Ultrastructure of denaturated potato proteins

The structure of potato protein precipitates formed by heat treatment or acidification of potato juice was investigated by transmission electron microscopy. Precipitates formed by heat denaturation (3 min at 100°C) consist of electron-dense particles of diameter < 300 nm, aggregated into three-dimensional open networks. Precipitates, formed by acidification to pH 3.0 and holding the acidified juice at room temperature, consist of network-like aggregates of electron-dense particles embedded in thin films which hinder the formation of a dense sediment. This structure explains the high sediment volumes which are typical of this kind of precipitate. By warming the acidified potato juice to 40°C, rapid flocculation and sedimentation of the suspended insoluble protein are observed within a few minutes and a dense sediment is finally obtained. This sediment consists mainly of the network-like aggregates of electron-dense particles and material of low electron density formed by the collapse of the films. Protein films have also been observed in precipitates of heat-denatured bovine serum albumin (BSA). High-methoxyl pectin promotes the formation of protein films on heat denaturation of potato proteins or BSA.     

Effect of heat shock on thermal tolerance and susceptibility of Listeria monocytogenes to other environmental stresses

In the present study, Listeria monocytogenes Scott A, V7 and CCRC 14930 were first subjected to heat shock at 45°C for 1 h or at 48°C for 10 min. Thermal tolerance at 55°C and survival of the heat shocked as well as non-heat shocked cells of L. monocytogenes in the presence of 25% NaCl, 0.01% crystal violet, 0.1% H₂O₂, and 18% ethanol were examined.It was found that heat shock response of L. monocytogenes varied with strains, the condition of heat shock treatment and type of subsequent stress. Compared with the non-heat shocked cells, the 45°C-1 h heat shocked cells of strains Scott A and V7 showed an increased survival after exposure to 55°C for 60 min. Meanwhile, survival of the 48°C-10 min heat shocked L. monocytogenes cells, regardless of strain, exhibited no significant difference (p>0.05) with their respective control cells. Generally, heat shocking at 45°C for 1 h increased the tolerance of L. monocytogenes to NaCl, ethanol and crystal violet. On the other hand, heat shocking at 48°C for 10 min although increased the resistance of L. monocytogenes to NaCl reduced its resistance to H₂O₂ and crystal violet.     

Inactivation and injury of Listeria monocytogenes as affected by heating and freezing

The effects of heating and freezing Listeria monocytogenes in tryptose phosphate broth an inactivation and injury were investigate. Four strains (Scott A, Brie-1, LCDC 81–861, and DA-3) were examined. Only slight decreases in the viable populations pf all four strains were detected after 75 min at 50°C. Populations of strains Scott A, LCDC 81–861, and DA-3 were reduced to non-detectable levels after 10 min at 60°C while viable cells of strain Brie-1 were recovered after heating at 60°C for 30 min. Viable populations of all four strains declined steadily at 52,54, and 56°C; similar inactivation rates were observed for strains Scott A, LcDC 81–861, and DA-3. Strain Brie-1 was consistently the most heat resistant. Substantial populations of viable cells of all strains heated at 52, 54, and 56°C were injured as evidenced by increased sensitivity to NaCl when plated on tryptose phosphate recover agar. Strain DA-3 was the most susceptible strain to sublethal heat injury. Viable populations of all four test strains were not appreciably reduced after 14 days at −18°C, although strain LCDC 81–861 lost viability at a more rapid rate at this temperature than did the other test strains. About 72, 82, 73, and 80% of Scott A, Brie-1, LCDC 81–861, and DA-3 cells, respectively, held at −18°C for 14 days were not receovered on tryptose phosphate agar (TPA) supplemented with 8% NaCl compared to populations recovered on TPA not supplemented with NaCl.     

Thermal Resistance of Egg-Associated Epidemic Strains of Salmonella enteritidis

Seventeen strains of Salmonella enteritidis isolated from patients and various implicated products from 5 egg-associated outbreaks of salmonellosis were investigated for plasmid content, phage type and thermal resistance. D values obtained at 57.2 and 60°C in liquid whole egg for all strains were within ranges reported by previous investigators. Sublethal heat shock at 40°C for 2 hr or 48°C for 30 min increased thermotolerance 2- to 3-fold. The induced thermotolerance decayed to control levels after 2–3 cycles of cell duplication at a lower temperature. Most strains contained a 39-Mda plasmid and were phage type 8. No correlation was observed between thermotolerance and presence or absence of plasmid or phage type.     

