen Xie ◽

Jie Xu ◽

Ren Yang ◽

Jaza Alshammari ◽

Mei-Jun Zhu ◽

...

Keyword(s):

Moisture Content ◽

Thermal Resistance ◽

Salmonella Enteritidis ◽

Thermal Inactivation ◽

Bacterial Pathogens ◽

Intrinsic Factor ◽

Inactivation Kinetics ◽

Bacterial Cells ◽

Freeze Dried ◽

Low Moisture Foods

Salmonella spp. are resilient bacterial pathogens in low-moisture foods. There has been a general lack of understanding of critical factors contributing to the enhanced thermal tolerance of Salmonella spp, in dry environments. In this study, we hypothesized that the moisture content (XW) of bacterial cells is a critical intrinsic factor influencing the resistance of Salmonella spp. against thermal inactivation. We selected Salmonella Enteritidis PT 30 to test this hypothesis. We first produced viable freeze-dried S. Enteritidis PT 30, conditioned the bacterial cells to different XW (7.7, 9.2, 12.4 and 15.7 g water/100g dry solids), and determined thermal inactivation kinetics of those cells at 80 °C. The results show that D-value (time required to achieve one-log reduction) decreased exponentially with increasing XW. We further measured water activities (aw) of the freeze-dried S. Enteritidis PT 30 as influenced by temperature between 20 and 80 °C. By using those data, we estimated the XW of S. Enteritidis PT 30 from the published papers that related D-values of the same bacteria strain at 80 °C with aw of five different food and silicon dioxide matrices. We discovered that the logarithmic D-values of S. Enteritidis PT 30 in all those matrices also decreased linearly with increasing XW of the bacterial cells. The findings suggest that the amount of moisture in S. Enteritidis PT 30 is a determinant factor on their ability to resist thermal inactivation. Our results may help future research into fundamental mechanisms for thermal inactivation of bacterial pathogens in dry environments. IMPORTANCE This paper established a logarithmic relationship between the thermal death time (D-value) of S. Enteritidis PT 30 and the moisture content (XW) of the bacterial cells by conducting thermal inactivation tests on freeze-dried S. Enteritidis PT 30. We further verified this relationship using literature data for S. Enteritidis PT 30 in five low moisture matrices. The findings suggest that XW of S. Enteritidis PT 30, which is rapidly adjusted by microenvironmental aw, or relative humidity, during heat treatments, is the key intrinsic factor determining thermal resistance of the bacterium. The quantitative relationships reported in this study may help guide future designs of industrial thermal processes for control of S. Enteritidis PT 30 or other Salmonella stains in low-moisture foods. Our findings highlight a need for further fundamental investigation into the role of water in protein denaturation and accumulation of compatible solutes during thermal inactivation of bacterial pathogens in dry environments.

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