Effects of cold storage and heat–acid shocks on growth and verotoxin 2 production ofEscherichia coliO157:H7

Escherichia coliO157:H7 was cold-stored (4°C) either in nutritious menstruum [buffered Brain Heart Infusion (BHI) broth] or with starvation (buffered saline) at pH 7.0 or 5.5. Cultures grown in BHI broth at 37°C for 24h served as non-cold-stored controls. After 4-weeks cold storage, bacterial cells were shocked by heat (45°C for 5min) and acid (pH 2.5 for 30min at 37°C) and subsequently moved to optimal conditions (BHI broth of pH 7.4 incubated at 37°C). The results showed: (a) both lag-phase duration and growth rate of this pathogen at 37°C significantly increased after cold-storage with starvation, but not after cold storage in the nutritious menstruum; (b) combined heat–acid shocks increased growth rates at 37°C of both previously cold-stored and non-cold-stored bacterial cells; (c) final concentrations of verotoxin produced by bacterial cells at 37°C were not affected by previous cold storage in the nutritious menstruum; (d) verotoxin production by bacterial cells at 37°C increased after cold storage with starvation, and heat–acid shocks further enhanced that production. Further research is needed to evaluate the food safety implications of these results, i.e. whether cells ofE. coliO157:H7 originating from nutrient-poor/lower-pH environments may be more harmful to humans than those from nutrient-rich/higher-pH foods.     

Thermal Inactivation of Clostridium perfringens After Growth at Several Constant and Linearly Rising Temperatures

Inactivation kinetics of Clostridium perfringens strains NCTC 8238 and NCTC 8798 vegetative cells were evaluated in autoclaved ground beef after growth at constant (37, 41, 45, or 49°C) or linearly rising temperatures (4.0, 6.0, or 7.5 C°/hr) representative of long-time, low-temperature (LTLT) cooking. Inactivation temperatures of 55, 57, 59, 60, and 61°C were used. D values and z values were determined. For strain NCTC 8798 cells grown at 45°C, the average D₅₉°C was 7.2 min and the z_D was 3.8 C°. Both strains exhibited greater heat resistance after growth at higher constant temperatures. Also, NCTC 8798 was more heat resistant than NCTC 8238. With linearly rising temperature, terminal growth temperatures appeared dominant in resistance to inactivation. These data will permit predictions of growth and survival of C. perfringens during LTLT cooking of beef roasts.     

Effect of temperature,calcium and protein concentration on aggregation of whey protein isolate: Formation of gel-like micro-particles

Protein aggregation occurs in biological systems and industrial processes, affecting protein solubility and functional properties. In this study, whey protein isolate (WPI) obtained from bovine milk was used as a model to study the dependence of aggregation on pre-heating temperature and on protein and calcium concentrations. WPI solutions (0.1–5.0%, w/v) were heated at 25–85 °C for 30 min prior to cooling and calcium addition. Tryptophan shifted to a more hydrophilic environment as WPI concentrations and pre-heating temperatures increased. Pre-heated WPI solutions yielded soluble particles, which aggregated to form porous gel-like particles by addition of calcium chloride. WPI microgel particles could be prepared by using a cold gelation method and preheated the protein above 65 °C. The particle size was monodisperse with sizes of about 190 nm and 255 nm, respectively in solutions pre-heated to 75 or 85 °C and containing 5 mm calcium.     

Sorbate-induced cross-protection in necrotoxigenic Escherichia coli

Three strains of necrotoxigenic Escherichia coli when pretreated with potassium sorbate at pH 7, when sorbate is predominantly in the anionic form, showed an elevated resistance to sodium chloride (27% w/v) compared to a control (no sorbate at pH 7). However, when the strains were subjected to sorbate at pH 5 and 4·75, when a large proportion of sorbate is protonated, the resistance to sodium chloride was reduced. Prior stress with sorbate at pH 7 also induced heat resistance (56°C for 80 min, one strain only tested). By using dialysis, the observed heat resistance was found to be mediated by an extracellular component that was identified tentatively as a protein with a molecular mass greater than 10 kDa. Although this protein was heat resistant at 75°C for 20 min, it was inactivated at 100°C for 20 min. Since necrotoxigenic strains of E. coli have been isolated from cheese and the preservatives sorbate and salt are sometimes used in cheese, this work emphasizes the need for further studies in this area to allow a more accurate risk assessment.     

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

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°C. From the D values of the spores determined in the temperature range of 113–121°C, z value (±SD) and D_90°C value were estimated to be 10.16±0.90°C and 1,071.52 min, respectively, and the inactivation rates were predicted to be only approximately 2% at 90°C for up to 10 min. Meanwhile, the viable count of spores was significantly higher when activated under a heat-shock condition of 90°C for over 9 min than those activated for shorter time periods. The heat tolerance of vegetative cells was extremely low, showing a D_90°C value (±SD) of 0.21±0.01 min. Finally, 3 different heat-shock conditions were compared: 70°C for 30 min, 80°C for 20 min, and 90°C for 10 min, and the experimental comparative data showed no significant differences in viable spore counts. Consequently, these results support that the heat-shock treatment at 90°C for 10 min is suitable to activate spores and to inactivate vegetative cells of C. sporogenes.     

Changes in heat tolerance of Escherichia coli O157:H7 after exposure to acidic environments

Acid-adapted bacterial cells are known to have enhanced tolerance to various secondary stresses. However, a comparison of heat tolerance of acid-adapted and acid-shocked cells of Escherichia coli O157:H7 has not been reported. D - and z -values of acid-adapted, acid-shocked, and control cells of an unusually heat-resistant strain (E0139) of E. coli O157:H7, as well as two other strains of E. coli O157:H7, were determined based upon the number of cells surviving heat treatment at 52, 54 or 56°C in tryptic soy broth (pH 7·2) for 0, 10, 20 or 30 min. The unusual heat tolerance of E. coli O157:H7 strain E0139 was confirmed. D -values for cells from 24-h cultures were 100·2, 28·3, and 6·1 min at 52, 54 and 56°C, respectively, with a z -value of 3·3°C. The highest D -values of other E. coli O157:H7 strains were 13·6 and 9·2 min at 52 and 54°C, respectively, whereas highest D -values of non-O157:H7 strains were 78·3 and 29·7 min at 52 and 54°C. D -values of acid-adapted cells were significantly higher than those of unadapted and acid-shocked cells at all temperatures tested. In a previous study, we observed that both acid-adapted cells and acid-shocked cells of strain E0139 had enhanced acid tolerance. This suggests that different mechanisms protect acid-adapted and acid-shocked cells against subsequent exposure to heat or an acidic environment. The two types of cells should be considered separately when evaluating survival and growth characteristics upon subsequent exposure to different secondary stress conditions.     

Elevation of heat resistance of Clostridium sporogenes following heat shock

Heat shock of strains of spores of Clostridium sporogenes at 80 or 100°C increased their apparent resistance to heating at 121·1°C, with the exception of NCIB 10696. The presence of glucose (0·5 – 1·0%) in the sporulation medium reduced the percentage of spores which could be activated by heat. Within individual spore populations, some spores required more heat than others to induce activation. Spores of PA 3679 remained activated for 18 months at 4°C and heat shock did not alter the z value. Experiments using dimethylglutaric acid/NaOH buffer suggest that heat shock in acid conditions has maximum effect on heat resistance.     

GABA shunt mediates thermotolerance in Saccharomyces cerevisiae by reducing reactive oxygen production

The GABA shunt pathway involves three enzymes, glutamate decarboxylase (GAD), GABA aminotransferase (GAT) and succinate semialdehyde dehydrogenase (SSADH). These enzymes act in concert to convert glutamate (α‐ketoglutarate) to succinate. Deletion mutations in each of these genes in Saccharomyces cerevisiae resulted in growth defects at 45°C. Double and triple mutation constructs were compared for thermotolerance with the wild‐type and single mutant strains. Although wild‐type and all mutant strains were highly susceptible to brief heat stress at 50°C, a non‐lethal 30 min at 40°C temperature pretreatment induced tolerance of the wild‐type and all of the mutants to 50°C. The mutant strains collectively exhibited similar susceptibility at 45°C to the induced 50°C treatments. Intracellular reactive oxygen intermediate (ROI) accumulation was measured in wild‐type and each of the mutant strains. ROI accumulation in each of the mutants and in various stress conditions was correlated to heat susceptibility of the mutant strains. The addition of ROI scavenger N‐tert‐butyl‐α‐phenylnitrone (PBN) enhanced survival of the mutants and strongly inhibited the accumulation of ROI, but did not have significant effect on the wild‐type. Measurement of intracellular GABA, glutamate and α‐ketoglutarate during lethal heat exposure at 45°C showed higher levels of accumulation of GABA and α‐ketoglutarate in the uga1 and uga2 mutants, while glutamate accumulated at higher level in the gad1 mutant. These results suggest that the GABA shunt pathway plays a crucial role in protecting yeast cells from heat damage by restricting ROI production involving the flux of carbon from α‐ketoglutarate to succinate during heat stress. Copyright © 2013 John Wiley & Sons, Ltd.     

Effects of the repair treatment on improving the heat resistance of Lactiplantibacillus plantarum LIP-1

To improve the heat resistance of lactobacilli during spray-drying, we first investigated the effect of heat shock and repair treatment on the heat resistance of Lactiplantibacillus plantarum LIP-1, and the specific cell repair mechanism. Compared with control group, the reduction of the strain after the heat treatment (75 °C for 40 s) decreased from 1.05 to 0.36 log CFU/mL (initial cell counts 9.30 log CFU/mL) by heat shock (44 °C for 10 min). The residuals of the strain after heat-treated increased by 0.90 log CFU/mL by heat shock firstly, and then the repair treatment (30 °C for 10 min).During recovery period, the relative content of unsaturated long-chain fatty acids (C18:1n9 & C18:2n6) induced by heat shock proteins increased by 7.0%, and the amount of DnaK protein anchored on the cell membrane increased by 0.17 pg/mL. Cell membrane damage caused by heat shock was reduced and strain heat resistance was improved.Industrial relevanceOur research found that the repair treatment after heat shock reduced cell membrane damage caused by heat shock, which is a very promising technology for improving the heat resistance of L. plantarum LIP-1. This technology is also expected to be widely used in the preparation of LAB powders by spray-drying technology.     

Short communication: Salt tolerance of Lactococcus lactis R-604 as influenced by mild stresses from ethanol,heat, hydrogen peroxide,and UV light

Lactococcus lactis is a culture widely used in salt-containing dairy products. Salt hinders bacterial growth, but exposure to environmental stress may protect cells against subsequent stress, including salt. The objective of this study was to evaluate the salt tolerance of L. lactis R-604 after exposure to various stresses. The culture was subjected to 10% (vol/vol) ethanol for 30 min, mild heat at 52°C for 30 min, 15 mM hydrogen peroxide for 30 min, or UV light (254 nm) for 5 min and compared with a control. Starting with 5 log cfu/mL for all treatments, growth was determined in M17 broth with 5 NaCl concentrations (0, 1, 3, 5, and 7% wt/vol). Plating was conducted daily for 5 d. Salt tolerance was enhanced with mild heat exposure before growth in M17 broth with 5% (wt/vol) NaCl on d 3, 4, and 5, and with exposure to hydrogen peroxide and ethanol stresses before growth in M17 broth with 5% (wt/vol) NaCl on d 4 and 5. Exposure of this culture to mild heat, hydrogen peroxide, or ethanol before growth in M17 broth containing 5% (wt/vol) salt can enhance its survival, which could be beneficial when using it in salt-containing dairy products.     

Heat resistance of Byssochlamys nivea, Byssochlamys fulva and Neosartorya fischeri isolated from canned tomato paste

Cultures of heat resistant molds (20) were isolated from spoiled canned tomato paste in order to estimate the pasteurization efficiency applied to commercially canned products. Ascospores of nine strains grown on malt extract agar for 30 days at 30°C, survived heating at 85°C for 20 min when initial numbers were near 10⁵/mL. Of these heat resistant strains were identified: two Byssochlamys nivea, three Byssochlamys fulva and four Neosartorya fischeri strains. Ascospores of all cultures were more heat resistant in tomato juice than in phosphate buffer. Thermal death rate curves were nonlogarithmic but approached logarithmic death rates at higher temperatures. The thermal destruction time for 1 log₁₀ at 90°C was 1.5 min for a Byssochlamys nivea strain, 8.1 min for a Byssochlamys fulva strain and 4.4 to 6.6 min for Neosartorya fischeri strains.     

Heating and Storage Conditions Affect Survival and Recovery of Listeria monocytogenes in Ground Pork

The effect of heating rate, heating atmosphere, and meat age on survival of Listeria monocytogenes was examined in ground pork, as well as the ability of injured cells to recover during storage under air and vacuum packaging at 4 and 30°C. Significantly more survivors were observed when samples were heated at 1.3°C/min than at 8.0°C/ min. Cells inoculated into 3-month-old pork were more sensitive to heating than cells inoculated into fresh ground pork (D_62°C= 5.2 min and 7.7 min, respectively). More survivors were detected when the meat was heated aerobically than anaerobically. However, storage under vacuum at 4 or 30°C resulted in faster recovery compared with cells packaged in air. Thus, heating and packaging conditions affected ability of this pathogen to survive and recover after a heat process in pork.     

Differences in thermotolerance of variousEscherichia coliO157:H7 strains in a salami matrix

Three strains ofEscherichia coliO157:H7 (ATCC 43895, Ent C9490 and 380–94) were inoculated into salami and heated in water baths at 50, 55 or 60°C. At intervals between 1 and 360 min, salami samples were removed from the water bath and examined for the presence of survivingE. coliO157:H7. Samples were directly plated onto sorbitol MacConkey (SMAC) agar, and onto tryptone soya agar (TSA) with SMAC overlay. The number of sub-lethally damaged cells in each sample was estimated from the differences between the resultant direct (uninjured cells only) and overlay (total recovery) counts. In samples heated at 50°C, the percentage of cell injury ranged from 71·8–88% for all strains. In samples heated at 55°C the percentage of sub-lethally damaged cells in strains ATCC 43895 and Ent C9490 was significantly higher (P< 0·001) at 97% than that observed in strain 380–94 (64%). Cell injury was not measured at 60°C. There were significant differences between the derived decimal reduction times (D-values) related to the different strains ofE. coliO157:H7, the heat treatment applied and the recovery/enumeration agars used. Significant interstrain differences (P< 0·05) in thermotolerance were noted. Strain Ent C9490 was significantly more heat resistant at 50°C and 60°C (D-values of 116·9 and 2·2 min, respectively), while at 55°C strain 380–94 was more thermotolerant (D-value of 21·9 min). The implications of these findings for the design of studies investigating the heat resistance ofE. coliO157:H7 in fermented meat environments are discussed.     

Calcium-Aggregated Milk: a Potential New Option for Improving the Viability of Lactic Acid Bacteria Under Heat Stress

Heat-induced inactivation of viable cells restricts a wide range of application of spray drying in producing dried lactic acid bacteria (LAB) products. In the present study, an effective method to enhance the stability of LAB under heat stress has been identified. This was done by enhancing the heat stability of skim milk as the carrier. Here, skim milk was supplemented with 10 mM CaCl₂ and heated to 90 °C for 10 min to induce protein aggregation. Using this Ca-aggregated skim milk as carrier, the survival of five LAB strains tested was found two orders of magnitudes higher than that of an untreated milk after the heating at a rising temperature from about 25 to 70 °C within 45 s. Possible mechanisms of the protection were explored by comparing the residual viability, microstructure of the cell-contained milk, and changes of suspension particle sizes caused by heat treatment of the four carriers, i.e., untreated milk, Ca-added milk, heat-treated milk, and Ca-aggregated milk. Ca-aggregated milk induced the highest microbial heat stability among them, providing a thick and compact encapsulation around LAB cells before the heat treatment for inactivation. The viable cells could stay in a comparatively more stable extracellular environment. This work reveals potentially a new option for using milk protein aggregates as a protectant of microorganisms. A series of calcium-enriched probiotic products may be developed based on the described principles of the finding of the Ca-aggregated milk.     

Resistance of Yeast to Dry Heat

The dry heat resistance of 10 strains of yeast was investigated to develop data useful for the evaluation of aseptic systems for packaging acid products and which sterilize containers with hot air. Although three of the strains tested showed little survival at 110°C, four other strains had Duo-c values between 1 and 4 min. Torulopsis glabrata had a D_126.7o.C of 0.78 min. Saccharomyces strains showed the highest dry heat resistance, with the most heat resistant strain tested having a D_126.7o of 5 min. The z values for these strains ranged from 9.1° to 13.3°C.     

Heat resistance in Escherichia coli and its implications on ground beef cooking recommendations in Canada

This study assessed the adequacy of the current cooking recommendations in relation to heat resistant Escherichia coli by evaluating eight potentially heat resistant E. coli strains (four generic and four E. coli O157:H7) along with AW1.7. The D_60°C-values for these strains varied from 1.3 to 9.0 min, with J3 and AW1.7 being the least and most heat resistant strains, respectively. The D_60°C-values for E. coli 62 and 68 were similar and were not affected by growth medium, while the heat resistance of C37, J3, and AW1.7 varied with the growth medium. When heated in extra lean ground beef (100 g) in vacuum pouches, the mean D_54°C, D_57°C, and D_60°C-values were 44.8, 18.6, and 2.9 min for C37, 13.8, 6.9, and 0.9 min for J3, and 40.5, 9.1, and 6.1 min for AW1.7. Burger temperatures continued to rise after being removed from heat when the target temperature was reached, by 3–5°C, and resting of 1 min would result in a destruction of 133, 374 and 14 log C37, J3 and AW1.7. These findings along with the very low occurrence of heat resistant E. coli expected in ground beef show that cooking ground beef to 71°C should be adequate.